Glucosinolate Breakdown Products Research Articles

Insights into the phytochemical composition of selected genotypes of organic kale (Brassica oleracea L. var. acephala)

Seven genotypes of kale (B. oleracea L. var. acephala), selected from a collection set up in the framework of the BRESOV H2020 Project, aimed at breeding under organic conditions, were analysed for the content in the characteristic phytochemicals of Brassica spp. The presence of prebiotic oligosaccharides was an important characteristic of this crop, with values ranging from 4.2 to 10.8 g/100 g d.w. The other detected soluble sugars were sucrose, glucose, and fructose, with glucose predominating (3.9–9.0 g/100 g d.w.). Sulphur compounds, such as sulphoxides, were detected in the form of pyruvic acid, their catabolic product (36–154 mg/100 g d.w.). In addition, the levels of main breakdown products of glucosinolates, such as sulforaphane and indole-3-carbinol, were found to be in average contents of 6.7 and 3.9 μmol/g d.w., respectively. Finally, the presence of major phytochemicals in kale, such as polyphenols and carotenoids, reported considerable concentrations (around 1400 mg/100 g d.w. and 19 mg/100 g d.w, respectively), typical for this Brassica crop.

Determination of glucosinolates and breakdown products in Brassicaceae baby leafy greens using UHPLC-QTOF/MS and GC/MS

Brassica vegetables (Brassicaceae) produce a unique class of compounds known as glucosinolates. The breakdown products of glucosinolates, including isothiocyanates, are recognized as the primary sources of bioactivity and characteristic taste, flavor and aroma of these vegetables. An emerging market trend is to harvest leafy greens at the immature stage known as “baby greens”, however, much is still unknown about the phytochemistry at this earlier stage of development. Glucosinolates and their breakdown products were examined in three members of the Brassicaceae family: Eruca vesicaria subsp. sativa ‘Arugula’, Brassica juncea ‘Garnet Giant’ (mustard greens) and Brassica oleracea var. acephala ‘Red Kale’. A total of 16 glucosinolates were tentatively identified using UHPLC-QTOF/MS and seven volatile breakdown products were identified using GC/MS. Nine putative glucosinolates and one isothiocyanate are reported for the first time in these plant species and varieties. Glucobarbarin, glucocheirolin, glucoiberin, gluconapin and neoglucobrassicin were newly identified in Eruca vesicaria subsp. sativa. Glucoalyssin, glucobarbarin, glucobrassicanapin and glucotropaeolin were newly identified in B. oleracea var. acephala, and butane isothiocyanate was newly identified in B. oleracea. Future work will focus on correlating chemistry data with sensory analyses to determine the impact of these specific glucosinolates and breakdown products on taste, flavor and aroma.

Glucosinolates and Cytotoxic Activity of Collard Volatiles Obtained Using Microwave-Assisted Extraction.

Glucosinolates (GSLs) in Brassica oleracea L. convar. acephala var. viridis (collard) flower, leaf, stem, and root were analyzed qualitatively and quantitatively via their desulfo-counterparts using UHPLC-DAD-MS/MS. Twelve GSLs were identified, including Met-derived GSLs (sinigrin, glucoibervirin, glucoerucin, glucoiberin, glucoraphanin, progoitrin), Trp-derived GSLs (4-hydroxyglucobrassicin, glucobrassicin, 4-methoxyglucobrassicin, and neoglucobrassicin), and Phe-derived GSLs (glucotropaeolin and gluconasturtiin). Total GSL content was highest in the root, having 63.40 μmol/g dried weight (DW), with gluconasturtiin (34.02 μmol/g DW) as the major GSL, followed by sinigrin and glucoibervirin (12.43 and 7.65 μmol/g DW, respectively). Total GSL contents in the flower, leaf, and stem were lower than in root, having 6.27, 2.64, and 1.84 μmol/g DW, respectively, with Trp and/or Met-derived GSLs as the predominant ones. GSL breakdown products were obtained via microwave hydrodiffusion and gravity (MHG) and volatile breakdown products were analyzed using GC-MS techniques. Volatile isolates were tested for their cytotoxic activity using MTT assay. MHG volatile extract from the root demonstrated the best cytotoxic activity against human bladder cancer cell line T24 and breast cancer cell line MDA-MB-231 during an incubation time of 72 h (IC50 21.58, and 11.62 μg/mL, respectively). The activity of the root extract can be attributed to its major volatile, 2-phenylethyl isothiocyanate (gluconasturtiin breakdown product).

Correlations between flavor and glucosinolates and changes in quality-related physiochemical characteristics of Guizhou suancai during the fermentation process

The flavor of Guizhou suancai fermented by the emerging varieties Zhuchang–2 (ZC–2) and Zhuchang Red (ZC Red) is superior to that of ZC–1, a typical local variety of leaf mustard in Guizhou Province, China. Here, changes in phytochemical qualities during the fermentation of three Guizhou suancai were characterized to identify the causes underlying differences in flavor. The content of pigments, antioxidants, gluconapin, gluconasturtiin, and total isothiocyanates, and antioxidant capacity were highest in fresh ZC Red. The content of sinigrin, indolic glucosinolates, and total glucosinolate breakdown products was highest in fresh ZC–2. The content of phytochemicals, with the exception of carotenoids, was significantly decreased after fermentation. Odor was correlated with gluconapin, while taste was correlated with sinigrin. These findings provide detailed insights into the sensory and phytochemical properties of three Guizhou suancai that could facilitate the selection of raw material varieties.

Variation in the main health-promoting compounds and antioxidant activity of different organs of Wasabi (Eutrema japonicum) from two producing areas.

Wasabi (Eutrema japonicum), also known as Japanese horseradish, is a perennial herb widely used in Japanese cuisine for its special flavour. The health-promoting phytochemicals and antioxidant capacity of four organs (leaf, petiole, rhizome, and root) of two cultivars (Chuankui-1 and Chuankui-2) of wasabi from two producing areas, Leibo and Guangyuan in Sichuan Province, China, were investigated in this study. The results showed that leaves were rich in pigments, soluble protein, ascorbic acid, and total phenolics and had the highest antioxidant capacity. Soluble sugars were highest in the petioles and were 1.1- to 5-fold higher than those in the other three organs. Glucosinolates and glucosinolate breakdown products (GBPs) were the most abundant in rhizomes, and their maximum values were 271.61 mmol kg-1 DW and 249.78 mmol kg-1 DW, respectively. The rhizomes of Chuankui-1 in Leibo and the leaves of Chuankui-1 in Guangyuan were superior in terms of glucosinolates and GBPs. These findings provide new insights that will aid the use of wasabi cultivars; they also have implications for the environmental characteristics needed to obtain better quality wasabi products. In the future, metabolome and transcriptome can be used to analyze the potential mechanism of differences among typical varieties, origins and parts.

Plant matrix concentration and redox status influence thermal glucosinolate stability and formation of nitriles in selected Brassica vegetable broths

Brassica vegetables are frequently consumed foods of nutritional interest, because they are rich in glucosinolates (GLSs). Among GLS breakdown products, especially isothiocyanates are known for their health-beneficial effects, while nitriles are less beneficial. To increase the understanding of the plant matrix’s influence on GLS degradation, differently concentrated vegetable broths were prepared from selected Brassica vegetables (kohlrabi and red cabbage) and subsequently boiled. Altogether, heat stability and conversion of GLSs to the corresponding nitriles were both strongly influenced by vegetable type and plant matrix concentration in the broths. After boiling kohlrabi broths for 120 min, recovery of 4-(methylthio)butyl-GLS as nitrile was 55.5 % in 1 g/mL broth and 8.4 % in 0.25 g/mL broth. In follow-up experiments, a pronounced influence of the matrix’s redox status was identified, with H2S being an important factor. A better understanding of these processes will help to preserve health-promoting effects of GLSs in Brassica vegetables in the future.

Effect of main vegetable ingredient on the glucosinolate, carotenoids, capsaicinoids, chlorophylls, and ascorbic acid content of kimchis

Kimchi, a traditional Korean food manufactured using various vegetables and other ingredients, such as garlic, ginger, and red pepper, imparts several health benefits. However, the variation in the bioactive compounds responsible for the health benefits of various kimchis is not well-characterized. Here, we analyzed the bioactive profile of eight kimchis, prepared using different vegetables as the main ingredient. We compared the major phytochemical compositions, including glucosinolates, carotenoids, capsaicinoids, chlorophylls, and ascorbic acid contents in Baechu-kimchi (BC) and Baek-kimchi (BK) prepared using napa cabbage, Gat-kimchi (GT) prepared using mustard leaf, Kkakdugi (KD), Chonggak-kimchi (CGK), and Yeolmu-kimchi (YM) prepared using radish, Pa-kimchi (PA) prepared using green onion, and Oi-sobagi (OB) prepared using cucumber. Among glucosinolates, 4-methoxyglucobrassicin was abundant in BC and BK, wherein 4-methoxyglucobrassicin and glucoraphasatin were dominant in CGK, YM, and KD. The glucosinolate breakdown product ascorbigen was abundant in all kimchi. Carotenoids, particularly capsanthin from red chili pepper, were the highest in PA. YM and PA showed high chlorophyll contents, and ascorbic acid contents were almost two-fold higher in GT than in OB and BK. Total capsaicinoids, which confer pungency, were highest in OB. PA, GT, and no capsaicinoid were detected in BK, which contained no red chili pepper powder.

Impact of heat stress of varying severity on papaya (Carica papaya) leaves: Major changes in stress volatile signatures, but surprisingly small enhancements of total emissions

Tropical plants are considered to be highly heat stress resistant, but heat waves of different frequency and duration induced by global climate change can exceed the plant heat stress tolerance in the tropics. To gain insight into the overall heat resistance, leaves of papaya were exposed to mild (37 and 41 °C), moderate (46 °C) and severe (49 °C) heat stress (applied for 5 min), and plant recovery was monitored for 48 h upon return to pre-stress conditions. Leaf photosynthetic characteristics were reduced immediately after stress in all cases, but photosynthesis rate almost fully recovered after mild heat treatment, and partly recovered in 48 h after moderate stress, while no recovery was observed under severe stress. Constitutive BVOC emissions dominated by long-chained aldehydes, and glucosinolate breakdown products, attained the emission maxima at 46 °C. Acute heat stress led only to a minor induction of BVOC emissions, and the greatest share of induced emissions was observed in leaves exposed to 46 °C. Induced emissions were dominated by oxygenated monoterpenes, indicating an increase in leaf oxidative status. Our results demonstrate that papaya could withstand moderately severe heat stress of up to 46 °C, but this level of stress will have negative impacts on plant carbon gain due to delayed photosynthetic recovery after heat stress. Although volatile profiles sensitively responded to heat stress, the magnitude and degree of elicitation were surprisingly small throughout the recovery, suggesting that papaya is relatively heat resistant and possess a suite of heat-protective traits. • Papaya photosynthetic recovery was rapid under mild heat stress, delayed under moderate stress and absent under severe stress. • Heat stress of different severity had only moderate effects on volatile emissions, but the composition of emitted volatiles was strongly altered. • Volatile emission blend rather than the absolute amount characterizes the impact of heat stress on leaves of papaya.

Genotypic variation of glucosinolates and their breakdown products in mustard (Brassica juncea) seeds

The glucosinolates and glucosinolate breakdown products (GBPs) in the seeds of three types of mustard (root, stem, and leaf mustards) from a core collection of 50 cultivars were studied. Nine glucosinolates and ten GBPs were identified, with sinigrin and 2-propenyl isothiocyanate (SIN-ITC) being the dominant glucosinolate and GBP, respectively. Among the tested cultivars, the contents of total glucosinolates and GBPs ranged from 46.84 to 89.30 μmol g−1 FW and from 23.07 to 82.20 μmol g−1 FW, respectively. Leaf mustard had both the highest average levels of glucosinolates and GBPs (65.04 μmol g−1 FW and 55.97 μmol g−1 FW, respectively) and the greatest variation in total GBP content compared with root and stem mustards. L8 had the highest content of SIN-ITC (72.06 μmol g−1 FW). These findings indicate that mustard seeds, especially cultivar L8, can be good candidates for functional foods since they contain extremely high levels of SIN-ITC, a potent anti-carcinogenic isothiocyanate.

Influence of isolation techniques on the composition of glucosinolate breakdown products, their antiproliferative activity and gastrointestinal stability of allyl isothiocyanate

Influence of isolation techniques on the composition of glucosinolate breakdown products, their antiproliferative activity and gastrointestinal stability of allyl isothiocyanate

Determination of Isothiocyanate-Protein Conjugates in a Vegetable-Enriched Bread.

Vegetables of the plant order Brassicales are believed to have health-promoting properties, as they provide high contents of glucosinolates (GLS) and deriving from these, enzymatically and heat-induced breakdown products, such as isothiocyanates (ITC). Besides their positive physiological effects, ITC are electrophilic and can undergo reactions with food components such as proteins. Following the trend of improving traditional food products with GLS-rich ingredients, interactions of ITC with proteins can diminish the properties of both components—protein’s value and functionality as well as ITC’s bioactivity. In vegetable-enriched bread, where cresses (Lepidium sativum L. or Tropaeolum majus L.) are added to the initial dough, together with benzyl cyanide, benzyl isothiocyanate (BITC) is formed during the baking process. The aim of the present study was to investigate the possible migration behavior of the GLS breakdown products and the formation of ITC-wheat protein conjugates. After the baking process, the breads’ proteins were enzymatically hydrolyzed, and the ITC-amino acid conjugates analyzed using a LC-ESI-MS/MS methodology. In all samples, BITC-protein conjugates were detected as thiourea derivatives, while formation of dithiocarbamates could not be detected. The study showed that GLS and their breakdown products such as ITC migrate into the surrounding food matrix and undergo reactions with proteins, which could in turn lead to modified protein properties and reduce the bioavailability of ITC and lysine.

The flavor of Chinese kale sprouts is affected by genotypic variation of glucosinolates and their breakdown products

Metabolic profiling of glucosinolates and their breakdown products in sprouts of 22 Chinese kale (Brassica oleracea var. alboglabra, BOA) varieties were investigated by using high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC–MS). Relationships between glucosinolate metabolites and flavor of Chinese kale sprouts were also analyzed. Results showed that compositions and contents of both glucosinolates and their breakdown products varied greatly among different varieties of Chinese kale sprouts. Gluconapin and 4,5-Epithio-pentanenitrile were the dominant glucosinolate and glucosinolate breakdown product in Chinese kale sprouts, respectively. Gluconapin and glucobrassicin were significantly related to bitterness (r = 0.577, 0.648, respectively; p < 0.05). BOA 1 and BOA 13, BOA 3 and BOA 10 are good candidates for future breeding programs since the former two varieties have light bitterness and pungency, and the latter two varieties contain high levels of glucosinolate breakdown products such as isothiocyanates and epithionitriles in sprouts.

Immunological Analysis of Isothiocyanate-Modified α-Lactalbumin Using High-Performance Thin Layer Chromatography.

Undirected modifications between food proteins and secondary plant metabolites can occur during food processing. The results of covalent interactions can alter the functional and biological properties of the proteins. The present work studied the extent of which covalent conjugation of the bioactive metabolite benzyl isothiocyanate (BITC; a glucosinolate breakdown product) to the whey protein α-lactalbumin affects the protein’s allergenicity. Additional to the immunological analysis of native untreated and BITC-modified α-lactalbumin, the analysis of antigenic properties of proteolytically digested protein derivatives was also performed by high performance thin layer chromatography and immunostaining. As a result of the chemical modifications, structural changes in the protein molecule affected the allergenic properties. In this process, epitopes are destroyed or inactivated, but at the same time, buried epitopes can be exposed or newly formed, so that the net effect was an increase in allergenicity, in this case. Results from the tryptic hydrolysis suggest that BITC conjugation sterically hindered the cleavage sites for the enzyme, resulting in reduced digestibility and allergenicity. Residual antigenicity can be still present as short peptide fragments that provide epitopes. The desire to make food safer for allergy sufferers and to protect sensitized individuals from an allergenic reaction makes it clear that the detection of food antigens is mandatory; especially by considering protein interactions.

Phytochemical characterization of turnip greens (Brassica rapa ssp. rapa): A systematic review.

ObjectiveThe Turnip (Brassica rapa L. ssp. rapa) is a leaf and root vegetable grown and consumed worldwide. The consumption of Turnip has been associated with beneficial effects on human health due to their phytochemicals that may control a variety of physiological functions, including antioxidant activity, enzyme regulation, and apoptotic control and the cell cycle. The current systematic review of the literature aims to evaluate both the profile and quantity of phytochemicals commonly found in Turnip greens and to provide perspectives for further investigation.MethodsThis review was conducted following the PRISMA guidelines. Four bibliographic databases (PubMed, Embase, Web-of-Science and Cochrane Central Register of Controlled Trials) were searched to identify published studies until April 8th, 2020 (date last searched) without data and language restriction. Studies were included if they used samples of Turnip greens (the leaves), and evaluated its phytochemical content. Two reviewers independently evaluated the titles and abstracts according to the selection criteria. For each potentially eligible study, two reviewers assessed the full-texts and independently extracted the data using a predesigned data extraction form.ResultsBased on the search strategy 5,077 potentially relevant citations were identified and full texts of 37 studies were evaluated, among which 18 studies were eligible to be included in the current review. The majority of included studies were focused on identification of glucosinolates and isothiocyanates (n = 14, 82%), four studies focused on organic acids, and five studies reported phenolic component profile in Turnip greens. Among included studies nine studies (50%) provided information on phytochemical’s content. We found 129 phytochemicals (19 glucosinolates, 33 glucosinolate-breakdown products, 10 organic acids and 59 polyphenolic compounds) reported in Turnip greens. Flavonoids were mainly present as quercetin, kaempferol and isorhamnetin derivatives; while aliphatic forms were the predominant glucosinolate (gluconapin was the most common across five studies, followed by glucobrassicanapin). In general, the phytochemical content varied among the leaves, tops and Turnip roots.ConclusionsEmerging evidence suggests the Turnip as a substantial source of diverse bioactive compounds. However, detailed investigation on the pure compounds derived from Turnip green, their bioavailability, transport and metabolism after consumption is further needed. Additional studies on their biological activity are crucial to develop dietary recommendations on the effective dosage and dietary recommendation of Turnip greens for nutrition and health.

Overcoming Glucosinolate-Myrosinase-Isothiocyanate Defense System by Plant Pathogenic Fungi

Natural compounds play an important role in shaping living plant responses. The resistance of plants is dependent on the formation and production of antimicrobial compounds of secondary metabolites. Glucosinolates (GSLs) are the main source of phytoanticipin in Brassicaceae and other plant families. The biological activity of glucosinolates are dependent on the release of various toxic compounds due to hydrolysis by myrosinase, isothiocyanate (ITC) is glucosinolate-breakdown products that inhibit the growth of microorganisms. In this review, we aim to understand how fungal pathogens overcome the glucosinolate-myrosinase-isothiocyanate system. The pathogens overwhelm the plant's defense system in various ways and disable each component of the system. Many plant pathogens may not cause tissue damage or activation of the glucosinolate-myrosinase-isothiocyanate system, others degrade or transforms the intact GSLs to less or non-toxic products, or inhibit the hydrolysis of GSLs catalyzed by myrosinase, or formed special mechanisms to detoxify toxic GSLs degradation products.

Transcriptome responses to different herbivores reveal differences in defense strategies between populations of Eruca sativa

BackgroundIntraspecific variations among induced responses might lead to understanding of adaptive variations in defense strategies against insects. We employed RNA-Seq transcriptome screening to elucidate the molecular basis for phenotypic differences between two populations of Eruca sativa (Brassicaceae), in defense against larvae of the generalist and specialist insects, Spodoptera littoralis and Pieris brassicae, respectively. The E. sativa populations originated from desert and Mediterranean sites, where the plants grow in distinct habitats.ResultsResponses to elicitation of the plants’ defenses against wounding and insect herbivory resulted in more upregulated transcripts in plants of the Mediterranean population than in those of the desert. PCA analysis differentiated between the two populations and between the elicitation treatments. Comprehensive analysis indicated that defense responses involved induction of the salicylic acid and jasmonic acid pathways in plants of the desert and Mediterranean populations, respectively. In general, the defense response involved upregulation of the aliphatic glucosinolates pathway in plants of the Mediterranean population, whereas herbivory caused downregulation of this pathway in desert plants. Further quantitative RT-PCR analysis indicated that defense response in the desert plants involved higher expression of nitrile-specifier protein (NSP) than in the Mediterranean plants, suggesting that in the desert plants glucosinolates breakdown products are directed to simple-nitriles rather than to the more toxic isothiocyanates. In addition, the defense response in plants of the desert population involved upregulation of flavonoid synthesis and sclerophylly.ConclusionsThe results indicated that differing defense responses in plants of the two populations are governed by different signaling cascades. We suggest that adaptive ecotypic differentiation in defense strategies could result from generalist and specialist herbivore pressures in the Mediterranean and desert populations, respectively. Moreover, the defense responses in plants of the desert habitat, which include upregulation of mechanical defenses, also could be associated with their dual role in defense against both biotic and abiotic stresses.

Natural Variation of Glucosinolates and Their Breakdown Products in Broccoli (Brassica oleracea var. italica) Seeds.

Seeds of 32 pure lines and 6 commercial broccoli cultivars were used to investigate variation in glucosinolates and their breakdown products. The aliphatic glucosinolate content was 54.5-218.7 μmol/g fresh weight, accounting for >90% of the total glucosinolates. The major glucosinolates found were glucoraphanin and glucoerucin in 27 samples and progoitrin in 7 samples. A gas chromatography-flame ionization detector (GC-FID) method was used to identify glucosinolate breakdown products; nine products were directly determined using standards. Using Arabidopsis thaliana lines myb28myb29 and Landsberg erecta to hydrolyze each reference glucosinolate, seven products were tentatively identified. 4-(Methylsulfinyl)butyl isothiocyanate and 5-(methylsulfinyl)pentanenitrile contents were 2.6-91.1 μmol/g fresh weight and 0-35.4 μmol/g fresh weight, respectively, with epithionitriles being more common than nitriles in accessions rich in alkenyl glucosinolate. Additionally, (S)-5-vinyl-1,3-oxazolidine-2-thione was detected in accessions rich in progoitrin. Specific lines with altered glucosinolate profiles and breakdown products were obtained and discussed according to the putative glucosinolate metabolism pathway.

MFS transporter from Botrytis cinerea provides tolerance to glucosinolate-breakdown products and is required for pathogenicity

Glucosinolates accumulate mainly in cruciferous plants and their hydrolysis-derived products play important roles in plant resistance against pathogens. The pathogen Botrytis cinerea has variable sensitivity to glucosinolates, but the mechanisms by which it responds to them are mostly unknown. Exposure of B. cinerea to glucosinolate-breakdown products induces expression of the Major Facilitator Superfamily transporter, mfsG, which functions in fungitoxic compound efflux. Inoculation of B. cinerea on wild-type Arabidopsis thaliana plants induces mfsG expression to higher levels than on glucosinolate-deficient A. thaliana mutants. A B. cinerea strain lacking functional mfsG transporter is deficient in efflux ability. It accumulates more isothiocyanates (ITCs) and is therefore more sensitive to this compound in vitro; it is also less virulent to glucosinolates-containing plants. Moreover, mfsG mediates ITC efflux in Saccharomyces cerevisiae cells, thereby conferring tolerance to ITCs in the yeast. These findings suggest that mfsG transporter is a virulence factor that increases tolerance to glucosinolates.

Boiling and steaming induced changes in secondary metabolites in three different cultivars of pak choi (Brassica rapa subsp. chinensis)

Pak choi (Brassica rapa subsp. chinensis) is a leafy vegetable that is widely available in Asia and consumed in rising quantities in Europe. Pak choi contains high levels of secondary plant metabolites, such as carotenoids, chlorophylls, glucosinolates, phenolic compounds, and vitamin K, which are beneficial for humans if consumed on a regular basis. The evaluation of the genotype-induced variation of secondary plant metabolites revealed that the cultivar ‘Amur’ contained the highest concentration of secondary plant metabolites. Furthermore, steaming retained more chlorophylls, glucosinolates, phenolic acids and flavonoid compounds than boiling. In contrast, both domestic cooking methods – boiling, and steaming – reduced the formation of glucosinolate breakdown products, especially the undesired epithionitriles and nitriles but less of the health-beneficial isothiocyanates.

Brassica-enriched wheat bread: Unraveling the impact of ontogeny and breadmaking on bioactive secondary plant metabolites of pak choi and kale

Consumption of Brassica vegetables is linked to health benefits, as they contain high concentrations of the following secondary plant metabolites (SPMs): glucosinolate breakdown products, carotenoids, chlorophylls, and phenolic compounds. Especially Brassica vegetables are consumed as microgreens (developed cotyledons). It was investigated how different ontogenetic stages (microgreens or leaves) of pak choi (Brassica rapa subsp. chinensis) and kale (Brassica oleracea var. sabellica) differ in their SPM concentration. The impact of breadmaking on SPMs in microgreens (7 days) and leaves (14 days) in pak choi and kale as a supplement in mixed wheat bread was assessed. In leaves, carotenoids, chlorophylls, and phenolic compounds were higher compared to those of microgreens. Breadmaking caused a decrease of SPMs. Chlorophyll degradation was observed, leading to pheophytin and pyropheophytin formation. In kale, sinapoylgentiobiose, a hydroxycinnamic acid derivative, concentration increased. Thus, leaves of Brassica species are suitable as natural ingredients for enhancing bioactive SPM concentrations in bread.