Abstract

Glucosinolate derived products are well known for their bioactive properties. These properties are defined by the type of glucosinolate product, and depend on the myrosinase catalysed reaction. The types of properties comprise; nutritional beneficial-, anticancerogenic-, antinutritional-, and pesticide effects. It is thus important with knowledge to these properties and access to the individual compounds. Myrosinase, glucosinolates and other biocactive compounds occurring in cruciferous seeds have now been isolated in Pilot Plant scale. Various types of glucosinolate derived products have been produced in pure state, and it is found that some of these products have potential applications as anticancerogenic compounds, and as biodegradable biocides, antifungal-, nematocidal-, insecticidal agents.

Introduction

Glucosinolates are plant products, which have been known for a long time (Roubiquet and Boutron, 1830; Gadamer, 1897) and they are now known to have well defined structures consisting of a side chain (R-group) and D-glucopyranose as β-thioglucoside, attached to the carbon (No. 0) in Z (cis)-N-hydroxime sulphate esters (Figure 1) (Ettlinger and Lundeen, 1956; Olsen and Sørensen, 1981; Sørensen, 1990). These compounds co-occur with isoenzymes of thioglucoside glucohydrolase (EC 3.2.3.1; myrosinase; Buchwaldt et al., 1986) in all plants of the order Capparales and in some few other plants (Kjær, 1960; Ettlinger and Kjær, 1968; Dahlgren, 1974; Rodman, 1981; Bjerg and Sørensen, 1987).

Figure 1. Structure of glucosinolates. R = side chain with structural resemblance to the parent amino acids; R₂ and R₆ = H or acyl derivatives; M⁺ = cation.

The structural variations among the more than 120 known glucosinolates are caused by the variations in the R-groups and owing to acyl substituents on the thioglucose part (Sørensen, 1990 and refs. cited therein). The latter group of glucosinolates has been overlooked by most researchers and analytical groups owing to limitations in the often used analytical techniques based on use of myrosinase or sulfatase (Bjerg and Sørensen, 1987; Sørensen, 1990). In cruciferous crops, as those belonging to the genera Brassica, the majority of glucosinolates are biosynthetic derived from the amino acid methionine (Nos. 1-14; Figure 2), tryptophan (Nos. 23-27; Figure 2) or phenylalanine (Nos. 16-22, 30-32 and 35; Figure 2). Other amino acids may also be precursers, e.g. in other cruciferous genera, in Resedaceae, Capparidaceae and other glucosinolate containing plants (vide supra).

Figure 2. Structures and trivial names of selected glucosinolates important for the quality of rapeseed and other cruciferous crops are shown in figure 1. and this figure.

The structures of the glucosinolates are defining their properties including the type of products formed as result of glucosinolate degradation (Bjerg and Sørensen, 1987; McDanell et al., 1988; Sørensen, 1990; Bjergegaard et al., 1994). The various types of degradation products are responsible for great variations in both wanted and unwanted proporties they give to important cruciferous crops, technically and economically important oils, proteins and other products obtainable therefrom (Bjergegaard and Sørensen, 1996; Hansen et al., 1997; Bagger et al., 1998). It is thus for various reasons important with specific knowledge to the actual glucosinolate structure, including stereochemistry, and to the mechanism of the myrosinase catalysed glucosinolate hydrolysis (Bjergegaard et al., 1994), which is the basis for production of added value cruciferous oilseed products (Bagger et al., 1998) and the wanted bioactive glucosinolate products (Agerbirk et al., 1998; Palmieri et al., 1998).

Experimental

The experimental procedures used for analytical determination of glucosinolates and myrosinase isoenzymes are described elsewhere (Sørensen et al., 1999). Description of the reactivity and transformation of alifatic and aromatic glucosinolate products into various types of derivatives as well as methods of analysis used for their determination are described recently (Agerbirk et al., 1998; Sørensen et al.,1999). The pilot plant techniques developed as ”Green Chemistry” and used for isolation of great amounts of glucosinolates, myrosinases and other products from oilseed crops have been presented previously (Bagger et al., 1998) and these technigues are now further developed in the EU supported BOP project (Bagger et al., 1999).

Results and Discussion

Myrosinases exhibit pronounced specificity toward the β-D-thioglucopyranoside part of glucosinolates (Sørensen, 1990), and the sulfate group seems also to be essential as hydrolysis of desulfoglucosinolates are not catalysed by myrosinases. These observations and results from enzyme kinetic and spectroscopic investigations have resulted in proposals for the mechanisms of myrosinase catalysed glucosinolate hydrolysis (Bjergegaard et al., 1994; Palmieri et al., 1998; Figure 3).

Figure 3. Proposed reaction mechanisms for myrosinase catalysed degradation of glucosinolates.

Cinnamoylglucosinolates are thus not transformed by catalysis of myrosinase isoenzymes and sulfatase, but glucosinolates with unsubstituted glucose part give with the exception for indol-3-ylmethyl glucosinolates and some few of other glucosinolates (vide infra; Figure 5), degradation products as shown in Figure 4.

Figure 4. Products from enzyme catalysed transformation of glucosinolates (alifatic; compose text and Figure 5)

With respect to the effects of ascorbic acid (Vit. C) in the myrosinase catalysed glucosinolate hydrolysis, it has been known for many years (Ettlinger and Kjær, 1968), that Vit. C is important for optimal catalytic activity. High concentration of Vit. C is in favour of nitrile formation (Olsen and Sørensen, 1981), and this is also the case with ferro ion as shown in figure 5 (Agerbirk et al., 1998). These redox reactions give thus basis for transfer of redox equivalents required for nitrile formation. Otherwise the formation of thiocyanates is possible (need for modifier protein) even though isothiocyanates most often are initial products. With the isothiocyanate group on an aliphatic carbon chain (Figure 2-4) the results can be volatile/lipofilic compounds (Nos. 1-3; Figure 2 and 3-methylthiopropyl (glucoibervirin; No. 7), 4-methylthiobutyl (glucoerucin; No. 8) and 5 methylthiopentyl (glucoberteroin; No. 9)). Less volatile compounds are the result when the molecular weigth increase and when it is more hydrophilic compounds as those with methylsulfinyl groups (Nos. 10-13; Figure 2). 2-Hydroxysubstituted compounds give in fast reactions the oxazolidine-2-thiones and isothiocyanates are also reactive toward nucleophiles as amino groups resulting in binding to proteins (Figure 5)(Bjergegaard et al., 1999).

Figure 5. Structures for some of the products formed as a result of glucosinolate degradation with illustration of the differences in products formed from aliphatic and aromatic glucosinolates, which can give stabilised carbonium ions.

Indol-3-ylmethylglucosinolates (Nos. 23-27; Figure 2) and other aromatic compounds which give a stabilised carbonium ion by release of the thiocyanate ion (Figure 5) give a great number of products resulting from reactions with nucleophiles. The carbonyls are among the products, but these compounds do not seem to be a direct intermediate, as often stated in the litterature, in the formation of oligomers and ascorbigens (Figure 5; Agerbirk et al., 1998).

With the techniques now available, it is possible to produce great amounts of individual glucosinolates and myrosinases (Bagger et al., 1998; 1999 this volume). The technigues and know-how also allow production of the individual bioactives from glucosinolate degradation products (ongoing developmental work). Thereby, it give the potentialities for well defined anticancerogenic compounds (Stoewsand, 1995; Agerbirk et al., 1998; Bonnesen et al., 1999) and biodegradable biocides comprising antifungal-, nematocidal- and insecticidal agents (ongoing test; Kirkegaard et al., 1996; Tsao et al., 1996; Brown and Morra, 1997; Manici et al., 1997; Pinto et al., 1998; Potter et al., 1998; Sarwar et al., 1998) which can be used in concentration levels below the levels creating antinutritional effects when used to monogastic animals (Sørensen, 1988).

Acknowledgements

The Danish Ministry of Food, Agriculture and Fishery, Strukturdirektoratet, and the Commission of European Union (Contract FAIR CT 95-0260) are greatefully acknowledged for financial support to this work.

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