Glucosinolates in seeds, sprouts and seedlings of cabbage and black radish as sources of bioactive compounds

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1 Glucosinolates in seeds, sprouts and seedlings of cabbage and black radish as sources of bioactive compounds Gölge Sarıkamış, Arda Yıldırım, and Didem Alkan Ankara University, Faculty of Agriculture, Department of Horticulture, 06110, Dışkapı, Ankara, Turkey ( Received 24 November 2014, accepted 6 March Published on the web 10 March Sarıkamıs, G., Yıldırım, A. and Alkan, D Glucosinolates in seeds, sprouts and seedlings of cabbage and black radish as sources of bioactive compounds. Can. J. Plant Sci. 95: The glucosinolate content of seeds, sprouts and seedlings of white head cabbage and black radish were analyzed in order to determine changes in aliphatic and indole glucosinolates during seed germination and early seedling growth. Both species with distinct glucosinolate profiles exhibited higher levels of aliphatics and to a much lesser extent indoles. Glucosinolate content decreased in germinating seeds and seedlings upon imbibition, followed by a slight increase after 48 h of germination. Total glucosinolates were highest in young seedlings at true leaf stage compared with seeds and sprouts in both species. The findings of the present study demonstrate changes in glucosinolates from seed to sprout and seedling during germination and early seedling growth as a particularly prominent factor in maximizing the concentration of the bioactive compounds available for improved health benefits. Key words: Cabbage, radish, glucosinolates, seeds, sprouts, seedlings Sarıkamıs, G., Yıldırım, A. et Alkan, D Les glucosinolates dans les graines, les germes et les plantules de chou et de radis noir, source de compose s bioactifs. Can. J. Plant Sci. 95: Les auteurs ont analysé la teneur en glucosinolates des graines, des germes et des plantules de chou blanc et de radis noir pour savoir si des changements affectent les compose s aliphatiques et ceux a` groupe indole pendant la germination et au de but de la croissance. Les deux espe` ces ont un profil diffe rent sur le plan des glucosinolates et contiennent plus de composés aliphatiques que de compose s à groupe indole. La teneur en glucosinolates diminue dans les graines en germination et les plantules en raison de leur inhibition, puis elle augmente légèrement 48 h apre` s la germination. Chez les deux espe` ces, la concentration totale de glucosinolates est plus e levée chez les jeunes plantules parvenues au stade de la vraie feuille que dans les graines et les germes. Les constatations de cette e tude indiquent que les glucosinolates changent lorsque la plante passe de la graine a` la plantule, a` la germination et au de but de sa croissance, ce qui pourrait eˆtre un important facteur à conside rer lorsqu on souhaite optimiser la concentration de compose s bioactifs disponibles pour la santé. Cabbage (Brassica oleracea var. capitata L.) and black radish (Raphanus sativus L.) are members of the Brassicaceae and like all other members of the family, they contain a special group of phytochemicals known as glucosinolates. Glucosinolates are broken down into isothiocyanates or nitriles and to indoles by plant endogenous myrosinase (b-thioglucosidase) upon cellular disruption or by the intestinal microflora upon consumption. Several studies suggest that isothiocyanates and indoles may contain chemopreventive activity providing healthpromoting properties (Aggarwal and Ichikawa 2005; Liu et al. 2013; Caruso et al. 2014). Glucosinolates constitute a large group, including several different forms with different side chain structures. Consequently, the break-down compounds are also diverse. Among these compounds, isothiocyanates, particularly sulforaphane (1-isothiocyanato-4-methylsulphinylbutane) of the aliphatic group derived from glucoraphanin (4- methylsulphinylbutyl), have been extensively studied as bioactive compounds (Sakao and Singh 2012; Houghton et al. 2013; Li and Zhang 2013). The biological properties of isothiocyanates and their protective effects Mots clés: Chou, radis, glucosinolates, graines, germes, plantules through multiple mechanisms that include induction of NF-E2-related factor-2 (Nrf2)-mediated gene transcription, cell cycle arrest and induction of apoptosis were described (Navarro and Lampe 2011). Isothiocyanates such as raphasatin (4-methylsulfanyl-3-butenyl isothiocyanate), the break down molecule of glucoraphasatin from radish, reported as an inducer of rat hepatic phase II enzymes (Razis et al. 2012), reveal a direct antioxidant activity by effectively quenching oxidant molecules such as hydrogen peroxide and organic hydroperoxides (Valgimigli and Iory 2009). Zanichelli et al. (2012) reported a dose-dependent effect of sulforaphane and raphasatin, for protection against oxidative damage in human mesenchymal stem cells. They suggested that low doses of isothiocyanates revealed better results in terms of reducing oxidative damage than higher doses, which may rather have adverse effects. Brassica sprouts are becoming a popular fresh food as a fast and easy way to substantially increase the intake of Abbreviation: DW, dry weight Can. J. Plant Sci. (2015) 95: doi: /cjps

2 682 CANADIAN JOURNAL OF PLANT SCIENCE health-promoting glucosinolates. Many studies, therefore, focus on determining glucosinolate content in sprouts as a valuable source of bioactive compounds (Baenas et al. 2012; De Nicola et al. 2013). It is important to elucidate changes in glucosinolates occurring during germination and the early seedling growth period in order to maximize their amount for increased health benefits. The current study revealed changes in glucosinolates from seed to sprout and seedling of two Brassica species with distinct glucosinolate profiles in order to gain an overall insight into changes at these early growth stages. MATERIALS AND METHODS Plant Material Seeds of white head cabbage (Brassica oleracea var. capitata L.) cv. Yalova 1 and black radish (Raphanus sativus L.) cv. Karagulle, two cultivars grown commonly in Turkey, were purchased from commercial seed companies. Laboratory Germination Tests A germination test was conducted as four replicates (300 mg each) for each time point. Seeds of each replicate were weighed, surface sterilized in 1.5% sodium hypochloride, rinsed three times in distilled water and placed on filter paper (Filtrak, Germany) in a petri dish (9 cm) moistened with 4 ml of distilled water. The dishes were placed in an incubator at 228C in the dark. Samples were taken at each time point and their glucosinolate content was analyzed. Cabbage seeds and germinating seeds and sprouts were collected at 0 (seeds), 1 (24 h), 2 (48 h), 3 (72 h), 5 (120 h),7 (168 h), and 10 d (240 h); black radish seeds and germinating seeds and sprouts were collected at 0 (seeds), 1 (24 h), 2 (48 h), 3 (72 h), and 5 d (120 h). Laboratory Seedling Emergence Test White head cabbage and black radish seeds were sown in peatmoss (Plantaflor-Humus,Verkaufs-GmBH, Germany) and perlite (2:1) containing seedling trays (32 cm 20 cm6 cm) and irrigated with distilled water. The trays were transferred to a growth room with a controlled temperature of 22928C. Light was provided at seedling level by cool fluorescent lamps (72 mmm 1 s 1 ) with a 16-h light/8-h dark cycle. The relative humidity in the growth room was maintained over 75% to reduce evaporation from the surface. Samples were collected upon emergence at different time points (days 5, 7, 10, 13, 16, 20 and days 7, 10, 14, 17, 21 for white head cabbage and black radish, respectively), taking four replicates for each time point. Plant age (days) indicate days elapsed from sowing. Young seedlings were harvested from the surface, weighed (fresh mass), flash frozen in liquid nitrogen, and stored at 808C until lyophilized in a freeze drier prior to analyses. Analysis of Glucosinolates Glucosinolates were extracted and analyzed according to ISO , developed for rapeseed with some modifications, as described by Sarıkamıs et al. (2006). Accordingly, g of lyophilized and milled tissue was weighed and incubated in 70% (vol/vol) methanol. A volume of 3 ml of the extract was passed through an ion exchange column (DEAE SephadexTM A25, Amersham Biosciences) followed by a desulfation step with the enzyme sulphatase. Desulfoglucosinolates were analyzed by HPLC-UV (Shimadzu ) detection at Ankara University, Department of Horticulture. Waters Spherisorb 5mM ODS 2, mm analytical cartridge was used as the column and the analyses was carried out on a gradient of 99% water and 1% acetonitrile, at a flow rate of 1 ml min 1 for 24 min. The detection was performed at a wavelength of 229 nm. The peaks were identified using commercial pure standards of sinigrin, progoitrin, glucotrapeolin, glucoraphenin purchased from PhytoLab, Germany. Sinigrin and glucotrapeolin (16 mm) were used as the internal standards for the quantification of the peaks. Glucosinolates were quantified according to internal standards and expressed as mmol g 1 dry weight (DW). Correction factors were used during quantification for each compound as listed by Brown et al. (2003). Statistical Analysis The experiment was conducted according to completely randomized design. Experimental data were expressed as the mean9standard error of the mean with four replications (n 4). One-way analysis of variance and Duncan s multiple range test was used to determine the significance of differences at different time points during the growth period using SPSS 18.0 (SPSS Inc., Chicago, IL). Significant differences were evaluated at P B0.001 error level. RESULTS Growth and Developmental Parameters Changes in Fresh and Dry Weights of Germinating Seeds, Sprouts and Young Seedlings of White Head Cabbage The fresh weights of germinating seeds and sprouts increased with germination time (Fig. 1a, b). A rapid and significant weight increase was observed after 72 h (day 3) of germination to g, increasing about twofold at 120 h (day 5) to g and threefold at 168 h (day 7) reaching its highest level at g (PB0.001). The dry weights varied from g to g (P B0.001) (Fig. 1a). The fresh weights of young seedlings increased linearly post-emergence. The increase was statistically significant at each time point throughout the growing period and reached g (Fig. 1b). The dry weights increased significantly from to g in cabbage (Fig. 1b).

3 SARIKAMIS ET AL. * GLUCOSINOLATES IN CABBAGE AND RADISH 683 Fig. 1. Changes in fresh and dry weights (g) of (a) germinating seeds and sprouts (b) seedlings of white head cabbage. *Values are the means of four replicates. Error bars show the standard error of the mean. Changes in Fresh and Dry Weights of Germinating Seeds, Sprouts and Young Seedlings of Black Radish A similar trend was observed with black radish. The fresh weights of germinating seeds and sprouts increased significantly with germination time from to g at 120 h (P B0.001) (Fig. 2a). Significant differences were not observed in dry weights, which varied between and g (P 0.05) (Fig. 2 a). The fresh weights of black radish seedlings increased regularly post-emergence (P B0.001). The fresh weight reached g at the end of 3 wk (Fig. 2b). The dry weight on the other hand increased from to g in black radish (Fig. 2b). Analysis of Aliphatic and İndole Glucosinolates Changes in Glucosinolate Content of Seeds, Sprouts and Young Seedlings of White Head Cabbage Glucosinolate profiling of white head cabbage identified glucoraphanin (4-methylsulphinylbutyl) of the aliphatic group, the precursor of sulforaphane (1-isothiocyanato- 4-methylsulphinylbutane), as the predominant glucosinolate in seeds, sprouts and young seedlings of white head cabbage followed by glucoiberin (3-methylsulphinylpropyl), the precursor of iberin (1-isothiocyanato-3- methylsulfinylpropane) and the alkylsulphinyl homologue of glucoraphanin. Indole glucosinolates glucobrassicin (3-indolylmethyl), 4-methoxyglucobrassicin (4-methoxy-3-indolylmethyl), neoglucobrassicin (1-methoxy-3-indolylmethyl) and 4- hydroxyglucobrassicin (4-hydroxy-3-indolylmethyl) were also identified in white head cabbage seeds, sprouts and young seedlings. Quantification of glucosinolates in germinating cabbage seeds and sprouts revealed the highest total aliphatic glucosinolate content as mmol g 1 DW in nongerminated seeds, glucoraphanin being the predominant glucosinolate in seeds, representing 83.9% of the total glucosinolates in seeds. Upon imbibition, total aliphatic glucosinolate content in seeds decreased to mmol g 1 DW at 24 h (day 1) followed by a marked decrease reaching mmol g 1 DW at 48 h (day 2) (P B0.001). This pattern remained fairly stable until 120 h (day 5), followed by a slight increase at 168 h (day 7) and 240 h (day 10) of the germination period (Table 1 and Fig. 3a). A similar trend was observed with glucoraphanin as the predominant aliphatic glucosinolate. Young cabbage seedlings revealed a pattern of increasing aliphatic glucosinolates from emergence quantified as mmol g 1 DW at day 5 to the highest level reached at day 13 quantified as mmol g 1 DW, followed by a decrease to mmol g 1 DW at day 20 (Table 2 and Fig. 3b) (PB0.001). A similar Fig. 2. Changes in fresh and dry weights (g) of (a) germinating seeds and sprouts (b) seedlings of black radish. Values are the means of four replicates. Error bars show the standard error of the mean.

4 684 CANADIAN JOURNAL OF PLANT SCIENCE Table 1. Glucosinolate content (mmolg 1 DW) in germinating seeds and sprouts of cabbage z Germination time (h) Glucoiberin Glucoraphanin Total aliphatics Glucobrassicin 4-MetGBS y 1-MetGBS y 4-OHGBS y Total indoles a a a c d a ab ab a c d ab ab b a c c b ab b a c b b ab b a b a b ab b a a a b b b a a a b y 4-MetGBS, 4-methoxyglucobrassicin; 1-MetGBS, 1-methoxyglucobrassicin; 4-OHGBS, 4-hydroxyglucobrassicin. ad Different letters within the same column indicate statistically significant differences (PB0.001). trend was observed with glucoraphanin as the predominant aliphatic glucosinolate representing 8085% of the total glucosinolates in seedlings, while glucoiberin was detected at very low levels accounting for 3% of the total glucosinolates. Indole glucosinolates were at lower levels compared with aliphatics in seeds, sprouts and seedlings (Tables 1 and 2). In seeds, indole glucosinolates accounted for 10% of the total, while in germinating seeds, sprouts accounted for 1025% of the total glucosinolates. Although an increase of up to threefold was observed in cabbage seedlings at the end of 3 wk, towards the end of experimental period, reaching mmolg 1 DW, indoles still accounted for 25% of the total glucosinolates (Table 2, Fig. 3a, b). Comparing the results obtained from two concurrent experiments (laboratory germination and laboratory emergence tests) it was determined that young but fully established seedlings at true leaf stage contained higher amounts of total aliphatic and indole glucosinolates. Changes in Glucosinolate Content in Seeds, Sprouts and Young Seedlings of Black Radish Glucosinolate profiling of black radish revealed that glucoraphenin (4-methylsufinyl-3-butenyl) of the aliphatic group, the precursor of sulforaphene (4-methylsulfinyl- 3-butenyl isothiocyanate), was the predominant glucosinolate in black radish seeds and sprouts with lower levels of glucoraphasatin (4-methylsulfanyl-3-butenyl), also known as dehydroerucin, glucodehydroerucin, the Fig. 3. Aliphatic and indole glucosinolate content in cabbage (a) seeds and sprouts (b) seedlings; in radish (c) seeds and sprouts (d) seedlings. Values are the means of four replicates. Error bars show the standard error of the mean.

5 SARIKAMIS ET AL. * GLUCOSINOLATES IN CABBAGE AND RADISH 685 Table 2. Glucosinolate content (mmolg 1 DW) in young cabbage seedlings upon emergence z Plant age (d) y Glucoiberin Glucoraphanin Total aliphatics Glucobrassicin 4-MetGBS x 1-MetGBS x 4-OHGBS x Total indoles a c c c a ab a c c c ab b a b c c c ab a a b b b a a b ab a b ab a b a a ab b y Plant age (days) indicate number of days elapsed upon sowing. x 4-MetGBS, 4-methoxyglucobrassicin; 1-MetGBS, 1-methoxyglucobrassicin; 4-OHGBS, 4-hydroxyglucobrassicin. ac Different letters within the same column indicate statistically significant differences (PB0.001). precursor molecule of raphasatin (4-methylsulfanyl-3- butenyl isothiocyanate). Indole glucosinolates glucobrassicin, 4-methoxyglucobrassicin, neoglucobrassicin and 4-hydroxyglucobrassicin were also determined in black radish seeds, sprouts and young seedlings. Quantification of glucosinolates in germinating black radish seeds and sprouts revealed the highest total aliphatic glucosinolate content as mmol g 1 DW in nongerminated seeds. Among aliphatics, glucoraphenin, the predominant glucosinolate, was at its highest level in nongerminated seeds, quantified as mmol g 1 DW, representing almost 80% of the total glucosinolates in seeds, while glucoraphasetin ( mmol g 1 DW) accounted for only 3% of the total glucosinolates (Table 3). Upon imbibition, while glucoraphenin content gradually decreased until the end of the experimental period at 120 h (day 5), glucoraphasatin content increased from mmol g 1 DW at 24 h (day 1) to mmol g 1 DW at 120 h (day 5) in sprouts (Table 3, Fig. 3c) (P B0.001). Although total aliphatic glucosinolate content decreased for a while until 48 h (day 2) upon imbibition, due to the substantial increase in glucoraphasatin, total aliphatic glucosinolate content exhibited an increasing pattern from 72 h (day 3) till the end of the experimental period at 120 h (day 5) reaching mmolg 1 DW. Therefore, at the end of the germination period, while the glucoraphenin content decreased to mmol g 1 DW representing 52% of the total, glucoraphasatin reached mmol g 1 DW accounting for 37% of the total glucosinolates (Table 3, Fig. 3c). Black radish seedlings contained higher levels of glucorapahasatin (61% of the total glucosinolates) compared with glucoraphenin (35% of the total glucosinolates) at day 7. However, the amount of both compounds, and hence total aliphatics, tended to decrease in time from mmol g 1 DW to mmol g 1 DW, decreasing threefold at day 21 (Table 4, Fig. 3d). The decrease in both glucoraphenin and glucoraphasatin revealed a similar pattern. In terms of total aliphatic glucosinolates, the highest level was quantified in seedlings at days 7 and 10 (Table 4). In terms of indoles, neoglucobrassicin (1-methoxy-3- indolylmethyl) level was highest in nongerminated seeds and sprouts, followed by 4-hydroxyglucobrassicin at lower levels. Total indoles were highest in nongerminated seeds, quantified as mmolg 1 DW, increased slightly upon imbibition at 24 h (day 1) then decreased from 48 h (day 2) to the end of the germination period (Table 3, Fig. 3c). Indole glucosinolates were higher in radish seeds and sprouts than seedlings (Table 4, Fig. 3c, d). Similar to our findings with cabbage, fully established radish seedlings at true leaf stage contained higher amounts of aliphatic glucosinolates and low levels of indoles compared with black radish seeds and sprouts. DISCUSSION The chemoprotective effects of Brassica species are related to their glucosinolate content. Aliphatic glucosinolates are hydrolyzed to their corresponding isothiocyanates, which have been associated with health-promoting Table 3. Glucosinolate content (mmolg 1 DW) in germinating seeds and sprouts of black radish z Germination time (h) Glucoraphenin Glucoraphasetin Total aliphatics Glucobrassicin 4-MetGBS y 1-MetGBS y 4-OHGBS y Total indoles a c b a a a a c b a a a ab c b a a a ab b b a a a b a a a b a y 4-MetGBS, 4-methoxyglucobrassicin; 1-MetGBS, 1-methoxyglucobrassicin; 4-OHGBS, 4-hydroxyglucobrassicin. ac Different letters within the same column indicate statistically significant differences (PB0.001).

6 686 CANADIAN JOURNAL OF PLANT SCIENCE Table 4. Glucosinolate content (mmolg 1 DW) in young black radish seedlings upon emergence z Plant age (d) y Glucoraphenin Glucoraphasetin Total aliphatics Glucobrassicin 4-MetGBS x 1-MetGBS x 4-OHGBS x Total indoles a a b b a a b b ab a a a c c ab a a b c cd a a a b d d a a a b y Plant age (days) indicate number of days elapsed upon sowing. x 4-MetGBS, 4-methoxyglucobrassicin; 1-MetGBS, 1-methoxyglucobrassicin; 4-OHGBS, 4-hydroxyglucobrassicin. ad Different letters within the same column indicate statistically significant differences (PB0.001). properties. Therefore, higher levels of aliphatic glucosinolates are desirable. However, variation in the levels of glucosinolates exists, depending upon plant tissues and organs, developmental stage of the plant, environmental factors, and various stress factors during the growing period or post-harvest and processing, as reviewed by Sarıkamıs (2009). The amount of glucosinolates in broccoli sprouts has been found to be higher than in fully grown plants (Fahey et al. 1997). However, rapid changes in glucosinolates during germination and early seedling growth need to be evaluated as a particularly relevant factor in maximizing the concentration of the bioactive compounds. In the current study, unlike many other studies reported here, we used two concurrent experiments, including germination and emergence tests, to gain an overall insight into variations in glucosinolates at different time points, representing germination and early seedling growth. Glucoraphanin, of the aliphatic group, was identified as the major glucosinolate in cabbage seeds, sprouts and seedlings. Aliphatic glucosinolate synthesis is usually associated with the genetic background of individuals, resulting in different glucosinolate profiles among species and cultivars. The influence of genetics on aliphatic glucosinolate production in broccoli was extensively studied by Mithen et al. (2003) and Sarıkamıs et al. (2006) via a breeding program through the introgression of genomic segments from a wild Brassica to a commercial broccoli cultivar, also leading to the development of highglucosinolate broccoli. Indole glucosinolates were at much lower levels compared with aliphatics contrary to our previous field experiments with fully grown white head cabbage plants of the same cultivar, indoles being the predominating glucosinolates. Indole glucosinolates are usually associated with stress factors as a part of plant defence. Therefore, high levels of indole glucosinolates in field experiments were attributed to the exposure of plants to high temperatures during the growth period (Sarıkamıs et al. 2009). Germination begins with water absorption (imbibition) followed by activation of stored substances leading to radicle and hypocotyl growth. Significant changes occur during germination, including the interconversion and synthesis of new compounds. Decreases in glucosinolates upon imbibition were determined in both species. Consistent with our findings, Gu et al. (2011) reported a decrease in germinating broccoli seeds and sprouts to the lowest value at 48 h and an increase at 60 h, remaining constant until the end of the experiment at 72 h. Baenas et al. (2012), who studied intact glucosinolates in seeds and sprouts of some Brassica vegetables, reported that the general trend was a decrease over germination time. The decrease in glucosinolate levels has been suggested to be a consequence of selective glucosinolate metabolism as well as dilution of glucosinolate concentration during tissue expansion (Chen and Andreasson 2001). In Arabidopsis thaliana, Brown et al. (2003) reported a decrease in glucosinolate content during the germination period, suggesting and providing evidence for the catabolism of glucosinolates. While glucoraphenin was the major glucosinolate in seeds and sprouts, glucoraphasetin was identified as the major glucosinolate, together with substantial amounts of glucoraphenin in black radish seedlings, both compounds associated with potential health benefits (Montaut et al. 2010; Razis et al. 2012; Zanichelli et al. 2012; Song et al. 2013). Decrease in glucoraphenin in radish sprouts has previously been reported to be due to the action of glucosinolate reductase converting glucoraphenin to glucodehydroerucin (glucoraphasatin). However, in a recent report on protective health benefits of white radish prepared by O Hare et al. (2011) decrease in glucoraphenin levels during sprout growth is attributed partly to dilution in percent dry matter, and partly to cultivar effects. On the other hand, the increase in glucorapahasatin is more likely to be due to de novo synthesis of the compound according to evidence that increase in glucoraphasatin exceeded decrease in glucoraphenin levels, and the sum of glucoraphenin and glucoraphasatin increased with sprout growth (O Hare et al. 2011). Overall, both species with distinct glucosinolate profiles exhibited higher levels of aliphatics and to a much lesser extent indoles and are potential sources of bioactive compounds. Comparing glucosinolate contents, young seedlings contained higher levels of total glucosinolates as potential sources of health-promoting compounds.

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