Profiling of Redox-Active Lipophilic Constituents in Leaf Mustard (Brassica juncea (L.) Czern.) Cultivars Using LC-MS and GC-MS

Leaf mustard is an important commercial and culinary vegetable. However, only limited information is available on the content and composition of the nutritionally important lipophilic constituents in these leaves. This research presents information on the contents and composition of carotenoids, tocols, phytosterols, and fatty acids in four cultivars of leaf mustard. The carotenoids and tocols were analyzed utilizing liquid chromatography (LC)–mass spectrometry (MS) with single ion monitoring (SIM), while phytosterols and fatty acids were analyzed using gas chromatography (GC)–MS and GC-flame ionization detection (FID), respectively. The LC-MS results revealed the dominance of (all-E)-lutein, within the range of 37.12 (cv. Asia Curled)—43.54% (cv. Jeok) of the total carotenoids. The highest amount of all of the individual carotenoids and total carotenoids (143.85 µg/g fresh weight; FW) were recorded in cv. Cheong. Among the studied leaf samples, 67.16 (cv. Asia Curled)—83.42 µg/g FW (cv. Cheong) of α-tocopherol was recorded. Among the phytosterols, β-sitosterol was the most dominant one among the studied mustard leaves, accounting for 80.42 (cv. Jeok)—83.14% (cv. Red frill) of the total phytosterols. The fatty acid analysis revealed the presence of a significant amount of rare hexadecatrienoic acid (C16:3n3) in the studied mustard leaves, which accounted for 27.17 (cv. Asia Curled)—32.59% (cv. Red frill) of the total fatty acids. Overall, the cv. Cheong represented the highest contents of carotenoids, tocols, and phytosterols. Moreover, cv. Red frill contains the highest amount of n-3 PUFAs and antioxidant compounds. Thus, these cultivars can be promoted in cuisines which can be eaten to obtain the highest health benefits.


Introduction
The epidemiological and clinical studies have shown the inverse relationship between the dietary consumption of fruits and vegetables and the prevalence of chronic diseases such as cancer, cardiovascular diseases (CVD), neurodegenerative diseases, obesity, type 2 diabetes (T2D), as well as all-cause mortality [1][2][3]. The bioactive antioxidant compounds, including the carotenoids and tocols present in fruits and vegetables, neutralize the cellular system's free radicals, thus preventing the oxidative damage of cells, proteins, and DNA, resulting in a low incidence of related diseases [4][5][6].
Brassica vegetables are a rich source of carotenoids [7], tocopherols [8], and several other bioactive compounds [9]. Brassica juncea (L.) Czern., which is commonly known as leaf mustard, brown mustard, Korean leaf mustard, Oriental mustard, and vegetable mustard, is an annual herb belonging to the Brassicaceae family. The leaf mustard is widely used in Asian, African, Southern, and American cuisines.
Carotenoids are tetraterpenoid pigments that are universally synthesized by all photoautotrophs. In animals, provitamin A carotenoids (e.g., αand β-carotene and presence of foreign particles, and stored in a −80 • C deep freezer (Ilshin Biobase Co., Ltd., Dongducheonsi, Gyeonggido, Republic of Korea) until their analysis. Figure 1 shows the phenotypic diversity among the mustard cultivars used in this investigation.
All of the organic solvents used for extraction were of LC grade, and they were obtained from J.T. Baker ® (Avantor Performance Materials Korea Ltd.), Suwon-Si, Republic of Korea.

Extraction of Crude Lipids (Lipophilic Compounds)
The lipophilic compounds, including the carotenoids, tocols, sterols, and fatty acids, were extracted simultaneously from fresh mustard leaves following our optimized protocol [17] with minor modifications. The detailed extraction procedure is given in Appendix A Figure A1. Synthetic antioxidant butylated hydroxytoluene (BHT) was added to the extraction solvent (0.1% w/v) to prevent the degradation of the lipophilic compounds [26].
The tocols and carotenoids were simultaneously analyzed by liquid chromatography (LC)-selected-ion monitoring (SIM)-based tandem mass spectrometry (MS/MS) methods without hydrolysis as it can degrade these compounds [27].
An aliquot of the extracted sample was hydrolyzed and converted to fatty acid methyl esters (FAMEs) ( Figure A2) and trimethylsiloxy [−O-Si(CH3)3; TMS] derivatives ( Figure A3) [27], and these were utilized for the GC-FID and GC-MS analyses of the fatty acids and phytosterols, respectively.

LC-SIM-MS Analysis of Tocols and Carotenoids
The major redox-active lipophilic constituents were analyzed by LC-SIM-based mass spectrometry method utilizing LCMS-9030 quadrupole time-of-flight (Q-TOF) mass spectrometer (Shimadzu, Tokyo, Japan). The LC-MS parameters used for the analysis of carotenoids and tocols are shown in Table 1. Additionally, the selected ion monitoring (SIM) parameters used for the analysis of carotenoids and tocols are shown in Table 2.

Fames Determinations by GC-FID and GC-MS
The FAMEs were qualitatively analyzed by GC (Agilent 7890B, Agilent Technologies Canada, Inc., Mississauga, ON, Canada). The analytical conditions are shown in Table 3. For the accurate identification of the FAMEs, the mass spectrum was recorded using the QP2010 SE GS-MS (Shimadzu, Japan) following the GC-FID thermal program. The mass fragmentation pattern was compared with the authentic standards as well as reference databases (NIST08, NIST08S, and Wiley9) to confirm the identity of the FAMEs.

GC-MS Analysis of Sterols
The sterols were analyzed after silylation utilizing QP2010 SE GC-MS (Shimadzu, Tokyo, Japan). The analytical conditions are shown in Table 4. The identity of the sterols was confirmed by comparing their mass fragmentation patterns with the authentic standards as well as reference databases (Wiley9, NIST08, and NIST08S).

Antioxidant Activity
The antioxidant activities were analyzed using two different types of extracts. Extract one (containing lipophilic compounds) was prepared (without adding the BHT), as mentioned in Section 2.2. The second extract was prepared using aqueous methanol. To prepare this extract, two grams of fresh leaves of each cultivar were extracted with 25 mL of aqueous methanol (80% v/v) as described previously [17]. The ABTS •+ and DPPH • radical scavenging abilities were determined according to our optimized method [28], based on Thaipong et al. [29]. The results were expressed in mg Trolox equivalents (TE)/100 g fresh weight (FW).

Statistical Analysis and Quality Control
A total of four replicate extractions and analyses were performed for each leaf sample. The one-way analysis of variance (ANOVA) was performed using IBM SPSS statistics (version 25), taking into account a significance level of 0.05 (Tukey HSD).
The limits of detection (LOD) and limits of quantitation (LOQ) were determined according to the signal-to-noise (S/N) ratio between >3 and >10, respectively, for the quantitative analysis utilizing LC-MS [30].
The GC-MS method used for phytosterols analysis was recently validated [31]. The recoveries of the phytosterols were accurately monitored and normalized using 5β-cholestan-3α-ol as the internal standard (IS).

Carotenoids and Tocols Composition
The solvent mixture with acetone/ethanol/hexane is most frequently employed to extract the polar and nonpolar carotenoids simultaneously [30]. In the preliminary investigations, we found that this solvent combination is effective for the simultaneous extraction of carotenoids, tocols, phytosterols, and fatty acids from mustard leaves. Moreover, considering the health and environmental hazards associated with hexane, this solvent was replaced with cyclohexane [32], which is safer and provided a similar yield of these lipophilic compounds from the studied samples (data not shown).
Among the studied mustard leaves of four cultivars, (all-E)-lutein was the most dominating carotenoid, ranging between 37.12 (cv. Asia Curled) and 43.54% (cv. Jeok) of the total carotenoids, which was followed by (all-E)-β-carotene and (all-E)-violaxanthin. The highest amount of all of the individual carotenoids and total carotenoids (143.85 µg/g fresh weight; FW) were recorded in cv. Cheong.
In the present investigation, among all of the studied cultivars, the lutein contents were 2.1-2.6 times higher than the β-carotene ones were. However, previous studies on B. juncea leaves recorded significantly varied β-carotene and lutein levels. For instance, Frazie et al. [33] analyzed the carotenoid profile of leaves of eight B. juncea cultivars that were grown, including the Jeok cultivar analyzed in the present investigation. In this study, the β-carotene contents were 1.14 times higher than the lutein ones were in the mature leaves of the Jeok cultivar. Similarly, Farnham et al. [7] recorded 88.0-129.2 µg/g FW of β-carotene and 61.2-88.6 µg/g FW of lutein in the field-grown mature leaves of the B. juncea cultivars. In contrast, Zeb [34] recorded 124 µg/g FW of β-carotene and 184 µg/g FW of lutein in the B. juncea leaves marketed in Pakistan. oxidative stress-related diseases, including CVD, T2D, neurodegenerative disorders, and various types of cancer [35][36][37].  Values (µg/g FW) are the mean ± standard deviation of four replicates. Different superscript letters (i.e., a, b, and c) indicate statistically significant differences among the different cultivars (p < 0.05, Tukey HSD).
We previously analyzed the carotenoid profile of 23 diverse lettuce cultivars and recorded 54.4-129.8 µg/g FW of the total carotenoids. Moreover, in that study, the (all-E)-β-carotene content ranged between 4.24 and 12.9 µg/g FW (7.30-13.03% of the total carotenoids), while in the present study, the (all-E)-β-carotene contents ranged between 21.34 and 24.45 µg/g FW (16.00-17.98% of the total carotenoids). This shows that mustard leaves are a richer source of provitamin A carotenoid (β-carotene) and total carotenoids than lettuce is.
The tocols include four naturally occurring tocopherols (δ-, γ-, β-, and α-) and four tocotrienols (δ-, γ-, β-, and α-) [13]. This study screened the mustard leaves for their tocols composition using LC-SIM-MS. In all of the studied samples, 67.16 (cv. Asia Curled)-83.42 µg/g FW (cv. Cheong) of α-tocopherol was recorded (Table 5), while other forms were not detected in a substantial amount. Only a few studies are available on the tocopherol contents of B. juncea leaves as most of the studies were focused on seed oil. Zeb [34] recorded 13.5 µg/g FW of α-tocopherol in the B. juncea leaves marketed in Pakistan. In contrast, Xiao et al. [8] recorded 221 µg/g FW of α-tocopherol in the commercially grown B. juncea microgreens.
Attributable to the presence of high contents of bioactive antioxidant compounds, such as carotenoids and tocopherols in mustard leaf, their intake can minimize the oxidative stress-related diseases, including CVD, T2D, neurodegenerative disorders, and various types of cancer [35][36][37].
In the present study, β-sitosterol was the most dominant phytosterol among the studied mustard leaves, ranging between 151.39 (cv. Red frill) and 240.34 (cv. Cheong) µg/g FW, which accounted for 80.42 (cv. Jeok)-83.14% (cv. Red frill) of the total sterol (Table 6). Limited studies are available on the sterol composition of leaf mustard and other brassica family vegetables. Among the vegetables and fruits commonly consumed in China, β-sitosterol was the most dominating phytosterol, with the highest concentration of it (µg/g FW) in pea (414), cauliflower (408), and broccoli (345), while in rape (probably Brassica napus), 85 and 14 µg/g of β-sitosterol and campesterol was recorded, respectively [38].  ., a, b, and c) indicate statistically significant differences among the different cultivars (p < 0.05, Tukey HSD).
The results of the present investigation and previous report suggest that the Brassica family vegetables, including leaf mustard, are good sources of bioactive phytosterols.

Fatty Acids
Herbs (photosynthetic tissue) are not a significant source of fatty acids as they are generally deficient in total lipids (2-4%, dry weight) [18]. However, herbs contain high proportions of health-beneficial omega-3 (n-3) fatty acid in the form of α-linolenic (ALA; C18:3n3).
In the present study, six major fatty acids were identified from the mustard leaf, and their relative occurrence (percentages of the total fatty acids) were estimated (Figure 3; Table 7). Among the studied mustard leaves, ALA was the most dominant fatty acid, accounting for 36.09 (cv. Cheong)-38.98% (cv. Asia Curled) of the total fatty acids. The previous reports also state that ALA is the most abundant fatty acid in herbs (e.g., green leafy vegetables), such as cabbage and spinach [41], perilla (Perilla frutescens Britt.) [39], lettuce [17], and Komatsuna (Japanese mustard spinach; Brassica rapa var. perviridis), and Tatsoi (Brassica rapa var. rosularis) [42].
In contrast to other herbs, a significant amount of hexadecatrienoic acid (C16:3n3; allcis 7,10,13) was recorded from the studied mustard leaves, accounting for 27.17 (cv. Asia Curled)-32.59% (cv. Red frill) of the total fatty acids. The GC-mass spectrum of the hexadecatrienoic acid identified from the mustard leaves is given in Figure A6.
The presence of a substantial amount of hexadecatrienoic acid is rare in higher plants. However, it has been previously recorded in the photosynthetic tissue of Brassica napus L. and Brassica oleracea L., which are classified as "16:3" plants (containing >2% of C16:3 fatty acids in total lipids), which is part of primitive lipid metabolism [43]. Interestingly, the contents of polyunsaturated fatty acids (PUFAs) in the photosynthetic leaves of B. napus L. are largely influenced by the environmental temperatures [24]. A low temperature induces fatty acid desaturation in B. napus L., resulting in an enhanced accumulation of C18:3 and C16:3 fatty acids, which helps maintain the fluidity in the membrane at a low temperature [24]. A substantially high amount of hexadecatrienoic acid recorded in the present investigation was probably a result of the low temperature (mean temperature of 10-15 • C) during the growth of the mustard plants.
From the nutritional perspective, the dominance of ALA and hexadecatrienoic acid in the mustard leaves makes them exceptionally rich in total PUFAs, containing 74.99-78.28% of the total fatty acids. Moreover, the total PUFAs/total saturated fatty acids (SFAs) were high (5.63 in cv. Cheong-7.26 in cv. Red frill).
Omega-3 (n-3) PUFAs are essential for normal growth and development. Moreover, they have vital positive effects on the brain, eyes, heart, joints, skin, mood, and behavior [18]. Thus, consuming mustard leaves for major nutritionally vital phytoconstituents (e.g., vitamins) and high proportions of n-3 PUFAs (63.89-69.74% of total fatty acids) may be one of their health benefits. Antioxidants 2022, 11, x FOR PEER REVIEW 11 of 20  Table 7. * Not a FAME.  Table 7. * Not a FAME. Values (mean ± standard deviation) are percentages of the total fatty acids from four determinations. FAME: fatty acid methyl ester; RT: retention time; PUFAs: total polyunsaturated fatty acids; SFAs: total saturated fatty acids. Different superscript letters (i.e., a, b, and c) indicate statistically significant differences among the different cultivars (p < 0.05, Tukey HSD).

Antioxidant Activity
In the present study, the antioxidant activities were analyzed from two different types of extracts. Interestingly, the aqueous methanolic extract showed 2-5-fold more ABTS •+ and DPPH • radical scavenging activities compared to the lipophilic extract prepared using acetone/ethanol/cyclohexane (1:1:2, v/v; recovered in acetone) ( Table 8). The previous studies have also revealed that methanol/water extract provides better ABTS •+ and DPPH • radical scavenging activities from herbs than the extracts do which were obtained with acetone [44,45]. In the investigations of the antioxidant activity of Daphne gnidioides L. and Daphne sericea L. leaves, the methanol extracts showed 2-3-fold more ABTS •+ and DPPH • radical scavenging compared to the acetone extract. Similarly, from the Amaranthus leaf, the methanol/water extract showed significantly higher antioxidant activities and total phenolic contents (TPC) than the extract did that was prepared in acetone. Values (mg of Trolox equivalent/100 g FW) are mean ± standard deviation of four replicates. Different superscript letters (i.e., a, b, and c) indicate statistically significant differences among the different cultivars (p < 0.05, Tukey HSD). 1 Activity from 80% methanolic extract (v/v); 2 Antioxidant activity from lipophilic extract prepared using acetone/ethanol/cyclohexane (1:1:2, v/v).
In herbs, the phenolic compounds (flavonoids and phenolic acids), tocopherols, ascorbic acid, and carotenoids are major antioxidants [46]. The present study used an acetone/ethanol/cyclohexane-based solvent to extract the lipophilic compounds, including carotenoids, tocopherols, sterols, and fatty acids. However, the significantly highest radical scavenging activities obtained from the aqueous methanolic extract were probably the results of a higher extraction of polyphenolic compounds.
The present study recorded the highest contents of carotenoids and α-tocopherol from cv. Cheong. However, the aqueous methanolic prepared from cv. Red frill showed significantly more ABTS •+ (640.66 mg TE/100 g FW) and DPPH • radical scavenging (284.96 mg TE/100 g FW) activities (Table 8). In contrast, among the lipophilic extracts prepared using acetone/ethanol/cyclohexane, cv. Cheong showed significantly higher ABTS •+ activity values of 148.27 mg TE/100 g FW (Table 8). Interestingly, the α-tocopherol content ( Table 5) was also highest in this cultivar (cv. Cheong). Moreover, the α-tocopherol contents showed a significant positive correlation (R = 0.796) with the ABTS •+ activity of the lipophilic extract (Table 9), which suggests that the α-tocopherol contents present in the mustard leaves are efficient scavenger of ABTS •+ radicals. In addition, the phytosterol contents correlate well (R = 0.949) with the α-tocopherol contents (Table 9). Surprisingly, in the present study, the total carotenoid contents were not correlated with the antioxidant activities ( Table 9), suggesting that bioactives other than the carotenoids are more potent antioxidants in the mustard leaves. Table 9. The correlation coefficient (R) between total carotenoids, α-tocopherol, and phytosterol contents and the antioxidant potential of leaves of various mustard cultivars. Both the ABTS •+ and DPPH • radical scavenging assays employed in the present study are based on the electron transfer (ET) mechanism. In agreement with previous reports [29,47], a significant correlation was obtained between these assays ( Table 9).
The Trolox equivalent antioxidant capacity (TEAC) in Brassica vegetables has shown a significant positive correlation with the anthocyanins contents [48]. Thus, the significantly highest radical scavenging activities obtained from cv. Red frill was probably the result of a high accumulation of anthocyanin pigments-the intense red color of cv. Red frill leaves, compared to other studied cultivars (Figure 1), also suggest a presence of a substantial amount of anthocyanins.

Conclusions
The results of the present study indicate that the contents of carotenoids, α-tocopherol, phytosterols, and fatty acids varied significantly among the cultivars. Among the mustard leaves of four cultivars studied, the highest contents of the total carotenoids (143.85 µg/g FW), α-tocopherol (83.42 µg/g FW), and total phytosterols (294.30 µg/g FW) were recorded in cv. Cheong.
Interestingly, the fatty acid analysis revealed the presence of a significant amount of rare hexadecatrienoic acid (C16:3n3) in the studied mustard leaves, accounting for 27.17 (cv. Asia Curled)-32.59% (cv. Red frill) of the total fatty acids. Moreover, the dominance of α-linolenic (ALA; C18:3n3) and hexadecatrienoic acid in the mustard leaves makes them exceptionally rich in total omega-3 (n-3) PUFAs, with the highest contents of 69.74% in cv. Red frill.
Overall, the cv. Cheong represented the highest contents of carotenoids, tocols, and phytosterols. Moreover, cv. Red frill contains the highest amount of n-3 PUFAs and antioxidant compounds. Thus, the use of these cultivars can be promoted in cuisines which can be eaten to obtain the highest health benefits. Further studies may be beneficial in revealing the hydrophilic constituents, including anthocyanin, in leaf mustard cultivars.            The representative gas chromatography (GC)-mass spectrometry (MS)-total ion chromatograms (TIC) of phytosterols identified in mustard leaves of various cultivars. IS: internal standard.