Chemical Hazards and Their Control: Endogenous Compounds€¦ · isolated from plants, fungi, and animals is high. Luckner worked with 26 biosynthetic groups di-vided into 107 subgroups,

CHAPTER 4

Chemical Hazards and Their Control:Endogenous Compounds

Leon Brimer

INTRODUCTION

Raw materials of vegetable origin may con-tain natural toxic or antinutritional compounds,endogenous constituents that are synthesized bythe plant itself. Antinutritional means a deleteri-ous effect due to the hindrance of uptake or useof other components in the diet. Examples ofantinutritional compounds include tannins,which among others bind to proteins, makingthem unacceptable as substrates for proteases;proteinase inhibitors, which inhibit proteinasessuch as trypsin and chymotrypsin; phytate, whichbinds a number of minerals, making them unavail-able for uptake; and thiaminase, which degradesvitamin B1. An effect may be due to the parentcompound or to metabolites that are formed in thegut or in the organism after absorption.

Although a few toxic and antinutritional com-pounds found in plants are proteins, most arelow molecular weight compounds. Examples ofproteins are ricin,53 which is found in the seedsofRicinus communis L. (castor bean); lectins,213

found especially within the legumes; and theproteinase inhibitors, which are also common inlegumes.62 The smaller molecules with deleteri-ous effects belong to the groups of compoundsthat are normally classified as secondary me-tabolites. The number of secondary constituentsisolated from plants, fungi, and animals is high.Luckner worked with 26 biosynthetic groups di-vided into 107 subgroups, many of which con-tained more than 1,000 structures.155 The major-ity of these compounds was found in plants. The

alkaloids, for example, have had the attention ofphytochemists for more than 150 years. In 1950,approximately 2,000 alkaloids were recognized;by 1970, the number had increased to approxi-mately 4,000; 20 years later, approximately10,000 were known.217 It is necessary, then, to fo-cus on the most important endogenous plant toxinsas seen from a food and feed point of view.

The most prominent constituents known to re-strict the nutritional value of food or fodder in-clude certain nonprotein amino acids, alkaloidsand glycosides, together with the tannins. How-ever, knowledge concerning the influence offer-mentation on these agents is very limited exceptfor certain of the glycosides. Because a numberof very important commodities of food and fod-der worldwide do contain toxic glycosides,46 theoccurrence and effects of toxic glycosides, andtheir fate during food fermentations, will be pre-sented in this chapter.

TOXIC AND ANTINUTRITIONALGLYCOSIDES IN FOOD AND FEED

Glycosides consist of one or more genins (ag-lycones) to which one or more mono- or oli-gosaccharides are linked. The glycosidiclinkage(s) may differ (i.e., one differentiates be-tween O-, S-, and C-glycosides) (Figure 4—1). Ifthe sugar part is a glucose moiety, it is called aglucoside. A number of different glycosides andoligosaccharides causing physiological effects(toxins) or reduced uptake or use of nutrients af-ter ingestion are known in the plant kingdom. So

Figure 4-1 (a) The general structure of O-, S-, and C-glucosides as representatives of the broader groups ofO-, S-, and C-glycosides, respectively. At top, an O-glucoside; middle, an S-glucoside; bottom, a C-glucoside.(b) Examples of naturally occurring O-, S-, and C-glucosides. Top (Linamarin—a cyanogenic glucoside found incassava), middle (Sinigrin—a glucosinolate), and bottom (Barbaloin—an anthrone C-glucoside from Aloe spp.;laxative). Note: R = aglycone (= genin).

are a number of bitter-tasting glycosides that re-duce the palatability of the plant (Table 4-1). Afew of these glycosides have been shown to beprotective to the plant;129'205 however, most haveonly been recognized as toxic to domestic animalsor humans. The broad range of compounds listedin Table 4-1 illustrates the diversity of chemicalstructures found even within the restricted field oftoxic and antinutritional glycosides and oligosac-charides. Because this diversity also means abroad range of different mechanisms of action, themost important compounds are described in thefollowing paragraphs.

Cyanogenic Glycosides

Cyanogenesis (cyano — Greek [kyanos =blue] and genesis — Greek [creation]) means theformation of cyanide/hydrogen cyanide, orHCN. Organisms that possess the ability to re-lease cyanide may be termed cyanogenic orcyanophoric (phoros — Greek [bearing]). If thecyanide is formed from the breakdown of an-other compound, this compound is called a cya-nogenic (cyanogenetic) compound, or simply, acyanogen. Cyanogens include cyanogenic gly-cosides, cyanogenic lipids, cyanohydrins, and

Table 4-1 Toxic, Antinutritional, and Bitter-Tasting Glycosides and Oligosaccharides

Compound group orcompound

Cyanogenic glycosides

Glycoalkaloids

Glycosides of organicnitriles

Glycosides and sugaresters of aliphaticnitrocompounds

Methylazoxymethanol(MAM) glycosides

Naringin

Oligosaccharides

Platyphylloside

Polyphenols

Ptaquiloside

Saponins

Vicine and convicine

Carboxyatractyloside(CAT) and relatedcompounds

Examples Toxicity, taste, etc.

O-Glycosides, Sugar Esters, and OligosaccharidesIn sources of food and feed

Linamarin in Manihot esculenta,Euphorbiaceae, in general wide-spread in the plant kingdom(Tracheophyta andSpermatophyta)52-129'182

Chachonin and solanin in Solanumtuberosum, Solanaceae(Angiospermae)140'206

Simmondsin in Simmondsia californica(Jojoba), Buxaceae(Angiospermae)2'277

Miserotoxin in Astragalus spp.,Fabaceae (= Leguminosae;Angiospermae)160'204

Cycasin in Cycasspp., Cycadaceae(Gymnospermae)22'154

In Citrus spp., espec. C. paradisi(grapefruit), Rutaceae(Angiospermae)227

In seeds of several legume spp.,Fabaceae ( = Leguminosae;Angiospermae)94'215

In Betula pendula, Betulaceae(Angiospermae)262

2-hydroxyarctiin in Carthamus tinctorius(Safflower), Asteraceae( = Compositae; Angiospermae)98'203

In Pteridium aquilinum, Polypodiaceae(Tracheophyta)249'250

Triterpene or steroid saponins inQuinoa spp., Borassus flabellifer,Glycyrrhlzae glabra and Balanitesspp. (Angiospermae)74'95'125

In Vicia faba (faba bean), Fabaceae( = Leguminosae; Angiospermae)276

In medicinal and toxic plants

CAT in Atractylis gummifera,Asteraceae (= Compositae;Angiospermae)49'187

Acute and chronic toxicity due torelease of HCN; neurotoxicityof intact glycosides dis-cussed; bitter taste

Corrosive to the gastrointestinaltract; upon absorption, acutelytoxic due to several mecha-nisms; bitter taste

Causes chronic toxicity ofunknown mechanism

Acutely toxic to ruminants;inhibit the TCA-cycle of thecells

Carcinogenic

Bitter taste

Flatulence-producing

Antinutritional (deterrrent) toseveral animal species

Cathartic (laxative); bitter taste

Acutely toxic and carcinogenic

Some atoxic, other mildly tostrongly toxic; several arebitter tasting

Acutely toxic to glucose-6-phosphate dehydrogenase-deficient individuals

Acutely toxic; inhibit mitochon-dria! oxidative phosphorylation

continues

Table 4-1 continued

Compound group orcompound

Cardeno- andbufodienolides

Cucurbitacins

Glycosides of Vitamin Ds

Ranunculin

Examples

"Digitalis" glycosides in Digitalis spp.(cardiac glycosides),Scrophulariaceae(Angiospermae)161'225

Cucurbitacin L in Citrullus colocynthis,Cucurbitaceae (Angiospermae),some cucurbitacins also present infood plants (ref. text below)103-126

Glycosides of 1oc,25-(OH)2D3 inSolanum glaucophyllum, Solanaceae(Angiospermae)279

In Ranunculus and Caratocephalusspp., Ranunculaceae(Angiospermae)180'195

Toxicity, taste, etc.

Acutely toxic to the heart

Intensely bitter substances,some of which are acutelytoxic

Chronic toxicity (vitamin Dintoxication — calcinoses)

Acutely toxic; irritant to mucousmembranes; Upon absorption,it affects several organs suchas the heart, the lungs, etc.

C-Glycosides (Some Also Occurring as O-Glycosides)In medicinal plants

Anthraquinone, an-throne, and dianthroneglycosides

Glucosinolates

Sennosides in Cassia angustifolia,Fabaceae (= Leguminosae;Angiospermae)152'280

S (Thio)-GlycosidesIn food and feed resources

In many species within the families ofCapparales (Angiospermae)23

Laxative effect; some com-pounds are drastica

Chronic toxicity due to releaseof thiocyanate and othercompounds; sharp (burning)taste

cyanogenic epoxides.31'182 Cyanogenesis has beendetected in prokaryotes, fungi, plants, and ani-mals. Cyanogens have been isolated from a greatnumber of organisms; the glycosides, however,have been isolated only from plants and insects.182

The release of cyanide from a cyanogen im-plies the degradation of the compound, a reac-tion that may be either spontaneous or enzymecatalyzed.52'182 Cyanogenic lipids and cyano-genic glycosides are broken down to cyanohy-drins (hydroxynitriles); these are cyanogens inthemselves (Figure 4-2). The cyanogenesisstarts on crushing of the tissue, the cyanogens,

and the degradative enzymes being compart-mentalized either at the subcellular level or attissue level in the intact plant.211

The glycosides are the most common cyano-gens, and comprise more than 60 structures.146'241

They were recognized early as substances that arepoisonous to animals.86 Cyanogens are of somesystematic importance at the level of higher planttaxa,182'265 and within certain families and gen-era 182,194,242 JJ16 ingestion of cyanide and cyano-genic compounds may lead to acute172 as well aschronic intoxications, the latter including the cen-tral nervous system (CNS) syndrome, konzo.269'270

2,3-Epoxynitrile Cyanogenic glycoside/lipid

Figure 4—2 The interrelationship between cyanogenic compounds and cyanide/hydrogen cyanide. In a cyano-genic glycoside, R1 is a saccharide moiety; in a cyanogenic lipid, an acyl moity. Hydrolases: glycosidase(s)—Refer also to Figure 4—3—or lipase. Note that the Cyanohydrin formed upon hydrolysis of one of the three typesof cyanogens (epoxynitriles, glycosides, or lipids) is a cyanogen itself.

Glycoalkaloids

Steroidal alkaloids and alkaloid glycosidesoccur throughout the genus Solanum (Solan-aceae). The common potato (S. tuberosum) con-tains in its edible tuber the two compounds a-chaconin and a-solanin.140 The total content mayvary from 10 mg/kg to 390 mg/kg, with a meanof 73 mg/kg.96 In other species of Solanum andclosely related genera, different glycosides andfree genins may dominate.92'206 Gastrointestinalabsorption of steroidal alkaloid glycosides var-ies between animal species. Some hydrolysis ofthe glycosidic bond and further metabolismseem to occur in different animal species, asjudged from analyses comparing the serum levelof oc-chaconin and/or a-solanin to that of totalalkaloids at different times after ingestion.140

The toxicity of the potato glycosides to humansincludes gastrointestinal upset with diarrhea,vomiting, and abdominal pain. In severe cases,neurological symptoms, some of which areclearly a result of the acetylcholinesterase in-

hibitor activity of these glycosides,216 are seen.140

Both a-chaconin and a-solanin, together withtheir aglycones, are teratogenic in one or moreanimal models.96 However, Kuiper-Goodman &Nawrot140 did not find the suggested associationof the consumption of blighted potatoes duringpregnancy with increasing incidences of spinabifida substantiated.

Methylazoxymethanol Glycosides

Glycosides of memylazoxymethanol (MAM)have been found only in cycads (Macrozamiaand Cycas spp). The concentration is high inseeds; smaller quantities are found in stems andleaves.154 Extensive losses of sheep have oc-curred in Australia due to consumption ofMacrozamia and Cycas spp.113 The first isola-tion of a MAM glycoside, macrozamin (the P-primeverosid of MAM), was from seeds of M.spiralis Miq., an Australian cycad. Today, otherMAM glycosides are known, among these cyca-sin (the (3-D-glucopyranosid of MAM), which

Cyanohydrin Cyanohydrin

Hydroxynitrilelyase or

Hydrogen Cyanide

Hydrolases Cyanide

was shown to be characteristic of, and exclusiveto, all the genera of cycads.154 The relative con-centrations of cycasin to macrozamin in ripeseeds differ within the cycad genera.177 The gly-cosides release MAM on the hydrolysis, whichis catalyzed by (3-glycosidases. MAM is a mu-tagenic and carcinogenic alkylating agent.163'178

Oligosaccharides

Flatulence is a common phenomenon that isassociated with the ingestion of legumes, amongothers, and caused by the microbial fermentationof low molecular weight sugars. Many of thesesugars are a-galactosides because humans donot have oc-galactosidase in their digestivetract.104 The legume oligosaccharides, raffmose,stachyose, and verbascose,215 are of particularinterest. Soybeans contain (by weight) approxi-mately 1% of raffmose and 2.5% of stachyose;215

the winged bean contains 1-2% of raffmose, 2-4% of stachyose, and 0.2-1% of verbascose.94

Ptaquiloside

Bracken fern(s) (Pteridium spp.) foundthroughout the world causes cancer of the uri-nary bladder in ruminants and is the only higherplant shown to cause cancer naturally in ani-mals.249 Enzootic hematuria, the clinical namegiven to the urinary bladder neoplasia of rumi-nants, tends to occur persistently in localizedbracken-infested regions. The major carcinogenof bracken is the mutagenic and clastogenicnorsesquiterpene glucoside, ptaquiloside, whichin laboratory animals has been shown to be carci-nogenic per a?.1 1U90'249 Bracken has further beenassociated with carcinoma of the upper digestivetract of cattle, where it is believed to transform thebovine papilloma (type 4) to a malign tumor. Afterhydrolysis of the glucoside, the genin is partlyconverted under alkaline conditions to adienone, which can then undergo further reac-tions to form adducts with DNA bases. A pre-liminary investigation of the alkylation patternsproduced has been presented.250 Bracken fern isacutely toxic to several farm animals such ashorses, cattle, and sheep, the syndromes being

different for the different animal species.82 Theadministration of pure ptaquiloside to a calf re-sulted in the same symptoms as known for thebracken intoxications of this species, thus dem-onstrating that the causative principle of cattlebracken poisoning is ptaquiloside.112

Saponins

A great number of food and feed plants con-tain saponins. Saponins may belong either to thegroup of pentacyclic triterpenoid saponins or tothe steroidal saponins. The latter include in abroad sense the steroidal alkaloid glycosides thatare found, for example, in potatoes. Althoughcertain saponins such as the medicinally usedquillaja saponin have been known for centuriesto damage mucous membranes,125 most saponinsare considered quite unproblematic when theyare administered orally. Saponin fractions fromcertain Yucca spp. have even been used as a feedadditive to promote growth of, for example, tur-keys.71 However, concerns have been raised re-cently that saponins in food or feed may promoteoral sensitisation to allergens through theirmembranolytic action in the gastrointestinaltract, resulting in enhanced uptake of the aller-gens.128 This concern is based on the fact that sa-ponins have been shown to act as oral adju-vants.59'132'158 Food plants that may containconsiderable amounts of saponins include theseeds ofQuinoa spp., fruits of Borassus flabellifer(palmyrah) and Balanites spp., and roots and sto-lons ofGlycyrrhizaeglabra (licorice) (Table 4-1).The palmyrah fruits are fermented to wine (palmwine) in Sri Lanka, whereas experimental solid-state tempeh fermentations have been describedfor quinoa. However, no information is availableconcerning the fate of the saponins in any of theseproducts.

Vicine and Convicine

Vicia faba (faba/fava bean), V. harbonensis,and V. sativa contain the two glycosides, vicineand convicine,219 which after hydrolysis in theintestine and uptake of the genins (divicine andisouramil) cause hemolytic anemia (favism) in

glucose-6-phosphate dehydrogenase-deficientindividuals.166'284 Together with condensedtannins, these two glycosides limit the use of theproteinaceous raw faba beans as feed for mono-gastric animals.167'276 Vicine and convicine havenot been detected in significant concentrationsin other plant species.

Cucurbitacins

Cucurbitacins were first characterized as thebitter compounds of cucumbers, marrows, andsquashes (Cucurbitaceae). The Cucurbitacins asa group are thought to be among the most bittersubstances known to man. Cucurbitacin B canbe detected in dilutions as low as 1 ppb, and theglycosides of cucurbitacin E at 10 ppb.175

Cucurbitacins make up a group of oxygenatedtetracyclic triterpenes, some of which occur asglycosides.103 Some Cucurbitacins are not onlybitter, but also toxic. Thus, the lethal dose for10% of a test group of mice (LD10 orally mice) ofcucurbitacin B is approximately 5 mg/kg b.w.103

This is quite strong toxicity, as seen from thefact that it is equal to the lowest dose used in theInternational Organization for Economic Coop-eration and Development Guidelines test foracute oral toxicity (Fixed Dose procedure,guideline no. 420).

Glucosinolates

In 1990, more than 100 glucosinolates werealready known.252 They occur in Capparales,Salvadorales, Violates, Euphorbiales, andTropaeolales within Violiflorae sensu Dahl-gren.57'58 Reasons for interest in glucosinolatesor glucosinolate-containing plants are the vari-ous antinutritional and toxic effects, the flavors,and the positive physiological effects associatedwith these constituents and their byproducts.23

Rape (Brassica napus, B. campestris, and B.juncea) is among the most important crop con-taining glucosinolates. Seeds of these speciescontain approximately 400 g of oil and approxi-mately 250 g of protein per kg. However, the useof rapeseed meals as a protein source in live-stock rations and human diets is limited because

of compounds associated with the protein frac-tions. These include phytic acid, phenolic com-pounds, and glucosinolates. Rapeseed that isbred to contain less than 2% erucic acid in its oiland less than 30 |ig/g of aliphatic glucosinolatesis termed "double low" or "canola." All pureglucosinolates tested in animal diets have causedantinutritional or toxic effects even when theywere in concentrations relevant to levels basedon double-low rapeseed as the protein source.243

RISKS ASSOCIATED WITH THEOCCURRENCE OF TOXICGLYCOSIDES IN DIFFERENTCOMMODITIES

Regarding toxins in food, the compounds thatcall for discussions in further detail are the cya-nogenic glycosides, but also the MAM glyco-sides, ptaquiloside, the saponins, the favismagents (vicine and convicine), and theglucosinolates.

Cyanogenic Glycosides

Although discussions concerning a toxicity ofintact cyanogenic glycosides may be found, theliterature at present concludes that known in-toxication syndromes, whether acute or chronic,are mainly due to HCN that is formed from thecompounds.235'236

A plant containing cyanogenic glycosidesmay or may not contain enzymes that catalyzetheir breakdown (i.e., hydrolases [(3-glycosi-dases] and cyanohydrin lyases). These are storedseparately from the glycosides.211 When a tissuecontaining both cyanogenic glycosides andthese enzymes is crushed, enzyme(s) andsubstrate(s) are brought together and hydrolysisand further lysis (i.e., cyanogenesis) starts. Thus,the intake of raw or processed cyanogenic mate-rial normally will mean an intake of a mixture ofthe genuine glycoside(s) and accompanying hy-drolysis products. Tissues that only contain theglycosides (and not the enzymes) will only giverise to exposure to the genuine glycoside(s).

Although cyanogenic glycosides may un-dergo acid hydrolysis,30'83 the conditions in the

stomach of a nonruminant, together with thevery short residence time, will let the main frac-tion pass to the intestine. In the intestine, the gly-cosides will be absorbed, as shown for linamarinin a number of animal species and in hu-mans,19'37'110'208 and for prunasin and amygdalinin different animal species,44'220'221'235 or it will behydrolyzed by microorganisms.28'44'210 In hu-mans, Carlson et a/.43 very recently found thatapproximately 25% of linamarin ingested in astiff porridge prepared from cassava flour wasabsorbed and excreted unchanged in the urine,whereas a little less than 50% was converted tocyanohydrin or cyanide and absorbed as such.The rest could not be accounted for. Most or allof the absorbed glycosides will be excreted inthe urine, as shown for both linamarin andamygdalin in animals and humans.7'37'110'173 HCNas well as cyanohydrins will give rise to cyanideexposure through absorption and nonenzymaticlyses of the cyanohydrins.

Whereas the absorbed glycosides will be ex-creted unchanged in the urine, the HCN will betotally or partly metabolized, the main metabo-lite being the goitrogenic compound, thiocyan-ate. The rate of this conversion will depend onthe nutritional status of the individual. Currentknowledge concerning the known biomarkersfor cyanide exposure (acute and long term) andtheir use in clinical and experimental toxicologywas reviewed by Rosling.231 The detoxificationprocesses (metabolization) and methods for theestimation of the sulphane sulphur pools availablefor this were reviewed by Westley.281 Acute hu-man intoxications have been described as a resultof the intake of cassava products and almonds,whereas sorghum and cyanogenic acacia leavesand pods have caused veterinary intoxications.

Acute Intoxication

Acute intoxications in humans caused by theintake of insufficiently processed cassava mealshave been reported from nearly all parts of the cas-sava consuming area, although it must be empha-sized that the published reports are very scarce inrelation to the extensive use of cassava as humanfood.5'76'78'172 The symptoms of acute intoxication

include vomiting, nausea, headache, dizziness,difficulty with vision, and collapse. 172

Chronic Intoxication Syndromes

Evidence has accumulated that cyanide expo-sure from the diet is a causative factor inkonzo,115'269 and may aggravate iodine defi-ciency disorders.60 The influence, if any, on thedevelopment of special types of diabetes re-mains a matter of discussion.3'263 Symptoms anddiagnosis of konzo have been described byRosling & Tylleskar.232

Based on the knowledge available concerningthe toxicity of cyanide and cyanogenic glyco-sides, the Joint World Health Organization(WHO)/Food and Agricultural Organization(FAO) Expert Commitee on Food Additives andContaminants (JECFA) tried to estimate a safelevel for the intake of cyanogenic glycosides byhumans. The committee concluded that "be-cause of lack of quantitative toxicological andepidemiological information, a safe level of in-take of cyanogenic glycosides could not be esti-mated." However, the committee also concludedthat "a level of up to 10 mg HCN/kg of productis not associated with acute toxicity."253(P332)

Thus, no authority has yet felt confident to setscientifically based safe levels for the intake ofone or more of the known cyanogenic glycosides(or their products of degradation), that is, levelsthat take the risk(s) for the development ofchronic intoxications into consideration. Inacknowledgement of this, the "InternationalWorkshop on Cassava Safety," held in Ibadan,Nigeria in 1994, concentrated on making recom-mendations concerning steps to be taken in re-search; in breeding programs; and in informa-tion to extension workers in the agricultural,food, and nutrition sectors.10

Long before humans knew the identity of cya-nide, they did know that bitter cassava is a goodstarch crop, but that it must be detoxified beforeconsumption.67'69 Today, we know that this isbecause of its content of the cyanogenic gluco-sides, linamarin and lotaustralin. Overviews ofthe occurrence of cyanogenic glycosides inplants used for human or animal consumptionare provided in Conn51 and Jones.129 Some of the

important species of plants have been subjectedto selection/breeding for a low total cyanogenicpotential (TCP). Examples of the constituentsand the TCP of economically important cropsare provided in the following sections, togetherwith some remarks concerning their importanceas food or feed commodities.

Phaseolus lunatus (Seeds) and Other Beans.Seeds from several species of legumes are usedfor human consumption, many of which containtoxic and antinutritional substances. Thus, seedsfrom, for example, P. lunatus, P. aureus,Cajanus cajan, Canavalia gladiata, and Vignaunguiculata have been examined due to con-cerns about the possibility for cyanide intoxica-tions.63'192 All of these species are known to becyanogenic in one or more tissues.242 P. lunatuscontains linamarin as its main cyanogenic con-stituent; the cyanogens have not been identifiedin the other species.242 Only P. lunatus has beensubjected to investigations concerning the varia-tion in the cyanogenic potential.21 However, sev-eral of the other species certainly may containtoxic amounts of cyanogens in the seeds.192

Prunus Species (Seeds). Peach, plum, cherry,apricot, and almond (family Amygdalaceaesensu Dahlgren ) are all drupes (stone fruits) ofgreat importance to man. Cyanogenic glycosidestypical for Amygdalaceae are phenylalanine de-rived.182 Thus, the ripe seeds of P. persica(peach), P. domestica (plum), P. avium/cerasus(cherry), P. dulcis (P. amygdalus) (almond), andP. armeniaca (apricot) all contain amygdalin asthe major cyanogenic constituent. The total cya-nogenic potential per gram dry weight of wholefruit rises during the early development, and therelative composition of cyanogens changes from100% prunasin in the beginning to nearly 100%of amygdalin in the ripe seed.90'170'189 Amygdalinand different Prunus seeds have, in spite of theirineffectivity, been commercially promoted foryears as medicines to treat different cancers.108

• Almond — This tree is very widely culti-vated around the Mediterranean. The nam-ing of the species and its varieties/cultivars

has changed through time.99 The tree comesin two varieties, var. dulcis and var. amara,of which var. amara contains high concen-trations of amygdalin in its ripe seeds (alsodenoted "bitter almonds").39'50'99 The seedsare used in confectionary and bakery.99

They contain approximately 50% of lipids,the oil being used in cosmetics and derma-tology.39'99 Bitter almonds (but also, e.g.,apricot seeds) are also used to produce anessential (volatile) oil called "oil of al-monds." This competes with synthetic ben-zaldehyde as a source of flavor.39 Only fewreferences exist concerning the content ofcyanogenic glycosides in almonds.50'90

Conn50 found bitter almond seeds to release290 mg of HCN/100 g of seed. Accordingto Sturm,260 commercial sweet almondsfrom California in general contain lessbitter seeds (approximately 1%) than the2-3% that is normally found in the Medi-terranean ones.

• Apricot — Apricots have considerable eco-nomic importance for several countriessuch as Italy, the production of which wasapproximately 200,000 tons in 1988.259

Different products are marketed from apri-cots, including fresh, dried, and cannedfruits; nectar; jam; and distilled liqueur.176

The number of varieties and hybrids ofapricots are numerous.174 Thus, Audergonet al. 14 tested more than 400 varieties aspart of a physicochemical characterizationprogram. Several marketed products ofapricots require destoning,55 leaving thestones as a byproduct from which oil can beextracted. The use of the seed/presscake is,however, restricted by the toxicity.266 De-pending on the variety and type of apricot,the apricot stone is relatively small, repre-senting 6-8% of the fruit weight, even if itcan sometimes reach 10%.176 To the bestknowledge of the present author, no inves-tigations have been published concerningthe variation in content of amygdalin inseeds of different cultivars. However, aspart of investigations concerning the mi-crobial degradation of cyanogens in such

seeds, Tuncel et al. 266'267 analyzed twobatches of bitter and one of sweet Turkishapricot seeds, obtained on the commercialmarket. The bitter ones were found to con-tain approximately 52 and 92 |imol/g d.w.,respectively; the sweet ones contained ap-proximately 2.5 |imol/g.

• Peach — Much of the same that has beensaid for apricot can be said for peach. Seedsfrom P. persica Batsch (peach) also containamygdalin as their major cyanogenic con-stituent.193 Kupchella & Syty141 analyzedthe total cyanogenic potential of the seedsfrom an undefined cultivar and found it tocorrespond to a content of amygdalin of ap-proximately 2.45% w/w.

Linum usitatissimum (Seeds = Linseed/Flax-seed). Flax is grown for two main purposes, fi-bers and seeds. Different cultivar s are used forthe two products. Whole seeds are used as alaxative due to the swelling seed coat polysac-charides.200 Both full-fat flaxseed flour and de-fatted meal from the oil extraction are on thecommercial market, the latter in two qualities,with 30% and 40% protein, respectively.198 Flaxis one of the major industrial oilseeds traded inworld markets. Global production for crop year1994-1995 was 2.44 million metric tons, withCanada contributing a major share. Flaxseed oilis used for a multitude of purposes, the oil beingpriced up to four times that of the whole seed.198

The extraction cake (linseed meal) is tradition-ally used for fodder purposes. Recently, researchinto the refinement of flax products hasaccelarated. Thus, two patents have been issuedfor the use of flaxseed polysaccharide (gum) forcosmetic and medical preparations,13'196 and anoptimization of protein extraction from defattedflaxseed meal has been presented.199

Until 1980, linamarin was thought to be themain cyanogen in linseed. However, looking forthe factor(s) in linseed meal responsible for itsprotective effect against selenium toxicity,Smith et a/.251 isolated two new cyanogenic gly-cosides (linustatin and neolinustatin). A laterTLC-based investigation concerning the con-centrations of different cyanogenic glycosides in

a linseed sample gave the following |umol/g:linustatin+neolinustatin 4.6, linamarin 0.46, andlotaustralin 0.36,32 pointing to linustatin andneolinustatin as the major cyanogenic constitu-ents. This was further confirmed by a high per-formance liquid chromatography (HPLC) analy-sis of 48 samples, which on the other hand onlyfound traces of linamarin and lotaustralin.240

However, a recent investigation showed quitesome variation between 10 cultivars. Two con-tained no linamarin, whereas in the cultivarVimy, 7.8% of the weight of the total cyano-genic glycosides were linamarin.201 This is closeto the findings of Brimer et al}2 for an unspeci-fied sample. Frehner et a!.90 analyzed both thecyanogenic potential and the relative cyanogencomposition during fruit development — one cul-tivar. As in Prunus seeds, the monoglucosidespredominated at anthesis, shifting towarddiglycosides during maturation. Rosling230

found the cyanogenic potential of a nonspecifiednumber of commercially sold linseed in Swedento range from 4 mmol/kg to 12 mmol/kg (112-336 mg kg-1 HCN). The acute lethal dose is lessthan 2 mmol in 24 hours in sick and malnour-ished patients.270

Manihot esculenta (Roots and Leaves). Thegenus Manihot (Euphorbiaceae) incorporatesmore than 200 species, all originating in tropicalAmerica, from where several have been spreadto other continents. Thus, M. esculenta Crantz(cassava) is today grown as a major source ofstarch in tropical Africa, India, Indochina, Indo-nesia, and Polynesia.184 As early as 1605,Clusius reported that cassava could be toxic toman. The two cyanogenic glucosides, linamarinand lotaustralin, are responsible for this.70'183 Thecyanogenic potential (CNp) of several cassavagermplasm collections has been investigated.Thus, Aalbersberg & Limalevu1 analyzed 28cultivars grown in Fiji, and found a variationfrom approximately 15 mg to 120 mg HCNequivalents/kg f.w. Dufour67'69 looked at 14 cul-tivars of the Tukanoan Indians in northwestAmazonia and found very high levels (310-561mg HCN eq./kg f.w.) in KU (toxic cultivars) and171 mg HCN eq./kg f.w. in the only Makasera

(nontoxic/safe cultivar) grown. The TukanoanIndians clearly expressed that they preferredtoxic varieties as the main staple (70% of calorieintake) component of their diet. In this connec-tion, it should be noted that the so-called "safe"(Makaserd) cultivar had a higher CNp than the100 ppm (f.w.) that was proposed as the upperlimit by Koch67'69 based on acute toxicity. Fi-nally, Bokanga24 examined 1,768 different cas-sava collections and found that the content of thecentral pith of the root varied from approxi-mately 1 mg to more than 530 mg HCN equiva-lent/ kg d.w. The peel surrounding the pith has amuch greater content, as have the leaves.24 Noacyanogenic cassava was found. While discuss-ing the cyanogenic potential in this precise way, itshould be born in mind that variations of up to100% may be recorded between roots of the sameplant.24 It has also been shown that age, agricul-tural practices,73 and environment may have astrong influence on its cyanogenic potential.24'26

The leaves of M esculenta also serve as foodand feed.25 The cyanogenic potential of leavesfrom the same plant is less variable than that ofthe roots,24 and is usually 5 to 20 times higher ona fresh weight basis.25 The high content in theleaves normally does not present a problem fortheir use in food, given the methods generallyused in their preparation.25 In contrast, the rootsof many cultivars, if not properly processed,have actually caused both acute and chronic in-toxications worldwide. However, it should beemphasized again that the cassava root (evenhighly cyanogenic types) is a very valuable andirreplaceable crop. To ensure its safe use in ev-ery community under all conditions, the effec-tiveness of the different processing techniques(under rural as well as industrialized conditions)needs to be verified and the knowledge spread.10

Sorghum Species (Leaves and Seeds). Seed-lings of S. bicolor (Poaceae) and other Sorghumspecies synthesize the cyanogenic glucosidedhurrin that is localized in the ariel shoots of theplant.101 Thus, three-day-old etiolated seedlingsof S. vulgare (i.e., the name for any cultivatedgrain sorghum) was found to contain up to 15(imoles/g.4 The content in mature leaves is much

lower. The concentration depends on species,subspecies, and race/cultivar, and is also influ-enced by ecological factors.65 Although most in-toxications are seen in cattle browsing a newlysprouted field, forage may not be totallysafe.65'282 Grain sorghums constitute an impor-tant part of human nutrition in several semi-aridareas of the world.61'88 Generally, the grains areconsidered completely safe for human consump-tion,88'136 although the digestibility and biologi-cal value are not always high as a result of theoccurrence of quite high concentrations ofphytate and polyphenolics in several cultivatedtypes.88'120 Especially in Sudan, sorghum is irre-placeable, being the traditional stable food.64 Al-though sorghum seeds in general are safe, ger-minated seeds are not. In certain Africancountries, germinated sorghum seeds are usedtraditionally for the production of malt,88 whichin turn is used for the brewing of alcoholic bev-erages64 and for the production of the bakedproducts called Hulu-mur.64 According toFAO,88 the traditional methods of preparation ofthese products remove the dhurrin effectively;however, it is stressed that the existence of theseproducts must not be seen as an indication ofsprouted sorghums being safe — they are not.88

MAM Glycosides

A metabolic fate and mechanism of toxicity,including the same alkylating end product aswith dimethylnitrosamine, has been proposedfor the MAM that is released from the MAMglycosides.209 Thus, cycasin has been shown tobe toxic to a number of animals, causing hepaticlesions and demyelination with axonal swellingin the spinal cord.22' 246 Cow's milk may be a vec-tor of transmission of plant toxins. Thus,Mickelsen et a/.168 showed that MAM can passinto the milk of lactating rats, causing tumors inthe offspring. The seeds of several Cycas spp.are traditionally eaten in Australia22 and on cer-tain islands.150'254 A special neurological syn-drome occurring on the island of Guam, andtermed Guam ALS-PDC, has been hypothesizedto be due to the intake of seeds of C.circinalis.150'254 In 1987, Spencer et at.254 pro-

posed that the causative factor of this syndromewas the neuroexcitotoxic amino acid p-N-methylamino-L-alanine (BMAA). However, anumber of subsequent investigations doubtedthis, as reviewed by Stone258 in an article on thegradual disappearance of this disease. Thus, itmay never be known whether the MAM glyco-sides could have a role in this disease, though itremains a possibility given the spinal cord le-sions reported in goats as a result of chronic in-take of cycasin.246

Ptaquiloside

The carcinogenicity of ptaquiloside demon-strated in feeding experiments with rats, mice,hamsters, guinea pigs, and cattle, among others,is alarming because the young shoots of brackenfern are highly regarded as a tasty dish in Ja-pan.111 Hence, this intake of bracken has beenlinked to high incidences of stomach cancer inJapan,111 and in Costa Rica among people whohave been exposed to milk that was produced inbracken-infested grasslands.6 The theory hasbeen supported by the finding of a high tumorincidence in rats and mice that were fed milkfrom cows that had been fed with dietarycomplements of bracken, and by the subsequentdemonstration of ptaquiloside in bovine milk.6

Saponins

Food and feed containing saponins includesoybean, guar, quinoa, balanites fruits, and oth-ers. Besides the membranolytic action of manysaponins, certain of these compounds exert spe-cial effects due to the structure of their agly-cone.286 Such effects include (1) lowering ofblood cholesterol;144 (2) reversible sodium reten-tion and potassium loss leading to hypertension,water retention, and electrolyte imbalance (e.g.,glycyrrhizinic acid found in licorice root, theroots and stolons from Glycyrrhiza glabra, andfor products to which licorice root extract, orglycyrrhizinic acid, has been added);100'133'239'256

and (3) crystal formation in the liver and biliarysystem, which may inhibit the excretion ofphylloerythrin (from chlorophyll degradation),

causing a subsequent photosensitation as seen in"Geeldikkop" (a Tribulus terristris intoxica-tion).131 A number of saponins are bitter. Theoccurrence of bitter saponins in palmyrah(Borassus flabellifer L) fruit pulp thus reducesthe use of juices based on this fruit.74 Likewise,seeds of Chenopodium spp. used for human con-sumption (C. quinoa [quinoa], C. pallidicaule[canihua], and C. berlandieri ssp. nuttaliae[Safford] Wilson and Heiser [huauzontle]) containbitter saponins,95'107'226 most of which are concen-trated in the outer layers of the grain.40'41'223

Vicine and Convicine

Favism is characterized by anemia, jaundice,and hemoglobinuria, and may develop in sub-jects with glucose-6-phosphate dehydrogenase(G6PD) deficiency as a consequence of fababean intake. Favism has also been reported inbreast-fed infants whose mothers had eaten fababeans, and in newborn infants.54 More than 300variants of G6PD are known.273 In addition, anassociation between the genotype OfACP1 (hu-man red cell acid phosphatase) and favism hasbeen shown, and a possible biochemical mecha-nism has been proposed.27 Most cases of G6PDdeficiency described in the past were from Italyand other countries around the Mediterranean,that is, patients with the common MediterraneanB-form of G6PD, rather than the common AfricanA (-) form.273 However, recent investigations haveshown that subjects with variants that result in arelatively mild G6PD deficiency may also developfavism.93'181 Preventive measures and treatmentshave been described elsewhere.102'162'188

Glucosinolates

The most prominent toxic manifestation ofglucosinolates in humans is the occurrence ofgoiter.214 In animal experiments, this and othereffects were generally more pronounced whenmyrosinases were included in the diet.252 The ef-fects seen were related to differences in the sidechains and to chirality.252 The fact that there areseveral mechanisms behind the toxic andantinutritional effects has also been very re-

cently stressed by the results of the most detailedstudies on the degradation products of variousglucosinolates.23 These authors presented anoverview of the different degradation productsformed from glucosinolates, which also include,for example, oligomers. From the degradation ofglucobrassicin (an indole glucosinolate),indolyl-3-methanol is formed in considerableamounts, but it disappears very quickly, givingrise to, among others, appreciable amounts ofthiocyanate ion. No organic isothiocyanates andthiocyanates are formed. In contrast, the degra-dation of various aliphatic glucosinolates resultsin the formation of nitriles as well asisothiocyanates and thiocyanates.23 Toxic effectsof glucosinolates in B. oleracea have been re-viewed by Stoewsand257 and those of crambe(Crambe abyssinicd) meal fed to broiler chicksby Kloss et al. 137 The mechanism behind the ob-served decrease in cancer risk for people on dietswith a high content of cruciferous vegetables hasbeen investigated by Wallig et a!.214

VARIATION IN TOXINCONCENTRATION AMONGVARIETIES AND CULTIVARS: THEINFLUENCE OF TRADITIONALDOMESTICATION AND MODERNBREEDING

Several toxic glycosides (including varioussaponins and cyanogenic glycosides, etc.) areknown to be bitter tasting in addition to toxic.Hence, the term "bitter," as opposed to "sweet,"has been used traditionally to designate naturallyoccurring or selected groups within a plant spe-cies that contain high amounts of the toxic (andbitter) substance. Depending on the view of thebotanical author, the groups in question may bedivided on the level of variety, form, or cultivar.Examples of plant species for which the divisionbitter/sweet has been used are P. dulcis and otherPrunus spp. (containing amygdalin), as well asM. esculenta (cassava, containing linamarin),and in quinoa.139 In most such cases, a correla-tion between the toxicity (content of glycoside)and the degree of bitterness of the plant part hasbeen established. However, it is only seldom that

a proper investigation concerning the degree towhich this correlation holds has been performed.Thus, a positive correlation, but with exceptions,was found in a number of smaller studies on cas-sava roots.247 Hence, King & Bradbury135 tookup the challenge of investigating in more detailthe bitter-tasting substances in cassava paren-chyma and cortex. Linamarin was found to bethe sole contributer to bitterness present in theparenchyma; a new structure (isopropyl-p-D-apiofuranosyl-( 1— 6)-(3-D-glucopyranoside) con-tributing in the cortex of some cultivars. This isin agreement with a very recent study fromMalawi, 234 which compared the content of cya-nogenic glucosides in the cortex of 492 cassavaroots with their taste as estimated by a tastepanel. The correlation had an r2 = 0.96 whenlooking at the cultivar level.

It is well documented, at least for a number ofcyanogenic plant species, that the concentrationof both the glycosides and the enzymes degrad-ing them can show a discrete variation (poly-morphism) as well as a continous one. The poly-morphism is genetically based, whereas thecontinous variations observed may be both ge-netically and environmentally influenced.24'26'116-119'130'159'186The genetic polymorphism (discretevariation, chemical races) with respect to the oc-currence of both cyanogenic constituents andhydrolytic enzymes makes it difficult to definewhat is meant by a "cyanogenic species." Fur-thermore, it should be noted that the cyanohy-drin lyase, which cleaves the cyanohydrinsformed after the hydrolysis of the glycoside(s),may be expressed in certain organs and not inothers. Thus, White et a/.283 recently showed thatthis enzyme, although present in the leaves, isnot expressed in the roots of cassava. This obser-vation explains why very high intermediate con-centrations of cyanohydrins are formed during theprocessing of cassava roots. The environmentalinfluences mentioned above may furthermoremean that certain plants will be found positive atsome times of the year and negative at others.

Increased use of more highly cyanogenic cul-tivars of cassava among small farmers has beenreported from several places. Thus, Dufour re-ported on a clear preference for Kii (toxic variet-

ies) for most purposes by the Tukanoan Indi-ans,67'69 whereas Aalbersberg & Limalevu1

stated that planting of the toxic (bitter) cultivarsincreased in New Guinea. Also, Onabolu et al 197

found that the three most commonly grown cul-tivars in Ososa (a semi-urban farming commu-nity approximately 80 km east of Lagos, Nige-ria), where cassava has been the main staple fordecades, were all stated to be poisonous and toneed processing. However, this was not re-garded as a disadvantage. Farmers' reasons forpreferentially growing cassava cultivars provid-ing bitter roots were studied in Malawi.48 Inmany traditional agricultural communities, thefarmers (often the women) judge the "safeness"of the roots by chewing a small piece. Accordingto Dufour, 67'69 the Tukanoan Indians appear to beable to distinguish accurately less from more poi-sonous cultivars by the taste. Very recent studiesfrom Malawi prove such a procedure to exist, andto be very effective (Chiwona-Karltun, personalcommunication, October 2000).

Several of the species within the familyCucurbitaceae, which are used as human food,naturally contain cucurbitacins in amounts thatare unacceptable to the market. However, in-tense domestication and breeding have resultedin cultivars low in bitter compounds.126'127

Breeding programs for curcurbits are constantlyaware of the bitterness.84

Great variations (0-13000 M£/g) may also befound in the content of ptaquiloside in brackenfern as a result of both ecological and geneticvariation, a tendency for higher contents beingreported when originating in relatively colderclimates.249 In addition, P. esculentum containsthe cyanogenic glucoside, prunasin, the concen-tration of which similarly has been related to cli-matic conditions.153

Also, quinoa cultivars vary concerning thequantitative content of saponins, and the tradi-tion has, as for other crops, been working withso-called sweet and bitter varieties.139

For V. faba, it should be mentioned that, al-though Due et al 66 gave the first report of a genethat codes for nearly a zero content of vicine andconvicine, present-day cultivars contain ap-proximately 7 and 2.5 mg g-1 respectively.276

REMOVAL OF TOXINS THROUGHPROCESSING

Traditional Household versus ModernIndustrial Processing

When discussing the removal of toxic andantinutritional constituents, one must distin-guish between traditional household processingand industrial processing. The two proceduresmay use different starting materials and willhave different means of analyzing these and dif-ferent methods available for processing. The pri-orities may indeed be very different when choos-ing between slow versus fast processingmethods, and between processes that require lowinput of water and/or energy as compared tomethods requiring a high input. The generaltrend of a greater number of traditional food fer-mentation processes being industrialized hasbeen discussed recently by Rombouts & Nout.228

A number of industrial processing methods orlaboratory methods meant for industrial devel-opment were investigated quite early on for soy-bean (e.g., removal of oligosaccharides and pro-teinase inhibitors)47'56'91'109'224'245'248'261'264 and forcruciferous plants (glucosinolates).20'72'85'151'255

Later, linseed (flax, cyanogenic glyco-sides),149'156'157'275 jojoba meal (organic nitrileglycosides),2 citrus juices (limonoids andnaringin),105'145'212'229'244 cotton seeds (gossy-pol),179 and quinoa seeds (saponins)95'226 werefocused on. For commodities such as cassavaroots and leaves, lima beans, cycas seeds,bracken leaves, and yam tubers (alkaloids aswell as terpenes), most methods described andinvestigated scientifically are actually house-hold processing methods. A look at the process-ing of cassava roots will help illustrate the im-portant characteristics for each of the twosectors.

Household Processing (Cassava Roots)

Both in South America and in sub-SaharanAfrica, sweet and bitter roots are generally re-garded as two well-known different crops, andmost traditional methods of processing that havebeen studied have proven very effective in re-

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