The main goal of the current study was to assess metabolites heterogeneity in four organs derived from two different varieties in Indian mustard (Brassica juncea) in the context of their nutrient and sulfur aroma profiles using GC–MS-based metabolomics approach. Seed, leaf, flower, and stem of B. juncea obtained from the two varieties (RH-725 and RH-761) were included for comparative metabolites profiling analysed using chemometric tools.Primary metabolites profiling in B. juncea organs via GC–MS analysis (post-silylation)To assess variation in primary nutritive metabolites among B. juncea plant parts from two different varieties, GC–MS analysis was employed post-silylation. A total of 101 peaks (Table 1, Fig. 1) were annotated, including sugars (mono- and disaccharides), sugar alcohols, sugar acid, fatty acids/esters, organic acids, amino acids, nitrogenous compounds, sterols, phenolics and alcohols.Table 1 Quantification of silylated metabolites in Brassica juncea organs from two different varieties (µg/mg) analyzed using GC–MS, n = 3.Figure 1Representative GC–MS chromatograms of identified primary metabolites in Brassica juncea organs from different varieties post-silylation.SugarsSugars were detected as the most dominant primary metabolites represented by 26 peaks identified in B. juncea different organs viz. leaf, stem, flower, and seed in both varieties. The highest levels of sugars represented by monosaccharides were detected in brassica stem RH-761 amounting for ca. 263.2 µg/mg, followed by leaf stem, and flower collected from RH-725 at 240.8, 197.65, and 147.7 µg/mg. In contrast, the highest levels of disaccharides were detected in seed from both varieties at 110–140 µg/mg, respectively. In contrast, the lowest sugar level was detected in the leaf RH-761 at ca. 4.3 µg/mg. Such variation in sugar levels indicate that B. juncea organs from the variety RH-725 are in general higher in sugar content, except only for the stem than RH-761. Glucose was the major sugar in leaf RH-725, stem and flower from both RH-761 and RH-725 detected at 26.7–126.4 µg/mg. Next to glucose, fructose and galactose were abundant in B. juncea leaf RH-725, stem and flower from both RH-761 and RH-725 at 30.3–62.03 and 10.5–97.5 µg/mg, respectively. Seeds from both varieties showed trace levels of glucose, fructose, and galactose, likely as storage organ to be richer in fatty acids. Interestingly, in seeds major sugar was represented by sucrose detected at much higher level in seeds with higher level in variety RH-725 at ca. 133.02 µg/mg compared to 98.6 µg/mg in RH-761. The abundance of sucrose in B. juncea seed pose it as the most palatable and edible part. Conclusively regarding sugar profile, organs from B. juncea variety RH-725 showed higher levels of monosaccharides than variety RH-761, except stem RH-761 that encompassed higher level of sugars among all organs. Among organs from both varieties, seeds contained higher levels of disaccharides. Free sugars such as stachyose, raffinose, melibiose, galactose, glucose and fructose were previously detected in B. juncea13.Unlike free sugars, sugar alcohols were detected at much lower levels in B. juncea organs from different varieties being detected at comparable levels of 19.9 and 16.6 µg/mg in leaf and flower RH-725, respectively. Glycerol was detected as the most abundant sugar alcohol in leaf and flower for variety RH-725 at 18.3 and 15.9 µg/mg, respectively. Likewise, sugar acids were detected at high level in B. juncea flowers from the two varieties with higher abundance in variety RH-725 at ca. 33.2 compared to 14.9 µg/mg in RH-761. Gluconic acid, an important sugar acid used in food and pharmaceutical industry14 was detected as the most abundant sugar acid in brassica flower at ca. 30.3 and 13.4 µg/mg in RH-725 and RH-761, respectively. Retrieving sugars results and comparison among brassica organs from the two varieties revealed that variety RH-725 was the richest in free sugars and its derivatives represented by glucose, fructose, galactose, sucrose, glycerol, and gluconic acid.Amino acids/nitrogenous compounds/phenolicsAmino acids play a pivotal role in human health owing to their nutritional and medicinal importance in the regulation of anti-oxidative, metabolic, and immune responses8. Amino acids were represented by 17 peaks detected at comparable higher levels in leaf and flower from both varieties compared to other organs and detected at higher level in leaf RH-725 and flower RH-725 at ca. 23.3 and 10.6 µg/mg, respectively. Such high amino acids level in leaf and flower from RH-725 pose it for improved nutritional makeup compared variety RH-761. Alanine, valine, and leucine were the most abundant amino acids detected among brassica organs, specially leaf and flower form RH-725 variety. Compared to the food sources of amino acids including meat, milk, cheese, and grains, B. juncea leaf and flower are considered as important source for nutritionally important amino acids. Amino acids were previously reported in Indian mustard seeds at high levels including phenylalanine, tyrosine, methionine, cystine, leucine, valine, and lysine15.Nitrogenous compounds were detected at trace levels in all examined organs ranging from 0.2–2.5 µg/mg and 0.3–0.9 µg/mg in organs from RH-725 and RH-761, respectively, with only leaf RH-725 and flower RH-725 that showed relatively higher levels. Likewise, phenolics were detected at trace levels in organs from the two varieties except a comparable level was detected in seed from RH-725 and RH-761 at ca. 3–4µg/mg, though it should be noted that GC/MS is not suited for profiling of phenolics considering their polar nature and warranting for using LC/MS technique16. Several polyphenols were previously reported in B. juncea using high-performance liquid chromatography (HPLC)17. Sinapinic acidwas the major phenolic acid in B. juncea seed specially in variety RH-725 (3.03 µg/mg). Sinapinic acid is a chief phenolic acid previously identified in rapeseed with potential health benefits including anti-inflammatory, hepatoprotective, cardioprotective, anti-cancer, and anti-diabetic18.Fatty acids/esters/sterolsFatty acids/esters represented by 22 peaks were detected at relatively high levels in all organs of B. juncea with higher levels in leaf RH-725 at 56.6 µg/mg represented by saturated, monounsaturated and polyunsaturated fatty acids. α-Linolenic acid is a poly-unsaturated ω-3 fatty acid with positive effects on atherosclerosis incidence risk and hence reduce cardiovascular diseases risk19 found at high level in leaf RH-725 at 12.5 µg/mg. Palmitic acid was detected as the major saturated fatty acids in leaf RH-725 and seed RH-725 at 8.4 and 7.02 µg/mg, respectively. In a previous study, fatty acids were previously detected in B. juncea specially unsaturated fatty acids including oleic acid and palmitoleic acid20. Moreover, B. juncea oil was reported to contain a balanced levels of both saturated and unsaturated fatty acids specially both omega-6 and omega-3 polyunsaturated fatty acids13.Regarding fatty acyl esters, 1-monopalmitin, an esterified product of palmitic acid previously identified in a variety of plant extracts and to exhibit antibacterial activities19, was detected at highest levels in leaf RH-725 of 10.2 µg/mg. Unlike fatty acids, sterols were detected at much lower levels in B. juncea represented mainly by two peaks identified as campesterol and β-sitosterol with relative high level in seed RH-725 at 2.4 µg/mg compared to other accessions. Such results indicated that B. juncea organs from variety RH-725 is the richest in fatty acids and sterols compared to that from RH-761.Organic acids/alcohols/lactonesOrganic acids were detected in all organs from the two varieties at comparable levels, with highest level found in leaf RH-725 at 176 µg/mg followed by flower RH-725 at 41.7 µg/mg. Organic acids are important in food products as natural preservative and can promote food digestion as well as aiding in protein utilization12. Malic acid was detected as the major form in B. juncea leaf RH-725 at ca. 128.2 µg/mg. Malic acid exhibits potential antioxidant capacity asides from its action as food preservative21. Such high level of malic acid in leaf from the variety RH-725 could be a marker for genetic variation between the two varieties RH-725 and RH-761 which has yet to be confirmed using QTL sequencing. Malic acid was previously detected in B. juncea flower in a study done by El Majdoub et al.22. Next to malic acid, hexanoic acid, a short-chain monocarboxylic acid was detected at high level in leaf RH-725 29.01 µg/mg. Compared to acids, alcohols were detected at trace levels in most samples represented mainly by phytol, a diterpene alcohol with potential health benefits23. Likewise, lactones were detected at trace levels in all organs, except in flower of RH-725 at 6.9 µg/mg versus 3.8 µg/mg in RH-761. Tetrahydroxypentanoic acid-1,4-lactone and gluconolactone were the two identified lactones found most abundant in flower RH-725.
