STEROIDAL GLYCOALKALOIDS IN SOLANUM SPECIES: CONSEQUENCES FOR POTATO BREEDING AND FOOD SAFETY
CENTRALE LANDBOUWCATALOQUS
0000 0352 3277 Promotoren: dr. J.J.C. Scheffer bijzonder hoogleraar in de kruidkunde dr. J.H. Koeman hoogleraar in de toxicologie 0ù&K>\\ZO\
W.M.J. VAN GELDER
STEROIDAL GLYCOALKALOIDS IN SOLANUM SPECIES: CONSEQUENCES FOR POTATO BREEDING AND FOOD SAFETY
Proefschrift ter verkrijging van de graad van doctor in de landbouwwetenschappen, op gezag van de rector magnificus, dr. H.C. van der Plas, in het openbaar te verdedigen op woensdag 11 oktober 1989 BIBLIOTHEEK des namiddags te vier uur in de aula VVUNIVERSITEI T van de Landbouwuniversiteit te Wageningen r '^* tttJoJlO' , f301
STELLINGEN
1.D eveelvuldi g gehanteerde limietva n 200m g steroidalkaloidglycosiden per kg verse ongeschilde aardappel, als criterium voor consumenten- veiligheid, berust noch op toxicologische studies noch op kennis over het voorkomen van steroidalkaloidglycosiden inwild e Solanum-soorten en dient derhalve als arbitrair teworde nbeschouwd . Dit proefschrift.
2. Voor de analyse van steroidalkaloidglycosiden in Solanum-soorten is capillaire gaschromatografie in combinatie met simultane vlamionisatie- detectie (FID) en specifieke-stikstofdetectie (NPD) minimaal noodzake lijk;bi j voorkeur dientmassaspectrometri e teworde n toegepast. Ditproefschrift .
3.He tvermoge nva n aardappelen tothe t accumulerenva n steroidalkaloid glycosiden onder praktijkcondities van teelt, bewaring en verwerking, dient een van de belangrijkste criteria te zijn bij het beoordelen van nieuwe rassen ophu n geschiktheidvoo r consumptie. Ditproefschrift .
4. Op grond van de huidige inzichten in de chemie enhe t voorkomen van steroidalkaloidglycosiden in Solanum-soorten kan geconcludeerd worden dat veel fytochemische en toxicologische studies tot misleidende onderzoekresultaten kunnenhebbe n geleid. Bushway RJ. (1983)A m PotatoJ 60:793-797 . Renwick, JH, Ciaringbold WDB, Earthy ME, Few JD, McLean ACS. (1984) Teratology 30:371-381. Ditproefschrift.
5. De conclusie van Osman et al. dat inmicrosomal e fracties uit blad materiaal van Solanum chacoense een isomeer van hydroxysolanidine gevormd zou zijn uit 14C-solanidine, is niet in overeenstemming met de gepresenteerde resultatenva nd emassaspectrometrisch e analyse. Osman S, Sinden SL, Deahl K, Moreau R. (1987) Phytochemistry 26:3163-3167. 6. Voor het bestuderen van de genetische variatie voor hoogmoleculaire gluteninesubunits in Triticum aestivum dienen naast natriumdodecyl- sulfaat-polyacrylamidegelelectroforese tevens andere techniekenvoo r het analyserenva n eengenotyp e teworde n toegepast. Kolster P,Krechtin gCF ,Va nGelde rWMJ . (1988)Euphytic a 39S:141-145.
7. Voor het verbeteren van de kwaliteit van tarwe als grondstofvoo r de broodbereiding dient te worden gestreefd naar eenvoudige methoden voor het selecteren van nieuwe tarwelljnen op de kwantitatieve samenstelling van dehoogmoleculair e gluteninesubunits.
8.He t introducerenva n genenva nwild e plantesoorten invoedselgewasse n dient alleen teworde n toegestaan, indienvoldoend e wetenschappelijke en experimentele ondersteuning door fytochemici en/of toxicologen is gegarandeerd.
9. Omdat tomaten tegenwoordig vaak in het "licht-kleurstadium" worden geoogst, is biochemisch onderzoek naar de degradatie van a-tomatine gedurende de doorrijping gewenst; dit geldt in het bijzonder wanneer genetisch materiaal van wilde Lvcopersicon-soorten in de cultuurtomaat wordt geïntroduceerd. VanGelde rWMJ , De PontiOMB . (1987)Euphytic a36:555-561 .
10. De gebruiksvriendelijkheid van electronisch tekstverwerken leidt niet tot een vermindering maar veeleer tot een toename van het papierverbruik en vergroot daardoor de perspectieven voor hennep als grondstofvoo r de papierindustrie.
11. Degenen die "natuurlijk" synoniem achten met "gezond", dienen te beseffen dat "natuurlijke toxinen" eenuitzonderin g op die regel zijn.
12. Genetische manipulatie: van een mug een olifant maken is gemakke lijker danhe t omgekeerde.
W.M.J. vanGelder . Proefschrift: Steroidal glycoalkaloids in Solanum species: consequences forpotat obreedin g and food safety. Wageningen, 11oktobe r 1989. CONTENTS
VOORWOORD/ACKNOWLEDGEMENTS 6
GENERAL INTRODUCTION 9
CHAPTER I OCCURRENCE OF STEROIDAL GLYCOALKALOIDS AND THEIR DISTRIBUTION AND ACCUMULATION IN THE POTATO TUBER 13
CHAPTER II EVALUATION OF THE TOXICITY OF STEROIDAL GLYCOALKALOIDS 35
CHAPTER III TWO-PHASE HYDROLYSIS OF STEROIDAL GLYCOALKALOIDS FROM SOLANUM SPECIES 69
CHAPTER IV CAPILLARY GAS CHROMATOGRAPHY OF STEROIDAL ALKALOIDS 85
CHAPTER V CAPILLARY GAS CHROMATOGRAPHY OF STEROIDAL ALKALOIDS: RETENTION INDICES AND SIMULTANEOUS FLAME IONIZATION/NITROGEN-SPECIFIC DETECTION 105
CHAPTER VI ANALYSIS OF STEROIDAL GLYCOALKALOIDS IN POTATO GENOTYPES CONTAINING GERMPLASM FROM WILD SOLANUM SPECIES 119
CHAPTER VII GLYCOSIDIC-BOUND STEROIDAL ALKALOIDS IN TUBERS AND LEAVES OF SOLANUM SPECIES USED IN POTATO BREEDING 135
CHAPTER VIII CHARACTERIZATION OF NOVEL STEROIDAL ALKALOIDS FROM TUBERS OF SOLANUM SPECIES BY COMBINED GAS CHROMATOGRAPHY AND MASS SPECTROMETRY 151
CHAPTER IX TRANSMISSION OF STEROIDAL GLYCOALKALOIDS FROM SOLANUM VERNEI TO THE CULTIVATED POTATO (5. TUBEROSUM) 163
SUMMARY AND CONCLUDING REMARKS 173
SAMENVATTING EN SLOTOPMERKINGEN 179
CURRICULUM VITAE 184 VOORWOORD/ACKNOWLEDGEMENTS
Iedereen die heeft bijgedragen aan het totstandkomen van dit proefschrift wil ik bedanken, temeer omdat de wijze van samenwerking en de kontakten altijd uiterst prettig waren. Allereerst dank ik mijn promotoren, Hans Scheffer en Jan Koeman, voor hun inspirerende begeleiding en daarnaast Albert Eenink die als directeur van de Stichting voor Planten veredeling SVP een bijzonder stimulerende invloed heeft gehad. Voorts dank ik de co auteurs Henk Huizing, Lidwine Dellaert, Henk Vinke, Louis Tuinstra en Jan van der Greef, voor hun waardevolle bijdragen. Dank ook aan Wim Traag, Paul Kienhuis, de heer L.G. Gramberg en Drs. CA. Landheer voor hun bijdragen op het gebied van massaspectrometrie, aan Ben Jansen voor zijn hulp bij het hydrogeneren en aan Drs. P. Plieger en Ir. A.L.M. Sanders voor het beschikbaar stellen van standaardalkaloïden. Thanks are due to Prof. Dr. Rodney Bushway, Dr. William Gaffield, Dr. Richard Keeler, Prof. Dr. D.N. Kirk, Dr. Stanley Osman and Dr. Steve Sinden for providing standard alkaloids. Verder dank ik Saskia van Erkelens, Gea Jonker, Marion Janssen, Marleen de Jongh de Leeuw en Pieter de Smidt voor hun hulp o.a. bij het tekstverwerken en het verzamelen van literatuur. Tenslotte dank ik vooral Harry Jonker en Kees Krechting voor hun vakbekwame assistentie en de bijzonder prettige werksfeer.
Het totstandkomen van dit proefschrift is gesubsidieerd door het Directoraat Generaal voor de Landbouw van de Europese Gemeenschap en door de Stichting 'Fonds Landbouw Export Bureau 1916/1918' (LEB-Fonds). AAN MARIE-CHARLOTTE, JEROEN EN PHILIP GENERALINTRODUCTIO N
The potato (Solanum tuberosum L.) which isgrow n in 80%o f allcoun tries, is one of the world's most important crops forhuma nconsump tion. In terms of globalproduction ,potat o ranks fourth afterwheat , maize and rice.Abou t 300 million metric tonnes ofpotatoe s arepro duced annually, of which about 70% in the developed and 30% in the developingcountrie s (FAO,1987) . Usuallypotatoe s aremerel y seena sa nenerg y sourceonly .However , when potatoes are compared with other plant foods on acooke dbasis , 'as eaten', it becomes clear that potatoes are also important as a source of minerals, vitamins and protein (Woolfe, 1987). The protein has ahig h nutritive quality because of itshig h content ofessentia l aminoacids ,especiall y lysine (VanGelde ran dVonk , 1980),whic hmake s it outstanding for complementing proteins from other plant food.Th e potato is superior to almost every other crop in foodproductio npe r hectarepe rda y (CIP,1982) . Inorde r to reduce yield losses and tuber injuries causedb ypara site infestations of the potato crop, considerable amounts offungi cides, insecticides andnematocide s areapplied .Th e disadvantages,i n termso fecologica ldamag ean dfinancia lcosts ,o fth echemica lcontro l ofparasite sbein grecognized ,th eattentio nfo rth egeneti cresistanc e ofth epotat o isstrongl y increasing.Th ebreedin go fne wpotat oculti - varswit ha nenhance d resistance toparasite s requires theus eo fwil d Solanumspecies ,a sthes especie sposses sa broa dspectru mo fresistan ces (Ross, 1986). They are also a source ofvaluabl e genes forresis tance to frost and drought, and for quality traits such ashig hcon tentso fprotei nan dstarc han dlo wcontent so freducin gsugars .A dis advantage of Solanum species,fro m a consumers point ofview , istha t theycontai nsteroida lglycoalkaloid s (SGAs). SGAs are natural toxins generally occurring in all parts of many Solanaceous plants. The SGAs consist of aC27-steroida l alkaloid (SA) anda suga rmoiety ,ofte na di- ,tri -o rtetrasaccharide .Th etuber so f the cultivated potato usually contain small quantities of a class of SGAs, the solanidine glycosides,whic h are believed not to present a health hazard to the consumer. However, potato tubers contain the entire enzyme systemnecessar y for synthesiso fSGAs ,an dman yfactor s 10 during growthan dpost-harves thandlin g ofpotatoe s caninduc e denov o synthesiswhic hma y leadt oaccumulatio n ofundesirabl yhig h levelso f SGAs.Man y cases ofpoisonin g thathav e occurred after consumptiono f potatoes, especially in the nineteenth and in the firsthal f ofthi s centurybu tals omor erecentl y (seeChapte r II),hav ebee nascribe dt o excessive levels of solanidine glycosides, resulting from abnormal growthan dstorag econdition so fth etubers .Thes ecase sgenerate dmuc h research onth epotat o SGAs,bu t the general interest inSGA si sstil l growing due to several other developments during this century, as is indicatedbelow . The discovery that SGAs from wild Solanum species are involved in the resistance of such species to certain parasites, initiated much research on this topic (for literature reviews see: Tingey, 1984; Roddick,1987) . Supplyan dpric efluctuation sbetwee n196 0an d1975 ,o f the steroidal sapogenin diosgenin, the startingmateria l for thepro duction ofsteroi dhormones ,prompte d largeprogramme s for thescreen ingo fSolanu m species for thepresenc e ofth e SAsolasodine ,tha tca n beuse da sa nalternativ e (Mann,1978 ;Ripperge r and Schreiber, 1981). A hypothesis, suggesting a toxinpresen t in certain imperfect potato tubers to be involved inhuma n birth defects (Renwick, 1972), caused greatconcer nan d ledt oa stron g increase ofth eresearc ho nth etox icityan dteratogenicit yo fth epotat oSGAs . The withdrawal from commerce in the USA in 1970,o f the cultivar Lenape because of excessive levels of solanidine glycosides in its tubers (Zitnak and Johnston, 1970), prompted studies on factors affecting the SGA accumulation in potato tubers. The evidence that 'Lenape' inherited the ability to synthesize large amounts of sola nidine glycosides from itswil d ancestor S.chacoens eBitt . (Sindene t al., 1984), forces potato breeders to realize that the introgression of genes from wild species into the cultivated potato,ca n result in the introduction of undesired levels or types of SGAs (VanGelde r et al.,198 7an d1988) .
AIMO FTH ESTUD YPRESENTE DI NTHI STHESI S
Tuberiferous wild Solanum species are increasingly being used in potato breeding (Ross, 1986). New techniques incel lbiology , sucha s 11 somatic hybridization, enable the potato breeder to recombine genes from tuberiferous and genetically strongly different,nontuberiferou s Solanum species (Fish et al., 1988). The preceding facts imply that there is a growing need for knowledge on the SGAs in Solanum species which are being used or are potentially useful in potato breeding. However, the qualitative and quantitative SGA compositions of wild speciesuse d inpotat obreedin gprogramme san do fthei roffspring ,hav e received littleo rn oattentio nunti lnow .Thi sca npartl yb eascribe d to the complex methodology of the analysis of the SGAs (VanGelder , 1989). The aim of this study was to develop and apply ametho d forqual itative and quantitative analysis of the SGA compositions of Solanum species, and topu t thecollecte d information into theperspective so f potatobreedin gan do ffoo dsafety ,i norde rt opoin tou tth epotentia l consequences of the introduction ofundesire d levels or types ofSGA s intoth ecultivate dpotato .Fo rthi spurpos eattentio nha sbee npai dt o thefollowin gtopics . -Reviewo fth eliteratur eo nth edistributio nan daccumulatio no fsola - nidine glycosides in the cultivated potato; SGAs from wild Solanum specieswil lalmos tcertainl yaccumulat esimilarl ywhe nintroduce dint o thehousehol d potato,becaus e thebiosyntheti c pathways of thevariou s SGAsar eclosel yrelated . -Evaluation ofth e toxicity of theSGA sa sth ecurren t so-calledsafe ty limits for solanidine glycosides inpotatoes ,reporte d in thelit erature,ar econflictin gan dno tbase do nsuc ha nevaluation . -Development of an advanced procedure for the comprehensive and quantitative analysis of the SGA composition of Solanum species.Muc h emphasis has been given to identification and/or characterization of (novel)aglycones . -Determination of the SGA composition of a number of wild Solanum species used in breeding programmes, and tentative study of the potential transmission of SGAs from wild Solanum species to the cultivatedpotato . -Based on these studies,recommendation s aremad ewit h respect toth e control ofth elevel san d thetype so fSGA s inpotatoe st ob euse dfo r humanconsumption . 12
REFERENCES CIP (1982)Worl dPotat oFacts .Internationa lPotat oCenter ,Lima . FAO (1987) 1986 FAO Production Yearbook, Vol. 40, FAO Statistics Series No.76 .FAO ,Rome . Fish N, Karp A, Jones MGK. (1988) Production of somatic hybrids by electrofusion inSolanum .Theo rApp lGene t76:260-266 . MannJD . (1978)Productio no fsolasodin e forth epharmaceutica lindus try.Ad vAgro n30:207-245 . RenwickJH . (1972)Anencephal yan dspin abifid aar eusuall ypreventabl e byavoidanc eo fa specifi cbu tunidentifie dsubstanc epresen ti ncer tainpotat otubers .Bri tJ Pre vSo cMe d26:67-88 . Ripperger H, Schreiber K. (1981) Solanum steroid alkaloids. In The Alkaloids XIX,RG A Rodrigo (Ed).Academi c Press,Ne w York, pp.81 - 192. Roddick JG. (1987) Antifungal activity of plant steroids. In Lipid Interactionsamon gPlant san dthei rPest san dPathogens ,G Fuller ,W D Nes (Eds).A mChe mSoc ,Washingto nDC ,pp .286-303 . RossH . (1986)Potat obreedin g -Problem san dperspectives .Advance si n plantbreedin g (SupplJ Plan tBreeding )13:1-130 . Sinden SL,Sanfor d LL,Web b RE. (1984)Geneti c and environmentalcon trolo fpotat oglycoalkaloids .A mPotat oJ 61:141-156 . Tingey WM. (1984)Glycoalkaloid s aspes tresistanc e factors.A mPotat o J61:157-167 . Van Gelder WMJ. (1990) Steroidal glycoalkaloids: consequences for potatobreedin g and foodsafet y ofutilizin gwil d Solanum speciesi n breeding programmes. In Handbook of Natural Toxins, Vol.'6 , RF Keeler,A TT u (Eds).Marce lDekke rIne , NewYork ,i npress . Van Gelder WMJ, Hopmans CWJ, Jonker HH, Van Eeuwijk FA. (1987)Bio synthesis and metabolism of C27-steroidal alkaloids in leaves of potato. Proc 10th Trienn Conf Eur Assoc Potato Res, Aalborg, pp. 276-277. VanGelde rWMJ ,Vink eJH ,Scheffe rJJC . (1988)Steroida lglycoalkaloid s intuber san d leaveso fSolanu m speciesuse d inpotat obreeding .Eu - phytica39S:147-158 . Van Gelder WMJ, Vonk CR. (1980)Amin o acid composition of coagulable protein from tuberso f3 4potat ovarietie s and itsrelationshi pwit h proteincontent .Potat oRe s23:427-434 . Woolfe JA. (1987)Th e Potato in the Human Diet. Cambridge University Press,Cambridge . Zitnak A, Johnston GR. (1970)Glycoalkaloi d content of B5141-6pota toes.A mPotat oJ 47:256-260 . 13
CHAPTERI *
OCCURRENCE OF STEROIDAL GLYCOALKALOIDS AND THEIR DISTRIBUTION AND ACCUMULATION INTH EPOTAT OTUBE R
INTRODUCTION -HISTOR YO FTH EPOTAT O
Thegeographica lorigi no fth epotat o isth eAnde so fBolivi aan dPeru , where many wild tuber-bearing Solanum species occur, from one ormor e of which the cultivated potato can have been derived (Wright,1977 ; Hawkes, 1978). Detailedstudie so fstarc han dothe rcel lstructure senable dremain s ofearl y foodplant s from theChilc aValle ynea rLim abein gidentifie d as potatoes, which were radiocarbon dated at an age of 8000 years (Engel, 1970). At that time, potato tubers were collected from wild growing plants, as the cultivation of the potato started about 5000 years ago (Cohen, 1977).However ,a tth e timeo fth eSpanis hconquest , thepotat oseem st ohav ebee na nintensivel ycultivate dcro pi nBolivi a andPeru . Thepotat owa sfirstl y introducedint oEurop e inabou t1570 ,i.e .i n Spain, fromwher e itdiffuse d through continental Europe and partso f Asia (Hawkes, 1978). A second introduction tookplac e inEngland ,be tween158 8an d 1593,fro mwher e itsprea d to Ireland,Scotland ,Wales , andt opart so fnorther nEurop ean dt omos to fth eBritis hcolonies .I n most European countries the potato started as a botanical curiosity and, for a long time, itwa shardl y grown as a food crop,excep t for southern France,wher e potatoes were eatenb y thepoores t people,an d for England and Russia,wher e theywer e esteemed asa delicac y atth e royal courts. Between 1750 and 1800 the potato became increasingly popular as a field crop inman y countries within and outsideEurope ,
*Thischapte r isbase do nth einvite drevie wpaper : Van Gelder WMJ. (1990) Steroidal glycoalkaloids: consequences for potato breeding and food safety of utilizing wild Solanum species in breeding programmes.I nHandboo k ofNatura lToxins ,Vol .6 ,R FKeeler , ATT u (Eds).Marce lDekke rInc. ,Ne wYork ,i npress . 14 andwithi n ahundre d years entirepopulation s became dependent onpo tatoesa sthei rprincipa lsourc eo fnourishment .
DISCOVERY,OCCURRENC EAN DCHEMISTR YO FSGA s
In 1820, Desfosses reported the discovery of an organic base, sola- née, isolated from the berries of black nightshade (Solanum nigrum L.). A 100 mg of this compound administered orally to a dog caused considerable vomiting and unconsciousness. A compound similar to 'solanée'wa s found inpotat o andwa sname d solanine (Baup, 1826).Th e discovery ofthi s toxini nthi sprincipa l foodcro p generatedmuc hre search,whic h isongoin gfo rove r16 0year snow . Important contributions to the knowledge of the chemistry and the occurrence of the SAs and SGAs havebee nmad ebetwee n 1861 and1955 . Zwenger andKin d (1861)foun d thatsolanin e frompotatoe swa sa glyco - alkaloid and they named the aglycone solanidine (Fig. 1).Th e final establishment of the formula of solanidine as C27H43NO (Schöpf and Herrmann,1933 )lea d toth e generalacceptanc e ofth e formulao fsola - nine as C45H73NO15 (Henry, 1949). About 130year s after the discovery of solanine, its compositionwa s finally revealed as amixtur e oftw o series of glycoalkaloids, the a-, ß- and 7-forms of solanine and of chaconine (Table 1), which have the same aglycone (solanidine) but different sugar moieties (Kuhn and Low, 1954;Kuh n et al., 1955aan d 1955b). About 90-95% of the solanidine inpotat o was found tob egly - cosidic-bound ina-solanin e anda-chaconin e (Kuhnan dLow , 1954;Kuh n et al., 1955b;Pasesnishenk o andGuseva , 1956). The ß- and7-form sde tected may have been artefacts resulting from the extraction proce dures (Prelog and Jeger, 1960) or intermediates in SGA metabolism. Since1820 ,hundred so fSGA shav ebee ndetecte d inabou t35 0specie s ofth e families Solanaceae andLiliaceae .Th e chemistry and theoccur renceo fth eSA san dSGA si nth egenu sSolanu mhav ebee nreviewe du pt o 1981 (Prelog and Jeger, 1953 and 1960;Schreiber , 1968;Ripperge r and Schreiber, 1981);mor e than8 0SA spossessin g theC27-skeleto no fcho - lestane have been described. They have been divided into five groups representing different aglycone structures, namely solanidanes (e.g. solanidine), spirosolanes (e.g. tomatidine and solasodine), solano- capsines,3-aminospirostane s (e.g.paniculidine ) andepiminocholesta - Table 1. Steroidal glycoalkaloids of tuber-bearing Solanum species.
Steroidal glycoalkaloid1 Sugar moiety Glycoside structure^
Solanidine glycosides a-Solanine Solatriose A: R-Gal<^
/3-Solanine:i Solabiose B: R-Gal-Glu 7-Solanine-5 Galactose C:R-Ga l Chacotriose D: R-Glu<^am-a a-Chaconine ^ Rham-b o Chacobiose R-Glu-Rham-a /3^-Chaconine Chacobiose R-Glu-Rham-b y92"Chaconine Glucose R-Glu 7-ChaCOnine Dehydrocommersonine Commertetraose R-Gal-Glu Demissidine glycosides Glu Demissine Lycotetraose I: R-Gal-Glu^ Xyl Commersonine Commertetraose As H Leptinidine glycosides Leptinine I Chacotriose As D Leptinine II Solatriose As A Acetylleptinidine glycosides Leptine I Chacotriose As D Leptine II Solatriose As A Tomatidenol glycosides a-Solamarine Solatriose As A /9-Solamarine Chacotriose As D Solasodine glycosides Solasonine Solatriose As A Solamargine Chacotriose As D Tomatidine glycosides a-Tomatine Lycotetraose As I Sisunine (neotomatine) Commertetraose As H LAglycone, structures are given in Fig. 1. -R= aglycone; Gal = galactose; Rham = rhamnose; Glu= glucose; Xyl =xylose . %inor steroidal glycoalkaloids may be artefacts ormetabolites . 16 Solanidine C27 H43N O Solanthrene C27 H41 N OH Cï3 H ICH HO ^ ^ HO Rubijervine C27 H43 N02 Isorubijervine C27H43N02 O CH3 H 0-C-CH3 CH3 V HO HO Leptinidine C27H43NO2 Acetylleptinidine C2g H45 N03 CH3H CH3 '* - Demissidine C27H45NO Tomatidine C27H45N02 Fig. 1. Structural formulas of C27-steroidal alkaloids (SAs)divide d into five groups: solanidanes, spirosolanes, solanocapsines, 3-amino- spirostanesan depiminocholestanes . 17 Tomatidadiene C27 H41 NO Tomatidenol C27H43N02 Solasodiene C27 H41 NO Solasodine C27H43N02 CH3 \ CH3 * > Solanocapsine C H N 0 Soladulcidine C27H45N02 27 46 2 2 CH3 CH3 H9N Solafloridine C27H45N02 Paniculidine C27H45N03 18 nes (e.g. solafloridine) (Fig. 1).Othe r structures such as that of the jervanes occur additionally inVeratru m species,famil y Liliaceae (Rippergeran dSchreiber ,1981) . Inth egenu s Solanum,th e SAsar eglycosidic-boun dvi a thehydroxy l group at the C-3 atom. They occur incombinatio nwit h differentsuga r moieties, e.g. solatriose, chacotriose, lycotetraose etc.A s aresul t numerous SGAs have been reported for Solanum species, but until now only a limited number of themhav e been detected inth etuber-bearin g species (Table1) . New SAs and SGAs are still being reported, for example sisunine (Osmane tal. , 1986),7/9-hydroxy-O-methylsolanocapsin e (Chakravartyan d Pakrashi, 1988), and solanudine (Usubillaga, 1988). New solanidane- type SAshav e recentlybee ndetecte d inSolanu mspecie suse d inpotat o breeding (VanGelde re tal. ,198 7an d1989 ;se ehereafter) . BIOSYNTHESISAN DDEGRADATIO NO FSGA s Thebiosyntheti c pathways ofth eSA sar eclosel y related toeac hothe r and to those of the steroidal sapogenins,whic h also occur inSolanu m species.The yal l followth e generalpathwa y ofth e steroidbiosynthe sis, starting from acetyl-coenzym A via the usual intermediates me valonic acid,squalene ,cycloarteno l andcholesterol .Th e steps inth e pathwaysfro mcholestero lt oth eSA shav eonl ypartiall ybee nelucidat ed. Fig. 2 deliniates the main steps of the (partially hypothetical) pathways for thebiosynthesi s ofth e SAs.Dormantino lan ddormantinon e have been isolated from a solanidine synthesizing Veratrum species (Kaneko et al., 1977), but theyhav e notye tbee nprove n tob einter mediates by tracer experiments. The amino acid arginine was suggested as the nitrogen source for solanidine in this biosynthesis route (Kanekoe tal. , 1976). The enzymatic glycosylation of the aglycones to the 7-, ß- andfi nally thea-form so fth e SGAsoccur s stepwise,a t least inth ecas eo f the solanidine and solasodine glycosides (Liljegren, 1971;Jadha v and Salunkhe, 1973; Lavintman et al., 1977; Osman et al., 1980). Solanidinewa sfoun dt ob erapidl yconverte d intoit sglycosylate dpro ducts,whic h ismentione d as apossibl e explanation for the facttha t free solanidine doesnormall yno toccu r inhealth y potato tubertissu e 19 O OH 3[CH3-C-SCOA] HOCH2-CH2-C -CH2-COOH —• Cl "^ CH3 Acetyt-CoA Mevalonic acid Farnesyl pyrophosphate Cholesterol Cycloartenol Squalene Dormantinol Dormantinone Verazine Soianidine Teinemine Etioline Solasodine Tomatidine Fig. 2.Biosyntheti c pathways of C27-steroidal alkaloids. 20 (Osmane tal. , 1980). InSolanu m species,th eaglycone s shown inTabl e 1 areth eend-product so fth eS Abiosynthesi swhic har eglycosylate dt o SGAs,wherea si nothe rgener ao fth eSolanaceae ,Solanu maglycone shav e been detected as precursors of other SAs. For example, in Veratrum species, solanidine was converted intojervin e andveratramin e (Kaneko et al., 1972) and in Fritillaria species, solanidine, solasodine and tomatidenol seemed tob emetabolize d tocamtschatcanidine ,hapepunine , anrakorininean d27-hydroxyspirosolan e (Kanekoe tal. , 1981). Potato tissues contain enzymes that can degrade a-solanine anda - chaconine by cleaving one or more sugars from their sugar moieties (Swain et al., 1978). Bushway et al. (1988) characterized an enzyme from potato as a rhamnosidase; it formed mainly /S^-chaconine and a smallamoun to f^2" cnacon:'-ne• Thes eenzyme sbecom eactiv eonl ywhe nth e tissue is disrupted, probably because the enzymes and substrates are present in different compartments within the cell. In analyses of potato tissues this enzymatic degradation of SGAs may be a source of artefacts. Since thebiosyntheti c pathways ofth evariou s SAsar e stronglyre lated, it is tob e expected that factors influencing the accumulation of the solanidine glycosides in household potatoes, will similarly affectalie nSGA si fthe ywoul dhav ebee nintroduce d intocultivars .I t is thus necessary to know the distribution of the SGAs in thepotat o plant,an dth efactor swhic hma yaffec tth eaccumulatio no fth eSGA si n itstubers . DISTRIBUTIONO FSGA sI NTH EPOTAT OPLAN T SGAs are found inal l organs of thepotat oplant ,th ehighes tconcen trations inpart s ofhig hmetaboli c activity, such as flowers,unrip e berries, young leaves and sprouts. The pure S. tuberosum cultivars containonl ysolanidin eglycosides .Th emos textensiv estud yu pt onow , on the distribution of the solanidine glycosides in different organs and tuber tissues ofpotat o was carried outb y Lampitte t al. (1943), who investigated different cultivars; their results are summarized in Table 2. Inadditio n data from other reports are compiled. Thevaria tion in the concentrations of the solanidine glycosides within a certainorga no rtissu e canb e ascribed toth e different cultivars «r-* ov r^ r^ i-i 00 00 ^ CTi CTv bû C 3 y—s r*—o \ O .-H oo r« OO erv erv r-i iH s_x •o eu eu *-• tca eu eu r* •H •d 3 3 ta ta 0 X .M G o 3 3 4-1 4J Es N N •H CU <*-N •~N 0 O NI CT\ r-~ ï*, i~- ^-00 \ .- r- « « ..» ta ON 00 <^f\ a\ .- .- •* •-V U i-i er> CM i-i y-v S-*. CM /-^ E 3 60 - 20 20 40 60 80 100 120 140 160 180 200 solanidine glycosides mg/kg freshtube r Fig. 3. Frequency distribution of the contents of solanidine glycosides (mg/kg fresh tuber) in 521 samples of potato tubers. These contents were reported in the literature and include those of many registered household potato cultivars grown in Australia, England, India, The Netherlands, New Zealand, Scotland, Sweden and theU.S.A . (the cultivar Lenape and breeding clones were excluded from this frequency distri bution). then remove only 30-35% of the total amount of SGAs present (Zitnak, 1961; Wood and Young, 1974;Verbis t andMonnet , 1979). Morris and Petermann (1985) found that the amount of a-solanine varied from 28%t o 57%o f the total amount of the solanidine glycosides in potato tubers. Free solanidine up to 33% of the total amount (free and glycosidic-bound solanidine)present ,ha s been reported to occur in bitter potato tubers (Zitnak, 1961), but Lampitt et al. (1943) could not detect free solanidine in tubers of nine cultivars, not even when the tubers contained large amounts of solanidine glycosides due to light exposure for 18weeks . 24 FACTORS AFFECTING THE ACCUMULATION OF SOLANIDINE GLYCOSIDES INPOTAT O TUBERS Geneticvariatio nan dgrowt hcondition s The contents ofsolanidin e glycosides infield-grow ntuber so fcommer cial potato cultivars can vary widely for individual samples. Values between 10mg/k gan d65 0mg/k ghav ebee nreporte d (Baerug,1962 ;Cron k et al., 1974; Fitzpatrick et al., 1978; Verbist and Monnet, 1979; Daviesan dBlincow ,1984 ;se eals oth estudie so fwhic hth evalue shav e beensummarize d inFig .3) .Th egeneti cvariatio ni sth elarges tsourc e ofvariatio n of the contents of solanidine glycosides,bu t thesecon tentsma yals ovar yamon gcrop so fa singl ecultiva rgrow na tdifferen t locations or indifferen tyears ,du et oth evaryin g environmentalcon ditions (Sanford and Sinden, 1972; Sinden and Webb, 1972 and 1974; Parnelle tal. ,1984 ;Va nGelde ran dDellaert ,1988) . Although interactions between genotype and environment have been demonstrated, the ranking order of genotypes as to their SGA contents remains quite constant under different environmental conditions (Lepper,1949 ;Sinde nan dWebb ,1974 ;Patchet te tal. ,1977 ;Lammerink , 1985; Olsson, 1986). In a recent study on 27 genotypes grown infou r seasons at two locations, a strong correlation (r = 0.86) was found between the genotype-averageso fth econtent s ofsolanidin e glycosides (10-650mg/kg ;measure d over theyear s and locations)an d thecoeffi cients of variation calculated for each genotype (Van Gelder and Dellaert,1988) .Thi sma yexplai nwh ycultivar swit hgeneticall ydeter mined 'high'content so fsolanidin eglycoside s inth etuber s (above10 0 mg/kg)accumulate d easily excessive amounts (above 200mg/kg )compare d withcultivar swit hlo wcontents . Most of the environmental variation seems to result from climatic influences (Maga, 1980;Jadha v et al., 1981). Especially anunusuall y coolgrowin gseaso naccompanie dwit ha nabnormall yhig hnumbe ro fover cast days, probably resulting in an immature potato crop, has been suggested tob e thecaus e ofexcessiv e levelso f solanidine glycosides which were held responsible for cases of acute poisoning (Bömer and Mattis,1924 ;Norberg , 1987). Immaturityha sbee nassociate dwit hhig ho reve nexcessiv elevel so f solanidine glycosides (Bömer andMattis ,1924 ;Wol f and Duggar,1946 ; 25 Patchett et al., 1977; Ahmed and Müller, 1979; Verbist and Monnet, 1979). Tuber size aswel l asmaturity , are inversely relatedwit hth e content of solanidine glycosides within acultivar , as these SGAsar e formed earlydurin g tuberdevelopmen t anda s theyar e "diluted"durin g the process of enlarging and maturation of the tubers.Moreover , the SGAsar econcentrate d inth epeel ,whic hi sa relativel y largerpar to f thetuber swhe nthe yar esmaller .Verbis tan dMonne t (1979)foun di n2 0 tuber samples ofeigh tcurren tcultivar s contents ofsolanidin eglyco sidesvaryin g from 96-448mg/kg ; the average tuber weights varied for thedifferen tsample s from9-4 0 g.The y supposedtha tsuc hsmal lpota toes can be responsible for cases of gastroenteritis. Other factors thathav ebee n investigated inrelatio n toSG Aaccumulatio n duringth e growth ofpotat o tubers inth efield ,ar eplantin g date,fertilizatio n with major elements and soil type.Thes e factors exerted only little effect on the accumulation of solanidine glycosides and are notasso ciated with potentially hazardous levels of these compounds (Maga, 1980; Jadhave tal. ,1981 ;Sinde ne tal. , 1984). Lightexposur ean dstorag econdition s Whenharveste dpotatoe sar eexpose dt olight ,thei rcontent so fsolani dine glycosidesma y increase considerably.Th e increasedepend s onth e cultivaran di spositivel yrelate dt oth eligh tintensit yan dth edura tion of the exposure (Jadhav and Salunkhe, 1975; Maga, 1980; Zitnak 1981; Jadhav et al., 1981;Sinde ne t al., 1984;Uppal , 1987).Upo nex posuret obrigh tsunligh t (35000-50000lx )fo r6 h ,th esolanidin egly coside content of freshly harvested tubers increased from 50mg/k gt o above 200mg/k g (Baerug, 1962). Levels ashig h as45 0mg/k ghav ebee n reported for tubers exposed to intense solar irradiationwhe n left in the field for 72h (Zitnak,196 1an d 1977). During lightexposure ,th e solanidine glycoside contents of immature and small tubers increased more strongly (up to 480mg/kg ) than those ofmatur e and largetuber s (up to 200mg/kg ) (Bömer andMattis ,1924 ;Wol f andDugar ,1946 ;Lep - per, 1949; Sinden and Webb, 1974;Patchet t et al., 1977). Artificial light during storage or marketing also induces de novo synthesis of solanidine glycosides. The increases are usually not as dramatic as thoseresultin gfro msunlight ,an dtuber so fcultivar snormall yshowin g contentsbelo w10 0mg/kg ,d ousuall yno texcee d15 0mg/k gupo nexposur e 26 toartificia l light (Ahmedan dMüller ,1981 ;Sinde ne t al., 1984;Lara - merink,1985 ;D eMain ee tal. , 1988). Light-induced synthesis of SGAs isofte n accompanied with greening ofth etuber sa sa resul to fth esynthesi so fchlorophyl l inth epotat o peel. Greened potatoes are often associated with bitterness andhig h SGA levels.Althoug h thespectra l responses toligh to fth emechanism s synthesizing the chlorophylls a and b and the SGAsa-solanin e and a- chaconine were similar (Petermannan dMorris ,1985) , thesebiochemica l processes seem to be able to proceed independently of each other (Grison,1987 ;D eMain ee t al., 1988). Potatoeswit h levelso fsolani - dine glycosides as high as 600-1000 mg/kg, did not show any greening (Zitnak,1977) .Thes edat aclearl ysho wtha tgreenin g isno ta reliabl e indicatoro fto ohig hSG Alevel si nlight-expose dpotatoes . Storage of potato tubers can increase their contents of solanidine glycosides (Cronk et al., 1974;Ahme d andMüller , 1981), but thelit térature iscontradictor ywit hrespec t toth e influence of thetemper ature during, and the duration of the storage period. Storage inth e darka tvariou stemperature sbetwee n0° Can d28° Cfo r1 2week s (Naire t al., 1981;Linnema ne tal. , 1985)o rbetwee n 10°Can d15° Cfo ru pt o7 months (Zitnak,1953 ,quote dfro mJadha ve tal. ,1981 ;Lammerink ,1985 ) hardly affected the accumulation of solanidine glycosides. Storage at 4°C resulted ina slight increase after 3month s ina stud yb yW uan d Salunkhe (1976), but innon-elevate d levels after 3-8 months inothe r studies (Wolf and Duggar, 1946; Fitzpatrick et al., 1977;Bostoc k et al., 1983). Storage at 4°C under humid conditions resulted in high contentso fsolanidin eglycoside safte r6 week s (Zitnak,1953) . A largenumbe r ofphysica l andchemica l treatments tocontro lpost - harvest accumulation of SGAs have been described. Among these are vacuum packaging andpackagin g incoloure dpolyethylen e bags, ionizing radiation, submerging in water to which often chemicals were added, heatingo ftuber san dtreatin gthe mwit hwaxes ,chemical so roils .Som e treatments simultaneously inhibited SGA accumulation in, and greening and/or sprouting of the tubers. For information on the efficacy and limitationso fthes etreatment sreview sb ySalunkh ean dW u (1979),Mag a (1980)an dJadha ve tal . (1981)ar ereferre dto . 27 Tuberinjur y Damage to tubers resulting from dropping,puncturing , cutting,hammer ing, brushing,o rcause db y diseases orb yanimals ,stimulate d theSG A synthesis (Sinden and Webb, 1974; Wu and Salunkhe, 1976; Ahmed and Müller,1978 ;Olsson ,1986 ;Mond ye tal. , 1987).Thi seffec ti sthough t tob edu et oa physiologica ldefens emechanis m inth etuber .I nslight ly injured tubers the contents of solanidine glycosides can increase 200-300% (Sinden and Webb, 1974). Severely damaged tubers contained levels of theseglycoside s sometimes exceeding 200mg/kg ,wit hextrem e values of 319 and 530 mg/kg (Olsson, 1986). Th e accumulation of the glycosides occurred inth epee l aswel l as inth eflesh ,mainl y inth e firstweek s ofstorag eafte r the inductiono fth e synthesisb ymechan icalinjur y (Wuan dSalunkhe ,1976) .Th eexten to fth eSG Aaccumulatio n depends strongly on the type of injury, on the cultivar and on the storagecondition safte rth einjury .Damage dtuber sstore da tc a5° Ci n the dark accumulated less solanidine glycosides than tubers stored at ca 20°C (Wuan d Salunkhe, 1976;Ahme d andMüller ,1978 ;Mond y etal. , 1987). Probably , the tuber defense mechanism ismor e active athighe r temperatures.Accordin g toFitzpatric k et al. (1978), damagedpotatoe s purchased on the retail market contained levels of SGAs up to 193 mg/kg. Olsson (1986) observed ahig h correlation between the initial solanidine glycoside contents of cultivars and breeding clones and their elevated contents after damage,an d recommended potato breeders to select for low SGA contents topreven t accumulation ofSGA sdu et o damage. Potatoprocessin g SGAs are fairlyheat-stabl e compounds,thei rmeltin g points,a twhic h someSGA sma ystar tdecomposing ,var yi ngenera lfro m230-280° C (Prelog and Jeger, 1960; Schreiber, 1968;Ripperge r and Schreiber, 1981). At these temperatures theiraglycone s dono t showdecomposition ,an deve n during gaschromatograph y at280-320° Cthe yhardl y decompose (VanGel der, 1985b). It is therefore not surprising that steaming, boiling, baking, cooking, frying and microwaving of potatoes did not affect theircontent so fsolanidin eglycoside s (Bakere tal. ,1955 ;Zitna kan d Johnston,1970 ;Bushwa yan dPonnampalam ,1981) . When during the manufacturing of potato chips (Eng. = crisps)an d 28 Frenchfrie s (Eng.= chips) ,hal fproduct ssuc ha sslice so rstrip sar e left for some time (3h to 3days )befor e frying or cooking, theSG A contents can increase with 100% (Ahmed andMüller , 1978;Maga , 1981). In aged potato slices of the cultivar Kennebec,whic h contains germ- plasmo fS .demissu mLindl. , thetomatideno l glycosides a- and/3-sola - marine,no toriginatin gfro mS .tuberosum ,wer edetecte d inadditio nt o a-solanine and a-chaconine (Shih and Kuc, 1974; Fitzpatrick et al., 1977). The above results are especially important in the lighto fth e results of Sizer et al. (1980), who showed that during the fryingo f potato chips water was lost, which resulted in a three- to fourfold concentrationo fSGAs .The yfoun dstrongl yvaryin gSG Acontents ,95-72 0 mg/kg, in commercially purchased chips containing most of the potato skin.Th eexcessiv e contentdi dno tcaus e anoticeabl ebitterness ,bu t theauthor ssuggeste dtha tth ebitternes scoul dhav ebee nmaske db yth e salton ,o r thehig hoi lconten to fth echips .Bitternes sca npossibl y also be masked by other flavour additives.A s especially infants and adolescents sometimes consume relatively largeamount s ofchips , iti s advisable that potatoes arepeele dwel l and that delays inprocessin g ofslice so rotherwis enonintac tpotatoe sar eavoided . CONCLUSIONS Itca nb e stated that invie wo fth edat agive ni nth eliteratur eman y factors influencing the accumulation of solanidine glycosides in potatoesshoul db ekep ti nmind . In order to control the accumulation of these SGAs resulting from the growth conditions, it is important that potato tubers are well- coveredwit h soildurin ggrowth ,an dtha t theyar eharveste dwhe nthe y are mature. When the crop has been exposed to physiological stress, especially unusual climatic conditions, it is advisable to check the SGAconten to fth etuber safte rharvest .Exposur eo fpotatoe s tobrigh t light,especiall y sunlight,mus tb eminimized . Potato tubersca nb estore dfo ra considerabl eperio do ftime ,prob ably up to eight months or even longer,withou t amarke d increase in thecontent so fsolanidin eglycosides .However ,i nsuc hcase sth estor agecondition smus tb eoptimum .Indication stha tstorag e inth edar ka t ca9-15° Can da ta lo wai rhumidit y ist ob epreferred ,ca nb efoun di n 29 the literature,bu t further study ofth eeffect so fstorag econdition s on theaccumulatio n ofSGA s isneeded .Thi s isth emor eso ,i nvie wo f the currently increasing interest forcol d storage (2-4°C)o fpotatoe s asa nalternativ e tochemica lsproutin ginhibitors . Growers of potatoes and those involved inmarketin g usually avoid injury thatdeteriorate s theoutwar d appearance ofpotatoes ,bu t iti s importantals ofro m theviewpoin t offoo dsafety ,tha tan ykin do fin juryt opotatoe sshoul db eavoided . 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(1986)Sisunine , a glycoalkaloid found in hybrids between Solanum acaule and Solanum x ajanhuiri. Phytochemis try25:967-968 . Parnell A, Bhuva VS, Bintcliffe EJB. (1984) The glycoalkaloid content V of potato varieties.J Natn InstAgri c Bot16:535-541 . Pasesnishenko VA, Guseva AR. (1956) Quantitative determination of po- 32 tato glycoalkaloids and theirpreparativ e separation.Bioche m (USSR) 21:606-611. PatchettBJ ,Cunningha mPS ,Lil lRE .(1977 )Glycoalkaloi d levels inNe w Zealandpotatoes .N ZJ Ex pAgri c5:55-57 . Petermann JB,Morri s SC. (1985)Th e spectral responses of chlorophyll and glycoalkaloid synthesis in potato tubers (Solanum tuberosum). PlantSe i39:105-110 . Ponnampalam R,Mond yNI .(1986 )Effec to f foliarapplicatio n ofindol e acetic acido nth e totalglycoalkaloid s andnitrat e nitrogenconten t ofpotatoes .J Agri cFoo dChe m34:686-688 . PrelogV , Jeger 0. (1953)Th e chemistry of Solanum andVeratru malka loids. InTh eAlkaloids , III,RH FManske ,H LHolme s (Eds). Academic Press,Ne wYork ,pp .247-312 . PrelogV ,Jege r0 . (1960)Steroi dalkaloids :th eSolanu m group.I nTh e Alkaloids,VII ,RH FMansk e (Ed).Academi c Press,Ne wYork , pp.343 - 361. Ripperger H, Schreiber K. (1981) Solanum steroid alkaloids. In The Alkaloids,XIX ,RG A Rodrigo (Ed).Academi c Press,Ne wYork ,pp .81 - 192. Salunkhe DK,W uMT . (1979)Contro l ofpostharves t glycoalkaloidforma tioni npotat otubers .J Foo dPro t42:519-525 . V Sanford LL,Sinde nSL . (1972)Inheritanc e ofpotat o glycoalkaloids.A m ^ PotatoJ 49:209-217 . Schöpf C, Herrmann R. (1933) Zur kenntnis des Solanidins. Chem Ber 66:298-305. SchreiberK . (1968)Steroi dalkaloids :th eSolanu mgroup .I nTh eAlka loids,X ,RH FMansk e (Ed).Academi c Press,Ne wYork ,pp .1-192 . ShihM-J , KucJ . (1974) a- and/3-Solamarin e inKennebe c Solanumtube rosumleave san dage dtube rslices .Phytochemistr y13:997-1000 . Sinden SL, Sanford LL,Web b RE. (1984)Geneti c and environmentalcon trolo fpotat oglycoalkaloids .A mPotat oJ 61:141-156 . Sinden SL, Webb RE. (1972) Effect of variety and location on the glycoalkaloidconten to fpotatoes .A mPotat oJ 49:334-338 . SindenSL ,Web bRE . (1974)Effec to fenvironmen t onglycoalkaloi dcon tent of six potato varieties at 39 locations.U S Dept Agric Techn Bull1472 . Sizer CE,Mag aJA , Craven CJ. (1980)Tota l glycoalkaloids inpotatoe s andpotat ochips .J Agri cFoo dChe m28:578-579 . SwainAP ,Fitzpatric kTJ ,Talle yEA ,Her bSF ,Osma nSF .(1978 )Enzymat ic hydrolysis of a-chaconine and a-solanine. Phytochemistry 17:800- 801. UppalDS . (1987)Varieta lan denvironmenta leffec to nth eglycoalkaloi d content ofpotat o (Solanum tuberosum L.). Plant Foods forHuma nNut r 37:333-340. Usubillaga A. (1988) Solanudine, a steroidal alkaloid from Solanum nudum.Phytochemistr y27:3031-3032 . VanGelde rW JM. (1985a)Plan tbreedin g asa too l for improvingproduc t quality.Bedrijfsontwikkelin g16:474-47 8 (inDutch) . Van GelderW JM. (1985b )Determinatio no f thetota lC27-steroida lalka loid composition ofSolanu m speciesb yhigh-resolutio nga schromato graphy.J Chromatog r331:285-293 . Van Gelder WJM, Dellaert LMW. (1988)Alkaloid s in potatoes. Prophyta 425:236-238 (inDutch) . VanGelde rWMJ ,Hopman sCWJ ,Jonke rHH ,Va nEeuwij k FA. (1987)Biosyn thesisan dmetabolis mo fC27-steroida lalkaloid s inleave so fpotato . 33 Proc 10th Trienn Conf EurAsso c Potato Res,Aalborg , pp. 21b-211. Van Gelder WJM, Tuinstra LGMTh, Van der Greef J, Scheffer JJC. (1989) Mass spectrometric characterization of novel steroidal alkaloids in Solanum: implications for potato breeding. J Chromatogr, inpress . Van Gelder WJM, Vinke JH, Scheffer JJC. (1988) Steroidal glycoalka- loids in tubers and leaves of Solanum species used in potato breed ing. Euphytica 39S:147-158. Verbist JF, Monnet R. (1979) A propos de la teneur en solanine des petits tubercules nouveaux de pomme de terre (Solanum tuberosum L.). Potato Res 22:239-244. Wolf MJ, Duggar BM. (1946) Estimation and physiological role of sola nine in the potato. J Agric Res 73:1-32. Wood FA, Young DA. (1974) TGA in potatoes. Canada Department of Agri culture Publication no.1533 . Wright Jr, HE. (1977) Environmental change and the origin of agricul ture in the Old and New Worlds. In Origins of Agriculture, CA Reed (Ed). Mouton Publishers,Th e Hague,Paris ,pp .282-318 . Wu MT, Salunkhe DK. (1976) Changes in glycoalkaloid content following mechanical injuries topotat o tubers.J A m Soc Hort Sei101:329-331 . Zitnak A. (1961)Th e occurrence and distribution of free alkaloid sola- nidine inNette d Gem potatoes. CanJ Biochem Physiol 39:1257-1265. ZitnakA . (1977)Steroid s and capsaicinoids of Solanaceous foodplants . In Nightshades and Health, NF Childers, GM Russo (Eds). Somerset Press Inc, Somerville,N.J. , pp. 41-91. Zitnak A. (1981)Photoinductio n of glycoalkaloids in cured potatoes.A m Potato J 58:415-421. Zitnak A, Johnston GR. (1970) Glycoalkaloid content of B5141-6 pota toes. Am Potato J47:256-260 . Zwenger C, Kind A. (1861) Ueber das Solanin und dessen Spaltungspro- s/dukte. Ann Chem Pharm 118:129-151. 35 CHAPTERI I EVALUATIONO FTH ETOXICIT YO FSTEROIDA LGLYCOALKALOID S INTRODUCTION The objective ofthi schapte r ist oevaluat e the toxicological datao n SGAs in order to assess possible consumer hazards and to derive safe levels forhousehol d potatoes.Als o an assessment will be made ofth e acceptablelevel so rcomparabl estandard spropose di nth eliteratur es o far. TOXICOLOGYO FSGA s Toxicokinetics Humandat a Harvey et al. (1985a, 1985b and 1986) demonstrated that solanidine glycosides as well as free aglycone were present in all sera of the several hundreds ofhuma nbeing s (womenan dmen )teste d inth ecours e of a number of separate experiments. Calculations based on the data presented in these studies showed that the concentrations varied from about 2-110 ngexpresse d as solanidine glycosidespe rm l serum.Harve y etal . (1985aan d1985b )foun dtha tth econcentration s ofthes eSGA si n the serawer e proportionally related to the SGA contents of thepota toes, an dt oth eamoun to fpotatoe s (whichha dbee nprepare d invariou s ways), consumed by the individuals. The relationship between the SGA concentration in the serum and themonthl y potato intake found inth e male subjects was different from that found in the female subjects (Fig. 1),whic h was ascribed to a greater dayt oda yvariatio n inth e size of potato portions eaten by the female subjects (Harvey et al., 1985a). Upon abstention of potatoes and potato products by two sub jects, their serum SGA concentrations decreased with 35-55% after the firstwee kan d 'becameminimal 'afte r2- 3week s (Harveye tal. , 1985a). Claringbolde tal . (1982)administere d tritiatedsolanidin e tohuma n volunteers (twomen ,on ewoman ) byintravenou s (iv)injections . Such 36 ?Q 25 30 35 Monthly intake (units) 15 20 25 30 35 Monthly intake (units) Fig.1 .Relationshi pbetwee ndietar ypotat o intakean dconcentratio no f SGAsi nseru mo ffemal e (A) andmal e (B) subjects. A:n = 18 ; y= 2.11x -18.36 ;r = 0.70 .B :n = 15 ;y - 0.91 x+ 3.11 ;r = 0.88 . AfterHarve ye tal . (1985a). 37 experiments are relevant from a consumers point of view, in that they provide valuable information on the elimination of the SGAs. The excre tion rates of the tritiated solanidine were low; during the first 24h , 3-7% of label was excreted and during the following week about 2% of the dose daily. A half-time of excretion of 34-68 days was calculated. The authors also examined human post-mortem liverswhic h were of normal histological appearance and originated from subjects who died suddenly from cardiovascular disease. They detected free solanidine as well as solanidine glycosides in total concentrations up to 106 mg solanidine (230 mg a-solanine equivalents) per kg liver (calculated from the data reported). Claringbold et al. (1980) studied the kinetics of tritiated solaso- dine after iv administration to twomen . The menha d excreted about16 % of the labeled solasodine after 24h and about 25% after 3days . After 8 days, labeled compound was still present in the erythrocytes and the plasma of the humans. These studies show that after consumption ofpotatoe s or potato pro ducts, human beings absorb the potato SGAs from the gastrointestinal tract in amounts proportionally related to the amounts ingested. After the SGAs have entered thebloo d stream they are eliminated only slowly. This implies that a certain level of accumulation of SGAs may occur in the human body, which is confirmed by the relatively high SGA levels that were found inpost-morte m livers.Th e experiments also showed that a-solanine and a-chaconine are partly hydrolysed liberating the aglycone solanidine. The long retention times of the SGAs in the human body concur with the finding of Rühl (1951)wh o detected 'solanine' in post-mortem urine 14 days after itha d been ingested. Experimental-animal data Nishie et al. (1971) studied the kinetics of a-solanine in the rat. After oral administration of 5 mg of tritiated a-solanine per kg body weight (bw), this SGA was poorly absorbed from the gastrointestinal tract; 78% was excreted after 24 h and 94%, (of which 84% with the faeces and 10%wit h the urine)afte r 4 days. The highest concentrations of a-solanine were found after 12 h in spleen, kidney, liver, lung, fat, heart,brai n andblood , in descending order. After intraperitoneal (ip)administratio n of tritiated a-solanine the excretion rate was much 38 lower:abou t34 %afte r2 4h a ta dos erang eo f5-1 5mg/k gbw .A ta hig h dose of 25 mg/kg bw the excretion almost stopped, resulting inaccu mulation of labeled compound in liver, spleen,kidne y and intestine. After oral as well as ip administration, solanidine was formed upon hydrolysis. Comparable results were obtained by Norred et al. (1976) who studied thekinetic susin g tritiateda-chaconine .Thu sbot hpotat o SGAs, a-solanine and a-chaconine, are poorly absorbed from the gas trointestinal tract in the rat, and the patterns of distribution and excretion of these compounds are similar. Solanidine was formed also uponhydrolysi so fa-chaconine . Alozie et al. (1978)studie d thekinetic s of tritiated a-chaconine inhamsters .Contrastin g to the rat,th ehamste r absorbed the labeled compound readily from the gastrointestinal tract after oraladminis trationo f1 0mg/k gbw .Afte r7 day sonl y24 %wa sexcreted ,muc ho fth e labeled compound persisted in spleen,liver , lung, testes,kidne y and brain, inconcentrations , indescendin g order,whic hwer e 3-6 timesa s high as that in the blood. After ip administration, only 3% of the labelwa sexcrete dafte r7 days . Claringbold et al. (1980) found tritiated solasodine to be still present in the livers ofhamster s 22day s after (intracardial)admin istration. The animal studies are generally inagreemen t with the findings in humans. The solanidine glycosides are (partially) absorbed from the gastrointestinal tractan donc epresen t inth ebloo d stream thesecom poundsar eabsorbe db yman yorgan san dar ethe nonl yslowl yeliminated . The amounto fsolanidin e glycosides found inth ebloo d representsonl y a relatively small fraction of the total amount present in thebody . The SGAs and the aglycones, solanidine as well as solasodine (which result fromhydrolysi s ofth eglycoside s inth ebody) , areretaine di n thebod yfo rsevera lweeks . The experiments with rats andhamster s show that considerabledif ferences may exist between species, in absorption and elimination of the solanidine glycosides. They further suggest that the hamsterre semblesma n more closely with respect to these kinetics than therat , and thus the hamster may possibly be a more adequate test model for studyingth ekinetics . 39 Toxicodynamics Acutean dsubacut etoxicit y Humandat a Potato SGÀs. In the literature case studies as well as experimental data on the acute and subacute toxicity of potato SGAs in man are available. The two sources of information will be dealt with subse quently. Inth eepidemiologica l literature alarg enumbe r ofcase so fpotat o poisoning havebee ndescribed .Th emos textensiv e and carefulexamina tiono fa nepidemi co fpotat opoisonin g isfoun di nMcMilla nan dThomp son(1979 ). Th eepidemi c involved7 8boy sbetwee n1 1year san d1 3year s old that became ill after eating a lunch at school, which included potatoes. Seventeen of the boys required hospital admission; three being dangerously ill.Th eauthor spresen tdetaile d clinicalfindings , including a description of the symptoms of the suspected potato poi soning,whic h started 4-14 h after themeal . The commonsymptom swer e gastrointestinal, such asvomitin g (for 1-3 days), diarrhoea (for 2-6 days)an d abdominalpain ;an dneurological ,suc ha srestlessness ,con fusion, delirium, stuporose, drowsiness orhallucination .Mos t ofth e boysha d fever sometimes ashig ha s40-40.5°C .Othe r symptomsrecorde d werenausea ,malaise ,a rapi dbu tver ywea kpuls e (160/min),difficul t butaccelerate drespiratio n (48/min),a lo wo runrecordabl ebloo dpres sure,unconsiousness ,ski nlesion san ddisturbanc eo fvision . After 6-11 days of admission, the boys were fit tob e discharged. Some still complained of visual disturbance, dizziness or pain on walking,bu twithi nth enex tfe wweek sal lo fthe mrecovered . Elaborate microbiological,bacteriologica l andvirologica l examina tions,an dchemica ltest sfo rnumerou sorgani can danorgani ccompounds , were carried outb y experts onblood , serum,vomit ,urin e andfaeces , aswel la so nkitche nequipmen tan d sampleso ffoo drecovere dfro mth e meal. Itwa s concluded that the illnesses were due to theconsumptio n of arelativel y smallnumbe r of toxicpotatoes ,whic hha dbee n inth e foodstores of the school for some months. The unpeeled potatoescon tained 330m gsolanidin e glycosidespe rk gwhils t toxicconcentration s ofman yothe r substances forwhic h thepotatoe swer e analysedwer eno t detected. Sixday safte r themeal ,plasm aCholinesteras e levelso fth e boyswer eo naverag e about 25%belo wnormal .Te nboy sha dlevel sbelo w 40 thelowe rlimi to fth enorma lrang eo fchildre nu pt o1 4year sold ,bu t one month later the values of allbu t onebo y werewithi n thenorma l range.A spotat o SGAsar e inhibitors ofCholinesteras e (Orgelle tal. , 1958;Orgell ,1963 ;Pati le tal. ,1972 ;Bushwa ye tal. , 1987),extract s of the toxic potatoes were tested for their inhibitor activity,whic h appeared three timeshighe r thanth eanticholinesteras e activity ofa n extract of potatoes which did not cause symptoms of illness. From a comparison of the anticholinesterase activity of extracts ofprepare d butuneate nbatche so fth etoxi cpotatoe swit hth eactivit yo fth epur e SGAs,th econten to fsolanidin eglycoside s inth eprepare dpotatoe swa s estimated tob e about 250-300mg/kg . Finally the conclusionwa s drawn that "the gastrointestinal, circulatory, neurological and dermatolog- ical findings and the results of laboratory investigations were in keepingwit hsolanin epoisoning" . A fatal case ofpoisonin g of a twoyea rol dgir l that ingested (at least)on epotat ofrui t (berry)wa sdescribe d indetai lb yRüh l (1951). Potato fruitshav e been shown to contain amounts of solanidineglyco sides varying from 420-1080 mg/kg fresh weight (Chapter I, Table2) . Considering all evidence (the nature of the plant material ingested, the clinical symptoms observed and the course of the illness,post mortem and histological examinations, pathological information froma similar case (Terbruggen, 1936), toxicological information in the literature,an d thepresenc e of 'solanine'i nth epost-morte m urine1 4 days after the potato fruit was ingested), Rühl concluded that death wascause db y "solaninepoisoning" . A massive outbreak of poisoning in Scotland in 1917 involved 61 persons; one subject,a five year oldboy , died (Harris andCockburn , 1918). Symptoms reportedwer eheadache ,vomiting ,diarrhoe a anddebil ity. The onset of symptoms varied from almost immediately after the meal to 2 days after the meal; the duration of the illnesses varied froma fe whour st o3 days .Investigation sshowe dconclusivel y thatal l the persons that became ill had consumed potatoes obtained from one source, whilst those persons (often in the same households) who did not partake of the potatoes were unaffected. The toxic potatoes were normal in appearance, although quite a number of them showed sprouts about 0.6 cm in length. Analyses showed that the toxic potatoescon tained 410 mg solanidine glycosides per kg, whilst normal potatoes 41 'considered nontoxic' showed contents of about 80mg/kg . The authors concluded: "theoutbrea ko fpoisonin gwa sdu et oth eeatin go fpotatoe s containinga nexcessivel y largequantit yo fsolanine" . Table 1 presents a historical overview of cases of poisoning in humans reported in the literature and ascribed to the consumption of tubers or other parts of thepotat o plant.I nal l thecase s thesymp toms of poisoning were comparable to those reported by McMillan and Thompson (1979).Fro mthes ecases ,i tca nb econclude d thati ngeneral , the onseto fsymptom svarie s from 1-2h toabou t2 day safte rconsump tion of the potatoes; th e duration of the illness varies from a few hours to about 2weeks . Inmos t cases,circumstantia l evidencejusti fied the conclusion that thepotat o materialwa s thecaus e of thein toxication. The epidemic ascribed to the rotten potatoes (Reelah and Keem,1958 )ca nhav ebee ncause db y avariet y ofsubstance sdu et oth e microbial contamination and cannot be ascribed with certainty to the SGAs. In the cases of poisoning due to consumption ofpotat o fruits, foliage andgreene d tubers,th eSG Acontent swer eno t reported,bu ti t isknow n that such tissues and also small tubers usually containhig h levelso fsolanidin eglycoside s (seeChapte rI ,Tabl e2) . It should be noted ingenera l that inman y casesdescribe d inthi s chapter the potatoes involved in poisoning showed a normal outward appearance. In addition to the case studies referred to above, there are some datafro mexperiment swit hhuma nvolunteer s (summarized inTabl e 2). A recent experiment involved six male volunteers who consumed potato tuberscontainin g32 0m gsolanidin eglycoside spe rk gfres hweight .Th e tuberswer ebake d andconsume d including the skin.Th e amounts ofSGA s ingested varied for differentvolunteer s from 1.75-2.58mg/k gbw .Al l persons showed symptoms of poisoning such as nauseating and severe distress, which started after about 2 h; one person showed no other symptoms;on evomite dafte r4 h ;an dfou rsuffere dfro mdiarrhoe awhic h startedafte r4 h .Symptom stapere dof fafte r8-1 0h (Bushway,1987) . The cases described above,clearl y indicate thatpotat o tubers,ma y cause poisoning in humans, due to their high levels of solanidine glycosides. 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Symptoms of acute toxicity caused by feeding potato mate rial or potato SGAs to animals are described in detail for the cow, sheep, horse and pig by Rühl (1951) and by König (1953) and for the rabbit, mouse, rhesus monkey and rat by Patil et al. (1972) and by Swinyard and Chaube (1973). The symptom complex is essentially the same in all species studied and can be summarized as follows. Neurological effects were prominent in all species and included apathy, drowsiness, weakness or paralysis, tachycardia, dyspnoea, asphyxia, cyanosis and unconsiousness. Dilated pupils or tachypnoea may occur. Gastrointes tinal effects such as vomiting were observed in the cow, sheep, horse and pig and diarrhoea in the cow, sheep, horse and mice. In some cases (cow) exanthematic symptoms such as skin eruptions were reported. Autopsy findings included mild to severe haemorrhagic gastroenteritis, haemorrhagic damage in mouth, nose, lungs, liver, spleen, kidneys, lymph nods, adrenals and salivary glands. Other effects observed were hepatic leucocytic infiltration in the mouse (Sharma et al., 1979), accumulation of pleural and peritoneal sanguineous fluids in the rat and the rhesus monkey (Swinyard and Chaube, 1973; Chaube and Swinyard, 1976), Cholinesterase inhibition in vivo in the rabbit and the dog (Patil et al., 1972) and increased body temperature up to 40.3°C in sheep (König, 1953). An extensive gross and microscopic examination of the pathologic effects of gavaging potato sprouts and potato SGAs to the hamster has been reported by Baker et al. (1987). In this experi ment, 9ou t of 10hamster s receiving dried potato sprout material and 3 out of 5 hamsters receiving potato SGAs died within 72h after dosing. At autopsy the most important effects were severe gastric and intesti nal mucosal necrosis andheamorrhage . The lesions observedwer e indica tive for exposure of the gastrointestinal epithelium to an irritant poison. The hamsters also showed congestion of the kidneys and lungs and gross and microscopic lesions of a variety of tissues. All lesions in the hamsters that were fed sprout material were similar to those in thehamster s that received extracted SGAs. The 3hamster s that survived to the time of euthanasia showed similar but milder symptoms. Gavaging sprout material from which the SGAs had been eliminated by extraction (5 hamsters), or gavaging water (3 control animals), did not produce abnormalities. These experiments showed that the SGAs were responsible fi o •l-l 00 4-1 Ov cd ^N •"N /-N <^v i-i x^l •-s u l-l ^—x O m i-i o 00 •U r- CN r-» r-. vo • ' oo r-. en cr\ r^ CT\ 0\ co o •i-l r-l o CT\i-l i-i •-I cu CT\r-l i-i C N_^ r-i ••—' •«-• •w* l-l v—' v—' •H p. eu t-t 1-4 i-i l-l CU l-l l-l et) i-i cd cd cd Pk cd cd cd 4J 4J 4J 4-> X) 4-> 4-1 eu 4-> CU CU cu CU cu cu e eu cd CU cd co eu •i-l •-I •l-l oe •l-l 3 •H U) 43 •r-l t-el 43 co o N en 4-1 cd co l-l r>H •o 0 O •H cd 43 •H •H cd l-l r-l c Z Pu co < Z rt o M o O a o 3 O < O < 03 P3 w (U ca ca co ca ca co ca CO ca 01 01 co pq o Q fn •x s-l < < < < <: < < <: < w o < < < <: 4J C bO •H C C •H cu O CO O M •r-l •l-l C CU O C4-I 4J CU S-l 4-1 •r-l cd 4-1 d O ü u c^u C ea E CU 4-1 •r-l 01 p. l-l •§ E 4J cu ca ca •I-I •r-l C S-l 3 CH /~S r* /•—\ s~*. 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II X cu H X CU C/5 S-l cd X P-> z Qi co CM oo ^o o oo cd cd 4-1 cd 0) 4-> 4J CU O 0) CD I-I 4-) C CU cu Q> C O IM •|M O bft •H u CU X! co •H e F. i* e e S CD 4-1 UI I-I Cl) Cl) cu cu Cl) M CU ScO "0 •ö Cle) x> "0 Tl cd Cd M M i—i M M cd ^ tó rt e cd e o 4-> a 3 3 o XI X) •H cd cd •U N—' cd ^^ , v l-i ». •o cd bO 4-> X>- , cu M 4-) ro o cd i X! m ft CNI u cd u l-l I-I 6 cd •O - O ••-I cd cd 4-1 X) X X •Ö eu e ^^ X O co - cd ^^ ^^ ^^ 4-1 CM r-~ c •l-l CU l-l 4.) 4-1 4-1 c r^ U for the toxic effects. In sheep, rats, rabbits and rhesus monkeys, feeding of potato foliage, sprouts, greened or sprouted tubers, pro duced the same effects as administration ofpurifie d potato SGAs (Rühl, 1951; König, 1953; Swinyard and Chaube, 1973). From these findings it canb e concluded that the SGAs were responsi ble for the toxic effects caused by the potato material. The clinical and autopsy findings reported for potato SGA poisoning in animals are comparable to those reported for poisoning in human beings caused by potatoes or potato SGAs. Acute toxic effects reported in the above-mentioned studies are included in Table 3 (parental administration) and Table 4 (oral admin istration). An interesting study on the acute and subacute toxicity of potato SGAs is that of Chaube and Swinyard (1976)wh o studied the lethality of a—solanine and a-chaconine after ip administration to female Wistar rats (Table 5). The authors reported that the signs of toxicity were externally: nasal, oral and periorbital haemorrhage; and internally: sanguineous ascitic and pleuritic fluid. A clear dose-response (lethality) relationship for a-solanine and a-chaconine was found. The authors also reported a study on the subacute toxicity of a-solanine and a-chaconine in pregnant and nonpregnant female Wistar rats. The animals received daily 5, 10 or 20 mg a-solanine or a-chaconine per kg bw ip for 8 days, or 40 mg/kg bw ip for 2 days. The results are shown inTabl e 6. External symptoms were similar to those described for acute toxicity. Sanguineous ascitic fluidwa s recovered from all animals that died and from about half of those that survived the dosages of 20-40 mg SGA per kg bw. Histological changes associated with the treatment were limited to moderate congestion of lung, spleen, and liver. The results indicate that in the rat the subacute lethality of the SGAs is quantitatively related to the daily dose and not to the total dose; for instance two daily injections of 40 mg/kg bw (80 mg/kg bw total) were at least as effective as 8 daily doses of 20 mg/kg bw (160 mg/kg bw total). The authors also concluded that pregnant rats are more sensi tive toa-chaconin e thannonpregnan t female rats. 50 Table5 .Acut etoxicit yo fa-solanin ean da-chaconin ei nth era tafte r intraperitonealadministration 3. a-Solanine a-Chaconine Dose Lethality LD50 Dose Lethality LD50 (rag/kg) (rag/kg) (mg/kg) (mg/kg) Dead/total % Dead/total% 20 0/22 0 84.0 20 0/15 0 67.0 50 3/11 27b 40 1/9 llb 75 2/6 33c 60 3/8 38c 85 5/10 50c 85 6/6 100c 100 4/4 100c aFemalerat sreceive dsingl ei pinjections . "Ratsdie d3- 7day safte rinjection . cRatsdie dwithi n2 4h afte rinjection . AfterChaub ean dSwinyard , (1976) Table 6. Subacute toxicity of a-solanine and a-chaconine in the rat after intraperitoneal administration to nonpregnant-female and to pregnantanimals . Dose (mg/kg) Lethality Daily Total a-Solanine a-Chaconine Nonpregnant Pregnant NonpregnantPregnan t Dead/total Dead/total Dead/total Dead/total 5a 40 0/5 0/7 0/6 0/6 10a 80 0/10 0/8 0/11 3/7c 20a 160 0/5 0/6 3/7d 2/4c 40b 80 l/6d 0/6 2/5d 4/6c a,bRat sreceive d8 (a )o r2 (b )consecutiv e ipinjections . 51 Although the data on the oral toxicity in experimental animals are scarce, they suggest that the sensitivity to potato SGAs of human beings corresponds to a larger extent with that of the hamster than with that of the rat or the mouse. Therefore for future experiments one should consider thehamste r as an adequate model. SGAs from wild Solanum species. Wilson et al. (1961) studied the acute toxicity of a-tomatine. A single oral dose of 900-1000 mg/kg bw to rats (5 test animals) was fatal within 24 h. Histologic examination showed an acute gastric mucosal erosion with a mild acute inflammation in the underlying submucosa. This inflammation had produced spasm of the pylorus, which in turn was considered the cause of the stomach to be distended with much fluid. After iv administration of a lethal dose in mice, death occurred within 0.5-2 min, due to a severe disturbance of the central nervous system. There was often bloody nasal discharge. Doses (iv)o f 15mg/k g bw orbelo wwer e always nonfatal; a dose of 17.5 and 20 mg/kg bw produced mortality in 11 out of 17 and 7 out of 11 mice, respectively, whereas doses above 22.5 mg/kg bw were always fatal. Thus, in this experiment the maximum nonfatal dose differed only slightly from the always fataldose . Nishie et al. (1975) compared the acute toxicity of a-tomatine with that of a-solanine and a—chaconine (Table 3).Th e authors concluded that these three SGAs were similarly toxic except for the lethal doses in rabbits; that of a-tomatine was more than twice as high than those of the potato SGAs. The data on the acute toxicity of a-tomatine are tabulated in the Tables 3an d4 . Reproduction and developmental toxicity Human data Potato SGAs. Studies that demonstrate effects of SGAs on reproduction in human beings have not been reported. However, potato SGAs have been associated indirectly with teratogenicity. Iii1972 ,Renwic k reported a correlation between seasonal, geographical and year-to-year variation in the incidence of anencephaly and spina bifida (ASB) in humans and the severity of blight-infestation (Phvtophthora infestans) in pota toes. He guessed that a teratogenic substance in the blight-infected potatoes might be responsible for these severe birth defects, which 52 occur relatively frequently in Britain; the incidence varying for different regions from 2-10 per 1000 births. He suggested that the teratogen could be a potato SGA. On the basis of this rather weak evidence Renwick (1972a; 1972b) evenmad e the suggestion that avoidance of potatoes by pregnant women would virtually eliminate these severe malformations. A subsequent short-term avoidance trial of potatoes and potato- containing food products showed that ASB occurred in 2 out of 23preg nancies in a potato-free group, whereas in a potato-consumers group 2 out of 56 pregnancies resulted in an ASB infant (Nevin and Merret, 1975). This report is regularly referred to in the literature as being evidence against Renwick's hypothesis, but it can only be concluded that thisver y limited study doesno t provide any evidence which canb e used to throw light on this problem. It is concluded that not any evidence of a causal relationship existing between ingestion of blighted potatoes or potato SGAs and ASB inhuma nbeing s has ever been presented. The literature reports on this subject are suggestive and dono t allow conclusions tob e drawn. Experimental-animal data Potato SGAs. Renwick's hypothesis initiated much research to test whether blighted or otherwise imperfect potatoes or potato SGAs, can induce ASB in experimental animals. Many studies provided negative evidence; those that were of a good standard are summarized hereafter. Neither tuber material infested with Phytophtora infestance or with other potato pathogens, nor aged tubers showing high SGA contents, were teratogenic upon gavaging in rats, marmosets,hamsters ,mic e or rabbits (Poswillo et al., 1973; Chaube et al., 1973; Ruddick et al., 1974; Keeler et al., 1975). Administration of o-solanine or a-chaconine, either in pure form or in potato extracts, did not result in birth defects in the following experimental animals: rats, given daily doses up to 25 mg/kg bw orally during day 8-11 or day 6-15 of gestation (Ruddick et al., 1974) or of 40 mg/kg bw ip on day 5 and 6 or 5-20 mg/kg bw ip during day 5-12 (Chaube and Swinyard, 1976); hamsters, given single doses varying from 20-40 mg/kgb w orally on day 7 or day 8 (Keeler et al., 1978); mice, given 20mg/k gb w ip daily during day 7-11 (Bell et al., 1976); and chick embryos, after injection into the yolk 53 sack of amounts up to 30 mg/kg egg (LD50 19 mg/kg egg) (Nishie et al., 1971 and 1975). In several other papers however, it was reported that a-solanine, a-chaconine or potato preparations, some of which were derived from blighted potatoes or from potato sprouts, can produce teratogenic effects. These positive studies must be considered carefully. The few referred to hereafter do not provoke major critical comments with respect to the experimental design. Mun et al. (1975) injected into the yolk sack of fertile chicken eggs an amount of 0.26 mg SGA/egg. The SGAswer e isolated from potatoes infected with blight (Phytophthora infestance). This preparation pro duced 26% embryonal mortality (out of 92 embryos) and 29% abnormal ities, mainl y tail, trunk and rump defects. Control groups showed 1% (out of 88) and 8% (out of 125)mortalit y and 9% and 10%abnormalities , respectively. For an assessment of the hazard of the SGAs inpotatoes , the information on the teratogenicity of the blight infected potato preparations cannot be used, because other compounds formed in the in fected potatoes canb e responsible for the effect as well. A 'solanine' preparation isolated from potato flowers produced 0-27% mortality and 15-25% abnormalities when doses of0.015-0.2 6mg/eg g were injected into the yolk sack. These effect levels were very low compared with those reported by Nishie et al. (1971 and 1975). However, it cannot be ex cluded that this preparation from flowers contained SGAs not found in potato tubers. Mun et al. (1975) did not find a dose-effect relation ship. In summary it can be stated that these authors showed that certain SGA preparations from potato material can induce malformations in chicken embryos, but it is not clear whether their results are of any significance for humanbeings . Renwick et al. (1984) reported cranial defects and exencephaly in hamsters given orally on day 7 or 8 of gestation, either a-solanine or a-chaconine in single doses varying from 165-200 mg/kg bw, or SGAs isolated from potato sprouts in a single dose of 240 mg/kg bw. The authors also reported a dose-related teratogenicity of potato sprouts in hamsters, but upon a closer look this relationship appeared to be based on the highest dose level only. This level, which was equivalent to about 225 mg SGA per kg bw, was not a realistic dose as it produced 31% maternal lethality. 54 Keelere tal .(197 6an d1978 )foun dpotat osprouts ,fro ma numbe ro f cultivars,whic h contained 220-400 times asmuc h SGAs as tubers tob e teratogenic inhamsters .However ,th edose sadministere d caused11-48 % maternal mortality. The terata included spinabifid a and exencephaly. Potato tubers or peelswer e not teratogenic at dosesu p tofou rtime s as high as the sprout dosages (5-21% maternal lethality) (see also Keeler, 1979). With respect to the birth defects in hamsters, it should bemen tioned that a particular strain of hamsters (Simonsen)wa s chosen as test animal to study teratogenicity of SGAs and SAs, because this strainha dprove n tob e sensitive tolo wdose s ofS Ateratogen san dt o produce a high incidence of deformities (Keeler et al., 1976). The oral doses of isolated SGAs or of sprouts,whic h produced teratogenic effects in thehamster s in the above studies,wer e quitehigh ,a swa s apparent from maternal mortality (see also Table 4 Renwick et al., 1984). Inthi srespec ti tshoul db enote dtha trecently ,th eWH OExper t TaskGrou po nUpdatin gth ePrinciple sfo rth eSafet yAssessmen to fFoo d Additives and Contaminants in Food (WHO,1987 )recommende d that: "for assessing, whether the organism is more sensitive to the agent under test during its reproductive and developmental stages than during its adultphase ,th ehighes tdos e offoo dchemica l that isadministere di s generally the amount that would be expected to cause slight maternal toxicity".Th egrou pfurthe rstate dthat :"I fth etes tsubstanc einjur es reproduction or development at levels comparable with levels that causetoxicit y inadults ,the nn ospecia lconcer nshoul db eattache dt o the results of the reproduction/developmental toxicity studies" (WHO, 1987). Consequently,on eca nno tconclud e from thehamste r trialstha t potatomateria lo rpotat oSGA sshoul db econsidere da steratogens . Considering all evidence, it can not be excluded that very high doses ofpotat o SGAsma yproduc ebirt h defects.However , there isfai r evidence from experience with known teratogens (e.g. thalidomide, vitaminA )tha ta 'noeffec tlevel 'concep tapplie st othes echemicals . Therefore,i tdoe sno tsee mlikel ytha tth epotat oSGA sar eteratogeni c atlevel spresen t inth ecurren tpotat ocultivars . Most of the above studies,regardles s ofwhethe r orno tbirt hdefect s were induced, showed that potato SGAs and potato preparations con- 55 taining the SGAs adversely affected the reproduction rate inexperi mental animals. Especially foetal mortality and resorption, but also abortion and sometimes decreased fertility of proven breeders, were reported. The lowest ip administered doses reported in the literature to produce effects on the reproduction of mammals are 5-20 mg/kg bw (Chaubean dSwinyard , 1976;Bel le tal. , 1976).Th erelevan tdat afro m theseliteratur ereport sar etabulate di nTabl e7 . Kline et al. (1961)reporte d a low oral doseo f 'solanine't opro duceeffect so nth ereproductio no fth erat .I nthi sstudy ,fiv egroup s ofpregnan trat swer e feda dlibitu m throughoutpregnanc y acommercia l laboratory diet or this diet mixed either with ground frozen potato sprouts or with 'solanine' in concentrations of 30 or 40mg/k gdiet . Unfortunately, the food consumption was not reported. The results of thisexperimen t (Table8 )showe dtha tth enumbe ro fpup spe rlitte rdi d notdiffe rbetwee nth etreatments ,bu ttha tbot hth epotat osprout san d 'solanine' caused a high incidence of neonatal mortality. Death oc curredwithi n3 day so fbirt hdu e tostarvation .A s thepup stha tdie d did not have milk in their stomachs, the authors presumed that the potato SGAs "may be toxic for certain functions such as lactation, possibly by virtue of an anti-hormone effect."Furthe r informationt o support this presumption was not given. As the amount of 'solanine' ingested by the animals isno tknown , thisrepor tca nno tb euse dfo r an appropriate hazard assessment, but anyhow the oral dose level of 'solanine', 30mg/k g in the diet,wa s low. The relatively low levels (oralan dip )o fSGA stha tproduce deffect so nth ereproductio n inrat s andmice ,sho wtha tthi saspec tdeserve sfurthe rattention . u CU x) CU o e 4-1 fi cd x) o 4-1 CU S-l S-l cd S-l CU cd 4-1 (U £1 ^VO^ r-l ^~^v£> 4-1 3 C>- , r-~ r-l cd e» X) CU cd •H C3> •-I CJN CU X S iH CU 4-1 1-4 4-1 o C/1 pa cu cd *—' ^^ r-l cd o 6 en 4-1 •r-l G O •r-l •r-l 4-1 O o o 1-4 LO CU i-l ••-4 cu o o o O o C CU G o •r-cl 0 W) C o o o O O •r-l m> •r-l ort v v v V V oc 4J -1 U) O p-i Oi p.. eu eu o ü cd 3 rC x) 4-1 4J M-l 4-1 ü 0 0 G O G !-l cd cd ö P. G G 0) O<* > bO O<* > bO X) U O N .- o eu o CU G 4-1 O 1-1 S-i CM 1-1 S-i cd G CN 1 P. VO 1 P- O O \X ) vo r-l vr> CU CU \C N CM e» CM er. G U) S->l . 4-1 dp c*> r-l \r ^ • H 4-1 rQ O ^5 m 0<*0> 4-1 e\ n 4-1 <\ f C o eu r-l VO r-l 4-1 O 4J o cd IV eCU 4-1 G C C C r-l 4-1 4-1 c G e cd G •r-l cd G •r-l O 4-1 cd o O O G O C o en CU oc •1-1 •1-1 •1-1 bû •r-l cn bû •r-l cn S-l 4-1 4J 4-1 eu 4-1 cu eu 4-1 cu ö CU 4J CU P. P. P. S-l P. cn S-i p. cn O o S-l S-l S-l P. S-l 3 S-l 3 4-1 3 eu x>g O o o O 4-1 a o 4-1 O -o 4-1 en en en 4-1 en CU 4-1 en CU o 4-1 •z3 eu eu CU O CU O O eu O CU u W erf ai erf S-l CH S-l 4-1 S-l p. z z 3 4-1 o X 4-1 •H X) a CU r-l 4-1 cd cd S-l 4-> S-l O S-4 3 P. u O 4J CU eu cd !-l rfi s C 4J 01 O S-l eu CU S eu £. XI r0 4-1 •r-l UI S-l o cd o C ü z 0 E •r-l •H 1-^1 CU- 4-1 M c eu G S-l •r-l O et) •H eu 4-> o CU XI G en eu 4-1 4-1 4-1 o cd cd •H eu P. cd cd cd •1-1 r-l X) H C/3 erf erf erf O eu o ••r-ol a en en 4-1 G 3 cd Ofi CU eu ö CU •H •H CU G C CU en 3 o 4J e CU •i-i •r-l S-i cd xs U) cd ••-I x> •re4 CU S S-l •1-1 oe oe rC en 4-1 •U •1-1 en o o ced 4-1 r-l S-l O en C o cd co r-l •H cd o •H cd u 4= ,C O CU •r-l rC U) C 1-4 r»1 o o C/} S-l 4J eu •1-1 O 1-1 O o 4-1 eu 3 M C/5 Ml co cd 4-1 cd o xe) rÖ cd X) cd 4-1 S eu 4-> r-4 en , s 4-1 en Cd G XS 4-> 0) CU UI •i-I CU ced G O ? C S-i CU cd 4-1 o O c^d Te3 eu E r-l r-l u Q cd 4-) P. bO •r-l eu )-l G ^—' •fiH r^ tu •r-l O X) p. 'S) C 4-1 S-l eu ed w cd cd o I—1 s-i r->4 , CU .* r-l 4J Ü £i 4-i •i-I en \W > O O o ed C cd O o C/3 P. H 'r-l Q •O •^—•e* CM -1 < CM 57 Table8 .Effec to fpotat osprout san do f 'solanine'o nneonata l mortality inra tpups . Testdie t Number Numbero fpup s Numbero f of litterswit h litters Born Weaned (%) 100%mortalit y Control (C) 11 115 95 (82.6) 1 C+10%potat osprout s 9 83 42 (50.6) 5 C+30mg/k gsolanine 1 4 41 8 (19.5) 2 C+30mg/k gsolanine ^ 10 100 31 (31.0) 6 C+40mg/k gsolanine' ' 10 106 33 (31.1) 5 Isolatedfro mpotat osprouts . o Commercially obtainedsolanin estandard . AfterKlin ee tal . (1961). SGAs from wild Solanum species.Wit h respect to teratogenicity ofth e SGAs from wild Solanum species, only few studies have been reported. Tomatine injected into theyol k sacko fchicke negg s inconcentration s varying from0.5-2 5mg/k geg gweigh tproduce d asignifican t (P= 0.05) embryomortalit y inchicke neggs ,bu ti twa sno tteratogeni c (Nishiee t al., 1975). Aspar to fa stud yfo rrevealin gstructura lrequirement sfo rterato genicity of Veratrum and Solanum SAs, the aglycones solasodine and tomatidine were tested (Keeler et al., 1976). A single high dose of solasodinebetwee n118 0an d163 0mg/k gbw ,gavage dt ohamster sa tda y7 or 8 of gestation, resulted in a large increase in foetal resorption (total resorption in21/11 5pregnan t dams; intw ocontro l groups 1/135 and2/57 )an da hig hincidenc eo fmalformation s (grossbirt hdefect si n 33% oflitter s involving 50/727 foetuses;i ntw ocontro l groups 3/1400 and 2/548foetuses) ,amongs twhic hneura l tubedefects .Tomatidin ewa s notteratogeni cno rdi di tproduc eresorption sa ta singl edos eo f120 0 or270 0mg/k gbw .Bot hthes eapola raglycone sproduce dlittl eo rn o 58 maternal mortality, probably due to their poor absorption from the gastro-intestinaltract- 1-. It can be concluded that tomatidine does probably not present a teratogenic hazard to humans. However, it can not be excluded that solasodine athig h dosage levels is teratogenic. As there areuncer tainties towards theoccurrenc e ofsolasodin e incurren tpotat oculti - vars (Chapter IX),on e remark should bemade .Th e aglycone solasodine isprobabl ypoorl yabsorbe dfro mth egastrointestina l tractcompare dt o its naturally occurring (glycosidic) form, which will be much better solubilized in the stomach. Therefore, it canno tb e totally excluded thatth esolasodin eglycoside sar eteratogeni c inhumans . EVALUATIONO FTH ETOXICOLOGICA LLITERATUR EDAT A Evidencefo rtoxicit yo fsolanidin eglycoside s inhuman s The studieso n thetoxicokinetic s showed thatSGA sar eabsorbe db yth e humanbod y inamount sproportionall y relatedt oth eamount so fpotatoe s (or potato products) consumed. Upon regular consumption ofpotatoes , thesolanidin e glycosides aswel l asfre eaglycon ema y accumulate toa certain level in various organs. In many countries potatoes are an important component of the diet and they are consumed almost daily. Thismean stha tchroni cexposur ema yoccur . Two sourceso f informationo n the toxicity ofSGA sar eavailabl ei n theliterature :firstly ,cas estudie so fpoisonin g inman ;secondly , 1In a similar study (Brown and Keeler, 1978) four synthetic solani- danes, the 22S.25R epimer of solanidine and the 22R.25S, the 22S,25S and the 22S.25R dihydro-5a forms of solanidine were tested, indose s varying from 39-343 mg/kg bw. All four solanldanes produced dose- related foetal resorption. The 22R.25S and 22S.25S solanidanes were not, but the two 22S.25R solanidanes were highly teratogenic, pro ducingmalformation s atdose sabov e 80mg/k gbw .Thi s isa lo weffect - level, considering that probably also these aglycones are poorly absorbed from the gastrointestinal tract. These data show that SAs possessing the 22S.25R configuration, including solasodine, are more likely to produce birth defects than 22R.25S SAs, such as solanidine andtomatidine .However ,th estructura lan dconfigurationa lspecificit y forteratogenicit yamon gSA san dSGA si sstil lno tclea r (Keeler,1986 ; Gaffield, 1986). Especially the reported teratogenicity of thepotat o SGAs (solanidine glycosides)canno tb e explained by the currenthypo thesisbase do nth estereochemica lconfiguration . 59 data from experiments, a few of which carried out with human volunteersan dthos einvolvin gexperimenta lanimals . Inth efirs tsourc eo finformation ,man ycase so facut ean dsubacut e poisoning of human beings due to consumption of sprouted, greened, small,o rotherwis e imperfectpotatoe s oro fothe rpart s ofth epotat o plant havebee n described. The statement oftenmad e in the literature for over 60year s now, that qualitatively imperfect potatoes arepoi sonous due to contents of solanidine glycosides exceeding 200mg/kg , is obviously based on this information. It can not be refuted that potatoes of inferior quality can cause poisoning inhuma nbeings .Th e literaturedat ashowe dtha tman ycase so facut epoisonin ghav eoccurre d after consumption of potatoes containing solanidine glycosides in amounts of 230 mg/kg or above. In several reports,th e facts clearly pointed towards these SGAs asbein g responsible for the cases ofpoi soning, although the evidencewa scircumstancia l;causa lrelationship s haveno tbee ndemonstrated .Th e dataarguin g onth epossibl eteratoge nicity ofpotatoe s inma n are suggestive andn o otherdat a onchroni c adverseeffect s inhuma nbeing shav ebee nreported .Therefore ,fro mth e first source of information it can only be concluded that sprouted, greened, smallo rotherwis e imperfectpotatoe s cancaus epoisonin gdu e toexcessiv elevel so fsolanidin eglycosides . The second source of information, the literature describing the experimentswit hhuma nvolunteer san dwit hexperimenta lanimals ,showe d thatpurifie d solanidineglycoside san dpotatoe scontainin ghig hlevel s ofthes eSGA sca nexer tacut etoxi ceffects .I nvariou sanima lspecies , a causal relationship between administration ofpurifie d potato SGAs, a-solaninea swel l asa-chaconine ,an d theproductio no f toxiceffect s was demonstrated. The symptom complex, the clinical as well as the autopsy findings, in human beings of so-called 'potato poisoning' or 'solanine poisoning' is essentially the same as the symptom complex produced in test animals that received purified potato SGAs orpotat o materialcontainin ghig hSG Alevels .Thes efact ssuppor tth ehypothesi s thatth eSGA sar eresponsibl efo rcase so fpotat opoisonin g inman . Acceptable levelfo rSGA si npotatoe s Many scientistswh o carriedou t studieso ncontent s of solanidinegly cosides in potatoes (see Chapter I) generally accepted the idea that 60 excessive levels of these SGAs cause poisoning inhuma n beings.Mos t of them refer in their papers to a so-called upper safety limit (accept able level) for contents of solanidine glycosides inpotatoes . Different safety limits have been proposed in the literature, but the one most often referred to is that of Borner and Mattis (1924). These authors analysed batches of potatoes involved in cases of acute and/or subacute poisoning of human beings in different parts of Ger many, and batches which did not cause symptoms of illness. The toxic tubers contained levels of solanidine glycosides varying from 257-583 mg/kg (Tabel 1),whils t the nontoxic potatoes contained levels of20 - 150 mg/kg. At that time, Griebel (1923) had reported that potatoes containing 380-790 mg 'solanine'/kg (Tabel 1) caused nauseating, vom iting and a strong irritation in the neck region in a large number of adults. Potatoes, which had been grown on the same site as the toxic potatoes, but which did not cause noticeable symptoms of illness con tained 196 mg 'solanine'/kg. From Griebel's and their own results, Bömer and Mattis (1924) concluded: "potatoes with a solanine content exceeding 200mg/k g seem to cause adverse effects onhuma nhealth" . Since then, many authors have assumed without further evidence that levels below 200 mg/kg are safe. They ignore the fact that the 200 mg/kg level only relates to acute and/or subacute effects and not to possible chronic effects. It should also be mentioned in this context that most of the researchers concerned are not toxicologists, but chemists, geneticists and plantphysiologists . Other authors have proposed lower acceptable levels because they took additional relevant facts into consideration. Ross et al. (1978) stated that as the contents of solanidine glycosides in potatoes vary with the year and location of growth, an upper limit of 60-70 mg/kg is to be applied for cultivars to be selected for human consumption. Recently, the National Institute of Agricultural Botany (NIAB) at Cambridge (UK) recommended, that the overall amount of SGAs ingested by the public should not be allowed to rise. NIAB therefore suggested 'the average SGA content over all eight maincrop recommended potato varieties', which was calculated (over years and locations) to be 60 mg/kg, as a guideline for potato breeders to bear in mind when submit ting new potato cultivars for National List testing (Parnell et al., 1984). 61 More recommendations on safety limits have been made in the lit erature, bu t none of them is based on an appropriate toxicological evaluation. Morris and Lee (1984) calculated the toxic doses for potato SGAs from thecase so fpotat opoisonin greporte d inth eliterature .Accord ingt othes e authors toxic dosesamounte d toabou t 2-5mg/k gbw .Thei r estimate was based on the upper SGA levels reported for the toxic potatoes (cf.Tabl e 1). Theyassume dth eamoun to fpotatoe sconsume dt o be 500 g (except for one epidemic for which 250 g was taken,a sth e serving had been specified being small) and the body weights of the persons affected tob e 80k g for adults,6 0k gfo rchildre nan d4 0k g forsmal lchildren .Th eauthor sstate dtha tth eestimate swer edesigne d toprovid e conservative values for the toxic doses, and that theSGA s couldb etwic ea stoxi ca scalculated . Lepper (1949),Viollie r (1941)an dAlf aan dHey l (1923)reporte dSG A levels oftoxi cpotatoe s (Table 1)no t included inth e studyo fMorri s andLe e (1984). Inthes ecase sth etoxi cdoses ,estimate db yth emetho d ofMorri san dLe e (1984),rang efro m2- 3mg/k gbw . Pfuhl (1899),wh oexamine da sever eepidemi co f 'solanine'poisonin g in the German army, stated that 200-400 mg ofpur e 'solanine'cause d symptoms of poisoning in human subjects. These amounts corresponded with his calculations of the amounts of potato SGAs ingested by the poisonedsoldiers ,whic hwer ea tmaximu m30 0mg ,correspondin gt oabou t 4mg/k gbw . In the experiments with human volunteers, amounts of 200 mg pure potato SGAs or more were reported to cause symptoms of illness in adults,whic h means that about 3mg/k g bw isa nacut e toxic dose.Th e acute toxic doses of potato SGAs consumed in their natural matrix, calculated from experiments with volunteers and from cases of potato poisoning,wer e between 1.75 mg/kg bw and 5mg/k gbw .Th e lowestdos e reported to cause acute toxic effects in human beings was thus 1.75 mg/kgbw . This means that an amount of10 5m gpotat o SGAs ingestedb y an average person (60kg )ma y potentially cause poisoning. This dose is ingestedwhe n 525g o fpotatoe s containing 200m g solanidineglyco sidespe rk gi sconsumed .Thi si sa fairl ylarg eamoun to fpotatoe sbu t consumptiono fsuc hamount s isno texceptional . Although an acceptable level for solanidine glycosides inpotatoe s 62 canno tb e derived fromdat ao nacut e toxicity, thesedat aappea ruse ful in showing that the so-called safety limit of 200 mg/kg, often referred to in the literature, is inadequate as virtually no margin existsbetwee n amounts ofpotatoe s thatma y cause acute toxic effects and amounts that are being consumed. It must therefore be concluded that the levels of solanidine glycosides inhousehol d potatoes should remain considerably below 200mg/kg ; this applies of course also for newcultivar scontainin ggermplas mfro mwil dSolanu mspecies . Literatureo nchroni ceffect s inhuma nbeing s isno tavailabl e thusth e no-adverse effect level is unknown. Consequently, anacceptabl e level forsolanidin eglycoside s inhousehol dpotatoe sca nno tb ederive dfro m thedat ao nhuma ntoxicity . Much literature on the toxicity of the potato SGAs in experimental animals is available. However, many studies show shortcomings from a toxicological point of view and/or with respect to essential infor mationo no rth equalit yo fth epotat omateria lo rth eSG Apreparation s thatwer etested .Th eresult so fsuc hstudie sca ntherefor eno tb euse d fora nadequat e toxicologicalevaluatio neither . Theanima lstudie so nth eacut ean dsubacut e toxicityd ono tprovid e information on the no-adverse effect level.Th e datapresente d inth e (sub)chronic studyb yKlin ee t al. suggesting thatrelativel y lowora l doses of 'solanine'ma yproduc e neonatalmortalit y inth e rat,ca nno t be used either because data on the 'solanine' intake are lacking. Therefore, it must unfortunately be concluded that, because of the virtual absence of chronic toxicity data, an adequate no-adverse effect level for potato SGAs can not be assessed. Thus there is no appropriate startingpoin tfo rth eestablishmen to fa so-calle daccept able daily intake (ADI)figur e forman ,an d consequently, theaccept ableleve lfo rsolanidin eglycoside s inpotatoe sca nno tb ederived . Hardlyan yinformatio no nth etoxicit yo fth eSGA sfro mwil dSolanu m speciesuse d inpotat obreedin g programmes isavailabl e inth elitera ture. Consequently, appropriate ADI figures forma n canno tb eestab lished for these SGAs, and theymus t therefore notb e introduced into thehousehol dpotato . In conclusion, it canb e stated that the 'safety limit'o f 200m g solanidine glycosides perkg ,propose d inth e literature forhousehol d 63 potatoes aswel la swit h respect toth eregistratio n ofne wcultivars , must be considered potentially acute toxic. The ingestion by the public ofth e amounto fsolanidin e glycosides shouldno tb eallowe dt o risebu tshould ,o nth econtrary ,preferabl yb ereduced .Therefore ,th e SGA contents of new household potato cultivars should not be higher than the average level of the current cultivars; preferably their contents should be lower. SGAs alien to S. tuberosum must not be introduced into thehousehol d potato.A scertai ncondition s duringth e growthan dpost-harves tperio do fpotatoe sca nresul ti na considerabl e accumulation of these SGAs (Chapter I), i t is advisable that forth e selection ofne w cultivars the guideline of about 60-70m gSG Ape rk g potatoes -recommended by Ross et al. (1978) and by NIAB (Parnell et al., 1984)- isuse di npotat obreeding ,unti la nappropriat eacceptabl e levelha sbee nassessed . Quantitative data reported in the following literature have beencon sulted for the present study but were not included for reasons such as: improper experimental design of the study, lack of informationo n essential experimental details, questionable quality of the potato material orSG Apreparation s tested,recantatio no f theresult sb yth e authors, or insignificance of results for human beings; Azim et al. (1982), Clarkee tal . (1973), ElShabraw y andNeg m (1980),Fontain ee t al. (1947), Gull etal . (1970),Harve y etal . (1986), Königan dStaff e (1953), Lorber et al. (1973), Pierro et al. (1977), Poswillo etal . (1972), Sackmanne tal .(1959 )an dSwinyar dan dChaub e (1973). REFERENCES AldousCN ,Sharm aRP ,Salunkh eDK . (1980)Effect so fchaconin eo nbrai n biogenic amines,electroencephalogram , cardiac rates andrespirator y response inrats .J Foo dSafet y2:20-33 . AlfaJ ,Hey lE .(1923 )Z Unter sNahr -Genuss m46:30 6 (Quotedfro mBöme r andMattis ,1924) . 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(1963)Inhibitio no fhuma nplasm aCholinesteras e invitr ob y alkaloids, glycosides, and other natural substances. Lloydia 26:36- 43. Orgell WH, Vaidya KA, Dahm PA. (1958) Inhibition of human plasma Cholinesterase in vitro by extracts of Solanaceous plants. Science 128:1136-1137. Parnell A, Bhuva VS, Bintcliffe EJB. (1984)Th e glycoalkaloid content ofpotat ovarieties .J Nat nIns tAgri cBo t16:535-541 . Patil BC, Sharma RP, Salunkhe DK, Salunkhe K. (1972) Evaluation of solanine toxicity.Foo dCosme tToxico l10:395-398 . Pfuhl E. (1899)Uebe rein eMassenerkrankun g durchVergiftun gmi tstar k solaninhaltigenKartoffeln .Dtsc hMe dWochensch r25:753-754 . Pierro LJ, Haines JS,Osma n SF. (1977)Teratogenicit y and toxicity of purifieda-chaconin ean da-solanine .Teratolog y15:31A . Poswillo DE, Sopher D, Mitchell S. (1972) Experimental induction of foetal malformation with 'blighted' potato: a preliminary report. Nature239:462-464 . 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Terbruggen A. (1936)Tödlich e Solaninvergiftung, zugleich einBeitra g zurBedeutun gde rakute nHirnschwellun gbe ivorzeitige r Synostosede r Schädelnähte und verringerter Schädelkapazität. Beitr Path Anat 97:391-395. Viollier R. (1941) Bericht über Lebensmittel-Kontrole imKanto nBase l Stadtwähren dde sJahre s1939 .Zeitsc hLebens mUnter sForsc h81:192 . WHO (1987)Principle s for the Safety Assessment of FoodAdditive s and Contaminants inFood .WHO ,Geneva . Willimott SG. (1933) An investigation of solanine poisoning. Analyst 58:431-439. Wilson GS. (1959)A small outbreak of solanine poisoning. Mon. Bull. Minis.Healt hPubli cHealt hLa bSer v18:207-210 . WilsonRH ,Pole yGW ,DeEd sF . (1961)Som epharmacologi c andtoxicologi c properties of tomatine and its derivatives. Toxicol Appl Pharmacol 3:39-48. Zettel H. (1937) Über Erkrankungen durch solaninreiche Kartoffeln. Ernährung2:71-75 . 69 CHAPTERIII * TWO-PHASEHYDROLYSI SO FSTEROIDA LGLYCOALKALOID SFRO MSOLANU MSPECIE S INTRODUCTION Wild andcultivate d primitive Solanum speciesar ebein guse d inpotat o breeding to introduce avariet y of desirable characteristics intoth e cultivated potato, for instance: disease and insect resistances;to lerances to frost, heat and other physiological stresses; improved yield,starc hconten tan dprocessin gquality . The cultivar Lenape inherited from its wild ancestor S. chacoense several of the mentioned useful characteristics but in addition the potencyt oaccumulat elarg eamount so fSGA s (Sindene tal. , 1984).Thi s was a serious warning that the utilization of germplasm from wild Solanum speciesmus t be approached with caution (see also ChapterI) . Other examples that this utilization may lead to new cultivars with high contents of SGAs have also been reported. In The Netherlands, genotypescontainin ggermplas m fromvariou swil dSolanu mspecies ,whic h had been developed for industrial starch production only, showedcon tents of solanidine glycosides up to 630mg/k g (VanGelder , 1982). In Scotland many advancedbreedin g clonesderive d fromS .verne iBitt .e t Wittm.,whic h is a source of resistance against the potato cystnema tode, appeared useless due to contents of solanidine glycosides upt o 1570mg/k g (Coxon et al., 1983). As aresult ,potat obreeder s areno w becoming morean dmor eawar e that introductiono falie ngermplas m into potato cultivars mustb e supportedb y SGA analyses.Gregor y thepres ent Scientific Director of the International Potato Center at Lima (Peru) stated: "Absence of glycoalkaloid assessment from a breeding programmeca nresul ti nwaste deffort ,tim ean dmoney " (Gregory, 1984). ChapterI Ishowe dtha tdu et oth escarc einformatio no nth etoxicit y Thischapte r isbase do nth epaper : Van Gelder WJM. (1984)A new hydrolysis technique for steroid glyco- alkaloids with unstable aglycones from Solanum spp. J Sei Food Agric 35:487-494. 70 of the SGAs fromwil d Solanum species, their acceptable levels for food products can not be established, and that consequently these SGAs must not be introduced into the cultivated potato. It is therefore necessary to analyse wild species for their SGA composition before they are used ina breedin g programme. A common step in the methods for SGA analysis is acidhydrolysi s of the SGAs in order to cleave the sugar moieties from the aglycones. The aglycones are subsequently extracted with an organic solvent and usu ally quantified as 'total glycoalkaloids' by a colorimetric or titri- metric procedure (Coxon, 1984). For determining SGAs in (wild) tuber- bearing Solanum species these quantitative techniques have been em ployed in combination with separate qualitative analyses of the main aglycones by chromatographic techniques (Osman et al., 1978;Gregor y et al., 1981). The methodology for the analysis of SGAs has recently been reviewed (VanGelder , 1989). However, the conventional acid hydrolysis of SGAs, applied in the studies reported until now, yields low and irreproducible recoveries, varying from 35-90%, at least for the solanidine glycosides and a- tomatine, even under optimized hydrolysis conditions (Coxon et al., 1979; Mackenzie and Gregory, 1979; Blincow et al., 1982; Davies and Blincow, 1984). The initial aim of the present study was to investigate thehydro lysis of glycosides of solanidine, demissidine, tomatidine and sola- sodine in order to select a procedure giving optimum recoveries, equal for the various aglycones. As solasodine glycosides were not avail able, only their aglycone could be tested for its stability under the hydrolysis conditions. The results showed that the conventional acid hydrolysis methods were not satisfactory because of degradation of aglycones. Alkaline hydrolysis was not promising either, because alkalis do not hydrolyse solanine (Henry, 1949). Therefore a new technique was developed, al lowing a simultaneous quantitative recovery of the aglycones from different types of SGAs. It exploits the differences in polarity be tween the SGAs and their aglycones. The hydrolysis medium consists of an aqueous acid and a nonpolar organic solvent, resulting in two immiscible phases. The polar SGAs are hydrolysed in the acid phase and the nonpolar aglycones formed are continuously withdrawn from the acid 71 into the organic phase, inwhic h they areprotecte d from furtherdis integration.Thu sth ehydrolysi sca nprocee dunti lth evariou sSGA sar e quantitatively cleaved. Afterwards, the aglycones can easily be recovered from the organic phase. The significance of this procedure foranalytica lan dpreparativ eapplication s isdiscussed . EXPERIMENTAL Standardalkaloid san dothe rchemical s Solanidine glycosides were kindly supplied by Mr P. Plieger (Proef station voor Aardappel Verwerking-TNO, Groningen, The Netherlands), solasodine and solasodiene by Mr A.L.M. Sanders (Diosynth, Oss,Th e Netherlands). a-Tomatine was obtained from Roth GmbH & Co (Karlsruhe, F.R.G.), a-solanine from Sigma (St.Louis ,MO ,U.S.A. ) and tomatidine from Fluka AG (Buchs, Switzerland). Solanidine and solanthrene were preparedb yhydrolysin gth esolanidin eglycosid emixture ,an dseparate d and purified as described in the Thin-layer chromatography paragraph. Demissinewa sextracte d fromleave so fS .demissu mLindl .b yth emetho d ofWan ge t al. (1972).Afte rconcentratin g theextract ,th ep Hwa sad justed to 10b y adding ammonia (5mol/1 )a t 70°C.The n demissinewa s allowed toprecipitat e overnight at2°C .Th eprecipitat ewa scollecte d by centrifugation for 20mi n at 11000 g,washe dwit h 10m l ofaqueou s ammonia (1 mmol/1)/methanol (1:1 v/v),an d centrifuged again. The demissine precipitate was recrystallized twicefro mho tethano l (96%). The purity ofth epreparatio nwa schecke db y thin-layerchromatograph y (TLC), as described hereafter for SGAs. The identity ofdemissin ewa s checkedb ycompariso no fit saglycon ewit hdemissidin e (Roth)usin gTL C and mass spectrometry (MS) (Mr CA. Landheer, Department of Organic Chemistry,Agricultura l University,Wageningen , TheNetherlands) .Als o the identity of solanidine and solanthrene was confirmed by MS. All otherchemical swer eanalytica lgrad e (E.Merck ,Darmstadt ,F.R.G.) . Plantmateria l Tuber samples of thepotat ohousehol d cultivar Bintje and ofth ecul - tivar Astarte, which is used only for industrial starch production, wereobtaine dfro mth ePotat oBreedin gDepartmen to fth eFoundatio nfo r AgriculturalPlan tBreedin gSV P (Wageningen,Th eNetherlands) . 72 Hydrolysiso fpur eSGA s Standard solutions were made up of,accuratel y weighed, 4m gSG A or2 mg SA per ml solvent. SGAs were dissolved in methanol/aqueoushydro chloric acid (0.25 mol/1) (1:1 v/v),an d SAs in methanol/chloroform (2:1 v/v). For conventional acidhydrolysis , 2.0 ml samples of the SGAan dS A standard solutions were added to 100m l of aqueous hydrochloric acid (Table 2).Durin ghydrolysis ,th etemperatur eo fth eaci dwa s97°C . Forhydrolysi s ina two-phas e system,1. 0m l samples ofth eSG Aan d SA solutions were pipetted into flasks, towhic h were added 50m l of hydrochloricaci di nth econcentration sgive ni nTabl e2 ,an d10 0m lo f carbon tetrachloride. During hydrolysis, the temperature of the lower (organic) layer was 80°C and that of the upper (aqueous) layer85°C . Hydrolyses were carriedou t induplicate ,usin g reflux condensers,fo r thereactio nperiod sgive ni nth eTable s2 an d3 . Estimationo frecover yo ftota laglycone s For theestimatio no f therecover y afterconventiona l acidhydrolysis , 4.0 mlo f thehydrolysat e werepipette d intoa 1 0m lvolumetri cflask . Theaci dwa sneutralize dwit haqueou ssodiu mhydroxid e (12mol/1) ,the n 2.0 ml of glacial acetic acidwer e added,an d the flaskwa s filledt o themar kwit h demineralizedwater .T oestimat e totalaglycones ,5. 0m l of this solution were transferred to a separatory funnel for complex formationwit hmethy l orange inacetat ebuffer ,a sdescribe db yBirne r (1969). After hydrolysis in the two-phase system, 10.0 ml of the hydro chloric acid phase and 20.0m l of the carbon tetrachloride phasewer e used to estimate the aglycone recovery. The former phase was adjusted top H1 0wit haqueou ssodiu mhydroxid e (12mol/1 )an dextracte dwit h1 0 mlo fchloroform .Th eextrac twa scombine dwit hth e2 0m lcarbo ntetra chloride phase and the whole washed with 20m l of aqueous ammonia (1 mmol/1).Th eorgani c liquidwa sevaporate d todrynes san dth eaglycone s weredissolve d in10. 0m lo faqueou saceti caci d (50%v/v) .The n2. 0m l of this solution and 3.0 mlo fdemineralize dwate rwer e transferredt o a separatory funnel to estimate the aglycones by complex formationa s above. •o G 4-1 4-> et) C CU cCU cu C O) 4-> 3 u O cu u r-l w ta cu o 43 CU cu S-l 4-1 a S-l S-i bO O 4J 0 CU O \ ,0 r^ 42 3 4-1 3 C>U , >, es >•CU> CU bO r-l •rl r-l 54 cu •o S-l S-l •rl 4-1 r; 4-1 0 S-i 0 ü rJ V 3 *—' O >CU u CU U) 43 o CU + 4-> cu CU S-l u bO cu u a cu \>- Ï >i r^ CU , *~N ,-1 r-l o 4-> *•—4J \ 44 /—s s~\ o C 43 44 4J cu cu cu bO (3 (3 cu 3 0 O •r-l cu CU 44 3 r-l UI ui r-l ü C!*U> r->.l 0 43 r-l 42 cu CU UI UI 5-1 4-1 UI bß 43 S-l S-l cu bO 43 cu o •rl 4-1 0 CU 0 —^ S-l S-l cd M r-l 4>i CU 43 3 4-1 3 >1 0 \C U •H 0 S-l 3 bO •H •rl r-i CU 3 3 i-I 3 p> ct) r-l •rl 4-1 f-; 4-1 S-l r-l r-l U) 1-1 a 03 rJ v-^ *—' 0 4-1 4-1 S-l o !=> 5 « — 3 CM 43 P. r-i G 3 •H M r>ï CU a 44 CU cu 3 O 42 S-l r-l X) 5-1 c-o bO bO r-, 43 43 CU S-l 4-1 \ \ 4-i CU CU 4-J M CU Thin-layerchromatograph y ForTLC , theaglycone swer e recovered from thehydrochlori c acidphas e and the carbon tetrachloride phase as described above.Afte revapora tion of the organic solvent, they were dissolved in 100 pi methanol/ chloroform (2:1v/v )an d2 pi ofthi ssolutio nwer eapplie dt oa What man TLC plate LK5D 20x 20 cm, togetherwit h 10 fig of standardalka loids. After equilibration of the TLC chamber for 30mi nwit h 100m l cyclohexane/ethyl acetate (55:45 v/v),th e plate was developed to a height of 14 cm. The SAs were detected by spraying the chromatogram with a solution of iodine in carbon tetrachloride (1g/10 0ml ). Fo r preparative TLC, 1m g samples of the SAswer e applied toWhatma nPL K plates. After the brown/yellow colour of the iodine-sprayed SAs had disappeared, theSA swer e recoveredunchange d (Schreiber et al., 1963) by elution with methanol/chloroform (2:1 v/v).Identificatio n of the SAs was performed by comparison of their Rp values and their colour development with those of standard compounds. For this colourdevel opment the TLC plates were sprayed with a saturated solution of cerium(IV) sulphate in concentrated sulphuric acid. Subsequently, observations of the coloured SAswer e made at room temperature,afte r heatingth eplate s ina nove na t120° Cfo r3 min ,an dafte rheatin gth e plates asecon d time for 10min .Th eR pvalue san dth ecolou rdevelop mento fth eSA sar epresente d inTabl e1 . TLC was also used in preliminary experiments carried out to study the partitioning of a-solanine and its hydrolysis products over the two-phase systems hydrochloric acid and carbon tetrachloride,hydro chloric acid and chloroform, and hydrochloric acid and n-hexane. The aglycones were recovered as described above and the SGAwa s recovered from both separated phases of each of the systems as follows.Afte r washing with aqueous ammonia (1mmol/1 ), th e organic phasewa sevapo rated to dryness and the residue dissolved in5 0/i lo fpyridine .A 1 0 /J1sampl ewa sapplie dt oa Whatma nLK5 Dplat efo rTLC .Th ehydrochlori c acid phase was made alkaline (pH 10), kep t at 70°C for 30 min and stored overnight at2°C .a-Solanin ewa sprecipitate db y centrifugation for 20mi na t1100 0g ,washe dwit h 10m lo faqueou sammoni a (1mmol/1 ) andcollecte db ycentrifugatio nagain .Subsequentl y itwa sdissolve di n aminimu m ofpyridine ,an d samplescontainin g 10-20 fig ofth eSG Awer e then applied to aLK5 Dplat ewhic hwa sdevelope d toa heigh to f1 4c m 75 using methanol/chloroform/water (5:5:1 v/v) as eluent (Sachse and Bachmann, 1969). Visualizatio n of the spots was as described for the aglycones. Hydrolysis of SGAs inpotat o tuber extracts Fresh potato tubers of 'Bintje' and 'Astarte', with a low and high content of solanidine glycosides respectively, were used. From samples of ten tubers of each, longitudinal sections were cut, to obtain sub- samples of 300 g. The subsamples, which were representative of each tuber in the sample as was tested earlier, among others in an inter- laboratory study (Morgan et al., 1985), were homogenized with 75 ml of water in a Waring Blendor. While homogenizing, nine samples each of 25 g of brei (corresponding to 20 g of fresh tubers)wer e taken for anal ysis, and to each sample 100 ml methanol/chloroform (2:1 v/v) were added (immediately) in order to prevent enzymic action. For extraction of the glycosides, a modification of the extraction procedure of Wang et al. (1972) was used. The suspension of tuber brei in methanol/ chloroform was transferred to a Waring Blendor using 25 ml of the methanol/chloroform for rinsing the glasswork. The suspension was ho mogenized for 2 min and then filtered through a Büchner funnel fitted with filter paper (Type 589/5, Schleicher and Schüll, Dassel, F.R.G.). The remaining pulp and the filter were re-extracted for 2mi n using 75 ml of the methanol/chloroform mixture. The combined filtrates were transferred to a separatory funnel, and 80 ml of a sodium sulphate so lution (55 mmol Na2SÛ4 per 1)wer e added. The mixture was gently shak en and then left overnight to allow complete separation of the layers. The chloroform (lower) layer was discarded and 15 ml of the sodium sulphate solution were added to the remaining liquid. After gently shaking, this mixture was left for 4 h and then the newly formed chloroform layer was discarded. The remaining (aqueous) methanolic layer was used for analysis. Allowing the solvent layers to separate for a sufficiently long period of time isessential . To study the influence of tuber extracts on the recovery of the aglycone solanidine after the two-phase hydrolysis, 2 mg accurately weighed solanidine glycosides dissolved in 1.0 ml methanol/chloroform (2:1 v/v) were added as a spike to the methanolic layers of three of the nine brei samples of each cultivar. 76 The methanolic layer was concentrated to near dryness in a water bath at 50°C and the residue transferred to an Erlenmeyer flask using 50 ml of aqueous hydrochloric acid. The conditions for conventional hydrolysis were: hydrochloric acid 1 mol/1, reaction period 2 h; and for the two-phase hydrolysis:hydrochlori c acid 2mol/ 1 towhic h 100m l of carbon tetrachloride were added, reaction period 2.5 h. After conventional acid hydrolysis, 5.0 ml of the hydrolysate were transferred to a 10 ml volumetric flask and processed further as de scribed for estimation of the recovery of total aglycones. After the two-phase hydrolysis, the system was allowed to cool and the pH of the aqueous phase was adjusted to 10wit h ammonia (7 mol/1). After separation of the layers, the aqueous phase was washed with 20m l of carbon tetrachloride and this carbon tetrachloride layer was added to the 100 ml carbon tetrachloride phase; then this total amount was washed with 25 ml of aqueous ammonia (1 mmol/1). From the total volume of carbon tetrachloride, amounts of 12.0 ml and 6.0 ml, for the 'Bintje' and the 'Astarte' samples respectively, were evaporated to dryness. The aglycones were redissolved in 5.0 ml of aqueous acetic acid (20% v/v),an d these solutions were transferred to a separatory funnel to estimate the SAs as described above. RESULTS AND DISCUSSION Hydrolysis ofpur e SGAs Table 2 shows that the acid hydrolysis of solanidine glycosides re sulted in varying losses up to 59%. Cleavage of these SGAs yields the aglycone solanidine and the sugar moieties solatriose and chacotriose (Chapter I, Fig. 1 and Table 1).Monitorin g by TLC of a mild and a vigorous hydrolysis of a-solanine showed that solanidine was converted to solanthrene. This dehydration product was very unstable and disap peared with concomitant formation of 2-12 degradation products. When solanidine was subjected to the hydrolysis conditions, the solanthrene formed was disintegrated even more than during the hydrolysis of a- solanine. Probably, the presence of the sugar moiety retards the for mation and through that the disintegration of solanthrene, and thus influences the recovery of aglycones. 77 Table 2. Recovery ofaglycones , includingdehydrate d derivatives,upo n conventional acidhydrolysi s of steroidalglycoalkaloids ,an drecover y ofsteroida lalkaloid ssubjecte dt oth esam econditions-*- . Steroidal Reaction Concentration Recovery (glyco)alkaloid period(h ) ofHC L(mol/1 ) (%) Solanidine 0.5 77 glycosides 1 83 2 78 2 78 2 73 4 64 6 41 Solanidine 55 37 16 Solasodine 2 97 3 98 4 92 Demissine 2 102 3 98 4 99 2 90 4 93 o-Tomatine 2 82 3 90 4 86 2 0 4 0 -•-SGAs,8 mg ;SAs ,4 mg ;aqueou shydrochlori cacid ,10 0m la t97°C . 78 As solasodine glycosides (solamargine, solasonine) were not avail able, experiments were carried out with the aglycone solasodine. Sola sodine plus its dehydration product solasodiene could be recovered almost quantitatively after a period of 2-3 h under rather mildhydro lysis conditions. These data suggest that the hydrolysis of the sola sodine glycosides may proceed without significant losses, because both aglycones canb e quantified together. Hydrolysis of demissine yields the aglycone demissidine; this SA was stable even after a prolonged hydrolysis period under vigorous condi tions. About 90% of tomatidine, the aglycone resulting from the cleav age of a-tomatine, could be recovered after employing rather mild hy drolysis conditions, but this SA was totally disintegrated when a higher acid concentration was used. Preliminary tests using TLC indi cated that differences existed between demissine and a-tomatine, as to reaction rate of the glycosidic cleavage. However, these SGAs possess the same tetrasaccharide moiety, lycotetraose (Chapter I, Table 1). Thus the difference between these SGAswit h respect tohydrolysi s seems to be due to differences in the chemical structures of the aglycones. This, together with the demonstrated influence of the sugar moiety on the stability of solanidine, suggests that hydrolysis of the SGAs is influenced by the chemical structures on both sides of the glycosidic bond. After the studies described here were carried out, Davies and Blincow (1984) reported that losses of a-solanine upon acid hydrolysis varied largely, when in different laboratories the same method was applied for assessing the content of solanidine glycosides inpotatoes . The losses depended on the concentration of solanidine glycosides and on the presence or absence of potato tuber extract in the hydrolysis medium. Those results and the results reported in this chapter, show that conventional acidhydrolysi s isno t suitable for thehydrolysi s of mixtures of SGAs. Table 3 shows the aglycone recoveries after hydrolysis of glyco- sidic-bound solanidine and tomatidine in a two-phase system of carbon tetrachloride and aqueous hydrochloric acid as well as the recovery of solasodine after being subjected to these hydrolysis conditions. Compared to conventional hydrolysis, recovery of the unstable aglycones was little affected by the reaction period and the acid concentration. 79 Table 3.Recover y of aglycones, including dehydrated derivatives, upon hydrolysis of steroidal glycoalkaloids using a two-phase system consisting of carbon tetrachloride and hydrochloric acid, and recovery of solasodine subjected to the same conditions . Steroidal Reaction Concentration Recovery (glyco)alkaloid period (h) ofHC L (mol/1) (%) Solanidine 1.5 2 70 glycosides 2 2 95 2.5 2 96 4 2 93 1.5 4 95 2 4 90 2.5 4 92 a-Tomatine 2 94 3 96 4 92 Solasodine 1.5 103 2 97 2.5 98 •'•SGAs,4 mg; solasodine, 2mg ; hydrochloric acid, 50m l at 85°C; carbon tetrachloride, 100ml . As was to be expected, recovery of the stable SA solasodine was also very high. The low recovery of solanidine after 90mi n in the two-phase system (hydrochloric acid concentration 2mol/1 ) was due to incomplete hydrolysis and not to aglycone degradation. The optimum conditions for the two-phase hydrolysis of the solanidine glycosides and a-tomatine were a reaction period of 2.5-3 h and an acid concentration of 2mol/1 , giving a recovery of about 96%. The lower reaction temperature (85°C) compared to the conventional procedure (97°C) may also have had some favourable influence on the recoveries. The high recoveries were largely explained using TLC experiments, a- 80 Solanine stillyielde d (viasolanidine )solanthrene ,bu t thiscompoun d didno t disintegrate.a-Solanin e and themajo r part of the solanidine remained in the aqueous acid phase,bu t the unstable solanthrene all remained in theprotectiv e organic phase. Inexperiment s withchloro form instead ofcarbo ntetrachloride ,TL C showed thata-solanin estil l remained in the acid phase,bu t that solanidine almost quantitatively remained in the protective chloroform phase, resulting in much less dehydration to solanthrene. Using a two-phase system of n-hexane and aqueous hydrochloric acid (2 mol/1), a-solanine, solanidine and the major part of the solanthrene were found in the acid phase.Thi sre sulted in a very low recovery because of disintegration of theagly - cones. Hydrolysiso fSGA si npotat otube rextract s Table 4 shows that the precision (coefficient of variation) and the yield in the determination of solanidine glycosides in tubers of the household potato cultivar Bintje and the industrial starch cultivar Astarte, improved considerably byusin g thetwo-phas ehydrolysi stech nique. Again low coefficients ofvariatio nwer e obtained; the average recoveries of the theoretical amounts of solanidine glycosides were about 95% and 101% for 'Bintje' and 'Astarte', respectively. These valuesar ehighe r thanthos ereporte db yothe rresearcher swh oaccepte d recoveries of 70%an d coefficients ofvariatio n of 15% (Coxone tal. , 1979; Coxon, 1984;Davie s and Blincow, 1984). Inman y otherpaper so n this topic,dat a on accuracy andprecisio n of themethod s appliedar e worse (seeCoxon ,1984 )o rno teve npresented . The recoveries in Table 5 compare well with the optimum values in Table 3.Thi smean s that thesolanidin e glycosides canb e convertedt o their aglycones reproducibly and sufficiently quantitatively, bytwo - phase hydrolysis of pure SGAs aswel l as of SGAs inpotat o tuberex tracts. A probable explanation is that the nonpolar compounds inth e potato tuber extract thatca npotentiall y interferewit h theaglycone s during hydrolysis are discarded with the chloroform layer after the extraction step with the methanol/chloroform mixture. The polar com pounds which can potentially interfere with the aglycones are sepa rated from the aglycones liberated during two-phase hydrolysis, as a resulto fth edifference s inpolarity . 81 Table 4. Comparison of conventional acid hydrolysis-1- (CAH) and two- phase hydrolysis^ (TPH) in the determination of solanidine glycosides in tubers of twopotat o cultivars. Cultivar Hydrolysis Solanidine glycosides in fresh tuber Yield^CAH technique Yield TPH Absolute Average Coefficient of (%) (mg/20 g) (mg/20 g) variation (%) Bintje CAH 0.56 0.61 25.61 71.7 0.48 0.78 TPH 0.88 0.85 11.16 0.74 0.92 Astarte CAH 3.66 3.83 10.38 83.7 4.28 3.54 TPH 4.48 4.57 2.49 4.70 4.54 ^CAH conditions: 50m lhydrochlori c acid 1mol/1 , reaction period 2h , reaction temperature 97°C. TPH conditions: 50m l hydrochloric acid 2mol/1 , 100m l carbon tetra chloride as protective phase, reaction period 2.5 h, reaction temperature 85°C. The procedure used for extraction of SGAs from potato tubers as introduced by Wang et al. (1972) has been criticized by some authors (Mackenzie and Gregory, 1979; Speroni and Pell, 1980), but proved tob e most satisfactory when applied in the present as well as in other studies (Clement and Verbist, 1980; Morgan et al., 1985). The present study showed that reproducible and quantitative results canb e obtained by this extraction procedure,provide d that the tuber homogenate isex tracted twice and the solvent layers of the chloroform/methanol/sodium o 4-1 £ O ^-v •u 4-1 <*> 4-1 G o eu C •H O o •1-1 U) G ••-I 4-1 4-1 •H 4-1 ta u 4-1 •H m CJN ta Ma eu S-l CM r^ S-4 eu O ca 4-1 4-1 u > S-l M Q) CD eu •X3 M -39 •i-l ea 01 eu •H UI G eu >s •H 1—1 > o •o •i-i u •CH 4-> oo ci CN m in ed cfl T3 i-l 1-1 m ON r-l i-i CM O eu dP CJN o CJN o CJ\ O c/> as ^ i-i i-l 0) U) U) M h~ oo VO ix> ro r-- G rO 6 O *w CM CM CM vO VD vc a < 3 «1 dl "0 •H 0) O Ü r-l ,—i ta txO b0 VI O S CD ••-I ••—' sulphate mixture are allowed to separate for the periods of time described in the Experimental section. Although the data presented in the Tables 4 and 5 can have been affected by anumbe r of analytical errors, such as inhomogeneity of the tuber brei, weighing errors, losses due to extraction, hydrolysis, clean-up and quantification of the solanidine glycosides, the overall error of the analytical procedure now presented is rather small, es pecially in comparisonwit h those of the reports mentioned before. To obtain optimum results with the two-phase system, the unstable aglycones must almost totally remain in the organic phase, and to limit the duration of hydrolysis, the substrate has to remain largely in the acid phase. Variation in liquid phases, especially by combining dif ferent organic solvents, enables one to obtain an optimum partition coefficient for almost any compound. So this technique may be appli cable to other chemical procedures where a hydrolysis step leads to side reactions or degradation. For instance, the estimation of cardiac glycosides, which is disturbed by side reactions during the hydrolysis necessary for genin formation (Meilink, 1980), may be improved by ap plication of a two-phase system. The production of solasodine from Solanum spp. for the pharmaceutical industry is attended by a consid erable loss, resulting from the formation of solasodiene,whic h can not be used for steroid hormone synthesis (Mann, 1978).Thi s dehydration is analogous to the solanthrene formation, which was shown in this study to be controllable. Therefore, solasodine dehydration may be prevented by using a two-phase hydrolysis with a carefully selected organic phase. In conclusion it can be stated that the two-phase hydrolysis tech nique can succesfully be applied to different SGAs, and that it offers prospects for developing a comprehensive and quantitative method for determining the SGA compositions of Solanum species and their hybrid offspring. The procedures for sample preparation and clean-up seem useful too,bu t quantitation, separation and identification of SGAs are rather complicated and time-consuming. Application of more advanced chromatographic techniques will perhaps be more efficient. REFERENCES Birner J. (1969) Determination of total steroid bases in Solanum spe cies. J Pharm Sei 58:258-259. 84 Blincow PJ, Davies AMC, Bintcliffe EJB, Clydesdale A, Draper SR, AllisonMJ . (1982)A screening method for the determination ofgly - coalkaloids in tubers of potato cultivars. J Natn Inst Agric Bot 16:92-97. Clement E,Verbis t JF. (1980)Dosag e de la solanine dans letubercul e de Solanum tuberosum L.: étud e comparative de neufméthode scolori - métrique.Lebens mWis sTechno l13:202-206 . Coxon DT. (1984) Methodology for glycoalkaloid analysis.A m PotatoJ 61:169-183. Coxon DT,Davie sAMC ,Morga nMRA . (1983)Specia lRepor tNo .7 :Glyco - alkaloids inpotatoes .AR CFoo dResearc hInstitute ,Norwich . CoxonDT ,Pric eKR ,Jone sPG . (1979)A simplifie dmetho dfo rth edeter mination of total glycoalkaloids inpotat o tubers.J Sei FoodAgri c 30:1043-1049. Davies AMC, Blincow PJ. (1984)Glycoalkaloi d content of potatoes and potatoproduct ssol di nth eUK .J Se iFoo dAgri c35:553-557 . Gregory P. (1984)Glycoalkaloi d compositiono fpotatoes :diversit yan d biological implications.A mpotat oJ 61:115-122 . Gregory P, Sinden SL,Osma n SF,Tinge y WM, Chessin DA. (1981)Glyco alkaloids of wild tuber-bearing Solanum species. J Agric Food Chem 29:1212-1215. HenryTA . (1949)Th ePlan tAlkaloids .J an dA Churchil lLtd ,London . Mackenzie JD, Gregory P. (1979)Evaluatio n of a comprehensive method fortota lglycoalkaloi ddetermination .A mPotat oJ 56:27-33 . MannJD . (1978)Productio n ofsolasodin e forth epharmaceutica lindus try.Ad vAgro n30:207-245 . Meilink JW. (1980) Gas chromatography and cardenolides. PhD Thesis, StateUniversit yLeiden ,Th eNetherlands . MorganMRA ,Coxo nDT ,Bramha mS ,Cha nHW-S ,Va nGelde rWMJ ,Alliso nMJ . (1985)Determinatio no fth eglycoalkaloi dconten to fpotat o tubersb y threemethod s includingenzyme-linke dimmunosorben tassay .J Se iFoo d Agric36:282-288 . Osman SF,Her b SF,Fitzpatric kTJ ,Schmiedich eP . (1978)Glycoalkaloi d composition of wild and cultivated tuber-bearing Solanum species of potential value in potato breeding programs. J Agric Food Chem 26:1246-1248. Sachse J, Bachmann F. (1969) Über die Alkaloidbestimmung in Solanum tuberosumL .Z Lebens mUnter sForsc h141:262-274 . Schreiber K,Auric h 0, Osske G. (1963)Solanum-Alkaloid e XVIII.Dünn schichtchromatographie von Solanum-Steroidalkaloiden und Steroid- sapogeninen.J Chromatog r12:63-69 . Sinden SL,Sanfor d LL,Web b RE. (1984)Geneti c and environmentalcon trolo fpotat oglycoalkaloids .A mPotat oJ 61:141-156 . SperoniJJ ,Pel lEJ . (1980)Modifie dmetho dfo rtube rglycoalkaloi dan d leafglycoalkaloi d analysis.A mPotat oJ 57:537-542 . Van Gelder WJM. (1982) Alkaloids in potatoes. SVP Berichten No.17 . Foundation forAgricultura l PlantBreedin g SVP,Wageningen ,pp . 1-12 (inDutch) . Van Gelder WMJ. (1990) Steroidal glycoalkaloids: consequences for potatobreedin g and foodsafet y ofutilizin gwil d Solanum speciesi n breeding programmes. In Handbook of Natural Toxins, Vol. 6, RF Keeler,A TT u (Eds).Marce lDekke r Ine,Ne wYork ,i npress . Wang SL, Bedford CL, Thompson NR. (1972)Determinatio n of glycoalka loids inpotatoe s (S. tuberosum)wit ha bisolven t extractionmethod . AmPotat oJ 49:302-308 . 85 CHAPTERIV * CAPILLARYGA SCHROMATOGRAPH YO FSTEROIDA LALKALOID S INTRODUCTION Themethodolog y for SGAanalysi s isver y complex,whic h isillustrate d by the hundreds ofpublication s describing thedevelopmen t ormodifi cation of methods for SGA analysis, of which more than 60 appeared during the past decade only, and which may alsob e apparent from the structures of these compounds (Chapter I, Fig 1an dTabl e 1).Man yo f thesemethod s havebee n developed for determining the contents ofso - lanidine glycosides in potatoes. Such methods, which do not apply to SGAs alien to S.tuberosum ,hav e been used for determining the total SGAcontent so fwil dSolanu mspecie san dthei roffsprin g (Coxone tal. , 1983; Grassertan dLellbach , 1987).Thi sma ylea dt ounderestimatio no f these contents, and possibly the introduction of undesired types of SGAs intone wpotat ocultivar swil lb eoverlooked .Th eai mo fth estud y presented in this chapter was to develop ametho d for the separation and individual quantification of the SGAs of wild Solanum species. Several authors recently stated that there isa nurgen t need forsuc h a method which must be at the same time comprehensive,quantitative , and efficient enough, for routine incorporation into breeding pro grammes (Gregory,1984 ;Sinde ne tal. ,1984 ;Tingey ,1984) . SELECTIONO FCHROMATOGRAPHI CTECHNIQU E Techniques that can be applied for analysis of SGA compositions are TLC, high-pressure liquid chromatography (HPLC), gas chromatography Thischapte r isbase do nth epaper s: Van Gelder WJM. (1985)Determinatio n of the total C27-steroidalalka loid composition of Solanum species by high-resolution gaschromato graphy.J Chromatog r331:285-293 . VanGelde rWMJ ,Scheffe rJJC . (199*)Strateg y foranalysi so fsteroida l glycoalkaloids inpotat o and tomato genotypes containing wild Solanum andLycopersico ngermplasm ,i npreparation . 86 (GC)usin g packed columns,an d capillary GC. In this section theap plicability of these techniques for analysis of the SGAs isevaluate d inorde rt oselec tth emos tpromisin gtechnique(s )fo rfurthe rstudy . TLCi sespeciall yvaluabl efo rqualitativ eanalyse so fSA san dSGAs . Many TLC systems for the separation and detection of these compounds have been developed, and are reviewed by Baerheim Svendsen andVer - poorte (1983). Sensitive reagents for visualization of SAs and SGAs areals oavailabl e (Jellemae t al., 1980)an dquantificatio nb ydensi tometry, although generally quite difficult, is feasible (Ahmed and Müller, 1978; Caddie et al., 1978;Jellem a et al., 1981 and 1982). A disadvantage ofTL C is that for the separation of complexmixture so f SAso rSGAs ,differen tsystem shav et ob eapplie d insuccession . Inth epas tdecade ,TL Canalysi so fSA san dSGA sha sbee nsupersede d byHPL Can dGC ,becaus eth eresolutio no fthes einstrumenta ltechnique s improved greatly and they can be automated for sample injection and quantification. Moreover, microprocessor-controlled programming allows variables such as column temperature, flow rate,mobil e phase,o rde tector sensitivity, to be changed automatically during analyses.HPL C and GC are complementary techniques. For separation of thermolabile, strongly polar,high-boilin g or nonvolatil e compounds,HPL C isofte n preferable.A nadvantag e ofG C isth eavailabilit y ofhighl y sensitive andgenerall yapplicabl edetectors . HPLC andG Co npacke d columnsca nb eapplie d for theseparatio nan d Individualquantificatio no fSGA san dthei raglycone swhe nonl y asin gle SGA is present in plant material,o rwhe n only a single aglycone is present glycosidically bound to different sugar moieties such as solanidine in a-solanine and a-chaconine. HPLC methods forquantifi cation ofa-solanin e and ofa-chaconin e inextract s ofpotat otubers , havebee n described (Morris and Lee,1981 ;Bushwa y et al., 1986),an d such methods specifically developed for the solasodine glycosides in Solanum species of potential use to the pharmaceutical industry, are available too (Crabbe and Fryer, 1980; Eldridge and Hockridge,1983 ; Cham andWilson , 1987). However, inman y Solanum species,severa l SAs may occur simultaneously, and each of them may be bound to various sugar moieties (Chapter I); the n many SGAs occur ina single species andth eseparatio nefficienc yo fHPL Cwil lb einsufficient .A G Cmetho d for the separation of SGAs on a 3%OV- 1colum nprogramme d up to350° C 87 has been described (Herb et al., 1975), which could potentially be applied to mixtures of SGAs such as those occurring inwil d Solanum species (Osmane t al., 1979). However, thismetho d suffered fromsev eral disadvantages. The samples had tob e derivatized bypermethyl - ation, which is difficult and laborious, and a chromatographic run lasted foralmos t twohours .Onl y relative quantificationwa spermit ted, thusa separate analysis forth etota l SGAconten twa srequired . Moreover, due toth ehig hG Coperatio n temperature, columndeteriora tion occurred already after 100analyse s (Herbe t al., 1975;Osma ne t al.,1978 ;Gregor ye tal. , 1981). •A nalternativ e approach consistso fextractio nan dtwo-phas ehydro lysis of the SGAs, separation and individual quantification ofth e aglycones,an dexpressio no fthei rcontent sa sglycosidic-boun dSAs . Forth eseparatio no faglycones ,HPL Ci sno tpreferabl ea stw otime - consuming analyses,on efo rth emor e polar andon efo rth eles spola r SAs were required (Hunter et al., 1980). Moreover, although HPLCwa s notapplie dquantitativel yb yHunte re tal . (1980), theirdat areveale d that theultraviolet-detecto r responsesvarie d largely forth ediffer entSAs .Thi slimit sth epossibilitie sfo rquantificatio no fth eSA sb y this technique. In general, the ultraviolet and refractive-indexde tectors used inHPL C are insensitive to saturated SAs,wherea s flame ionisation detection (FID)use d inG C isver y sensitive andgenerall y applicablet oSA san dSGAs . Quantificationb yFI Do fsolanidin e injecteddirectly , i.e.underi - vatized, into the gas Chromatograph has been reported (Osman and Sinden,1977 ;Coxo ne tal. ,1979 ;King ,1980) .However ,th eA5 -an d5a - aglycone pair solanidine and demissidine could notb e separated.Fo r the quantification of these aglycones, a separate analysis had tob e carried out in which solanidine was converted into solanthrene and demissidine remainedunchanged . A recently developed GC technique which is potentially useful to analysis of SAs, is capillary GC, also called high-resolution GC (HRGC). This technique enables efficient separations of complexmix tures inrelativel y shortperiod s oftime ,becaus e open tubeswit ha n internal diameter (I.D.)o f0.1-0. 3m mar euse d inwhic h thestation aryphas e isdirectl y coated asa thin film,usuall y0.1-0. 4/xm ,ont o thewal l ofth ecolumn ;a sa resul ta lo wcarrie r gaspressur e canb e 88 applied enabling the use ofver y long columns (10-100 m). With increas ing length and decreasing I.D. of the column, the separation efficiency increases proportionally. However, these improvements are attended by a proportionate increase in the complexity of the technique and of the number of problems which can be encountered. Therefore, capillary GC demands a much more careful approach than GC onpacke d columns. The literature referred to above as well as preliminary experiments showed that with respect to detection and quantification, GC of agly- cones liberated during two-phase hydrolysis of SGAs was the most prom ising approach, and that with respect to the separation efficiency, capillary GC offered prospects for developing a method for determina tion of the SGA composition of Solanum species. Therefore GC analyses of SAs using packed as well as capillary columns were compared and the most promising technique was further optimized. EXPERIMENTAL Apparatus A Packard 439 microprocessor-controlled gas Chromatograph was used, equipped with two flame ionization detectors and two injectors for splitless and split sampling techniques. One injector was used for a glass column (1m x 2m m I.D.) packed with 10%SE-3 0 on Chromosorb W HP (80-100 mesh) and the second one for fused-silica columns. The fused- silica columns of 50 m x 0.22 mm I.D. were coated with CP-Sil 5, film thickness 0.12 /im,o r CP-Sil 19 CB, film thickness 0.19 /um.Al l columns were obtained from Chrompack Nederland (Middelburg, The Netherlands). The maximum operating temperatures of the SE-30 and CP-Sil 5 columns were 325°C (isothermal) and 350°C (programmed), and of the CP-Sil 19 CB column, 300°C and 325°C, respectively. Other conditions were: injector, 325°C; detector, 350°C; flow-rates for FID, hydrogen 25 ml/min, air 250 ml/min; detector sensitivity, 1 pA/mV, unless stated otherwise. The injectionvolume s and splitting ratios are given either in the text or in the captions of tables and figures. Chromatographic data were recorded and calculated using a Shimadzu C-R2A data processor. Standard compounds and other chemicals 5a-Cholestane was obtained from Sigma and toluene (analytical grade) 89 from E.Merck . All other chemicals were as described inChapte rIII . Standardswer edissolve d inmethanol/toluen e (1:1v/v) . Plantmateria l Tubersample sfro mhousehol dpotat ocultivars ,cultivar s forindustria l starchproduction ,progenie s fromcrosse sbetwee nSolanu m tuberosumL . and S.verne i Bitt. etWittm . and accessions of theprimitiv eculti vated speciesS .phurej aJuz . etBuk .wer e studied.The ywer eobtaine d from thePotat oBreedin g Department ofth eFoundatio n forAgricultura l PlantBreedin gSVP . Quantificationo fsolanidin eglycoside s Potato tuberswer e sampled andhomogenized , and the SGAs extracted as described in Chapter III. The aqueous methanolic layer obtained was evaporated tonea rdryness . Forth equantification sb ycapillar yG Cth econcentrate dextrac twa s hydrolysed using the two-phase technique. The hydrolysis conditions were: 50m l ofhydrochlori c acid (2mol/1 )an d 100m lo fcarbo ntetra chloride;reactio nperio d 3h .Afte rhydrolysis ,th ephase swer ealka - linized and the carbon tetrachloride phase containing the aglycones was separated, and the aqueous phase washed with 25m l of chloroform and thendiscarded . Thecombine dorgani cphase swer ewashe dwit h2 5m l ofammoni a (1mmol/1 )an devaporate d tonea rdryness .Subsequently ,th e aglycones were transferred quantitatively tocrim p topvial s andafte r evaporation of the solvent, theywer e redissolved in 1.0 mlmethanol / toluene (1:1v/v ) or diluted further as required. The additionalcap illary GC conditions were as follows: carrier gas, helium, linear velocity 24.3 cm/s; oven temperature, 280°C; injection volume, 2 pi; splitting ratio, 1:100. For quantification of solanidine and itsde hydration product solanthrene, their (combined) peak areas were com pared with those of a calibration line that was constructed using amounts of 0.1, 0.5, 1.0, 2.0 and 3.0 mg solanidine in 1.0 mlmetha nol/toluene (1:1v/v) . 5a-Cholestanewa suse da sinterna lstandard .Th e concentration of an SGA was calculated from the concentration of the corresponding aglycone (including its dehydration product) by multi plyingth elatte rconcentratio nb yth erati oo fth emolecula rmasse so f theSG Aan dit saglycone . 90 For quantification of solanidine glycosides by a colorimetric meth od, the concentrated aqueous methanolic layer was transferred to a 30 ml centrifuge tube and the beaker was rinsed twice with 5m l of acetic acid (0.8 mol/1). The solution was brought to pH 10wit h about 3m l of ammonia (7 mol/1) and heated to 70°C. The solanidine glycosides were left overnight at 2°C and were then collected by centrifugation for 15 min at 11000 g. The dried precipitates were redissolved in 1 ml of phosphoric acid (0.5 mol/1), and subsequently 10 ml freshly prepared Clarke reagent, containing 300 mg paraformaldehyde in phosphoric acid (8.5 mol/1), were added.Afte r 30min , the absorbances were measured at 600nm . The concentrations of the solanidine glycosides were calculated using a calibration line that was constructed using amounts of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 mg of a-solanine in 1.0 ml of phosphoric acid (0.5 mol/1). Depending on the colour intensities of the solutions, absorbances were measured using spectrophotometer cells of 5.0 mm or 10.0 mm path length. An inter-laboratory study (Morgan et al., 1985), in which this colorimetric method was compared with two other methods for quantification of solanidine glycosides in potato tubers, showed that the colorimetric method used here was sufficiently accurate andprecis e inorde r tob e used as a reference method. RESULTS AND DISCUSSION GC analysis of SAs using packed and capillary columns In studies on the GC separation of SAs using packed columns, attempts to separate the A5- and 5a-aglycone pair solanidine and demissidine were not successful, although various column types and GC conditions were applied (King, 1980). The closely related chemical structures of A5- and 5a-aglycone pairs (Chapter I, Fig. 1) result in only minute differences between their boiling points. Therefore, separation of these compounds will be very difficult. Fig. 1A shows a gas chroma- togram obtained for a mixture of SA standards, the solanidanes solani dine and demissidine, and the spirosolanes tomatidine and solasodiene, using a 1 m SE-30 column. Calculations using data from the individu ally chromatographed SAs gave plate numbers, N, for these compounds varying from 1877 to 2656 plates/m. The plate number required for the separation of solanidine, demissidine and solasodiene with resolution 91 v... -1 «— 10 15 10 20 min Fig.1 .Ga schromatogram sobtaine dfo rsteroida lalkaloid susin gpacke d (A)an d capillary (B)columns . Packed column:glass ,1 m x 2 m m I.D., 10% SE-30 on Chromosorb W HP (80-100 mesh). Carrier gas: nitrogen, flow-rate1 5ml/min .Ove ntemperature :260-300° Ca t5°C/min ,the n300° C for 10 min. Detector sensitivity: 10 pA/mV. Injection volume: 1ji l containing2 fig ofeac hcomponent .Capillar ycolumn :fuse dsilica ,5 0m x 0.22m m I.D., CP-Sil5 ,fil m thickness0.1 2 pm. Carriergas :helium , linear velocity 24.5 cm/s. Oven temperature: 290°C. Injectionvolume : 1 /ulcontainin g 1 pg ofeac hcomponent ;splittin g ratio:1:130 .Peaks : 1 = solvent; 2 = solanidine; 3 = demissidine; 4 = solasodiene; 5= tomatidine; 6 = solasodine. For further details see Experimental section. R = 1 (98%baselin e separation for solanidine-demissidine and demis- sidine-solasodiene) was 678000. Thus the theoretically requiredmini mum column length was 305 m. Consequently, no further attempts with thistechniqu ewer emade . Fig. IBshow sa chromatogra m ofth e samemixtur e ofSA sobtaine db y capillary GCusin g a5 0m CP-Sil 5column .Th eretentio ntimes ,t^ ,o f solanidine (peak 2) and demissidine (peak 3)wer e still very close, CR 2 = 13.10 min and tj^3 = 13.29 min respectively, but due to the smallpea kwidth s agoo d separation,R = 1.3,wa sachieved .Solasodin e (22aN-spirosol-5-en-3-ol, peak 6),whic h was added to the alkaloid mixture, was well separated from its dehydration product solasodiene (peak 4) and from tomatidine (22/3N-spirosolan-3-ol,pea k 5), Thepea k 92 symmetry appeared tob equit e good,althoug h some tailing of thepeak s 5 and 6 was noticeable. This tailing effect decreased uponoptimiza tiono fth eG Cparameters ,bu ti tneve rtotall ydisappeared . 240 260 280 300 320 columntemperatur e (X)) Fig. 2.Effec to fGC-ove n temperature on theresolution ,R ,o fsolani - dine-demissidine (R2 3), demissidine-solasodiene (R34 )an dsolasodine - tomatidine (R5 g). Becauseo fpea kasymmetry ,n ovalu efo rR g5 wa sob tained at240°C .Th e splitting ratioan d thecarrie r gasvelocit ywer e kept constant at 1:130 and 24.3± 0.2 cm/s, respectively. Forfurthe r detailsse eExperimenta lsection . Optimizationo fG Ccondition s To improve thechromatographi c results,th eG Ccondition swer estudie d using the separation of solanidine and demissidine as themai ncrite rion.A t an oven temperature of 290°C, the separationo f thisA5 -an d 5a-aglycone pair, expressed as the resolution of the peaks 2 and 3, was optimum, R23 = 1.7, at a carrier gasvelocity , u, of 24.3cm/s . Duringfurthe roptimization ,u wa skep ta tthi svalue . 93 Theeffec to fth eove ntemperatur eo nth eresolutio nan dpea karea s was investigated at 240, 260, 280, 290, 300 and 320°C. The optimum resolution,R 23 = 1.9,wa sobserve da t280° C (Fig.2) .A thighe rtem peratures the resolution decreased because of the decrease inparti tion coefficient,k . At lower temperatures,k increases,bu t thisdi d not result in an improved resolution because even at 260°C a strong fronting behaviour (up to 52% asymmetry of leading peaks) and peak broadening set in, which was probably caused by condensation of the sample in the column.A t the same time,th e retention times increased exponentially.A t240°C ,t Rfo rtomatidin ewa s9 9min . The peak areas of solanidine, demissidine and solasodiene showed very little variation at the different oven temperatures.Thus , there was hardly any decomposition of these compounds upon increasing the temperature from 260°Ct o 320°C.Unde r thesam econditions ,solasodin e and tomatidine underwent up to 12%decomposition ,whic h could be seen fromth egradua l increaseo fsmal lpeak si nfron to fth esolasodin ean d tomatidine peaks.A t 280°C, theoptimu m temperature for separationo f theSAs ,decompositio no fsolasodin ean dtomatidin ewa sles sthe n3% . Table 1 shows the retention time, resolution and separationnumbe r forsi xSA schromatographe da tth eoptimu move ntemperatur ean dcarrie r gasvelocity .Value sfo rth einterna lstandar dar eals ogiven . The retention timeo fth e lastelute d component (tomatidine)wa s2 7 min at280°C .Whe nanalysin g Solanum specieshavin gunknow n SGAcompo sitions, the total analysis may last longer because the retention times of SAs not tested in this study may exceed that oftomatidine . Forexample , tj^o fjervine ,a nS Afro mVeratru m species,wa s39. 8min , whenchromatographe dunde rth esam econditions . The SAs tested were well separated. The resolution of twoconsec utive peaks was alwaysbette r thanR = 1.5 (baseline separation).Al thoughonl yon eA5 -an d 5a-aglyconepai rwa savailabl e for thisstudy , it is likely from the relatively high resolution of solanidine and demissidine, R23 = 1.9, that other A5-5apair s like tomatidine-toma- tidenol andsolasodine-soladulcidin e (Chapter I,Fig . 1)wil lb esepa rated as well. Both tomatidenol (22/9N-spirosol-5-en-3-ol) and sola- dulcidine (22aN-spirosolan-3-ol) are expected to be eluted between solasodine (22aN-spirosol-5-en-3-ol)an dtomatidin e(22/3N-spirosolan-3 - ol). The separationnumber ,SN ,show stha ta maximu m of fourpeak sca n 94 Table 1.Retentio n time, t^, resolution, R, and separation number, SN, of steroidal alkaloids and of 5a-cholestane obtained by capillary GC using a fused-silica column (50m x 0.22 mm I.D.) coated with CP-Sil 5 (film thickness 0.12 /im); the tgs on a similar column coated with CP-Sil 19 CB are also given. Compound CP-Sil 5 CP-Sil 19 CB tR (min) R SN tR (min) 5a-Cholestane 11.60 13.17 6.2 4 Solanthrene 12.28 16.57 35 29 Solanidine 16.94 29.52 1.9 0 Demissidine 17.23 29.87 3.1 1 Solasodiene 17.78 31 31 24 Solasodine 25.22 >90 6.1 4 Oven temperature: 280°C. Linear carrier gas (helium)velocity : 24.3 cm/s. Injection volume: 1 fim; splitting ratio 1:100. be separated between the last two components. Attempts to improve the GC results using a more polar liquid phase (CP-Sil 19 CB) were not successful. The retention times increased drastically (Table 1),an d solasodine and tomatidinewer e still not eluted after 90mi n at 280°C. Attempts to reduce the analysis time were made by oven temperature programming. The best of the programmes tested, permitted the analysis time to be reduced by 9 min. However, this resulted in a 15-20% de crease in resolution and, due to the use of column temperatures up to 320°C, in an increased decomposition of solasodine and tomatidine. SAs not available for this study or novel SAs may also be present in Solanum species. Therefore, in studying the SA composition of Solanum 95 species, the resolution must be optimum, enabling detection of such compounds. Consequently, the following temperature programme was cho sen: 280°C for 28 min (for optimum separation and quantification of the known SAs from tuber-bearing Solanum species); temperature in crease, 8°C/min to 320°C, and then isothermal for at least 5 min (for detection of SAswhic h might elute after tomatidine). -1— -1— —I— 10 15 20 25 30mi n Fig. 3. Capillary gas chromatogram showing the steroidal alkaloid profile of an extract from tubers of S. tuberosum cv Elkana, spiked with 1 /ig//ilo f components 2, 3, 4, 5, 6 and 8. Column: fused silica, 50 m x 0.22 mm I.D., CP-Sil 5, film thickness 0.12 /im. Carrier gas: helium, linear velocity 24.3 cm/s. Oven temperature: 280°C for 28 min, then 280-320°C at 8°C/min, finally 320°C for 5 min. Injection volume: 1 /il; splitting ratio: 1:100. Attenuation: 2 .Peaks : 1 = solvent; 2= solanidine; 3= demissidine; 4= solasodiene; 5= tomatidine; 6= sola- sodine; 7 = solanthrene; 8 = 5a-cholestane; peaks not marked represent unidentified (minor) compounds. For further details see Experimental section. Validation of the capillary GC method Fig. 3 shows a chromatogram obtained by capillary GC of a spiked potato extract. The extract was obtained from tubers of the cultivar Elkana, which is used for potato-starch production. 'Elkana' contained a high concentration (460 mg/kg) of solanidine glycosides, determined as solanthrene. Prior to injection, 5a-cholestane, solanidine, demissi- 96 dine, solasodiene, solasodine andtomatidin ewer e added toth epotat o hydrolysate. Thechromatogra m shows that allcompound s were separated and thatcompound s interferingwit h theresolutio no fth eSA swer eno t presenti nth epotat o tuberextract . To investigate theprospect s ofth ecapillar y GCmetho d forquali tative and quantitative analysis of the SGAs ofvariou s Solanum spe cies, the contents of solanidine glycosides determined by acolori - metric reference methodwer e comparedwit h those determinedb ycapil lary GC of the aglycone of which the concentration was expresseda s solanidine glycosides. Ten genotypes covering a wide range in these contents were used (Table 2). Although the methods differ strongly (precipitation,centrifugation ,Clark ereactio nan dcolorimetr yo fSGA s versus two-phase hydrolysis ofSGAs , liquid-liquid extractionan dcap illaryG Co fth eaglycone) , theyyielde d similar results.Th ecorrela tioncoefficient ,r ,wa s0.97 6an dth eregressio nequatio nwa s y= 0.95 x+ 2.9 8 where y applies toth e colorimetric method andx toth ecapillar yG C method. Calculationo fth eSG Aconten t Inmethod suse dfo rdeterminin g thetota lSG Acontent ,whic hhav ebee n reported inth eliteratur e (Fitzpatrick andOsman ,1974 ;Coxo ne tal. , 1979; Bushwaye tal. , 1980), theaglycone s liberated after acidhydro lysisar edetermined , andthe nthe yar eexpresse da sSGA sb ymultiply ing the concentration ofth e aglycones by the ratio ofth emolecula r masseso fth ecorrespondin g SGAsan daglycones .I nthi sway ,onl yaver ageconversio n factors,whic hneglec t thevariatio n inchai n lengtho f the sugar moieties, canb e applied. However, when the aglycones are separatedb ychromatograph yan didentifie dan dquantifie d individually, accurate conversion factors canb eused . Forthi s purpose, itmus tb e taken into account that intuber-bearin g Solanum species,solanidine , solasodine, tomatidenol, leptinidine andacetylleptinidin e occurboun d to trisaccharides, and demissidine and tomatidine totetrasaccharide s (Chapter I,Tabl e 1),althoug h exceptionsma yexist .Suc ha nexceptio n isdehydrocommersonine , composed ofsolanidin ean dth etetrasaccharid e commertetraose, which has been detected inth e foliage of S.commer - soniian dS .toralapanu m (Gregorye tal. ,1981 ;John san dOsman ,1986) . 97 Table2 .Content so fsolanidin eglycoside so fte npotat ogenotypes , determinedb ycapillar yG Can db ya colorimetri creferenc emethod . Genotype Solanidineglycoside s (mg/kgfres hweight ) CapillaryG C Colorimetry 1 10 20 2 10 40 3 20 60 4 150 150 5 190 210 6 220 290 7 360 310 8 380 460 9 410 400 10 470 470 Average 222 241 Injectionvolume :2 pi; splittin gratio :1:50 .Fo rothe rexperimenta l conditionsse eFig .3 . 1Thecontent shav eoriginall ybee ndetermine dan dreporte da smg/10 0 gfres hweigh tan dhav eno wbee nmultiplie db ya facto r1 0becaus e ofstandardization . In that case, the results would have been calculated 14.6% too low. Evenmor eaccurat edat aca nb eobtaine dwhe nth econcentration s areex pressed asmoles/kg .The nth evalue swil lb e independent ofth emolec ular mass of the aglycone,an d of thepresenc e of a sugar moiety and itsnumbe ro fsugars . In conclusion, the capillary GCmetho d described here, incombina tionwit h theextractio n and two-phasehydrolysi s described inChapte r III, offersprospect s forquantitativ e analysis ofth e SGAcompositio n of Solanum species.Furthe r sample clean-up and derivatization ofth e SAsi sno trequired .Fo roptimu maccuracy ,a calibratio nlin eshoul db e constructed foreac hS Aan dth econtent sexpresse da smoles/kg . 98 REFERENCES Ahmed SS,Mulle r K. (1978) Effect of wound-damages on the glycoalka- loidconten t inpotat otuber san dchips .Lebens mWis sTechno l11:144 - 146. Baerheim Svendsen A, Verpoorte R. (1983)Chromatograph y ofAlkaloids . PartA :Thin-laye rChromatography .Elsevier ,Amsterdam . Bushway RJ, BureauJL ,Kin g J. (1986)Modificatio n of therapi dhigh - performance liquid chromatographic method for the determination of potatoglycoalkaloids .J Agri cFoo dChe m34:277-279 . Bushway RJ, Wilson AM, Bushway AA. (1980) Determination of total glycoalkaloids inpotat o tubersusin ga modifie dtitratio nmethod .A m PotatoJ 57:561-565 . Caddie LS,Stelzi g DA,Harpe r KL,Youn g RJ. (1978)Thin-laye r chroma tographic system for identificationan dquantitatio n ofpotat o tuber glycoalkaloids.J Agri cFoo dChe m26:1453-1454 . Cham BE, Wilson L. (1987) HPLC of glycoalkaloids from Solanum sodo- maeum.Plant aMe d53:59-62 . Crabbe PG, Fryer C. (1980)Rapi d quantitative analysis ofsolasodine , solasodineglycoside san dsolasodien eb yhigh-pressur e liquidchroma tography.J Chromatog r187:87-100 . Coxon DT, DaviesAMC ,Morga nMRA . (1983)Specia lRepor tNo .7 :Glyco alkaloids inpotatoes .AR CFoo dResearc hInstitute ,Norwich . Coxon DT, Price KR, Jones PG. (1979)A simplified method for thede termination of total glycoalkaloids in potato tubers. J Sei Food Agric30:1043-1049 . Eldridge AC, Hockridge ME. (1983) High-performance liquid chromato graphic separation of eastern black nightshade (Solanum ptvcantunO glycoalkaloids.J Agri cFoo dChe m31:1218-1220 . Fitzpatrick TJ,Osma n SF. (1974)A comprehensiv e method for thedeter minationo ftota lpotat oglycoalkaloids .A mPotat oJ 51:318-323 . Grassert V, Lellbach H. (1987)Untersuchunge n desGlykoalkaloidgehalt s von Kartoffelhybriden mit Resistenz gegen die Kartoffelnematoden Globoderarostochiensi san dGloboder apallida .Bioche mPhysio lPflan z 182:473-479. Gregory P, Sinden SL,Osma n SF,Tinge y WM, Chessin DA. (1981)Glyco alkaloids of wild tuber-bearing Solanum species. J Agric Food Chem 29:1212-1215. Gregory P. (1984)Glycoalkaloi d compositiono fpotatoes :diversit yan d biologicalimplications .A mPotat oJ 61:115-122 . Herb SF, Fitzpatrick TJ,Osma n SF. (1975)Separatio n ofpotat oglyco alkaloidsb yga schromatography .J Agri cFoo dChe m23:520-523 . Hunter IR,Walde nMK ,Heftman nE . (1980)High-performanc e liquidchro matography of Solanum and Veratrum alkaloids.J Chromatogr198:363 - 366. JellemaR ,Elem aET ,Malingr eThM . (1980)Optica lbrightener s asthin - layer chromatographic detection reagents for glycoalkaloids and steroidalkaloid s inSolanu mspecies .J Chromatog r189:406-409 . Jellema R, Elema ET, Malingre ThM. (1981)Fluorodensitometri c deter mination of potato glycoalkaloids on thin-layer chromatograms. J Chromatogr210:121-129 . Jellema R, Elema ET,Malingr e ThM. (1982)A rapid quantitative deter mination of the individual glycoalkaloids in tubers and leaves of Solanum tuberosumL .Potat oRe s25:247-255 . Johns T,Osma nSF . (1986)Glycoalkaloid s ofSolanu m seriesMepistacro - lobuman drelate dpotat ocultigens .Bioche mSyste mEco l14:651-655 . 99 King RR. (1980)Analysi s ofpotat o glycoalkaloids by gas-liquidchro matography of alkaloid components. J Assoc Off Anal Chem 63:1226- 1230. MorganMRA ,Coxo nDT ,Bramha mS ,Cha nHW-S ,Va nGelde rWMJ ,Alliso nMJ . (1985)Determinatio no fth eglycoalkaloi dconten to fpotat o tubersb y threemethod s includingenzyme-linke d immunosorbentassay .J Se iFoo d Agric36:282-288 . Morris SC,Le eTH . (1981)Analysi s ofpotat o glycoalkaloids withradi ally compressed high-performance liquid chromatographic cartridges andethanolamin ei nth emobil ephase .J Chromatog r219:403-410 . Osman SF,Her b SF,Fitzpatric kTJ ,Schmiedich eP . (1978)Glycoalkaloi d composition of wild and cultivated tuber-bearing Solanum species of potential value in potato breeding programs. J Agric Food Chem 26:1246-1248. Osman SF, Sinden SL. (1977) Analysis of mixtures of solanidine and demissidineglycoalkaloid s containing identicalcarbohydrat eunits .J AgricFoo dChe m25:955-957 . Osman SF, Zacharius RM, Kalan EB, Fitzpatrick TJ, Krulick S. (1979) Stressmetabolite s ofth epotat oan dothe rSolanaceou splants .J Foo d Prot42:502-507 . Sinden SL, Sanford LL,Web b RE. (1984)Geneti c and environmentalcon trolo fpotat oglycoalkaloids .A mPotat oJ 61:141-156 . TingeyWM . (1984)Glycoalkaloid s aspes t resistance factors.A mPotat o J61:157-167 . 101 CHAPTERV * CAPILLARY GAS CHROMATOGRAPHY OFSTEROIDA LALKALOIDS :RETENTIO N INDICES ANDSIMULTANEOU SFLAM EIONIZATION/NITROGEN-SPECIFI CDETECTIO N INTRODUCTION Capillary GCanalyse susin g themetho ddescribe d inChapte r IV,showe d that SGAs, such as solanidine, demissidine, solasodine or tomatidine glycosides,occu r intuber san d leaveso fwil d Solanum speciesuse di n potatobreedin gan di nleave so fprogen yderive dfro msuc hspecie s (Van Gelderan dJonker ,1986 ;Va nGelde re tal. , 1987). Inadditio nt opeak s ofth eaglycone so fthes eSGAs ,th echromatogram sshowe dpeak so fothe r compounds with retention times within the range of those of the SAs. These peaks could not be identified by comparison of their retention timeswit h those of the standard SAs available. Inth eframewor k ofa tomato breeding programme on resistance to the glasshouse whitefly (Trialeurodes vaporariorum Westw.), a study on the SGA composition of the wild tomato species Lvcopersicon hirsutum glabratum C.H.Muller , ando ftomat obreedin gline sderive dfro mthi sspecies ,wa scarrie dou t (VanGelde ran dD ePonti ,1987) .Als o inthi sstudy ,som eunidentifie d (minor)compound swer edetected ,whic helute dsomewha tbefor ean dafte r tomatidine. Preliminary experiments in which chromatograms were recorded by nitrogen-specific detection (NPD) using a thermoionic nitrogen-phosphorus detector, indicated that some of theunidentifie d compounds wereprobabl y SAs. In ascreenin g ofbreedin gmateria lfo r SAs, the characterization of each unknown peak in a chromatogram by costly spectrometric techniques ishardl y feasible.Therefor e ametho d wasdevelope d thatenable sdifferentiatio nbetwee npeak so funknow nSA s and peaks of nonnitrogen-containing (non-N)compounds .Th e methodin - *Thischapte r isbase do nth epaper : Van GelderW JM, Jonke r HH,Huizin g HJ, Scheffer JJC. (1988)Capillar y gas chromatography of steroidal alkaloids from Solanaceae. Retention indices and simultaneous flame ionization/nitrogen-specific detection. J Chromatogr442:133-145 . 102 volves a capillary column connected by a splitting device to a dual detector system for FID and NPD,whic h is connected to a two-channel systemfo rinteractiv eprocessin go fth etw oset so fdata .A sa furthe r aid for identificationo fth e SAs,a retentio n index systemwa sintro duced. The chromatographic conditions were optimized andhydroge nwa s used ascarrie r gas inorde r todecreas e theduratio no fth eanalyses . Spectrometric techniques willhav e tob eemploye d forcharacterizatio n onlywhe nne wSA sar edetected . EXPERIMENTAL Apparatus The gas Chromatograph described in Chapter IV was equipped with detectors forFI Dan dNP Dan dwit h two split/splitless-typeinjectors . One injector was in the split sampling mode, splitting ratio 1:50 unless otherwise stated; thevaporizatio n tubewa sa modifie dJenning s tube (Freeman, 1981)provide d with a fritted glass filter and partly filled with deactivated quartz-wool. The other injector was used for controlledsuppl yo fnitrogen ,2 0ml/min ,a smake-u pga sfo ra four-wa y effluent-splitting device, which consisted of a quartz tube (9 mm x 1.3 mm I.D.) and two two-hole vespel ferrules in a stainless-steel housing.Th eeffluen tsplitte rconnecte d thecolum nwit hbot hdetector s via two deactivated fused-silica restriction tubes (20 cm x 0.22 mm I.D.). The effluent-splitting ratio for FID and NPD which depends on the length ratio of the restriction tubeswa skep ta t1: 1 inorde rt o obtain identical retention times for theFI Dan d theNP D traces.Othe r GC conditions were as follows. Column: fused silica, 50 m x 0.22 mm I.D., CP-Sil 5 CB, film thickness 0.12 /um(Chrompac k Nederland); oven temperature,270° Co r280°C ;detector ,325°C ;injector ,initiall y300° C but after the optimization experiment 350°C; carrier gas,hydrogen , linear velocity 49 cm/s, unless stated otherwise; flow-rates forFID , hydrogen 25ml/min ,ai r 250ml/min ; and for NPD,hydroge n 5.0ml/min , air 50 ml/min. The detectors were supplied with nitrogen, 30 ml/min each, as make-up gas (unless stated otherwise), including 10 ml/min each of themake-u p gas from the effluent splitter. For theinjector , Chromsep Red, high-temperature (up to 450°C) septa (Chrompack) were employed. Two Shimadzu C-R3A data processors were used for recording 103 the FID and NPD signals and for interactive processing of the data. The processors were interconnected by a PC14N current loop interface, an RS232C and aMIC-loop . Software For communication between the data processors, a programme was written partly in BASIC and partly inmachin e language. After the introduction of the identification system using NPD/FID response ratios and reten tion indices, a BASIC programme was written for automated calculation of these values. Integrated into the BASIC programme, concentrations were measured with the ROM-software of the dataprocessors . Standard alkaloids and other chemicals Solanidine, solanthrene, demissidine, solasodiene, solasodine, toma- tidine, 5cc-cholestane, the solvents and other chemicals, were as described in the Chapters III and IV. For comparison of SAs and test compounds with respect to the relationship between injection volumes and peak areas, a commercially available test mixture (Chrompack, Cat.No. 6606) was used. This contained n-octanal, n-octanol-1, n-undecane, 2,6-dimethylphenol, methyl n-octanoate, 2,6-dimethyl- aniline, naphthalene, n-dodecane, n-tridecane and methyl n-decanoate, each in a concentration of 1% in cyclohexane. Cholesterol and stigma- sterol were obtained from Roth, diosgenin, n-octacosane and n-tetra- triacontane from Sigma and jervine and tigogenin from ICN Pharma ceuticals, (Plainview, NY, U.S.A.). Calculation of retention indices Retention indices, I, on CP-Sil 5 CB at 270°C were calculated using the formula: 1 t t: lo (t t ) îCP-Sil 5 CB _ l00.c1-H00.(c2-C1^. °g( R,i- R,0)- 5 R,l- R,0 270 iog^R^-tR.ûMog^R.l-tR.o) where C^= the carbon number of the first reference compound (n-octa cosane, C28H58), C2 = the carbon number of the second reference com pound (n-tetratriacontane, C34H70), CR 0 = tne retention time of an unretarded component (methane), t^ ^ and t^^ an d t^2 = tne retention bû e ON ro r-l CM CM co cd co UI G < CU co bO O co S-l •ü •o •H H Bi i O O 33 4-J Ai CO M ON Cl) CN C Ai VO O •H Bi O (-1 x CU c O •U CU e" co bX) 00 •i-I O O CM CM S-l 00 o o ON O •o 00 o CO o 4-1 CU ai co P-i 3) co O n) bO !-l O co l-i cu oo co 1^- co o CN CM co •i-l 00 •O CU Pd c S-l H 33 4-) o O co Bi cfl CU 4-> u o e 3 o VC f-- CU 4-1 bO CÖ IT) o S-l ai CO 1-1 CU •o P< O E c O m CU cu 4-> bü m O oo m o C c J-l co CM un oo cd CU 4J r-l •iH •H O CM ON 4-> > ai — É5 3 •u co CO C 5 O CU 4-1 r-l •O CU CU cu Cd CU co cu 3 e S3 CM •l-l r-l a CU 0) •1-1 •o bl) co Tj o CU o co o co 4-J r-l S-l •r-l co n) cfl rO 4-> E r-l E •r-l o O O m cu o H < co H O e C/2 a co 105 times of the component ofwhic h Iha s tob e determined, of the first referencecompoun dan do fth esecon dreferenc ecompound ,respectively . RESULTSAN DDISCUSSIO N Separationo fSA s The effecto f the injector temperature onth eretentio n time,relativ e response, chromatographic stability andresolutio nwa s investigatedb y increasing this temperature from 300°C to450° Cb y increments of25°C . The temperature increase gradually resulted inshorte r retentiontime s of the SAs but the effect was negligible (< 2%) even at 450°C. The compound ratios, for instance that of solanidine and tomatidine,di d not change at thevariou s injector temperatures,whic hmean t thatal l the SAs were vaporized to the same extent at the various injector temperatures. Thus, the injector temperature did not influence the possible discrimination of the SAs. However, increasing the injector temperature deteriorated thebaselin e stability andpea k symmetry,an d caused ghostpeaks ,especiall y attemperature s above 375°C.Thes ephe nomena probably resulted from septum bleeding and/or sample decom position. The resolutiono fth eearl y elutingcompound s solanidinean d demissidine was optimum at an injector temperature of 350°C,wherea s forth elat eelutin gcompound ssuc ha ssolasodin ean dtomatidin e itwa s optimum at 375-400°C. Because the chromatographic stability deteri orateda tth ehighe r temperatures anda noptimu m resolutiono fth eA5 - and 5a-aglycone pair solanidine and demissidine was important, 350°C waschose na sth einjecto rtemperature . Table 1 shows the retention times and the resolution of the SAs chromatographed on the CP-Sil 5 CB column at an oven temperature of 280°Cusin ghelium ,nitroge nan dhydroge na sth ecarrie rgase sa topti mized average linear velocities. The highest resolution was obtained with nitrogen, but this was attended with long retention times. The separationefficiencie sobtaine dwit hheliu man dhydroge nwer esimilar , but usinghydroge n the analysis durationwa shal f as long. Comparison of the separation efficiencies with hydrogen at oven temperatures of 260, 270, 280 and 290"C, showed that the optimum temperature forth e separation of solanidine, demissidine and solasodienewa s 270°C.Com paredwit hheliu m asth ecarrie rga san d theove ntemperatur eo f280° C 106 usedbefore ,270° Cwa s favourablewit hregar d toth ecolum ndurabilit y (maximum isothermal operating temperature 300°C), and a 30% shorter analysisduratio nwa sobtaine d (Table1) . Splitinjectio no fSA s In isothermal capillary GC, the split injection technique isprefer able, especially when automated injection is applied. However, when quantititive results are required, many problems may arise (GrobJr . andNeukom ,1982) .Therefore ,th esplittin gproces swa sinvestigate di n order to study whether quantitative results could be obtained forth e individualSAs . For quantitative analyses it is necessary that the splitting is linear both for various concentrations and sample sizes. This was checked by constructing FID calibration curves for the internalstan dard 5a-cholestane, using two injection volumes, 0.9 pi and 1.9 /il. Forbot hcurves ,th epea karea swer e foundt ob edirectl yproportiona l to the concentrations. So,splittin gwa s independent ofconcentration . However, the normalized FID responses of 5o:-choiestane found for the 0.9 pi injectionvolume swer e only80 %o fthos e foundfo r1. 9 pi. This suggestedtha tth esplittin grati ovarie ddependin go nth esampl esize . For quantitative analyses, the normalized peak areas must be similar for thevariou s samplesize san d foreac hSA .Therefor e theconcentra tions of the compounds were kept constant, whilst the sample sizes werevaried .Tabl e 2give s theaverag e ofth enormalize d peakarea so f solanidine, demissidine, solasodiene, solasodine and tomatidine for each sample size.Th enormalize d peakarea swer eexpresse d aspercent ages of thepea k areas,average d for the SAs,a tth e injectionvolum e of 2.4 pi. For each sample size the variation (range and % S.D.) in normalized peak areas between the individual SAs is also given. The experiments showed that the normalized peak areas decreased with decreasing sample sizes. The linearity of the calibration plots (see hereafter) showed that thiswa s not due to irreversible adsorptiono f thecomponent sont oth ecolumn .Therefore ,i ti smos tprobabl e thatth e pressure pulse causedb y flashvaporizatio no f thesampl e decreasesa t smaller sample sizes, and as a consequence, the splitting ratio is lowered,whic h in turn results ina reduction of thepea k areas.Con comitantly with thisreductio n innormalize d peakareas ,th evariatio n 107 Table 2. Variation in normalized peak areas for steroidal alkaloids (SAs)an dfo rtes tcompound sdependen to nth einjectio nvolume . Injection Relativenormalize dpea karea s (%) volume (/il) SAs Testcompound s Average Range %S.D . Average Range %S.D . 2.4 100 - - 1.9 98 95-103 4.3 100 - - 1.4 94 91-9 8 3.1 100 99-107 2.4 1.0 67 59- 80 9.9 85 78-8 6 2.7 0.8 53 46- 57 10.7 80 77-8 1 1.5 0.4 30 17-3 9 31.6 76 72-7 9 3.1 0.3 69 64-7 2 4.1 SAs: solanidine, demissidine, solasodiene, solasodine and tomatidine; forth ecompositio no fth etes tmixtur e seeExperimenta lsection . Normalizedpea karea si npercentage so fth eare aa t2. 4ju lfo rSA s anda t1. 9/i lfo rth etes tcompounds ,respectively . in these areas between the individual SAs increased, as is shownb y the range and % S.D. From replicate analyses it was clear that this variationwa sno tdu e toa lac k inprecisio no foperatio no rdetectio n (n= 5 ;% S.D . forth epea karea so fsolanidin e2.22 ;demissidin e2.06 ; solasodiene 2.15; solasodine 4.31 and tomatidine 3.08). Comparisono f the peak areas of the individual SAs showed that at injectionvolume s < 1/i lth e relative peak areas of the compounds decreased with their order of elution. This can be ascribed to discrimination of thecom pounds according to their boiling points. Discrimination is due to variationo fsplittin grati ofo rth edifferen tcompound spresen t inth e splitter (Grob Jr. and Neukom, 1982). This nonlinearity of thesplit tingproces s apparently increased atsmalle r sample sizes.So ,discri minationoccurre d inspit eo fth eprecaution s takenwit hrespec tt oth e configurationo f thevaporizatio n tube,t o ensure that the samplewa s heated sufficiently rapidly for acomplet e vaporization during injec- 108 tion (seeExperimenta lsection) . A similar experiment was done using a commercially availablemix ture of testcompound s ofwel l definedchromatographi c behaviour.Als o in this experiment, although to a lesser extent, the normalized peak areas decreased (relative to 1.9 /il) with decreasing sample sizes (Table 2). Thisshow sth echaracteristic so fth esplitte rconfiguratio n used.However ,th edecreas e inth epea k areaswa s similar foral lcom pounds of the test mixture. The results obtained for the SAs andfo r the test compounds show that the SAs are difficult compounds forcap illary GC.Fo r quantitative results,extr a care isneeded . A constant injectionvolum e isessential ,whic hdisallow scorrectio no fpea karea s bychangin gth einjectio nvolum ewhe nth ecolum ni soverloaded ,o rwhe n the peaks are too small to be detected. Also an assessment of the smallest volume that gives uniform results for the different SAs is required. In our study 1.4 /ilwa s found tob e appropriate, evenmor e so because at this volume the results for 5a-cholestane agreed well with those for the SAs,allowin g this compound tob euse d as internal standard. Nitrogen-specificdetectio no fSA s The selectivity and sensitivity ofa N-specifi c detector depend onth e position of its alkali metal source, on the supply of thermal energy for emission of the alkali metal ions and on thecompositio n andflo w of the gas surrounding the alkali source (Sevcik, 1976;Verga ,1983 ; Shmidel et al., 1986). Each of these factors isth eresultan tbot ho f detector characteristics determined by the instrumental design ando f adjustments of chromatographic conditions and detector variables.Th e selectivity and sensitivity of the detector used in this study were mainly determined by the distance between the rubidium bead and the detectorjet ,b y theflo wo fhydroge nuse d forcombustion ,b yth eflo w of the nitrogen make-up gas and by the current of the electrically heated bead foradjustmen t ofth ebackgroun d ionizationcurren t (BIC). As an instrumentalprerequisite ,th epositio no f therubidiu mbea dwa s carefully tunedan d thehydroge n flowadjuste d to5. 0 ml/min.The nth e influence of the flow-rate of the make-up gas and of the BIC on the selectivity and sensitivity were studied. In studying the SA compo sitiono f Solanaceae, theforme r ismor eimportan t thanth elatter , 109 Table 3. Influence of make-up gas flow and background ionization current (BIC)o nth eselectivit yan dsensitivit yo fNP Dfo rsolasodin e anddiosgeni ni ncompariso nt oth eFI Dresponse . BIC Nitrogen Relative sensitivity Selectivity-'- (PA) make -up NPD/FID Solasodine/Dios genin gas flow (ml/min ) Solasodine Diosgenin NPD FID 80 20 3.2 0.119 25 0.91 80 25 3.1 0.019 139 0.87 80 30 2.9 0.006 418 0.88 80 35 2.4 0.005 462 0.86 40 20 1.5 0.082 16 0.90 40 25 1.5 0.011 117 0.88 40 30 1.1 0.003 389 0.90 40 35 0.9 0.002 409 0.91 Injectionvolume :3 /il . ••-Normalizedfo rconcentratio no fdiosgeni n= 7.11 »concentratio no f solasodine. because the selectivity allows differentiation between SAs andnon - nitrogen-containin g compounds (non-N compounds), whilst forquantifi cationo fSA sth esensitivit yo fFI Di susuall ysufficient .A numbe ro f experiments were carried out,usin g SAsan d several more orles sre latednon- Ncompounds ,t ocompar edifferen tBIC san dmake-u pga sflows . An example isgive n inTabl e 3.Th erelativ e sensitivityo fNP D(NPD / FID)wa sdefine d asth erati oo fth eNP Dan dFI Dresponse st oth esam e amount ofa compound . Theselectivit ywa sdefine d asth erati oo fth e response toa compoun dunde r study,i.e .solasodin e andth enormalize d response to a reference compound, i.e. diosgenin. At nitrogen flow- rates of 20,2 5 and 30m l ml/min and a BIC of 80pA , the relative sensitivity of NPD to solasodine (22aN-spirosol-5-en-3-ol) remained similar,whils tth erelativ esensitivit yt odiosgenin ,th enon- N(22aO ) analogue, strongly decreased when the flow-rate was increased. Asa resultth e selectivityo f NPD forth e N-containingcompoun dgreatl y 110 FID Att NPD Fig. 1. Gas chromatograms obtained simultaneously by FID and NPD, showing the selectivity of NPD for solasodine, 22aN, compared to the 22aO analogue diosgenin. Column: fused silica, 50 m x 0.22 mm I.D., CP-Sil 5 CB, film thickness 0.12 /im. Carrier gas: hydrogen, linear velocity 53 cm/s. Oven temperature: 270°C. Injection volume: 3 /il; splitting ratio: 1:50; effluent splitting ratio: 1:1 (FID:NPD). Detector sensitivity: lpA/mV. Attenuation: 2 .Fo r further details see Experimental section. improved. At 35 ml/min and 80 pA the selectivity was further improved, but the sensitivity to solasodine became rather low for subtraction of FID from NPD traces (see hereafter). Lowering the BIC to 40 pA reduced the sensitivity of NPD to solasodine more than to diosgenin, and thus the selectivity slightly decreased. Increasing the BIC above 80 pA negatively affected thebaselin e stability and the noise level. In summary, the make-up gas flow especially influenced the NPD sensitivity to non-N compounds and so it had a strong effect on the selectivity for SAs, whereas the BIC mainly influenced the sensitivity ofNP D to the SAs.Afte r these experiments amake-u p gas flow ofnitro - Ill gen of 30 ml/min and a BIC of 80p A were chosen as optimum for the presentstudy . The relative sensitivity and the selectivity of NPD for the SAs were rather low compared with the corresponding values forpyridine : NPD/FID response ratio 22;selectivit ypyridine/cyclohexan e 2900.Thi s mightb eexplaine db yth elo wN/ Crati oo fth eSAs ,i.e .1:27 . Fig. 1 shows the gas chromatograms of amixtur e of solasodine and diosgenin recorded simultaneously by FID and NPD.Onl y high levels of the non-N compound, far above the theoretical sample capacity of the column, were able to interfere with the N-specific detection. This means that simultaneous detectionb yFI D andNP Dunambiguousl ydiffer entiatesbetwee nSA san dnon- Ncompounds . Quantificationo fSA s Calibration lines were constructed for FID andNPD .A s samples,solu tions ofsolanidine ,o fdemissidin eplu s tomatidine and ofsolasodien e plussolasodin ewer eused .Eac hcompoun dwa suse di n1 1concentrations , varying from0.05-1 0mg/ml .Althoug hconcentration sbetwee n4 mg/m lan d 10mg/m l may cause overloading of the column, theywer e usedbecaus e preliminary experiments showed that tuberso fwil d Solanum speciesma y containlevel so fsolanidin eglycoside sabov e600 0mg/kg . Table 4.Slope ,m , intercept,c ,an dcoefficien t ofcorrelation ,r ,o f the calibrationsy = m x -c fo rsteroida l alkaloids (SAs)wit hFI Dan d NPD,wher ey = respons e (counts•1 0) an dx = concentratio n (mgSA/ml) . SA FID NPD r.10 4 r• 10 4 Solanidine 70 4.4 9997 190 15.5 9997 Demissidine 70 5.3 9996 186 1.4 9984 Solasodiene 70 6.7 9996 183 8.0 9983 Solasodine 58 4.0 9999 149 11.7 9994 Tomatidine 62 13.6 9980 153 9.5 9994 112 The calibration lines for FID and for NPD were linear over the entire concentration range, for all SAs tested (Table 4).Unde r the conditions described for sample preparation (Chapter IV) and chroma tography, 50-an d 150-fold concentrations for the individual SAs could be quantified by FID and NPD respectively, without exceeding the col umn capacity. Under these conditions andusin g theNP D calibrations of Table 4, 2.7 mg/kg was the minimum content for accurate quantification. The actual detection limit that canb e achieved using modified procedures for sample preparation is lower, but it was not relevant to determine this limit. The concentrations between 4 mg/ml and 10mg/ml , which exceeded thecolum n capacity, hadn o effect onth e peak symmetry. Thus, when closely eluting SAs are not present, extremely high levels of SAsca nb e quantified without dilution ofth e samples. Identificationo fSA s The reproducibility of the retention times expressed as % S.D. (n= 6) was 0.045 for solanthrene and 0.037 for solanidine; for demissidine, solasodiene, solasodine and tomatidine it varied from 0.040-0.068. These values agreed with those reported for accurate analyses (Rooney and Freeman, 1981). However, such determinations areusuall y made under ideal conditions. This means that injections are made successively within one day and using one concentration. In studying the SAcompo sition of Solanaceae, samples with widely different concentrations of SAs must be analysed in the course of weeks or months. Progressive phase stripping of the column due to the high oven temperature, and especially a large variation in peak heights due to variation inth e concentrations of the SAs,migh t result in less reproducible retention times. Therefore, we also determined routinely the reproducibility of the retention times of the SAs. Nine different tuber samples of six Solanum species, obtained from the Potato Breeding Department of the Foundation forAgricultura l Plant Breeding SVPwer e analysed for their SA composition using theextractio n andhydrolysi s procedures described in the Chapters III and IV. The analyses were carried out between routine analyses in the course of a five-day period. Most of the samples contained only solanidine glycosides -their contents varying widely: 600-6200 mg/kg- so calculations were made only for the corre- 113 Table 5. Retention times, t^, retention indices, I, and NPD/FID response ratios of steroidal alkaloids, 5a-cholestane, sterols and steroidal sapogenins. Peak Compound tR I NPD/FID number response ratio 1 n-Octacosane (C28H5 8)2 5.87 2800.0 - 2 5a-Cholestane 6.61 2866.5 - 3 Solanthrene 7.61 2941.8 3.04 4 Cholesterol 10.13 3068.3 - 5 Solanidine 11.07 3129.3 2.96 6 Demissidine 11.25 3136.8 3.01 7 Solasodiene 11.59 3151.1 3.06 8 Stigmasterol 13.22 3212.9 - 9 Diosgenin 13.43 3220.4 - 10 Tigogenin 13.78 3232.4 - 11 Solasodine 17.36 3337.6 3.02 12 Tomatidine 18.65 3369.8 2.88 13 n-Tetratriacontane (C34H70)2 19.96 3400.0 - 14 Jervine 28.73 3559.4 3.43 Column: fused silica, 50m x 0.2 2m mI.D. ,CP-SI L5 CB ,fil m thick ness 0.12 /im. Oven temperature: 270°C. Carrier gas: hydrogen, linear velocity 49cm/s . •'•Peaknumber s correspond with thoseo fFig .2 . Reference compound. sponding aglycones solanthrene and solanidine. The reproducibility of the retention times deteriorated strongly; the % S.D. was now 0.864 and 0.690, respectively. Duet oth elarg e variations inretentio n time, itwa sno tpossibl e tose ta time window forsolanidin e along withon e for demissidine, inorde r toidentif y these peaksb yretentio n times. Att. 26 B[ i Retention time (min) 115 Fig. 2. Gas chromatograms obtained simultaneously by FID (A) and NPD (B) for a potato extract spiked with steroidal alkaloids, and with 5a-cholestane, sterols and steroidal sapogenins. Post-analysis repro cessing of the raw data from A and B (subtraction of the FID trace from the NPD trace) is shown in C. Carrier gas: hydrogen, linear velocity 49 cm/s. Injection volume: 1.4 /il. Attenuation: FID 2 ,NP D 2 . Further details as in Fig. 1. Amounts (ng) of compounds in the column: 1 = n-octacosane, 19.0; 2 = 5a-cholestane, 13.7;3 = solan- threne, 12.9; 4 = cholesterol, 14.4; 5 = solanidine, 15.2; 6 = demissidine, 16.0; 7 = solasodiene, 13.6; 8 = stigmasterol, 14.1; 9 = diosgenin, 20.9; 10 = tigogenin, 14.2; 11 = solasodine, 15.7; 12= tomatidine, 17.4; 13 = n-tetratriacontane, 17.9; 14= jervine , 31.3. For certain Solanum species, complex chromatograms showing large numbers of SAsma yb e obtained. As potato breeding may require many SA analyses, the characterization and identification of compounds by costly spectrometric techniques is hardly feasible. Therefore a cheap and relatively simple system for characterization and identification using retention indices andNPD/FI D response ratioswa s developed. The retention indices shown in Table 5 were sufficiently reproduc ible. Under ideal conditions, the % S.D. of the indices for the SAs varied from 0.006-0.010. In the above mentioned routine experiment the % S.D. of the retention index was 0.046 for solanthrene and0.02 8fo r solanidine. Tests with widely different concentrations of solanidine and demissidine showed that typical identification windows for the retention indices of these closely eluting compounds would be 3129.3 ± 1.7 and 3136.8 ± 1.7 units, respectively. This meant that the reten tion index system could be used as anai d inth e identificationo fth e SAs. For automated identification in our laboratory, the indices are compared, using the BASIC programme, with the values of stored reten tion index windows. These values have been determined for individual SAs, using samples ofplan t material or standard solutions,wit h awid e concentration range. The accuracy and precision of the retention indicesma yb e further improvedb yusin g foreac h SAtw oreferenc e com pounds differing by only one inthei r number ofcarbo n atoms (Schomburg 116 and Dlelman, 1973). For practical reasons this was not done. Future studies on the SA composition of Solanaceae will reveal whether improvemento fth eretentio ninde xsyste mi srequired . TheNPD/FI Drespons e ratioso fth eSA swer equit e similar (Table5 ) and they will thus be useful in the characterization ofunknow ncom pounds. As the NPD/FID response ratios of the SAs differed markedly from thato fpyridine ,whic hwa s22 ,i tshoul db eworthwhil e toinves tigate whether these ratios are sufficiently specific for different types of N-containing compounds todiscriminat e between SAs andothe r N-containing compounds whichma yb epresen t inplan t extracts. Ifso , thiscoul dallo wa considerabl e simplificationo fth ecomple xprocedur e for sample preparation currently in use. Fig. 2 shows gas chromato- grams of apotat o extract spikedwit h SAs and relatednon- Ncompound s (5a-cholestane, sterols and steroidal sapogenins)obtaine d by FID (A) and NPD (B). Th e peak numbers correspond with those of the compounds listed in Table 5. The FID chromatogram shows that,unde r thecondi tions applied, all compounds were well separated. The Solanum SAs tested were eluted within 19min .Th eVeratru m SAjervin e (C27H39NO3) had a retention time of almost 29min .Thi s shows the necessity ofa temperatureris eafte relutio no ftomatidin e (orth ereferenc ecompoun d tetratriacontane) to facilitate the detection of other, lateeluting , SAs as stated in Chapter IV. Comparison of the FID chromatogram with the NPD chromatogram reveals the peak vacancies in the latter,whic h correspond with the non-N compounds of Table 5. Fig. 2C shows the result of post-analysis reprocessing of the chromatograms, which was realized by the software mentioned in the Experimental section. By subtraction of the FID chromatogram from the NPD chromatogram, the peakvacancie s intr eNP D tracear econverte d intonegativ epeaks .So , the differentiation between SAs and non-N compounds is unambiguously achieved. In conclusion, quantification of individual SAsusin g capillary GC canb e done routinely eitherb y FID orb yNPD ,provide d extra carei s taken over the injection of the samples. Usually FID is more stable thanNPD ,bu t the latter willb e more sensitive.Th eNPD/FI D response ratios can be used together with retention indices in the identifi cation and characterization of SAs. GC-MS analysis can probably be restricted to compounds with NPD/FID response ratioswhic h correspond 117 to thoseo f theSA san dwit hretentio n indiceswhic hd ono tagre ewit h thoseo fth eknow nSAs . REFERENCES Freeman RR. (1981) Split injection. In High Resolution Gas Chromato graphy,2n dEd ,R RFreema n (Ed).Hewlett-Packar dCompany ,pp .54-59 . GrobJ rK ,Neuko mHP . (1982)Dependenc e ofth e splitting ratioo ncol umn temperature in split injection capillary gas chromatography.J Chromatogr236:297-306 . Rooney TA, FreemanRR . (1981)Retentio n time stability. InHig hReso lution Gas Chromatography, 2nd Ed,R R Freeman (Ed).Hewlett-Packar d Company,pp .131-139 . Schomburg G, Dielman G. (1973) Identification by means of retention parameters.J Chromatog rSe i11:151-159 . Sevcik J. (1976)Detector s inGa sChromatography . Elsevier,Amsterdam , NewYork . ShmidelEB ,Kalabin aLI ,Kolomiet sLN .(1986 )Comparativ estud yo fsom e thermoionicdetecto rdesigns .J Chromatog r365:353-358 . VanGelde rWMJ ,Hopman sCWJ ,Jonke rHH ,Va nEeuwij k FA. (1987)Biosyn thesisan dmetabolis mo fC27-steroida lalkaloid s inleave so fpotato . Proc10t hTrien nCon fEu rAsso cPotat oRes ,Aalborg ,pp .276-277 . Van Gelder WMJ, Jonker HH. (1986) Steroidal alkaloid composition of tubers of exotic Solanum species ofvalu e inpotat o breedingdeter mined by high-resolution gas chromatography. In Potato Research of Tomorrow,AG BBeekma n (Ed).PUDOC ,Wageningen ,pp .166-169 . Van Gelder WMJ, De Ponti 0MB. (1987) a-Tomatine and other steroidal glycoalkaloids infruit so ftomat oline sresistan t to theglasshous e whitefly (Trialeurodesvaporarioru mWestw.) .Euphytic a36:555-561 . Verga GR. (1983)High-resolutio n gas chromatographic determinationo f nitrogen and phosphorus compounds incomple x organic mixtures by a tunableselectiv ethermoioni cdetector .J Chromatog r279:657-665 . 119 CHAPTERVI * ANALYSIS OF STEROIDAL GLYCOALKALOIDS IN POTATO GENOTYPES CONTAINING GERMPLASMFRO MWIL DSOLANU MSPECIE S INTRODUCTION The procedures for bisolvent extraction, two-phase hydrolysis and capillary GC were developed using pure SAs and SGAs, and tubers of commercial potato cultivars. Preliminary experiments carried out for studies on tomato fruits (Van Gelder & De Ponti, 1987) and potato leaves (VanGelde re tal. , 1987),showe dtha tth ehydrolysi scondition s may require optimization for analyses of differentplan t organs.Thi s can be explained by the differences in the chemical composition of diverse plant tissues which may vary considerably in their concen trations of starch, proteins, organic acids, sugars, fats and other constituents. Suchvariatio nals oexist sbetwee ntuber s fromdifferen t Solanumspecie san dfro mthei rhybri doffspring . SGAs containing solanidine, solasodine and tomatidine as aglycones have been detected in tubers of wild Solanum species (VanGelde r and Jonker, 1986)an d in the plant material analysed in the studiesmen tionedabove .However ,no tal lo fthes eglycoside scoul db euse ddurin g the development of the various analytical procedures.Recen tprepara tion of sufficient amounts of standard SGAs and of plant materials containing them,enable d theseprocedure s tob eoptimize d forapplica tiont olargel ydivers esamples ,amon gother sincludin gdifferen tplan t organs. A large number of standard SAsbecam e available recently,an d enabled their potential separation tob e studied and their retention indices and NPD/FID response ratios tob e determined. The results of theseexperiment sar epresente d inthi schapter . Procedural difficulties of literature methods for SGA analysis, Thischapte r isbase do na par to fth epaper : VanGelde rWMJ ,Scheffe rJJC . (199*)Strateg y foranalysi so fsteroida l glycoalkaloids inpotat o and tomato genotypes containing wild Solanum andLvcopersico ngermplasm ,i npreparation . 120 whichma yeasil y introduce experimental errorswhe napplie d todivers e breeding material, are discussed inrelatio n to themetho d developed inth estudie sdescribe di nthi sthesis . EXPERIMENTAL Plantmateria l For determining the number of bisolvent extractions required, tuber samples were obtained from the Potato Breeding Department of the Foundation forAgricultura l PlantBreedin gSVP .Thes e samples included field-grown tubers ofth ehousehol d potato cultivar Bintje ando fth e potatocys tnematod eresistan tSV Pclon eA M78-377 8whic hha sS .verne i Bitt.e tWittm .a sprogenitor ,an dglasshouse-grow ntuber so fS.speeaz - ziniiBitt .an do ftw oclones ,hybrid s 1an d2 ,fro ma cros sbetwee na diploidgenotyp eo fS .tuberosu mL .an dS .leptophye sBitt . The samples fordeterminin g therecoverie so fSGAs ,afte rtwo-phas e hydrolysis andcapillar y GCwer e tubers and/or leaves ofS . tuberosum ssp. andigena (Juz.e tBuk. )Hawkes ,S .brevicaul e Bitt. and S.ven - turii Hawkes et Hjerting collected from plants grown in 1986 under controlled short-day conditions (seeChapte r VII),field-grow n tubers of thehousehol d potato cultivar Pimpernel,gree nberrie s (fruits)o f S.nigru mL .collecte d inth efield ,an dfruit so fth etomat ocultiva r Allround (Lvcopersiconesculentu mL. )an do fth ewil d species L.hir - sutumglabratu mC.H .Muller . For comparing the capillary GC method and a colorimetric method, selected asreference , tuber samples ofS . gourlavi Hawkes,S .lepto phyes. S .spegazzini i andS .sucrens eHawkes ,o f 'Bintje'an d'Pimper nel',o fth eSV Pclone sA M78-377 8an dA M78-378 7 ('Arabesque')an do f clones from crosses between diploid genotypes of S. tuberosum and accessionso fS .spegazzinii .S .vernei .S .sparsipilu m (Bitt.)Juz .e t Buk. and S. leptophyes were used. The samples were from different locations,includin gexperimenta lfield ,glasshous ean dgrowt hcabinet . For the comparison of the GC method, tuber samples containing only solanidineglycoside swer eselected . Standardalkaloid san dothe rchemical s Solasodine,solasodiene ,solanthrene ,a-tomatine ,tomatidin ean ddemis - 121 sldine and a-solaninewer e as described inChapte r III,5a-cholestan e was as inChapte r IV,an d diosgenin andjervin ewer e as inChapte rV . Solanidine was from Roth.Tomatidenol ,solanocapsin e andsolanid-4-en - 3-onewer eobtaine dfro mProf .Dr .D.N .Kirk ,Steroi dReferenc eCollec tion (University of London, U.K.), veratramine, rubijervine,isorubi - jervine and cyclopaminefro mDr .R.F .Keeler ,Poisonou s PlantResearc h Laboratory (Logan,UT ,U.S.A.) ,N-acetyltomatidin efro mDr .S.F .Osman , Eastern Regional ResearchCente r (Philadelphia,PA ,U.S.A. )an ddeace - tylmuldamine (teinimine) from Dr. W. Gaffield, Western Regional Research Center (Albany, CA, U.S.A.). Soladulcidine was prepared by hydrogénation, at room temperature for 24h , of 100m g solasodine in 20m lo fglacia laceti cacid ,usin ghydroge na t20 0kPa ,an d2 5m gpal ladium 10%o ncarbo na scatalyst .Tomatidan-3-on e and demissidan-3-one (5a-solanidanone)wer eprepare db ydehydratio no ftomatidin ean ddemis - sidineaccordin gt oSchreibe re tal . (1963).Tomatidadien ewa sobtaine d by hydrolysis of tomatidenol using the two-phase system described in Chapter III. Solasodine glycosides were isolated from 300 g of green berrieso fS .nigru mb ybisolven textraction ,usin gadjuste dvolume so f solvents, and alkali precipitation as described for the colorimetric method (Chapters III and IV). The SGA precipitate was washed with ammonia (1mmol/1 )an d recentrifuged, then dissolved in 100m l ofho t ethanol (70%)an d leftfo rcrystallizatio n at -20°C.Th e crystalswer e collected by centrifugation at 20000 g for 15min ,an dwashe dwit hn - pentane, after which the procedure for crystallization and centrifu gation was repeated twice.Abou t 160m g of dried glycoalkaloids were obtained. TLC on HPTLC plates Silica gel 60 (E.Merck ) using ethyl acetate/pyridine/water (30:10:30 v/v) (Boll, 1962) and visualization by Clarke reagent containing 300 mg paraformaldehyde in 10 ml of phosphoric acid (8.5 mol/1), showe d two spots (Rp = 0.30 and 0.48), which corresponded with those of the standard compoundsa-solasonin e anda-solamargin e obtained fromDr .S.L . Sinden,Agricultura lResearc h Service (Beltsville,MD ,U.S.A.) .Upo ntwo-phas ehydrolysis ,th eglyco alkaloids produced solasodienewhe ncarbo n tetrachloridewa sused ,an d solasodiene and solasodine when chloroform was used as organicphase . The identity of these SAswa sconfirme db y their retention indices,b y their NPD/FID response ratios determined by capillary GC (Chapter V) and by GC-MS, using standard compounds. The extract of S.nigru mwa s 122 thenregarde da mixtur eo fa-solasonin ean da-solamargine ;thi smixtur e ofsolasodin eglycoside swa suse da ssuch . Solventsan dothe rchemical swer eanalytica lgrade . Samplepreparatio n For the analysis of tubers, at least 15 tubers were taken from each genotype. Smalltuber s ofwil d speciesan dhybrid swer ebrough t intoa Waring Blendor and homogenized using an amount of water equal to 1/4 ofth esampl eweight .Larg e tubers,suc ha sthos e ofcommercia lculti - vars,wer e cut into longitudinal sections representative toeac htube r inorde r toobtai nsubsample s of24 0g ,whic hwer e thenhomogenize di n 60m lo fwater . While homogenizing, 6.25 g aliquots of the tuber brei were taken foranalysi sb y theG Cmethod ,an d 12.5g aliquot s foranalysi sb yth e colorimetric method. These aliquots were transferred to a Waring Blendor with 75 ml of methanol/chloroform (2:1 v/v) as described in Chapter III. When the tubers of the wild Solanum species were very small -their weight may vary from 0.2-10 gunde r the conditions used forthei rpropagation -an dth eweigh to fa sampl eo f1 5tuber swa s2 5g or less, the samples were directly brought into the blender together withadjuste dvolume so fth emethanol/chlorofor mmixture . For the analysis of leaves, samples of 10g wer ebrough t into the blendertogethe rwit h7 5m lo fth emethanol/chlorofor mmixture . The tomato samples consisted of ten fruits which were homogenized until a fine homogeneous slurry was obtained. While spinning this slurry,2 5g sample swer e takenfo r 'Allround'an d 10g sample sfo rL . hirsutum glabratum. which were each transferred to a Waring Blendor with10 0m lo fth emethanol/chlorofor mmixture . The samples of S. nigrum consisted of 10g of greenberrie swhic h were brought into the blender with 75 ml of the methanol/chloroform mixture. Allsample swer eprepare di nduplicate ,unles sotherwis estated . The plant material was extracted with methanol/chloroform during homogenization (bisolvent method; Chapter III).Afte r concentrating the methanolic layer obtained by this extraction, the residue was either worked up for quantification of the solanidine glycosides by colorimetry as described in Chapter IV, or analysed by capillary GC 123 aftertwo-phas ehydrolysis . Optimizationo ftwo-phas ehydrolysi s For optimization of the two-phase hydrolysis procedure described in Chapter III, the following organic phases were tested: benzene; n-butanol; n-butanol/chloroform (1:1, 1:3, 1:5, 1:7 and 1:9 v/v); n-butanol/chloroform/acetic acid (2:4:1 v/v); carbon tetrachloride; chloroform; dichloromethane; dichloromethane/chloroform (1:1 v/v); diethyl ether and 2-propanol.Variou shydrochlori c acidconcentration s (0.5, 2,4 ,an d6 mol/1 )an dhydrolysi s durationsvaryin g from1-1 8h , dependingo nth ehydrochlori caci dconcentrations ,wer eapplied . The residue from thebisolven t extractionwa s transferredquantita tively to a 500m l flask using 100m l ofth eaci dphas e applied;the n 100 ml of the organic phase tested were added and thehydrolysi s was carried out on aho t plate. Subsequently, the twophase swer eallowe d to cool and the pH was adjusted to 10 with ammonia (7mol/1) . The aqueousphas ewa s separatedof f ina separator y funnel andwashe dwit h 20 ml of chloroform, which were thenadde d to the organic phase.Th e combined organic phases werewashe dwit h 25m l of ammonia (1mmol/1 ), and evaporated to dryness, and the residue was transferred quantita tivelyt oa crim pto pvia lan dfinall ydissolve d in1. 5m lo fmethanol / toluene (2:1v/v )containin g 1mg/m lo f5o-cholestan ean ddiosgeni na s referencecompounds . In the recovery experiments carried out for selectiono f theopti mum two-phase system, 1.6 mga-solanine , 1.7 mg solasodine glycosides and/or 2.2 mg a-tomatine, in 2 ml of methanol/chloroform (2:1v/v) , were added to the methanolic layers after their separation from the bisolventextractio nmixtures . For determining the recoveries for avariet y of Solanaceous plant samples, using the bisolvent extraction and the optimized two-phase hydrolysis system consisting of 100 ml of chloroform and 100 ml of hydrochloric acid (4mol/1) ,an dapplyin ga hydrolysi sduratio no f5 h , 2.3 mga-solanine , 2.3 mg solasodine glycosides and/or 3.2 mga-toma tine in2 m lo fmethanol/chlorofor m (2:1v/v) ,wer e added toth ehomo - genatesafte radditio no fth e7 5m lo fmethanol/chlorofor m usedfo rth e bisolventextraction . GC, determination ofretentio n indices andNPD/FI D responseratios , 124 and quantification of SAswer e carried out as described inChapte rV . Theconten to fa specifi cSG Awa scalculate db ymultiplyin g theconten t ofit saglycon eb yth erati oo fth emolecula rmasse so fth eSG Aan dth e aglyconemoiety . RESULTSAN DDISCUSSIO N Quantitativedeterminatio no fSA san dSGA s Fordeterminin g thenumbe ro frepetitiv eextraction srequire dfo rquan titative analyses,homogenate swer eprepare d intriplicat e fromtuber s of five markedly different potato genotypes. Each homogenate was extracted four times using the bisolvent extraction method and after each extraction the amount of solanidine glycosides extracted wasde termined. Thecontent so fsolanidin e glycosides ofth e fivegenotypes , calculated from the combined results after four repetitive extrac tions, were on average 26, 168,235 ,39 1an d 1180mg/k g freshweigh t for 'Bintje', the hybrids 1 and 2, AM 78-3778 and S. spegazzinii respectively. On average,84. 3 ± 3.1%, 96.5 ±0.8 %an d> 99.5%o fth e total amounts of solanidine glycosides were extracted from these diverse tuber samples after the first, second and third extraction, respectively. It was concluded that two repetitive extractions will usuallyb esufficient ,provide dth esolven tlayer sar eallowe d tosepa ratefo rsufficientl y longperiod so ftim ea sdescribe d inChapte rIII . Most of the organic solvents and mixtures of solvents, tested as organicphase si ncombinatio nwit hhydrochlori caci dfo rth ehydrolysi s of extracts from tubers, leaves and fruits spiked with a-solanine, solasodine glycosides and/or a-tomatine, resulted in low recoveries. This was due either to improper partition coefficients, which often caused degradation of SAs, or to the formation of an SA absorbing emulsionbetwee n the organic and the acid phases,o r toproblem swit h respect toth esolubilit y ofth eglycoalkaloid s oraglycones .Onl yth e chlorinated hydrocarbons yielded acceptable orgoo d recoveries.There fore, thesephase swer e further testedusin gvariou shydrochlori c acid concentrations andvariou s hydrolysis durations.Th eoptimu m two-phase system for thehydrolysi s of SGAs inextract s from different typeso f plant material consisted ofchlorofor m andhydrochlori c acid (4mol/1 ) anda hydrolysi s durationo f5 h . Subsequently, thissyste mwa suse d 4-1 1 O c CU 3 C UI 4- •r-l 4J XI Cd cd E C ect O CU H bO p CM >»140 0 CD E _o o ü 200 400 600 800 1000 1200 1400 capillary GC Fig. 1. Contents of solanidine glycosides (mg/kg fresh weight) in tuberso fpotat ocultivars ,wil dSolanu mspecie san dhybrid sdetermine d bycapillar yG Can da colorimetri creferenc emethod . the corresponding SGAs were all similar. Besides, adequate sets of samplescontainin gthes eSGAs ,whic hwer eneede dfo rsuc ha comparison , wereno tavailable . The contents of solanidine glycosides of individual potato tubers canvar y considerably (ChapterVII) , showingcoefficient s ofvariatio n up to 60% (Ross et al., 1978). Analysis of individual tubers of two potato cultivars indicated that for obtaining a representative value for abatc h of a given genotype, samples should preferably consisto f at least 15 tubers (Van Gelder, 1987). However, inman y studiesre ferred to in Chapter I, samples consisted of 4-6 tubers only. It is thusadvisabl e that before the capillaryG C method isapplie dfo r 129 Fig. 2. Chromatogram of a potato tuber extract spiked with steroidal alkaloids, obtained by post-analysis reprocessing of FID and NPD data from capillary GC. Peaknumber s and GC conditions as inTabl e 2. studying genetic variation or the inheritance of individual SGAs, or for selection purposes, the minimum sample size (number of tubers) is assessed. The same applies to analysis of other plantparts . Separation and identification of SAs Fig. 2 shows the result of post-analysis reprocessing of the simulta neously obtained NPD and FID data after GC of a potato tuber extract spiked with SAs on a CP-Sil 5 CB column, 50 m x 0.22 mm I.D., with a film thickness of 0.12 /im. The separation efficiency towards this complex SA mixture was very good; a similar separation has not been reported before. Separation of the mixture using the same column type with a film thickness of 0.4 /xm, further improved the resolution, but then the retention times were longer: t^'s for the last-eluting peak 130 Table 2. Retention indices, I, and NPD/FID response ratios of steroidal alkaloids from Solanaceae and Liliaceae determined by capillaryGC 1. Peak Compound I NPD/FID number^ responserati o 1 5a-Choiestane^ 2867 - 2 Solanthrene 2943 2.98 3 Solanidine 3130 2.96 4 Demissidine 3137 3.04 5 Solasodiene 3152 3.01 6 Demissidan-3-on e 3167 3.05 7 Tomatidadiene 3172 3.02 8 Diosgenin-* 3221 - 9 Solanid-4-en-3-one 3232 2.96 10 Rubijervine 3305 3.01 11 Solasodine 3338 3.00 12 Soladulcidine 3349 3.03 13 Tomatidenol 3361 2.93 14 Tomatidine 3370 2.88 15 Isorubijervin e 3374 3.00 16 Tomatidan-3-on e 3400 2.99 17 Solanocapsine 3409 3.80 18 Cyclopamine 3422 3.20 19 Veratramine 3475 3.13 20 Deacetylmuldamine 3544 3.26 21 Jervine 3560 3.35 22 N-acetyltomatidine 3598 2.60 ^•Column: fused silica, 50 m x 0.22 mm I.D., CP-Sil 5 CB, film thickness0.1 2/im .Ove ntemperature :270°C . ^Peak numbers correspond with those of Fig. 2; structural for mulasar egive ni nFig .3 an di nChapte rI ,Fig .1 . •^Referencecompound . 131 (N-acetyltomatidine)wer e 29.85mi no n0.1 2 /uman d84. 5mi no n0. 4 ßm, respectively. Sucha colum nca nb eusefu lwhe na nextremel yhig hreso lution is required. This resolution is about the ultimate which can presently be achieved, as application of the same column typewit ha film thickness of1. 2/x mdi dno tresul t ina furthe r increasebu ti na decrease of the resolution and, in adddition, in extremely long retentiontimes . Retention indices and NPD/FID response ratios are useful for the identificationan dcharacterizatio no fSA s(Chapte rV an dVII) .Tabl e2 shows these indices and ratios determined for a large number of SAs. Because thestructura lrelationshi pbetwee ndiosgeni nan d5a-cholestan e and the SAs is favourable with respect to theaccurac yo f theidenti fication and quantification, these steroidal compounds were used as reference compounds instead of n-octacosane and n-tetratriacontane used before. All the solanidane and spirosolane type SAs, which are commonly found in tuber-bearing Solanum species (see Chapter I, Fig1 andTabl e 1), showednormalize dNPD/FI Drespons eratio so fabou t3 .SA s which are structurally different with respect to the E-ring or toth e N-containing F-ring, especially N-acetyltomatidine, jervine andcyclo - pamine (Fig.3) ,showe dsomewha tdeviatin gNPD/FI Drespons eratios .Th e latter two compounds and also theVeratru m SAsveratramin e anddeace - tylmuldamine have not (yet) been found in Solanum and Lycopersicon species.Th ehig hvalu eo fsolanocapsin e isdu et oth epresenc eo ftw o nitrogenatoms . Therear ea numbe ro fN-containin gplan tcompound swhic hpotentiall y couldb ecoextracte d with the SAsan dhenc e could interferewit hthei r identification. However, compounds of this nature have not been de tected so far.Thi s is due to the currentprocedur e for sampleprepa ration, which is rather specific for the SAs because the bisolvent extraction and two-phasehydrolysi s exploit thedifferenc e inpolarit y between the glycoalkaloids and their aglycones.Moreover , the SAsre quirehig h temperatures for theirelutio ndurin g capillaryGC ,s omos t compounds present together with the SAs were found to pre-elute, and simultaneous NPD and FID unambiguously differentiate between SAs and steroidal sapogenins,whic hmigh thav ebee n (partly)isolate d together withth eSA san dwhic hma yhav esimila rretentio ntime s (Fig. 2). 132 H 5a-cholestane C27H48 Diosgenin C27H42O3 Demissidan-3-one C27 H+3 NO Solanid-4- en-3-one C27H41NO (Solanidan-3-one) Tomatidan-3-one C27 H43 N02 Cyclopamine C27 H+1 N02 H CH3 CH3 CH3 CH3 Veratramine C27 H38 N02 Jervine C27 H3g N03 n „,„*,;„,, iw„m;„„ r u MO N-acetyltomatidine CiqH47N04 Fig. 3. Structural formulas of 5a-cholestane, diosgenin and steroidal alkaloids. 133 Inconlusion ,th eabov eresult s showed that themetho d describedI n thisthesis ,i susefu lfo rqualitativ ean dquantitativ eanalysi so fth e SAsi ndivers esample so fSolanaceou splan tmaterial . When wild Solanum species are utilised in potato breeding pro grammes, iti sadvisabl e tomak ea qualitativ e andquantitativ einven tory of the SAs present in the potential crossing parents. For this analysis, a carefully developed procedure for sample preparation followed by capillary GC should be applied, because other procedures for samplepreparation ,reporte d inth eliterature ,ca nlea d tolosse s of various SGAs or SAs, and other analytical techniques do notoffe r the separation efficiency which can be achieved to date by capillary GC. REFERENCES ArnesonPA ,Durbi nRD . (1968)Studie so nth emod eo factio no ftomatin e asa fungitoxi cagent .Plan tPhysio l43:683-686 . Baker LC, Lampitt LH,Meredit h OB. (1955) Solanine, glycoside of the potato. III.-An improved method of extraction and determination.J SeiFoo dAgri c6:197-202 . Blincow PJ, Davies AMC, Bintcliffe EJB, Clydesdale A, Draper SR, AllisonMJ . (1982)A screenin gmetho dfo rth edeterminatio no fglyco - alkaloids intuber so fpotat ovarieties .J Nat nIns tAgri cBo t16:92 - 97. BollPM . (1962)Alkaloida lglycoside sfro mSolanu mdulcamara .II .Thre e new alkaloidal glycosides and a reassessment of soladulcamaridine. ActaChe mScan d16:1819-1830 . DeahlKL ,Sinde nSL . (1987)A technique forrapi ddetectio no fleptin e glycoalkaloids inpotat ofoliage .A mPotat oJ 64:285-290 . Gregory P, Sinden SL,Osma n SF, Tingey WM, Chessin DA. (1981)Glyco alkaloids of wild tuber-bearing Solanum species. J Agric Food Chem 29:1212-1215. Heftmann E. (1967)Biochemistr y ofsteroida l sapogenins andglycoalka loids.Lloydi a30:209-230 . KuhnR , Low I. (1961)Zu rKonstitutio n der Leptine. ChemBe r94:1088 - 1095. Olsson K. (1986) The influence of genotype on the effects of impact damageo nth eaccumulatio no fglycoalkaloid s inpotat otubers .Potat o Res29:1-12 . OsmanSF .(1983 )Glycoalkaloid s inpotato .Foo dChe m11:235-247 . Roddick JG. (1974) The steroidal glycoalkaloid a-tomatine. Phyto- chemistry13:9-25 . Roddick JG. (1979) Complex formation between Solanaceous steroidal glycoalkaloids and free sterols in vitro. Phytochemistry 18:1467- 1470. Roddick JG. (1980) A sterol-binding assay for potato glycoalkaloids. Phytochemistry19:2455-2457 . Rooke HS,Bushil l JH,Lampit t LH,Jackso n EM. (1943)Solanine ,glyco side of the potato. I. Its isolation and determination.J So c Chem IndLondo n62:20-24 . 134 Ross H, Pasemann P, Nitsche W. (1978) Der Glykoalkaloidgehalt von Kartoffelsorten in seiner Abhängigkeit von Anbauort und -Jahr und seinerBeziehun gzu mGeschmack .Z Pflanzenzücht g80:64-79 . Sachse J, Bachmann F. (1969)Übe r die Alkaloidbestimmung in Solanum tuberosumL .Z Lebens mUnter sForsc h141:262-274 . Schreiber K,Auric h0 ,Ossk eG . (1963)Solanum-Alkaloide .XVIII .Dünn schichtchromatographie von Solanum-Steroidalkaloiden und Steroid- sapogeninen.J Chromatog r12:63-69 . Schwarze P. (1962)Methode nzu m Solaninnachweisun dzu rSolaninbestim - mungi nKartoffelzuchtmaterial .Züchte r32:155-160 . SchwarzeP . (1963)Übe rde nGlykoalkaloidgehal tun ddi eZusammensetzun g des Glykoalkaloidkomplexes in Nachkommen der Artkreuzung Solanum tuberosumx Solanu mchacoense . Züchter33:275-281 . Sinden SL,Sanfor d LL,Deah lKL . (1986)Segregatio n of leptine glyco- alkaloids inSolanu mchacoens eBitter .J Agri cFoo dChe m34:372-377 . Slijm P,Weinan sR . (1973)Content s of solanine andprotei n inpotat o varieties.Repor t21103 ,5 Decembe r1973 ,Avebe ,Veenda m (InDutch) . Van Gelder WMJ, Jonker HH. (1986) Steroidal alkaloid composition of tubers of exotic Solanum species ofvalu e inpotat o breedingdeter mined by high-resolution gas chromatography. In Potato Research of Tomorrow,AG BBeekma n (Ed).PUDOC ,Wageningen ,pp .166-169 . Van GelderWMJ . (1987)Researc ho nth eC27-steroida l alkaloidcomposi tion of potato. Development of an efficient and quantitative method for determining the totalC27-steroida lalkaloi dcompositio n ofwil d species, interspecific hybrids and cultivars of Solanum. Research Report EC, Contract No 4910, Directorate-General for Agriculture, EuropeanCommunities ,Brussels . Van Gelder WMJ, De Ponti 0MB. (1987). a-Tomatine and other steroidal glycoalkaloids infruit so ftomat o lines resistant toth eglasshous e whitefly (Trialeurodesvaporarioru mWestw.) . Euphytica36:555-561 . VanGelde rWMJ ,Hopman s CWJ,Jonke rHH ,Va nEeuwij k FA. (1987)Biosyn thesisan dmetabolis mo fC27-steroida lalkaloid s inleave so fpotato . Proc10t hTrien nCon fEu rAsso cPotat oRes ,Aalborg ,pp .276-277 . 135 CHAPTERVII * GLYCOSIDIC-BOUND STEROIDAL ALKALOIDS IN TUBERS AND LEAVES OF SOLANUM SPECIESUSE DI NPOTAT OBREEDIN G INTRODUCTION Inmos tstudie so nSA so rSGA spresen ti nSolanu mspecies ,aeria lplan t parts have been analysed, usually for compounds of pharmaceutical interest (Petroshenko, 1956; Schreiber et al., 1961;Schreiber ,1963 ; BognärandMakleit ,1965 ;Schreiber ,1968 ;Bradle ye tal. ,1978 ;Weile r et al.,1980 ;Ripperge r and Schreiber, 1981). Most attentionha sbee n paid tosolasodine ,whic h isa ra wmateria lfo rth e industrialproduc tiono fsteroi dhormones .Onl yfe wan dlimite dsurvey so nth eS Ao rSG A compositionso ftuber so fSolanu mspecie shav ebee npresente d (Osmane t al., 1978; Van Gelder and Jonker, 1986; Johns and Osman, 1986). In these studies, only the main SAs or SGAs have been detected and/or quantified. In this chapter, the entire composition of glycosidic-bound SAs, including the minor SAs, of tubers and in some cases of leaves ofa number ofSolanu m specieswhic h arebein guse d inpotat obreeding ,ar e reported. EXPERIMENTAL Plantmateria l The species studied were: S. acaule Bitt., S.berthaulti i Hawkes,S . brevicaule Bitt., S.bukasovi i Juz., S. canasense Hawkes,S .pourlav i Hawkes, S. leptophves Bitt., S. multidissectum Hawkes, S. oplocense Hawkes, S. sparsipilum (Bitt.)Juz .e t Buk., S. spepazziniiBitt. ,S . sucrense Hawkes,S .venturi iHawke s etHjertin g andS .verne iBitt .e t Thischapte r isbase do nth epaper : VanGelde rWMJ ,Vink eJH ,Scheffe rJJC . (1988)Steroida lglycoalkaloid s intuber san dleave so fSolanu mspecie suse d inpotat obreeding .Euphy - tica39S:147-158 . 136 Wittm. The taxonomie nomenclature is according to Hawkes (1978). Clones of these species were derived from accessions of the Dutch- German potato collection at Braunschweig Genetic Resources Centre (BGRC, Braunschweig, F.R.G.), except for S. berthaultii of which the provenance is not known. The first number of the accession numbers mentioned in this study, corresponds to the BGRC accession number (Hoekstra and Seidewitz, 1987), the second number refers to theclon e number.Th eplan tmateria l tob eanalyse dwa sproduce d ina glasshous e undercontrolle dconditions ,whic har eroutinel yuse da tth eFoundatio n forAgricultura l Plant Breeding SVP to induce tuber formation inwil d species.Th egrowin gperio dwa s frommi dMarc h tomi dAugust .Th eday - lengthwa s 12h from 6.00 h to18.0 0h ; itwa s controlledb y anauto mated PVC awning, which covered and darkened the growing area ofth e plants completely. In 1985,th e sums of solar radiation (350-3000nm ) were for March (two weeks), April, May, June, July and August (two weeks): 11, 36, 48, 46, 51 and 23 kJ/cm ,respectively , and for the same period in 1986: 11, 26,59 ,56 ,5 9 and 24kJ/cm^ , respectively. The temperature was kept at ca 18°C from 7.30 h to 18.00h and atc a 13°C from 18.00h to 7.30 h.Th erelativ e airhumidit ywa skep ta tc a 60-70%. If necessary for keeping the temperature at 18°C, the glass housewa ssomewha t shaded onho tbrigh t days. In1985 ,th eplant swer e grown inearthenwar e pots of 11c m diameter forpropagatio n oftuber s as part of a breeding programme; in 1986 the plants were especially grown for SA analysis, for which plastic pots of 18c m diameterwer e used. To enable a comparison of the contents of solanidine glycosides inth esmal ltuber so fth ewil dspecie swit hthos eo fcultivars ,plant s of 'Bintje', 'Pimpernel'an d of the SVPprogenitor s AM 78-3778 andA M 78-3787 ('Arabesque')wer e grownunde r thesam econdition s as thoseo f thewil d species in the glasshouse in 1986.Afte r harvesting, thetu bers were stored in the dark at 5-7°Cfo rsi xweeks ,befor e theywer e analysed. Chemicalsan dprocedure s Standard SAs, solvents and solutions for extraction and hydrolysis, sample preparation, capillary GC,an d identificationan dcharacteriza tion of SAsb y retention indices and simultaneous NPD andFI Dwer ea s described in the Chapters V and VI. The compounds 5a-cholestane and 137 diosgeninwer euse da sreferenc ecompounds .Th eNPD/FI Drespons eratio s were calculated from the peak areas, but when integration of small overlapping peakswa s inaccurate,th epea kheight swer emeasured .Con tents ofth e individual SGAswer ecalculate d from theG Cpea karea so f the corresponding aglycones (and also of theirA3,A5-dehydratio npro ducts in the case of A5-unsaturated aglycones) and were expressed on the basis of solatriose in the case of solanidine, solasodine and unidentified SAs, and of lycotetraose in the case of demissidine and tomatidine. The contents of the solanidine glycosides of the field- grown tubers of the cultivars were determined using thecolorimetri c methoddescribe d inChapte rIV . RESULTSAN DDISCUSSIO N TheSG Aconten to fsmal ltuber s Thewil dspecie sproduce d tubersvaryin gi nfres hweigh tfro m0.2-1 5g . Such small sizes might influence the SGA contents of the tubers.I n order to get an impression of the magnitude of this effect, tubers corresponding insmal l sizet othos eo fth ewil d specieswer eselecte d from the glasshouse-grown tubers of the four cultivars. For each cul- tivar, the contents of the solanidine glycosides of seven individual tuberswer e determined. Theresult s areshow n inTabl e 1.Th e"normal " contents ofth ecultivar s aregive na swell .Thes e figuresar eaverag e valuesdetermine dfo rbatche so f2 0field-grow nmatur e tuberscollecte d over atleas t threeyear s attw o locations.Th econtent s ofsolanidin e glycosides inth eglasshouse-grow nsmal ltuber swer etw ot othre etime s higher thanthos e ofth e field-grownnorma l tubers.Withi na cultivar , the smallest tubers showed, in general, the highest contents. The ranking ordero fth ecultivar s ast othei rcontent swa sequa lfo rbot h environments. These datamigh t be useful for placing the SGA contents ofth eexoti c tubersi na mor erealisti cperspective .However ,a ssmal l tubers ofwil d species canb ematur e and suchtuber so fcultivar sar e usually immature, wild species may show a different (less strong) correlation between tuber size and SGA content compared to theculti vated potato. Therefore, further studies on the relation between the SGAconten tan dth esiz ean dmaturit yo ftuber s shouldb ecarrie dout . 138 Table 1. Freshweigh t (g)an d content of solanldlne glycosides (mg/kg fresh weight) of seven individually analysed small tubers of the cultivars Bintje,A M 78-3778,A M 78-3787 ('Arabesque'), andPimpernel , grown in pots in a glasshouse. In comparison, the contents offield - grownmature-harveste dtuber sar egive n. Cultivar Tuberfres hweigh t Solanidineglycoside s Range Average Range Average Bintje glasshouse 1.0-15 8.2 73-18 8 126 field 139 40 AM78-377 8 glasshouse 0.2-15 5.7 321-1484 721 field 140 360 AM78-378 7 glasshouse 3.9-12 7.1 95-26 5 155 field 152 58 Pimpernel glasshouse 0.3-17 8.6 132-1287 522 field 122 146 •'-Averagevalue sdetermine d forsample s of2 0tuber scollecte d overa t leastthre eyear sa ttw olocations . TheSG Acompositio no fSolanu mspecie s Table 2show s theS Acompositions ,expresse d asglycoalkaloids ,o fth e tubers (T)an d in some cases ofth eleave s (L)o fa numbe r ofSolanu m speciesuse d inpotat obreeding .Unti lnow ,suc hcomposition shav eno t been reported for the species S. brevicaule. S. bukasovii. S. cana- sense. S. gourlavi.S .leptophyes .S .multidissectum .S .oplocense .S . sparsipilum.S .spegazzini ian dS .venturii . Schreibere tal .(1961 )reporte dfo rth efoliag eo fS .berthaulti ia contento f1200 0m gsolanidin eglycoside spe rk gdr yweight .Gregor ye t al. (1981)foun dsolasodin eglycoside s ina concentratio no f100 0mg/k g dry foliage in this species.Th e S.berthaulti i accession we analysed 139 contained 1090 mg solasodine glycosides per kg fresh weight, which corresponded to about 8000mg/k g dry foliage (Fig. 1A).W e couldno t detect anysolanidine ,no teve nwhe nth edetectio n limitwa sa slo wa s 0.2 mg/kg. In the tubers ofS .berthaultii . forwhic h theS Acomposi tion has not been reported before, we found solanidine but nosola sodine glycosides (Fig. IB). Small amounts of other compounds were present in the foliage aswel l as inth etubers . Severalo f thesear e mostprobabl y SAsa s is indicatedb y theNPD/FI D response ratioswhic h areshow na tth epea ktop si nth efigures .Superimpositio no fth echro - matograms of the leaf and tuber samples and comparison ofth eNPD/FI D response ratios, showed that none of the leaf SAs corresponded toan y of the tuber SAs,possibl y except for the compound which elutedafte r 11.00min .M S may showwhethe r these twopeak scorrespon d to thesam e compound. Grafting experiments usingcombination s ofspecie s fromSolanu man d other genera of Solanaceae,hav e suggested that the SAs inth eleave s are synthesized independently from the SAs inth e root system (Petro- shenko, 1956), and thatn o translocation of SAs from theroot s toth e leavesoccurs .Ou rdat aobtaine d forS .berthaulti i indicated thatth e SAs inth eleave sar e synthesized also independently from theS Ameta bolism in the tubers (stem system) and vice versa,whic h means that the SA synthesis in S. berthaultii is organ specific or regulatedb y light. The literature on the SA composition of S.acaul e is contradictory as to the presence of demissidlne and tomatidine glycosides (forde tails see Gregory, 1984). Both our present and earlier analyses (Van Gelder andJonker ,1986 )showe d that inth eaccession s studied,demis - sidineglycoside sa swel la stomatidin eglycoside swer ealway spresent , but we also found that in addition solanidine glycosides can be present. The discrepancies between the compositions described for Solanum speciesb y differentauthor sma yb e duet o intraspecificheterogeneit y and/or differences in the growing conditions applied, but probably, imperfection of the analytical techniques,wit h respect to the iden tificationaccurac yan ddetectio nlimits ,ha splaye da nimportan trole . to 01 r-l CU ••H xl 4J Ü G c CU 3 3 P. O o Ol p. H B cd 0 o i-i cd ro Xl •u o en o ï-l vi i • Vi Vi • • Vi Vi i i m i i m o o CU o S •i-I xl X) •1-1 rt0} i-I 01 r-l 4J o cd 0 X! S in co o o 1->1 , r~- m ecd H bO /*^ i-I H CD 01 C CU •H X) X! •H O 01 01 O eu et) o 1-1 o CTv O 1->,1 4J t/2 bO o M-l CU i-l O C 01 •H CU 0] •o X) X) •i-l •1-1 •H 01 01 O 01 o i-l •1-1 o cd S M m n U CU 1-^1 in i-i o i-i Q bO i-i i-i m ed r-l i-i CV 01 cd C CU •i-l Xl X) •.H •oH 01 o Vi O etc) o i 00 •4tinOOOeNr0O 1->-1 , bO Oc CM r-> O CTv •1-1 Tl in O O O noooooo u u u u u u u C NOini^OHOvOoocoroinHHHHHHH uo o o u td PI oo M M r-.v0.3-CM co H coi- IO H in tfl NuiiniDinminaiNvomj-j-J^^Nn n) >N •ü r-l •H a> O J-l •>H ai 4-> u O 1 1 1 i T-l co CM S-l !-l - o <*> o o ta in csiC M oo o voCT-or^cr\oocMr^a\CMCMCMOooiHm^icM r~~Y"v'— rvrH Iff B 143 Fig. 1.Steroida l alkaloidcompositio no fleave s (A)an d tubers (B)o f S.berthaulti i shownb ypost-analysi s reprocessingo fNP Dan d FIDdat a simultaneously obtained by capillary GC.Column : fused silica, 50m x 0.22 mm I.D., CP-Sil 5 CB, film thickness 0.12 ftm. Carrier gas: hydrogen, linear velocity 50 cm/s. Oven temperature: 270°C. Injection volume: 1.4 /il. Reference compounds: a-cholestane and diosgenin. Positive peaks represent nitrogen-containing compounds with NPD/FID response ratios as shown at the peak tops, negative peaks represent nonnitrogen-containingcompounds . For S. vernei. 2000-5000 mg solanidine glycosides per kg dry weight havebee nreporte dfo rfoliag esample s (Schreiber,1963) .Althoug hmor e data on the SAs of S.verne ihav e notbee n reported since then,thi s speciesha sbee nwidel yuse di npotat obreedin g (Ross,1986) , forexam ple as a source of resistance to the potato cystnematode .Probably , it was assumed that the data reportedb y Schreiber wouldb e valid in general forfoliag ea swel la stuber so fal lS .verne iaccession suse d inbreedin gprogramme s (cf.Holden ,1981) .Ou ranalyse sshowe dtha tth e leaves and tubers of clones from S.verne i accession 15451, contained highlevel so fsolasodin eglycoside s (Fig.2 Aan dB) .Thi si sth efirs t timesolasodin e isreporte dt ooccu r intuber so fwil dSolanu mspecies . Solasodineha sbee nreporte d toposses s awea k teratogenic activity in hamsters, but the activity of the solasodine glycosides is not known (seeChapte r II).I ti stherefor eadvisabl et ostud ywhethe r theseSGA s aresynthesize d intuber so fhybri doffspring . In addition to the solasodine glycosides, only one other SA was presenti ntrac eamount s inth eleave so fS .vernei .wherea sth etuber s containedhig hlevel so fsolanidin eglycoside san dconsiderabl eamount s of unidentified compounds. The NPD/FID response ratios indicated that the unidentified peaks represented SAs.Th e tubers of the accessions 15451 (Fig. 2B) and 8240 (Fig. 3A)showe d different SAcompositions , especially with respect to some of the major unidentified compounds. Thisindicate s thatintraspecifi cvariatio nexists . S. sparsipilum accession 8206 showedremarkabl e differencesbetwee n the SA composition of clone 7, grown in 1985,an d that of clone15 , grown in 1986. (Table 2 and Fig. 4A and B). The origin of these differences isno tclear . Iti sno tlikel y fromth eviewpoin to fth e 144 )t--^y^r-. JW_ B Fig. 2.Steroida lalkaloi dcompositio no fleave s (A)an d tubers (B)o f S.verne i15451-12 .Experimenta lcondition sa si nFig .1 . Fig. 3. Steroidal alkaloid composition of tubers of S.verne i 8240-10 (A)an dS .bukasovi i 1033-20 (B).Experimenta lcondition sa si nFig .1 . 146 - rM csi "c^ 1 o —CC TOooo n ^ n • f\ y k.nn B Fig. 4. Steroidal alkaloid composition of tubers of S. sparsipilum 8206-7grow ni n198 5 (A)an d8206-1 5grow ni n198 6 (B). 147 biogenesis of SAs, that such differences within accessions aregenet ically determined. On the other hand, it is not unlikely that the growing conditions may influence the SA composition. In 1985, a low solanidine contentwa s attended by thepresenc e of a relatively large numbero funidentifie dSAs .Th esmal learthenwar epot suse dfo rgrowin g theplant s in198 5migh thav e inducedsom estres swhic hcoul dhav ebee n (partly) responsible for this. The same was true for S. spegazzinii. butth ephenomeno nwa sno tobserve dfo rtuber so fothe rspecie ssuc ha s S.bukasovi i (Fig. 3B)an d S.venturii . growni n198 5unde r thesam e conditions. Anyhow, the occurrence of markedly different SAcomposi tions within accessions is most interesting. Further studies on the structure of theunidentifie d compoundsma ypossibl yyiel d information onthei rnature . Except for S. acaule. the tubers of all species studied contained solanidine glycosides as main components. S. acaule belongs to the seriesAcaulia .whils tth eothe rspecie sbelon gt oth eserie sTuberosa . Inmos ttube rsamples ,unidentifie dcompound swer edetected ,whic hmos t likely were SAs as was shown by their NPD/FID response ratios. Such compounds,whic hwer e latercharacterize d asSA susin g GC-MS,ha dbee n found earlier in foliage of S. vernei offspring (Van Gelder et al., 1987)an d in fruits ofLycopersico n species (VanGelde r andD ePonti , 1987). Basedo nth eprocedur e forthei r isolation,i tca nb econclude d that such SAs occurred in the leaves and tubers as glycosides (see Chapter VI).I n the presently analysed tubers,th e individualconcen trations of the unidentified SAs,expresse d as glycoalkaloids,varie d fromles stha n2. 5mg/k g (traces)t omor etha n20 0mg/kg ,an dth etota l amountsvarie d fromabou t2.5-95 0mg/kg . Most ofth e species showedhig h totalSG Acontents .Unde r thegrow ing conditions applied,al lspecie sproduce d smalltubers ,whic hmigh t have beenpartl y responsible for theirhig h contents,a s shownabove . However, even when this is taken into consideration, several species showed totalcontent swhic hwer e extremelyhigh .Thus ,analysi s ofth e hybridprogen yo fsuc hspecie si snecessary . The SVP progenitors AM 78-3778 and AM 78-3787 ('Arabesque')wer e both derived from S. vernei accessions by backcrossing with S.tube rosum. Bothgenotype s showed ahig h levelo fresistanc e topotat ocys t nematodes (Dellaert andVinke , 1987). The clone AM 78-3787whic hcom - 148 bines ahig h level ofnematod e resistance with a lowconten t ofSGAs , hadbee n selected inspit eo fth eextremel yhig h SGAcontent snormall y found inS .vernei . However, in the course of thebreedin gprogramme , manyhybrid swer e discardedbecaus e ofthei rhig hSG Acontents .I nth e tubers of 'AM 78-3787'whic h were analysed for their SAcompositions , glycosidic-boundsolanidin ewa sfound .I nth eleaves ,solasodin ean da n unidentified compound, which was shown by GC-MS to be possibly an isomer of solanidine,wer e also present (VanGelde r et al., 1987).W e didno tye tinvestigat ewhethe r thesefolia rSA sar eorga nspecific ,o r whether they may be synthesized in the tubers,dependen t on theenvi ronmentalconditions . Until now studies on the composition of glycosidic-bound SAs of Solanum species have been carried out from aphytochemica lviewpoint , inwhic h genetic and environmental aspects havebee n disregarded. Our resultssho wtha tstudie so nth einheritanc eo fth evariou sSAs ,o nth e inter- and intraspecificvariatio n in SA compositions,an d on thein fluence of the environment on the SA composition, will be useful for efficiently utilizing wild germplasm. Intraspecific variation may be used profitably by selecting crossing parents which combine adesire d traitwit hth eleas tunfavourabl e SAcomposition . Inconclusio n it canb e stated thathig h concentrations ofsolani dine glycosides and a number of other SGAs, can be present inwil d Solanum species.A s in some cases the SGA compositionvarie dwithi na species or between different organs of one plant, the limited data available in the literature can not be used as a general guide in potatobreeding .I ti stherefor erecommende d toanalys eth eSA so fwil d crossing parents, and to identify or characterize the unknown SAs, before they are used in a breeding programme. Those genotypes, that combine a desired trait with the least unfavourable SA composition, canthe nb eselecte da scrossin gparents .Whe nspecie sshowin gextreme lyhig hlevel so fSGA so rcontainin gSA swhic hshoul dno tb eallowe di n potatotubers ,ar euse di nbreedin gprogrammes ,th ehybri dprogen ymus t alsob e analysed. Studies on the effect of tuber sizean dmaturit yo n the SGA contents of wild species, cultivars and their hybrids,wil l showwhethe r iti spossibl e toselec tfo ra lo wSG Aconten t ina nearl y stageo fa breedin gprogramme . 149 REFERENCES Bognâr R, Makleit S. (1965) Steroidalkaloidglykoside. 8. Mitteilung: Zusammenfassung der eigenen bisherigen Untersuchungsergebnisse über das Vorkommen von Steroidalkaloidglykosiden in Pflanzen der Gattung Solanum.Pharmazi e20:40-42 . Bradley V, Collins DJ, Crabbe PG, Eastwood FW, Irvine MC, Swan JM, Symon DE. (1978)A survey of Australian Solanum plants for poten tiallyusefu lsource so fsolasodine .Aus tJ Bo t26:723-754 . Dellaert LMW, Vinke JH. (1987) Testing potatoes for resistance to Globodera pallida pathotype Pa-3;resistanc e spectra ofplan tgeno types and virulence spectra of Pa-3 isolates.Revu e Nématol10:445 - 453. Gregory P. (1984)Glycoalkaloi d composition ofpotatoes :diversit yan d biological implications.A mPotat oJ 61:115-122 . Gregory P, Sinden SL, Osman SF, Tingey WM, Chessin DA. (1981)Glyco - alkaloids of wild tuber-bearing Solanum species. J Agric Food Chem 29:1212-1215. HawkesJG . (1978)Biosystematic so f thepotato .I nTh ePotat oCrop ,P M Harris (Ed).Chapma nan dHall ,London ,pp .15-69 . Hoekstra R, SeidewitzL . (1987)Evaluatio n datao ntuber-bearin gSola num species, 2nd Ed. Inst für Pflanzenbau und Pflanzenzüchtung der FAL, Braunschweig, and Stichting voor Plantenveredeling SVP, Wageningen. HoldenJHW . (1981)Th e contribution ofbreedin g to the improvement of potato quality. In Survey Papers 8th Trienn Conf Eur Assoc Potato Res,Munich ,pp .37-53 . Johns T, Osman SF.(1986) Glycoalkaloids of Solanum seriesMepistacro - lobuman drelate dpotat ocultigens .Bioche mSyste mEco l14:651-655 . Osman SF,Her b SF,Fitzpatric kTJ , Schmiediche P. (1978)Glycoalkaloi d composition of wild and cultivated tuber-bearing Solanum species of potential value in potato breeding programs. J Agric Food Chem 26:1246-1248. Petroshenko EI. (1956)Glycoalkaloid s incro p plants.Us p Sovrem Biol 42:19-32 (inRussian) . Ripperger H, Schreiber K. (1981) Solanum steroid alkaloids. In The Alkaloids,XIX ,RG A Rodrigo (Ed).Academi c Press,Ne wYork ,pp .81 - 192. RossH . (1986)Potat obreedin g -Problem san dperspectives .Advance si n plantbreeding ,Supp lJ Plan tBreedin g13:1-130 . Schreiber K. (1963)Übe r die Alkaloidglykoside knollentragender Sola- num-Arten.Kulturpflanz e11:422-450 . Schreiber K. (1968) Steroid alkaloids: the Solanum group. In The Alkaloids,X ,RH FMansk e (Ed).Academi cPress ,Ne wYork ,pp .1-192 . SchreiberK ,Hamme rU ,Itha lE ,Ripperge r H,Rudolp hW ,Weissenbor nA . (1961) Über das Alkaloid-Vorkommen verschiedener Solanum-Arten. TagungsberDtsc hAka dLandwirtschaftswis sBer l27:47-73 . VanGelde rWMJ ,Hopman sCWJ ,Jonke rHH ,Va nEeuwij k FA. (1987)Biosyn thesisan dmetabolis mo fC27-steroida lalkaloid s inleave so fpotato . Proc10t hTrien nCon fEu rAsso cPotat oRes ,Aalborg ,pp .276-277 . Van Gelder WMJ, Jonker HH. (1986) Steroidal alkaloid composition of tubers of exotic Solanum species ofvalu e inpotat o breedingdeter mined by high-resolution gas chromatography. In Potato Research of Tomorrow,AG BBeekma n (Ed).PUDOC ,Wageningen ,pp .166-169 . Van Gelder WMJ, De Ponti 0MB. (1987) a-Tomatine and other steroidal glycoalkaloids infruit so f tomatoline s resistant to theglasshous e 150 whitefly (Trialeurodesvaporarloru mWestw.) . Euphytlca36:555-561 . Weiier EW, Krüger H, Zenk MH. (1980)Radioimmunoassa y for thedeter mination of the steroidal alkaloid solasodine and related compounds inlivin gplant san dherbariu m specimens.Plant aMe d39:112-124 . 151 CHAPTERVIII * CHARACTERIZATION OF NOVEL STEROIDAL ALKALOIDS FROM TUBERS OF SOLANUM SPECIESB YCOMBINE DGA SCHROMATOGRAPH YAN DMAS SSPECTROMETR Y INTRODUCTION Tubers of Solanum species contained, inadditio n toknow nglycosidic - bound SAs, unidentified compoundswhic hwer e tentatively characterized as SAs by their NPD/FID response ratios determined by capillary GC. Based onth eprocedur e for their isolation itwa s concluded thatthes e SAsoriginate dals ofro mglycoside s (ChapterVII ). This chapter describes the characterization of such unidentified compoundsusin gretentio nindices ,GC-MS ,high-resolutio nM S (HRMS)an d hydrolysis in two-phase systems. By combining the data obtained by these techniques, a number of novel SAs were characterized. The SA composition (including main and minor SAs) of tubers of three wild Solanumspecie san don eprimitiv ecultivate d subspecies,widel yuse di n potato breeding, is presented. The implications of the results for potatobreedin gar ediscussed . EXPERIMENTAL Plantmateria l Tubers of Solanum chacoense Bitt.accessio n 8054, S. leptophvesBitt . accession 27208, and S. tuberosum ssp. andigena (Juz.e tBuk. )Hawke s accession 1024, were produced by cultural techniques described in ChapterVI Ifo r1986 ,a swer eth etaxonomi enomenclatur ean dth eprove nance ofth e accessionnumbers .Th eanalyse s ofS .sparsipilu m (Bitt.) Juz. etBuk .wer ecarrie d outo nth eextrac t from thetuber so facces - Thischapte r isbase do nth epaper : Van Gelder WMJ, Tuinstra LGMTh,Va n der GreefJ , SchefferJJC . (1989) Characterization of novel steroidal alkaloids from tubers of Solanum species by combined gas chromatography and mass spectrometry: impli cationsfo rpotat obreeding .J Chromatogr ,i npress . 152 sion820 6genotyp enumbe r7 grow ni n1985 ,a sdescribe d inChapte rVII . Chemicalsan dsampl epreparatio n Standard SAs,solvents ,extractio no fth etubers ,two-phas ehydrolysis , sample clean-up, and capillary GCusin g retention indices andNPD/FI D responseratios ,wer ea sdescribe d inth eChapter sV an dVI . GC-MSan dHRM S AFinniga n450 0ga sChromatograph-mas sspectromete rcouple d toa nInco s data system was used to record the mass spectra of the SAs. The SAs were gas chromatographed using a fused-silica column, 25m x 0.22 mm I.D., CP-Sil 5 CB, film thickness 0.12 /zm (Chrompack Nederland). The temperatureprogramm eo fth eove nwas :280° C (20min) , increase8°C/mi n for 5 min, then 320°C for 5min .Othe r GC conditionswere :injector , 300°C; interface,290°C ;carrie r gas,helium , linearvelocit y 30cm/s ; injectionvolume , 2 pi, splitting ratio 1:50. The conditions ofelec tron impact ionization (EI) were: ion source temperature 230°C, emission current 0.25 mA, ion source energy 70 eV; those ofchemica l ionization (CI):ionizatio n gasmethane ,io nsourc e temperature180°C , emission current 0.25 mA, ion source energy 100 eV, ionizer pressure 67 Pa.Th emas s rangewa s monitored from 50 to 500mas s units inth e EImod ean dfro m9 0t o45 0mas sunit si nth eC Imode .Th esca nrat ewa s 1scan/0. 5s . The peaks of the total ioncurren t (TIC)profil e obtainedb yGC-M S were comparedwit h thepeak s ofth emas schromatogram s recordeda tm/ z 150an dm/ z204 ,whic har especifi cfo rth esolanidanes ,an da tm/ z11 4 and m/z 138,whic h are specific for the spirosolanes (Ripperger and Schreiber, 1981). For each tuber extract, the TIC profile and mass chromatograms were compared with the chromatogram of the N-containing compounds,obtaine db ycapillar yG Cusin gsimultaneou sdetectio nb yNP D and FID, inorde r to investigate whether other typeso fSA swer epre sent.Coelutio no fcomponent swa schecke db y comparing theTI Cprofil e andmas schromatogram srecorde da tm/ z15 0an dm/ z204 ,wit hmas schro matogramsrecorde da tspecifi cm/ zvalues . HRMSwa suse d forconfirmatio n ofth emasse sobtaine db yGC-MS ,fo r determinationo fth eexac tmasse so findividua lcomponent san dfo rcal culationo fthei relementa lcompositions .A Finniga nMAT-71 1mas sspec - 153 trometer coupled to a Tracor TN-1750multichanne l analyser was used. The EIcondition swere : ionsourc e temperature 240°C,emissio ncurren t 800 fiA, ion source energy 100 eV, and accelerationvoltag e 8kV .Th e resolutionwa s2000 0 (10%valle ydefinition) ,th eprob etemperatur ewa s increasedfro m50° Ct o350° Ci n50 0s ,an dth emas srang ewa sfixed .A n amount of 1-5 /il of tuber extract was introduced into the mass spectrometer via a direct insertionprobe .Onl y the tuber extractso f S.chacoens ean dS .sparsipilu mwer eanalyse db yHRMS . RESULTSAN DDISCUSSIO N Identificationan dcharacterizatio no fSA s HRMS of the tuber extracts of S. chacoense and S. sparsipilum showed abundant ions with the masses 150.1283 and 204.1752, for which the formulas C^gH^N andC14H22 Nwer e calculated,respectively . Thesefor mulas agree with those of the diagnostic fragments at m/z 150 (base peak)an dm/ z 204o f the solanidanes (Ripperger and Schreiber, 1981). Fragments with exact masses corresponding to the base peaks of other groups of SAs, such as spirosolanes, epiminocholestanes, aminospiro- stanesan dsolanocapsines ,wer eno tpresent . Allcompound s inth etube rextract so fth efou rSolanu m(sub)specie s thatwer echaracterize d asS Ab y theirNPD/FI Drespons e ratios,showe d mainion sa tm/ z15 0(bas epeak )an dm/ z20 4i nth espectr aobtaine db y GC-MS (EImode) . Thismean s thatthe yal lwer e solanidaneswit hunsub - stituted Ean d F rings (Ripperger and Schreiber, 1981;Kanek oe tal. , 1981). Fig. 1 shows the TICprofil e (D)an d themas s chromatogramsa t the m/z values of the diagnostic ions of the solanidanes (B and C). Base peaks corresponding toothe r groups of SAswer e notdetected ,a s is illustrated for the spirosolanes by themas s chromatogram recorded atm/ z11 4(Fig .1A) . Substitutions in the steroid skeleton (rings A-D) do not markedly influence the fragmentation patterns (Budzikiewics, 1964). However,a comparison of the EI mass spectra of nine standard alkaloids showed that differentiation between the saturated SAs (demissidine,soladul - cidine and tomatidine), theA5-unsaturate dSA s (solanidine,solasodin e andtomatidenol )an dthei r3,5-dien e dehydrationproduct s (solanthrene, solasodienean dtomatidadiene )wa spossibl eb yusin gth eratio so fth e 154 7.9 A m/zII A Splrosolanesno tdetecte d 100-°1B m/z15 0/ lSolanidane s 38.9 Fig. 1. Mass chromatograms recorded at the diagnostic m/z values of spirosolanes (A:m/ z 114,bas epeak )an d solanidanes (B:m/ z 150,bas e peak; C:m/ z 204)an d the total ioncurren t (TIC) profile,obtaine d by GC-MS (EImode ) analysis ofa steroidal alkaloid extract from S.sparsipilu mtubers . abundances of the ions atm/ z 91an dm/ z 93,whic hwer eabou t 0.8 for thesaturate d SAs,1. 3 forth eA5-unsaturate dcompound san d2. 0 forth e 3,5-dienes, respectively. Similar ratios for the three respective SA groupsteste dwer efoun dfo rth eabundance sa tm/ z10 5an dm/ z107 . GC-MS (CImode )o fth e SAsshowe d (M+l)+ ionswit ha relativ eabun dance of 30-100%. SAs containing ahydroxy l group showed in addition (M+l-18)+ ionswit h arelativ e abundance of 20-50%,du e toth elos so f waterfro mth eprotonate dmolecule s (Fig. 2). m co •o co c oo o cd 33 co CM co- co 4-> co + a /^ co o 1-1 o\- m + co S-l + •H o O co- 0) CM CO p. X w UI + UI cd B 4-> e 4 CM C 55 •t-i L CM 13 CM •* •H K O- o •* CM C in .-I cd i-i O o U) <4-l O cd txO HO •p o e a) .-I o. •H en •d o •Ö <1> •U .E i-3l ui O o 0) o m O 2 1-1 x> O •cH O UI •H xt c CO •ioH 4J r>H^ O 0c) l-l 4-1 •ö u u 01 •. Z Z 1-1 X z ^ ai •O ui 0) T3 x: 01 P. N •r-l • m in co c^ 1-1 o 0) 4-»1 in r- m in m m i-t 00 4-1 cyi cTi i—i er» l-l CM Upon two-phase hydrolysis using chloroform as organic phase, theA5 - unsaturated SGAs produce 5-ene-3/9-ol aglycones as well as thecorre sponding 3,5-diene dehydration products,bu t using carbon tetrachlo ride, they produce almost entirely 3,5-dienes (Chapter III).Thi s phenomenon (shift from the concentration of the 5-ene-3^-ol to the concentrationo fth e3,5-diene )wa suse dfo rth edetectio no fth eenol s and thedehydratio nproduct sb y capillary GC.Th e finalidentificatio n ofeac h5-ene-3ß-o lan d3,5-dien e pairwa sachieve dusin gth edat afro m EImod eGC-M S (ratioso f theabundance s atm/ z 91/93an dm/ z105/107) , from CI mode GC-MS (presence of (M+l-18)+ ions), and from the HRMS (absence ofoxyge natom s inth edehydratio nproducts ,an da differenc e of 18.0105 between the masses of the SAs and their dehydration pro ducts). Table 1 shows the main physical and chemical datawhic h enableda characterizationo fth eSAs .Onl yth enaturall yoccurrin gSA sar egive n andno tthei rdehydratio nproducts .Compoun d1 ,a solanida-dien-ol ,ma y be dehydrosolanidine according to its retention time and that of its dehydration product C27H39N (mass 377.3082), which can be dehydro- solanthrene.Compoun d 3i sa saturate d SA,whic hcontain s twohydroxy l groups and isprobabl y an isomer of compound 8.Compound s 4an d 5ar e isomers ofcompoun d 1.Compoun d 7wit hM 4" atm/ z427 ,showe dth elos s ofon emolecul eo fwate rupo nhydrolysi s (difference inmas so f18.010 5 with itsdehydratio nproduct) ,a n (M+l-18)+pea k inC Imod eGC-MS ,an d an (M-31)+ peak in the EI mode.Therefor e compound 7ma y bemethoxy - solanidine orhydroxymethylsolanidine .Th edat a from theHRM Sreveale d thepresenc e ofa nS Ashowin g themas s427.345 0whic hcorrespond swit h the formula C28H45NO2; this supported this hypothesis.A second com- poundwit hM atm/ z42 7wa sdetecte db yGC-MS ,namel ycompoun d6 ,o f which the mass spectrum differed from that of compound 7. HRMS also revealedth epresenc eo fa compoun dwit ha mas so f427.3086 ,an dconse quently, itwa s assumed that thismas s corresponded tocompoun d 6,fo r which the formulaC27H41NO 3wa scalculated .Althoug h thecompoun dcon tained three oxygen atoms, it lost only one molecule of water upon hydrolysis (massdifferenc e 18.0105); iti stherefor e supposed tob ea solanid-en-ol containing no second enol group but more likely keto and/or hydroxy groups.Compoun d 10ma yb emethylsolanidin e as itpro duced adehydratio nproduc t C28H43N (mass 393.3395),whic h isprobabl y 158 a methylsolanidadiene (methylsolanthrene).On e solanidane-typeS Awit h a molecular mass of 423wa s detected by the EImod e GC-MS. Thiscom pound (11)coul deithe rb eC27H37NO 3o rC28H41NO2 ,becaus eexac tmasse s corresponding to both formulas were detected by the HRMS. Low in tensities of the molecular ions 411.3137 (C27H41NO2) and 425.2930 (C27H39NO3) were also measured by the HRMS, but corresponding peaks were not detected by GC-MS; these masses probably correspond totrac e compounds. Biological implications A large number of naturally occurring steroidal sapogenins, a well- studied class of compounds closely related to the SAs, have beende scribed in a recent review (Patel et al., 1987). Among these were methyl-, di- and trihydroxy-, and mono- and diketo-sapogenins aswel l asene -an d diene-forms.Also ,thoug h toa lesse r extent,a variet yo f SAs has been described; until 1981,mor e than 80 aglycones have been reported for the genus Solanum only (Ripperger and Schreiber, 1981). Regularly, novel SAs and SGAs are being detected in Solanum species, mostly in aerial plant parts, as a result of qualitative studies on pharmacologically interesting compounds (Chakravarty and Pakrashi, 1987; Chun-Nan Lin et al., 1987;Chakravart y and Pakrashi, 1988;se e also Chapter I).Onl y fewstudie so n theS Acompositio no ftuber shav e beencarrie dout ,an dconsequentl y onlyoccasionall y novel SAso rSGA s have been found since 1981 (Osman et al., 1986; Van Gelder et al., 1987; seeals oChapte rVII) .Unti lnow ,si xSA so fth esolanidan egrou p havebee ndescribe d forSolanu m species,o fwhic h only two,solanidin e anddemissidine ,hav ebee ndetecte di ntubers . Table 2 shows that avariet y of (minor)SA soccurre d inth etuber s of theSolanu m (sub)speciesuse d inthi s study.Th e detectiono fthes e SAswa sachieve db y theapplicatio no fcapillar y GCusin g simultaneous FID andNPD .I nadditio n tosolanidine ,a tleas t fouro fthes eSA sma y be regarded as (minor)compound s common to the genus Solanum,a sthe y occurred in species which belong to different series of this genus, namely theserie sCommersonian a (S.chacoense )an dTuberosa . The nature of the novel SAs of the Solanum (sub)species studied could be revealed, as was described above. They all belonged to the solanidanegrou p andwer emos tprobabl y substituted, dehydrogenated, 159 Table 2. Contents-*-o f steroidal alkaloids (SAs)o f tubers of Solanum (sub)species.Al lSA spossesse da solanidan eskeleton . SA(glycosidi - Solanum Solanum Solanum Solanumtuberosu m callybound ) chacoense leptophve;3 sparsiDilu m ssp.andipen a 1C2 7H41NO 30 61 200 19 2Solanidin e 2121 199 255 450 3C 27H45N02 40 12 35 14 4C 27H41NO 30 125 5C 27H41NO 100 6C 27H41N03 25 7C 28H45N02 5 18 100 12 8C 27H45N02 15 9 C28H43N02 5 40 10C 28H45NO 17 78 200 31 11C 27H37N03o r 75 C28H41N02 Unidentified Tr2 Tr 140 Tr Total 2228 403 1300 526 SAs expressed as glycosides on a trisaccharide basis in mg/kg fresh weight. 'Tr traces. or substituted saturated forms of solanidine, inwhic h for instance a hydrogen has been replaced by a hydroxyl or methyl group. However, their origin is not yet clear. In the S. sparsipilum sample studied, elevated levelso fnove lSA swer epresent .Thi sma yhav ebee n (partly) theresul to fth edifferen tgrowin gcondition so fthes etuber s (Chapter VII), asi tha sbee nshow ntha tenvironmenta lcondition sma yaffec tth e biosynthesis of SAs in Solanum species in many ways (Kuc, 1984). In Veratrum species, solanidine accumulated, orwa s converted tojervin e andveratramine ,dependin g on theabsenc eo rpresenc e ofillumination , respectively (Kaneko et al., 1978). Solanidine,solasodin e andtomati - denol, common end-products in the SA biogenesis in Solanum species, seemedt ob eprecursor s inth ebiogenesi so fcamtschatcanidine ,hapepu - 160 nine, anrakorinine and 27-hydroxyspirosolane,whic h areSA so fFritil - lariaspecie s (Kanekoe t al., 1981). Thus the SAs found inth etuber s ofth ewil dan dprimitiv especie sstudied ,migh tb eintermediate s inS A metabolismand/o rstres smetabolites . Thespecifi cbiologica lactivitie so fSGA sar eofte nassociate dwit h structural characteristics of theiraglycones .Fo r instance,th eanti cholinesterase activity of SGAs (Bushway et al., 1987) depended much more on the type of SA than on the composition of the sugarmoiety . Furthermore, a comparison between the solanidine-type glycoalkaloids leptine I, leptinine I and a-chaconine,whic h possess the same sugar moietyan ddiffe ronl ywit hrespec tt oth esubstituen t (acetyl,hydrox - ylan dhydrogen ,respectively )a tC2 3o fth esolanidan e skeleton(Chap terI ,Fig .1 an dTabl e 1),showe dtha tth eC23-acety lgrou p isrespon sible for thehig hantifeedan t activityo fleptin e Iagains t theColo rado potato beetle (Kuhn& Low, 1961). Thus,unknow n and/ormino rSA s as detected in this study must not be overlooked. However, this can easily happen when imperfect analytical techniques are used, orwhe n insufficient attention ispai d tothe m since theirbiologica lsignifi cancei sno tclear . For a complete structure elucidation, the SAs and their glycosides needt ob eisolate dan dcostl yan dtime-consumin g studiesb yMS ,infra red and nuclear magnetic resonance spectroscopy must be carried out. Once theentir e structure ofth eindividua l compounds isknown ,infor mation on their toxicity will still be lacking. It is therefore more efficient to investigatewhethe r these (minor)compound s occur incul - tivars,eithe rnormall yo rinduce db yenvironmenta l (stress)condition s duringcultivatio nan dstorag eo fth etubers .I fthes ecompound sd ono t occur inth ecurren thousehol dpotato ,i ti sadvisabl et opreven tthei r introductionint oth efutur ecultivars . REFERENCES Budzikiewics H. (1964)Zu mmassenspektroskopische nFragmentierungsver haltenvo nSteroidalkaloiden .Tetrahedro n20:2267-2278 . BushwayRH ,Savag eSA ,Ferguso nBS .(1987 )Inhibitio no facety lCholin esterase by Solanaceous glycoalkaloids and alkaloids. Am Potato J 64:409-413. Chakravarty AK, Pakrashi SC. (1987) Solanocastrine, a unique 16,23- cyclo-22,26-epiminocholestane from Solanum capsicastrum. Tetrahedron Letters28:4753-4756 . ChakravartyAK ,Pakrash iSC . (1988)7/5-Hydroxy-O-methylsolanocapsine ,a 161 new 3-amlno steroidal alkaloid from Solanum capsicastrum. Phyto- chemistry27:956-958 . Chun-Nan Lin, Mei-Ing Chung and Son-Yuan Lin. (1987) Steroidal alka loidsfro mSolanu mcapsicastrum .Phytochemistr y 26:305-307 KanekoK ,Kawamur aN ,Kuribayash iT ,Tanak aM ,Mitsuhash iH ,Koyam aH . (1978)Structure s of twocevanin e alkaloids,shinonomenin e andvera - florizine, and acevanidan e alkaloid,procevine ,isolate d fromillu minatedVeratrum .Tetrahedro nLetter sNo .48:4801-4804 . Kaneko K, Tanaka M, Nakaoka U, Tanaka Y, Yoshida N, Mitsuhashi H. (1981) Camtschatcanidine, an alkaloid from Fritillaria camtschat- censis.Phytochemistr y20:327-329 . Kuc J. (1984) Steroid glycoalkaloids and related compounds as potato quality factors.A mPotat oJ 61:123-139 . KuhnR , Low I. (1961)Zu rKonstitutio n der Leptine. ChemBe r94:1088 - 1095. OsmanSF ,John sTA ,Pric eKR . (1986)Sisunine ,a glycoalkaloi d foundi n hybrids between Solanum acaule and Solanum x aianhuiri. Phytochem istry25:967-968 . PatelAV , Blunden G, Crabb TA, Sauvaire Y, BaccouYC . (1987)A revie w ofnaturally-occurrin g steroidalsapogenins .Fitoterapi a58:67-107 . Ripperger H,Schreibe rK . (1981)Solanu m steroid alkaloids.I nTh eAl kaloids,XIX ,RG ARodrig o (Ed).Academi cPress ,Ne wYork ,pp .81-192 . VanGelde rWMJ ,Hopman sCWJ ,Jonker ,HH ,Va nEeuwij kFA . (1987)Biosyn thesisan dmetabolis mo fC27-steroida lalkaloid s inleave so fpotato . Proc10t hTrien nCon fEu rAsso cPotat oRes ,Aalborg ,pp .276-277 . 163 CHAPTERIX * TRANSMISSION OF STEROIDAL GLYCOALKALOIDS FROM SOLANUM VERNEI TO THE CULTIVATEDPOTAT O (S.TUBEROSUM ) INTRODUCTION S.verne i is widely being used in potato breeding programmes inman y countries, mainly as a source of resistance to potato cyst nematodes (Globodera species)an dbecaus e of itshig h starchcontent .Commercia l household cultivars containing S. vernei germplasm have already been released in several countries (Ross, 1986) and an increasing number will be released in the near future. Until now itwa s believed that thiswil dspecie scontain sonl ysolanidin eglycoside s (seeChapte rVII ) and in some reports itha sbee nmentione d that itsoffsprin gwa sana lysedfo rthes eSGA s (Coxone tal. ,1983 ;Grasser tan dLellbach , 1987). As a result, advanced breeding clones were discarded because of the high levels of solanidine glycosides -u p to 1570mg/k g (seeChapter s IIIan dVII) . However,tuber s ofS .verne iwhic hw eanalyse dcontaine d in addition to the solanidine glycosides, solasodine glycosides and several unidentified SAs (Chapter VII).Compare d with the potential acute toxic levelo f20 0mg/k go f thesolanidin e glycosides (seeChap ter II), th e contents of these compounds in tubers of S.verne iwer e extremelyhig h (2742-6093mg/kg) .Th econtent so fsolasodin eglycoside s wereals ohig h (675-3483mg/kg) .Toxicologica linformatio no nth esola sodineglycoside sa swel la so nothe rSGA sfro mwil dSolanu mspecie si s lacking and therefore itha s been stated that alien SGAsmus t notb e introduced intoth ehousehol dpotat o (ChapterII) . The aim of the study described in this chapterwa s to identifyan d quantify the SAs in tubers of S.verne i and inoffsprin g ofS .verne i and S. tuberosum in order to investigate whether the ability tosyn - Thischapte r isbase do nth epaper : Van Gelder WMJ, SchefferJJC . (199*)Transmissio n of steroidal glyco- alkaloids from Solanumverne i toth ecultivate dpotat o (S.tuberosum) , submitted forpublication . 164 thesize solasodine or other alien aglycones canb e transmitted from thiswil d species topotat o cultivars.Som eo fth edat apresente dher e arepar to fa study onth einheritanc eo flea fan dtube r SGAsan dth e relationships between the contents of these SGAs and important agri culturalcharacteristics ,usin gvariou sSolanu mspecies .Th eresult so f thatstud ywil lb epublishe di na separat epaper . EXPERIMENTAL Plantmateria l Theprovenanc eo fS .verne iBitt .e tWittm .accessio n 8240,clon enum ber 19,an d accession 15451, clone number 12,an do f theSV Ppotat o clones AM 78-3778 andA M 78-3787 ('Arabesque')ha sbee n described in Chapter VII.Th ehybrid s 24an d3 2wer e parto fth eoffsprin g froma crossbetwee nS .verne i15451-1 2an dth ediploi dS .tuberosu mL .clon e SH 78-202-3265 grown in a study on the inheritance ofth e SGAs.Th e tubers of S. vernei grown under a short-day (12h light-12 h dark) photoperiod,wer eproduce d ina glasshous eunde rth econtrolle dcondi tions as described in Chapter VII for theplant s grown in1986 .Th e sums of the solar radiation (350-3000 nm) were inth epresen t study forApril ,May ,June ,Jul yan dAugus t (2weeks) : 34,54 ,34 ,5 5an d1 7 kJ/cm , respectively. The plants were grown in a randomized block designwit hsi xreplicates . The tuberso fth egenotype s grownunde ra long-da yphotoperio dwer e also produced ina glasshouse .Th egrowin g periodwa sfro mApri ltil l the thirdwee k ofAugus t 1987;th eaverag e monthly temperature inth e glasshousewa s17 ,18 ,19 ,2 4an d 33°C, respectively, andth eminimu m night temperature 15°C.Th ephotoperio dwa sth enatura l daylength.Th e relative airhumidit ywa skep ta t60-70% .Th esum so fth esola rradia tionwer ea sabove .Th eplant swer e growni na randomize dbloc kdesig n withte nreplicates . Chemicalsan dprocedure s The tuberbatche so feac hreplicat ewer eanalyse d separately. Standard SAs, andextractio n anddeterminatio n ofth eSA swer e asdescribe di n theChapter sV an dVI .Column suse dfo rcapillar yG Cwer efuse dsilica , 50m x 0.2 2m mI.D. , CP-Sil5 CB ,fil m thickness 0.12/i man d0. 4/xm , 165 and CP-Sil 19 CB, film thickness 0.19 /im (Chrompack Nederland). SA concentrations were calculated from thepea k areas of the5-ene-3/8-o l and 3,5-dienepairs , and expressed as SGAs on a trisaccharidebasis . Solanidine epimers synthesized according to Brown and Keeler (1978) were kindly supplied by Dr. W. Gaffield. Identification of SAs by comparison of retention indices, NPD/FID response ratios,hydrolysi s behaviouran dmas sspectr ao fth eSA san dthei rcorrespondin g 3,5-diene dehydrationproduct swit hthos eo freferenc ecompound swa sa sdescribe d before (ChapterVIII) . ForTL Ccompariso no fth e synthetic 22R.25Rsolanidin e and thesup posed natural 22R.25R solanidine isolated from S. vernei tubers and from leaves of 'AM 78-3787',high-performanc e TLC plates, 5x 10cm , silica gel 60 (E. Merck) were used. Volumes of 1 /il of the appropriately dilutedtube rextract sprepare dfo rcapillar yG Can do fa methanol/toluene (2:1 v/v) solution containing 5 /igo f the synthetic epimerwer eapplie dt oa TL Cplate .Plate swer edevelope dusin gt-buty l methyl ether/methanol (95:5 v/v) as eluent (Gaffield, 1988). A satu rated solutiono fcerium(IV )sulphat e insulphuri c acid (65%)wa suse d as visualization reagent. The spots became brown, and turned to grey afterheatin g theplate sfo r5 mi na t120°C . Identification ofth ediagnosti c ionso fGC-M S (EImode ,7 0eV )wa s as described in Chapter VIII andb y fragmentation mapping (Barber et al., 1968). GC-MS diagnostic ions: solanthrene (I= 2943) and solan- + + threne isomer (I = 2889) (C27H41N)m/ z 379 M *, 364 [M-CH3], 20 4 [C14H22N1+. 15° base Peak tc10H16N]+; solanidine (I= 3130), 22R,25R epimer of solanidine (I - 3121) and solanidine isomer (I — 3058) + + (C27H43NO)m/ z 397M *, 382 [M-CH3] , 204,15 0bas e peak; solasodiene + + + and tomatidadiene (C27H41N0)m/ z 395M *, 380 [M-CH3] , 367 [M-C2H4] , + + + 138 [C9H16N] , 114bas epea k [C6H12NO] , 113 [C6Hi1N0] ;solanid-5-ene - 3y9-olderivativ e (C27H41NO3)m/ z 427M +*, 412 [M-CH3]+, 204,15 0bas e peak;solasodin ean dtomatideno l (C27H43NO2)m/ z41 3M +*,39 8 [M-CH3]+, + + 385 [M-C2H4] , 271 [C9H27NO] , 138,11 4bas epeak . RESULTSAN DDISCUSSIO N Table 1show s the contents of thevariou s SAs,expresse d asSGA so na trisaccharide basis, oftuber so fth e S.verne iclone s 8240-19 and 166 Table 1.Contents 1 (+standar d errors)o fsteroida lalkaloid s (SAs)o f tubers of Solanum vernei grownunde r short-day (SD)an d long-day (LD) photoperiods. Plants were grown in six replicates which were analysed separately. Steroidalalkaloi d Reten- S.verne i S.verne i15451-1 2 tion 8240-19 index SD SD LD Solanidine isomer 3058 63± 10.4 25± 11.7 Trz 22R.25RSolanidin e 3121 Nd3 45± 9.9 Tr Solanidine4 3130 4961± 48 4 2769± 553 4595+ 87 9 C27H41N035 3195 12± 6.2 Nd Nd Solasodine 3338 495± 71 1301+ 366 105± 62 Tomatidenol 3360 455± 25 6 291± 184 Nd Total 5986+ 71 3 4431+ 108 8 4700+ 93 1 SAsexpresse da sglycoside so na trisaccharid ebasi s inmg/k gfres h weight. 2Tr= trac e (<2. 5 mg/kg). •%d= no tdetected . 422R,25Sconfiguration . -"Substitutedsolanideno l (seeChapte rVIII ). 15451-12, obtained by capillary GC on a CP-Sil 5 CB fused-silica column,5 0m x 0.2 2m mI.D. , filmthicknes s0.1 2j*m .Thes econtent sar e averages of six separately analysed tuber batches obtained from six plantswhic hha dbee ngrow na sreplicates ,a sth etube rSG Acontent so f differentplant so fa singl egenotyp egrow ni nth esam ecompartmen tma y varyconsiderably . Highconcentration so fsolanidine ,solasodine ,an dtomatideno lwer e found. In clone 8240-19, low concentrations of asolanid-5-ene-3/3-o l derivativewit ha molecula rweigh t of42 7an da retentio n index ,1,o f 3195 were found. This compound was identical to the SA which had previouslybee ndetecte d inothe rSolanu mspecie san dwa sidentifie da s C27H41N03 (ChapterVIII) . Twosolanidane swit hmolecula r ionpeaks ,M ,a tm/ z37 9 (I= 2889 ) 167 Table 2.Retentio n indices,I ,obtaine d by capillary GC andR pvalue s obtainedb yTL Co fsolanidin e (22R,25S),o fit ssuppose dnatura lepime r (22R,25R) isolated from tubers of S.verne i 15451-12 and from leaves ofth epotat oprogenito rA M78-3787 ,an do fit ssyntheti cepimers . Configuration I(Capil l .ary GC) RF (TLC) ofsolanidin e CP-Sil5 CB CP-Sil5 C B CP-Sil1 9C B Silica 0.12/u m 0.4/i m 0.19/i m gel6 0 22R,25R;Natura l 3120.9 3122.2 3130.0 0.80 22R.25R;Syntheti c 3120.6 3122.8 3129.9 0.81 22R,25S;Solanidin e 3129.8 3131.1 3134.3 0.66 22S.25R;Syntheti c 3246.3 3248.0 3227.3 - 22S.25S;Syntheti c 3250.1 3253.3 3228.0 - and m/z 397 (I= 3058), respectively, were identified as a 3,5-diene and 5-ene-3/9-olpair .Thes ecompound s appeared tob e isomers ofsolan - threne (solanida-3,5-diene) and solanidine, respectively. The dienes areproduce db ydehydratio no fth e5-ene-3/3-ol supo nhydrolysi s andar e an aid in the identification of SAs (Chapter VIII). In the accession 15451, another isomer of solanidine (I= 3121)whic h had earlierbee n detected in leaves of the potato clone AM 78-3787 ('Arabesque')wa s present (Van Gelder et al., 1987; cf. also Chapter VII).Th e mass spectra and the retention indices of the two just-mentioned natural solanidine isomers were compared with those of the synthetic 22R.25R, 22S.25R, and 22S,25S epimers.Th e solanidine isomer with I= 3058di d not correspond to any of the epimers, but the results strongly sug gested that the isomer with I= 3121 thatwa s found in the tubers of theS .verne iclon e15451-1 2an di nth eleave so fth eclon eA M78-3787 , was the 22R,25R epimer of solanidine (solanidine shows the 22R,25S configuration). Further comparison of these compounds by capillary GC using the same column typewit h a film thickness of0. 4 /tm,whic hin creased the resolution by almost 40%,an d using amor e polar column (CP-Sil 19 CB) provided additional evidence that this natural isomer was identicalwit hth esyntheti c 22R.25Repime r (Table2) . 168 The results were confirmed by TLC on silica gel 60 using t-butyl methyl ether/methanol (95:5 v/v) as eluent (Gaffield, 1988). To our knowledge this is the first report on anaturall y occurring epimero f solanidine. The 22S.25R and 22S.25S epimers were not detected in S. verneino r inan yothe rgenotyp e investigateds ofar . A short-day regimewa s applied in this studybecaus e it isfavour able for the tuberization of S.vernei .Earlie r studies stronglysug gested that the environment may influence the SA composition, both qualitativelyan dquantitativel y (VanGelde re tal. ,1987 ;Chapte rVII , seeals oChapte rVIII) .Therefor eth eindividua lS Acontent s (expressed on a glycoside basis)o f tubers of the S.verne iclon e 15451-12grow n under a long-day photoperiod, which was applied in the inheritance study mentioned above, are given for comparison (Table 1).Th e total contents of the SGAs in the tubers did not differ between the two regimes (P= 0.05), and they were as (extremely)hig h as those found before intuber so fS .verne i (ChapterVII) .Unde rlong-da yconditions , theconten to fsolanidin e glycosideswa smuc hhigher ,wherea s thecon tents of the other SA glycosides were significantly lower (P= 0.05). Such concentration shifts resulting from differences in photoperiods have also been observed forvariou s sterols in leaves ofS .tuberosu m ssp. andigena (Baean dMercer ,1970) . The aglycones inS .verne i areal lA5-unsaturate dSA san d theyhav e teinemine as common precursor (Chapter I).I t seems therefore most likely that the environment changes the expression of the genesregu lating theconversio no fteinemin e toth evariou s aglycones.Unde rth e long-day conditions,th eprecursor ,th esynthesi s rateo fwhic happar ently did not change,wa s almost entirely converted into solanidine, whereas under the short-day conditions considerable amounts of other teinemine-derived SAswer e synthesized. The influence ofenvironmenta l conditions on the biosynthetic pathways of the SAs has been reported earlier. For instance, conversion of solanidine to jervine andvera - tramine in a Veratrum species was regulated by light (Kaneko et al., 1978), and the ability of tubers of the potato cultivar Kennebec to synthesize tomatidenol glycosides,becam e specifically expressed when sliced tuberswer e stored (Shih and Kuc, 1974;Chapte r I).Storag eo f slices isno tuncommo ni nth epotat oprocessin gindustry . 169 Table 3.Contents 1 (+standar d errors)o f themai nsteroida lalkaloid s (SAs)o ftuber s ofS .verne i 15451-12,o fS . tuberosum SH78-202-3265 , of two of their hybrids, and of three cultivars,grow nunde r along - dayphotoperiod . o Genotype N^ Solanidine Solasodine glycosides glycos ides S.verne i 6 4595+ 87 9 105± 62 Hybrid2 4 5 897± 15 7 20+ 5 Hybrid 32 9 1370+ 14 1 22+ 5 S.tuberosu m 8 92+20 Nd3 AM78-377 8 10 825+ 86 7+ 1 AM78-378 7 7 149 +22 3+ 0.4 Bintje 9 153+ 12 Nd •'-SAsexpresse da sglycoside so na trisaccharid ebasi s inmg/k gfres h weight. o •'Numbero freplicate swhic hproduce dtuber san dwer eanalysed . Nd= no tdetected . Although intuber so fSolanu mspecies ,solanidin ea swel la ssolasodin e and tomatidenolhav ebee nfoun d sofa r linked tosolatrios e andchaco - triose (ina-solanin e and a-chaconine, in solasonine andsolamargine , and in a-solamarine and /3-solamarine, respectively), these aglycones canoccu rboun d toothe r sugars (Chapters Ian d IV). Thereforea stud y onth esuga rmoietie so fth eSGA so fS .verne i isi nprogress . S.verne i belongs to the Solanum series Tuberosa.However , theSG A composition ofth e tuberso fS .verne iwa sdifferen t from thoseo fal l other species of this series which we investigated (Van Gelder and Jonker, 1986;Chapter sVI Ian d VIII). This isquit e interesting froma chemotaxonomie point ofview . Further study of this series,fo rwhic h 68wil dspecie swer edescribe db yHawke s (1978),wil lrevea l itsdiver sitywit hrespec tt oth eSGAs . Table3 show sth econtent so fth emai nSGA so ftuber so fth eparent s and oftw ohybrid s ofa cros sbetwee nS .verne i anda diploi dS .tub e- rosum. For comparison, the contents of the cultivar Bintje and ofth e 170 clonesA M 78-3778 andA M 78-3787,whic hbot hhav e aS .verne iclon ea s progenitor,ar egiven .Th etuber swer eproduced *i na glasshous eunde ra naturallong-da yphotoperiod . The contents ofth e solanidine glycosides of 'Bintje', 'AM78-3778 ' and 'AM 78-3787' agreed fairly with those of an earlier study which showed thatunde r the growing conditions applied, the contents ofth e solanidine glycosides were two to three times higher than in normal field-grown tubers (Chapter VII).Fo r instance,field-gro'w ntuber so f 'Bintje' contain usually about 40 mg solanidine glycosides per kg. However, even when this aspect is taken into consideration, the contents of the solanidine glycosides of the hybrids are above the potentialacut etoxi clevel . Also solasodine glycosideswer e found inth e tuberso f thehybrids ; fortunately the concentrations were low. Solasodine hasbee n reported to cause birth defects when a single high dose of 1180mg/k g bw was gavaged to hamsters of a strain that is considered sensitive to SA teratogens (see Chapter II). To administer an equivalent amount of this aglycone, about 10 kg of tubers of thehybrid s would have tob e gavaged to a single hamster, and thus the level of solasodine inth e hybridsseem sirrelevan tfro mth eviewpoin to fteratogenicity .However , init snatura lmatrix ,solasodin e isingeste dglycosidicall ybound ,an d toxicological information on the solasodine glycosides, administered either as pure compounds or in the natural matrix, isno tavailable . Most probably the glycosides are absorbed to a larger extent fromth e gastrointestinaltrac ttha nth eaglycon ean dthu sthe yma yperhap ssho w a higher teratogenic potency.As ,unfortunately , thenecessar ytoxico logical data are lacking, it can not be concluded unequivocally that solasodine glycoside levels as found in the hybrids dono t present a hazardt ohuma nbeings . Also inth etuber so f 'AM78-3778 'an d 'AM78-3787' ,lo wcontent so f solasodine glycosides were detected. These SVP-progenitors have been derivedfro mS .verne iinterspecifi chybrid swhic hha dbee nbackcrosse d three times using S. tuberosum cultivars and species in which only solanidane-typeSGA shav ebee ndetecte d inth etubers .Therefor e itwa s notexpecte d that solasodinewoul db estil lsynthesize d inthes egeno types. Toxicological informationo n theothe r SAsdetecte d inS .verne ii s 171 not available.Wit h respect to the relationship between the structure of SAs or SGAs and their teratogenic potency, itshoul db enote dtha t induction of congenital malformations by solanidanes and spirosolanes has been associated with the 22S.25R configuration occurring for instance in solasodine (see Chapter II).Demissidin e (22R.25S) and tomatidine (22R,25S)di dno tinduc emalformations .Thu si fthi sstereo chemical concept appears to be valid, tomatidenol (22R.25S)woul d be beyondsuspicion . Studies on the teratogenicity ofSA s showing the 22R.25Rconfigura tion have not been reported. However, these SGAs were found in low concentrations in the tubers of the S. vernei clones, and they were noteve ndetecte d inth eoffspring . In conclusion it can be stated that the considerably reduced con tents of the solanidine and solasodine glycosides in thehybrid scom paredwit h the S.verne iparen tan d theabsenc e ofothe rSGA sfoun di n S.vernei . is reassuring for the potatobreeder , as iti slikel ytha t genotypes that are safe for consumption canb e produced by (repeated) backcrossing. However, it must not be overlooked that S. vernei off spring canpossibl y accumulate larger amountso fsolasodin e glycosides orsynthesiz ealie nSGAs ,inherite dfro mth ewil dspecies ,unde rculti vation conditions differing from those applied here. Furthermore,t o ourknowledge ,i nno ton ecountr ywher e S.verne iha sbee nutilize di n breeding programmes, its offspring or new cultivars containing its germplasm, have been analysed for their total SGA composition. It is therefore advisable toanalys e the SGAso fsuc hne w cultivarsyet ,an d to carry out the analysis on tubers produced under various environ mentalconditions . REFERENCES BaeM ,Merce r EI. (1970)Th eeffec to flong -an dshort-da yphotoperiod s on the sterol levels in the leaves of Solanum andigena. Phyto- chemistry9:63-68 . Barber M,Gree nBN ,Wolstenholm eWA ,Jenning sKR . (1968)Fragmentatio n maps and their use in structural analysis. Adv Mass Spectrometry 4:89-98. BrownD ,Keele rRF . (1978)Structure-activit y relationo fsteroi dtera togens.3 .Solanida nepimers .J Agri cFoo dChe m26:566-569 . CoxonDT ,Davie sAMC ,Morga nMRA . (1983)Specia l Report No.7 :Glyco - alkaloids inpotatoes .AR CFoo dResearc hInstitute ,Norwich . Gaffield W. (1988) Personal communication Dr.W . Gaffield, W RegRe s Cent,Albany ,CA ,U.S.A . GrassertV ,Lellbac hH . (1987) Untersuchungende sGlykoalkaloidgehalt s 172 von Kartoffelhybriden mit Resistenz gegen die Kartoffelnematoden Globodera rostochiensis and Globodera pallida. Biochem Physiol Pflanz182:473-479 . HawkesJG . (1978)Histor y ofth epotato .I nTh e Potato Crop,P MHarri s (Ed).Chapma nan dHall ,London ,pp .1-14 . KanekoK ,Kawamur aN ,Kuribayash iT ,Tanak aM ,Mitsuhash iH ,Koyam aH . (1978)Structure s oftw ocevanin e alkaloids,shinonomenin e andvera - florizine, and a cevanidane alkaloid, procevine, isolated from illuminatedVeratrum .Tetrahedro nLetter sNo .48:4801-4804 . RossH . (1986)Potat obreedin g -Problem san dperspectives .Advance si n plantbreeding ,Supp lJ Plan tBreedin g13:1-130 . ShihM-J , Kuc J. (1974) a- and/3-Solamarin e inKennebe c Solanumtube rosumleave san dage dtube rslices .Phytochemistr y13:997-1000 . VanGelde rWMJ ,Hopman sCWJ ,Jonke rHH ,Va nEeuwij k FA. (1987)Biosyn thesisan dmetabolis mo fC27-steroida lalkaloid s inleave so fpotato . Proc10t hTrien nCon fEu rAsso cPotat oRes ,Aalborg ,pp .276-277 . Van Gelder WJM, Jonker HH. (1986) Steroidal alkaloid composition of tubers of exotic Solanum species ofvalu e inpotat obreedin gdeter mined by high-resolution gas chromatography. In Potato Research of Tomorrow,AG BBeekma n (Ed). PUDOC,Wageningen ,pp .166-169 . 173 SUMMARY ANDCONCLUDIN G REMARKS Tuberiferous and nontuberiferous wild Solanum species are increasingly being used in potato breeding as a source of genes for disease and pestresistance san dfo rothe rvaluabl echaracteristics .A disadvantag e of Solanum species,fro m a consumers point ofview , istha t theycon tain steroidal glycoalkaloids (SGAs), which are natural toxinsoccur ring inal lpart so f theseplants .Th eSGA s consisto fa C27-steroida l alkaloid (SA)an da suga rmoiety ,ofte na tri -o rtetrasaccharide .Th e tuberso fth ecultivate dpotat ousuall ycontai nsmal lquantitie so fon e typeo fSGAs ,th esolanidin eglycosides . Evidenceha sbee npresente dtha tutilizatio no fwil dSolanu mspecie s can result in the introduction ofhazardou s levels of solanidine gly cosides into the cultivated potato. However, little is known of the qualitative and quantitative SGA composition of wild Solanum species used in potato breeding. Such information on hybrid offspring or on cultivarscontainin ggermplas mfro mwil dspecie si sentirel ylacking . The aimo f thestudie s described inthi s thesiswa s toevaluat eth e possible health hazards of the SGAs from the genus Solanum and to develop and apply methods for analysis of the SGA compositions of Solanumspecies .Th ecollecte d informationwa splace d intoth eperspec tives of potato breeding and of food safety, in order to point out possible consequences of introducingundesire d levels ortype s ofSGA s intoth ecultivate dpotato . InChapte rI ,th eliteratur eo nth edistributio nan daccumulatio no f solanidine glycosides inth ecultivate dpotat o ando n thebiosynthesi s of the SGAs is reviewed. It was shown that many factors during the growth andpost-harves tperio do fpotatoe s can lead tolevel so fsola nidineglycoside sexceedin g20 0mg/k gfres hweight .Thi sinformatio ni s important alsowit h respect to introduction of SGAs fromwil d Solanum species into thehousehol dpotato ,a salie nSGA swil l almostcertainl y accumulate similarly,becaus e thebiosyntheti c pathways of thevariou s SGAsar eclosel yrelated . The objective of Chapter II was to evaluate the toxicity of the SGAs,base do nth edat aavailabl e inth eliterature ,i norde rt oasses s possibleconsume rhazard san dt oderiv esaf elevel sfo rhousehol dpota - 174 toes. The data showed that acute poisoning in man may occur due to consumption of potatoes with a solanidine glycoside content above 200 mg/kg freshweight .Thi s level is only two to three timeshighe r than levels that are considered normal for tubers of current cultivars. Because of the virtual absence of chronic toxicity data, an adequate 'no-adverseeffec tlevel 'fo rpotat oSGA scoul dno tb eassessed .Conse quently an acceptable daily intake (ADI)figur e forma n ora naccept ableleve lfo rpotatoe scoul dno tb ederived .I twa sconclude d thatth e SGAconten to fne whousehol d potatocultivar s shouldno tb eallowe dt o rise above the average level of the current cultivars; preferably it shouldb elower .SGA salie nt oS .tuberosu mmus tno tb eintroduce dint o thehousehol dpotat oa sacceptabl elevel shav eno tbee nestablished . The methods for SGA analysis described in the literature are not adequate for determining -qualitatively and quantitatively- the SGA composition of breeding material. Therefore, a comprehensive,quanti tative and efficient method applicable todivers e plant material,wa s developed. In Chapter III, a new hydrolysis technique employing a two-phase system is described. The technique was developed in order to prevent the losses of aglycones which do usually occur during conventional hydrolysis of different SGAs. The two-phase system consists of an aqueous acid phase, in which the glycosides are hydrolysed, and an immiscible nonpolar organic phase,whic h serves as aprotectiv e phase for the unstable nonpolar aglycones.Usin g the technique quantitative recoveries of various SAs after simultaneous hydrolysis of different SGAswer eobtained . In Chapter IV a capillary gas chromatography (GC) method is de scribed,whic h enabled -for the first time- the separation of theA5 - and 5a-aglycone pair solanidine and demissidine aswel l as separation of the other SAs studied. Derivatization of the SAswa sno trequired . The capillary GC method in combination with the bisolvent extraction andtwo-phas ehydrolysi sdescribe d inChapte rII Ioffere dprospect sfo r quantitativeanalysi so fcomple xSG Acomposition so fSolanu mspecie si n asingl erun . Chapter V deals with the development of a method that enabled an unambiguous differentiation during capillary GC between peaks ofun known SAs and peaks of closely related nonnltrogen-containing (non-N) 175 compoundssuc ha ssterol san dsteroida lsapogenins .Th emetho dinvolve s a splitting device thatconnect s acapillar y columnt oa dua ldetecto r system, for flame ionization detection (FID)an dN-specifi c detection (NPD), respectively. This system isconnecte d to a two-channel system for interactiveprocessin g ofth e twoset s ofdata .Th eapplicatio no f hydrogen as carrier gas was introduced to achieve a short analysis duration.A s tubers ofwil d Solanum species appeared tovar y strongly in SGA contents, identification by retention times was notpossible . Therefore a retention index system for the SAs was introduced. The retention indices were sufficiently reproducible, even for identifi cation of the closely eluting A5- and 5a-aglyconepai r solanidine and demissidine. Application of NPD/FID response ratios together withre tention indices proved tob e useful in the identificationan dcharac terization of SAs. Costly spectrometric techniques will have to be employedfo rcharacterizatio nonl ywhe nne wSA sar edetected . Chapter VI dealswit h an evaluation of the developed methods using diverse plant tissueso fwil d Solanum andLvcopersico nspecies ,culti - vars andhybri dprogeny .Th e resultsshowe d thatth emethod swer euse fulfo rqualitativ ean dquantitativ eanalysi so fth eSA si nth edivers e samples.A potato extract spikedwit h 20SAs ,includin g threeA5 -an d 5a-aglyconepairs ,coul db e analysed ina single GC run.A comparison with procedures described in the literature showed that theprocedur e developed for sample preparation followed by capillary GC should be applied,becaus e literatureprocedure s for samplepreparatio n canlea d to losseso fvariou s SGAs or SAs, andothe r chromatographic techniques dono toffe rth eefficienc yrequire dfo rseparatio no fth evariou sSAs . In Chapter VII, the SGA compositions of a large number of wild Solanum species used inpotat o breeding, arepresented . The tuberso f most ofth especie s containedhig hconcentration so fsolanidin eglyco sides. Some species contained glycosidic-bounddemissidine ,solasodin e and/or tomatidine. Unidentified compounds were also detected, which were most probably SAs aswa s shownb y their NPD/FID responseratios . The total SGA contents of the tubers of thewil d speciesvarie d from 123 to 7348 mg/kg. In order to place these contents in a realistic perspective, tubers ofcultivars ,correspondin g insmal lsiz ewit han d grownunde rth esam econdition sa sth etuber so fth ewil dspecies ,wer e analysed. The contents of solanidine glycosides of these tubers were 176 two to three times higher than those of field-grown normal tubers. Thus, even when this factor was taken into consideration, the SGA contents of the tubers of several wild species were extremely high. This study also revealed that samples of some wild species contained high orlo wlevel so fon eo rman yothe r glycosidic-bound SAs,an dtha t the SGA composition can vary within species and between different organs of one plant. The results further suggested that the SGA synthesis canb e organ specific or regulated by light and thattrans locationo fSGA sfro mleave st otuber san dvic evers adi dno toccur .I t wasconclude d thatwil dcrossin gparent sshoul db eanalyse da st othei r SAcomposition ,befor e theyar euse di na breedin gprogramme . A procedure developed for the characterization ofunidentifie d SAs presenti nplan textract swit hcomple xS Acompositions ,i sdescribe di n Chapter VIII. Thecombine d resultso fGC-mas s spectrometry (MS)apply ingelectro nimpac tan dchemica lionization ,an dhigh-resolutio nMS ,o f the application of retention indices andNPD/FI D response ratios,an d of a comparison of the degree of dehydration ofSA si ndifferen ttwo - phasehydrolysi s systems,wer e used. This study revealed that theSA s all possessed a solanidane skeleton; they were characterized assub stituted,e.g .hydroxylate do rmethylated ,forms ;dehydrogenate dforms ; orsubstitute dsaturate dform so fsolanidine .Mos to fthes eSA sha dno t been reportedbefore .Th e complete SAcomposition , including themino r new SAs,o f Solanum (sub)speciesuse d inpotat obreeding , ispresente d in this chapter and the importance of analysis of the minor SAs is indicated. S.verne i iswidel ybein guse d inpotat obreeding .Althoug h theSG A composition of its tubersha d notbee n reported, cultivars containing S.verne igermplas mhav ebee nrelease dan da nincreasin gnumbe rwil lb e releasedi nth efuture . Chapter IXdescribe s the identification andcharacterizatio n ofth e SAs of S.verne i tubers produced under various growth conditions,an d dealswit ha tentativ e studyo nth etransmissio no fthes eSA st ohybri d offspring. High levels of solanidine and solasodine glycosides were foundtogethe rwit htomatideno lan dnove lglycosidic-boun d SAs,amongs t which the 22R.25Repime r ofsolanidine ,a structura l configurationno t reported before for naturally occurring solanidanes. It was revealed that the SA composition can vary significantly in tubers grown under 177 differentcultivatio nconditions . In the tubers of S. vernei offspring, high levels of solanidine glycosideswer efoun dbu tth enewl y identifiedan dcharacterize d SAso f S. vernei were not detected. Solasodine was also found in the off spring,eve n intuber so fcultivar sobtaine d after several timesback - crossing,bu t fortunately, thelevel swer e low.However ,i tca nno tb e totally excluded that S. vernei offspring may synthesize hazardous levelso fsolasodin eglycoside sunde rparticula rgrowt ho rpost-harves t conditions, and in this respect the possible teratogenic potency of solasodineshoul db ekep ti nmind . Inconclusio n itca nb e stated that theingestio nb y thepubli co fth e amount of solanidine glycosides should not be allowed to rise but shouldpreferabl yb e reduced. SGAsalie nt oS .tuberosu m shouldno tb e introduced intoth ehousehol dpotato .Th ebasi sfo rth eproductio no fa potato crop safe for consumption is, to grow current cultivars andt o breed new ones which accumulate low levels of only solanidine glyco sides under various growthan dpost-harves t conditions.Utilizatio no f germplasm from wild Solanum species must be approached with caution. Therefore it is recommended to analyse the SGAs of potential wild crossingparent sbefor ethe yar euse di na breedin gprogramme ,i norde r to select the genotypes that combine a desired trait with the least unfavourable SGA composition. Depending on the SGA composition ofth e parents,th eoffsprin gshoul db emonitore dtoo . For analysis of SGA compositions, the newly developed procedures for separation, quantification and identification/characterization, described inthi s thesis,mus tb e applied,a sth econventiona lmethod s described inth eliteratur ed ono tmee tth erequire dstandards . The accumulation of SGAs in new cultivars containing wild-species germplasm should be studied under various environmental conditions before such cultivars are registered. The guideline of 60-70m gsola nidineglycoside spe rk gfres htube rrecommende d inth eliteratur efro m the viewpoint of consumer safety (see Chapter II), shoul d be used in potatobreedin gunti la nadequat eacceptabl e levelfo rthes ecompound s hasbee nestablished . 179 SAMENVATTING ENSLOTOPMERKINGE N In deaardappelveredelin gword t intoenemend emat egebrui k gemaaktva n knoldragendee nniet-knoldragend ewild eSolanum-soorten ,o mbelangrijk e eigenschappenzoal sresistenti etege nziekte ne nplage nt eintroducere n ind egecultiveerd e aardappel.Vanui the toogpun tva nvoedselveilighei d hebben Solanum soorten als nadeel dat zij steroidalkaloidglycosiden bevatten (SGAs). Di t zijn toxinen die van nature in alle delen van plantenva nhe tgeslach tSolanu mvoorkomen .SGA szij nopgebouw dui tee n C27-steroidalkaloide (SA) en een suikergroep, meestal een tri- of tetrasaccharide. De knollen van de gecultiveerde aardappel bevatten slechts één type SGAs, de solanidineglycosiden, gewoonlijk inklein e hoeveelheden. Het benutten van wilde Solanum-soorten kan in bepaalde gevallen leiden tot nieuwe aardappelrassen met ongewenst hoge gehalten aan solanidineglycosiden. Er is echter weinig bekend over dekwalitatiev e enkwantitatiev e SGA-samenstellingva nd ewild e Solanum-soortendi ei n deaardappelveredelin g gebruiktworden .Informati eove rdez esamenstel lingva nnakomelinge nva nhybride ne nva nrasse ndi eerfelij kmateriaa l vanwild esoorte nbevatten ,ontbreek tgeheel . Hetonderzoe kda ti ndi tproefschrif tbeschreve nwordt ,ha dto tdoe l de potentiële gezondheidsrisico's van de SGAs te schatten enmethode n te ontwikkelen, ten einde de SGA-samenstellingva n Solanum-soortent e kunnen analyseren. De verzamelde informatie werd geëvalueerd, vanuit het oogpuntva nzowe ld eaardappelveredelin g alsva nd evoedselveilig heid, om de mogelijke gevolgen aan te gevenva nhe t introducerenva n ongewenstegehalte no ftype nSGA si nd egecultiveerd eaardappel . InHoofdstu k Iword tbeschreve nwaa r envi awelk ebiosynthese-rout e de SGAs ind eaardappe lworde ngevormd .He tblijk t datvelerle ifacto ren tijdensd egroe iva nd eaardappe l entijden sopslag ,verwerkin ge n distributie vanaardappelen ,kunne n leidento ttoenam eva nhe tgehalt e aan solanidineglycosiden tot boven 200 mg/kg vers gewicht. Omdat de biosynthese van de verschillende SGAs uit wilde Solanum-soortenver loopt via routes die nauw verwant zijn aan dieva n de solanidinegly cosidenui taardappelen ,i she taannemelij kda tdez esoortvreemd eSGAs , 180 indien geïntroduceerd in de aardappel, op een zelfde wijze zullen accumuleren. Hetdoe lva nHoofdstu k II isd e toxiciteitva n deSGA st eevaluere n op basis van de beschikbare literatuurgegevens, teneinde de risico's voor deconsumen tvas t testelle ne nSGA-gehalte nvoo r deaardappe lt e kunnen afleiden, die voor de consument als veilig beschouwd kunnen worden. Uit de gegevens blijkt dat acute vergiftiging bij demen ska n optreden alsgevol gva nd e consumptieva naardappele nme tgehalte naa n solanidineglycosiden boven 200 mg/kg vers gewicht. Dit gehalte is slechtstwe eto tdri ekee rz ohoo gal sd egehalte ndi evoo raardappele n alsnormaa lworde nbeschouwd .Omda tgegeven sove rchronisch e toxiciteit nagenoeg ontbreken, kon een geen-nadelig-effect niveau (no-adverse effect level)nie tworde nvastgesteld . Als gevolg daarvan konnoc hd e aanvaardbare dagelijkse opname (acceptable daily intake, ADI),noc h een geschikt maximum aanvaardbaar gehalte (acceptable level) van solanidineglycosiden in aardappelen worden berekend. Op grond van de toxiciteitsgegevens werd gesteld, dat het gehalte aan solanidinegly cosiden van nieuwe consumptierassen niet hoger dient te zijn danhe t gemiddelde gehalte van de huidige rassen -bij voorkeur zou het lager moeten zijn- enda tvoorkome ndien tt eworde nda t soortvreemdeSGA si n deconsumptie-aardappe lworde ngeïntroduceerd . Demethode ndi ei nd eliteratuu rbeschreve nzij nvoo rd eanalys eva n SGAszij nnie tgeschik tvoo rhe tbepale nva nd ekwalitatiev ee nkwanti tatieve SGA-samenstelling van veredelingsmateriaal. Daarom werd een efficiënte analysemethode ontwikkeld, dieoo kbi j ander plantmateriaal danaardappele nka nworde ntoegepast . In Hoofdstuk III wordt een nieuwe hydrolysetechniek beschreven, waarbij gebruik wordt gemaakt van een twee-fasensysteem: een zure waterfase waarin de glycosiden worden gehydrolyseerd, en een daarmee nietmengbar eapolair eorganisch efase ,di edien tal sbeschermend efas e voord einstabiel eapolair eaglyconen .Dez etechnie kwer dontwikkel do m de gebruikelijke dochongewenst everlieze naa nSGA sdi ebi jconventio nelezur ehydrolys eoptreden ,t evermijden . In Hoofdstuk IV wordt beschreven hoe met behulp van capillaire gaschromatografie (GC) het A5- en 5a-aglyconen-paar solanidine en demissidine alsmede andere SAs kon worden gescheiden. Een dergelijke scheiding was niet eerder gerapporteerd. Derivatiserenva n de SAswa s 181 niet nodig. De capillaire GCmethode , in combinatie met debisolvent - extractie enhe t twee-fasensysteemvoo rhydrolys ebeschreve n inHoofd stuk III,boo d goede perspectieven voor het efficiënt enkwantitatie f analyserenva ncomplex eSGA-samenstellinge nva nSolanum-soorten . HoofdstukV handel t overhe t ontwikkelenva nee nmethod e metbehul p waarvan tijdens capillaire GC, piekenva n onbekende SAs onderscheiden kunnenworde nva npieke nva nnauwverwant eniet-stikstofhoudend e (non-N) verbindingen zoals sterolen en steroidsapogeninen. Bij deze methode werden simultaanvlamionisatie-detecti e (FID)e nN-specifiek e detectie (NPD) toegepast, alsmede twee-kanaals gegevensverwerking. Voor het verkorten van de analyseduur werd waterstof gebruikt als dragergas. Omdat inwild e Solanum-soorten de SA-concentraties aanzienlijk bleken te variëren, kon de retentietijd niet voor identificatie worden gebruikt. Daarom werd een retentie-indexsysteem geïntroduceerd dat voldoendereproduceerbar ewaarde ngaf ,zelf svoo rhe t identificerenva n directn aelkaa reluerend everbindingen ,zoal she tA5 -e n5a-aglyconen - paar solanidine en demissidine. Het toepassenva n kostbare spectrome- trische technieken kan daardoor beperktblijve n tothe t identificeren en/ofkarakterisere nva nnieuw eSAs . De evaluatieva nd eontwikkeld emethode nword tbeschreve n inHoofd stukVI .Ui t de resultatenblee k dat demethode n toepasbaarware nbi j verschillend materiaal van Solanum- en Lvcopersicon-soorten. van rassen, en van hybriden. Een aardappelextract waaraan 20 SAs waren toegevoegd,ko ni nee nenkel eGC-ru nworde ngeanalyseerd . In Hoofdstuk VII wordt de SGA-samenstelling van een groot aantal wilde Solanum-soorten weergegeven. De knollen van de meeste wilde soortenbevatte nhog egehalte naa nsolanidineglycoside n invergelijkin g met de huidige consumptie-aardappelrassen. Daarnaast kunnen wilde soorten uiteenlopende concentraties aan diverse andere glycosidisch gebonden SAs bevatten. De SGA-samenstelling bleek binnen een ende zelfde Solanum-soort te kunnen variëren maar ook tussenverschillend e organen van dezelfde plant. Het is daarom raadzaam wilde kruisings- ouders te analyseren voordat zij in een veredelingsprogramma worden gebruikt. Omdat inwild e Solanum-soortenonbekend e SAsbleke nvoo r tekomen , werd eenprocedur e ontwikkeld omdergelijk e SAs indienaanwezi g inee n extract,t ekarakterisere nen/o ft eidentificeren .Dez eprocedur eword t 182 beschreveni nHoofdstu kVIII .D egecombineerd eresultate nva nGC-massa - spectrometrie (GC-MS),waarvoo r 'electron impact'e nchemisch eionisa - tiewerde n toegepast,e nva nhoge-resoluti eMS ,va nhe t toepassenva n retentie-indices en NPD/FID responsverhoudingen, en van eenvergelij kingva n de graad van dehydratie van SAs inverschillend e twee-fasen- systemen voor hydrolyse toonden het volgende aan.All e onbekende SAs bezatenee nsolanidaanskele t enware ndu sverwan taa nsolanidin eui td e aardappel; zij bevatten bijvoorbeeld een extra hydroxyl- of methyl- groep.D emeest eva ndez everbindinge nware nnie teerde rgerapporteerd . De totale SA-samenstelling, inclusief de afzonderlijke concentraties vand enieuw e SAs,va nenkel eSolanum-(onder )soortendi e ind everede lingworde ngebruikt ,word t indi thoofdstu kgepresenteerd . S. vernei wordt veelvuldig in de aardappelveredeling gebruikt. Hoewel de SGA-samenstellingva n deknolle nva n deze soort nietbeken d is, zijn er rassen ontwikkeld die erfelijk materiaal van deze soort bevatten.Ee ntoenemen daanta lva ndergelijk erasse nza li nd etoekoms t invel elande no pd erassenlijste nverschijnen . Hoofdstuk IXbeschrijf t de identificatie enkarakteriserin g vand e SAs uit knollen van S.vernei . enva n enkelehybrid e nakomelingen en rassen die erfelijk materiaal van deze soort bevatten. In de wilde soort werden hoge gehalten aan solanidine- en solasodineglycosiden gevonden. Daarnaast kwamen, glycosidisch gebonden, tomatidenol en nieuwe SAs voor, waaronder de 22R,25R-epimeer van solanidine, een verbindingme tee nruimtelijk estructuu rdi eno gnie teerde r isvermel d voor natuurlijke solanidanen. In dit onderzoek bleek ook dat de SA- samenstelling van knollen aanzienlijk kanvariëre n afhankelijk vand e teeltconditiesva nd eplant . Knollenva nhybrid enakomelinge n enva nrasse ndi eerfelij kmateri aalva nS .verne ibevatten ,kunne n lageo fhog e gehaltenaa nsolanidi - neglycosiden bevatten. Daarnaastwerde n solasodineglycosiden gevonden, zelfsi nrasse ndi emeerder emale nware nteruggekruist ,maa rd econcen traties waren gelukkig laag. Het kan echter niet worden uitgesloten, dat nakomelingen van S.verne i onderbepaald e groei-omstandighedeno f tijdens na-oogstbehandelingen potentieel schadelijke hoeveelheden aan solasodineglycosiden ind ekno l synthetiseren. Indi tverban dverdien t demogelijk eteratogen eactivitei tva nsolasodin ed enodig eaandacht . 183 Samenvattend kan worden gesteld dat de opname van de hoeveelheid solanidineglycosiden door de consument niet dient toe te nemen,maa r bijvoorkeu r gereduceerd dient teworden . SGAs dieva n oorsprong niet in S. tuberosum voorkomen dienen niet in de consumptie-aardappel te worden geïntroduceerd. Het introduceren van erfelijk materiaal van wilde Solanum-soorten in de cultuuraardappel dient behoedzaam te geschieden. Het is daarom aan tebevele n de SGAsva npotentiël ewild e kruisingsouders te analyseren,voorda t zij inee nveredelingsprogramm a wordengebruikt ,o mzodoend edi egenotype nt ekunne nselectere ndi eee n gewensteeigenscha pbezitte ni ncombinati eme td emins tongunstig eSGA - samenstelling. Afhankelijk van de SGA-samenstelling van de ouders dienen vervolgens de nakomelingen geanalyseerd te worden. Voor deze analyses zijn de in dit proefschrift beschreven nieuwe methoden geschikt;d emethode ndi ei nd eliteratuu rzij nbeschreve nvoldoe nnie t aand et estelle neisen . Rassen die erfelijk materiaal van wilde Solanum-soortenbevatten , dienen voordat zij als consumptieras worden toegelaten,onderzoch t te worden op een eventuele accumulatie van SGAs onder diverse teelt-, opslag-e nverwerkingsomstandigheden .D erichtlij nva n60-7 0m gsolani dineglycosiden perk gvers e aardappel,di e uithe t oogpuntva nconsu - mentenveiligheid ind e literatuurword t aanbevolen (zieHoofdstu kII) , dient in de aardappelveredeling te worden aangehouden totdat een geschiktmaximaa ltoelaatbaa rgehalt evoo rdez estoffe ni svastgesteld . 184 CURRICULUMVITA E W.M.J,va nGelde rwer dgebore nt e 's-Hertogenboscho p1 0maar t1947 .N a zijnHBS- B opleiding enhe tvervulle nva nzij nmilitair edienstplicht , volgde hij aand eHoger eLaboratoriumschoo lt eOs sd eavondopleidinge n HBO-Ianalytisch echemi ee nHBO- Bbiochemie ;einddiplom a1972 .Va n197 1 tot 1985wa shi jwerkzaa m bij de Stichting voor Plantenveredeling SVP te Wageningen, als hoofd van de Sectie Chemie. Tijdens deze periode volgde hij colleges (kandidaats-B en doctoraal) en (PAO)cursussen, onder andere ind emoleculair e biologie,toxicologie ,e n instrumentele analysemethoden, aan de Landbouwuniversiteit en aan de Technische Universiteit te Eindhoven. Hij verrichtte onderzoek naar de relatie tussen mitochondrion-activiteit en heterosis bij mais, en naar de samenstelling van eiwit inaardappelen .Va n 1985 tot 1989wa shi jbi j deSV Pwerkzaa mal shoof dva nd eAfdelin gBiochemie .Tijden sdez eperi ode verrichtte/leidde hij onderzoek onder andere naar debiochemisch genetischeachtergron dva nd ebakkwalitei tva ntarwe ,onderzoe kgerich t op de ontwikkeling van snelle en gevoelige immunochemischediagnosti sche technieken en onderzoeknaa rkwaliteitsaspecte n bijverschillend e gewassen.He tonderzoe kda t indi tproefschrif tword tbeschreve nheef t hijtusse n198 3e n198 8bi jd eSV Pverricht .Me tingan gva n1 septembe r 1989i shi jaangestel dbi jhe tAgrotechnologisc hOnderzoekinstituu tAT O te Wageningen, als hoofd van de Hoofdafdeling Agrificatie, Nieuwe Gewassene nProdukten .