Glucosinolates in Seeds, Sprouts and Seedlings of Cabbage and Black Radish As Sources of Bioactive Compounds

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

Go¨lge Sarıkamıs¸, Arda Yıldırım, and Didem Alkan

Ankara University, Faculty of Agriculture, Department of Horticulture, 06110, Dıs¸kapı, Ankara, Turkey (e-mail: [email protected]). Received 24 November 2014, accepted 6 March 2015. Published on the web 10 March 2015.

Sarıkamıs¸, G., Yıldırım, A. and Alkan, D. 2015. Glucosinolates in seeds, sprouts and seedlings of cabbage and black radish as sources of bioactive compounds. Can. J. Plant Sci. 95: 681687. 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. 2015. 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: 681687. Les auteurs ont analyse´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 compose´s aliphatiques que de compose´sa` groupe indole. La teneur en glucosinolates diminue dans les graines en germination et les plantules en raison de leur inhibition, puis elle augmente le´ge` rement 48 h apre` s la germination. Chez les deux espe` ces, la concentration totale de glucosinolates est plus e´leve´e 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 a` conside´rer lorsqu’on souhaite optimiser la concentration de compose´s bioactifs disponibles pour la sante´.

Mots cle´s: Chou, radis, glucosinolates, graines, germes, plantules For personal use only.

Cabbage (Brassica oleracea var. capitata L.) and black through multiple mechanisms that include induction of radish (Raphanus sativus L.) are members of the NF-E2-related factor-2 (Nrf2)-mediated gene transcrip- Brassicaceae and like all other members of the family, tion, cell cycle arrest and induction of apoptosis were they contain a special group of phytochemicals known described (Navarro and Lampe 2011). Isothiocyanates as glucosinolates. Glucosinolates are broken down into such as raphasatin (4-methylsulfanyl-3-butenyl isothio- isothiocyanates or nitriles and to indoles by plant endo- cyanate), the break down molecule of glucoraphasatin genous myrosinase (b-thioglucosidase) upon cellular dis- from radish, reported as an inducer of rat hepatic phase ruption or by the intestinal microflora upon consumption. II enzymes (Razis et al. 2012), reveal a direct antioxidant Several studies suggest that isothiocyanates and indoles activity by effectively quenching oxidant molecules may contain chemopreventive activity providing health- such as hydrogen peroxide and organic hydroperoxides promoting properties (Aggarwal and Ichikawa 2005; (Valgimigli and Iory 2009). Zanichelli et al. (2012) re- Liu et al. 2013; Caruso et al. 2014). ported a dose-dependent effect of sulforaphane and Glucosinolates constitute a large group, including

Can. J. Plant Sci. Downloaded from pubs.aic.ca by Chinese Academy of Agricultural Sciences on 12/09/15 raphasatin, for protection against oxidative damage in several different forms with different side chain structures. human mesenchymal stem cells. They suggested that low Consequently, the break-down compounds are also diverse. doses of isothiocyanates revealed better results in terms Among these compounds, isothiocyanates, particularly sulforaphane (1-isothiocyanato-4-methylsulphinylbutane) of reducing oxidative damage than higher doses, which of the aliphatic group derived from glucoraphanin (4- may rather have adverse effects. methylsulphinylbutyl), have been extensively studied as Brassica sprouts are becoming a popular fresh food as bioactive compounds (Sakao and Singh 2012; Houghton a fast and easy way to substantially increase the intake of et al. 2013; Li and Zhang 2013). The biological properties of isothiocyanates and their protective effects Abbreviation: DW, dry weight

Can. J. Plant Sci. (2015) 95: 681687 doi:10.4141/CJPS-2014-412 681 682 CANADIAN JOURNAL OF PLANT SCIENCE

health-promoting glucosinolates. Many studies, therefore, Analysis of Glucosinolates focus on determining glucosinolate content in sprouts as Glucosinolates were extracted and analyzed according a valuable source of bioactive compounds (Baenas et al. to ISO 9167-1, developed for rapeseed with some 2012; De Nicola et al. 2013). It is important to elucidate modifications, as described by Sarıkamıs¸et al. (2006). changes in glucosinolates occurring during germina- Accordingly, 0.30.4 g of lyophilized and milled tissue tion and the early seedling growth period in order to was weighed and incubated in 70% (vol/vol) methanol. maximize their amount for increased health benefits. A volume of 3 mL of the extract was passed through The current study revealed changes in glucosinolates an ion exchange column (DEAE SephadexTM A25, from seed to sprout and seedling of two Brassica species Amersham Biosciences) followed by a desulfation step with distinct glucosinolate profiles in order to gain an with the enzyme sulphatase. Desulfoglucosinolates were overall insight into changes at these early growth stages. analyzed by HPLC-UV (Shimadzu†) detection at Ankara University, Department of Horticulture. Waters Spherisorb† 5mM ODS 2, 4.6250 mm analytical car- MATERIALS AND METHODS tridge was used as the column and the analyses was carried out on a gradient of 99% water and 1% acetonitrile, at a Plant Material 1 Seeds of white head cabbage (Brassica oleracea var. flow rate of 1 mL min for 24 min. The detection was capitata L.) cv. Yalova 1 and black radish (Raphanus performed at a wavelength of 229 nm. The peaks were sativus L.) cv. Karagulle, two cultivars grown commonly identified using commercial pure standards of sinigrin, in Turkey, were purchased from commercial seed progoitrin, glucotrapeolin, glucoraphenin purchased companies. from PhytoLab, Germany. Sinigrin and glucotrapeolin (16 mM) were used as the internal standards for the quantification of the peaks. Glucosinolates were quan- Laboratory Germination Tests tified according to internal standards and expressed as A germination test was conducted as four replicates mmol g1 dry weight (DW). Correction factors were (300 mg each) for each time point. Seeds of each repli- used during quantification for each compound as listed cate were weighed, surface sterilized in 1.5% sodium by Brown et al. (2003). hypochloride, rinsed three times in distilled water and placed on filter paper (Filtrak, Germany) in a petri Statistical Analysis dish (9 cm) moistened with 4 mL of distilled water. The experiment was conducted according to completely The dishes were placed in an incubator at 228C in the randomized design. Experimental data were expressed dark. Samples were taken at each time point and their as the mean9standard error of the mean with four glucosinolate content was analyzed. Cabbage seeds replications (n4). One-way analysis of variance and and germinating seeds and sprouts were collected at 0 Duncan’s multiple range test was used to determine the

For personal use only. (seeds), 1 (24 h), 2 (48 h), 3 (72 h), 5 (120 h),7 (168 h), significance of differences at different time points during and 10 d (240 h); black radish seeds and germinating the growth period using SPSS 18.0 (SPSS Inc., Chicago, seeds and sprouts were collected at 0 (seeds), 1 (24 h), IL). Significant differences were evaluated at PB0.001 2 (48 h), 3 (72 h), and 5 d (120 h). error level.

RESULTS Laboratory Seedling Emergence Test Growth and Developmental Parameters White head cabbage and black radish seeds were sown in peatmoss (Plantaflor-Humus,Verkaufs-GmBH, Germany) Changes in Fresh and Dry Weights of Germinating and perlite (2:1) containing seedling trays (32 cm Seeds, Sprouts and Young Seedlings of White Head 20 cm6 cm) and irrigated with distilled water. The Cabbage trays were transferred to a growth room with a con- The fresh weights of germinating seeds and sprouts in- trolled temperature of 22928C. Light was provided at creased with germination time (Fig. 1a, b). A rapid and seedling level by cool fluorescent lamps (72 mMm1 s1) significant weight increase was observed after 72 h (day 3) with a 16-h light/8-h dark cycle. The relative humidity of germination to 1.1590.13 g, increasing about two- in the growth room was maintained over 75% to reduce fold at 120 h (day 5) to 2.4390.05 g and threefold at Can. J. Plant Sci. Downloaded from pubs.aic.ca by Chinese Academy of Agricultural Sciences on 12/09/15 evaporation from the surface. Samples were collected 168 h (day 7) reaching its highest level at 3.3690.138 g upon emergence at different time points (days 5, 7, 10, 13, (PB0.001). The dry weights varied from 0.1590.01 g to 16, 20 and days 7, 10, 14, 17, 21 for white head cabbage 0.2790.00 g (PB0.001) (Fig. 1a). and black radish, respectively), taking four replicates for The fresh weights of young seedlings increased linearly each time point. Plant age (days) indicate days elapsed post-emergence. The increase was statistically significant from sowing. Young seedlings were harvested from the at each time point throughout the growing period and surface, weighed (fresh mass), flash frozen in liquid reached 13.0691.22 g (Fig. 1b). The dry weights in- nitrogen, and stored at 808C until lyophilized in a creased significantly from 0.1290.01 to 0.7190.07 g in freeze drier prior to analyses. cabbage (Fig. 1b). 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 Indole glucosinolates glucobrassicin (3-indolylmethyl), Seeds, Sprouts and Young Seedlings of Black Radish 4-methoxyglucobrassicin (4-methoxy-3-indolylmethyl), A similar trend was observed with black radish. The neoglucobrassicin (1-methoxy-3-indolylmethyl) and 4- fresh weights of germinating seeds and sprouts increased hydroxyglucobrassicin (4-hydroxy-3-indolylmethyl) were significantly with germination time from 0.390.00 to also identified in white head cabbage seeds, sprouts and 1.4790.21 g at 120 h (PB0.001) (Fig. 2a). Significant young seedlings. differences were not observed in dry weights, which Quantification of glucosinolates in germinating cab- varied between 0.2690.01 and 0.3090.01 g (P0.05) bage seeds and sprouts revealed the highest total (Fig. 2 a). aliphatic glucosinolate content as 4.6590.62 mmol g1 The fresh weights of black radish seedlings increased DW in nongerminated seeds, glucoraphanin being the regularly post-emergence (PB0.001). The fresh weight predominant glucosinolate in seeds, representing 83.9% reached 14.0791.02 g at the end of 3 wk (Fig. 2b). The of the total glucosinolates in seeds. Upon imbibition, dry weight on the other hand increased from 0.1390.03 total aliphatic glucosinolate content in seeds decreased to 0.8290.05 g in black radish (Fig. 2b). to 3.7191.14 mmol g1 DW at 24 h (day 1) followed by a marked decrease reaching 1.8690.09 mmol g1 DW at ˙ Analysis of Aliphatic and Indole Glucosinolates 48 h (day 2) (PB0.001). This pattern remained fairly Changes in Glucosinolate Content of Seeds, Sprouts stable until 120 h (day 5), followed by a slight increase and Young Seedlings of White Head Cabbage at 168 h (day 7) and 240 h (day 10) of the germination For personal use only. Glucosinolate profiling of white head cabbage identified period (Table 1 and Fig. 3a). A similar trend was ob- glucoraphanin (4-methylsulphinylbutyl) of the aliphatic served with glucoraphanin as the predominant aliphatic group, the precursor of sulforaphane (1-isothiocyanato- glucosinolate. 4-methylsulphinylbutane), as the predominant glucosi- Young cabbage seedlings revealed a pattern of increas- nolate in seeds, sprouts and young seedlings of white ing aliphatic glucosinolates from emergence quantified head cabbage followed by glucoiberin (3-methylsulphi- as 5.0590.46 mmol g1 DW at day 5 to the highest level nylpropyl), the precursor of iberin (1-isothiocyanato-3- reached at day 13 quantified as 12.8190.59 mmol g1 methylsulfinylpropane) and the alkylsulphinyl homologue DW, followed by a decrease to 8.6590.17 mmol g1 DW of glucoraphanin. at day 20 (Table 2 and Fig. 3b) (PB0.001). A similar Can. J. Plant Sci. Downloaded from pubs.aic.ca by Chinese Academy of Agricultural Sciences on 12/09/15

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. 684 CANADIAN JOURNAL OF PLANT SCIENCE

Table 1. Glucosinolate content (mmolg1 DW) in germinating seeds and sprouts of cabbagez

Germination time (h) Glucoiberin Glucoraphanin Total aliphatics Glucobrassicin 4-MetGBSy 1-MetGBSy 4-OHGBSy Total indoles

0 0.2790.01a 4.3890.43a 4.6590.42 0.1290.01a 0.0590.00c 0.0090.00d 0.3990.04a 0.5790.05 24 0.2690.05ab 3.4591.14ab 3.7191.14 0.1190.02a 0.0490.09c 0.0190.01d 0.3190.08ab 0.4690.12 48 0.1690.04ab 1.7090.09b 1.8690.09 0.1290.02a 0.0490.00c 0.1190.04c 0.2490.02b 0.5190.02 72 0.1690.02ab 1.6490.08b 1.8090.06 0.1290.04a 0.0690.02c 0.2090.03b 0.2390.01b 0.6190.05 120 0.1490.04ab 1.6790.14b 1.8190.11 0.1690.01a 0.1090.01b 0.3090.02a 0.2490.02b 0.8090.05 168 0.1390.01ab 1.9190.34b 2.0490.35 0.1490.01a 0.1590.01a 0.3390.03a 0.2090.02b 0.8190.06 240 0.1190.04b 2.1490.14b 2.2590.17 0.1190.01a 0.1790.01a 0.3290.01a 0.1890.01b 0.7890.03

zThe glucosinolate content data represent the mean9SE of four replicates expressed as mmol of glucosinolate g1 of dry weight. y4-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 predomi- Comparing the results obtained from two concurrent nant aliphatic glucosinolate representing 8085% of the experiments (laboratory germination and laboratory total glucosinolates in seedlings, while glucoiberin was emergence tests) it was determined that young but fully detected at very low levels accounting for 3% of the total established seedlings at true leaf stage contained higher glucosinolates. amounts of total aliphatic and indole glucosinolates. Indole glucosinolates were at lower levels compared with aliphatics in seeds, sprouts and seedlings (Tables 1 Changes in Glucosinolate Content in Seeds, and 2). In seeds, indole glucosinolates accounted for 10% Sprouts and Young Seedlings of Black Radish of the total, while in germinating seeds, sprouts ac- Glucosinolate profiling of black radish revealed that counted for 1025% of the total glucosinolates. Although glucoraphenin (4-methylsufinyl-3-butenyl) of the aliphatic an increase of up to threefold was observed in cabbage group, the precursor of sulforaphene (4-methylsulfinyl- seedlings at the end of 3 wk, towards the end of experi- 3-butenyl isothiocyanate), was the predominant gluco- mental period, reaching 3.0690.21 mmolg1 DW, in- sinolate in black radish seeds and sprouts with lower doles still accounted for 25% of the total glucosinolates levels of glucoraphasatin (4-methylsulfanyl-3-butenyl), (Table 2, Fig. 3a, b). also known as dehydroerucin, glucodehydroerucin, the For personal use only. Can. J. Plant Sci. Downloaded from pubs.aic.ca by Chinese Academy of Agricultural Sciences on 12/09/15

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. SARIKAMIS˛ ET AL. * GLUCOSINOLATES IN CABBAGE AND RADISH 685

Table 2. Glucosinolate content (mmolg1 DW) in young cabbage seedlings upon emergencez

Plant age (d)y Glucoiberin Glucoraphanin Total aliphatics Glucobrassicin 4-MetGBSx 1-MetGBSx 4-OHGBSx Total indoles

5 0.2090.08a 4.8590.49c 5.0590.46 0.2690.02c 0.1190.01c 0.2690.02a 0.4790.03ab 1.1090.07 7 0.2390.04a 5.0490.95c 5.2790.95 0.2590.03c 0.1390.01c 0.2190.02ab 0.4390.05b 1.0390.10 10 0.1590.02a 8.5390.65b 8.6990.67 0.3890.04c 0.2090.02c 0.0790.01c 0.5290.08ab 1.1890.14 13 0.2790.03a 12.5490.60a 12.8190.59 1.1490.07b 0.5290.03b 0.1890.01b 0.6890.02a 2.5290.08 17 0.2590.12a 10.0590.42b 10.3090.46 1.4490.08ab 0.6290.02a 0.1790.01b 0.6190.03ab 2.8490.11 20 0.1390.08a 8.5190.22b 8.6590.17 1.7690.21a 0.7090.05a 0.2090.02ab 0.4090.06b 3.0690.21

zThe glucosinolate content data represent the mean9SE of four replicates expressed as mmol of glucosinolate g1 of dry weight. yPlant age (days) indicate number of days elapsed upon sowing. x4-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- Black radish seedlings contained higher levels of butenyl isothiocyanate). Indole glucosinolates gluco- glucorapahasatin (61% of the total glucosinolates) comp- brassicin, 4-methoxyglucobrassicin, neoglucobrassicin ared with glucoraphenin (35% of the total glucosinolates) and 4-hydroxyglucobrassicin were also determined in at day 7. However, the amount of both compounds, and black radish seeds, sprouts and young seedlings. hence total aliphatics, tended to decrease in time from Quantification of glucosinolates in germinating black 78.1295.39 mmol g1 DW to 21.5492.50 mmol g1 radish seeds and sprouts revealed the highest total ali- DW, decreasing threefold at day 21 (Table 4, Fig. 3d). phatic glucosinolate content as 42.9992.15 mmol g1 The decrease in both glucoraphenin and glucoraphasatin DW in nongerminated seeds. Among aliphatics, glucor- revealed a similar pattern. In terms of total aliphatic aphenin, the predominant glucosinolate, was at its highest glucosinolates, the highest level was quantified in seed- level in nongerminated seeds, quantified as 41.569 lings at days 7 and 10 (Table 4). 2.19 mmol g1 DW, representing almost 80% of the total In terms of indoles, neoglucobrassicin (1-methoxy-3- glucosinolates in seeds, while glucoraphasetin (1.439 indolylmethyl) level was highest in nongerminated seeds 0.14 mmol g1 DW) accounted for only 3% of the total and sprouts, followed by 4-hydroxyglucobrassicin at lower levels. Total indoles were highest in nongermi- glucosinolates (Table 3). Upon imbibition, while glucor- 1 aphenin content gradually decreased until the end of the nated seeds, quantified as 9.2190.08 mmolg DW, experimental period at 120 h (day 5), glucoraphasatin increased slightly upon imbibition at 24 h (day 1) then 1 decreased from 48 h (day 2) to the end of the germina- content increased from 1.0590.03 mmol g DW at 24 h 1 tion period (Table 3, Fig. 3c). Indole glucosinolates were (day 1) to 20.4392.35 mmol g DW at 120 h (day 5) higher in radish seeds and sprouts than seedlings (Table 4, For personal use only. in sprouts (Table 3, Fig. 3c) (PB0.001). Although total Fig. 3c, d). aliphatic glucosinolate content decreased for a while Similar to our findings with cabbage, fully established until 48 h (day 2) upon imbibition, due to the substantial radish seedlings at true leaf stage contained higher increase in glucoraphasatin, total aliphatic glucosinolate amounts of aliphatic glucosinolates and low levels of content exhibited an increasing pattern from 72 h (day 3) indoles compared with black radish seeds and sprouts. till the end of the experimental period at 120 h (day 5) reaching 48.7493.67 mmolg1 DW. Therefore, at the end of the germination period, while the glucoraphenin DISCUSSION content decreased to 28.3191.67 mmol g1 DW repre- The chemoprotective effects of Brassica species are related senting 52% of the total, glucoraphasatin reached to their glucosinolate content. Aliphatic glucosinolates 20.4392.35 mmol g1 DW accounting for 37% of the are hydrolyzed to their corresponding isothiocyanates, total glucosinolates (Table 3, Fig. 3c). which have been associated with health-promoting

Table 3. Glucosinolate content (mmolg1 DW) in germinating seeds and sprouts of black radishz

y y y Can. J. Plant Sci. Downloaded from pubs.aic.ca by Chinese Academy of Agricultural Sciences on 12/09/15 Germination time (h) Glucoraphenin Glucoraphasetin Total aliphatics Glucobrassicin 4-MetGBS 1-MetGBS 4-OHGBS Total indoles

0 41.5692.19a 1.4390.14c 42.9992.15 0.7490.07b 1.0690.07a 6.1190.05a 1.2990.03a 9.2190.08 24 39.1292.01a 1.0590.03c 40.1892.03 0.7790.03b 1.0390.14a 6.2790.29a 1.3590.07a 9.4190.49 48 34.8091.25ab 3.1790.19c 37.9791.42 0.7290.03b 0.9990.04a 5.4190.46a 1.4290.05a 8.5490.55 72 33.3593.44ab 8.4690.59b 41.8193.89 0.7690.02b 0.9690.08a 5.4790.64a 1.4090.14a 8.5990.87 120 28.3191.67b 20.4392.35a 48.7493.67 1.0890.11a 0.8290.05a 2.8190.59b 1.3290.14a 6.0390.69

zThe glucosinolate content data represent the mean9SE of four replicates expressed as mmol of glucosinolate g1 of dry weight. y4-MetGBS, 4-methoxyglucobrassicin; 1-MetGBS, 1-methoxyglucobrassicin; 4-OHGBS, 4-hydroxyglucobrassicin. ac Different letters within the same column indicate statistically significant differences (PB0.001). 686 CANADIAN JOURNAL OF PLANT SCIENCE

Table 4. Glucosinolate content (mmolg1 DW) in young black radish seedlings upon emergencez

Plant age (d)y Glucoraphenin Glucoraphasetin Total aliphatics Glucobrassicin 4-MetGBSx 1-MetGBSx 4-OHGBSx Total indoles

7 28.1691.69a 49.9695.31a 78.1295.39 0.7890.66b 0.3090.16b 0.1690.08a 2.1490.05a 3.3990.95 10 21.6091.05b 37.6492.65b 59.2593.56 1.8990.11ab 1.3290.15a 0.1490.10a 2.1390.10a 5.4990.26 14 15.7590.37c 24.0190.52c 39.7691.12 1.7290.86ab 1.0790.06a 0.1290.06a 1.0690.01b 3.9890.13 17 12.9690.70c 19.4491.33cd 32.4091.91 1.9690.09a 1.2390.16a 0.2090.07a 1.3590.07b 4.7590.32 21 8.5690.970d 12.9891.61d 21.5492.50 1.9990.24a 1.1890.21a 0.0690.03a 1.3490.09b 4.5790.44

zThe glucosinolate content data represent the mean9SE of four replicates expressed as mmol of glucosinolate g1 of dry weight. yPlant age (days) indicate number of days elapsed upon sowing. x4-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 glucosi- occur during germination, including the interconversion nolates are desirable. However, variation in the levels and synthesis of new compounds. Decreases in glucosi- of glucosinolates exists, depending upon plant tissues nolates upon imbibition were determined in both species. and organs, developmental stage of the plant, environ- Consistent with our findings, Gu et al. (2011) reported mental factors, and various stress factors during the grow- a decrease in germinating broccoli seeds and sprouts ing period or post-harvest and processing, as reviewed to the lowest value at 48 h and an increase at 60 h, by Sarıkamıs¸(2009). remaining constant until the end of the experiment at The amount of glucosinolates in broccoli sprouts has 72 h. Baenas et al. (2012), who studied intact glucosi- been found to be higher than in fully grown plants (Fahey nolates in seeds and sprouts of some Brassica vegetables, et al. 1997). However, rapid changes in glucosinolates reported that the general trend was a decrease over during germination and early seedling growth need to be germination time. The decrease in glucosinolate levels evaluated as a particularly relevant factor in maximizing has been suggested to be a consequence of selective the concentration of the bioactive compounds. glucosinolate metabolism as well as dilution of glucosi- In the current study, unlike many other studies re- nolate concentration during tissue expansion (Chen and ported here, we used two concurrent experiments, includ- Andreasson 2001). In Arabidopsis thaliana, Brown et al. ing germination and emergence tests, to gain an overall (2003) reported a decrease in glucosinolate content insight into variations in glucosinolates at different during the germination period, suggesting and providing time points, representing germination and early seedling evidence for the catabolism of glucosinolates. growth. While glucoraphenin was the major glucosinolate in Glucoraphanin, of the aliphatic group, was identified seeds and sprouts, glucoraphasetin was identified as the For personal use only. as the major glucosinolate in cabbage seeds, sprouts and major glucosinolate, together with substantial amounts seedlings. Aliphatic glucosinolate synthesis is usually of glucoraphenin in black radish seedlings, both com- associated with the genetic background of individuals, pounds associated with potential health benefits (Montaut resulting in different glucosinolate profiles among species et al. 2010; Razis et al. 2012; Zanichelli et al. 2012; Song and cultivars. The influence of genetics on aliphatic gluco- et al. 2013). Decrease in glucoraphenin in radish sprouts sinolate production in broccoli was extensively studied has previously been reported to be due to the action by Mithen et al. (2003) and Sarıkamıs¸et al. (2006) via a of glucosinolate reductase converting glucoraphenin to breeding program through the introgression of genomic glucodehydroerucin (glucoraphasatin). However, in a segments from a wild Brassica to a commercial broccoli recent report on protective health benefits of white radish cultivar, also leading to the development of high- prepared by O’Hare et al. (2011) decrease in glucoraphe- glucosinolate broccoli. nin levels during sprout growth is attributed partly to Indole glucosinolates were at much lower levels com- dilution in percent dry matter, and partly to cultivar pared with aliphatics contrary to our previous field effects. On the other hand, the increase in glucorapaha- experiments with fully grown white head cabbage plants satin is more likely to be due to de novo synthesis of the of the same cultivar, indoles being the predominating compound according to evidence that increase in glucor-

Can. J. Plant Sci. Downloaded from pubs.aic.ca by Chinese Academy of Agricultural Sciences on 12/09/15 glucosinolates. Indole glucosinolates are usually asso- aphasatin exceeded decrease in glucoraphenin levels, and ciated with stress factors as a part of plant defence. the sum of glucoraphenin and glucoraphasatin increased Therefore, high levels of indole glucosinolates in field with sprout growth (O’Hare et al. 2011). experiments were attributed to the exposure of plants to Overall, both species with distinct glucosinolate pro- high temperatures during the growth period (Sarıkamıs¸ files exhibited higher levels of aliphatics and to a much et al. 2009). lesser extent indoles and are potential sources of bioactive Germination begins with water absorption (imbibi- compounds. Comparing glucosinolate contents, young tion) followed by activation of stored substances leading seedlings contained higher levels of total glucosinolates to radicle and hypocotyl growth. Significant changes as potential sources of health-promoting compounds. SARIKAMIS˛ ET AL. * GLUCOSINOLATES IN CABBAGE AND RADISH 687

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