Effect of NaCl treatments on glucosinolate metabolism in broccoli sprouts

Effect of NaCl treatments on glucosinolate metabolism in broccoli sprouts 1 first-author 2 1 3 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:qmwang@zju.edu.cn †E-mail:qmwang@zju.edu.cn qmwang@zju.edu.cn Department of Horticulture, Zhejiang University, Hangzhou 310058, China Department of Horticulture, Zhejiang University, Hangzhou 310058, China School of Food and Pharmacy, Zhejiang Ocean University, Zhoushan 316000, China School of Food and Pharmacy, Zhejiang Ocean University, Zhoushan 316000, China Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Ministry of Agriculture, Hangzhou 310058, China Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Ministry of Agriculture, Hangzhou 310058, China To understand the regulation mechanism of NaCl on glucosinolate metabolism in broccoli sprouts, the germination rate, fresh weight, contents of glucosinolates and sulforaphane, as well as myrosinase activity of broccoli sprouts germinated under 0, 20, 40, 60, 80, and 100 mmol/L of NaCl were investigated in our experiment. The results showed that glucoerucin, glucobrassicin, and 4-hydroxy glucobrassicin in 7-d-old broccoli sprouts were significantly enhanced and the activity of myrosinase was inhibited by 100 mmol/L of NaCl. However, the total glucosinolate content in 7-d-old broccoli sprouts was markedly decreased although the fresh weight was significantly increased after treatment with NaCl at relatively low concentrations (20, 40, and 60 mmol/L). NaCl treatment at the concentration of 60 mmol/L for 5 d maintained higher biomass and comparatively higher content of glucosinolates in sprouts of broccoli with decreased myrosinase activity. A relatively high level of NaCl treatment (100 mmol/L) significantly increased the content of sulforaphane in 7-d-old broccoli sprouts compared with the control. These results indicate that broccoli sprouts grown under a suitable concentration of NaCl could be desirable for human nutrition. Glucosinolates Brassica oleracea Sulforaphane Myrosinase NaCl Introduction 1 Glucosinolates are a class of amino-acid-derived secondary metabolites, especially found in Arabidopsis thaliana and Brassica crops (Chen and Andreasson, 2001 2001 ; Fahey et al., 2001 2001 ). Under optimal metabolic conditions, vacuole-existed glucosinolates are generally spatially separated from their hydrolysis enzymes, known as myrosinases (thioglucoside glucohydrolase, EC 3.2.1.147), which are located in specific proteins of plant cells (van Eylen et al., 2006 2006 ; Kissen et al., 2009 2009 ). The myrosinases are distributed throughout the organs of the plant, including leaves, roots, flowers, fruit, and seeds (Textor and Gershenzon, 2009 2009 ). Glucosinolate-myrosinase is a unique substrate-enzyme system, in which myrosinases are responsible for glucosinolate turnover (van Eylen et al., 2006 2006 ). Glucosinolates themselves are biologically inactive and must be enzymatically hydrolyzed to produce bioactive compounds. When exposed to wounding or insect attack, the intact cells are disrupted, causing myrosinase to come into contact with glucosinolates and hydrolyze them to glucose and unstable intermediates. These unstable intermediates spontaneously decompose into hydrogen sulfate and isothiocyanates, nitriles, thiocyanates, or epithionitriles (van Eylen et al., 2006 2006 ). One of these decomposition products, sulforaphane, an isothiocyanate metabolite produced from glucoraphanin (4-methylsulphinylbutyl glucosinolate) by myrosinase, can protect rodents from chemically induced cancer and inhibit the development of cancer in transgenic mice. Sulforaphane is also found to prevent the growth of human cancer cells as well as cell cycle arrest and reactive oxygen species-dependent apoptosis (Sharma et al., 2010 2010 ). An appropriate consumption of cruciferous vegetables has been shown to be beneficial to human health (Okulicz, 2010 2010 ). The sprouts of broccoli ( Brassica oleracea var. italica ) are rich sources of glucosinolates and their hydrolysis products. For example, glucoraphanin, the main glucosinolate in broccoli sprouts, is present in sprouts in concentrations of 15 times of that in mature plants, while the content of sulforaphane, one of the degradation products of glucoraphanin, is 10 times higher than that of mature plants (Fahey et al., 1997 1997 ; Nakagawa et al., 2006 2006 ). Akhlaghi and Bandy ( 2010 2010 ) reported that eating broccoli sprouts for a relatively short time could notably reduce damage such as oxidative stress and cell death caused by ischemia-reperfusion on the heart (Akhlaghi and Bandy, 2010 2010 ). The glucosinolate-myrosinase system of plants is affected by various environmental and ontogenic factors. Temperature (van Eylen et al., 2008 2008 ), growth conditions such as nitrogen and sulfur assimilation (Pérez-Balibrea et al., 2010 2010 ), selenium application (Nyberg, 1991 1991 ), water deficiency (Schreiner et al., 2009 2009 ), and phenological stage (Velasco et al., 2007 2007 ) can all make an impact on the content of glucosinolates in plants. In addition, cooking methods may also affect the content of glucosinolates in Brassica rapa (Francisco et al., 2010 2010 ). The influence of salt stress on the primary metabolism such as photosynthesis and metabolism of antioxidants in mustard ( Brassica juncea L.) has been well documented (Khan et al., 2009 2009 ). Moreover, salt stress was also found to influence secondary metabolism in plants. It had also been demonstrated that the content of glucosinolates in radish ( Raphanus sativus L.) sprouts (Yuan et al., 2010 2010 ), canola ( Brassica napus L.) (Qasim et al., 2003 2003 ), and broccoli ( Brassica oleracea L.) florets (López-Berenguer et al., 2008 2008 ) can be changed by the application of NaCl. However, limited information is available about the influence of NaCl treatments on the metabolism of glucosinolates in broccoli sprouts. The aim of the present study was to evaluate the glucosinolate metabolism in broccoli sprouts under NaCl treatment by determining the glucosinolate composition and their contents. The activity of its hydrolysis enzyme, myrosinase, and the content of the main hydrolysis product, sulforaphane, in broccoli sprouts were also analyzed in the present study. Materials and methods 1 Plant materials and cultivation conditions 2 Seeds of broccoli ( Brassica oleracea var. italica cv. Youxiu) were ordered from the Sakata Seed Corporation (Japan). The seeds were cultivated and harvested as previously described (Yuan et al., 2009 2009 ) except that the treatment sprouts were grown on 0.5% agar (5 g/L) with 20, 40, 60, 80, or 100 mmol/L of NaCl added separately in culture flasks. For each treatment, three replicates were taken for analysis. Glucosinolate assay 2 Glucosinolates were extracted and analyzed as previously described with minor modifications (Yuan et al., 2009 2009 ). A total of 500 mg sprouts were boiled in 3 ml water for two times respectively, and 1 ml of the combined aqueous extract was applied to a DEAE-Sephadex A-25 (35 mg) column (GE Healthcare, Piscataway, NJ, USA). The desulfoglucosinolates were obtained according to the procedure of Yuan et al. ( 2009 2009 ). Then the extraction was analyzed by high performance liquid chromatography (HPLC). The glucosinolate concentration was expressed as μmol/g fresh weight of broccoli sprouts. Myrosinase activity determination 2 Myrosinase activity was determined using spectrophotometry as described previously by Yuan et al. ( 2009 2009 ). A total of 500 mg broccoli sprouts were ground with 1.8 ml of 50 mmol/L 2-( N -morpholino) ethanesulfonic acid (MES) buffer (pH 6.0) to homogenate at 0 °C, and then incubated at 25 °C for 5 min. After centrifuging at 10 000 r/min and 4 °C for 10 min, the supernatants were collected for measurements. The assay followed the procedure of Yuan et al. ( 2009 2009 ). The myrosinase activity was expressed as nmol glucose formed per minute and mg total protein. Sulforaphane measurement 2 Sulforaphane content was determined as described previously by Guo et al. ( 2011 2011 ). A total of 500 mg broccoli sprouts were homogenized with 2 ml of 50 mmol/L MES buffer solution (pH 6.0) at room temperature. After incubating for 5 min, the mixture was centrifuged at 8 000 r/min for 5 min, and then 400 μl supernatant was analyzed using a Shimadzu 2014 series gas chromatograph according to the procedure of Guo et al. ( 2011 2011 ). The sulforaphane concentration was expressed as mg/g fresh weight of broccoli sprouts. Statistical analyses 2 Statistical analysis was performed using the SPSS package program version 11.5 (SPSS Inc., Chicago, IL, USA). Data were analyzed by one-way analysis of variance (ANOVA), followed by Turkey’s Honestly Significant Difference (HSD) multiple comparison test. The values are reported as means with their standard error for all results. Differences were considered significant at P < 0.05. Results 1 Effect of NaCl on the germination rate and fresh weight in broccoli sprouts 2 The seed germination rate was not notably influenced by NaCl treatments at the concentrations of 20, 40, and 60 mmol/L, while it was inhibited at the concentrations of 80 and 100 mmol/L as compared to the control (Fig. 1a 1a ). Each data point is the mean of three replicates per treatment and time point (mean±standard error). Values not sharing a common letter are significantly different at P < 0.05 In short, the fresh weights of broccoli sprouts were markedly enhanced during the experimental period (Fig. 1b 1b ). The biomass of 3-d-old broccoli sprouts was significantly enhanced after treatments with 20, 40, and 60 mmol/L of NaCl, while it declined after application of 80 and 100 mmol/L of NaCl as compared to the control. The fresh weights of 5-d-old and 7-d-old broccoli sprouts were increased by applications of 20, 40, 60, and 80 mmol/L of NaCl. However, the fresh weights under treatment with 100 mmol/L of NaCl did not differ significantly from those of the control. Effect of NaCl on the content of glucosinolates in broccoli sprouts 2 The current experiment found seven kinds of glucosinolates in “Youxiu” broccoli sprouts (Table 1 1 ; Fig. 2 2 ). These included three aliphatic glucosinolates including glucoraphanin, glucoerucin, and glucoalyssin as well as four indole ones including 4-hydroxy glucobrassicin, glucobrassicin, 4-methoxy glucobrassicin, and neoglucobrassicin which were detected by HPLC. Among these glucosinolates, glucoraphanin was the most abundant one in “Youxiu” sprouts, accounting for 67% of total glucosinolate content. The most predominant indole glucosinolate was found to be 4-methoxy glucobrassicin (Table 2 2 ). Retention time (min) Abbreviation Trivial name Chemical name Relative response factor 7.787 GRA Glucoraphanin 4-Methylsulfinylbutyl- 0.9 12.410 GL Glucoalyssin 5-Methylthiopentyl- 0.9 15.669 4-OHIM 4-Hydroxy glucobrassicin 4-Hydroxy-indol-3-ylmethyl- 0.3 19.213 GER Glucoerucin 4-Methylthiobutyl- 0.9 20.951 IM Glucobrassicin Indol-3-ylmethyl- 0.3 22.928 4-IM 4-Methoxy glucobrassicin 4-Methoxy-indol-3-ylmethyl- 0.3 27.996 1-IM Neoglucobrassicin 1-Methoxyindol-3-ylmethyl- 0.2 * Standard: sinigrin (8.979 min) Each value is the mean of three replicates per treatment and time point (mean±standard error). Values not sharing a common letter are significantly different at P < 0.05. GLS: glucosinolate; GRA: glucoraphanin; GER: glucoerucin; GL: glucoalyssin; IM: glucobrassicin; 4-OHIM: 4-hydroxy glucobrassicin; 1-IM: neoglucobrassicin; 4-IM: 4-methoxy glucobrassicin 2 GLS 2 Time 6 Glucosinolate content (μmol/g fresh weight) 0 mmol/L NaCl 20 mmol/L NaCl 40 mmol/L NaCl 60 mmol/L NaCl 80 mmol/L NaCl 100 mmol/L NaCl 3 GRA Day 3 11.99±5.21a 9.57±0.59a 10.76±3.46a 10.50±3.43a 9.10±2.00a 8.91±4.13a Day 5 6.94±3.31a 5.12±0.40ab 5.89±0.95ab 6.20±0.95a 3.61±1.35b 2.43±0.71c Day 7 5.29±2.36a 2.58±1.04b 2.33±0.05b 2.36±0.05b 1.84±0.99b 2.70±0.83b 3 GL Day 3 0.12±0.03b 0.11±0.02b 0.12±0.03b 0.11±0.02b 0.19±0.03a 0.18±0.04a Day 5 0.07±0.02a 0.06±0.00ab 0.06±0.02ab 0.07±0.02a 0.06±0.00ab 0.05±0.02b Day 7 0.05±0.02a 0.03±0.00a 0.03±0.02a 0.03±0.02a 0.04±0.02a 0.06±0.02a 3 GER Day 3 5.42±0.95b 4.86±1.07b 5.65±2.15b 5.43±1.61b 12.30±3.71a 12.29±5.06a Day 5 4.34±1.33a 2.72±0.61a 3.09±0.81a 3.08±0.29a 5.30±2.94a 3.18±1.09a Day 7 2.09±1.30bc 1.39±0.36bc 1.16±0.43c 1.82±1.20bc 3.41±1.37b 5.76±2.41a 3 4-OHIM Day 3 0.85±0.52a 0.55±0.24a 0.72±0.21a 0.81±0.28a 1.02±0.26a 0.94±0.33a Day 5 0.30±0.31a 0.16±0.12a 0.13±0.05a 0.23±0.05a 0.22±0.02a 0.13±0.07a Day 7 0.10±0.07b 0.02±0.02c 0.02±0.02c 0.03±0.00c 0.07±0.02bc 0.20±0.02a 3 IM Day 3 0.41±0.05b 0.31±0.12b 0.34±0.05b 0.45±0.21b 0.94±0.31a 0.81±0.05a Day 5 0.16±0.14ab 0.10±0.03b 0.08±0.02b 0.15±0.07ab 0.22±0.07a 0.14±0.05ab Day 7 0.08±0.03b 0.02±0.00c 0.03±0.02c 0.04±0.03c 0.10±0.03b 0.29±0.02a 3 4-IM Day 3 0.15±0.05a 0.20±0.09a 0.15±0.05a 0.15±0.07a 0.21±0.05a 0.23±0.10a Day 5 0.32±0.10a 0.20±0.12ab 0.28±0.10ab 0.23±0.10ab 0.27±0.03ab 0.16±0.09b Day 7 0.35±0.09a 0.21±0.02b 0.19±0.05b 0.22±0.09ab 0.20±0.10b 0.27±0.16ab 3 1-IM Day 3 0.07±0.02a 0.04±0.00a 0.05±0.03a 0.06±0.03a 0.10±0.03a 0.09±0.00a Day 5 0.07±0.03a 0.03±0.02a 0.04±0.02a 0.04±0.00a 0.06±0.05a 0.03±0.02a Day 7 0.04±0.02c 0.02±0.00c 0.01±0.02c 0.02±0.02c 0.11±0.05b 0.17±0.02a 3 Total GLS Day 3 18.95±6.77a 15.58±0.74a 17.73±5.91a 17.46±5.44a 23.79±6.20a 23.38±9.62a Day 5 12.10±2.82a 8.32±0.81bc 9.48±1.66ab 9.92±0.97ab 9.66±2.56ab 6.05±1.97c Day 7 7.89±3.78ab 4.20±1.30b 3.71±0.38c 4.45±1.73b 5.72±2.49ab 9.35±3.36a Generally, total glucosinolates in 3-d-old broccoli sprouts treated with different concentrations of NaCl maintained at the same level as that in the control. However, the content of glucosinolates in 7-d-old broccoli sprouts was significantly decreased by 20, 40, 60, and 80 mmol/L of NaCl treatment and maintained the same level in broccoli sprouts treated by 100 mmol/L of NaCl as that in the control. Individual glucosinolate responded differently to the NaCl treatment. Glucoraphanin was decreased significantly in 7-d-old sprouts treated by different concentrations of NaCl. At the same time, glucoerucin and three kinds of indole glucosinolates, neoglucobrassicin, 4-hydroxy glucobrassicin, and glucobrassicin in 7-d-old sprouts were increased by relatively high concentration NaCl (100 mmol/L) treatment as compared to the control. On the other hand, low and medium concentrations of NaCl (20 and 40 mmol/L) treatments reduced the contents of glucobrassicin, 4-methoxy glucobrassicin, and 4-hydroxy glucobrassicin while they exerted no significant influence on the accumulations of neoglucobrassicin, glucoalyssin, and glucoerucin. Effect of NaCl on the activity of myrosinase in broccoli sprouts 2 As shown in Fig. 3 3 , the activity of myrosinase was increased with the growth of broccoli sprouts, which was consistent with the developmental decrease of the glucosinolates in sprouts. Treatment with 80 and 100 mmol/L of NaCl increased the activity of myrosinase in 5-d-old broccoli sprouts as compared to the control. However, the activity of myrosinase in 7-d-old sprouts was significantly inhibited after all the treatments with NaCl compared to the control. Each value is the mean of three replicates per treatment and time point (mean±standard error). Values not sharing a common letter are significantly different at P < 0.05 Effect of NaCl on the content of sulforaphane in broccoli sprouts 2 Sulforaphane, with glucoraphanin as substrate, was the main product of glucosinolate degradation catalyzed by myrosinase. The sulforaphane content in broccoli sprouts treated by various concentrations of NaCl during different developmental stages is shown in Fig. 4 4 and decreased with the growth of broccoli sprouts. The application of 100 mmol/L of NaCl significantly increased the content of sulforaphane in 3-d-old and 7-d-old broccoli sprouts over that of the control. The content of sulforaphane in 7-d-old broccoli sprouts treated by 100 mmol/L of NaCl was 2.1 times of that in the control sprouts. Each value is the mean of three replicates per treatment and time point (mean±standard error). Values not sharing a common letter are significantly different at P < 0.05 Discussion 1 The germination rate, fresh weight, contents of glucosinolates and sulforaphane, as well as myrosinase activity of broccoli sprouts under 0 (control), 20, 40, 60, 80, and 100 mmol/L of NaCl were investigated in the current study. Seed germination was inhibited by the application of 80 and 100 mmol/L of NaCl, which was similar to the results in other species of vegetable such as radish and beetroot (Zapata et al., 2004 2004 ; Yuan et al., 2010 2010 ). The fresh weights of 5-d-old and 7-d-old “Youxiu” sprouts treated with 20, 40, 60, and 80 mmol/L of NaCl were significantly heavier than that of the control. This was in accordance with the findings that 10 and 50 mmol/L of NaCl treatments increased the fresh weight of radish sprouts (Yuan et al., 2010 2010 ). This may be due to the nutrients that Na + and C1 − ions provide and the reserves of cotyledons which are sufficient to permit the sprouts to adjust to the osmotic stress, maintaining turgor and allowing the growth of sprouts (Scialabba and Melati, 1990 1990 ; Yuan et al., 2009 2009 ). The differences between the 3-d-old sprouts and 5- and 7-d-old sprouts treated with 80 and 100 mmol/L of NaCl indicated that the older the sprouts are, the more able to readily adapt to their environment. In the research by Aires et al. ( 2006 2006 ), the glucosinolates in broccoli include 3-methyl-sulfinylpropyl glucosinolate and 2-phenyl-ethyl glucosinolate, which were not found in the present study. However, two indole glucosinolates, 4-methoxy glucobrassicin (4-IM) and neoglucobrassicin (1-IM), were detected in our research. These discrepancies might be due to the different varieties and varied developmental stages used in our study. Moreover, glucoraphanin was still the most substantial glucosinolate in “Youxiu” sprouts. The glucosinolates in sprouts were found to decrease with sprout growth (Fahey et al., 1997 1997 ; Pereira et al., 2002 2002 ). This trend was also observed in the current survey. The contents of total as well as individual glucosinolates were significantly decreased with the growth of sprouts and the 3-d-old sprouts had higher contents of total as well as individual glucosinolates in our study. It had been observed that NaCl exerted a positive influence on the content of glucosinolates in canola ( Brassica napus L.) (Qasim et al., 2003 2003 ) and radish ( Raphanus sativus L.) sprouts (Yuan et al., 2010 2010 ). In the present study, the content of glucosinolates in 7-d-old broccoli sprouts under treatment of 100 mmol/L of NaCl stayed at the same level as that in the control. The different effects of NaCl on canola, radish, and broccoli sprouts could be due to the different glucosinolate profiles of these species. In the broccoli sprouts, glucoraphanin is the dominant glucosinolate while 2-hydroxy-3-butenyl glucosinolate and glucoraphasatin are dominant in Brassica napus and radish sprouts, respectively. The glucoraphanin content in 5- and 7-d-old broccoli sprouts was dropped significantly after treatment with NaCl. In contrast, the glucoerucin content in 7-d-old broccoli sprouts was raised by NaCl treatment. The rapid growth of sprouts benefiting from the nutrition of Na + and C1 − ions after treatment with NaCl of 40 mmol/L may be the reason for the decline of the glucosinolate contents in sprouts with the plant growth. Myrosinase was reported to be vulnerable to environmental variations such as phototropic stimulation (Yamada et al., 2003 2003 ) and methyl jasmonate treatment (Kim et al., 2006 2006 ). In our study, the myrosinase activity was increased in 5-d-old sprouts and inhibited in 7-d-old sprouts by 80 and 100 mmol/L of NaCl compared to the control, which was consistent with the change of glucosinolates in broccoli sprouts. Sulforaphane is affected by various factors, such as pressure (van Eylen et al., 2008 2008 ) and cooking methods (Jones et al., 2010 2010 ). The changing trend of sulforaphane content was similar with the content of glucosinolates, especially glucoraphanin in broccoli sprouts during developmental stages in the present study. The highest content of sulforaphane, the glucoraphanin degradation product, was found in 3-d-old broccoli sprouts and it decreased with the growth of broccoli sprouts. The reduction of sulforaphane content with the growth of sprouts could be due to the osmotic adjustment of glucosinolate degradation by myrosinase in the present study. Sulforaphane has been implicated in the cancer-protective effects of Brassica vegetables (Singh et al., 2009 2009 ; Sharma et al., 2010 2010 ). The accumulation of sulforaphane in 7-d-old sprouts treated with 100 mmol/L NaCl was 2.1 times of that in the control sprouts in the present study, suggesting that sprouts treated with high concentration of NaCl may be desirable from a health perspective. On the other hand, the myrosinase activity of sprouts treated by 100 mmol/L of NaCl was lower than that in the control. Therefore, this could be due to another mechanism for the sulforaphane formation from glucoraphanin. Conclusions 1 In conclusion, NaCl treatment has a concentration-dependent effect on glucosinolate-myrosinase system in broccoli sprouts. NaCl treatments at relatively low concentrations enhanced the growth of the broccoli sprouts. NaCl treatment at the concentration of 60 mmol/L for 5 d maintained higher biomass and comparatively higher content of glucosinolates in broccoli sprouts with decreased myrosinase activity. The application of high concentration, especially 100 mmol/L of NaCl, for 3 d can endow broccoli sprouts with abundant glucosinolates and sulforaphane. The 100 mmol/L NaCl treatment significantly increased the sulforaphane content in 7-d-old broccoli sprouts as compared to the control. 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Polyamines and ethylene changes during germination of different plant species under salinity 10.1631/jzus.B1200096 Project supported by the National High-Tech R&D Program (863) of China (No. 2008AA10Z111), the National Natural Science Foundation of China (No. 30900984), the Fok Ying Tong Education Foundation (No. 104034), and the Program for New Century Excellent Talents in University (No. NCET-05-0516), China 2012-03-26 2013-01-14 2012-07-02 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2013 2013 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 02 14