The chemistry and occurrence of glucosinolates and their breakdown products have been reviewed extensively by Fahey et al. (2001).
Several glucosinolates are isolated in the pure state. The first crystalline glucosinolate was isolated from the seed of white mustard in 1830 and since then the elucidation of their struc-tures and chemistry has continued (Gildemeister and Hofmann, 1927). The common structure of glucosinolates is shown in Fig. 3.1. The side chain determines the chemical and biologi-cal nature of glucosinolates. They are considered to be (Z)-cis-N -hydroximinosulfate esters possessing a side chain R and a sulfur-linkedD-glucopyranose moiety. Natural glucosinolates contain exclusively a β-D-glucopyranosyl linkage (Blanc-Muesser et al., 1990).
The side chain of the glucosinolates is variable and is the basis for their structural het-erogenity and for the biological activity of the enzymatic and chemical breakdown products.
C S
N O SO3 C6H11O5
R −
Fig. 3.1 General structure of glucosinolates.
Table 3.1 Glucosinolates commonly found in Brassica vegetables.
Trivial name Chemical name (side chain R)
Aliphatic glucosinolates
Glucoiberin 3-Methylsulfinylpropyl
Progoitrin 2-Hydroxy-3-butenyl
Sinigrin 2-Propenyl
Gluconapoleiferin 2-Hydroxy-4-pentenyl Glucoraphanin 4-Methylsulfinylbutyl Glucoalyssin 5-Methylsulfinylpentyl
Glucocapparin Methyl
Glucobrassicanapin 4-Pentenyl
Glucocheirolin 3-Methylsulfonylpropyl Glucoiberverin 3-Methylthiopropyl
Gluconapin 3-Butenyl
Indole glucosinolates
4-Hydroxyglucobrassicin 4-Hydroxy-3-indolylmethyl
Glucobrassicin 3-Indolylmethyl
4-Methoxyglucobrassicin 4-Methoxy-3-indolylmethyl Neoglucobrassicin 1-Methoxy-3-indolylmethyl Aromatic glucosinolates
Glucosinalbin p-Hydroxybenzyl
Glucotropaeolin Benzyl
Gluconasturtiin 2-Phenethyl
Table 3.1 gives an overview of the most commonly found glucosinolates in Brassica vegeta-bles, however an extensive list of 120 different glucosinolates identified in higher plants has been described by Fahey et al. (2001).
Glucosinolates are prevalent in about 16 botanical families of the order Capparales, such as the Capparaceae, Brassicaceae, Caricaceae and Resedaceae (Fahey et al., 2001). For the human diet, representatives of the Brassicaceae are of particular importance as vegetables (e.g. cabbage, Brussels sprouts, broccoli, cauliflower), root vegetables (e.g. radish, turnip and swede), leaf vegetables (e.g. rocket salad) and seasonings and relishes (e.g. mustard, wasabi) and sources of oil (Holst and Williamson, 2004). They occur in all parts of the plants, but in different profiles and concentrations. Usually, a single plant species contains up to four different glucosinolates in significant amounts while, as many as 15 different glucosinolates can be found in the same plant. The highest concentrations are usually found in the seeds, except for indol-3-ylmethyl and N -methoxyindol-3-ylmethyl glucosinolates, which are rarely found in seeds (Tookey et al., 1980). Several reviews have presented and discussed the variation in glucosinolate composition and profiles of various representatives of the Brassicaceae. Occurrence and concentrations of glucosinolates vary according to difference in species and varieties, tissue type, physiological age, environmental conditions (agronomic practices, climatic and ecophysiological conditions), presence of pest infestation (Rosa et al., 1997; Fahey et al., 2001; Holst and Williamson, 2004; Schreiner, 2005).
3.3 BIOSYNTHESIS
The pathway of glucosinolate biosynthesis has been studied since the 1960s and the iden-tity of many intermediates, enzymes and genes involved is now known. The biosynthesis of
glucosinolates was recently reviewed extensively by Halkier and Gershenzon (2006). Knowl-edge of biosynthetic pathways of glucosinolates has increased as research advanced from tra-ditional in vivo feeding studies and biochemical characterization of the enzymatic activities in plant extracts to identification and characterization of the biosynthetic genes encoding the involved enzymes. Especially the studies of glucosinolates in the model plant Arabidopsis facilitated the progress.
Kjaer and Conti (1954) suggested that amino acids may be natural precursors of the aglycone moiety of glucosinolates based on the similarities between the carbon skeletons of some amino acids and the glucosinolates. This hypothesis was confirmed by studies of the different biosynthetic stages. Most of these studies involved the administration of variously labelled compounds (3H, 14C,15N or 35S) to plants and the assessment of their relative efficiencies as precursors on the basis of the extent of incorporation of isotope into the glucosinolate. The classification of glucosinolates as shown in Table 3.1 depends on the amino acid from which they are derived; aliphatic glucosinolates derived from alanine, leucine, isoleucine, methionine or valine; aromatic glucosinolates derived from phenylalanine or tyrosine; and indole glucosinolates are derived from tryptophane (Sørensen, 1990).
The biosynthesis of glucosinolates from amino acids can be divided into three separate steps. The first step is the chain elongation of aliphatic and aromatic amino acids by inserting methylene groups into their side chains. Second, the metabolic modification of the amino acids (or chain-extended derivatives of amino acids) takes place via an aldoxime intermedi-ate. The same modifications also occur in the biosynthetic route of cyanogenic glycosides.
However, the co-occurrence of glucosinolates and cyanogenic glycosides in the same plant is very rare (an example is Carica papaya). The biosynthesis of the cyanogenic glycosides has been elucidated in more detail by Halkier and Lindberg-Møller (1991) and by Koch et al. (1992). Third, following the formation of the aldoxime, the glucosinolate is formed by various secondary transformations such as S-insertion, glucosylation and sulfation. Further modification of the side chain can occur in the formed glucosinolate by, for example, oxida-tion and/or eliminaoxida-tion reacoxida-tions. The different steps in the synthesis are discussed below in more detail.
3.3.1 Amino acid modification
The glucosinolates can be divided into two groups by origin: those derived from common amino acids and those derived from modified amino acids. The modification of common amino acids is mainly in the form of side chain elongation. A general route for this was pro-posed by Kjaer (1976). Various enzymes are involved in these steps (Halkier and Gershenzon, 2006). The parent amino acid is deaminated to form the corresponding 2-oxo acid. Next is a three-step cycle of (i) condensation with acetyl-CoA, (ii) isomerization and (iii) oxidation–
decarboxylation to yield a 2-oxo acid with one more methylene group than the starting com-pound. The resulting chain-extended 2-oxo acid can undergo additional chain-elongation cycles, each adding one further methylene group, or, following transamination, can enter the glucosinolate core biosynthetic pathway. Up to nine elongation cycles are known to occur in plants.
3.3.2 Conversion of amino acids
The glucosinolate core pathway converts the amino acid to an S-alkylthiohydroximate via two consecutive reactions that are catalysed by structurally specific cytochrome P450s,
R CH COOH
NH2 NOH
R CH R C
NOH S−
S
N C R
OSO3− Glucose Glucose
R C
NOH S
Amino acid Aldoxime Thiohydroximic acid
Desulfoglucosinolate Glucosinolate Fig. 3.2 The simplified biosynthesis of the glucosinolate core structure.
encoded by the CYP79 and CYP83 gene families. C-S lyase activity results in the for-mation of thiohydroximates that are converted to desulfo-glucosinolates by a non-specific S-glucosyltransferase. The final glucosinolate is produced by sulfation by one of three structurally specific sulfotransferases. Subsequently, various secondary side-chain modifica-tions can occur, including oxidation, hydroxylation, alkenylation, acylation or esterification (Halkier and Gershenzon, 2006).
3.3.3 Secondary transformations
The formed parent glucosinolate is subject to a wide range of further modifications of the R group. The R group of glucosinolates derived from methionine (and chain-elongated ho-mologs) is especially subject to further modifications, such as the stepwise oxidation of the sulfur atom in the methylthioalkyl side chain leading to methylsulfinylalkyl and methylsul-fonylalkyl moieties. Methylsulfinylalkyl side chains can be further modified by oxidative cleavage to produce alkenyl or hydroalkenyl chains. These reactions are of biological as well as biochemical interest because they influence the direction of glucosinolate hydrolysis and the resulting activity of the hydrolysis products (Halkier and Gershenzon, 2006).
The simplified biosynthetic pathway for glucosinolates is shown in Fig. 3.2 (based on Halkier and Gershenzon, 2006).
3.4 HYDROLYSIS
As mentioned before, hydrolysis products rather than intact glucosinolates are responsible for the various biological effects. Most glucosinolates are chemically stable and to lesser extent thermal stable. Therefore, hydrolysis is mainly enzymatically driven by the endogenous enzyme myrosinase. Upon consumption of intact glucosinolates without the presence of active myrosinase (e.g. cooked vegetables) glucosinolates can also be hydrolysed by ß-glucosidases from the intestinal flora (Shapiro et al., 1998, 2006).
3.4.1 Myrosinase
Myrosinase (thioglucoside glucohydrolase EC 3.2.3.1) is the trivial name for the enzyme (or group of enzymes) responsible for the hydrolysis of glucosinolates. All plants that contain
glucosinolates also contain myrosinase. Myrosinase is widely distributed, occurring in my-rosin cells of seeds, leaves, stems and roots of glucosinolate-containing plants and the activity appears to be higher in the young tissues of the plant (Bones and Rossiter, 1996).
Myrosinases have generally been well characterized by various approaches such as an-alytical gel electrophoresis, immunohistochemical techniques, light microscopy and elec-tron microscopy (Buchwaldt et al., 1986; Thangstad et al., 1990). The various studies have demonstrated the presence of several myrosinase isoenzymes (MacGibbon and Allison, 1970;
Buchwaldt et al., 1986). Different patterns were found depending on whether the extracts were made from the leaf, stem, root or seed. Little is known about the substrate specificity of myrosinase isoenzymes. There are two myrosinases isolated by James and Rossiter (1991) that degrade different glucosinolates at different rates. However, both isoenzymes show high-est activity against aliphatic glucosinolates and least activity against indole glucosinolates.
Members of a given class of glucosinolates are degraded at approximately the same rate in vitro. It is also possible that the specificity is affected by associated factors like epithiospec-ifier protein, myrosinase-binding protein or other myrosinase-associated proteins or compo-nents. Myrosinase activity varies by plant species, organ and stage of development (Bones, 1990), but activity is also affected by seasonal conditions and climatic factors (Charron et al., 2005).
Ascorbic acid has been shown to modulate myrosinase activity in some species; it inhibits at high concentrations and activates at low levels. Activation appears to be the result of a conformational change in the protein structure, leading to an enhanced reaction rate when the effector-binding sites are occupied (Ohtsuru and Hata, 1973).
The complexity of the glucosinolate/myrosinase system indicates an important role in cru-ciferous plants (Bones and Rossiter, 1996). The glucosinolate/myrosinase system may have several functions in the plant: (i) plant defence against fungal diseases and pest infestation;
(ii) sulfur and nitrogen metabolism; and (iii) growth regulation.
Plant breeding strategies over past decades have concentrated on reducing the glucosinolate content of rapeseed to improve the acceptability of rapeseed meal and meet the increasingly stringent requirements of the rapeseed processing industry. One approach to reducing the un-desired breakdown products of glucosinolates would be to change the amount of myrosinase available for hydrolysis of the glucosinolates.
3.4.2 Hydrolysis products
Hydrolysis products of glucosinolates contribute significantly to the typical flavour of Bras-sica vegetables. Myrosinase catalyse the hydrolysis of glucosinolates by splitting off the glucose. The unstable aglucone (thiohydroxymate-O-sulfonate) then eliminates sulfate by a Lossen rearrangement (Fig. 3.3). The structure of the resulting products depends on a variety of factors. Whether ITCs or nitriles are formed depends on the specific glucosinolates, the part of the plant where they are located, the treatment of plant material before the hydrolysis of glucosinolates and conditions during hydrolysis, especially pH. Isothiocyanates are usually produced at pH 5–7, while nitriles are the major degradation products under acidic conditions.
Most hydrolysis products are stable except for glucosinolates possessing a ß-hydroxylated side chain; ß-hydroxy-ITCs are unstable and spontaneously cyclise to oxazolidine-2-thiones (e.g. goitrin). Indole glucosinolates such as glucobrassicin form unstable ITCs and undergo further hydrolysis to give 3-indolemethanol, 3-indoleacetonitrile and 3,3diindolylmethane and subsequently condenses into dimers, trimers or tetramers (Holst and Williamson, 2004).
R S Glc
Myrosinase Glucose
N
OSO3−
R SH
N
ESP ESP
when R = when R =
OH Isothiocyanate
Nitrile
Epithionitrile
Thiocyanate R
n
S OSO3−
C
N C S
R N C S N
R
S
N n
C N
OH
NH
S Oxazolidine- O
2-thione
Fig. 3.3 Outline of glucosinolate hydrolysis (from Halkier and Gershenzon, 2006). ESP, epithiospecifier protein; R, variable side chain. Reprinted with permission, from the Annual Review of Plant Biology, Volume 57, copyright 2006, by Annual Reviews www.annualreviews.org
Hanley et al. (1990) isolated an indole isothiocyanate from neoglucobrassicin degradation under specific experimental conditions. Epithioalkanes are produced from the hydrolysis of alkenyl glucosinolates when myrosinase co-occurs with a labile protein known as epithiospec-ifier protein (ESP), a small protein that is not ubiquitous in the Cruciferae (Matusheski et al., 2006).
3.5 ANALYTICAL METHODS
Understanding the involvement of glucosinolates and their biological active breakdown prod-ucts in the many different scientific fields requires the development of rapid, reliable and re-producible methods for their identification and quantification. However, rarely does a method provide rapidity, simplicity, sensitivity and accuracy to discriminate among all compounds of interest. Especially since there occur a large amount of different glucosinolates in Brassicas and the fact that each glucosinolate can produce different breakdown products makes their analysis very complicated.
Analytical methods can be divided into those for total glucosinolates, individual glucosi-nolates and the breakdown products. Over the past four decades, increased knowledge of the diversity of the glucosinolates, their enzymatically released products and factors influencing their release have led to a multiplicity of analytical methods. Griffiths et al. (1998) and Kiddle et al. (2001) have presented an extensive overview of the wide variety of analytical methods of glucosinolates in plant tissue.
Since glucosinolates coexist with myrosinase in the plant, processes like grinding or cutting of fresh tissue in the presence of water will initiate a rapid hydrolysis of these compounds.
For analysis of intact glucosinolates, inhibition of myrosinase activity is therefore essential.
Before disruption of the material, samples should be completely dried by freeze-drying or frozen in liquid nitrogen. The use of aqueous methanol for extraction, in combination with high temperatures, also inhibits myrosinase (Heaney and Fenwick, 1993).
3.5.1 Total glucosinolates
Glucosinolates yield equimolar amounts of glucose on hydrolysis with myrosinase. This is true for almost all glucosinolates and spectroscopic methods for total glucosinolates based on the measurement of enzymatically released glucose or sulfate are relatively rapid and simple to apply (Schnug, 1987; Heaney et al., 1988). Van Doorn et al. (1998) described a more specific determination of the glucosinolates sinigrin and progoitrin by raising antisera to these glucosinolates.
Measuring total glucosinolate levels has received less attention because of the great progress in analytical methodology for individual glucosinolates that leads to much more useful information. However, the high costs and labour input required for obtaining glucosi-nolate data is a serious hinder to generating large sets of samples, which is usually necessary in screening breeding programmes. Therefore, the use of fast analytical techniques such as near-infrared spectroscopy (NIRS) result in many advantages since analysis can be carried out with a considerable saving of time and at relative low costs. This technique has mostly applied for glucosinolate content in seeds of Brassica vegetables where these compounds are present in significant higher concentrations than those usually found in leaves (Velasco and Becker, 1998; Font et al., 2004). Font et al. (2005) have described the quantification of glucosinolates in leaves of Brassica napa by NIRS. They demonstrated the potential of NIRS for predicting the total glucosinolate content as well as the major individual glucosinolates found in B. napus.
3.5.2 Individual glucosinolates
For more detailed analysis, researchers have used either HPLC-MS analysis for intact glu-cosinolates or GC-MS analysis for the hydrolysis products. GC-MS can be very sensitive,
but has some limitations as some glucosinolate side chains (e.g. indolyl or hydroxylated side chains) produce poorly volatile or unstable breakdown products.
Spinks et al. (1984) developed a reverse-phase HPLC method for quantitative analysis of desulfoglucosinolates which is, hitherto, most widely used. This method uses an on-column enzymatic desulfation treatment of plant extracts followed by HPLC detection of the resulting desulfoglucosinolates. For the analysis, desulfobenzyl or desulfosinigrin is used as internal standard and relative response factors are determined with purified standards. Validation of chromatographic profiles takes place by correspondence of glucosinolate retention times with standardized rapeseed extracts or other available standards. The intact glucosinolates can be analysed directly by HPLC, although this often gives a poor resolution. Desulfation results in better separation and cleaner samples and also makes the compounds easier to analyse by HPLC or mass spectrometric techniques. HPLC systems using a photodiode array (PDA) detector are very sensitive allowing detection of glucosinolate levels in the nano-molar range. Whilst spectral data of individual desulfoglucosinolates will allow initial confirmation of structural class, addition of MS detection increases the discriminatory power of the technique even further (Kiddle et al., 2001).
Nowadays, mass spectrometry has become a powerful technique for quantification of bioactive compounds in complex biological matrices; however, most methods were qualita-tive. Griffiths et al. (1998) reported a liquid chromatography–atmospheric pressure chemical ionization–mass spectrometric (LC-APCI/MS) method for determining quantitatively desul-foglucosinolates.
Song et al. (2005) described the quantitative analysis of glucosinolates in vegetable extracts and blood plasma by negative ion electrospray LC-MS/MS.
Both LC-thermospray-MS and LC-APCI-MS have been employed for desulfoglucosi-nolates (Mellon et al., 2002; Song et al., 2005) and the latter has been found to give the best sensitivity, with at least an order of magnitude improvement over UV detection and the added advantage of structural confirmation from the mass spectrum. The sensitivity can be further improved by the use of synthetic per-deuterated desulfoglucosinolates as internal standards.
Tian et al. (2005) improved the LC-APCI/MS method by using liquid chromatography–
electrospray ionization–tandem mass spectrometry (LC-ESI/MS/MS). This method has the advantage that the glucosinolates do not require desulfonation before the analysis, while the detection limits are 10-fold lower compared to conventional HPLC methods.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was introduced in 1987 and developed for use with nonvolatile and large biomolecules (Karas et al., 1987). It has some advantages over other methods including speed of analysis, high sensitivity, good tolerance toward contaminants, and the ability to analyse complex mixtures. MALDI-TOF MS thus has potential for the analysis of plant metabolites, both in the plant itself and in foodstuffs, as a profile of the sample can be ob-tained in only a few minutes (Karas, 1996). Botting et al. (2002) applied MALDI-TOF for the characterization of a number of intact glucosinolates. The method was used for crude plant extracts to rapidly examine glucosinolate profiles. The method is much faster compared to LC-MS methods and required far less sample, but has the drawback of not being a quantitative analysis method.
One of the major problems in the analysis of glucosinolates is the paucity of high purity chromatographic standard glucosinolates available to researchers. Only a limited number of glucosinolates are commercially available. The glucosinolate 2-propenyl (sinigrin) is not a suitable internal standard because of the presence of this compound in most brassicaceous
plants. On the other hand, benzylglucosinolate (glucotropaeolin) is commonly not present in Brassicas and has been used as an internal standard.
3.5.3 Breakdown products
The application of HPLC to the investigation of glucosinolate breakdown products has been limited due to the volatility of many compounds. Furthermore, thiocyanates and nitriles are not detectable spectrometrically.
Isothiocyanates and nitriles can be analysed by GC, HPLC with UV detection may be used for analysis of oxazolidinethiones and indoles. Quinsac et al. (1992) developed a method for analysing oxazolidinethiones in biological fluids with a high degree of selectivity. However, HPLC finds most use in the analysis of intact glucosinolates or desulfoglucosinolates. For identification and confirmation of structures both techniques can be coupled to mass spec-trometry. Mass spectroscopy has proved to be an important tool in the identification and structural elucidation of glucosinolates and their breakdown products.
Zhang et al. (1992a) developed a spectroscopic quantitation of organic ITCs. Under mild conditions nearly all organic ITCs react quantitatively with an excess of vicinal dithiols to give rise to five-membered cyclic condensation products, with release of the corresponding free amines. The method can be used to measure 1 nmol or less of pure ITCs or ITCs in crude mixtures.
Song et al. (2005) reported an LC-MS/MS method for the analysis of ITCs and amine degradation products. Isothiocyanates were pre-derivatized and quantified by positive ion electrospray LC-MS/MS.