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Dynamic variation of glucosinolates and isothiocyanates in broccoli sprouts during hydrolysis
Scientia Horticulturae 255 (2019) 128–133
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Dynamic variation of glucosinolates and isothiocyanates in broccoli sprouts during hydrolysis Chaoqun Leng1, Yuxuan Zhang1, Mian Wang, Pei Wang, Zhenxin Gu, Runqiang Yang
T
⁎
College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, People’s Republic of China
ARTICLE INFO
ABSTRACT
Keywords: Brassica oleracea var. italica Glucoraphanin Glucoerucin Sulforaphane Erucin 1-Isothiocyanato-butane
In this study, the glucosinolates (GLs) content and isothiocyanates (ITCs) formation in broccoli sprouts and their dynamic variation during hydrolysis were investigated. The results showed that 7 kinds of GLs were detected and glucoraphanin (GRA) was the dominating component. After hydrolysis, three kinds of ITCs and 2 kinds of nitriles were detected in broccoli sprouts, however, the corresponding nitriles of 4-isothiocyanato-1-butene and 1-isothiocyanato-butane were not detectable. This inferred that the inter-transformation among ITCs existed during sprouts hydrolysis. According to the identification, 4-isothiocyanato-1-butene had another source in addition to gluconapin (GNA), 1-isothiocyanato-butane stemmed from glucoerucin (GER). Moreover, the affinity of MYR in broccoli sprouts was stronger to GER than to GRA and other GLs.
1. Introduction Brassica vegetables sprouts have become popular because of their high levels of glucosinolates (GLs) (Guo et al., 2014b; Pająk et al., 2014). In intact Brassica vegetables sprouts cells, GLs are found in vacuoles, and spatially separated from myrosinase (MYR) (Guo et al., 2011). When tissue is damaged, GLs contact with MYR and are converted into isothiocyanates (ITCs), thiocyanates, nitriles, epithionitriles and oxazolidines (Tang et al., 2013). ITCs have been proven to present strong anti-cancer activities (Ferguson and Schlothauer, 2012; Traka and Mithen, 2009). In addition, they also have anti-inflammatory, antibacterial and cardioprotective activities (Traka and Mithen, 2009). Many researchers have focused on the investigation of the changes in GLs content and sulforaphane formation in broccoli during processing (Wang et al., 2012), cooking (Yuan et al., 2009) and seeds germination (Guo et al., 2014b). In broccoli, glucoraphanin (GRA) is the most important GL that could be converted into sulforaphane, comprising over 50% of the total GLs (Guo et al., 2018, 2011; Martinez-Villaluenga et al., 2010). Hence, as a natural and health-promoting food, broccoli sprouts have attracted increasing attention. Recently, researchers have mainly focused on the GLs profile and their distribution in Brassica vegetables (Bhandari et al., 2015; Guo et al., 2014b; Yang et al., 2016). They also showed that the category and content of GLs in Brassica vegetable sprouts were different from each other. Tong et al. (2015) detected 6 kinds of GLs, including two aliphatic GLs (4-Methylsulphinylbutyl and 4-Methylthiobutyl GLs),
⁎ 1
three indole GLs (3-Indolylmethyl, 1-Methoxy-3-Indolylmethyl and 4Methoxy-3-Indolylmethyl GLs) and one aromatic GL (2-Phenylethyl GL) in broccoli. These GLs are not the same as that indentified by Guo et al. (2014b). The results might be related to species and cultivars of Brassica vegetables. In addition to GLs, their hydrolysates, especially for ITCs, showed the important functions for human health. Usually, the kinds and the percentage of ITCs in hydrolysate of GLs determine the function. However, many factors such as EDTA (Guo et al., 2013), Fe2+ (Latté et al., 2011), vitamin C (Pérez-Balibrea et al., 2011) influenced the hydrolysates of GLs. Guo et al. (2013) detected obtained 7 kinds of ITCs in hydrolysates of broccoli sprouts (cv. Lvlingxiang). While only 3 kinds of ITCs were detected in the other broccoli cultivar (cv. Lvxiong), yet sulforaphane was the dominant ITC (Guo et al., 2016), suggesting that the types of ITCs are depend on cultivars of broccoli. In addition, our previous study showed that 6 GLs were identified from broccoli sprouts, while only 3 ITCs were identified and the precursor GL of 1isothiocyanato-butane was not detectable. ITCs or nitriles from other GLs that exist in broccolis sprouts were not detectable (Yang et al., 2016). These results imply that the vibration or reciprocal transformation of GLs or ITCs exist during broccoli sprouts hydrolysis. Hence, there is a hypothesis that GLs in broccoli sprouts would convert to each other and the formed ITCs are also unstable so as to convert into each other during hydrolysis. Thereby, the objectives of this study are to investigate the changes of the category and content of GLs and their hydrolysates during incubation trying to track the source of some ITCs including sulforaphane, 1-isothiocyanato-butane and
Corresponding author. Contributed equally.
https://doi.org/10.1016/j.scienta.2019.05.026 Received 7 December 2018; Received in revised form 8 May 2019; Accepted 9 May 2019 Available online 20 May 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
Scientia Horticulturae 255 (2019) 128–133
C. Leng, et al.
erucin.
Table 1 GLs and ITCs content in broccoli sprouts. GLs
Content (μmol/10 sprouts)
GLs hydrolysates
Relative content (%)
GRA GB 4MGB
2.38 ± 0.10a 0.05 ± 0.00d 0.30 ± 0.04c
63.31 6.76 23.35
GNA
0.01 ± 0.00e
GER 4HGB
0.55 ± 0.04b 0.03 ± 0.00e
Sulforaphane Sulforaphane nitrile 1-isothiocyanato-1butane 4-isothiocyanato-1butene Erucin nitrile
2. Materials and methods 2.1. Materials and reagents Broccoli seeds (Brassica oleracea var. italica cv. Youxiu) were ordered from Sakata Seed Corporation (Japan). Sulforaphane, 4-isothiocyanato-1-butene, 1-isothiocyanato-butane, benzonitrile, acetonitrile and methanol (HPLC grade) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). GRA (from broccoli) and glucoerucin (GER, from rocket) were purchased from Hangzhou Lin’an Tianhong Bio-Tech Co., Ltd. (Zhejiang, China). All other chemicals and reagents were of analytical grade and purchased from Shanghai Institute of Biochemistry (Shanghai, China).
2.43 4.17
GLS: glucosinolates, 4HGB: 4-hydroxy glucobrassicin, GB: glucobrassicin, 4MGB: 4-methoxyglucobrassicin, GNA: gluconapin, GRA: glucoraphanin, GER: glucoerucin. The data were analyzed using one-way ANOVA. The different letters in the same column represent significant differences at p < 0.05 .
2.2. Cultivation condition and treatments Seeds were rinsed in distilled water and sterilized with O3 for 15 min. They were then washed with distilled water and soaked in
Fig. 1. Changes of GLs content during hydrolysis of broccoli sprouts. GLS: glucosinolates, 4HGB: 4-hydroxy glucobrassicin, GB: glucobrassicin, 4MGB: 4-methoxyglucobrassicin, GNA: gluconapin, GRA: glucoraphanin, GER: glucoerucin. Control: The 10 broccoli sprouts were homogenized with 5 mL of distilled water. GER AD: The 10 broccoli sprouts were homogenized with 5 mL of GER solution (30 mmol/L). After, the homogenates were incubated in a water-bath at 37 °C for 0, 20, 40 and 60 min, respectively. ND: Not detectable. The data were analyzed using T-test. Asterisk represents significant differences between GER AD and the control at p < 0.05. 129
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Fig. 2. The degradation rate of GRA and GER during sprouts hydrolysis. Control: The 10 broccoli sprouts were homogenized with 5 mL of distilled water. GER AD: The 10 broccoli sprouts were homogenized with 5 mL of GER solution (30 mmol/L). After, the homogenates were incubated in a water-bath at 37 °C for 0, 20, 40 and 60 min, respectively.
distilled water at 30 °C for 4 h. Next, they were spread evenly on trays filled with vermiculite and irrigated with distilled water. The trays were transferred to a controlled environment chamber with a 16 h light/8 h dark cycle and air temperature of 25 °C. The sprouts were watered with 20 mL distilled water every 12 h. Finally, the 9-day-old sprouts were rapidly and gently collected and kept in polyethylene bags, then frozen in liquid nitrogen and stored at −70 °C for further measurements.
2.3. Broccoli sprouts hydrolysis Ten of 9-day broccoli sprouts were harvested and homogenized with 5 mL of cold distilled water (control) or 5.0 mL 30 mmol/L of GER (GER AD) on ice bath within a short time. The homogenates were transferred into a water bath to incubate at 37 °C for 0, 20, 40 and 60 min, respectively. After, the composition and levels of GLs and ITCs were analyzed. 130
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5975C Plus MS (Agilent Technologies Co. Ltd.) with a fused silica capillary Agilent Technology HP-5 ms (5% phenylmethyl siloxane) column (30 m ×0.25 mm i. d., 0.1 μm film thickness). The sulforaphane, 4-isothiocyanato-1-butene, 1-isothiocyanato-butane and nitriles content was measured according to the method found in Wang et al. (2012) with minor modifications. Sprouts hydrolysate was prepared as above. An Agilent 6890 N GC system (Agilent Technologies Co. Ltd., USA) with a flame ionization detector (FID) and a fused silica capillary Agilent Technology HP-5 ms (5% phenyl methyl siloxane) column (30 m × 0.25 mm i.d., film thickness 0.1 mm) was used for analysis. 2.6. Statistical analysis Experimental data were expressed as mean ± standard deviation (SD) with three replications (n = 3). SPSS 18.0 (SPSS Inc., Chicago, IL, USA) was applied for significant difference tests. Data were analyzed by Duncan’s multiple-range tests at p < 0.05. 3. Results 3.1. GLs composition and content of broccoli sprouts In this study, the six GLs were detected in broccoli sprouts (Table 1). There were three aliphatic GLs (GRA, GNA and GER) and three indole GLs (GB, 4MGB and 4HGB). Among them, aliphatic GLs accounted for 86.76% of the total GLs, especially GRA content was more than 70% of the total GLs. Five kinds of hydrolysates were detected in broccoli sprouts, including 3 kinds of ITCs and 2 kinds of nitriles (Table 1). Interestingly, the precursor of 1-isothiocyanato-butane was not detectable in sprouts hydrolysate. Erucin, the hydrolysate of GER (with high content in broccoli sprouts) was not detectable, either, but erucin nitrile was detected. Consequently, it could be deduced that 1-isothiocyanato-butane was originated from GER. In addition, 4-isothiocyanato-1-butene showed a high content while its precursor GL (GNA) content was low in broccoli sprouts, revealing the reciprocal transformation among GLs and ITCs, respectively, during hydrolysis. 3.2. Dynamic changes of GLs and ITCs in broccoli sprouts during hydrolysis In order to authenticate the reciprocal transformation among GLs in the process of hydrolysis and the source of 1-isothiocyanato-butane, exogenous GER was added during broccoli sprouts hydrolysis, and then the changes in categories and content of GLs and ITCs were investigated.
Fig. 3. ITCs formation during hydrolysis. Control: The 10 broccoli sprouts were homogenized with 5 mL of distilled water. GER AD: The 10 broccoli sprouts were homogenized with 5 mL of GER solution (30 mmol/L). After, the homogenates were incubated in a water-bath at 37 °C for 0, 20, 40 and 60 min, respectively. The data were analyzed using two-way ANOVA. The different lower case letters in each index represent significant differences at p < 0.05.
3.2.1. Changes of GLs degradation rate during hydrolysis Fig. 1 illustrates that GLs content drastically decreased with hydrolysis time. After 20 min of hydrolysis, GLs were hydrolyzed completely in the control sprouts except 4MGB and GRA. However, when GER was added (GER AD), hydrolysis rate of other GLs was inhibited. After 40 min of hydrolysis, only GNA was hydrolyzed completely. GLs were degraded significantly in the sprouts during homogenate (data not shown) and their content went down gradually during hydrolysis. All GLs were nearly degraded after 60 min of hydrolysis (Fig. 2A). It demonstrated that MYR, existed in broccoli sprouts, showed a high efficiency on GLs degradation. Nevertheless, when GER was added during hydrolysis process, hydrolysis rate of other GLs dropped rapidly (Fig. 2B). As shown in Fig. 2C, the degradation rate of GRA increased gradually with hydrolysis time, especially sharp degradation (65.37%) occurred at 0–20 min in control group. Adding GER, GRA degradation rate also showed a linear incremental trend with hydrolysis time, but was dramatically inhibited (38.51% at 60 min of hydrolysis). In comparison with GRA, GER was degraded rapidly with the hydrolysis time. Within 20 min of hydrolysis, the 92.78% of GER
2.4. GLs content determination GLs were extracted and analyzed as previously reported by Yang et al. (2015) Sinigrin (Sigma, St. Louis, MO, USA) was added to each sample as an internal standard before the first extraction. The GL content was expressed as μmol/10 sprouts of broccoli. 2.5. ITCs composition and content determination The 10 fresh broccoli sprouts were homogenized in 5 mL of distilled water. The mixture was incubated at 37 °C for 3 h. Then, to extract the ITCs, 3 mL of methylene chloride was added to the homogenized sprouts, vibrated for 5 min and allowed to incubate for 30 min. The mixture was centrifuged at 10,000×g for 15 min. The composition of the ITCs and nitriles was determined according to the method of Yang et al. (2016) The sample was analyzed using a 7890A GC system with a 131
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Fig. 4. Metabolic profile of GRA and GER in broccoli sprouts during incubation.
was degraded in the control group. Although the degradation rate of GER was inhibited after exogenous GER addition, it still presented a linear increase, and showed a 65.10% of hydrolysis at 60 min (Fig. 2D).
hydrolysate erucin was not detectable while erucin nitrile was detected after hydrolysis (Table 1). In hydrolysates, 1-isothiocyanato-butane had the abundant content. Hence, it can be deduced that ITCs were transformed each other during hydrolysis. In order to confirm this hypothesis, we designed the hydrolysis experiment. The results showed that GNA, the precursor of 4-isothiocyanato-1-butene, was hydrolyzed during the first 20 min, but the content of 4-isothiocyanato-1-butene increased gradually and kept the higher relative content during the whole period of hydrolysis (Fig. 2). It demonstrated that 4-isothiocyanato-1-butene had other sources in addition to GNA, but it was not explicit. When GER was added during hydrolysis, the content of 1isothiocyanato-butane increased rapidly, thus inhibited the rate of sulforaphane formation (Fig. 3). It could be demonstrated that, on the one hand, the affinity which MYR acted to GER was obviously higher than that acted to GRA and other GLs, which was also revealed in Figs. 1 and 2. On the other hand, it was illustrated that 1-isothiocyanato-butane was really from GER. However, literature confirmed that erucin and erucin nitrile were hydrolysates of GER (Abbaoui et al., 2012; Angelino and Jeffery, 2013), although erucin was not detected in this study. In addition, erucin nitrile was detected in hydrolysates of broccoli sprouts, demonstrating that it is probable that erucin transformed into 1-isothiocyanato-butane immediately after formation. The other possibility is that 1-isothiocyanato-butane could be produced directly from GER. Recently, it was found that sulforaphane and eurcin could transform each other in human intestinal tract (Abbaoui et al., 2012). In summary, sulforaphane was derived from GRA, while 1-isothiocyanato-butane was derived from GER during broccoli sprouts (Fig. 4).
3.2.2. The changes of ITCs formation during hydrolysis In this study, only 3 kinds of ITCs were detected in the hydrolysates of broccoli sprouts (Fig. 3). Corresponding to GRA degradation (Fig. 2C), sulforaphane formation increased gradually with hydrolysis time. However, it was inhibited significantly after adding GER. There were no significant changes of sulforaphane formation during 0–40 min incubation, while it presented a little increase at 60 min (Fig. 3A). In the control group, sulforaphane showed the highest content while 4-isothiocyanato-1-butene showed the lowest content (Fig. 3C). However, 1isothiocyanato-butane became the highest content after adding GER (Fig. 3B), and the formation of the other two ITCs was inhibited significantly (Fig. 3A and C). Nevertheless, there was an upward trend in all kinds of ITCs formation with the hydrolysis time expand. 4. Discussion According to hydrolysates identification, three kinds of ITCs were detected. The results were consistent with that of Guo et al. (2014b). In this study, sulforaphane and 4-isothiocyanato-1-butene were the dominant ITCs. However, Guo et al. (2013) obtained 7 kinds of ITCs in hydrolysates of broccoli sprouts including other 4 kinds of ITCs in addition to 3 kinds mentioned in the present research, yet sulforaphane and 4-isothiocyanato-1-butene were the dominant ITCs. It is probably related to broccoli cultivars and hydrolysis environment (Guo et al., 2014a, 2013). Other researchers also investigated the metabolism of ITCs from broccoli sprouts in both mouse and human (Oliviero et al., 2014; Saha et al., 2012), but they just focused on the metabolism of acknowledged anti-cancer substances such as sulforaphane and erucin. Interestingly, scanty content of GNA was observed in broccoli sprouts (Table 1), but its hydrolysate 4-isothiocyanato-1-butene formation increased significantly with hydrolysis time, and accounted for a great proportion in ITCs (Fig. 3). Broccoli is rich in GER, but its
5. Conclusion Based on this study, the affinity of broccoli sprout MYR to GER was stronger than to other GLs. During hydrolysis, ITCs transformed each other and 1-isothiocyanato-butane was derived from GER in broccoli sprouts. 132
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Conflicts of interest
Guo, L., Zhu, Y., Wang, F., 2018. Calcium sulfate treatment enhances bioactive compounds and antioxidant capacity in broccoli sprouts during growth and storage. Postharvest Biol. Technol. 139, 12–19. Latté, K.P., Appel, K.-E., Lampen, A., 2011. Health benefits and possible risks of broccolian overview. Food Chem. Toxicol. 49, 3287–3309. Martinez-Villaluenga, C., Penas, E., Ciska, E., Piskula, M.K., Kozlowska, H., VidalValverde, C., Frias, J., 2010. Time dependence of bioactive compounds and antioxidant capacity during germination of different cultivars of broccoli and radish seeds. Food Chem. 120, 710–716. Oliviero, T., Verkerk, R., Vermeulen, M., Dekker, M., 2014. In vivo formation and bioavailability of isothiocyanates from glucosinolates in broccoli as affected by processing conditions. Mol. Nutr. Food Res. 58, 1447–1456. Pająk, P., Socha, R., Gałkowska, D., Rożnowski, J., Fortuna, T., 2014. Phenolic profile and antioxidant activity in selected seeds and sprouts. Food Chem. 143, 300–306. Pérez-Balibrea, S., Moreno, D.A., García-Viguera, C., 2011. Genotypic effects on the phytochemical quality of seeds and sprouts from commercial broccoli cultivars. Food Chem. 125, 348–354. Saha, S., Hollands, W., Teucher, B., Needs, P.W., Narbad, A., Ortori, C.A., Barrett, D.A., Rossiter, J.T., Mithen, R.F., Kroon, P.A., 2012. Isothiocyanate concentrations and interconversion of sulforaphane to erucin in human subjects after consumption of commercial frozen broccoli compared to fresh broccoli. Mol. Nutr. Food Res. 56, 1906–1916. Tang, L., Paonessa, J.D., Zhang, Y., Ambrosone, C.B., McCann, S.E., 2013. Total isothiocyanate yield from raw cruciferous vegetables commonly consumed in the United States. J. Funct. Food 5, 1996–2001. Tong, Y., Gabriel-Neumann, E., Krumbein, A., Ngwene, B., George, E., Schreiner, M., 2015. Interactive effects of arbuscular mycorrhizal fungi and intercropping with sesame (Sesamum indicum) on the glucosinolate profile in broccoli (Brassica oleracea var. Italica). Env. Exp. Bot. 109, 288–295. Traka, M., Mithen, R., 2009. Glucosinolates, isothiocyanates and human health. Phytochem. Rev. 8, 269–282. Wang, G.C., Farnham, M., Jeffery, E.H., 2012. Impact of thermal processing on sulforaphane yield from broccoli (Brassica oleracea L. Ssp. italica). J. Agric. Food Chem. 60, 6743–6748. Yang, R., Guo, L., Zhou, Y., Shen, C., Gu, Z., 2015. Calcium mitigates the stress caused by ZnSO4 as sulphur fertilizer and enhances sulforaphane formation of broccoli sprouts. RSC Adv. 5, 12563–12570. Yang, R., Hui, Q., Zhang, W., Zhou, Y., Guo, L., Shen, C., Gu, Z., 2016. Effects of CaCl2 on the metabolism of glucosinolates and the formation of isothiocyanates as well as the antioxidant capacity of broccoli sprouts. J. Funct. Food. 156–163. Yuan, G.-f., Sun, B., Yuan, J., Wang, Q.-m., 2009. Effects of different cooking methods on health-promoting compounds of broccoli. J. Zhejiang Univ. SC B 10, 580–588.
All authors declare no conflicts of interest. Acknowledgements The authors gratefully acknowledge the financial support provided by the China Postdoctoral Science Foundation (2015M570455) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References Abbaoui, B., Riedl, K.M., Ralston, R.A., Thomas-Ahner, J.M., Schwartz, S.J., Clinton, S.K., Mortazavi, A., 2012. Inhibition of bladder cancer by broccoli isothiocyanates sulforaphane and erucin: characterization, metabolism, and interconversion. Mol. Nutr. Food Res. 56, 1675–1687. Angelino, D., Jeffery, E., 2013. Glucosinolate hydrolysis and bioavailability of resulting isothiocyanates: focus on glucoraphanin. J. Funct. Food 7, 67–76. Bhandari, S.R., Jo, J.S., Lee, J.G., 2015. Comparison of glucosinolate profiles in different tissues of nine brassica crops. Molecules 20, 15827–15841. Ferguson, L.R., Schlothauer, R.C., 2012. The potential role of nutritional genomics tools in validating high health foods for cancer control: broccoli as example. Mol. Nutr. Food Res. 56, 126–146. Guo, R., Yuan, G., Wang, Q., 2011. Effect of sucrose and mannitol on the accumulation of health-promoting compounds and the activity of metabolic enzymes in broccoli sprouts. Sci. Hort. 128, 159–165. Guo, Q., Guo, L., Wang, Z., Zhuang, Y., Gu, Z., 2013. Response surface optimization and identification of isothiocyanates produced from broccoli sprouts. Food Chem. 141, 1580–1586. Guo, L., Yang, R., Wang, Z., Guo, Q., Gu, Z., 2014a. Effect of NaCl stress on healthpromoting compounds and antioxidant activity in the sprouts of three broccoli cultivars. Int. J. Food Sci. Nutr. 65, 476–481. Guo, L., Yang, R., Wang, Z., Guo, Q., Gu, Z., 2014b. Glucoraphanin, sulforaphane and myrosinase activity in germinating broccoli sprouts as affected by growth temperature and plant organs. J. Funct. Food. 9, 70–77. Guo, L., Yang, R., Zhou, Y., Gu, Z., 2016. Heat and hypoxia stresses enhance the accumulation of aliphatic glucosinolates and sulforaphane in broccoli sprouts. Eur. Food Res. Technol. 242, 107–116.
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Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Dynamic variation of glucosinolates and isothiocyanates in broccoli sprouts during hydrolysis Chaoqun Leng1, Yuxuan Zhang1, Mian Wang, Pei Wang, Zhenxin Gu, Runqiang Yang
T
⁎
College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, People’s Republic of China
ARTICLE INFO
ABSTRACT
Keywords: Brassica oleracea var. italica Glucoraphanin Glucoerucin Sulforaphane Erucin 1-Isothiocyanato-butane
In this study, the glucosinolates (GLs) content and isothiocyanates (ITCs) formation in broccoli sprouts and their dynamic variation during hydrolysis were investigated. The results showed that 7 kinds of GLs were detected and glucoraphanin (GRA) was the dominating component. After hydrolysis, three kinds of ITCs and 2 kinds of nitriles were detected in broccoli sprouts, however, the corresponding nitriles of 4-isothiocyanato-1-butene and 1-isothiocyanato-butane were not detectable. This inferred that the inter-transformation among ITCs existed during sprouts hydrolysis. According to the identification, 4-isothiocyanato-1-butene had another source in addition to gluconapin (GNA), 1-isothiocyanato-butane stemmed from glucoerucin (GER). Moreover, the affinity of MYR in broccoli sprouts was stronger to GER than to GRA and other GLs.
1. Introduction Brassica vegetables sprouts have become popular because of their high levels of glucosinolates (GLs) (Guo et al., 2014b; Pająk et al., 2014). In intact Brassica vegetables sprouts cells, GLs are found in vacuoles, and spatially separated from myrosinase (MYR) (Guo et al., 2011). When tissue is damaged, GLs contact with MYR and are converted into isothiocyanates (ITCs), thiocyanates, nitriles, epithionitriles and oxazolidines (Tang et al., 2013). ITCs have been proven to present strong anti-cancer activities (Ferguson and Schlothauer, 2012; Traka and Mithen, 2009). In addition, they also have anti-inflammatory, antibacterial and cardioprotective activities (Traka and Mithen, 2009). Many researchers have focused on the investigation of the changes in GLs content and sulforaphane formation in broccoli during processing (Wang et al., 2012), cooking (Yuan et al., 2009) and seeds germination (Guo et al., 2014b). In broccoli, glucoraphanin (GRA) is the most important GL that could be converted into sulforaphane, comprising over 50% of the total GLs (Guo et al., 2018, 2011; Martinez-Villaluenga et al., 2010). Hence, as a natural and health-promoting food, broccoli sprouts have attracted increasing attention. Recently, researchers have mainly focused on the GLs profile and their distribution in Brassica vegetables (Bhandari et al., 2015; Guo et al., 2014b; Yang et al., 2016). They also showed that the category and content of GLs in Brassica vegetable sprouts were different from each other. Tong et al. (2015) detected 6 kinds of GLs, including two aliphatic GLs (4-Methylsulphinylbutyl and 4-Methylthiobutyl GLs),
⁎ 1
three indole GLs (3-Indolylmethyl, 1-Methoxy-3-Indolylmethyl and 4Methoxy-3-Indolylmethyl GLs) and one aromatic GL (2-Phenylethyl GL) in broccoli. These GLs are not the same as that indentified by Guo et al. (2014b). The results might be related to species and cultivars of Brassica vegetables. In addition to GLs, their hydrolysates, especially for ITCs, showed the important functions for human health. Usually, the kinds and the percentage of ITCs in hydrolysate of GLs determine the function. However, many factors such as EDTA (Guo et al., 2013), Fe2+ (Latté et al., 2011), vitamin C (Pérez-Balibrea et al., 2011) influenced the hydrolysates of GLs. Guo et al. (2013) detected obtained 7 kinds of ITCs in hydrolysates of broccoli sprouts (cv. Lvlingxiang). While only 3 kinds of ITCs were detected in the other broccoli cultivar (cv. Lvxiong), yet sulforaphane was the dominant ITC (Guo et al., 2016), suggesting that the types of ITCs are depend on cultivars of broccoli. In addition, our previous study showed that 6 GLs were identified from broccoli sprouts, while only 3 ITCs were identified and the precursor GL of 1isothiocyanato-butane was not detectable. ITCs or nitriles from other GLs that exist in broccolis sprouts were not detectable (Yang et al., 2016). These results imply that the vibration or reciprocal transformation of GLs or ITCs exist during broccoli sprouts hydrolysis. Hence, there is a hypothesis that GLs in broccoli sprouts would convert to each other and the formed ITCs are also unstable so as to convert into each other during hydrolysis. Thereby, the objectives of this study are to investigate the changes of the category and content of GLs and their hydrolysates during incubation trying to track the source of some ITCs including sulforaphane, 1-isothiocyanato-butane and
Corresponding author. Contributed equally.
https://doi.org/10.1016/j.scienta.2019.05.026 Received 7 December 2018; Received in revised form 8 May 2019; Accepted 9 May 2019 Available online 20 May 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
Scientia Horticulturae 255 (2019) 128–133
C. Leng, et al.
erucin.
Table 1 GLs and ITCs content in broccoli sprouts. GLs
Content (μmol/10 sprouts)
GLs hydrolysates
Relative content (%)
GRA GB 4MGB
2.38 ± 0.10a 0.05 ± 0.00d 0.30 ± 0.04c
63.31 6.76 23.35
GNA
0.01 ± 0.00e
GER 4HGB
0.55 ± 0.04b 0.03 ± 0.00e
Sulforaphane Sulforaphane nitrile 1-isothiocyanato-1butane 4-isothiocyanato-1butene Erucin nitrile
2. Materials and methods 2.1. Materials and reagents Broccoli seeds (Brassica oleracea var. italica cv. Youxiu) were ordered from Sakata Seed Corporation (Japan). Sulforaphane, 4-isothiocyanato-1-butene, 1-isothiocyanato-butane, benzonitrile, acetonitrile and methanol (HPLC grade) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). GRA (from broccoli) and glucoerucin (GER, from rocket) were purchased from Hangzhou Lin’an Tianhong Bio-Tech Co., Ltd. (Zhejiang, China). All other chemicals and reagents were of analytical grade and purchased from Shanghai Institute of Biochemistry (Shanghai, China).
2.43 4.17
GLS: glucosinolates, 4HGB: 4-hydroxy glucobrassicin, GB: glucobrassicin, 4MGB: 4-methoxyglucobrassicin, GNA: gluconapin, GRA: glucoraphanin, GER: glucoerucin. The data were analyzed using one-way ANOVA. The different letters in the same column represent significant differences at p < 0.05 .
2.2. Cultivation condition and treatments Seeds were rinsed in distilled water and sterilized with O3 for 15 min. They were then washed with distilled water and soaked in
Fig. 1. Changes of GLs content during hydrolysis of broccoli sprouts. GLS: glucosinolates, 4HGB: 4-hydroxy glucobrassicin, GB: glucobrassicin, 4MGB: 4-methoxyglucobrassicin, GNA: gluconapin, GRA: glucoraphanin, GER: glucoerucin. Control: The 10 broccoli sprouts were homogenized with 5 mL of distilled water. GER AD: The 10 broccoli sprouts were homogenized with 5 mL of GER solution (30 mmol/L). After, the homogenates were incubated in a water-bath at 37 °C for 0, 20, 40 and 60 min, respectively. ND: Not detectable. The data were analyzed using T-test. Asterisk represents significant differences between GER AD and the control at p < 0.05. 129
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Fig. 2. The degradation rate of GRA and GER during sprouts hydrolysis. Control: The 10 broccoli sprouts were homogenized with 5 mL of distilled water. GER AD: The 10 broccoli sprouts were homogenized with 5 mL of GER solution (30 mmol/L). After, the homogenates were incubated in a water-bath at 37 °C for 0, 20, 40 and 60 min, respectively.
distilled water at 30 °C for 4 h. Next, they were spread evenly on trays filled with vermiculite and irrigated with distilled water. The trays were transferred to a controlled environment chamber with a 16 h light/8 h dark cycle and air temperature of 25 °C. The sprouts were watered with 20 mL distilled water every 12 h. Finally, the 9-day-old sprouts were rapidly and gently collected and kept in polyethylene bags, then frozen in liquid nitrogen and stored at −70 °C for further measurements.
2.3. Broccoli sprouts hydrolysis Ten of 9-day broccoli sprouts were harvested and homogenized with 5 mL of cold distilled water (control) or 5.0 mL 30 mmol/L of GER (GER AD) on ice bath within a short time. The homogenates were transferred into a water bath to incubate at 37 °C for 0, 20, 40 and 60 min, respectively. After, the composition and levels of GLs and ITCs were analyzed. 130
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5975C Plus MS (Agilent Technologies Co. Ltd.) with a fused silica capillary Agilent Technology HP-5 ms (5% phenylmethyl siloxane) column (30 m ×0.25 mm i. d., 0.1 μm film thickness). The sulforaphane, 4-isothiocyanato-1-butene, 1-isothiocyanato-butane and nitriles content was measured according to the method found in Wang et al. (2012) with minor modifications. Sprouts hydrolysate was prepared as above. An Agilent 6890 N GC system (Agilent Technologies Co. Ltd., USA) with a flame ionization detector (FID) and a fused silica capillary Agilent Technology HP-5 ms (5% phenyl methyl siloxane) column (30 m × 0.25 mm i.d., film thickness 0.1 mm) was used for analysis. 2.6. Statistical analysis Experimental data were expressed as mean ± standard deviation (SD) with three replications (n = 3). SPSS 18.0 (SPSS Inc., Chicago, IL, USA) was applied for significant difference tests. Data were analyzed by Duncan’s multiple-range tests at p < 0.05. 3. Results 3.1. GLs composition and content of broccoli sprouts In this study, the six GLs were detected in broccoli sprouts (Table 1). There were three aliphatic GLs (GRA, GNA and GER) and three indole GLs (GB, 4MGB and 4HGB). Among them, aliphatic GLs accounted for 86.76% of the total GLs, especially GRA content was more than 70% of the total GLs. Five kinds of hydrolysates were detected in broccoli sprouts, including 3 kinds of ITCs and 2 kinds of nitriles (Table 1). Interestingly, the precursor of 1-isothiocyanato-butane was not detectable in sprouts hydrolysate. Erucin, the hydrolysate of GER (with high content in broccoli sprouts) was not detectable, either, but erucin nitrile was detected. Consequently, it could be deduced that 1-isothiocyanato-butane was originated from GER. In addition, 4-isothiocyanato-1-butene showed a high content while its precursor GL (GNA) content was low in broccoli sprouts, revealing the reciprocal transformation among GLs and ITCs, respectively, during hydrolysis. 3.2. Dynamic changes of GLs and ITCs in broccoli sprouts during hydrolysis In order to authenticate the reciprocal transformation among GLs in the process of hydrolysis and the source of 1-isothiocyanato-butane, exogenous GER was added during broccoli sprouts hydrolysis, and then the changes in categories and content of GLs and ITCs were investigated.
Fig. 3. ITCs formation during hydrolysis. Control: The 10 broccoli sprouts were homogenized with 5 mL of distilled water. GER AD: The 10 broccoli sprouts were homogenized with 5 mL of GER solution (30 mmol/L). After, the homogenates were incubated in a water-bath at 37 °C for 0, 20, 40 and 60 min, respectively. The data were analyzed using two-way ANOVA. The different lower case letters in each index represent significant differences at p < 0.05.
3.2.1. Changes of GLs degradation rate during hydrolysis Fig. 1 illustrates that GLs content drastically decreased with hydrolysis time. After 20 min of hydrolysis, GLs were hydrolyzed completely in the control sprouts except 4MGB and GRA. However, when GER was added (GER AD), hydrolysis rate of other GLs was inhibited. After 40 min of hydrolysis, only GNA was hydrolyzed completely. GLs were degraded significantly in the sprouts during homogenate (data not shown) and their content went down gradually during hydrolysis. All GLs were nearly degraded after 60 min of hydrolysis (Fig. 2A). It demonstrated that MYR, existed in broccoli sprouts, showed a high efficiency on GLs degradation. Nevertheless, when GER was added during hydrolysis process, hydrolysis rate of other GLs dropped rapidly (Fig. 2B). As shown in Fig. 2C, the degradation rate of GRA increased gradually with hydrolysis time, especially sharp degradation (65.37%) occurred at 0–20 min in control group. Adding GER, GRA degradation rate also showed a linear incremental trend with hydrolysis time, but was dramatically inhibited (38.51% at 60 min of hydrolysis). In comparison with GRA, GER was degraded rapidly with the hydrolysis time. Within 20 min of hydrolysis, the 92.78% of GER
2.4. GLs content determination GLs were extracted and analyzed as previously reported by Yang et al. (2015) Sinigrin (Sigma, St. Louis, MO, USA) was added to each sample as an internal standard before the first extraction. The GL content was expressed as μmol/10 sprouts of broccoli. 2.5. ITCs composition and content determination The 10 fresh broccoli sprouts were homogenized in 5 mL of distilled water. The mixture was incubated at 37 °C for 3 h. Then, to extract the ITCs, 3 mL of methylene chloride was added to the homogenized sprouts, vibrated for 5 min and allowed to incubate for 30 min. The mixture was centrifuged at 10,000×g for 15 min. The composition of the ITCs and nitriles was determined according to the method of Yang et al. (2016) The sample was analyzed using a 7890A GC system with a 131
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Fig. 4. Metabolic profile of GRA and GER in broccoli sprouts during incubation.
was degraded in the control group. Although the degradation rate of GER was inhibited after exogenous GER addition, it still presented a linear increase, and showed a 65.10% of hydrolysis at 60 min (Fig. 2D).
hydrolysate erucin was not detectable while erucin nitrile was detected after hydrolysis (Table 1). In hydrolysates, 1-isothiocyanato-butane had the abundant content. Hence, it can be deduced that ITCs were transformed each other during hydrolysis. In order to confirm this hypothesis, we designed the hydrolysis experiment. The results showed that GNA, the precursor of 4-isothiocyanato-1-butene, was hydrolyzed during the first 20 min, but the content of 4-isothiocyanato-1-butene increased gradually and kept the higher relative content during the whole period of hydrolysis (Fig. 2). It demonstrated that 4-isothiocyanato-1-butene had other sources in addition to GNA, but it was not explicit. When GER was added during hydrolysis, the content of 1isothiocyanato-butane increased rapidly, thus inhibited the rate of sulforaphane formation (Fig. 3). It could be demonstrated that, on the one hand, the affinity which MYR acted to GER was obviously higher than that acted to GRA and other GLs, which was also revealed in Figs. 1 and 2. On the other hand, it was illustrated that 1-isothiocyanato-butane was really from GER. However, literature confirmed that erucin and erucin nitrile were hydrolysates of GER (Abbaoui et al., 2012; Angelino and Jeffery, 2013), although erucin was not detected in this study. In addition, erucin nitrile was detected in hydrolysates of broccoli sprouts, demonstrating that it is probable that erucin transformed into 1-isothiocyanato-butane immediately after formation. The other possibility is that 1-isothiocyanato-butane could be produced directly from GER. Recently, it was found that sulforaphane and eurcin could transform each other in human intestinal tract (Abbaoui et al., 2012). In summary, sulforaphane was derived from GRA, while 1-isothiocyanato-butane was derived from GER during broccoli sprouts (Fig. 4).
3.2.2. The changes of ITCs formation during hydrolysis In this study, only 3 kinds of ITCs were detected in the hydrolysates of broccoli sprouts (Fig. 3). Corresponding to GRA degradation (Fig. 2C), sulforaphane formation increased gradually with hydrolysis time. However, it was inhibited significantly after adding GER. There were no significant changes of sulforaphane formation during 0–40 min incubation, while it presented a little increase at 60 min (Fig. 3A). In the control group, sulforaphane showed the highest content while 4-isothiocyanato-1-butene showed the lowest content (Fig. 3C). However, 1isothiocyanato-butane became the highest content after adding GER (Fig. 3B), and the formation of the other two ITCs was inhibited significantly (Fig. 3A and C). Nevertheless, there was an upward trend in all kinds of ITCs formation with the hydrolysis time expand. 4. Discussion According to hydrolysates identification, three kinds of ITCs were detected. The results were consistent with that of Guo et al. (2014b). In this study, sulforaphane and 4-isothiocyanato-1-butene were the dominant ITCs. However, Guo et al. (2013) obtained 7 kinds of ITCs in hydrolysates of broccoli sprouts including other 4 kinds of ITCs in addition to 3 kinds mentioned in the present research, yet sulforaphane and 4-isothiocyanato-1-butene were the dominant ITCs. It is probably related to broccoli cultivars and hydrolysis environment (Guo et al., 2014a, 2013). Other researchers also investigated the metabolism of ITCs from broccoli sprouts in both mouse and human (Oliviero et al., 2014; Saha et al., 2012), but they just focused on the metabolism of acknowledged anti-cancer substances such as sulforaphane and erucin. Interestingly, scanty content of GNA was observed in broccoli sprouts (Table 1), but its hydrolysate 4-isothiocyanato-1-butene formation increased significantly with hydrolysis time, and accounted for a great proportion in ITCs (Fig. 3). Broccoli is rich in GER, but its
5. Conclusion Based on this study, the affinity of broccoli sprout MYR to GER was stronger than to other GLs. During hydrolysis, ITCs transformed each other and 1-isothiocyanato-butane was derived from GER in broccoli sprouts. 132
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Conflicts of interest
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