The mechanism of deterioration of the glucosinolate-myrosynase system in radish roots during cold storage after harvest

Food Chemistry 233 (2017) 60–68

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

The mechanism of deterioration of the glucosinolate-myrosynase system in radish roots during cold storage after harvest Jeong Gu Lee a, Sooyeon Lim a, Jongkee Kim b, Eun Jin Lee a,⇑ a b

Department of Plant Science, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea Department of Integrative Plant Science, Chung-Ang University, Anseong 456-756, Republic of Korea

a r t i c l e

i n f o

Article history: Received 27 September 2016 Received in revised form 13 April 2017 Accepted 17 April 2017 Available online 19 April 2017 Keywords: Cold storage Glucosinolate Isothiocyanate Myrosinase Raphanus sativus

a b s t r a c t The hydrolysis of glucosinolates (GSLs) by myrosinase yields varieties of degradation products including isothiocyanates (ITCs). This process is controlled by the glucosinolatemyrosinase (G-M) system. The major ITCs in radish roots are raphasatin and sulforaphene (SFE), and the levels of these compounds decrease during storage after harvest. We investigated the GM system to understand the mechanism behind the decrease in the ITCs in radish roots. Six varieties of radish roots were stored for 8 weeks at 0–1.5 °C. The concentrations of GSLs (glucoraphasatin and glucoraphenin) were maintained at harvest levels without significant changes during the storage period. However, SFE concentration and myrosinase activity remarkably decreased for 8 weeks. Pearson correlation analysis between ITCs, GSLs, and myrosinase activity showed that a decrease of SFE during storage had a positive correlation with a decrease in myrosinase activity, which resulted from a decrease of ascorbic acid but also a decrease of myrosinase activity-related gene expressions. Ó 2017 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction Radish (Raphanus sativus L.), a popular vegetable cultivated around the world, is a root vegetable of the Brassicaceae family that has numerous varieties. Radish roots are good sources of vitamin C, folic acid, minerals, polyphenols, and glucosinolates (GSLs) (Hanlon & Barnes, 2011). Radish roots can be consumed in different forms, both processed and fresh. Before radish roots are consumed, sometimes they are stored for 1–4 months. For long-term storage, cold storage (Thompson, 2003) or modified atmosphere packaging (MAP) (Schreiner, Huyskens-Keil, Krumbein, Prono-Widayat, & Lüdders, 2003) is recommended. The MAP material can help maintain appropriate oxygen and carbon dioxide levels (Sandhya, 2010). GSLs are nitrogen and sulfurcontaining secondary metabolites found in the Brassicaceae family (Rosa, Heaney, Fenwick, & Portas, 1997). Although the isothiocyanates (ITCs) have been found to be more than one thousand times more cytotoxic than the GSLs (Musk, Smith, & Johnson, 1995), intact GSLs also have direct biological activity to impact on the chemopreventive activity linked to cruciferous vegetable consumption (Abdull Razis, Bagatta, De Nicola, Iori, & Ioannides, 2010). All GSLs have a core structure like a b-thioglucoside moiety, a sulfonated oxime aglycone, and a vari-

⇑ Corresponding author.

able side chain derived from amino acids. To date, approximately 130 different side chains of GSLs have been reported. GSLs are classified by precursor amino acids and types of side chains. GSLs derived from methionine, leucine, or isoleucine are classified as aliphatic GSLs, those derived from phenylalanine or tyrosine are classified as aromatic GSLs, and those derived from tryptophan are classified as indolyl GSLs (Fahey, Zalcmann, & Talalay, 2001). In radish roots, the major GSLs are glucoraphasatin (GRH) and glucoraphenin (GRE), which are derived from methionine. GRH is the predominant GSL that accounts for approximately 74% of the total GSLs while GRE is the second most common GSL and accounts for less than 10% of the total GSLs in radish roots (Hanlon & Barnes, 2011; Yi et al., 2015). When plant tissue is damaged by mechanical treatments (chewing, chopping, or cutting) by insects or humans, myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1), separated from GSLs into vacuoles of myrosin cells (Bones & Rossiter, 2006), hydrolyzes GSLs to form ITCs, which have various biological functions in humans and plants. This overall process to form ITCs is called the glucosinolatemyrosinase (GM) system and described in Supplemental Fig. S1. Myrosinase is the only known enzyme that cleaves a thio-linked glucose found in nature. In the presence of water, myrosinase cleaves off the glucose from GSLs (Holley & Jones, 1985). However, myrosinase and its substrate GSLs are stored in separate and different cell types (Winde & Wittstock, 2011). While GSLs are stored in

E-mail address: [email protected] (E.J. Lee). http://dx.doi.org/10.1016/j.foodchem.2017.04.104 0308-8146/Ó 2017 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

J.G. Lee et al. / Food Chemistry 233 (2017) 60–68

‘‘S-cells” (Koroleva et al., 2000), myrosinase is stored as the tonoplast-like membrane surrounding myrosin grains within myrosin cells (Höglund, Lenman, & Rask, 1992). The myrosinase enzyme is encoded by the thioglucoside glucohydrolase (TGG) gene family. TGG4 (Atlg47600) has been shown to encode functional myrosinase and seems to have root-specific expression patterns (Andersson et al., 2009). In addition, myrosinase-binding protein (MBP) can interact with myrosinase and form a myrosinase complex. MBP has been known to contribute to myrosinase activity in different Brassica species (Capella, Menossi, Arruda, & Benedetti, 2001; Rask et al., 2000). Ascorbic acid is a known cofactor of myrosinase, serving as a base catalyst in GSL hydrolysis. For example, myrosinase isolated from radish roots showed an increase in activity from 2.06 µmol/min per mg of protein in the absence of ascorbic acid to 280 µmol/min per mg of protein in the presence of 500 µM ascorbic acid on the substrate allyl glucosinolate (Shikita, Fahey, Golden, Holtzclaw, & Talalay, 1999). The hydrolysis of GSLs by myrosinase can yield a variety of degradation products including ITCs, nitriles, thiocyanates, and epithionitriles, depending on pH and the presence of certain cofactors (Kissen, Rossiter, & Bones, 2009; Winde & Wittstock, 2011). ITCs are the most functional phytochemicals among GSL degradation products. In plants, ITCs are considered to play a role in the defense against herbivores (Beekwilder et al., 2008). ITCs have biological activity, such as antimicrobial (Esaki & Onozaki, 1982; Fahey et al., 2002; Lim, Han, & Kim, 2016), antioxidant (Barillari et al., 2006; Katsuzaki, Miyahara, Ota, Imai, & Komiya, 2004; Takaya, Kondo, Furukawa, & Niwa, 2003), and anticarcinogenic properties (Barillari et al., 2007; Hanlon, Webber, & Barnes, 2007; Yang, Teng, Qu, Wang, & Yuan, 2016) in human cell line and in mice. The major ITCs of radish roots, raphasatin (RH) and sulforaphene (SFE) (Scholl, Eshelman, Barnes, & Hanlon, 2011), are converted from GRH and GRE, respectively, by the activation of the G-M system (Hanlon & Barnes, 2011). In our previous study (Lim, Lee, & Kim, 2015), an over 35% reduction in SFE in radish roots was observed during cold storage for 8 weeks. In this study, we first tried to clear up the cause of ITC reduction of harvested radish roots based on the G-M system. The objective of this study was to understand the changes of the G-M system in harvested radish roots. We analyzed the ITC concentrations of RH and SFE in six varieties of radish roots. Additionally, GRH and GRE concentrations, myrosinase activity, and myrosinase-related genes (TGG4 and MBP2) expression, and ascorbic acid were analyzed. 2. Materials and methods 2.1. Plant materials Six varieties of radish roots (Raphanus sativus L.), 4 cultivars (Seoho, Cheonghwang, Junmuhumu, and Alpine), and 2 breeding lines (15FH352-1 and 15RA11-1), were harvested from an experimental field in Jeonju, Korea (Supplemental Fig. S2). After removing the leaves, radish roots were immediately transported to the laboratory. 2.2. Storage of radish roots Harvested radish roots were washed with 0.01% sodium hypochlorite for 3 min, washed twice with tap water, wiped with a paper towel to dry up, and pre-cooled at 2 °C for 6 h. Radish roots were covered with polyethylene film during storage. The storage temperature was 0–1.5 °C, and the relative humidity was maintained at 85–90%. Three radish roots were sampled at 0 and

61

8 weeks, respectively, and analyzed for ITCs concentration, GSL concentration, myrosinase activity, ascorbic acid concentration, and expressions of myrosinase-related genes of TGG4 and MBP2. The middle area of the roots was sliced to a 1cmthick disk and sliced into quarters. Sliced radish disks were stored in plastic bags at 95 °C or freeze dried. 2.3. Extraction of ITCs The ITCs in the radish roots were extracted using liquid-liquid extraction methods (Kim, Kim, & Lim, 2015) with slight modifications. Eight milliliters of methylene chloride (MC) and distilled water and 1 mL of 100 ppm benzyl ITC in MC as an internal standard were added to 500 mg of lyophilized radish root powder. To hydrolyze endogenous GSLs to ITCs by myrosinase, a mixture was placed in a water bath at 37 °C for 30 min. The hydrolyzed sample was added to 10 mL of MC and centrifuged at 2000g for 10 min. The MC layer was collected and filtered with filter paper containing anhydrous sodium sulfate to remove any water. The MC extract was evaporated to remove the MC under a nitrogen evaporator at 30 °C, re-dissolved in 1 mL of MC, and finally filtered with a 0.45-lm syringe filter. 2.4. Analysis of ITCs The ITC contents were analyzed by GC-MS using the method described by Kim, Lee, and Kim (1997). The analysis was performed on a TRACE1310 GC system (Thermo, USA) with ISQ LT mass spectrophotometer (Thermo, USA) and a DB-5 fused silica capillary column (0.25  30 mm, Agilent Technologies, USA). The oven temperature was set to increase from 50 °C to 310 °C at a rate of 5 °C/min. The injector was used in a split-less mode at 250 °C. The flow rate of the helium was 1 mL/min. The range of mass scan was from 35 m/z to 550 m/z. 2.5. Extraction of crude myrosinase Crude myrosinase was extracted using a previously described method (Lim et al., 2015) with slight modifications. Fifty grams of radish root were homogenized with 80 mL of extraction buffer (10 mM potassium phosphate containing 3 mM DTT, 1 mM EDTA, and 5% glycerol, pH 7.2) for 1 min. The mixture was immediately filtered with six layers of gauze and centrifuged at 6700  g for 30 min at 4 °C. The supernatant was saturated with ammonium sulfate to 55% and centrifuged at 6700  g for 20 min at 4 °C. The supernatant was saturated with ammonium sulfate to 80% and centrifuged at 6700  g for 50 min at 4 °C. The pellet was dissolved in 2 mL of desalting buffer (50 mM Tris-HCl, pH 8.6) and loaded onto a PD-10 column (GE healthcare, USA) equilibrated by 25 mL of desalting buffer. The desalted myrosinase was eluted with 3.5 mL of desalting buffer. 2.6. Assay of myrosinase activity The myrosinase activity was investigated using a GO assay kit (Lim et al., 2015). One hundred microliters of crude myrosinase solution was added to 800 lL of 33 mM potassium phosphate buffer and 100 lL of 2 mM sinigrin. The mixture was incubated at 37 °C for 30 min, placed in a heat block at 95 °C for 10 min to stop enzyme activity, and centrifuged at 19,700  g for 20 min. Fifty microliters of the supernatant was mixed with 100 lL of mixture regent in a 96-well plate, incubated at 37 °C for 30 min, and added to 100 lL of 12 N sulfuric acid to stop the enzyme activity. The absorbance of the mixture was measured by a Multiple Plate Reader Victor 3 (Perkin Elmer, USA) at 540 nm.

62

J.G. Lee et al. / Food Chemistry 233 (2017) 60–68

2.7. Extraction of desulfo-GSLs GSL contents were determined by the purification and desulfation of GSLs (ISO 9167-1:1995/A1:2013) and by applying the desulfo-GSLs analysis method (Yi et al., 2015). Ten milliliters of boiled 70% methanol were added to 200 mg of lyophilized radish root powder and placed in a water bath at 90 °C for 30 min, and centrifuged at 6950  g for 30 min. The supernatant was evaporated under a nitrogen evaporator at 40 °C and re-dissolved in 1 mL of 30% methanol. The methanol extract was loaded onto an ion exchange column (5 mL of volume, Thermo Scientific, USA) filled with 100 mg of DEAE sephadex-A25 resin (GE Healthcare, USA) and 1 mL of 2 M acetic acid. Next, the column was washed twice with 1 mL of buffer (20 mM sodium acetate, pH 5.0). Purified sulfatase (75 lL, 150 U/mL) was added onto the top of the column and incubated at room temperature for 16 h. Desulfo-GSLs were eluted 3 times with 0.5 mL of distilled water and freeze dried. The lyophilized desulfo-GSLs were dissolved in

0.5 mL of distilled water and filtered with a 0.45-lm syringe filter.

2.8. Analysis of desulfo-GSLs The desulfo-GSL extracts were analyzed using a YL 9100 HPLC system (Younglin Instrument, Korea) equipped with UV detector set at 229 nm and eclipse plus C-18 column (4.6  250 mm, Agilent Technologies, USA). The initial conditions of the mobile phase were set up to 2% acetonitrile in water and maintained for 5 min. The flow rate was held constant at 1.0 mL/min. A gradient mobile phase of water (A) and acetonitrile (B) separated the compounds by increasing B from 2% to 20% in 25 min and then switched to an additional linear gradient of 20% to 100% B up to 35 min. For desulfo-GSLs quantification, sinigrin monohydrate (SigmaAldrich, USA) was used as an external standard. The absorbance at 229 nm was monitored for various concentrations (0.0–2.0 mg/

Fig. 1. Changes in raphasatin (A) and sulforaphene (B) concentrations in six varieties of radish roots during storage at 0 °C. Vertical bars are the means ± SD (n = 3). *, significant at P < 0.05.

J.G. Lee et al. / Food Chemistry 233 (2017) 60–68

mL) of desulfo-sinigrin, and each GSL content was calculated based on the UV response factors of other GSLs relative to sinigrin.

2.9. Identification of desulfo-GSLs Identification of desulfo-GSLs was performed using a Q Exactive Hybrid Quadrupole-Orbitrap instrument (Thermo Scientific Co., USA) equipped with a Dionex Ultimate 3000 UHPLC system (Thermo Scientific Co., USA) (Yi et al., 2015). Ten lL of samples were separated on an INNO 10 column (2.0  100 mm, Young Jin Biochrom, Korea) at a 150 lL/min flow rate and a 35 °C column temperature. Solvent A (0.1% formic acid) and B (acetonitrile) were used as mobile phase. The initial composition of the mobile phase consisted of 90% A. The A decreased from 90% to 20% in 14 min and was maintained for the next 15 min for washing. The portion of solution A was switched to an additional linear gradient of 20– 90% B for up to 16 min and equilibrated for 20 min. Desulfo-GSLs were ionized using electrospray ionization (ESI) in the capillary column. The temperature of the capillary column was maintained at 320 °C, and the voltage was set to 3.5 kV. The N2 gas flow was

63

12 L/min, and the nebulizer pressure was 35 psi. The average scan time was 0.01 min, and the average time to change polarity was 0.02 min. The system was operated in both negative and positive modes and scanned from m/z 50 to 1000. After injection into the UPLC-ESI-MS/MS system, additional confirmation on the identified desulfo-GSLs was carried out by performing a selective ion scanning and matching with mass fragments of reference information (Yi et al., 2015). The data set for the identification of separated peaks was generated using the UPLC-MS/MS (Thermo Scientific Co., USA) system connected to an Xcalibur2 data system. The fragment patterns of desulfo-GSLs were shown in Supplemental Fig. S3.

2.10. RNA extraction and qRT-PCR expression analysis Total RNA was extracted from radish roots using HiGene total RNA Prep kit (Biofact, Korea). Radish roots were macerated in liquid nitrogen with a precooled mortar and pestle. Five hundred milligrams of ground radish roots were added to extraction solution and centrifuged for 3 min at 21,000g. The supernatant was

Fig. 2. Changes in glucoraphasatin (A) and glucoraphenin (B) concentrations in six varieties of radish roots during storage at 0 °C. Vertical bars are the means ± SD (n = 3). *, significant at P < 0.05.

64

J.G. Lee et al. / Food Chemistry 233 (2017) 60–68

washed twice with RNA washing solution, and then RNA was extracted with DEPC water. cDNA synthesis was performed using a amfiRivert platinum cDNA synthesis master mix kit (GenDEPOT, USA) for qRT-PCR. qRT-PCR was performed in a CMQE 500 (Cosmogenetech, Korea) at a final volume of 20 lL. Relative expressions were calculated with normalization against the expression of the radish Actin2 gene. The primers used for the qRT-PCR were listed in Supplemental Table S1. 2.11. Analysis of total ascorbic acid Total ascorbic acid analysis was conducted according to the methods described by Chebrolu, Jayaprakasha, Yoo, Jifon, and Patil (2012) with some modifications. Five grams of frozen radish root were homogenized with meta-phosphoric acid extraction solution and centrifuged for 10 min at 7000g. Five hundred microliters of supernatant were added to 500 lL of 10 mM DTT solution. The mixture was analyzed using YL 9100 HPLC system

(Younglin, Korea) with a UV detector set at 252 nm and ZORBAX NH2 column (4.6  250 mm, Agilent Technologies, USA). The mobile phase was 10 mM ammonium dihydrogen phosphate (pH 2.6) and flow rate was 1.0 mL/min. 2.12. Statistical analysis The data were statistically evaluated using SPSS 22.0 statistical software. Experiments were conducted in randomized designs with three replicates. The means and standard deviations were calculated and the means were compared by t-test at P < 0.05 (⁄) and P < 0.01 (⁄⁄). Correlation analysis was conducted by calculating the change rates, which were calculated by dividing 8 weeks by 0 weeks in each parameter for ITCs, GSL concentrations, and myrosinase activity, respectively. Partial least squaresdiscriminant analysis (PLS-DA) was conducted using the R statistical analysis program (version 3.0.1) to evaluate similarities among groups of multivariate data.

Fig. 3. Changes in myrosinase activity (A) and correlation analysis between sulforaphene, glucoraphenin (B), and myrosinase activity (C) in six varieties of radish roots during storage at 0 °C. Vertical bars are the means ± SE (n = 3). The correlation plot was based on linear regression analysis. r, Pearson correlation coefficient. ns, non-significant; *, significant at P < 0.05; **, significant at P < 0.01.

J.G. Lee et al. / Food Chemistry 233 (2017) 60–68

3. Results and discussion 3.1. ITC concentration during cold storage The GC–MS analysis of ITCs showed RH and SFE at 22.2 min and 29.1 min, respectively (Supplemental Fig. S4). The changes in the RH and SFE concentrations in radish roots during cold storage are shown in Fig. 1. The concentrations of ITCs gradually decreased in all varieties during the entire storage period, and this trend was more clearly noticeable in the SFE concentration than RH. RH decreased from 0.076 to 0.025 mmol/g dry weight in ‘Seoho’ radish roots (Fig. 1A). Significant reductions in SFE concentrations were observed in ‘Seoho,’ ‘Cheonghwang,’ ‘Alpine’, ‘15RA11-1’ radish roots at 8 weeks, decreasing to 45%, 54%, 71%, and 30% of the harvest SFE concentrations, respectively (Fig. 1B). From these results, we clearly confirmed the reduction of SFE concentrations in radish roots during cold storage after harvest. Similar results were reported by Lim et al. (2015), who confirmed that there

65

was a 39% reduction in the harvest SFE concentrations in the ‘Chungwoon plus’ and 28% in the ‘Taebaek’ radish roots for 8 weeks of cold storage. The chemical structure of RH is very unstable and easily degraded with 30 min after exposure to an aqueous solution, whereas SFE stably remained for 24 h (Scholl et al., 2011). The precise quantification of RH is virtually impossible considering its extraction procedure. Perhaps this explains why we obtained more significant results for SFE reduction than for RH. 3.2. GSL concentration during cold storage The HPLC analysis of GSLs showed eight types of GSLs in radish roots (Supplemental Fig. S5). We only quantified GRH and GRE concentrations because GRH and GRE were precursors of RH and SFE, respectively. Changes of GRH and GRE concentrations in radish roots during cold storage are presented in Fig. 2. In contrast to ITCs shown in Fig. 1, the changes of both GSLs, GRH and GRE, showed a no clear trend of increasing or decreasing in all varieties of radish

Fig. 4. PLS-DA score plot (left) and loading plot (right) of glucosinolates (GRH, glucoraphasatin; GRE, glucoraphenin), isothiocyanates (RH, raphasatin; SFE, sulforaphene), and myrosinase activity (MYR). SH, Seoho; FH, 15FH352-1; RA, 15RA11-1; CH, Cheonghwang; JM, Junmuhumu; AL, Alpine.

Fig. 5. Changes in the ascorbic acid concentration in six varieties of radish roots during storage at 0 °C. Vertical bars are the means ± SD (n = 3). **, significant at P < 0.01.

66

J.G. Lee et al. / Food Chemistry 233 (2017) 60–68

roots. Except for ‘Seoho’ and ‘15RA11-1’ radish roots (Fig. 2A), the concentration of GSLs was maintained at the initial harvest levels for 8 weeks. These results agreed with those of Rodrigues and Rosa (1999), who reported that glucoraphanin, the major GSL in broccoli, the concentration decreased only 4% for 5 days at 4 °C, and Rangkadilok et al. (2002), who reported that the GSL concentration of broccoli stored at 4 °C with MAP for 25 days remained at the harvest level. We could assume that because aliphatic GSLs of GRH and GRE are chemically stable compared to other aliphatic and indolyl GSLs (Hanschen, Lamy, Schreiner, & Rohn, 2014), GRH and GRE synthesized in radish roots did not degrade for up to 8 weeks of cold storage after harvest.

respectively. In our study, the decreasing activity of myrosinase was evident with the reduction of ITC in cold-stored radish roots. At the end of the storage period, myrosinase activity has been reduced by about 20% of the initial activity level in all radish cultivars. Lim et al. (2015) reported that there was a 13% reduction in the myrosinase activity in the ‘Chungwoon plus’ and 15% in the ‘Taebaek’ radish roots for 8 weeks of cold storage. A decrease of myrosinase activity might be associated with the postharvest senescence of radish roots. We thus investigated the myrosinase activity-related factors of both ascorbic acid concentration (Fig. 5) and the expression of myrosinase-related genes of TGG4 and MBP2 (Fig. 6).

3.3. Myrosinase activity during cold storage

3.4. PLS-DA analysis between ITC and GSL concentrations and myrosinase activity

The changes in myrosinase activity are shown in Fig. 3A. The myrosinase activities decreased in all radish roots during storage. Significant decreases of myrosinase activities were observed in ‘Seoho’, ‘Cheonghwang’, and ‘15FH352-1’ radish roots, showing 10%, 11%, and 15% reductions at the harvest levels of 8 weeks,

We conducted Pearson’s correlation analysis and PLS-DA to confirm the cause of the significant decrease in ITCs in radish roots during storage. Supplemental Fig. S6 showed that the RH concentration had a negative correlation with the GRH concentration

Fig. 6. Relative expressions of the TGG4 (A) and MBP2 (B) genes in six varieties of radish roots during storage at 0 °C. Vertical bars are the means ± SE (n = 3). *, significant at P < 0.05.

J.G. Lee et al. / Food Chemistry 233 (2017) 60–68

(r = 0.204ns) and had a positive correlation with the myrosinase activity (r = 0.174ns) with no statistical significance. The SFE concentration had a weak negative correlation with the GRE concentration (r = 0.130ns) (Fig. 3B) but had a definite positive correlation with the myrosinase activity (r = 0.684, P < 0.01) (Fig. 3C). The ITC and GSL concentration and the myrosinase activity in radish roots during cold storage were examined by PLS-DA (Fig. 4), with two principal components explaining 65.34% of the overall variance (46.52% and 18.82% for component 1 and component 2, respectively). In the PLS-DA score plot, samples at 0 weeks were located on the right side, and samples at 8 weeks were located on the left side. Additionally, we confirmed that the horizontal discrimination between each group for 0 weeks and 8 weeks was almost affected by the SFE concentration and the myrosinase activity in the PLS-DA loading plot. These results suggested that the SFE concentration (Fig. 1B) and the myrosinase activity (Fig. 3A) clearly decreased in radish roots during cold storage and the decrease in the SFE concentration could be affected by the decrease of myrosinase activity more than the decrease of GSL concentration.

67

monly occur during cold storage after harvest. We first investigated the cause of decreased ITCs in terms of the G-M system of radish roots after harvest and concluded that the deterioration of the G-M system resulting from reduced myrosinase activity is the main cause. In more detail, both decreases of ascorbic acid content and the expression of the MBP2 gene were the primary causes of the reduced myrosinase activity and finally inhibited the formation of ITCs in radish roots. The loss of overall cell viability may be the result of the deterioration of the G-M system in the stored radish roots via accelerated water loss or physical damage. Appropriate postharvest technology to maintain the ITC concentration, myrosinase activity, and freshness should be developed. Additionally, studies of the GM system will provide an advanced view to improve the quality of radish roots during storage, focusing on ITC concentration, myrosinase activity, and myrosinase-related factors, but not GSL concentration. Our findings could be applied to develop specific postharvest technology to retain more nutrients or to breed highly nutritious radish cultivars. Conflict of interest The authors declare no competing financial interest.

3.5. Ascorbic acid concentration during cold storage Acknowledgments According to Burmeister, Cottaz, Rollin, Vasella, and Henrissat (2000), myrosinase is strongly activated by ascorbic acid because ascorbic acid is a known cofactor of myrosinase, serving as a base catalyst in glucosinolate hydrolysis. Changes in the ascorbic acid concentration in radish roots during cold storage are shown in Fig. 5. The concentration of ascorbic acid significantly decreased in all radish roots during cold storage (P < 0.01). The ascorbic acid concentration decreased to 35%, 17%, 10%, 18%, 20%, and 19% of the harvest levels for ‘Seoho,’ ‘Cheongwhang,’ ‘Junmuhumu,’ ‘Alpine,’ ‘15FH352-1,’ and ‘15RA11-1’, respectively. The decrease of ascorbic acid concentration is a common phenomenon by which fruits and vegetables lose antioxidant activity and freshness after harvest. del Aguila et al. (2006) reported that there is a 2.5% reduction in the ascorbic acid concentration in the radish roots for 15 days cold storage at 1 °C. We assumed that the decrease of myrosinase activity in radish roots during cold storage might have resulted from the decrease of ascorbic acid concentration as a cofactor. 3.6. Quantitative analysis of myrosinase-related gene expression during cold storage We conducted qRT-PCR to confirm the relative expressions of TGG4 and MBP2 genes, which are involved in the biosynthesis and activity of the myrosinase enzyme. The relative expressions of the TGG4 and MBP2 genes in radish roots during cold storage are shown in Fig. 6. In ‘Cheonghwang’ and ‘Alpine’ radish roots, the expressions of the TGG4 gene increased; however, it decreased in ‘15RA11-1,’ ‘15FH352-1,’ ‘Seoho,’ and ‘Junmuhumu’ radish roots with no statistical significance (Fig. 6A). However, the expression of the MBP2 gene significantly decreased in ‘15RA11-1,’ ‘Seoho,’ and ‘Junmuhumu’ radishes, reducing to 65%, 63%, and 61% of the harvest levels at 8 weeks, respectively (Fig. 6B). As a result, we expected that the significant decrease of myrosinase activity shown in Fig. 3A was related to the down-regulation of the MBP2 gene (Fig. 6B), which is involved in the activation of myrosinase but not the lack of biosynthesis of myrosinase. 4. Conclusion Radish roots are healthy vegetables for human consumption; however, large decreases in bioactive compounds of ITCs com-

This work was supported by Basic Science Research Program (NRF-2016R1A1A1A05919210) through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology and by Aspiring Researcher Program through the Seoul National University (SNU) in 2014. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2017. 04.104. References Andersson, D., Chakrabarty, D., Bejai, S., Zhang, J., Rask, L., & Meijer, J. (2009). Myrosinases from root and leaves of Arabidopsis thaliana have different catalytic properties. Phytochemistry, 70, 1345–1354. Barillari, J., Cervellati, R., Costa, S., Guerra, M. C., Speroni, E., Utan, A., & Iori, R. (2006). Antioxidant and choleretic properties of Raphanus sativus L. sprout (Kaiware Daikon) extract. Journal of Agricultural and Food Chemistry, 54, 9773–9778. Barillari, J., Iori, R., Broccoli, M., Pozzetti, L., Canistro, D., Sapone, A., et al. (2007). Glucoraphasatin and glucoraphenin, a redox pair of glucosinolates of Brassicaceae, differently affect metabolizing enzymes in rats. Journal of Agricultural and Food Chemistry, 55, 5505–5511. Beekwilder, J., Van Leeuwen, W., Van Dam, N. M., Bertossi, M., Grandi, V., Mizzi, L., et al. (2008). The impact of the absence of aliphatic glucosinolates on insect herbivory in Arabidopsis. PLoS ONE, 3, e2068. Bones, A. M., & Rossiter, J. T. (2006). The enzymic and chemically induced decomposition of glucosinolates. Phytochemistry, 67, 1053–1067. Burmeister, W. P., Cottaz, S., Rollin, P., Vasella, A., & Henrissat, B. (2000). High resolution X-ray crystallography shows that ascorbate is a cofactor for myrosinase and substitutes for the function of the catalytic base. The Journal of Biological Chemistry, 275, 39385–39393. Capella, A., Menossi, M., Arruda, P., & Benedetti, C. (2001). COI1 affects myrosinase activity and controls the expression of two flower-specific myrosinase-binding protein homologues in Arabidopsis. Planta, 213, 691–0699. Chebrolu, K. K., Jayaprakasha, G. K., Yoo, K. S., Jifon, J. L., & Patil, B. S. (2012). An improved sample preparation method for quantification of ascorbic acid and dehydroascorbic acid by HPLC. LWT-Food Science and Technology, 47, 443–449. del Aguila, J. S., Sasaki, F. F., Heiffig, L. S., Ortega, E. M. M., Jacomino, A. P., & Kluge, R. A. (2006). Fresh-cut radish using different cut types and storage temperatures. Postharvest Biology and Technology, 40, 149–154. Esaki, H., & Onozaki, H. (1982). Antimicrobial action of pungent principles in radish root. Journal of Japan Society of Nutrition and Food Sciences, 35, 207–211. Fahey, J. W., Haristoy, X., Dolan, P. M., Kensler, T. W., Scholtus, I., Stephenson, K. K., et al. (2002). Sulforaphane inhibits extracellular, intracellular, and antibioticresistant strains of Helicobacter pylori and prevents benzo [a] pyrene-induced stomach tumors. Proceedings of the National Academy of Sciences, 99, 7610–7615.

68

J.G. Lee et al. / Food Chemistry 233 (2017) 60–68

Fahey, J. W., Zalcmann, A. T., & Talalay, P. (2001). The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry, 56, 5–51. Hanlon, P. R., & Barnes, D. M. (2011). Phytochemical composition and biological activity of 8 varieties of radish (Raphanus sativus L.) sprouts and mature taproots. Journal of Food Science, 76, C185–C192. Hanlon, P. R., Webber, D. M., & Barnes, D. M. (2007). Aqueous extract from Spanish black radish (Raphanus sativus L. Var. niger) induces detoxification enzymes in the HepG2 human hepatoma cell line. Journal of Agricultural and Food Chemistry, 55, 6439–6446. Hanschen, F. 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. Höglund, A. S., Lenman, M., & Rask, L. (1992). Myrosinase is localized to the interior of myrosin grains and is not associated to the surrounding tonoplast membrane. Plant Science, 85, 165–170. Holley, R. A., & Jones, J. D. (1985). The role of myrosinase in the development of toxicity toward Nematospora in mustard seed. Canadian Journal of Botany, 63, 521–526. Katsuzaki, H., Miyahara, Y., Ota, M., Imai, K., & Komiya, T. (2004). Chemistry and antioxidative activity of hot water extract of Japanese radish (daikon). BioFactors, 21, 211–214. Kim, J. W., Kim, M. B., & Lim, S. B. (2015). Formation and stabilization of raphasatin and sulforaphene from radish roots by endogenous enzymolysis. Preventive Nutrition and Food Science, 20, 119. Kim, M. R., Lee, K. J., & Kim, H. Y. (1997). Effect of processing on the content of sulforaphane of broccoli. Journal of the Korean Society of Food Science and Nutrition, 13, 422–426. Kissen, R., Rossiter, J. T., & Bones, A. M. (2009). The ‘mustard oil bomb’: Not so easy to assemble?! Localization, expression and distribution of the components of the myrosinase enzyme system. Phytochemistry Review, 8, 69–86. Koroleva, O. A., Davies, A., Deeken, R., Thorpe, M. R., Tomos, A. D., & Hedrich, R. (2000). Identification of a new glucosinolate-rich cell type in Arabidopsis flower stalk. Plant Physiology, 124, 599–608. Lim, S., Han, S., & Kim, J. (2016). Sulforaphene identified from radish (Raphanus sativus L.) seeds possesses antimicrobial properties against multidrug-resistant bacteria and methicillin-resistant Staphylococcus aureus. Journal of Functional Foods, 24, 131–141. Lim, S., Lee, E. J., & Kim, J. (2015). Decreased sulforaphene concentration and reduced myrosinase activity of radish root during cold storage. Postharvest Biology and Technology, 100, 219–225. Musk, S. R. R., Smith, T. K., & Johnson, I. T. (1995). On the cytotoxicity and genotoxicity of allyl and phenethyl isothiocyanates and their parent glucosinolates sinigrin and gluconasturtiin. Mutation Research, 348, 19–23.

Abdull Razis, A. F., Bagatta, M., De Nicola, G. R., Iori, R., & Ioannides, C. (2010). Intact glucosinolates modulate hepatic cytochrome P450 and phase II conjugation activities and may contribute directly to the chemopreventive activity of cruciferous vegetables. Toxicology, 277, 74–85. Rangkadilok, N., Tomkins, B., Nicolas, M. E., Premier, R. R., Bennett, R. N., Eagling, D. R., et al. (2002). The effect of post-harvest and packaging treatments on glucoraphanin concentration in broccoli (Brassica oleracea var. italica). Journal of Agricultural and Food Chemistry, 50, 7386–7391. Rask, L., Andréasson, E., Ekbom, B., Eriksson, S., Pontoppidan, B., & Meijer, J. (2000). Myrosinase: Gene family evolution and herbivore defense in Brassicaceae. Plant Molecular Biology, 42, 93–113. Rodrigues, A. S., & Rosa, E. A. S. (1999). Effect of post-harvest treatments on the level of glucosinolates in broccoli. Journal of the Science of Food and Agriculture, 79, 1028–1032. Rosa, E. A. S., Heaney, R. K., Fenwick, G. R., & Portas, C. A. M. (1997). Glucosinolates in crop plants. Horticultural Review, 19, 99–215. Sandhya (2010). Modified atmosphere packaging of fresh produce: Current status and future needs. LWT-Food Science and Technology, 43, 381–392. Scholl, C., Eshelman, B. D., Barnes, D. M., & Hanlon, P. R. (2011). Raphasatin is a more potent inducer of the detoxification enzymes than its degradation products. Journal of Food Science, 76, 504–511. Schreiner, M., Huyskens-Keil, S., Krumbein, A., Prono-Widayat, H., & Lüdders, P. (2003). Effect of film packaging and surface coating on primary and secondary plant compounds in fruit and vegetable products. Journal of Food Engineering, 56, 237–240. Shikita, M., Fahey, J. W., Golden, T. R., Holtzclaw, W. D., & Talalay, P. (1999). An unusual case of ‘uncompetitive activation’ by ascorbic acid: Purification and kinetic properties of a myrosinase from Raphanus sativus seedlings. Biochemical Journal, 341, 725–732. Takaya, Y., Kondo, Y., Furukawa, T., & Niwa, M. (2003). Antioxidant constituents of radish sprout (kaiware-daikon), Raphanus sativus L.. Journal of Agricultural and Food Chemistry, 51, 8061–8066. Thompson, A. K. (2003). Postharvest technology of fruits and vegetables. Oxford: Harlow, Blackwell Science Ltd.. Winde, I., & Wittstock, U. (2011). Insect herbivore counteradaptations to the plant glucosinolate-myrosinase system. Phytochemistry, 72, 1566–1575. Yang, M., Teng, W., Qu, Y., Wang, H., & Yuan, Q. (2016). Sulforaphene inhibits triple negative breast cancer through activating tumor suppressor Egr1. Breast Cancer Research and Treatment, 158, 277–286. Yi, G., Lim, S., Chae, W. B., Park, J. E., Park, H. R., Lee, E. J., et al. (2015). Root glucosinolate profiles for screening of radish (Raphanus sativus L.) genetic resources. Journal of Agricultural and Food Chemistry, 64, 61–70.