*2.1. Glucosinolates*

The unique properties of glucosinolates and their breakdown products isothiocyanates, so called mustard oils, providing the typical flavour and (bitter) taste of mustard plants. Especially their seeds have been known for hundreds of years [21]. Since then, up to 200 different glucosinolates have been reported, predominantly in the plant families of the order Brassicales, but also in plants of the genus Drypetes [22,23].

Glucosinolates may be defined as amino acid-derived sulphur-containing glycosides sharing a generic chemical structure consisting of thiohydroximates carrying an S-linked bglucopyranosyl moiety and an O-linked sulfate residue, and with a variable side chain (R). Until now, more than 200 side-groups (alkyl, alkenyl, aryl, indolyl) have been identified. Occasionally, further substituents are attached to O, S or N atoms of the side chain or glucosyl moiety [22–24].

Glucosinolates can be divided into three chemical groups including aliphatic, indole and aromatic glucosinolates depending on the amino acid precursor. Aliphatic glucosinolates are derived from alanine, leucine, isoleucine, valine and methionine. Indolic group are derived from tryptophan and aromatic glucosinolates are derived from phenylalanine and tyrosine, respectively. The majority of those are the aliphatic glucosinolates derived from chain-elongated methionine [25–27].

Most of the hydrophilic glucosinolates are chemically and thermically stable. However, they can be converted into diverse breakdown products by specific ß-thioglucosidases, referred to as myrosinases, which are present in glucosinolate-producing plants but also in fungi and in bacteria commonly associated with gut microflora [28,29] (Figure 2).

**Figure 2.** Enzymatic processing of glucosinolates by myrosinase into hydrolysis products.

In that regard it is of interest, that significant amounts of metabolites of glucosinolates (largely dithiocarbamates) were excreted via the urine of healthy human volunteers after eating brassica vegetables, even when myrosinase has been completely heat-inactivated. The excretion falls to negligible levels when the microbiota was disturbed, e.g., due to antibiotic treatment [30]. Therefore, bacterial myrosinase activity can cause the breakdown of glucosinolates in the distal gut, leading to release of several glucosinolate metabolites, e.g., isothiocyanates into the faecal stream and/or the urine.

In intact plants, myrosinases are separated from its substrates, e.g., in different cells or in different intracellular compartments. Damage of the plant tissue in case of preparing or chewing results in enzyme release. In the presence of water myrosinase initiates upon catalyzation by ascorbic acid hydrolytic cleavage of the β-glucosyl moity forming glucose, hydrogen ion and an unstable aglycone (thiohydroxamate-O-sulfonate). It is of note, that myrosinases are activated to various degrees by ascorbic acid, and in some instances the enzyme is almost inactive in its absence [22]. After the non-enzymatically release of the sulfate residue various volatile and non-volatile compounds derive, which exert different biological activities such as plant defence against insects or phytopathogens, or as attractants, and also give the specific flavour and pungency of mustard [31,32].

The hydrolysis products include isothiocyanates, previously known as "thioglucosides" or "mustard oils", thiocyanates, nitriles, epithionitriles, oxazolidine-2-thiones (5 vinyl-2-oxazolidinethione and 5-vinyl-1,3 oxazolidine-2-thione), and indol derivatives depending on the aglycone structure and reaction conditions (e.g., pH value, ferrous ion concentration, specifier proteins) (Figure 2). Considering plant biology, these alternative breakdown products provide an additional level of plant defence [33,34].

Under physiological conditions, isothiocyanates contribute to 60–90% of the total glucosinolate breakdown products. After release of glucose, the thiohydroximate-O-sulfonate

is formed, which can be spontaneously degraded by a Lossen-like rearrangement to the relatively stable isothiocyanates.

Isothiocyanates are very reactive and toxic for microbes, nematodes, fungi and insects, however many species of the Brassicaceae are able to promote alternative activation pathways by the action of specifier proteins. In the presence of these proteins, formation of isothiocyanates is reduced in favour of alternative breakdown products [35].

For instance, the presence of nitrile specifier proteins (NSPs) leads to an increased formation of nitriles [36,37], whereas, the occurrence and activity of the epithiospecifier protein (ESP) leads to the generation of epithionitriles from alkenyl-glucosinolate aglycons as well as nitriles from non-alkenyl-glucosinolate-aglycons. Moreover, it was shown that at physiological pH, isothiocyanates are the major products, whereas nitriles are formed at more acid pH [38].

Each type of Brassica vegetables including mustard plants show a characteristic glucosinolate composition and most species contain a limited number of glucosinolates [22]. The amount and composition vary among the different plant organs such as roots, leaves, stems and seeds, mainly with the highest concentrations often found in the reproductive tissues (florets, flowers and seeds). Therefore, for quantification of glucosinolates and their corresponding breakdown products, the seeds seem to be the best bulk source. The glucosinolate profile also depends on various environmental and ecophysiological factors such as plant nutrition, water availability, plant age and plant cycle [39].

Tables 1 and 2 give an overview of glucosinolates and their corresponding desulfated breakdown products identified in the mainly cultivated mustard species *S. alba*, *B. nigra*, *B. juncea* [22,24,29,40]. The mustard species *S. alba, B. nigra* and *B. juncea* show large ranges between the highest and lowest levels of total glucosinolate content as well as different content of predominant glucosinolates, rise up to 200 μmol/g seed.

**Mustard Species Common Names Glucosinolates Identified Systematic Name Trivial Name (a-Glycone = R Side Chain)** *S. alba* white, yellow mustard Gluconapin <sup>a</sup> 3-Butenyl Progoitrin <sup>a</sup> 2R-2-Hydroxy-3-butenyl Glucobrassicanapin <sup>a</sup> Pent-4-enyl # (Gluco-)Sinalbin <sup>b</sup> 4-Hydroxybenzyl Glucotropaeolin <sup>b</sup> Benzyl Gluconasturtiin <sup>b</sup> 2-Phenylethyl Glucoerucin <sup>c</sup> 4-Methylthiobutyl Glucoibe(rve)rin <sup>c</sup> 3-Methylthiopropyl Glucoiberin <sup>c</sup> 3-Methylsulphinylpropyl 2-Methylpropyl Isobutyl <sup>d</sup> Glucobrassicin <sup>e</sup> 3-Indolylmethyl Neoglucobrassicin <sup>e</sup> N-Methoxy-3-indolylmethyl *B. nigra* wlack, shortpod mustard, moutarde noire # (Gluco-)Sinigrin <sup>a</sup> 2-Propenyl *B. juncea* brown, indian, asiatic, chinese, sarepta mustard # (Gluco-)Sinigrin <sup>a</sup> 2-Propenyl # Gluconapin <sup>a</sup> 3-Butenyl Progoitrin <sup>a</sup> 2*R*-2-Hydroxy-3-butenyl Epiprogoitrin <sup>a</sup> 2*S*-2-Hydroxy-3-butenyl Glucosinalbin <sup>b</sup> 4-Hydroxybenzyl

**Table 1.** Distribution of glucosinolates among mustard plant species.
