Effect of the Number of Dark Days and Planting Density on the Health-Promoting Phytochemicals and Antioxidant Capacity of Mustard (Brassica juncea) Sprouts

Mustard is an edible vegetable in the genus Brassica with tender and clean sprouts and short growth cycles that has become a rich source of nutrients required by humans. Here, the effects of dark exposure duration and planting density on the health-promoting phytochemicals and the antioxidant capacity of mustard sprouts were evaluated. The content of soluble sugar, soluble protein, chlorophyll, and carotenoids and the antioxidant capacity of mustard were higher in the two-day dark treatment; the content of indolic glucosinolates was also more affected in the dark day experiment than in the planting density experiment. The soluble sugar, soluble protein, and aliphatic and total glucosinolate levels were higher when sprouts were grown at high densities (6–7 g per tray); however, no significant variation was observed in the content of chlorophyll and carotenoids and the antioxidant capacity. The results of this study show that the optimum cultivation regime for maximizing the concentrations of nutrients of mustard plants is a planting density of 6 g of seeds per tray and a two-day dark treatment.


Introduction
Mustard (Brassica juncea) is a widely consumed, nutritious vegetable in the genus Brassica [1]. It is mostly cultivated for use as a fresh vegetable because of its high concentrations of bioactive components, such as chlorophyll, carotenoids, ascorbic acid, phenolic compounds, and glucosinolates, and many studies of these biochemical compounds in mustard have been conducted in recent years [2][3][4]. The consumption of sprouts is increasing, and this increase has been largely driven by a growing trend in healthy eating habits among consumers [5,6]. Sprouts have been shown in many studies to be more nutrient-dense than ungerminated seeds or even mature vegetables [5]. The levels of these health-promoting nutrients in sprouts are greatly affected by the growth environment and culture conditions [7,8]. For example, a previous study has shown that UV-B illumination 149.60 ± 6.29 b 109.68 ± 5.64 a 6.44 ± 0.24 c 0.61 ± 0.04 ab D4 136.87 ± 2.28 c 103.86 ± 0.76 bc 5.70 ± 0.14 d 0.60 ± 0.03 b Data are expressed as mean ± standard deviation. Same letter in the same column means no significant differences among values (p < 0.05) according to the LSD's test. D1: dark treatment for one day; D2: dark treatment for two days; D3: dark treatment for three days; D4: dark treatment for four days. 152.65 ± 6.73 a 108.09 ± 2.66 b 6.40 ± 0.08 a 0.68 ± 0.06 a Data are expressed as mean ± standard deviation. Same letter in the same column means no significant differences among values (p < 0.05) according to the LSD's test. P1: a sowing density of 4 g of seeds per tray; P2: a sowing density of 5 g of seeds per tray; P3: a sowing density of 6 g of seeds per tray; P4: a sowing density of 7 g of seeds per tray.

Chlorophyll and Carotenoids
The chlorophyll content was highest in D2 (7.29 mg g −1 ), and it was 5.72%, 13.17%, and 27.83% higher in D2 than in D1 (6.89 mg g −1 ), D3 (6.44 mg g −1 ), and D4 (5.70 mg g −1 ), respectively. The carotenoid content was highest in D1 (0.64 mg g −1 ), and there was no significant difference in the carotenoid content between D2 (0.63 mg g −1 ) and D1 (Table 1). D2 had a positive effect on the accumulation of photosynthetic pigments. However, there was no significant effect of planting density on the level of chlorophyll and carotenoids ( Table 2).

Ascorbic Acid
The ascorbic acid content was highest in D4 (1.41 mg g −1 ), followed by D2 (1.31 mg g −1 ), and there was no significant difference in the ascorbic acid content between D4 and D2 ( Table 3).
The ascorbic acid content was higher in P3 (2.52 mg g −1 ) and P4 (2.61 mg g −1 ) than in P1 (2.24 mg g −1 ) and P2 (2.07 mg g −1 ) (Table 4), which indicated that the ascorbic acid content was higher at high planting densities. Table 3. Ascorbic acid, proanthocyanidins, flavonoids, total phenolics, and antioxidant activity content in different days of dark treatment of mustard sprouts.

Proanthocyanidins, Flavonoids, and Total Phenolics
The content of proanthocyanidins (5.95 mg g −1 ), flavonoids (14.92 mg g −1 ), and total phenolics (16.05 mg g −1 ) was highest in D2 (Table 3), demonstrating that D2 was optimal for maximizing the content of the above three antioxidants.
The content of proanthocyanidins was highest in P3 (6.39 mg g −1 ), and no differences were observed in the content of flavonoids and total phenolics among the four planting density treatments (Table 4).

Antioxidant Activity
High levels of ABTS (47.97 mg g −1 ) and FRAP (0.15 mmol g −1 ) were observed in D4, and no significant differences were observed in ABTS and FRAP among the three remaining treatments (Table 3). Planting density barely made any difference to the antioxidant activity (Table 4).

Glucosinolates
Three aliphatic and four indolic glucosinolates were identified in mustard sprouts in our study. The content of total and individual glucosinolates varied among the dark day and planting density treatments (Figures 1 and 2).

PCA
To evaluate the effect of treatment more comprehensively and systematically, PCA was performed to characterize differences in the content of the main health-promoting phytochemicals and antioxidant capacity among dark day and planting density treatments.
The first principal component (PC1) and second principal component (PC2) explained 38.6% and 23.8% of the variance, respectively, for the dark day PCA. D2 and D4 were sepa-Plants 2022, 11, 2515 7 of 15 rated along PC1, and D4 was separated from D1 and D2 along PC2 ( Figure 3A). PLS-DA was also performed. PLS-DA1 and PLS-DA2 accounted for 30.8% and 27.9% of the variance, respectively, and their distributions were similar to PC1 and PC2 ( Figure 3B). According to the loading and VIP plots of the PLS-DA, the major contributors to D2 were soluble sugar, flavonoids, total indolic glucosinolates, glucobrassicin, total chlorophyll, and proanthocyanidins, and the major contributors to D4 were ABTS and 4-methoxyglucobrassicin ( Figure 3C).

PCA
To evaluate the effect of treatment more comprehensively and systematically, PCA was performed to characterize differences in the content of the main health-promoting phytochemicals and antioxidant capacity among dark day and planting density treatments.
The first principal component (PC1) and second principal component (PC2) explained 38.6% and 23.8% of the variance, respectively, for the dark day PCA. D2 and D4 were separated along PC1, and D4 was separated from D1 and D2 along PC2 ( Figure 3A). PLS-DA was also performed. PLS-DA1 and PLS-DA2 accounted for 30.8% and 27.9% of the variance, respectively, and their distributions were similar to PC1 and PC2 ( Figure  3B). According to the loading and VIP plots of the PLS-DA, the major contributors to D2 were soluble sugar, flavonoids, total indolic glucosinolates, glucobrassicin, total chlorophyll, and proanthocyanidins, and the major contributors to D4 were ABTS and 4-methoxyglucobrassicin ( Figure 3C).  PC1 and PC2 explained 42.9% and 13.2% of the variance, respectively, in the planting density PCA. P4 and P2 were separated along PC1, and P4 was separated from P3 along  Figure 4A). PLS-DA was also performed. PLS-DA1 and PLS-DA2 accounted for 42.4% and 12.6% of the variance, respectively. P4 and P3 could be discriminated from P1 and P2 along PLS-DA1, and P4 could be discriminated from P3 along PLS-DA2 ( Figure 4B). According to the loading and VIP plots of the PLS-DA, ascorbic acid, progoitrin, gluconapin, 4-methoxyglucobrassicin, sinigrin, total glucosinolates, and total aliphatic glucosinolates were the major contributors to P4, soluble protein was the main contributor to P3, and proanthocyanidins were the main contributor to P1 ( Figure 4C). glucobrassicin; GBS: glucobrassicin; 4-MGBS: 4-methoxyglucobrassicin; 4-HGBS: 4-hydroxyglucobrassicin; AGS: total aliphatic glucosinolates; IGS: total indolic glucosinolates; GS: total glucosinolates. D1: dark treatment for one day; D2: dark treatment for two days; D3: dark treatment for three days; D4: dark treatment for four days. PC1 and PC2 explained 42.9% and 13.2% of the variance, respectively, in the planting density PCA. P4 and P2 were separated along PC1, and P4 was separated from P3 along PC2 ( Figure 4A). PLS-DA was also performed. PLS-DA1 and PLS-DA2 accounted for 42.4% and 12.6% of the variance, respectively. P4 and P3 could be discriminated from P1 and P2 along PLS-DA1, and P4 could be discriminated from P3 along PLS-DA2 ( Figure  4B). According to the loading and VIP plots of the PLS-DA, ascorbic acid, progoitrin, gluconapin, 4-methoxyglucobrassicin, sinigrin, total glucosinolates, and total aliphatic glucosinolates were the major contributors to P4, soluble protein was the main contributor to P3, and proanthocyanidins were the main contributor to P1 ( Figure 4C).

Correlation Analysis
To investigate the correlations between the health-promoting phytochemicals and antioxidant activity, Pearson correlation coefficients were determined, and the correlation threshold R 2 > 0.65 was used. Three groups with significant positive correlations among variables were detected. The first group included correlations among sinigrin, progoitrin, gluconapin, total aliphatic glucosinolates, and total glucosinolates; the second group included correlations among total phenolics, ascorbic acid, proanthocyanidins, FRAP, and 4-methoxyglucobrassicin; the last group included correlations among total indolic glucosinolates and glucobrassicin. No correlations were observed among the above three groups.
In the first group, total glucosinolates were positively correlated with total aliphatic glucosinolates, sinigrin, progoitrin, and gluconapin, and this group had the highest number of correlations (four edges). In the second group, FRAP and total phenolics were significantly correlated with each other and with ascorbic acid and proanthocyanidins (three edges), whereas the latter two were irrelevant. In addition, 4-methoxyglucobrassicin was correlated with ascorbic acid. In the third group, total indolic glucosinolates and glucobrassicin were positively correlated with each other ( Figure 5). hydroxyglucobrassicin; AGS: total aliphatic glucosinolates; IGS: total indolic glucosinolates; GS: total glucosinolates. P1: a sowing density of 4 g of seeds per tray; P2: a sowing density of 5 g of seeds per tray; P3: a sowing density of 6 g of seeds per tray; P4: a sowing density of 7 g of seeds per tray.

Correlation Analysis
To investigate the correlations between the health-promoting phytochemicals and antioxidant activity, Pearson correlation coefficients were determined, and the correlation threshold R 2 > 0.65 was used. Three groups with significant positive correlations among variables were detected. The first group included correlations among sinigrin, progoitrin, gluconapin, total aliphatic glucosinolates, and total glucosinolates; the second group included correlations among total phenolics, ascorbic acid, proanthocyanidins, FRAP, and 4-methoxyglucobrassicin; the last group included correlations among total indolic glucosinolates and glucobrassicin. No correlations were observed among the above three groups.
In the first group, total glucosinolates were positively correlated with total aliphatic glucosinolates, sinigrin, progoitrin, and gluconapin, and this group had the highest number of correlations (four edges). In the second group, FRAP and total phenolics were significantly correlated with each other and with ascorbic acid and proanthocyanidins (three edges), whereas the latter two were irrelevant. In addition, 4-methoxyglucobrassicin was correlated with ascorbic acid. In the third group, total indolic glucosinolates and glucobrassicin were positively correlated with each other ( Figure 5).

Discussion
The health-promoting phytochemicals and antioxidant activity of mustard sprouts were greatly affected by the number of days they were exposed to darkness and planting density.
Dark conditions promote the skotomorphogenesis of sprouts. During this process, the hypocotyl rapidly elongates, the cotyledon slowly expands, and undifferentiated chloroplast precursors are produced [23]. This process is modulated by phytochrome-interacting factors (PIFs) to a large extent, suggesting that the length of exposure to dark treatment affects levels of photosynthetic pigments, such as chlorophyll and carotenoids [24]. Cotyledons are considered the main assimilatory organ in the early stage of sprouts, and their size, regulated by the length of dark exposure, is closely related to the accumulation of plant nutrients, such as soluble sugar and protein [23,25]. In the dark day experiment, D2 was optimal for maximizing the concentrations of chlorophyll, carotenoids, soluble sugar, and soluble protein in mustard sprouts. The content of chlorophyll and carotenoids did not vary significantly among planting density treatments; this might stem from the shading of tender leaves by neighboring plants in the early sprouting stage, which prevents light from being detected by the phytochrome photoreceptors [24,26]. Research has shown that planting density affects the content of soluble sugar and protein [27]. However, no linear relationship between planting density and the content of soluble sugar and protein in mustard sprouts was observed in our study.
The content of ascorbic acid and phenolic compounds (including total phenolics, flavonoids, and proanthocyanidins) and antioxidant activities were measured to characterize variation in antioxidant capacity among treatments [28]. In previous studies, radish sprouts grown under dark conditions were shown to have fewer total phenolic and flavonoid compounds than those grown under light conditions [29]. Phenolic compounds and antioxidant activities in broccoli and kale sprouts are lowest under constant darkness and increase with the duration of artificial light exposure [30]. The expression of the genes involved in polyphenol biosynthesis is highly upregulated by light stimulation when reactive oxygen species (ROS) accumulate under light stress [30,31]. This effect has also been observed in flavonoid compounds and other antioxidants [32]. However, in our study, the antioxidant capacity did not continuously decrease with the number of dark days. The content of ascorbic acid (1.41 mg g −1 ), ABTS + (47.97%), and FRAP (0.15 mg g −1 ) was highest in D4; however, D2 was the best overall treatment compared with D1. Therefore, we speculated that appropriate levels of dark exposure are needed to optimize antioxidant capacity, and this requires further study [31]. Planting density has been shown to affect the antioxidant capacity, including the content of ascorbic acid and total phenolics, in previous studies [20,33]. The same has been observed for glucosinolates; abiotic stress caused by planting density is the main factor affecting the antioxidant capacity of plants [17,22,33]. Competition for nutrients and space increases with planting density, and this is thought to increase the antioxidant capacity [33,34]. However, in the planting density experiment, ascorbic acid and proanthocyanidins were the only compounds that were most abundant at high planting densities: P3 (6.39 mg g −1 ) and P4 (2.61 mg g −1 ), respectively. No significant variation in the other compounds was observed among planting density treatments, and this might suggest that the effect of planting density is negligible in the early sprouting stage of mustard.
Glucosinolates are important secondary metabolites in cruciferous plants that have received much interest in recent years for their strong biological activity and anti-cancer function [1,35]. Seven glucosinolates of two classes, aliphatic and indolic, were detected in mustard sprouts in this study, with sinigrin being predominant. The abundance of these glucosinolates might contribute to the high nutritional value of mustard sprouts [2,3]. However, the content of glucosinolates is heavily affected by environmental conditions and cultivation methods; it thus responded strongly to these two factors examined in our study [35].
There has been much debate regarding whether glucosinolate biosynthesis is stimulated by light or dark treatment [7]. Sprouts grown in the dark have a higher content of indolic glucosinolates, whereas those grown under light have a higher content of aliphatic glucosinolates [36,37]. Dark treatment had the strongest positive effect on total indolic glucosinolates compared with aliphatic glucosinolates. Glucobrassicin, an important indolic glucosinolate, is most abundant in 7-day-old mustard sprouts under complete darkness [36]. Rapeseed sprouts have higher concentrations of 4-hydroxyglucobrassicin, neoglucobrassicin, and total indolic glucosinolates when they are grown in the dark than when they are grown under light [36,38]. However, light treatment enhanced the concentrations of glu-cosinolates (expressed as mg of sinigrin per 100 g) by 33% in broccoli sprouts over 7 days of development [39]. The extent to which glucosinalbin (the specific and decisive aliphatic glucosinolate in white mustard) was reduced during germination was mitigated by treatment with 24 h of darkness [7]. The close correlation of glucosinolates with dark/light treatment might be related to the R2R3-MYB gene subfamily, a complex group of transcription factors in Brassica plants. The expression of MYB34, MYB51, and MYB122, which have a major effect on indolic glucosinolate biosynthesis, is upregulated by dark conditions, whereas the expression of MYB28 and MYB29, which affect aliphatic glucosinolate biosynthesis, is upregulated under light exposure [37,40,41]. The expression levels of these transcription factors are also affected by the degree of light and dark regulation, depending on the intensity and length of darkness or light exposure [32,37,42]. Thus, for mustard sprouts, exposure to darkness for 2 d may maximize the upregulation of the expression levels of the transcription factors involved in glucosinolate regulation, especially indolic glucosinolates.
In the planting density experiment, the content of aliphatic and total glucosinolates increased with planting density. The concentrations of total aliphatic glucosinolates in P3 and P4 were 45.25% and 32.82% higher than that in P1 and 28.86% and 17.83% higher than that in P2, respectively; total glucosinolates were also significantly higher at high planting densities. The degradation of glucosinolate by myrosinase hydrolysis has been shown to be related to the response of plants to stress, and aliphatic glucosinolates are the most strongly affected by stress [43][44][45]. In the sprouts of polyploid Brassica juncea, the abundance of aliphatic glucosinolates is highest under biotic stress, such as glucose stress, and this is not the case with indolic glucosinolates [44]. An increase in glucosinolates, which is mainly determined by sinigrin, has been observed in broccoli in response to salt stress [45]. Additionally, as stress factors, nutrient and space deficiencies associated with high planting density are closely related to the content of glucosinolates. In six canola varieties, increases in plant densities result in a higher glucosinolate content [34]. Watercress grown under 20 cm spacing (72 plants per bed) is superior to 31 cm (36 plants per bed) spacing for maximizing the glucosinolate content [46]. Myrosinase-mediated glucosinolate degradation products can be rearranged to form different glucosinolate activation products, and this process is affected by planting density [43,47]. As planting densities increase, either direct elicitors from neighboring plants or indirect elicitors by competing nutrients and space lead to variation in the structural outcome of glucosinolate activation [43]. Thus, owing to nutrient and space stress, higher planting density (seeds at a density of 6-7 g) enhances yield as well as the nutritional value of mustard sprouts, and these changes are mostly associated with variations in glucosinolate concentrations [3,18].

Plant Material
The leaf mustard variant Brassica juncea var. rugosa, which is mostly cultivated for use as a fresh vegetable, was used as experimental material.
In the dark day experiment, 5 g of mustard seeds was evenly propagated in a seedling tray (32.6 cm × 22.4 cm × 4.1 cm) and covered with moist germinating paper. The mustard sprouts were exposed to darkness for 1 d (D1), 2 d (D2), 3 d (D3), and 4 d (D4) at a temperature of 25 • C and relative humidity (RH) of 70%. There were four replicates for each treatment, and one tray corresponded to one replicate. In the planting density experiment, 4 g (P1), 5 g (P2), 6 g (P3), and 7 g (P4) of mustard seeds were sown in each tray, and the plants were exposed to darkness for 2 d; all other conditions were the same among the treatments. After 7 d of growth, which corresponded to when the mustard sprouts reached the size at which they are typically harvested, the aerial parts were harvested, lyophilized, and stored at −20 • C for further analysis.

Soluble Protein Content
Fifty milligrams of freeze-dried powdered material was soaked in distilled water, and the solution was stirred for 30 s, settled for 30 min, and centrifuged for 5 min at 4000× g, and then 1 mL was transferred to a polypropylene tube. Coomassie brilliant blue G-250 was combined with 1 mL supernatant, and the absorbance was measured at 595 nm within 20 min after the reaction [4].

Soluble Sugar Content
Fifty milligrams of powder was extracted in 10 mL of distilled water for 20 min at 90 • C, and the homogenates were centrifuged at 4000× g for 5 min. A combination of 1 mL of sample extract, 0.5 mL of anthrone-ethyl acetate reagent, and 5 mL of concentrated sulfuric acid was homogenized and boiled for 5 min. The absorbance of the reaction mixtures was measured at 630 nm [4].

Chlorophyll and Carotenoids Content
An appropriate amount of sample was weighed, ground, and extracted with 10 mL of ethanol. The supernatant was collected and analyzed by the spectrophotometer, absorbance was detected at 665 nm, 649 nm, and 451 nm to measure the content of total chlorophyll and carotenoids, respectively [4].

Ascorbic Acid Content
Fifty mg of sample powder was extracted with 5 mL of 1.0% (w/v) oxalic acid and then centrifuged for 5 min at 4000× g. Each sample was filtered through a 0.45 µm cellulose acetate filter and analyzed by high-performance liquid chromatography (HPLC). The amount of ascorbic acid was calculated from absorbance values at 243 nm [4].

Proanthocyanidin Content
The powder of each sample was weighed, ground, and transferred to 4 mL of the extracting reagent. The solution was vortexed for 5 min, shaken for 1 h, and centrifuged at 4000× g for 5 min. The p-dimethylaminocinnamaldehyde reagent was added to 700 µL of supernatant. The absorbance of the mixture was spectrophotometrically detected at 640 nm after 20 min [48].

Flavonoid Content
Forty milligrams of sample powder was extracted in 50% ethanol and incubated at room temperature for 24 h in the dark. A 1.2 mL aliquot of the supernatant was mixed with 60 µL of 2% aluminum trichloride, 60 µL of 1 mol L −1 potassium acetate, and 1.68 mL of distilled water after being centrifuged. The absorption was read at 415 nm after 40 min [4].

Total Phenolic Content
Total phenolics were extracted with 50% ethanol, and the supernatant (300 µL) was mixed with 1.5 mL of 0.2 mol L −1 Folin-Ciocalteu reagent, and after 3 min, 1.2 mL of saturated sodium carbonate was added. The mixtures were allowed to stand for 20 min at room temperature, and the absorbance was measured at 760 nm with the spectrophotometer [4].

ABTS Assay
An aliquot of 300 µL of each extracted sample was added to 3 mL of ABTS + solution. The absorbance was measured spectrophotometrically at 734 nm after exactly 2 h, and then the value was calculated [4].

Ferric Reducing Antioxidant Power (FRAP)
The extracted samples (300 µL) were added to 2.7 mL of the FRAP working solution, incubated at 37 • C, and vortexed. The absorbance was then recorded at 593 nm after 10 min, and the result was expressed as mmol kg −1 of dry weight [4].

Glucosinolate Composition and Content
Powdered samples (100 mg) were boiled in 5 mL of water for 10 min, and the supernatant was collected and applied to a DEAE-Sephadex A-25 column. The glucosinolates were converted into their desulpho analogues by treatment with aryl sulphatase, and the desulphoglucosinolates were eluted. HPLC analysis of desulphoglucosinolates was carried out using an Agilent 1260 HPLC instrument equipped with a variable wavelength detector (VWD) detector. The samples were separated at 30 • C on a Waters Spherisorb C18 column (250 × 4.6 mm) using acetonitrile and water at a flow rate of 1.0 m Lmin −1 . The absorbance was detected at 226 nm [4].

Statistical Analyses
All assays were performed in four replicates and all results were shown as mean ± standard deviation. Statistical analysis used SPSS version 20 (SPSS Inc., Chicago, IL, USA). The data obtained were subjected to analysis of one-way ANOVAs. The histogram graphs were conducted using Origin 8.5.0 (OriginLab Corporation, Northampton, MA, USA). A principal components analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) were performed using SIMCA 14.1 Demo software (Umetrics, Malmö, Sweden) with unit variance scaling to determine the relationships among the samples. The correlation analyses were also performed and were visualized using Cytoscape v. 3.5.1 (The Cytoscape Consortium, New York, NY, USA) [49,50].

Conclusions
Dark treatment and planting density had marked effects on the health-promoting phytochemicals and antioxidant capacity of mustard sprouts in our study. D2 increased the content of soluble sugar and protein, chlorophyll, carotenoids, and indolic glucosinolates. High planting densities (i.e., seeds sown at a density of 6-7 g per tray (P3 and P4) significantly enhanced the content of soluble sugar, protein, and aliphatic and total glucosinolates, and no marked increases were observed in the content of chlorophyll and carotenoids and antioxidant capacity. Overall, a sowing density of 6 g of seeds per tray and dark treatment for two days are optimal for maximizing the nutritional quality of mustard sprouts and minimizing costs. In addition, the regulatory mechanisms of darkness and planting density on indolic and aliphatic glucosinolates, respectively, in mustard sprouts, should be studied further. It is also of great significance to establish a quantifiable formula to more systematically evaluate the treatment effect in the future.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants11192515/s1, Table S1: Correlation of the health-promoting phytochemicals and antioxidant capacity in mustard sprouts.