Figure 7.30 Biosynthesis of the alkaloid capsaicin. (From Medicinal Natural Products by P.M.
Dewick, 1998, reproduced with permission from John Wiley & Sons.)
Figure 7.30 Biosynthesis of the alkaloid capsaicin. (From Medicinal Natural Products by P.M.
Dewick, 1998, reproduced with permission from John Wiley & Sons.)
which is used to cure bronchial asthma. Theobromine is found in cocoa and related chocolate products (Dewick, 1998). Purine bases like adenine and guanine are transformed in these species to purine alkaloids. The biosynthesis of these compounds starts from xanthosine monophosphate methylation to give 7-methylxanthosine monophosphate. Alternatively the dephosphorylation of xanthosine monophosphate gives xan-thosine. The latter is the precursor of theophylline, whereas hydrolysis of the phosphate group of 7-methylxanthosine monophosphate gives 7-methylxanthosine. The latter compound undergoes cleavage of the ribose to yield 7-methylxanthine, which after successive methylations is transformed to theobromine and finally to caffeine. The hydrolysis of the phosphate ester of xanthosine monophosphate gives xanthosine. The latter compound, after cleavage of ribose and two methylations, is finally transformed to theophylline (Figure 7.31).
EPHEDRINE: MA HUANG
Dietary supplements of ephedrine plus caffeine for weight loss (weight loss being the current first-line recommendation of physicians for osteoporosis) show some promise, but are not sufficient in number of study subjects. Ephedrine is a sympathomimetic amine with effects similar to those of adrenaline; it is produced by Ephedra (or ma huang), which has been used by the Chinese for at least 5,000 years. Ephedrine has resulted in deaths and hence is worrisome as an over-the-counter dietary supplement (Fillmore et al., 1999). Ephedrine has been described as a causative factor of vasculitis but myocarditis has not yet been associated with either ephedrine or its plant derivative ephedra (Zaacks et al, 1999). The content of ephedra alkaloids in herbal dietary supplements containing Ephedra (ma huang) has been studied by Gurley and co© 2003 Taylor & Francis Ltd workers (2000). The Ephedra alkaloid content of 20 Ephedra-containing supplements was determined and found to contain: ( — )-ephedrine, ( + )-pseudoephedrine, ( — )-methylephedrine, ( — )-norephedrine and ( + )-norpseudoephedrine. Total alkaloid content ranged from 0.0 to 18.5 mg per dosage unit. Ranges for ( — )-ephedrine and ( + )-pseudoephedrine were 1.1—15.3 mg and 0.2—9.5 mg, respectively. ( + )-Norpseu-doephedrine, a Schedule IV controlled substance, was often present. Finally, half of the products exhibited discrepancies between the label claim for Ephedra alkaloid content and actual alkaloid content in excess of 20 per cent (Gurley et al., 2000). Norpseu-doephedrine and catinone are also contained in khat (Catha edulis), a small tree cultivated in Ethiopia. The leaves of khat are chewed for a stimulant effect (Dewick, 1998).
Biosynthesis of Ephedra alkaloids starts from condensation of benzoic acid with pyruvic acid to give dichetopropyl phenyl, which after transamination yields cathi-none. The latter is reduced to the diastereoisomers norephedrine and norpseu-doephedrine (cathine). Methylation of these two compounds gives ephedrine and pseudoephedrine, respectively (Figure 7.32).
The cyanogenic glycosides are glycosides of a-hydroxinitriles and they are amino-acid-derived plant constituents, present in more than 2,500 plant species. The generation of cyanide (HCN) from cyanogenic glycosides is a two-step process involving a degly-cosilation and a cleavage of the molecule (regulated by (3-glucosidase and a-hydroxy-nitrilase). Furthermore, on enzymatic hydrolysis, cyanogenic glycosides yield the
aglycone (which is an a-hydroxynitrile) and the sugar moiety. The aglycones can be grouped into aliphatic and aromatic compounds; the sugar is mostly D-glucose, but can be other sugars too, for example, gentiobiose or primeverose (Vetter, 2000).
HCN is toxic to most living organisms due to its ability to bind to the metal (Fe, Zn and Cu) functional groups or ligands of many enzymes. Inhibition of oxygen reduction in the respiratory electron chain is one example, but inhibition is also exerted towards plastocyanin reduction (during photosynthesis) and catalase activity. All plants produce cyanide as a by-product of ethylene synthesis (McMahon et al., 1995) and between 3,000 and 12,000 plant species produce cyanogenic compounds (Kakes, 1990).
Several food plants are cyanogenic, including white clover, flax, almonds, wild lima bean and, particularly, cassava. This last plant is the major source of calories for more than 500 million people, with over 150 million tons of cassava root being harvested annually (Boccas, 1987). Cassava is thus one of the major food crops in which cyano-genesis is a real problem. This tropical root crop is grown extensively in Africa, Asia and Latin America. Its use provides up to 60 per cent of dietary calories in some areas. Furthermore, cassava roots are an imporant insurance crop for subsistence farmers throughout the tropics. All tissues of cassava, particularly leaves and root peel, contain high amounts of the cyanogenic glycoside linamarin and lesser amounts of lotaustralin (Bradbury and Egan, 1992). Some culivars of cassava may contain up to 500 mg/kg-1 cyanogenic glycosides (McMahon et al., 1995).
In general, the dangers of long-term exposure to HCN are not completely understood, but toxic effects involving the central nervous system, the gastrointestinal tract and the thyroid have been observed. According to the hypothesis of Kamalu (1993), linamarin absorbed from cassava diets causes inhibition of Na-K-ATPase, giving rise to electrolyte imbalance with potassium depletion. This depletion causes cellular swelling, vacuolation and rupture of the epithelial cells of the proximal tubules, which results in proteinuria and causes low serum albumin concentration. Such conditions are endemic in areas where people use high amounts of food plants containing cyanogenic glycosides. The problem is even higher in areas where a low-protein diet is accompanied with the consumption of such plants. In fact, amino acids help detoxify cyanide once it has been ingested or released inside the body.
Determination of cyanogenic glycoside content in dietary supplements is done by means of qualitative, semi-quantitative or quantitative methods. The first group of determinations includes direct methods; the second group is based on preliminary hydrolysis and quantification of HCN. Because of the medical significance of cyanide, it is not surprising that most research data are reported in terms of potential cyanide yields rather than the glycoside content itself. A direct estimation of linamarin in beans, bean paste products and cassava flour has been preformed (Kawamura et al, 1993). A modern variant of the old picric acid method for the estimation of HCN has been developed by Hin et al, (1996). This system is based on the hydrolysis of linamarin by stabilized leaf linamarase with detection of the cyanide by an alkaline picrate reagent.
In the general pathway of biosynthesis of cyanogenic glycosides, the a-amino acids are hydroxylated to form an N-hydroxylamino acid, which is then converted to an aldoxime and this in turn to a nitrile. The nitrile is hydroxylated to form an a-hydroxy-nitrile, which is glucosylated to form the corresponding cyanogenic glycoside (McFar-lane et al, 1975). The precursor of the linamarin synthesis is the valine and the conversion of valine to acetone cyanohydrin (non-glycosylated form of linamarin) is catalysed by NADPH-dependent cytochrome P450. The initial step is the N-hydroxy-lation of valine followed by the formation of 2-methyl-propanal oxime and its dehydration to yield 2-methylpropionitrile. The addition of oxygen forms acetone cyanohydrin, which is then glycosylated to form linamarin (Koch et al, 1992). The term 'cyanogenesis' means not only the synthesis or presence of a cyanogenic glycoside, but the enzymatic hydrolysis producing free HCN and other compounds. Since no HCN is released from intact cyanogenic plants, the substrates (the cyanogenic gly-coside) and the enzymes must be located in different cell compartments (Vetter, 2000).
The generation of cyanide from linamarin is a two-step process involving the initial deglycosilation of linamarin and the cleavage of linamarin to acetone cyanohydrin to form acetone and cyanide. These reactions are catalysed by a (3-glucosidase (linama-rase) and by a-hydroxynitrile lyase (HNL). Since acetone cyanohydrin may enzymati-cally as well as spontaneously decompose, it has been generally assumed that the linamarase activity is the rate-limiting step. The second, i.e. the final, step of the cyanogenesis is the breakdown of acetone to cyanide and acetone. This can occur both spontaneously (at temperatures greater than 35 °C or at pH greater than 4.0) and enzymatically catalysed by a-hydroxynitrile lyase (Vetter, 2000).
Figure 7.33 summarizes the biosynthesis and the generation of HCN from cyanogenic glycosides.
148 Massimo Maffei Glucosinolates and isothiocyanates
Glucosinolates (or (3-thioglucose-N-hydroxysulphates), the precursors of isothio-cyanates, are present in sixteen plant families, including a large number of edible species. These compounds have recently attracted intense research interest because of their cancer chemoprotective attributes. Moreover glucosinolate/isothiocyanates possess antibacterial, fungicidal, nematocidal and allelopathic properties (Fahey et al., 2001).
The consumption of food plants, such as cruciferous vegetables, has been found to be linked to reduced incidence of many types of cancer (Michaud et al, 1999; Talalay, 1999). At least some of the cancer chemoprotective activity of these vegetables is widely believed to be due to their content of minor dietary components such as glu-cosinolates (Fahey et al, 2001). Some glucosinolates have been reported to induce mammalian Phase 2 enzymes of detoxication (Fahey et al, 1997). Conversion of glucosinolates to thiocyanates, nitriles and isothiocyanates by the enzyme myrosinase (which is present in the microflora of the human digestive tract) is the important step in the process of cancer prevention. In fact these molecules possess potential antiprolif-erative, apoptosis-promoting, redox regulatory and Phase 1 enzyme-inhibiting roles (Nakamura et al, 2000; Fahey et al, 2001). For an extended revision of cancer-preventive effects of glucosinolate/isothiocyanate see Fahey et al. (2001).
Glucosinolates are tasteless and odourless compounds, whereas isothiocyanates are liquids with a sharp smell and taste (mustard oils). Black mustard (Brassica nigra L.) contains sinigrin. The drug is used as a condiment because of the sharp taste of the allyl isothiocyanate, whereas white mustard (Sinapis alba) contains sinalbin and is used as a spice. Powdered black and white mustard can be stirred and taken as an emetic (Samuelsson, 1992; Mithen et al, 2000).
In the same way as for cyanogenic glycosides, the biosynthesis of glucosinolates starts from an a-amino acid. Elongation of the amino acid side-chains occurs before S-glycosylation, whereas side-chain modification probably occurs after addition of the aglycone moiety. In the same way as for cyanogenic glycosides, the initial step in the biosynthesis proceeds by N-hydroxylation of a precursor amino acid, followed by decarboxylation to form an aldoxime (Bennett et al, 1995; Mithen et al, 2000). In the biosynthesis of sinalbin, tyrosine is the precursor. Biosynthetic steps after aldoxime formation are believed to involve conversion to a thiohydoximic acid, introduction of the thioglucoside sulphur from cysteine, S-glycosyl transfer from UDP-glucose, and sulphation by the donor 3'-phosphoadenosine-5'-phosphosulphate (PAPS) (reviewed by Fahey et al, 2001).
Glucosinolates are very stable water-soluble precursors of isothiocyanates. Conversion of glucosinolates to isothiocyanates occurs upon wounding of the plant, mastication of fresh plants (i.e. vegetables), or by damage caused by shipping, handling or bruising. Tissue damage releases myrosinase, an enzyme sequestered within aqueous vacuoles that hydrolyses glucosinolates. After hydrolytic cleavage of glucose, the sulphate moiety is released non-enzymatically to form thiohydroxamate-O-sulphonate. This unstable intermediate then rearranges to form isothiocyanates, or other breakdown products (such as thiocyanates, nitriles, epithionitriles and oxazolidine-2-thiones) in a manner that depends upon glucosinolate substrate as well as the reaction conditions (Fahey et al., 2001). Figure 7.34 depicts the pathway of the glucosinolate sinalbin formation and the breakdown products of myrosinase activity.
Other bioactive compounds
Several other compounds, deriving from different pathways, have bioactive properties. Most of these molecules belong to the so-called acetate pathway, which leads to fatty acids and polyketides. Polyketides represent a large class of natural products that are grouped together on purely biosynthetic grounds. Included in such compounds are fatty acids, polyacetylenes, prostaglandins, macrolide antibiotics and many aromatic compounds (Dewick, 1998). Below are described biosynthetic patwhays to some of the most interesting compounds found in dietary supplements: from St John's wort hypericin to the widely studied omega-3 fatty acids.
Hypericin and hyperforin: St John's wort
There is a growing increase in the sale of herbal medicines. This is particularly the case for St John's wort (Hypericum perforatum), a herbal antidepressant whose sales in the USA in 1998 were estimated at $200 million, while in Europe in 1999 sales amounted to $6 billion (Mclntyre, 2000). Indeed, in Germany St John's wort is the leading treatment for depression, outselling fluoxetine (Prozac®) by a factor of four (Ernst, 1999). St John's wort contains numerous biologically active constituents, including naphthodi-anthrones (e.g. hypericin and its derivatives), phloroglucinol derivatives (e.g. hyper-forin) and flavonoids (e.g. rutin, quercetin, quercitrin and biapigenin). For the treatment of depression, standardized alcoholic (60 per cent ethanol or 80 per cent methanol) extracts are commonly used. These are prepared from the dried plant and formulated into tablets, capsules and syrups for oral administration. Alcoholic extracts can contain 0.1—0.3 per cent hypericin, 2—4 per cent flavonoids and up to 6 per cent hyperforin. Commercial extracts are standardized to 0.3 per cent hypericin (Di Carlo et al, 2001). Inhibition of monoamine oxidase (MAO) by hypericin was believed to be the primary mode of action of the antidepressant effect of St John's wort. However, this initial assumption has not been confirmed in several subsequent studies (Muller et al, 1998; Nathan, 1999). In fact, the current standardization of H. perforatum extracts based on hypericin content correlates poorly with clinical potency because the antide-pressant effect of H. perforatum extracts depends on their hyperforin content (Laakmann et al, 1998). St John's wort activates the pregnane X receptor (PXR, a member of the steroid thyroid hormone receptor family that serves as a key regulator of cytochrome P450 enzyme system transcription) and consequently induces the expression of cytochrome P450 in human hepatocytes. Hyperforin, but not hypericin, is the chemical component of St John's wort responsible for PXR activation (Moore et al, 2000). Hypericum extracts have only weak activity in assays related to mechanisms of the synthetic antidepressants, that is, inhibition of MAO, catechol O-methyltransferase or serotonin re-uptake. It has been postulated that the clinical efficacy of St John's wort could be attributable to the combined contribution of several mechanisms, each one too weak by itself to account for the overall effect. The recent demonstration of a significant affinity of hypericin for sigma receptors presents new possibilities for consideration (Bennett et al., 1998). However, recent findings indicate that acute or chronic treatment with Hypericum extract does not alter mouse brain MAO activity and extracts devoid of hypericin still retain antidepressant activity (Di Carlo et al, 2001).
Another matter of concern is the phototoxic effects of hypericin and its derivatives — a disease called hypericism. Pseudohypericin and hypericin, the major photosensitizing constituents of H. perforatum, are believed to cause hypericism. Since hypericin has been proposed as a photosensitizer for photodynamic cancer therapy, the photocyto-toxicity of its congener pseudohypericin has been investigated. Pseudohypericin, in contrast to hypericin, interacts strongly with constituents of fetal calf serum (FCS), lowering its interaction with cells. Since pseudohypericin is two to three times more abundant in Hypericum than hypericin and the bioavailabilities of pseudohypericin and hypericin after oral administration are similar, results from the work of Vandenbo-gaerde and collaborators (1998) suggest that hypericin, and not pseudohypericin, is likely to be the constituent responsible for hypericism. Moreover, the dramatic decrease of photosensitizing activity of pseudohypericin in the presence of serum may restrict its applicability in clinical situations. Recently, studies done by Bernd and co-workers (1999) have confirmed the phototoxic activity of Hypericum extract on human keratinocytes. However, the blood levels that are to be expected during antidepressive therapy are presumably too low to induce phototoxic skin reactions.
The biosynthesis of the naphthodianthrone hypericin starts from the cyclization of a polyketide containing eight C2 units. After several modifications and the aromatiza-tion of the molecule, several tetrahydronaphthalene intermediates are formed, to end up with the anthrone, emodin anthrone. A further oxidative step can create a dehydro-dianthrone, and then coupling of the aromatic rings through protohypericin gives the naphthodianthrone hypericin (Dewick, 1998). Hydroxylation of the latter gives rise to pseudohypericin (Wink, 1999).
The phloroglucinol derivative hyperforin is probably synthesized from chalcone derivatives (Torssell, 1997).
Figure 7.35 depicts the biosynthetic pathway to hypericin and the structural formula of hyperforin.
152 Massimo Maffei Resveratrol: grape and wines
Resveratrol (3,4,5-trihydroxy-trans-stilbene) is a phenolic compound of the stilbene family present in wines and various parts of the grape, including the skin, and shows antioxidant and antiproliferative activities. Given that it is present in grape skins but not in the flesh, white wine contains very small amounts of resveratrol, compared to red wine. In red wines the concentrations of the trans-isomer, which is the major form, generally ranges from 0.1 to 15 mg/L (Fremont, 2000). Several researchers have investigated the antioxidant and pro-oxidant activities of resveratrol and compared them with other antioxidants widely used in foods (Murcia and Martinez-Tome, 2001; Stivala et al., 2001). The results showed that the hydroxyl group in the 4 position is not the sole determinant for antioxidant activity. In contrast, the presence of 4-OH together with stereoisomery in the trans-conformation (4-hydroxystyryl moiety), was absolutely required for inhibition of cell proliferation. Enzymatic assays in vitro demonstrated that inhibition of DNA synthesis was induced by a direct interaction of resveratrol with a- and 8-DNA polymerases (Stivala et al, 2001). Moreover, a direct comparison of resveratrol with other antioxidants showed that the order of HOCl scavenging activity was prolyl gallate > resveratrol > a-tocopherol > phenol; however, resveratrol was found to be inefficient in scavenging OH* and H2O2 (Murcia and Martinez-Tome, 2001).
Recent studies indicate that resveratrol can block the process of multistep carcino-genesis through mitotic signal transduction blockade and can also reduce risk of cardiovascular disease owing to its phyto-oestrogenic activity. Furthermore, it has been suggested that the antitumour and antimetastatic activities of this molecule might be due to the inhibition of DNA synthesis in Lewis lung carcinoma cells (Kimura and Okuda, 2001). Resveratrol was also found to strongly inhibit nitric oxide (NO) generation and reduce the amount of cytosolic inducible nitric oxide synthase (Lin and Tsai,
Resveratrol is glucuronated in the human liver and glucuronidation may reduce the bioavailability of this compound. However, recent findings that the flavonoids quercetin, myricetin, catechin, kaempferol, fisetin and apigenin may inhibit resvera-trol glucuronidation suggest that such an inhibition might improve the bioavailability of resveratrol (De Santi et al., 2000).
Resveratrol biosynthesis starts from the phenolic compound building block trans-4-hydroxycinnamoyl CoA and proceeds through addition of 3-malonyl-CoA units to form a polyketide intermediate. The latter undergoes decarboxylation and cyclization to form the stilbene skeleton of resveratrol (Dewick, 1998) (Figure 7.36).
Figure 7.36 Biosynthetic pathway to the stilbene resveratrol.
Figure 7.36 Biosynthetic pathway to the stilbene resveratrol.
Future advances in clinical cancer research will come from an emphasis on prevention rather than the treatment of metastatic disease. This research effort encompasses epi-demiological studies, such as those responsible for the recognition that high consumption of vegetables and fruits is associated with a reduced risk of some cancers, laboratory experiments to evaluate potential natural and synthetic products as chemo-preventive agents, and the execution of clinical preventive trials (Greenwald et al., 1993). Experimental studies performed in several laboratories suggest that the omega-3 fatty acids (FAs) can have a chemosuppressive effect on the progression of microscopic metastatic foci (Rose et al, 1996). These remarkable nutrients have attracted interest because of their importance in normal brain development, as dietary supplements for the prevention and treatment of chronic cardiovascular disease, and in the treatment of arthritic disorders and diabetes mellitus (Rose and Connolly, 1999).
The unsaturated FAs comprise monounsaturates and polyunsaturates. The conventional chemical nomenclature is to begin the systematic numbering of carbon atoms from the carboxyl terminal group. The carbon atom numbers 2 and 3 from the car-boxyl group are referred to as the a and P carbons, respectively, the last carbon is the w- or n-carbon, and the position of a double bond is indicated by the symbol A, followed by a number: for example, A9 refers to a double bond between carbon atoms 9 and 10 from the carboxyl group. However, an accepted practice in describing the chemical structure of FA molecules is to start numbering the carbons at the methyl group (w- or n-). The omega-3 (n-3) and omega-6 (n-6) polyunsaturated FAs cannot be synthesized by mammals, and because they must be obtained from the diet, they are referred to as 'essential fatty acids'. The n-3 FAs are represented by a-linolenic acid (LNA) and the n-6 FAs by linoleic acid (LA). Both LNA and LA are metabolized to longer-chain FAs, largely in the liver; LNA is converted to eicosapentaenoic acid (EPA), and thence to docosahexaenoic acid (DHA), while LA is the metabolic precursor of arachidonic acid (AA) (Rose and Connolly, 1999). It has been demonstrated that while dietary LA may influence eicosanoid formation by increasing the tissue AA pool, this contribution diminishes as dietary AA intake increases (Whelan et al., 1993).
FAs are found naturally in high concentration in fish. In addition to fish and fish oils, soybean and canola (low erucic acid rapeseed) oils may provide a significant source of dietary n-3 FA in the form of LNA (Hunter, 1990), the major FA in chloroplast lipids. In North American diets, the principal food sources of LNA are salad and cooking oils and salad dressing products. The per capita intake in the United States has been estimated to be c. 16-20 g/day for men and 12 g/day for women (Kim et al, 1984). Rose and Connolly (1999) have recently reviewed the potential that long-chain n-3 FAs exhibit as breast and colon cancer chemopreventive agents.
In higher plants there are two sets of reactions leading to production and accumulation of polyunsaturated fatty acids. The set of reactions occurring solely within the chloroplast are termed the 'prokaryotic pathway'; those involving glycerolipid synthesis in the endoplasmic reticulum and subsequent transfer to the chloroplast constitute the 'eukaryotic pathway'. Figure 7.37 shows the early steps in the biosynthesis of fatty acids. Fatty acids grow by the addition of two-carbon units; acetyl-CoA is the building block for the synthesis of both malonyl-CoA and the condensation reactions that lead to chain elongation. The first product of condensation is 3-ketobutyryl-ACP, which is reduced to 3-hydroxybutyryl-ACP, a reaction catalysed by the enzyme
ketoacyl-ACP synthase (KAS) isoform III. The latter compound is dehydrated and then reduced to butyryl-ACP which undergoes condensation with malonyl-ACP to form 3-ketoacyl-ACP, a reaction catalysed by KAS I. For the next six turns of the cycle, the condensation reaction is catalysed by KAS I and, finally, the conversion of 16:0 to 18:0 is catalysed by isoform II of KAS. Each condensation is accompanied by a decarboxylation, and the reaction goes on by addition of C2 units (Somerville et al., 2000). Figure 7.38 depicts the several steps involved in FA synthesis in leaves of Ara-bidopsis thaliana.
The first genetically modified (GM) crops were put on the market in the mid-1990s. Since then, uneven developments have occurred in various parts of the world. In the
future, the proportion of acreage planted with transgenic crops and the range of trans-genic crops are sure to increase. As with other innovations, the rapid uptake of GM crops is driven by profitability expectations (EC Working Document, 2000).
Before entering the world of bioengineering let us recall some basic definitions; then we will move to the discussion of food biotechnology, international policy, regulation and public concern. We will end the chapter with the last frontier in agro-biotechnology: molecular farming.
This is the manipulation of an organism's genetic endowment by introducing or eliminating specific genes through modern molecular biology techniques. A broad definition of genetic engineering also includes selective breeding and other means of artificial selection.
GENETICALLY MODIFIED ORGANISMS (GMO)
These are organisms produced from genetic engineering techniques that allow the transfer of functional genes from one organism to another, including from one species to another. Bacteria, fungi, viruses, plants, insects, fish and mammals are examples of organisms whose genetic material has been artificially modified in order to change some physical property or capability. 'Living modified organism' (LMO), and 'transgenic organism' are other terms that are often used in place of GMO.
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