Sulforaphane and its antioxidative effects in broccoli seeds and sprouts of different cultivars

Journal Pre-proofs Sulforaphane and its Antioxidative Effects in Broccoli Seeds and Sprouts of Different Cultivars Xingang Lv, Guanli Meng, Weina Li, Daidi Fan, Xiao Wang, Cesar A. Espinoza-Pinochet, Carlos L. Cespedes-Acuña PII: DOI: Reference:

S0308-8146(20)30063-7 https://doi.org/10.1016/j.foodchem.2020.126216 FOCH 126216

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

12 April 2019 6 January 2020 13 January 2020

Please cite this article as: Lv, X., Meng, G., Li, W., Fan, D., Wang, X., Espinoza-Pinochet, C.A., CespedesAcuña, C.L., Sulforaphane and its Antioxidative Effects in Broccoli Seeds and Sprouts of Different Cultivars, Food Chemistry (2020), doi: https://doi.org/10.1016/j.foodchem.2020.126216

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Sulforaphane and its Antioxidative Effects in Broccoli Seeds and Sprouts of Different Cultivars Xingang Lv1#, Guanli Meng1#, Weina Li2, Daidi Fan2*, Xiao Wang1, Cesar A. EspinozaPinochet4, Carlos L. Cespedes-Acuña*3. 1

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College of Food Science and Technology, Northwest University, Xi’an 710069, P. R. China

Shaanxi Key laboratory of Degradable Biomedical Materials, School of Chemical Engineering, Northwest University, Xi’an 710069, P. R. China. 3

Chemistry and Biotechnology of Bioactive Natural Products, Department of Basic Sciences, Faculty of Sciences, Universidad del Bio Bio, 3800708, Chillan, Chile.

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Agroindustrial Department, School of Agricultural Engineering, University of Concepcion, Chillan, Chile. *Corresponding Author. E-mail: [email protected], (CL Cespedes-Acuña), [email protected], (DD Fan). Tel: +86-13809193640. #The authors contributed equally to this work

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ABSTRACT The purpose of this study was to clearly understand the health-promoting potentials of broccoli seeds and sprouts according to identify their representative bioactive compounds and antioxidant activities in six varieties. Sulforaphane (SF) extraction was firstly optimized from seeds and sprouts. Then SF extracted under optimized conditions from seeds and sprouts were compared. Most varieties obtained the maximum SF, total phenolic (TP) and flavonoid (TF) contents in sprouts on day 3. SF contents in sprouts were 46 % to 97 % of seeds, whereas TP and TF contents in sprouts were 1.12 to 3.58 times higher than seeds among varieties. After in vitro digestion, broccoli sprouts from MNL variety kept considerable SF, TF, and TP contents, as well as antioxidant capacities, with all values higher than seeds. Compared with seeds, sprouts after 3 days germination were also recommended as raw materials of functional foods that possess high health-promoting potential. Keywords: Broccoli seeds; Broccoli sprouts; Sulforaphane; Phenolic, In vitro antioxidant capacity; In vitro digestion

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1. Introduction Epidemiological studies indicated that the regular consumption of cruciferous plants obviously reduced the risks of various cancers, including breast cancer, bladder cancer, prostate cancer, gastric cancer, and colon cancer (Arumugam, & Razis, 2018). These positive and healthy effects were mainly attributed to sulforaphane [SF, 1-Isothiocyanato-4-(methylsulfnyl) butane], which had been demonstrated to curb or block tumors growth by inducing the phase II enzymes and inhibiting the phase I enzymes (Abdull Razis, De Nicola, Pagnotta, Iori, & Ioannides, 2012). In addition, SF possessed anti-arthritic and immuno-regulatory activity (Ko, Choi, Jeomg, & Im, 2013), and antibacterial activity (Benzekri, Bouslama, Papetti, Snoussi, Benslimene, et al., 2016). Broccoli, a member of the Brassica genus of cruciferous family, is a very popular vegetable as a rich source of nutritional components and bioactive compounds, such as vitamin C, dietary fiber, phenolic compounds and glucoraphanin, the precursor of sulforaphane (Mahn, & Perez, 2016; Ferreira, Passos, Cardoso, Wessel, & Coimbra, 2018). In recent years, broccoli seeds and sprouts have attracted great research interest due to the fact that they possess much higher glucoraphanin and endogenous myrosinase than mature plants, which could produce a great amount of sulforaphane after disruption of plant tissues and cells (Pérez-Balibrea, Moreno, & García-Viguera, 2011; Guo, Yang, Wang, Guo, and Gu, 2014). Besides glucoraphanin and myrosinase, the formation and yield of SF also rely on the selected incubation conditions like pH, time, temperature, ascorbic acid and ethylenediaminetetraacetic acid (Shen, Su, Wang, Du, & Wang, 2010; Guo, Guo, Wang, Zhuang, & Gu, 2013; Ku, Kim, Jeffery, Kang, & Juvik, 2016). To the best of our knowledge, previous studies had separately optimized the extraction conditions for SF formation from broccoli seeds and sprouts, which made it no way to reliably

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compare the amount of SF produced from seeds and sprouts (Guo et al., 2013; Wu, Mao, Mei, & Liu, 2013; Tian, Xu, Hu, Liu, & Pan, 2017). SF production potential between broccoli seeds and sprouts has been previously compared, but it is still unclear whether the amount of SF directly received from seeds of a special number is high or low than sprouts which germinated from seeds of the same amount. Previous studies mainly investigated the difference of SF yields from broccoli seeds and sprouts on the basis of equal quality, i.e. 1 g seeds and 1 g sprouts (fresh weight or dry matter weight) (Guo et al., 2013; Wu et al 2013; López-Cervantes, Tirado-Noriega, Sánchez-Machado, Campas-Baypoli, CantúSoto, & Núñez-Gastélum, 2013; Shams, Abu-Khudir, & Ali, 2017; Tian et al., 2017). There were varied results for SF yields between broccoli seeds and sprouts among studies attributing to the inconsistency of selected units. Shams et al. (2017) reported the SF contents from broccoli seeds and 7-day broccoli were 460.4 and 465.7 μg/g FW (fresh weight), respectively (no significant difference at the 0.05 level). However, results by López-Cervantes et al. (2013) indicated 11-day old sprouts yielded 1483.76 μg/g DW (dry matter) of SF (the highest yield during germination), only accounting for 40.8% of seeds (3631.91 μg/g DW). Additionally, SF yields during sprout germinating process varied in different studies. Besides of SF of high healthy benefits, recent reports indicated that broccoli seeds and young sprouts are also rich in other health-promoting phytochemical compositions and possess high antioxidant activities (Baenas, Gómez-Jodar, Moreno, García-Viguera, & Periago, 2017; Becker, Jeffery, & Juvik, 2017). Thus, the exhibited biological activities were perhaps not only due to SF itself, but other bioactive compounds, such as phenolic, also likely exert functional effects (Pasko et al., 2018; Wu, Xiao, Mao, Liu, Huang, & Mei, 2015). Just like SF, comparison results of other health-promoting compounds and antioxidant activities between broccoli seeds

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and sprouts were also complicated. Pérez-Balibrea et al. (2011) reported the total phenolic content and antioxidant capacity were higher in seeds than those in sprouts, and the phytochemical contents varied among varieties. However, these results are of less significance for representing the variation in bioactive components during the sprouting period as their contents were expressed on fresh weight basis. Contrary results indicated that the seeds contained less total phenolic and flavonoid contents and lower DPPH (2,2-diphenyl-1-picrylhydrazyl-hydrate) radical scavenging capacity than sprouts (López-Cervantes et al., 2013). According to above summary, from the perspective of comparison methods, broccoli varieties, and germination processes, there is still lack of a systematic comparison of SF yields and other bioactive compounds contents between broccoli seeds and sprouts. Thus, the objective of this study was, (1) to optimize the extraction conditions of SF from seeds and sprouts at the same time to ensure the maximum SF yields from them; (2) to compare the SF yields, total flavonoid (TF) contents , and total phenolic (TP) contents from broccoli seeds and sprouts (after 3, 5, and 7 days germination respectively) of six different cultivated varieties. To be comparable, the content of these bioactive compounds from 100 fresh sprouts was divided by the weight (gram) of 100 seeds, and then this value was compared with their content from one gram seeds; and (3) to evaluate and compare the the stability and bioaccessibility of SF, TF and TP from broccoli seeds and sprouts upon in vitro gastrointestinal digestion (GID); total antioxidant activities (TAC) of samples before and after digestion were also investigated in this section. The obtained results may provide a reference for the application of broccoli seeds and sprouts as raw materials of functional foods. 2. Materials and Methods 2.1. Plant material and germination conditions

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Broccoli seeds of six varieties were purchased from a local market (Lv Wa Wa, LWW; You Ji Qing Hua Cai, YJ; Yi Dai Lv Tan, YDLT; Ma Ni La, MNL; Xi Mei, XM; Lv Yu, LY). Broccoli seeds were germinated according to the method described by Gu, Guo, Zhang, Chen, Han, & Gu (2012) with minor modifications. Briefly, broccoli seeds were rinsed and immersed in distilled water and soaked at 30 °C for 2 h. After absorbing the soaking water, the seeds were uniformly sown in trays (5 g per tray) lined with absorbent gauze and germinated in incubators under the conditions of constant temperature of 25 °C and darkness. Distilled water was spayed automatically and intermittently. Broccoli sprout samples were carefully collected after 3, 5, and 7 days germination, weighed (fresh weight) after removing surface water, and then immediately stored at -80 °C for further phytochemical analysis. During collection each time, 100 samples were averagely taken from six trays and mixed together to reduce the inherent bias among trays and this process was conducted for three triplicates. For phytochemical analysis, samples were obtained from above three triplicates, respectively. 2.2. Extraction and measurement of SF Broccoli seeds were comminuted by analysis grinder. Seed powder (0.5 g) was immersed in distilled water at 55 °C for 5 min to inactivate the epithiospecifier protein. Sprouts (1 g fresh sprouts) were blanched at 55 °C for 15 min, cooled quickly under flowing water, and then homogenized in a porcelain mortar (The above conditions were obtained through preliminary tests and the detailed data were not shown here). Then the homogenization was mixed with phosphate buffer of different pH values to a certain solid-liquid ratio (the ratio of the weight of seed or fresh sprouts to the volume of phosphate buffer). The solution was then incubated in a shaking water bath at 37 °C, and every 30 min sonicated for 5 min. After incubation, 5 mL acetone was added to the sample for extraction under ultrasound condition and then centrifuged

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at 4000 rpm for 20 min. The residues were extracted with another 5 mL acetone and after centrifugation, the supernatant of two sources were mixed. The mixture solution of acetone and water was completely evaporated to dryness using a rotary evaporator at 30 °C. The residue was dissolved in methanol (5 mL) and then the solution was filtered through a 0.45 μm filter to further determine the content of SF. Specially, the pH value of phosphate buffer, the solid-liquid ratio, the incubation and extraction time were set in three levels during optimization process, but then was set under optimized conditions. The content of SF was measured based on a method described by Guo et al. (2013) with slight modification. The extracted samples were analyzed using a high-performance liquid chromatography (HPLC) system (UltiMate 3000, Thermo Scientific Co. Ltd.) with a Zobax SBC18 column (5 μm particle size 4.6 × 250 mm). The flow rate was 1.0 mL/min in isocratic mode of 28% (v/v) methanol. The injection volume was 10 μL and chromatogram was recorded with ultraviolet detection at 195 nm. SF standard (Sigma) was used as a reference to produce the standard curve. The SF contents were calculated and expressed by mg SF per gram of seeds or fresh sprouts. Furthermore, to be comparable with the seeds, the contents of SF and the following bioactive compounds in 100 fresh sprouts were divided by the weight of 100 seeds and then the contents of bioactive compounds in fresh sprout were expressed as mg per gram of seeds. 2.3. Response surface optimization Based on the results of the single factor experiment (data not shown), the variable factors and their levels were determined and listed in Table 1. Then a Box-Behnken design (BBD) was used to construct a four factor, three-level experimental model consisting of 29 experimental runs to optimize the extraction conditions of SF from broccoli seeds and sprouts, respectively.

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The SF yield (Y) was used as response variables. The predicted experimental data were fitted to a second-order polynomial model as follows (Eq.1): 3

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i 1

i 1

2

3

Y  A 0   A i X i   A ii X i    A ijX i Yj 2

(Eq.1)

i 1 j i 1

Where Y represents the response variable, Xi and Xj represent independent variables, represents the intercept coefficient, A1 represents the linear coefficient, quadratic coefficient, and

ij

ii

0

represents the

represent the cross-product coefficient. The predicted value and

coefficients were analyzed via Design-Expert.V8.0.6.1 software and the F value (p < 0.05) was used to analyze the significance of the dependent variables. 2.4. Measurement of seed and sprout weights An analytical balances was used to determine the weight of seeds and sprouts (3, 5, and 7 days) of six selected varieties (three replicates). The obtained values were calculated and expressed as the weight of 100 sprouts plants (100 seeds). 2.5. Measurement of TP and TF contents 0.5 g broccoli seeds and sprouts (3, 5, and 7 days) were ultrasonically extracted with 5.0 mL methanol for 30 min, respectively. Then, the solutions were centrifuged at 4000 rpm for 10 min and the supernatant was collected. TP content was assayed by Folin-Ciocalteu colorimetric. The diluted sample solution of 1 mL was added to 1 mL of Folin-Ciocalteu reagents (0.2 M). After incubation for 5 min, 2% sodium carbonate solution of 2 mL was added. The mixture was incubated at room temperature for 2 h in the dark and its absorbance was determined at 765 nm (Pajak, Socha, Galkowska, Roznowski, & Fortuna, 2014). Results were expressed as mg gallic acid equivalents (GAE) per gram of seeds or fresh sprouts (mg GAE/g). Furthermore, TP in broccoli sprouts was also transferred to the unit of mg GAE/g seed as the same as SF.

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TF content was determined as described by De Nicola et al. (2013) with slight modifications. 1 mL supernatant was mixed with 1 mL 5 % sodium nitrite and incubated for 6 min, and then 1mL 10 % aluminum nitrate was added. After 6 min incubation, 10 mL 4% sodium hydroxide was added for further 15 min incubation. Then, the absorbance of the mixture was measured at 510 nm. The TF content was expressed as mg of rutin equivalents (RE) per gram of seeds or fresh sprouts (mg RE/g). Furthermore, TF content in broccoli sprouts was also transferred to the unit of mg RE/g seed as the same as SF. 2.6. Measurement of antioxidant capacities The antioxidant capacites were determined by DPPH radical scavenging capacity assay and ferric reducing antioxidant potential (FRAP). The DPPH radical scavenging capacity assay, described by López-Cervantes et al. (2013) and Shams et al. (2017), was conducted with minor modifications. 3 mL of 0.1 mM DPPH solution was added to 1 mL of the appropriately-diluted extracted sample and the mixture was allowed to react under dark at room temperature for 1 h. Then, the absorbance of the solution was monitored at 517 nm and anhydrous ethanol was used as reference. The control consisted of 1 mL methanol and 3 mL DPPH solution while the blank consisted of 1 mL sample and 3 mL methanol. DPPH radical scavenging capacity was calculated by using ascorbic acid antioxidant activity as the standard curve and expressed as mg ascorbic acid equivalents (TEAC) per gram of seeds or fresh sprouts (mg TEAC/g). Furthermore, The DPPH radical scavenging capacity of broccoli sprouts was also transferred to the unit of mg TEAC/g seed as the same as SF. The FRAP was assayed according to the method by Yuan, Wang, Guo, and Wang (2010) with minor modifications. The FRAP working solution of 3.6 mL (0.3 M acetate buffer, 0.02 M ferric chloride, 0.01 M 2,4,6-tripyridyl-S-triazine in 0.04 M hydrochloric acid at the ratio of

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10:1:1) was added to diluted extracted sample (400 μL). After the mixture was incubated at 37 °C for 10 min, the absorbance was recorded at 593 nm. FRAP values in the sample were calculated using ferrous sulfate heptahydrate as standard curve and expressed as mg of Fe2+ per gram of seeds or fresh sprouts. Furthermore, the FRAR values of broccoli sprouts were also transferred to the unit of mg Fe2+/g seed as the same as SF. 2.7. In vitro gastrointestinal digestion (GID) According to SF,TP and TF contents in the seed and sprouts, the variety MNL was selected for in vitro GID, which was performed as described by Ortega-Vidal, Ruiz-Riaguas, Fernándezde Córdova, Ortega-Barrales, & Llorent-Martínez (2019) and Thomas-Valdés, Theoduloz, Jimé nez-Aspee, & Schmeda-Hirschmann (2019). The sprout after frozen in liquid N2 and seed were ground into powder, respectively. After incubation for SF production according to the method in 2.2, the solution and solid part were frozen dried together and then used for in vitro GID. All the simulated digestion steps were carried out in a water bath at 37 oC, under constant shaking in the dark. For the gastric phase (GD), 1g of incubated and lyophilized seed or equivalent sprout was added into 10 mL of gastric-simulated fluid for 2h. And then, the solution was centrifuged and the supernatant (GD-ST) and residues (GD-RS) were both frozen at -80 oC for subsequent analysis. For the GID process, 1g of frozen-dried seed or equivalent sprout was mixed with 10 mL of gastric-simulated fluid for 2 h, and then 10 mL of simulated intestinal fluid was added in the mixture for another 2 h. And then, the solution was centrifuged and the supernatant (ID-ST) and residues (ID-RS) were both frozen at -80 oC for subsequent analysis. GID process for all samples were conducted in triplicate. The absolute contents of SF, TP and TF in samples of GD-ST, GD-RS, ID-ST, and ID-RS obtained from 1 g of lyophilized raw materials were measured according to methods described

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above, and expressed as mg. An index of bioaccessibility was adopted to reflect the effects of in vitro GID on selected bioactive compounds. Bioaccessibility is defined as the percentage of these compounds that is solubilized in the gastrointestinal tract and becomes available for absorption (Correa-Betanzo, Allen-Vercoe, Mcdonald, Schroeter, Corredig, & Paliyath, 2014), which can be calculated following Eq.2 (taking SF as an example). Bioaccessi bility 

SFIDST SFND

(Eq.2)

where: SFID-ST is the content of SF (mg) in the soluble fractions after in vitro GID. SFND is the SF content (mg) in 1 g frozen dried samples before digestion. 2.8. Statistical analysis All experimental data were presented as means ± standard deviation (SD) of independent replicates in triplicate. After analysis of one-way variance (ANOVA), Duncan’s Multiple Range Test was performed (p < 0.05) using IBM SPSS Statistics 20 software to determine the level of statistics significance. 3. Results and Discussion 3.1. Optimization of the extraction conditions of SF from broccoli seeds and sprouts The results of BBD experiments and analysis of variance were shown in Table 2. The regression equation in terms of actual factors was generated to analyze the significant variable on the extraction yield of SF from broccoli seeds (Y1) and sprouts (Y2), which were presented as follows:

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Y1 = 3.990 + 0.169 X1 + 0.051 X2 + 0.050 X3 – 0.041 X4 – 0.033 X1X2 + 0.061 X1X3+ 0.222 X1X4 – 0.071 X2X3 – 0.269 X2X4 – 0.080 X3X4 – 0.424 X12 – 0.380 X22 – 0.209 X32– 0.135 X42. Y2 = 0.483 + 0.006 X1 – 0.066 X2 – 0.009 X3 – 0.015 X4 + 0.003 X1X2 + 0.068 X1X3 + 0.025 X1X4 + 0.037 X2X3 + 0.007 X2X4 – 0.044 X3X4 – 0.108 X12 – 0.128 X22 – 0.119 X32 – 0.063 X42. The data of ANOVA analysis showed that the models of seeds and sprouts were remarkably significant (p < 0.01). The coefficients of determination of both models were 0.9032 and 0.8392, respectively, suggesting excellent correlations between the actual and predicted production yield. Moreover, the lack of fit tests of both models were not significant (pseed = 0.2367, psprout = 0.0534), which indicates that the current models were adequate for predicting the SF yields from broccoli seeds and sprouts, further confirming the validity of these models. The optimal values of the SF production for independent variables were actualized using regression equations and contour plots. The theoretical yield of SF from seeds was 4.014 mg/g under the conditions of solid-liquid ratio of 1:8.32, enzymatic hydrolysis time of 4.11 h, extraction time of 28.2 min, and a pH value of 4.08. The actual experimental value under optimal conditions was 3.9618 mg/g FW, which is close to the predicted values and confirmed the validity and practicability of this model. In addition, the actual value of SF from broccoli sprouts was 0.4867 mg/g FW under the optimal extraction conditions of solid-liquid ratio of 1:5, enzymatic hydrolysis time of 5.46 h, 0.19 mg of ascorbic acids, and a pH value of 4.0, which was basically consistent with the predicted value (0.4931 mg/g FW). 3.2. Sprouts weight change during broccoli seed germination

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In this study, the properties of broccoli seeds during the germination were determined. Table 3 show the fresh weight of 100 seeds and sprouts of 100 strains after 3, 5, and 7days germination of the six varieties. From prolonged germination process, the weight of sprouts increased significantly and the weight of 7-day old sprouts was about 10 times that of seeds. There was significant difference in fresh weight of seeds and sprouts among varieties. The seed weight of XM was nearly twice that of other varieties and its sprouts weight also had the highest increasing rate. LWW and LY had the lowest weight of seeds as well as sprouts. 3.3. Evaluation and comparison on SF yields The SF yields from seeds and sprouts of 3, 5, and 7-day old of six broccoli varieties were determined under optimized conditions for extraction (Figure 1A). SF yields from seeds were significantly higher than that from sprouts for six broccoli varieties, ranging from 4.47 to 13.19fold. The SF yields among six varieties of broccoli seeds and sprouts were significantly different, which was in agreement with previous studies (Gu et al., 2012; Ku et al., 2016). MNL seeds exhibited the maximum SF level (12.07 mg/g FW) about 1.4-5 times higher than other varieties, followed by XM (8.43 mg/g FW ) and YDLT (7.65 mg/g FW) seeds, and YJ (3.96 mg/g FW), LWW(3.32 mg/g FW), and LY (2.43 mg/g FW) seeds displayed the lowest. It has been reported the SF yield from seeds was 3.63 mg/g FW (López-Cervantes et al., 2013) and 0.46 mg/g FW (Shams et al. 2017), suggesting great variations among broccoli varieties. The SF yields from sprouts decreased sharply during germination, and from day 3 to 7, MNL decreased from 2.70 to 0.61 mg/g FW, but YJ, LWW, and LY decreased as low as 0.05, 0.02, and 0.01 mg/g FW, respectively. Guo et al. (2014) also observed that SF yield from sprouts decreased by 91.02% from germination (6.21 mg/g FW) to the 7-day old age (0.83 mg/g FW). Because the moisture content of the sprouts increases during the germination process, it is difficult to determine how

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the total SF yield changes in this process and whether it is high or low compared to the SF yield from seeds. To be comparable with the seeds and clearly identify its changes during germination process, the amount of SF from 100 fresh sprouts were divided by the weight of 100 seeds in this study. As shown in Figure 1B, most varieties obtained the maximum SF yields from sprouts on day 3, whereas YDLT reached the maximum value on day 5, which was 1.7-fold of 3-day old sprouts. Gu et al. (2012) found the highest SF yield of LLX sprouts on day 2 with the value of 3.38 mg/g DW, while Tian et al. (2017) recorded that 5-day old sprouts obtained the highest yield of 0.253 mg/g FW, indicating a difference in maximum SF yield among varieties during germination process. Maximum SF yield was found in MNL sprouts of day 3, with the value of 11.70 mg/g Seed, showing no significant difference with its yield from MNL seeds (12.07 mg/g Seed). Other varieties all obtained 19.0 % to 53.9 % lower SF yields from sprouts (the maximum value during germination was selected) than their seeds. In the study by Gu et al. (2012), SF yield was the same based on dry weight between broccoli seeds and 2-day sprouts, but another study by López-Cervantes et al. (2013) indicated maximum SF yield from 11-day sprout was only 40.8% of that from seeds. Above results suggested the significant influence of broccoli varieties and germination time on SF production. And the difference between sprouts and seeds was mainly attributed to broccoli varieties. The selection of brassica sprouts for optimum phytochemical composition is the key to optimize new fresh foods enriched in bioactive compounds with health benefits (Baenas et al., 2017). On the other hand, knowledge concerning the bioavailability of plant ITCs is essential to predict the potential level of exposure as GL determination alone may not accurately reflect how much of the final active ITC can be formed from sprouts.

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3.4. Evaluation and comparison on TP and TF after extraction In this study, in order to clearly understand comprehensive health-promoting potentials of broccoli seeds and sprouts, TP and TF in seeds and sprouts (3, 5, and 7-day old) from the six investigated broccoli varieties were determined and compared (Tables 4). On the fresh weight basis, TP and TF contents in broccoli seeds were significantly higher than those in sprouts and variability was observed among varieties. During germination process, both values in sprouts of six varieties declined significantly and the highest values occurred on day 3, accounted for over 27.7 - 75.3 % and 20.5 - 53.4 % of the original seeds from different broccoli varieties for TF and TP, respectively. The minimum values of decline rates in TF and TP from germination to day 7 was LWW with 69.6 % and 76.4 %, and the maximum values was YDLT, reaching 87.5 % and 96.5 %. These results are consistent with those recorded by Pérez-Balibrea et al. (2011). From the viewpoint of the contents of these phytochemical compounds, they were comparable with other fruit or vegetables. For example, TP value ranged from 0.94 to 2.44 mg/g FW in 3-day sprouts, and in previous reports, TP contents were 5.97, 2.72, 1.82, 1.47, 0.66, 0.65, 0.57, 0.56, 0.53 and 0.40 mg/g FW in apple, red grape, strawberry, lemon, peach, orange, banana, pear, and pineapple, and were 3.10, 2.69, 2.06, 2.57, and 1.44 mg/g FW in red onion, Ceylon spinach, green pepper, beetroot, and bitter melon (Lin, & Tang, 2007), respectively. Therefore, fresh broccoli sprouts were raw materials of similar healthy benefits. For further analysis, the TP and TF contents in 100 fresh sprouts were divided by the weight of 100 seeds in this study for reasonable comparison between sprouts and seeds and clear characterization of content change of bioactive compounds during germination process. As shown in Table 4, the TP and TF contents in broccoli sprouts were significantly higher than those in seeds, and most varieties obtained the maximum TP and TF values in sprouts on day 3, except

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YJ sprouts on day 7. The broccoli sprouts increased in a proportion from 12 % to 154 % in TP and from 66 % to 259 % in TF in 3-day old sprouts, respectively, with reference to seeds. Maximum increment was found in 7-day old sprouts of YJ variety, with 193 % in TP and 276 % in TF, respectively, compared with seeds. López-Cervantes et al. (2013) found the highest concentrations of TP and TF in 11-day old sprouts, which were 767 % and 254 % higher than seeds. Fernandez-Orozco, Frias, Zielinski, Piskula, Kozlowska, & Vidal-Valverde (2008) also reported an enhanced of TP and TF in the older sprouts. Furthermore, changes of these bioactive compounds varied greatly among varieties during germination process. For example, LWW and YDLT had the same TP contents in seeds, but in their 3-day old sprouts, LWW kept two-fold higher TP than YDLT, and LWW kept stable level while YDLT showed significant decrease during germination. YJ and YDLT showed no significant difference in TF contents in seeds, whereas, TF in YJ sprouts increased from 10.18 mg RE/g Seed on day 3 to 14.32 mg RE/g Seed on day 7, but in YDLT sprouts decreased from 6.27 mg RE/g Seed to 5.67 mg RE/g Seed. Thus, through systematic analysis of results from previous research and this study, we could find that broccoli varieties and germination process are the main sources of these differences. In brief, although germination reduces SF yield to some extent, it is beneficial to the formation and accumulation of total phenol and flavonoids, ensuring the health properties of sprouts. 3.5. Evaluation and comparison on SF, TP, TF, and TAC after GID process Although this study and previous researches provide valuable information on the chemical properties of broccoli seeds and sprouts from a solvent extraction perspective, little is known about the stability and biological effects of these bioactive compounds after digestion. And this information is critical to the bioaccessibility and bioavailability of these compounds. Thus, by comparing the yields of SF, TP and TF after extraction, MNL seeds and 3-d sprouts were selected

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to further assess and compare the changes of these components after GID. SF was relatively stable during GID process, with no significant change in the soluble fraction (ST) from sprouts after digestion compared with ND sample (Figure 2A). For ST samples from seeds, GD resulted in a 16.9 % loss of SF, but after ID, the SF content showed an additional increase to 10.21 mg, resulting in no significant difference with ND sample. As the solid fraction (RS) kept SF contents from 1.39 mg to 3.22 mg, the stable level of SF in the soluble fraction might also attribute to its release from sample tissues in vitro GID. Thus, SF from seeds and sprouts both showed high bioaccessibility values of 0.91 and 1.00, respectively. The high bioaccessibility of SF in vitro experiments provide an additional evidence for its efficient utilization, as many previous researches have reported a high bioavailability of SF in vivo (Veeranki, Bhattacharya, Marshall, & Zhang, 2012; Oliviero, Verkerk, Vermeulen, & Dekker, 2014). Digestion phases significantly affected the stability of TP and TF in broccoli seeds and sprouts. After gastric phase, ST samples kept consistent TP and TF contents with ND ones (Figure 2B and C), except a significant increase of TP content in seed . As RS samples also contained TP and TF of certain amount, combined contents of TP and TF from ST and RS were significantly higher than ND, indicating GD was helpful for the release of these chemical compounds from plant tissues. No data in previous literature referring to broccoli were found about the GD effect on TP and TF, but similar results were found in other plant materials, such as in blueberry (Correa-Betanzo et al., 2014), persimmon flours (Lucas-González, Viuda-Martos, Álvarez, & Fernández-López, 2018), and millet grain (Chandrasekara, & Shahidi, 2012). The acid medium in the gastric phase promotes the break of bonds between polyphenolic compounds and fiber, protein or carbohydrates, benefiting for their release from food matrix (Alminger,

17

Aura, Bohn, Dufour, Gomes, Karakaya, Martínez-Cuesta, McDougall, Requena, & Santos, 2014). The reduction of phenolics and flavonoids after GID has been widely reported by scientific community (Lucas-González et al., 2018; Correa-Betanzo et al., 2014; Ortega-Vidal et al., 2019). Compared with ND sprouts, GID resulted in a significant reduction of 34.2 % for TF conents, but did not signifcantly afftect the TP contents. The changes of TP and TF contents were also found in seeds after GID process, but their differences were not significant between ND and ID samples (Figure 2B and C). Research by Rychlik, Olejnik, Olkowicz, Kowalska, Juzwa, Myszka, Dembczynski, Moyer, & Grajek (2015) reported about 47 % reduction of TP after GID in broccoli sprout. However, Gawlik-Dziki, Jeżyna, Świeca, Dziki, Baraniak, & Czyż (2012) found after GID process, TP contents in broccoli sprouts increased from 4.12 mg/g FW to 5.04 mg/g FW and TF contents from 2.36 mg/g FW to 5.11mg/g FW. The variation of these results might be due to the broccoli variety, tissue status (homogenization or powder, fresh or dried) in vitro GID or the response of these compounds to gastrointestinal conditions. Finally, the bioaccessibility values of TP were 1.20 and 0.76, and of TF were 0.72 and 0.66 for seeds and sprouts, respectively, which generally higher than other plant materials (Fernández-Poyatos et al., 2019; Ortega-Vidal et al., 2019; Thomas-Valdés., 2019). ID-ST samples from seeds showed a 41.3 % reduction for DPPH but held the same value of FRAP compare with ND ones and with no significant loss of TP or TF. DPPH and FRAP values in ID-ST samples from sprouts showed 28.1 % and 32.1 % lower than in ND ones, respectively. In contrast, only TF, and not TP, showed any significant change in value from ND to ID-ST for sprouts (Figure 2). Previous reports widely presented the reduction of antioxidant values (DPPH, FRAP), with the range from 41.3% to 224 % in samples of pomegranate peel (Gullon, Pintado,

18

Fernández-López, Pérez-Álvarez, & Viuda-Martos, 2015), table olive (Fernández-Poyatos,

Ruiz-Medina, & Llorent-Martínez, 2015), strawberry (Thomas-Valdés et al., 2019) and others, which were in agreement with our findings. However, the results obtained for the in vitro DPPH and FRAP assays (Figure 3) are inconsistent with the pattern of loss of TP and TF observed after the digestion process (Figure 2). As indicated above, for one thing, GD was helpful for the release of these chemical compounds from plant tissues, however, for another, GID could result in the significant loss of TP and TF. Another undeniable fact is that the GID had different repercussions in the stability of individual phenolics and flavonids (Lucas-González et al., 2018; Thomas-Valdés et al., 2019). Thus, after GID process, the individual phenolics or flavonids in seed and sprouts could be different from ND ones.These compounds might be different in terms of antioxidant activities, resulting in the inconsistent relationship between antioxidant values and TP or TF contents. After GID process, broccoli seeds and sprouts kept almost the same amount of SF. The values of DPPH and FRAP in young broccoli sprouts were significantly higher compared to that in seeds according to the data from ND samples. After digestion, sprouts also hold 44.0 % and 67.9 % higher contents of TP and TF, as well as 54.9 % and 31.7 % higher values of DPPH and FRAP than seeds, respectively. Thus, compared with seeds, sprouts from MNL variety possess more bioactive compounds with health benefits. 4. Conclusions From the changing tendency of investigated phytochemical compounds, genotype and germination process are two main factors influencing their values in seeds and sprouts. Based

19

on optimized extraction conditions and established comparison method, it is clear that higher SF yields could be obtained from seeds than sprouts. As SF yields decreased during germination process, young sprouts were the primary sources of SF, but the SF yields from different varieties greatly varied. TP and TF contents were higher in sprouts than seeds and they both increased to a relatively stable level during germination, but the increasing rate also differed among varieties. Thus, it is necessary to conduct practical experiments to determine and evaluate the nutritional status and values from seeds of different broccoli varieties and sprouts of different germination time. After in vitro digestion, broccoli sprouts kept considerable SF, TF, and TP contents, as well as antioxidant capacities, with all values higher than seeds. In conclusion, compared with the original seeds, broccoli sprouts after 3 days germination also rich in bioactive compounds, offering an advantage for the application in dietary supplement. Acknowledgements This work was supported by National Natural Science Foundation of China (21706213), National Natural Science Foundation of China (21706211), and Chinese Postdoctoral Science Foundation (2016M602853). Competing financial interests The authors declare no competing financial interest. Appendix A. Supplementary data Supplementary data to this article can be found online at

20

References Abbaoui, B., Riedl, K. M., Ralston, R. A., Thomas-Ahner, J. M., Schwartz, S. J., Clinton, S. K., & Mortazavi, A. (2012). Inhibition of bladder cancer by broccoli isothiocyanates sulforaphane and erucin: characterization, metabolism, and interconversion. Molecular Nutrition & Food Research, 56, 1675-1687. Abdull Razis, A. F., De Nicola, G. R., Pagnotta, E., Iori, R., & Ioannides, C. (2012). 4Methylsulfanyl-3-butenyl isothiocyanate derived from glucoraphasatin is a potent inducer of rat hepatic phase II enzymes and a potential chemopreventive agent. Archives of Toxicology, 86, 183-194. Alminger, M., Aura, A. M., Bohn, T., Dufour, C., El, S. N., Gomes, A., Karakaya S., MartínezCuesta M. C., McDougall G. J., Requena T., & Santos, C. N. (2014). In vitro models for studying secondary plant metabolite digestion and bioaccessibility.Comprehensive Reviews in Food Science and Food Safety, 13, 413-436. Arumugam, A., & Razis, A. F. A. (2018). Apoptosis as a mechanism of the cancer chemopreventive activity of glucosinolates: A review. Asian Pacific journal of cancer prevention: APJCP, 19(6), 1439.. Baenas, N., Gómez-Jodar, I., Moreno, D. A., García-Viguera, C., & Periago, P. M. (2017). Broccoli and radish sprouts are safe and rich in bioactive phytochemicals. Postharvest Biology and Technology, 127, 60-67. Becker, T.M., Jeffery, EH., Juvik, JA. (2017). Proposed method for estimating health-promoting glucosinolates and hydrolysis products in Broccoli (Brassica oleracea var. italica) using relative transcript abundance. Journal of Agricultural and Food Chemistry, 65, 301-308.

21

Benzekri, R., Bouslama, L., Papetti, A., Snoussi, M., Benslimene, I., Hamami, M., Limam, F. (2016). Isolation and identification of an antibacterial compound from Diplotaxis harra (Forssk.) Boiss. Industrial Crops and Products, 80, 228–234. Bessler, H., & Djaldetti, M. (2018). Broccoli and human health: immunomodulatory effect of sulforaphane in a model of colon cancer. International Journal of Food Sciences and Nutrition, 69, 946-953. Carbonell-Capella, J. M., Buniowska, M., Barba, F. J., Esteve, M. J., & Frígola, A. (2014). Analytical methods for determining bioavailability and bioaccessibility of bioactive compounds from fruits and vegetables: A review. Comprehensive Reviews in Food Science and Food Safety, 13(2), 155-171. Chandrasekara, A., & Shahidi, F. (2012). Bioaccessibility and antioxidant potential of millet grain phenolics as affected by simulated in vitro digestion and microbial fermentation. Journal of Functional Foods, 4, 226-237. Correa-Betanzo, J., Allen-Vercoe, E., Mcdonald, J., Schroeter, K., Corredig, M., & Paliyath, G. (2014). Stability and biological activity of wild blueberry (vaccinium angustifolium) polyphenols during simulated in vitro gastrointestinal digestion. Food Chemistry, 165, 522531. De Nicola, G. R., Bagatta, M., Pagnotta, E., Angelino, D., Gennari, L., Ninfali, P., Rollin P., & Iori, R. (2013). Comparison of bioactive phytochemical content and release of isothiocyanates in selected brassica sprouts. Food Chemistry, 141, 297-303. Fernández-Poyatos, M. P., Ruiz-Medina, A., & Llorent-Martínez, E. J. (2019). Phytochemical profile, mineral content, and antioxidant activity of Olea europaea L. cv. Cornezuelo table

22

olives. Influence of in vitro simulated gastrointestinal digestion. Food Chemistry, 297, 124933. Ferreira, S. S., Passos, C. P., Cardoso, S. M., Wessel, D. F., & Coimbra, M. A. (2018). Microwave assisted dehydration of broccoli by-products and simultaneous extraction of bioactive compounds. Food Chemistry, 246, 386-393. Gawlik-Dziki, U., Jeżyna, M., Świeca, M., Dziki, D., Baraniak, B., & Czyż, J. (2012). Effect of bioaccessibility of phenolic compounds on in vitro anticancer activity of broccoli sprouts. Food Research International, 49, 469-476. Gu, Y. J., Guo, Q. H., Zhang, L., Chen, Z. G., Han, Y. B., & Gu, Z. X. (2012). Physiological and biochemical metabolism of germinating broccoli seeds and sprouts. Journal of Agricultural and Food Chemistry, 60, 209-213. Gullon, B., Pintado, M. E., Fernández-López, J., Pérez-Álvarez, J. A., & Viuda-Martos, M. (2015). In vitro gastrointestinal digestion of pomegranate peel (Punica granatum) flour obtained from co-products: Changes in the antioxidant potential and bioactive compounds stability. Journal of Functional Foods, 19, 617-628. Guo, L. P., Yang, R. Q., Wang, Z. Y., Guo, Q. H., & Gu, Z. X. (2014). Glucoraphanin, sulforaphane and myrosinase activity in germinating broccoli sprouts as affected by growth temperature and plant organs. Journal of Functional Foods, 9, 70-77. Guo, Q. H., Guo, L. P., Wang, Z. Y., Zhuang, Y., & Gu, Z. X. (2013). Response surface optimization and identification of isothiocyanates produced from broccoli sprouts. Food Chemistry, 141, 1580-1586. Ko, J. Y., Choi, Y. J., Jeong, G. J., & Im, G. I. (2013). Sulforaphane-PLGA microspheres for the intra-articular treatment of osteoarthritis. Biomaterials, 34, 5359-5368.

23

Ku, K-M., Kim, M.J., Jeffery, E.H., Kang, Y.H., Juvik, JA. (2016). Profiles of glucosinolates, their hydrolysis products, and quinone reductase inducing activity from 39 Arugula (Eruca sativa Mill.) accessions. Journal of Agricultural and Food Chemistry, 64, 6524-6532. Lin, J. Y., & Tang, C. Y. (2007). Determination of total phenolic and flavonoid contents in selected fruits and vegetables, as well as their stimulatory effects on mouse splenocyte proliferation. Food Chemistry, 101(1), 140-147. López-Cervantes, J., Tirado-Noriega, L. G., Sánchez-Machado, D. I., Campas-Baypoli, O. N., Cantú-Soto, E. U., & Núñez-Gastélum, J. A. (2013). Biochemical composition of broccoli seeds and sprouts at different stages of seedling development. International Journal of Food Science & Technology, 48(11), 2267-2275. Lucas-González, R., Viuda-Martos, M., Álvarez, J. A. P., & Fern ández-López, J. (2018). Changes in bioaccessibility, polyphenol profile and antioxidant potential of flours obtained from persimmon fruit (Diospyros kaki) co-products during in vitro gastrointestinal digestion. Food chemistry, 256, 252-258. Mahn, A., & Perez, C. (2016). Optimization of an incubation step to maximize sulforaphane content in pre-processed broccoli. Journal of Food Science and Technology, 53(11), 41104115. Oliviero, T., Verkerk, R., Vermeulen, M., & Dekker, M. (2014). In vivo formation and bioavailability of isothiocyanates from glucosinolates in broccoli as affected by processing conditions. Molecular Nutrition & Food Research, 58(7), 1447-1456. Ortega-Vidal, J., Ruiz-Riaguas, A., Fern á ndez-de C ó rdova, M. L., Ortega-Barrales, P., & Llorent-Mart í nez, E. J. (2019). Phenolic profile and antioxidant activity of Jasonia

24

glutinosa herbal tea. Influence of simulated gastrointestinal in vitro digestion. Food chemistry, 287, 258-264. Pajak, P., Socha, R., Galkowska, D., Roznowski, J., & Fortuna, T. (2014). Phenolic profile and antioxidant activity in selected seeds and sprouts. Food Chemistry, 143, 300-306. Pasko, P., Krosniak, M., Prochownik, E., Tyszka-Czochara, M., Folta, M., Francik, R., Sikora, J.; Malinowski, M.; & Zagrodzki, P. (2018). Effect of broccoli sprouts on thyroid function, haematological, biochemical, and immunological parameters in rats with thyroid imbalance. Biomedicine & Pharmacotherapy, 97, 82-90. Pérez-Balibrea, S., Moreno, D. A., & García-Viguera, C. (2011). Genotypic effects on the phytochemical quality of seeds and sprouts from commercial broccoli cultivars. Food Chemistry, 125, 348-354. Rychlik, J., Olejnik, A., Olkowicz, M., Kowalska, K., Juzwa, W., Myszka, K.,Dembczynski, R., Moyer, M. P., & Grajek, W. (2015). Antioxidant capacity of broccoli sprouts subjected to gastrointestinal digestion. Journal of the Science of Food and Agriculture, 95, 1892-1902. Shams, R., Abu-Khudir, R., & Ali, E. M. (2017). Sulforaphane, polyphenols and related antiinflammatory and antioxidant activities changes of Egyptian broccoli during growth. Journal of Food Measurement and Characterization, 11, 2061-2068. Shen, L. Q., Su, G. Y., Wang, X. Y., Du, Q. Z., & Wang, K. W. (2010). Endogenous and exogenous enzymolysis of vegetable-sourced glucosinolates and influencing factors. Food Chemistry, 119, 987-994. Thomas-Valdés, S., Theoduloz, C., Jiménez-Aspee, F., & Schmeda-Hirschmann, G. (2019). Effect of simulated gastrointestinal digestion on polyphenols and bioactivity of the native

25

Chilean red strawberry (Fragaria chiloensis ssp. chiloensis f. patagonica). Food Research International, 123, 106-114. Tian, M., Xu, X. Y., Hu, H., Liu, Y., & Pan, S. Y. (2017). Optimisation of enzymatic production of sulforaphane in broccoli sprouts and their total antioxidant activity at different growth and storage days. Journal of Food Science and Technology, 54, 209-218. Vallejo, F., Gil-Izquierdo, A., P é rez-Vicente, A., & Garc í a-Viguera, C. (2004). In vitro gastrointestinal

digestion

study

of

broccoli

inflorescence

phenolic

compounds,

glucosinolates, and vitamin C. Journal of Agricultural and Food Chemistry, 52, 135-138. Veeranki, O. L., Bhattacharya, A., Marshall, J. R., & Zhang, Y. (2012). Organ-specific exposure and response to sulforaphane, a key chemopreventive ingredient in broccoli: implications for cancer prevention. British Journal of Nutrition, 109, 25-32. Wu, Y. F., Mao, J. W., Mei, L. H., & Liu, S. W. (2013). Studies on statistical optimization of sulforaphane production from broccoli seed. Electronic Journal of Biotechnology, 16. Wu, Y. F., Xiao, G. N., Mao, J. W., Liu, S. W., Huang, J., & Mei, L. H. (2015). Dietary sulforaphane inhibits histone deacetylase activity in B16 melanoma cells. Journal of Functional Foods, 18, 182-189. Yuan, G. F., Wang, X. P., Guo, R. F., & Wang, Q. M. (2010). Effect of salt stress on phenolic compounds, glucosinolates, myrosinase and antioxidant activity in radish sprouts. Food Chemistry, 121, 1014-1019.

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FIGURE CAPTIONS Figure 1. Sulforaphane yield from broccoli seeds and sprouts (after 3, 5, and 7 days germination respectively) of different broccoli varieties. (A) Sulforaphane yield was calculated and expressed by mg SF per gram seed or fresh sprout (mg/g FW). (B) Sulforaphane yield from fresh sprouts was calculated and expressed as mg per gram seed (mg/g seed). Data were means ± standard deviations of triplicate measures. The different lowercase letters indicated significant differences of values in sampling day in the same variety. The different uppercase letters indicated significant differences of values in variety in the same sampling day ( p < 0.05).

Figure 2. Impact of sulforaphane (A), total phenolic (B) and total flavonoid content (C) of MNL broccoli seeds and 3-day sprouts, following gastrointestinal digestion in vitro. The different lowercase letters indicated significant differences of values from all samples. The different uppercase letters indicated significant differences of values from seeds or sprouts ( p < 0.05). ND: not digested. GD:after gastric digestion. ID: after gastrointestinal digestion. ST: soluble fraction after GD or ID. RS: insoluble fraction after GD or ID.

Figure 3. Changes of DPPH radical scavenging capacity (A) and ferric reducing antioxidant power (B) from broccoli seeds and sprouts in vitro gastrointestinal digestion. The different lowercase letters indicated significant differences of values from all samples. The different uppercase letters indicated significant differences of values from seeds or sprouts ( p < 0.05). ND: not digested. GD:after gastric digestion. ID: after gastrointestinal digestion. ST: soluble fraction after GD or ID. RS: insoluble fraction after GD or ID.

27

28

Figure 1

29

Figure 2

30

Figure 3

31

Table 1. Independent variables and their levels for BBD Seeds

Sprouts Levels

Levels

Independent variables

Independent variables -1

0

1

-1

0

1

X1: Solvent to sample ratio (g/mL)

6

8

10

X1’: Solvent to sample ratio (m:v)

4

5

6

X2:Enzymatic hydrolysis time (h)

3

4

5

X2’: Enzymatic hydrolysis time (h)

4

6

8

X3: pH

3.5

4

4.5

X3’: pH

3.5

3

4.5

X4: Extraction time (min)

20

30

40

X4’: Ascorbic acid (mg)

0.1

0.2

0.3

32

Table 2. Experimental design and results of BBD Solvent to sample ratio (m:v)

Enzymatic hydrolysis time (h)

(X1; X1’)

(X2; X2’)

1

0

2

Run

pH

Extraction time (min) (X4)

Extraction percentage

Extraction percentage

(X3; X3’)

Ascorbic acid (mg) (X4’)

(Y1 mg/g FW)

(Y2 mg/g FW)

0

0

0

4.0164

0.4965

0

-1

0

1

3.3848

0.2947

3

1

0

1

0

3.6268

0.2268

4

0

0

-1

1

3.7525

0.3579

5

-1

0

0

-1

3.5912

0.3200

6

0

0

1

-1

3.7244

0.3053

7

0

1

0

-1

3.9463

0.2484

8

0

1

1

0

3.3930

0.2575

9

-1

-1

0

0

3.0254

0.2650

10

0

0

1

1

3.7013

0.2266

11

0

1

0

1

3.1408

0.1617

12

0

0

0

0

4.1457

0.5034

13

1

0

0

1

3.8484

0.4100

14

-1

1

0

0

3.1152

0.1201

15

0

0

-1

-1

3.3552

0.2609

16

1

-1

0

0

3.3451

0.3399

17

0

0

0

0

3.9670

0.5104

18

1

0

-1

0

3.0316

0.1239

19

0

0

0

0

3.8456

0.4378

20

0

-1

-1

0

3.4009

0.3446

21

0

1

-1

0

3.5322

0.1603

22

-1

0

0

1

3.4627

0.2690

23

0

-1

0

-1

3.1161

0.4104

24

1

0

0

-1

3.5214

0.3600

25

0

-1

1

0

3.5450

0.2927

26

1

1

0

0

3.3042

0.2086

27

-1

0

1

0

3.1204

0.2245

28

-1

0

-1

0

3.0546

0.3947

29

0

0

0

0

3.9762

0.4672

The extraction conditions of sulforaphane from broccoli seeds was optimized from following four independent

33

variables: the solvent to sample ratio (X1), the enzymatic hydrolysis time (X2), the pH value (X3), and the extraction time (X4). The extraction conditions of sulforaphane from broccoli sprouts was optimized from following four independent variables: the solvent to sample ratio (X1’), the enzymatic hydrolysis time (X2’), the pH value (X3’), and the amount of ascorbic acid addition (X4’). The SF yield from broccoli seeds (Y1) and sprouts (Y2) were used as response variables.

34

Table 3. Weight of seed (100 seed) and sprout (100 plants) of six cultivars LWW

YJ

YDLT

MNL

XM

LY

0.33±0.03aІAІІ

0.40±0.01aC

0.43±0.01aD

0.41±0.02aCD

0.84±0.00aE

0.35±0.01aB

3d

1.55±0.05bA

2.14±0.03bB

2.67±0.13bC

1.77±0.15bA

3.67±0.12bD

1.55±0.17bA

5d

2.62±0.21cA

3.66±0.25cBC

4.14±0.15cC

3.46±0.35cB

5.65±0.52cD

2.55±0.03cA

7d

3.20±0.28dA

5.35±0.26dC

5.36±0.74dC

4.27±0.12dB

6.00±0.28cC

3.06±0.19dA

Seeds/g

Sprouts/g

І Values within same columns (n = 3; ± SD); the different lowercase letter indicate differ significantly at p < 0.05 according to Duncan’s test. ІІ Values within same row (n = 3; ± SD); the different uppercase letter indicate differ significantly at p < 0.05 according to Duncan’s test.

35

Table 4. TP and TF contents in broccoli seeds and sprouts

Cultivar

LWW

time (d)

TP (mg GAE /g FW)

TF (mg RE/g FW )

TP (mg GAE /g seed)

TF (mg RE/g seed)

Seeds

4.57±0.11cІCІІ

4.01±0.01bB

4.57±0.11aІCІІ

4.01±0.01aB

3

2.44±0.43bD

3.02±1.19bB

11.62±2.06bD

14.36±5.65bB

5

1.43±0.10aD

1.45±0.20aC

11.54±0.81bC

11.67±1.62bC

7

1.08±0.04aE

1.22±0.02aE

10.58±0.43bC

11.93±0.23bC

4.20±0.09dB

3.80±0.09dB

4.20±0.09aB

3.80±0.09aB

3

1.71±0.03cC

1.88±0.07cA

9.26±0.18bC

10.18±0.39bAB

5

1.25±0.01bC

1.32±0.01bC

11.56±0.08cC

12.21±0.09cC

7

0.91±0.07aD

1.06±0.10aD

12.34±0.93cD

14.32±1.30dD

4.58±0.11dC

3.68±0.23cB

4.58±0.11cC

3.68±0.23aB

3

0.94±0.01cA

1.02±0.04bA

5.83±0.04dA

6.27±0.22bA

5

0.41±0.02bA

0.64±0.10aA

3.88±0.16bA

6.12±0.98bAB

7

0.16±0.02aA

0.46±0.06aA

1.98±0.20aA

5.67±0.68bAB

4.04±0.18bAB

2.86±0.62cA

4.04±0.18aAB

2.86±0.62aA

3

1.73±0.07abC

1.84±0.31bA

7.49±0.29bB

7.98±1.36cA

5

1.28±0.01aC

0.84±0.04aB

10.86±0.10dC

7.10±0.31bcB

7

0.94±0.04aD

0.60±0.04aB

9.87±0.43cC

6.31±0.39bB

3.89±0.04cA

2.60±0.01dA

3.89±0.04aA

2.60±0.01aA

3

1.00±0.02bAB

1.35±0.02cA

4.36±0.09aA

5.86±0.10cA

5

0.60±0.06aB

0.81±0.02bAB

4.02±0.43aA

5.42±0.14bA

7

0.60±0.05aC

0.74±0.02aC

4.26±0.35aB

5.24±0.15bAB

4.11±0.11dB

3.50±0.04dB

4.11±0.11bB

3.50±0.04aB

3

1.30±0.05cB

1.32±0.03cA

5.71±0.21dA

5.80±0.11cA

5

0.67±0.02bB

0.71±0.10bAB

4.84±0.12cB

5.13±0.70bcA

7

0.29±0.02aB

0.54±0.04aAB

2.49±0.15aA

4.71±0.33bA

Germination

Sprouts

Seeds YJ

Sprouts

Seeds YDLT

Sprouts

Seeds MNL

Sprouts

Seeds XM Sprouts

Seeds LY

Sprouts

І Within columns, values in sampling day (n = 3; ± SD) with different lowercase letters indicate significant differences at p < 0.05 according to Duncan’s test.

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ІІ Within columns, values in variety (n = 3; ± SD) with the different uppercase letters indicate significant differences at p < 0.05 according to Duncan’s test. TP:Total phenolic. TF:Total flavonoid.

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Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Highlights - Extraction process of SF from broccoli seeds and sprouts was separately optimized - SF yields decreased during germination process and varied among varieties. - Antioxidant capacity was significantly higher in sprouts than seeds. - 3-day-old broccoli sprouts were recommended as raw materials of functional foods.

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