Plant materials and chemicals

Broccoli seeds (Brassica oleracea L.), radish seeds (Raphanus sativus L. Mantanghong), rocket seeds (Eruca sativa Mill.), rape seeds (Brassica campestris L. Shanghaiqing) and mustard seeds (Brassica juncea L.) were kindly provided by Vegetables and Flowers Institute, China Academy of Agriculture Science. Acetonitrile and trifluoroacetic acid (TFA) were of HPLC grade and purchased from Fisher Scientific Co., LTD (Tustin, CA). Ultra-pure water was obtained by Q Millipore System (Millipore, USA). Sodium hypochlorite solution, dichloromethane were analytical grade and purchased from Beijing Chemical Works (Beijing, China), applied without further purification.

Seeds germination and sprouts cultivation

One hundred grams of broccoli (radish, rocket, rape or mustard) seeds were cleaned with deionized water and subsequently they were immersed in a 0.7% sodium hypochlorite solution for 30 min. Then those seeds were drained and washed with deionized water until the solution reached a neutral pH. Afterwards, they were soaked in 500 ml deionized water overnight. The imbibed seeds germinated on four layers of moist sterile gauzes in culture trays which were placed in 25 °C incubators, watered every 8 h to maintain a constant water content. During first 2 days, all seeds germinated in darkness conditions, after that they were cultivated under photoperiod conditions (16 h light and 8 h darkness). Sprouts were harvested daily during germination for up to 7 days. One half of the harvested sprouts were stored at − 20 °C refrigerator and the others were freeze-dried with lyophilizer.

Extraction of glucosinolates

The extraction of glucosinolates in each cruciferous sprout including broccoli, radish, rocket, rape and mustard sprouts were conducted according to previously published method with minor modifications [17]. Firstly, 10 mg of each freeze-dried sprouts were ground into powder with an analytical grinder and then 3 ml boiling water was added. The mixture was heated in a water bath set at 100 °C for 15 min before being centrifuged at 12,000 rpm for 5 min. Subsequently, the supernatant was decanted and the residue was extracted twice with 3 ml boiling water. Finally, the combined supernatants were concentrated to 3 ml using rotary evaporator and filtered with 0.22 μm nylon membrane.

Glucosinolates analysis by HPLC/Q-TOF/MS

The content of glucosinolate in each cruciferous sprouts extraction filtrate was analyzed by an Agilent 1290 HPLC (Agilent Technologies, Germany), coupled to an Agilent 6540 quadrupole-time of flight mass spectrometer (Agilent Technologies, Germany). The separation was carried out on a reversed phase C 18 column (2.1 × 150 mm, 3.5 μm; Agilent Technologies, Germany) set at a flow rate of 0.3 ml/min, eluting with a gradient elution of acetonitrile (mobile phase A) and 0.02% (v/v) TFA aqueous solution (mobile phase B) as follows: linear gradient from 1% A to 15% A for 10 min, 100% A kept for 2 min, then 1% A kept for 8 min. The injection volume was 10 μl and the detection wavelength was set at 235 nm. MS spectrometry data were acquired in the negative ionization mode for glucosinolates, and the separate glucosinolates were identified from the extracted samples according to their [M–H]− fragmentations.

Extraction of formed sulforaphane

Two grams of broccoli sprouts combined with 2 g of radish (rocket, rape or mustard) sprouts were juiced together with 20 ml deionized water for 3 min, using a juice centrifuge (Joyoung model JYL-C18D, China). Two grams of single broccoli sprouts acted as control. The obtained slurry was allowed to hydrolyze at room temperature for 1 h to facilitate product formation. Then the slurry was centrifuged at 12,000 rpm for 5 min and supernatant was extracted twice with 20 ml dichloromethane. Then the combined dichloromethane fraction was dried using rotary evaporator and the residue was dissolved in 20 ml water. Finally, the sample was filtered with 0.22 μm nylon membrane.

Sulforaphane formation analysis by HPLC/Q-TOF/MS

The sulforaphane formation in each combined cruciferous sprouts extraction filtrate was analyzed on an Agilent 1290 HPLC (Agilent Technologies, Germany), coupled to an Agilent 6540 quadrupole-time of flight mass spectrometer (Agilent Technologies, Germany). The separation was carried out on a reversed phase C 18 column (2.1 × 150 mm, 3.5 μm; Agilent Technologies, Germany) set at a flow rate of 0.3 ml/min, eluting with a gradient elution of acetonitrile (mobile phase A) and 0.02% (v/v) TFA aqueous solution (mobile phase B) as follows: linear gradient from 20% A to 80% A for 10 min, 100% A kept for 2 min, then 20% A kept for 8 min. The injection volume was 10 μL and the detection wavelength was set at 254 nm [18]. MS spectrometry data were acquired in the positive ionization mode for sulforaphane, and the sulforaphane was identified from the extracted samples according to its [M + H]− fragmentations.

Sulforaphane nitrile formation analysis by GC–MS

The extraction of sample was the same as that of sulforaphane analysis. Then the combined dichloromethane fraction was concentrated to 20 ml under vacuum. After that the dichloromethane concentrate was filtered with 0.22 μm nylon membrane and sulforaphane nitrile in it was analyzed on an Agilent 7890B GC system (Agilent Technologies, Germany), equipped with an Agilent 5977A mass spectrometer (Agilent Technologies, Germany). The separation was carried out on an Agilent HP-5 ms (5% phenyl methyl siloxane) column (30 m × 0.25 mm, 0.1 μm film thickness; Agilent Technologies, Germany). The temperature of injector and detector were set at 210 and 280 °C, respectively. Column oven temperature began at 40 °C for 2 min, then gradient (10 °C/min) increased to and kept at 260 °C for 10 min. Helium was used as carrier gas and the flow rate was 25 psi. Ion source temperature was 230 °C, and electron energy was 70 eV [19].

Statistical analysis

All analytical experiments were carried out in three replicates and the results were presented as a mean ± standard deviation (SD). One way analysis of variance (ANOVA) was employed to identify significant differences (p < 0.05) between data sets using software Origin 8.5.