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Ethnobotany and phytochemistry of Tropaeolum tuberosum and lepidium meyenii from Andean South America Johns, Timothy Allan 1980

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ETHNOBOTANY AND PHYTOCHEMISTRY OF TROPAEOLUM TUBEROSUM AND LEPIDIUM MEYENII TIMOTHY ALLAN JOHNS B.Sc .(Hons. 3, WcMaster U n i v e r s i t y , 1S72 A THESIS SUBMITTED IN FART IA L FULFILMENT CF THE BEQUIKEMENTS FOR TEE DEGREE OF WASTER OF SCIENCE i n THE FAC CITY CF GRADUATE STUDIES FROM ANDEAN SCUTH AMERICA by ( D e p a r t m e n t o l | B o t a n y ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY CF BRITISH COLUMBIA June 1980 Ti m o t h y A l l a n J o h n s , 1880 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f O 0 | QA\ The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 D a t e fl UQU.H l j / lUffi i A b s t r a c t A s y s l e a a t i c i n v e s t i g a t i o n o f t h e e t h n o p h a r m a c o l o g y o f T r o p a e o l u i n tuberosum S u i z fi Pavon and l e ^ i d j u m meyeni i Walp> t i n t h e T r o p a e o l a c e a e and E r a s s i c a c e a e r e s p e c t i v e l y , was u n d e r t a k e n t o d e t e r m i n e the p h y s i c a l . b a s i s f o r t h e m e d i c i n a l and n u t r i t i o n a l u s e s o f t h e s e s p e c i e s by n a t i v e s o f t h e Andes mountains* The d o m e s t i c a t i o n of T. t u b e r o s u m i n r e l a t i o n t o t h e s e u s e s was c o n s i d e r e d f r o m t h e p e r s p e c t i v e o f the g l u c o s i n o l a t e cherootaxcnomy of t h e two s u b s p e c i e s , tuberosum and s i l v e s t r e • H i g h P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h y (HPLC ) was u s e d a s t h e p r i m a r y t o o l f o r d e t e r m i n i n g t h e i s o t h i o c y a n a t e s h y d r o l y z e d e n z y m a t i c a l l y from g l u c o s i n o l a t e s o f the t u b e r s , s e e d s , f l o w e r s and l e a v e s o f b o t h s u b s p e c i e s o f T,. .tub^xosijn , and from t h e r o o t s o f L . nte.v_ejii.i • On the b a s i s of HPLC, p a p e r c h r o m a t o g r a p h y o f t h i o u r e a d e r i v a t i v e s , mass s p e c t r o m e t r y and n u c l e a r m a g n e t i c r e s o n a n c e s p e c t r o m e t r y i t was c o n c l u d e d t h a t t h e s o l e i s o t h i o c y a n a t e l i b e r a t e d f r o m T. • tvjbejjjogum s u b s p . tuberosum i s p—me th o x y b enzy 1 i s o t h i o c y a n a t e . T.. tuberosum s u b s p . s i l v e s t r e i s c h a r a c t e r i z e d by b e n z y l , 2 — p r o p y l and 2 — b u t y l i s o t h i o c y a n a t e s . T h i s d i f f e r e n c e i n g l u c o s i n o l a t e s s u p p o r t s th e e x i s t e n c e o f two d i s t i n c t s u b s p e c i e s . H y d r o i l y s a t es o f I, • meyeni i c o n t a i n b e n z y l i s o t h i o c y a n a t e as t h e p r i m a r y c o n s t i t u e n t , and p—methoxybenzy1 i s o t h i o c y a n a t e as a minor c o n s t i t u e n t . N,K,Di(methoxy, 4— b e n z y l ) t h i o u r e a was d e t e c t e d i n t h e i s o t h i o c y a n a t e e x t r a c t s o f T• t u b e r o s u m s u b s p . tuberosum . A s t a t i s t i c a l s u r v e y o f t h e e t h n o b o t a n i c a I u s e s o f g l u c o s i n o l a t e — c o n t a i n i n g p l a n t s f r o m a r o u n d t h e w o r l d was i i c a r r i e d o u t . The s i g n i f i c a n t m e d i c i n a l u s e s f o r g l u c o s i n o l a t e — c o n t a i n i n g p l a n t s i n g e n e r a l c o r r e l a t e s p o s i t i v e l y w i t h t h e u s e s f r o m t h e Andes o f T • t u b erosum and _L • mej^rjj.i . P h a r m a c o l o g i c a l s t u d i e s on c r u d e p l a n t o a t e r i a l and e x t r a c t s o f X» t u b e r o s u m * and on p u r e compounds were c a r r i e d out i n r e l a t i o n t o t h e r e p u t e d u s e s of t h e s e s p e c i e s * T r o pa e o I urn tuberosum and L e c i d i urn me.v_ej3.il. a r e b e l i e v e d t o a f f e c t human f e r t i l i t y . F e e d i n g s t u d i e s o f f e m a l e g u i n e a p i g s and i n vj>t£o s t u d i e s t o t e s t the 11 fi—estradiol b i n d i n g i n h i b i t i o n o f e x t r a c t s and o f p u r e i s o t h i o c y a n a t e s f a i l e d t o s u b s t a n t i a t e any e s t r o g e n i c a c t i v i t y of t h e s e t a x a . However» p r e l i m i n a r y r e s u l t s f o r N,N,Di(methoxy, 4 - b e n z y l ) t h i o u r e a s u g g e s t t h a t t h i s c o n f o u n d c o i t p e t i t i ve l y i n h i b i t s e s t r a d i o l b i n d i n g and may have e s t r o g e n i c a c t i v i t y . The a n t i a p h r o d i s i a c b e l i e f s a s s o c i a t e d w i t h T. t u b e r o s u m s u b s p . tuberosum were examined i n male r a t s f e d a d i e t c o n t a i n i n g t u b e r s o f t h i s t a x o n . E x p e r i m e n t a l a n i n a l s and c o n t r o l s showed e q u a l c a p a b i l i t y i n i m p r e g n a t i n g f e m a l e s , a l t h o u g h a n i m a l s f e d T. t u b e r o sjum showed a 45% d r o p i n t h e i r t l o o d l e v e l s o f t e s tost: e r o n e/d 1 hy r o t e s t e s t e rone . T h i s d r o p a p p e a r s t o be r e l a t e d t o the a n t i m e t a t o o l i c e f f e c t s o f i s o t h i o c y a n a t e s i n the t u b e r s . T u b e r s of X£0£aeoJLym tuberosum and p u r i f i e d i s o t h i o c y a n a t e s were shown to be a n t i b i o t i c b u t not p h o t o t o x i c a g a i n s t y e a s t and b a c t e r i a . B e n z y l i s o t h i c c y a n a t e was shown t o be n e m a t o c i d a l . T e s t s o f a t u b e r e x t r a c t a g a i n s t H e r p e s Type I v i r u s f a i l e d t o s u b s t a n t i a t e p o s s i b l e a n t i v i r a l a c t i v i t y f o r t h i s s p e c i e s . A n a l y s i s o f f r e e amino a c i d s i n t u b e r s of T. t u b e r o s u m f a i l e d t o d e t e c t any n o n — p r o t e i n o r u n u s u a l amino a c i d s . i i i l a b l e Of C o n t e n t s In t r o d u c t i o n • • • • • • • • • • • • • • • • • • • • • • • . • • • • • • • • « • « • « • • • • • • « • • 1 L i t e r a t u r e Beview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 E t h n o b o t a r y C f G l u c o s i n o l a t e C o n t a i n i n g P l a n t s : D a t a T a b u l a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 E t h n o b o t a n y And E t h n o p h a r n a c o l c g y Cf T r o n a e o l u m t u b e r o s u m And L e p i d i u m meyeni i . . . . . . . . . . . . . . . . . . . . . . 6 S y s t e m a t i c s And P o s s i b l e O r i g i n Of T r o n a e o lum t u b e r o s u m 15 A n d r o g e n s And P l a n t E s t r o g e n s . . . . . . . . . . . . . . . . . . . . . . . . . . 17 G L u c o s l n c l a t e s And T h e i r M e t a b o l i t e s As B i o l o g i c a l l y A c t i v e Compounds ..................................... 21 M a t e r i a l s And Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 O u t l i n e Cf B e s e a r c h • 28 S t a t i s t i c a l A n a l y s i s And E a t a C o l l e c t i o n . . . . . . . . . . . . . . . 29 P l a n t M a t e r i a l 34 C h e m i c a l s • . • • • • • • • • • • • • • • • • . • • • . • • • . • • • • » • • • » • • • • • • • • • • 36 G l u c o s i n o l a t e s . . . . . . . . . . . . 36 I s o t h i o c y a n a t e s • . • • • • . . • • • • • « • • • • • • • • • • • • • • • • • > • • • « • • 36 T h i o u r e a s • • • • • . • . • • • • • • • • « • . • • • • • • • • • • • • • • • • • • • • • • • • • 37 M a t e r i a l s Used In A s s a y s F o r E s t r o g e n i c And A n t i — a n d r o g e n i c A c t i v i t i e s 37 P r e p a r a t i o n Of P l a n t E x t r a c t s . . . . . . . . . . . . . . . . . . . . . . . . . . 37 G l u c o s i n o l a t e s And I s o t h i o c y a n a t e s . . . . . . . . . . . . . . . . . . 37 Use Cf Endogenous Enzyme ( C o l e , 1976) . . . . . . . . . . . . . . . 38 Use Of M y r o s i n a s c P r e p a r a t i o n . . . . . . . . . . . . . . . . . . . . . . . 38 F r e e Amino A c i d s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 i v D e t e c t i o n And D e t e r m i n a t i o n Of C o n s t i t u e n t s . . . . . . . . . . . . 39 G l u c o s i n o l a t e s . . . . . . . . . . * . . . . . . . . . « . . • . . . . . . . . • . . . . • . 39 I s o t h i o c y a n a t e s ...................................... 40 Mass S p e c t r a l A n a l y s i s ...••...*...........••.•....... 42 N u c l e a r M a g n e t i c Resonance S p e c t r o m e t r y 42 Amino A c i d A n a l y s i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 D e t e r m i n a t i o n C f Chromosome Number . . . . . . . . . . . . . . . . . . . . . 43 T e s t s F o r E i o l o g i c a l A c t i v i t y . . . . . . . . . . . . . . . . . . . . . . . . . . 45 A n t i b i o t i c A c t i v i t y 45 A n t i v i r a l A c t i v i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 N e m a t o c i d a l A c t i v i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 E s t r o g e n i c A c t i v i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 7 E f f e c t s Cf T r o c a e o l u m t u b e r o s u m On The E s t r u s C y c l e Of The G u i n e a F i g 48 R a d i o a c t i v e C o m p e t i t i v e B i n d i n g A s s a y . . . . . . . . . . . . . . . 4 9 D i x o n P l o t 50 A n t i — a n d r o g e n i c A c t i v i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 R e s u l t s 53 S t a t i s t i c a l A n a l y s i s Of D a t a Bank Of G l u c o s i n o l a t e -c o n t a i n l n g P l a n t s 53 D e t e c t i o n And D e t e r m i n a t i o n Of C o n s t i t u e n t s . . . . . . . . . . . . 55 TLC Cf G l u c o s i n o l a t e s Of T r o p a e o l u m tuberosum s u b s p . tuberosum And L e o i d i u m m e y e n i i ............ 55 P a p e r C h r o m a t o g r a p h y Cf G l u c o s 1 n o l a t e s . . . . . . . . . . . . . . 55 A n a l y s i s C f I s o t h i o c y a n a t e s .......... ~. «... .......... • 57 Amino A c i d A n a l y s i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 D e t e r m i n a t i o n Of Chrcmosome Number . . . . . . . . . . . . . . . . . . . . . 73 T e s t s F o r E i o l o g i c a l A c t i v i t y < 73 V A n t i b i o t i c A c t i v i t y . . ............................... 73 A n t i v i r a l A c t i v i t y 74 N e m a t o c i d a l A c t i v i t y . ............................... 76 E s t r o g e n i c A c t i v i t y . ................................ 76 A n t i — a n d r o g e n i c A c t i v i t y . . .......................... 79 D i s c u s s i o n . ............................................... S3 G l u c o s i n o l a t e s Of J.££na_eo JLum t u b e r o sum . . . . . . . . 8 3 G l u c o s i n o l a t e s Of L e p i d l u n me ye n i l . .................... 84 T h i o c a r b a m a t e s And T h i o u r e a s In G l u c o s i n o l a t e — c o n t a i n i n g P l a n t s * m 85 E v a l u a t i o n Cf HPIC Methods Of A n a l y s i s Of I s o t h i o c y a n a t e s . .................................... 86 S y s t e m a t i c a C f T r o p a eolutr t u b e r osum .................... 87 E t h n o p h a r m a c o l o g y Of T r opaeolum tub erogjum And L^-Pid ium meven i i .............................................. 90 L i t e r a t u r e C i t e d «• 97 S u p p l e m e n t a r y B i b l i o g r a p h y IS M e d i c a l And N u t r i t i o n a l E t h n o b o t a n y Of L a t i n A m e r i c a And The West I n d i e s .......106 S u p p l e m e n t a r y B i b l i o g r a p h y I I : S e l e c t e d M e d i c i n a l E t h n o b o t a n y I l l Vi L i s - t o f T a b l e s I A n a l y s i s of c o n t i n g e n c y t a b l e s ....................... 33 I I C o l l e c t i o n s o f T r c p a e o l u m tuberosum ..................35 I I I Use c a t e g o r i e s a n a l y z e d f o r s i g n i f i c a n t m e d i c i n a l e f f e c t s .....54 IV T h i n l a y e r and p a p e r c h r o m a t o g r a p h y o f g l u c o s i n o l a t e s 56 V P a p e r c h r o m a t o g r a p h y o f t h i o u r e a d e r i v a t i v e s .........58 VI HPLC a n a l y s i s of i s o t h i o c y a n a t e s ..................... 59 V I I Mass s p e c t r o m e t r y o f HPLC f r a c t i o n s .................. 64 V I I I NMR d a t a f o r p— m e t h o x y b e n z y I i s o t h i o c y a n a t e ..........67 IX A n t i b i o t i c a c t i v i t y 74 X A n t i v i r a l a c t i v i t y ...................................75 XI E f f e c t o f TrcpaeoIurn t u b e r o s u m on e s t r u s c y c l e •••••••77 X I I E s t r o g e n i c a c t i v i t y . . ................................ 78 X I I I R a t b r e e d i n g e x p e r i m e n t ..............................81 XIV L e v e l s o f t e s t o s t e r o n e + d i h y d r o t e s t o s t e r o n e i n r a t b l o o d serum ..........................................82 v i i L i s t o f F i g u r e s 1 l u b e r s c f T t o p a e o l u i t tybero^uro s u b s p . tuberosum ........ 6 2 I s o t h i o c y a n a t e s o f the genus I r o ^ a e o l u m ................12 3 E d i b l e r o o t s o f Lep idlurn meyeni i .......................13 4 V e g e t a t i v e p o r t i o n s o f Tropa.eolum tuberosum ............ 15 5 S t r u c t u r e s o f some known a n t l a n d r o g e n s . . . . . . . . . . . . . . . . . I S 6 Breakdown o f g l u c o s i n o l a t e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 7 Examples o f t h i o u r e a s and t h i o c a r b a m a t e s from g l u c o s i n o l a t e c o n t a i n i n g p l a n t s ...................................... .24 8 M e r c a p t u r l c a c i d o3f b e n z y l i s o t h i o c y a n a t e . . . . . . . . . . . . . . 26 9 T u b e r s o f T r o p a e o l u m t u b e r o sum s u b s p . s i I v e s t r e ........44 10 R e v e r s e p h a s e a n a l y s i s o f T r q p a e c l u m t u b e r o s u m s u b s p . s i l v e s t r e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 11 R e s o l u t i o n of b e n z y l and p—me t h o x y b e n z y l i s o t h i o c y a n a t e s by normal phase HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 12 R e v e r s e phase HPLC c f t u b e r s o f JT. tuberosum s u b s p . tube rosum •.•.•.•...••••.••••••••••••.•••••.•••••65 13 H1—NMR s p e c t r a of p—methoxybenzyI i s o t h i o c y a n a t e f r o m T• t ube rosum sub sp . t u b e rosum •••...•••••.....•••..••.••66 14 Normal phase HPLC o f t u b e r s o f J;. tutjeros,utn s u b s p . tub e rosum • •••••••.••••••••••••••••••*•••••••••••• 69 15 Normal phase HPLC o f T. tuberosum s u b s p . s i l v e s t r e ...••70 16 HPLC o f Lep i d ium meyen i i .71 17 D i x o n p l o t of i n h i b i t i o n o f e s t r a d i o l b i n d i n g by b e n z y l i s o t h i o c y a n a t e .... ....... .......................E0 v i i i Acknowle dgements T h i s work was i n s p i r e d and e n c o u r a g e d by my c o l l e g u e s i n P e r u * S p e c i a l t h a n k s i s e x t e n d e d t o I n g . A. C a u i n o , P o n t i f i c i a U n i v e r s i d a d C a t o l i c a d e l P e r u f o r c o l l e c t i n g t u b e r s o f T r o c a e o l u m tub erosum . I n g . C. Ochoa, I n t e r n a t i o n a l P o t a t o C e n t e r ( C . I . P . ) , f a c i l i t a t e d t h e e x p o r t o f t h e m a t e r i a l from P e r u . I n g . H. C o r t e s B r a v o . U n i v e r s i d a d N a c i o n a l d e l C u z c o p r o v i d e d s e e d s o f T. tuberosum • Dr. R. I . F o r d and M. F. Brown, , U n i v e r s i t y o f M i c h i g a n , g e n e r o u s l y s u p p l i e d i n f o r m a t i o n and r o o t s o f i e ^ i d i u m meyeni i . S t a f f o f the UBC and VanDusen B o t a n i c a l G a r d e n s s u p p l i e d o t h e r p l a n t s f o r s t u d y , and the C a n a d i a n G r a i n Commission, V a n c o u v e r w i l l i n g l y s u p p l i e d s e e d s o f y e l l o w m u s t a r d ( S i n a p i s a l t a ). Dr. D.L. B a i l l i e , Simon F r a s e r U n i v e r s i t y , p r o v i d e d nematodes ( Caeno rh a b d J, t i s e l e a a n s ). D r s . A. K J a e r , E.W. U n d e r b i l l and L.R. W e t t e r s u p p l i e d c h e m i c a l s t a n d a r d s . D r . G.B. S t r a l e y , o f VanDusen , and F. Newsome and B. J u d d o f UBC p a r t i c i p a t e d i n t h i s s t u d y i n ways t h a t a r e a c k o w l e d g e d t h r o u g h o u t the t h e s i s . E.A. N o r t o n c o n t r i b u t e d h i s e x p e r t i s e i n p h o t o g r a p h y . As w e l l E.A. Graham, Z. Abramowski, K. Shekhtman, S. G o p a u l and Do M i l e k p r o v i d e d v a l u a b l e t e c h n i c a l s u p p o r t . D.W. P h i l l i p s , C.—K. Wat, and F. E a l z a d e s e r v e s p e c i a l m e n t i o n f o r t h e i r d a y — t o — d a y a i d and a d v i c e . I would l i k e to thank D r . I.E.P. T a y l o r f o r t h e use o f amino a c i d a n a l y s i s f a c i l i t i e s , and Dr. G. E i g e n d o r f and t h e D epartment of C h e m i s t r y , UBC, f o r t h e use o f mass s p e c t r o m e t r y and NMR f a c i l i t i e s . S. E i t a and M. G r e i g o f t h e Computing C e n t r e , UBC, and J.D. Radke p r o v i d e d c o n s i d e r a b l e h e l p i n the s t a t i s t i c a l p a r t o f t h e r e s e a r c h . ix I am g r a t e f u l t o t i e NSERC, Canada f o r f i n a n c i a l s u p p o r t i n t h e form o f a p o s t g r a d u a t e s c h o l a r s h i p * The g r e a t e s t s u p p o r t e r , s c e p t i c and c r i t i c o f t h i s work has been my s u p e r v i s o r , I r . G.H.N. To w e r s . H i s s e n s e o f humour, e n t h u s i a s m , i d e a s and p a t i e n c e have been i n v a l u a b l e * As w e l l I am g r a t e f u l f o r the s u p p o r t and h e l p o f the r e s t my g r a d u a t e c o m m i t t e e , Drs • R.L. T a y l o r and F*R. G a n d e r s * D r . T a y l o r , D r . R * J . P e a r s o n , D r . H.V* S u h n l e i n and Dr. J . Maze have a l l been p a r t i c u l a r l y g e n e r o u s w i t h t h e i r t i m e and i d e a s * S u s a n L* Keen w i l l i n g l y p r o o f r e a d w h a t e v e r I p r e s e n t e d h e r w i t h and k e p t me g o i n g t o t h e end* 1 I n t r o d u c t i o n T r o n a e o l u n tuberosum R u i z S Pavon and L e o i d i u m meveni i Walp., i n t h e T r o p a e o l a c e a e and B r a s s i c a c e a e r e s p e c t i v e l y , a r e two s p e c i e s o f p l a n t s from Andean S o u t h A m e r i c a c u l t i v a t e d f o r t h e i r e d i b l e and m e d i c i n a l u s e s * To u n d e r s t a n d why t h e y a r e so u s e d r e q u i r e s i n s i g h t f i r s t l y i n t o t h e i r o r i g i n and t h e i r d o m e s t i c a t i o n , and s e c o n d l y i n t o t h e i r p h y s i c a l p r o p e r t i e s and how t h e s e p r o p e r t i e s have c o n t r i b u t e d t o the w e l f a r e o f t h e p e o p l e who use them* The l a t t e r n e c e s s i t a t e s i n v e s t i g a t i o n i n t o t h e c h e m i s t r y and p h a r m a c o l o g y o f the p l a n t s and w i l l c o m p r i s e t h e b u l k o f t h i s t h e s i s * The model o f e t h n o b o t a n i c a l i n v e s t i g a t i o n o u t l i n e d by S c h u l t e s ( 1 £ 6 0 , 1 9 7 2 ) p r o v i d e s t h e b a s i s , w h e t h e r s t a t e d o r i m p l i e d , f o r most s t u d i e s i n t o t h e p h a r m a c o l o g y o f f o l k m e d i c i n e * S c h u l t e s s t r e s s e s t h e i m p o r t a n c e o f e t h n o b o t a n i c a l work i n t h e s e a r c h f o r new d r u g s and s u g g e s t s the f o l l o w i n g m e t h o d o l o g y : 1* B i b l i o g r a p h i c s e a r c h o f e t h n o b o t a n i c a l and h e r b a l i s t l c l i t e r a t u r e of t h e p a s t * 2* S e a r c h o f h e r b a r i a f o r e t h n o b o t a n i c a l r e f e r e n c e s . 3* E t h n o b o t a n i c a l f i e l d work among p r i m i t i v e s o c i e t i e s • 4. P h y t o c h e m i c a l s c r e e n i n g and i n v e s t i g a t i o n * The c o n t r i b u t i o n o f e t h n o b o t a n y t o modern b o t a n i c a l , p h a r m a c o l o g i c a l and m e d i c a l s c i e n c e s has g e n e r a l l y been 2 n e g l i g i b l e or n e g l e c t e d b e c a u s e o f t h e o f t e n s u b j e c t i v e n a t u r e o f b o t h the way i n w h i c h p l a n t s a r e u s e d by g r o u p s o f human b e i n g s , and the way i n which a c q u i r e d i n f o r m a t i o n i s c o m p i l e d and e v a l u a t e d . C o n s i d e r i n g t h e v a r i e t y o f c u l t u r a l c o n t e x t s i n w h i c h the e m p i r i c a l knowledge o f m e d i c i n a l and e d i b l e p l a n t s i s f o r m u l a t e d and t h e n i n t u r n c o m p i l e d , and c o n s i d e r i n g t h e v a r i a t i o n i n c o n c e p t s o f h e a l t h and d i s e a s e , and o f c u r i n g , i t i s o f t e n d i . f f i c u l t t o t r a n s l a t e e t hnobo t a n i c a l d a t a c r o s s -c u l t u r a l l y i n t o i n f o r m a t i o n m e a n i n g f u l to t h e n a t u r a l s c i e n t i s t . In t h i s c o n t e x t t h e i n s i g h t s o f t h e a n t h r o p o l o g i s t a r e i n v a l u a b l e i n i n t e r p r e t i n g t h e m y t h o l o g i c a l and s o c i o l o g i c a l a s p e c t s of f o l k m e d i c i n e , and i n p r e v e n t i n g t h e p i t f a l l s of a s t r i c t l y e t h n o c e n t r i c a p p r o a c h i n e v a l u a t i n g a p a r t i c u l a r f o l k remedy. In g e n e r a l , the ir.eans o f e f f e c t i v e l y e v a l u a t i n g the e x t e n s i v e p u b l i s h e d i n f o r m a t i o n on the m e d i c i n a l use o f p l a n t s a r e l a c k i n g . P h y t o c h e m i c a l and p h a r m a c o l o g i c a l s c r e e n i n g o f p l a n t s i n r e l a t i o n t o t h e i r u s e s p r o v i d e s t h e o n l y r e a l t e s t o f t h e e f f i c a c y o f a r e p u t e d n a t i v e remedy. In many c a s e s the p a t h f r o m n a t i v e remedy t o known c o n s t i t u e n t and even p h a r m a c o l o g i c a l p r e p a r a t i o n i s s t r a i g h t f o r w a r d . Such c a s e s , however, become i n c r e a s i n g l y l e s s f r e q u e n t as t h e more d r a m a t i c f o l k r e m e d i e s a r e i n v e s t i g a t e d . C o n s i d e r i n g t h e d i v e r s e and u n s y s t e m a t i c n a t u r e o f e t h n o b o t a n i c a l l i t e r a t u r e a n o n — c r i t i c a l s e l e c t i o n o f p l a n t s f o r s t u d y i s l i t t l e more t h a n random p h y t o e h e m i c a I s t u d y i n i t s e l f . I f e t h n o b o t a n y i s t o make a c o n t r i b u t i o n t o p h a r m a c o l o g y and p h y t o c h e m i s t r y , i t must do so by i n c r e a s i n g the odds o f s u c c e s s when l o o k i n g f o r a c t i v e p r i n c i p l e s , and by p r e d i c t i n g beyond the l e v e l o f c h a n c e the d i s t r i b u t i o n o f 3 p a r t i c u l a r c h e m i c a l s * One method o f d o i n g t h i s i s t h r o u g h more s y s t e m a t i c e v a l u a t i o n of e t h n o b o t a n i c a l d a t a * I n t h i s t h e s i s e t h n o b o t a n i c a 1 i n f o r m a t i o n i s c o n s i d e r e d from a p l a n t t a x o n o m i c p e r s p e c t i v e w i t h a t t e n t i o n to b o t h c l a s s i c a l taxonomy and chemotaxonomy. W i t h i n t h i s f r a m e w o r k , d a t a c o m p i l e d and a n a l y z e d by modern c o m p u t i n g methods i s used to d e t e r m i n e p a t t e r n s o f use i n p l a n t t a x a and t o r e l a t e t h i s t o known p h y t o c h e m i s t r y . P l a n t s a r e t h e n t e s t e d i n r e l a t i o n to t h i s a n a l y s i s f o r c h e m i c a l c o n s t i t u e n t s and f o r b i o l o g i c a l a c t i v i t y . The p r i n c i p l e f o c u s o f t h i s s t u d y , T r o c a e o i u m t u b e r o s u m , i s an e d i b l e t u b e r — p r c d u e i n g p l a n t o f t h e h i g h Andes c u l t i v a t e d f o r i t s f o o d and m e d i c i n a l u s e s . P r e - C o l u m b i a n b e l i e f s i n t h e p l a n t ' s p r o p e r t i e s as a male a n t i a p h r c d i s i a c and f e m a l e f e r t i l i t y a g e n t have c o n t i n u e d to the p r e s e n t , and may be a s s o c i a t e d w i t h t h e o r i g i n a l d o m e s t i c a t i o n o f t h e s p e c i e s . I t s c o n t r i b u t i o n t o the a d a p t a t i o n o f Andean I n d i a n s t o t h e i r h a r s h h y p o x i c e n v i r o n m e n t has i n t e r e s t e d the a u t h o r s i n c e h i s s o j o u r n i n P e r u i n 1977. M a t e r i a l was c o l l e c t e d t h e n f o r s t u d y . C h e m i c a l and p h a r m a c o l o g i c a l i n v e s t i g a t i o n o f the p l a n t has been u n d e r t a k e n i n t h i s c o n t e x t . The p r i n c i p l e s e c o n d a r y c o n s t i t u e n t s o f t h e s p e c i e s a r e g l u e o s i n o l a t e s • G l u c o s i n o l a t e s c h a r a c t e r i z e s e v e r a l f a m i l i e s , i n c l u d i n g the T r o p a e c l a c e a e , whose a f f i n i t y i n t h e o r d e r C a p p a r a l e s has been u n d e r l i n e d by t h e p r e s e n c e o f t h i s c l a s s o f compound ( E a h l g r e n . 1S75). I n t h e C a p p a r a l e s , most o b v i o u s b i o l o g i c a l a c t i v i t y i s a t t r i b u t a b l e to the e n z y m a t i c breakdown p r o d u c t s o f g l u c o s i n o l a t e s , the i s o t h i o c y a n a t e s ( V i r t a n e n , 1 9 6 5). V a r i a t i o n i n c h e m i c a l p r o p e r t i e s w i t h i n t h e c l a s s o f compounds i s due to s i d e c h a i n d i f f e r e n c e s i n t h e a g l y c o n e s . 4 Chemical, i n v e s t i g a t i o n w i t h i n g l u c o s i n o l a t e — c o n t a i n i n g p l a n t s has been p r i n c i p a l l y o f a chemotaxonomic n a t u r e * Members o f t h e o r d e r a r e w i d e l y d i s t r i b u t e d and a r e r e p r e s e n t e d i n most f l o r a s and e t h n o b o t a n i c a l l i t e r a t u r e . Review of t h i s l i t e r a t u r e has s o u g h t p a t t e r n s of c o n s i s t e n c y o f m e d i c i n a l and n u t r i t i o n a l use i n r e l a t i o n to T, • tuberosum • One s p e c i e s , L e p i d i u m meyeni i , i n the B r a s s i c a c e a e , s t a n d s c u t * I t i s s y m p a t r i c w i t h T* t u b e r o s u m , i s c u l t i v a t e d f o r i t s e d i b l e r o o t and i s r e p o r t e d l y u s e d m e d i c i n a l l y t o a f f e c t human f e r t i l i t y i n a s i m i l a r way t o T* t u b e r o s u m • I t was a n a l y z e d p h y t o c h e m i c a l l y f o r t h i s r e a s o n * 5 L i t e r a t u r e r e v i e w E t h n o b o t a n v o f a l u c o s i n o l a t e c o n t a i n i n g p l a n t s : d a t a t a b u l a ti^ga R e f e r e n c e s u s e d i n t h e c o m p i l a t i o n o f a d a t a base on t h e e t h n o b o t a n i c a l u s e s o f the o r d e r C a p p a r a l e s i n C e n t r a l and S o u t h A m e r i c a a r e l i s t e d i n S u p p l e m e n t a r y B i b l i o g r a p h y I . T h i s p r o v i d e s a f a i r l y c o m p r e h e n s i v e b i b l i o g r a p h y o f the e t h n o b o t a n y o f L a t i n A m e r i c a and has been l i s t e d s e p a r a t e l y f o r t h i s r e a s o n . Most b o o k s o r a r t i c l e s a r e r e g i o n a l i n p e r s p e c t i v e , o f t e n r e f e r r i n g t o a s p e c i f i c c u l t u r a l g r o u p o r g e o g r a p h i c a l a r e a . R e f e r e n c e s w h i c h c o m p i l e i n f o r m a t i o n f r o m d i v e r s e s o u r c e s a r e p a r t i c u l a r l y u s e f u l . A l t s c h u l (1£73) l i s t s i n f o r m a t i o n g l e a n e d f r o m t h e Gray H e r b a r i u m and c o v e r s a wide g e o g r a p h i c a l a r e a . F o r d (1975) c o m p i l e s e x t e n s i v e e t h n o b o t a n i c a l d a t a f r o m t h e a r i d r e g i o n s of n o r t h e r n M e x i c o and s o u t h w e s t e r n USA, as u s e d by S p a n i s h s p e a k i n g p e o p l e s . R e f e r e n c e s on a g l o b a l s c a l e t h a t were r e v i e w e d i n r e l a t i o n t o t h e C a p p a r a l e s a r e l i s t e d i n S u p p l e m e n t a r y B i b l i o g r a p h y I I . Moerman's (1977) s u r v e y o f A m e r i c a n I n d i a n m e d i c a l e t h n o b o t a n y p r o v i d e s a b a s i s f o r any s t u d y i n Canada and the USA. P a r t i c u l a r l y u s e f u l f o r t r o p i c a l e t h n o b o t a n y were B u r k i l l ( 1966 ) and Watt and £ r e y e r - E r a n d w i j k ( 1 9 6 2 ) . 6 fi t hno b Q tajiv_and_e,thno. p h a r m a c c I gg y_ q .f T r o t a e o l u m t u b e r o s u m a.n.d Lgeidium. m e y e j l l The e x p l o i t a t i o n of T r o n a e o l u m t u b e r o s u i t a s an e d i b l e t u b e r ( F i g u r e 1.) has r e c e i v e d m i n i m a l a t t e n t i o n o t h e r t h a n t o r e c o r d i t s use i n i t s n a t i v e h a b i t a t . S u r v e y v o l u m e s o f u s e f u l p l a n t s i n v a r i a b l y r e c o r d t h e f a c t o f i t s c u l t i v a t i o n ( B r u c k e r , 1977; F i g u r e 1. T u b e r s of T r o c a e o l u m t u b e r o s u m s u b s p . t u berosum M o n t a l d o , 1977; Simmonds , 1976; T a n a k a , 1976; Uphof, 1968). V a r i o u s e t h n o g r a p h i c r e p o r t s ( B a k e r and L i t t l e , 1976; Gade, 1975; Mazess and B a k e r , 1964; S t e i n , 1961; V a s q u e z V a r e l a , 1952) m e n tion t h e use o f the p l a n t i n s p e c i f i c r e g i o n s i n t h e A n d e s . More d e t a i l e d d e s c r i p t i o n o f i t s c u l t i v a t i o n and u s e i s g i v e n by Hodge (1946, 1951) and Leon (1964). F e r n a n d e z (1973) p r o v i d e s 7 u s e f u l i n f o r m a t i o n i n t o t h e h i s t o r y o f i t s e x p l o i t a t i o n p a r t i c u l a r l y i n A r g e n t i n a * and W i l l i a m s (1978) g i v e s e v i d e n c e ( a l t h o u g h p e r h a p s q u e s t i o n a b l e ) f o r t h e p o s t — c o n q u e s t i n t r o d u c t i o n o f t h e p l a r . t i n t o . and c u r r e n t use i n . M e x i c o * B e c k e t t ( 1 9 7 9 ) documents c u r r e n t i n t e r e s t i n t h e p l a n t i n B r i t a i n , a l t h o u g h p r i m a r i l y as an o r n a m e n t a l . The n u t r i t i o n a l c o n t r i b u t i o n of t u b e r s o f T r o p a e o l u i n t u b e r o s u m i s p r i m a r i l y due to t h e i r h i g h c a r b o h y d r a t e c o n t e n t . Leung and F l o r e s ( 1961 ) r e c o r d 11 grams c a r b o h y d r a t e p e r 100 grams wet w e i g h t . A l t h o u g h no r e p o r t i s known d e f i n i n g t h e s t a r c h , l e a v e s o f T r c p a e o l u m ma.iu s a r e known t o c o n t a i n m a n n i t o l s ( G i b b s , 1974) and i t i s p o s s i b l e t h a t t h i s s u g a r a l c o h o l c o m p r i s e s the main low m o l e c u l a r w e i g h t c a r b o h y d r a t e i n t h e t u b e r s . The h i g h a s c o r b i c a c i d c o n t e n t (67.0 mg/lOOg wet w e i g h t ) ( L e u n g and F l o r e s , 1961 ) c o n t r i b u t e s t o t h e n u t r i t i o n a l v a l u e o f t h i s p l a n t . The p r o t e i n c o n t e n t o f t h e t u b e r s o f 1.6 g/lOOg wet w e i g h t ( L e u n g and F l o r e s , 1961), c o r r e s p o n d s t o the low v a l u e s of most p l a n t t u b e r s . P o t a t o e s c o n t a i n 2g/100g wet w e i g h t by c c m p a r i s c n . R e p o r t s o f m e d i c i n a l u s e s o f T r o p a e o l u m tuberosum c o i n c i d e w i t h r e p o r t s o f i t s use as a f o o d c r o p . In a r e a s o f the S o u t h e r n Andes where c u l t i v a t i o n o f the p l a n t i s ' o r n a m e n t a l and p s e u d o -o r n a m e n t a l ' , F e r n a n d e z ( 1 9 7 3 ) e x p l a i n s i t s p r e s e n c e as a r e l i c t o f a p r i m o r d i a l a g r i c u l t u r a l c o m p l e x . I t s p r o p a g a t i o n f o r n o n -economic r e a s o n s would u n d e r l i n e a s i g n i f i c a n t p l a c e i n t h e p a s t f o r t h i s p l a n t i n t h e c u l t u r e s o f t h e s e p e o p l e s . The c u r r e n t m e d i c i n a l u s e s and b e l i e f s s u r r o u n d i n g T. t u b e r o sum s u p p o r t t h i s t h e o r y • B e l i e f s c o n c e r n i n g t h e m e d i c i n a l u s e s o f ' i s a n o ' , 'anu' o r 'mashua' a r e f a i r l y d i v e r s e hut a few p a t t e r n s do emerge. G a r c i a 8 B a r r i g a (1975) r e p o r t s t h a t i n C o l o m b i a t h e t u b e r s a r e c o n s i d e r e d d i u r e t i c ; they s e r v e t c b r e a k b l a d d e r and k i d n e y s t o n e s and a r e u s e d t c t r e a t k i d n e y p a i n and o t h e r k i d n e y d i s e a s e s . Soukup ( 1 9 7 0 ) and O b l i t a s P o b l e t e ( 1 9 6 9 ) r e p o r t s i m i l a r u s e s In P e r u and B o l i v i a r e s p e c t i v e l y . Hodge ( 1946,1951 ) r e p o r t s t h a t i n Q u i t o an Ind'ian woman a s s u r e d him t h a t c o o k e d t u b e r s a r e e s p e c i a l l y good f o r t h e k i d n e y s and l i v e r . S t r o n g d i u r e t i c e f f e c t s i n r a t s have been r e p o r t e d f o r a l l y l i s o t h l ocy a n a t e ( M u z t a r .et a l . , 1 9 7 9 b ) . P o s s i b l e s i m i l a r e f f e c t s f o r o t h e r i s c t h i o c y a n a t e s p r o v i d e s t h e most immediate p h y s i o l o g i c a l e x p l a n a t i o n f o r any o f t h e s e f a i r l y c o n s i s t e n t u s e s . U r i n e e x c r e t e d by man and e x p e r i n e n t a l a n i m a l s a f t e r e a t i n g l e a v e s o f T r o ^ a e x l u i raa.ius^ , t h e g a r d e n n a s t u r t i u m , has been r e p o r t e d t o be a n t i b i o t i c (Watt and B r e y e r — B r a n d w i j k , 1962) and i t i s p o s s i b l e t h a t such a c t i v i t y may f a v o u r a b l y a f f e c t the u r i n a r y t r a c t i n some c a s e s . G a r c i a B a r r i g a ( 1 9 7 5 ) a l s o r e p o r t s t h a t t h e t u b e r s a r e e f f e c t i v e i n t r e a t i n g s k i n d i s e a s e s s u c h as eczemas and h e r p e s . V a l d i z a n and M aldonado, (1922) r e p o r t t h a t i n P e r u the f l o w e r s a r e r u b b e d on s p o t s ("empeines 1 ) on the f a c e and O b l i t a s P o b l e t e ( 1 9 6 9 ) l i s t s the t u b e r s as t r e a t m e n t f o r the s k i n u l c e r s c a u s e d by t r o p i c a l i n s e c t s . T h e s e c o n d i t i o n s a p p e a r t o be g e n e r a l l y b i o t i c o r v i r a l i n e t i o l o g y . E x t r a c t s and e s s e n t i a l o i l s o f T_ro.Eaeo.Lu.SI .EJLJ.y_g have been shown t o be a n t i b i o t i c a g a i n s t Ba.cJ_.lJ_.__LS. sub t i l i s . E s c h e r i c h i a c o l i , S t a c h y l o c o c c u s j a u r e u s ( W i n t e r and W i l l e k e , 1952); C a p d i da a l b i c a n s . M i c r o s p o r u m lanosum, T r i c h o p h y t o n gypseum ( V i c k k a n o v e t a l . , 1 9 6 9 ) ; S a c c h a r o mvc es spp . . L a c t o b a c t e r ium b u c h n e r i and A c e t o b a c t e r a c e t i ( S h c h e r b a n o v s k i a r d N l l o v , 1969 ). E a n n e n b e r g e_t a _ l . ( 1956 ) 9 showed t h a t the a c t i v e a n t i m i c r o b i a l p r i n c i p l e o f n a s t u r t i u m i s i d e n t i c a l w i t h b e n z y l i s o t h i o c y a n a t e , one o f the i s o t h i o c y a n a t e s p r e s e n t i n t h e s e e d s o f w i l d T. Jtub^xasum ( K j a e r e t a l . , 197S). G l u c o s i n o l a t e c o n t a i n i n g p l a n t s , i n c l u d i n g T r o o a e o l u m maJus a r e a l s o known t o be i n s e c t i c i d a l ( S e h g a l and U j a g i r , 1877; B l a u e t a l . i 1978). Such a c t i v i t y s u p p o r t s the r e p o r t e d use from B o l i v i a o f T. t u b e r osum f o r h i l l i n g l i c e ( O b l i t a s P o b l e t e , 1969 ). A. Caminc ( p e r s . comm., 1977 ), an a n t h r o p o l o g i s t w o r k i n g on t r a d i t i o n a l a g r i c u l t u r a l p r a c t i c e s i n C u y o — c u y o , P e r u s a y s t h a t t h e l o c a l f a r m e r s p l a n t * i s a n o * i n t e r s p e r s e d among o t h e r t u b e r c r o p s b e c a u s e t h e y c o n s i d e r i t r e s i s t a n t to p a t h o g e n a t t a c k and c a p a b l e o f p r o t e c t i n g o t h e r t u b e r s f r o m d e s t r u c t i o n . C e r t a i n l y a n t i f u n g a l and i n s e c t i c i d a l p r o p e r t i e s a r e s i g n i f i c a n t i n t h i s c a s e . Nematodes pose a p a r t i c u l a r p r o b l e m i n t h i s a r e a . Gommers (1973) i n a r e v i e w o f p l a n t n e m a t i c i d a l p r i n c i p l e s q u o t e s works t h a t show an i n h i b i t o r y e f f e c t o f I s o t h i o c y a n a t e c o n t a i n i n g p l a n t s on the emergence o f l a r v a e o f H e t e r o d e r a r o s t o c h i e q s i s V/ollenweber • O t h e r f o l k u s e s o f T r o o a e o l u m tubexojguj. l a c k t h e p o s s i b l e r a t i o n a l b a s i s o f t h e f a r m e r c a s e s . O b l i t a s P o b l e t e ( 1969 ) r e p o r t s i t s b e i n g u s e d t o t r e a t n e r v o u s d i s e a s e s ; a s an e x p e c t o r a n t and as an a n t i — c o u g h remedy; and t o combat p o l y c e t h e m l a , a common a i l m e n t o f h i g h a l t i t u d e s ( F u c h s , 1978). C o r t e s B r a v o ( 1 9 7 7 ) , i n a p e r s o n a l c o m m u n i c a t i o n , s t a t e d t h a t i n C u zco t h e t u b e r s a r e t a k e n i n soup t o t r e a t stomach u l c e r s . The most p e r v a s i v e and p e r h a p s t h e most p u z z l i n g o f a l l b e l i e f s a s s o c i a t e d w i t h T. tube ro sum i s t h a t o f the p l a n t ' s e f f i c a c y as an a n t i a p h r o d i s i a c o f m a l e s . The s i x t e e n t h c e n t u r y S p a n i s h c h r o n i q u l l i s t s document t h a t the I n c a s f e d 'anu• t o t h e i r t r o o p s so t h a t t h e y w o u l d f o r g e t t h e i r women w h i l e on 10 m i l i t a r y o p e r a t i o n s , and t h a t t h e t u b e r s u p p r e s s e s s e x u a l a p p e t i t e and d e c r e a s e s the r e p r o d u c t i v e p o t e n t i a l : " t i e n e v i r t u d e s t a r a i z de r e p r i m i r e l a p e t i t o v e n e r e o , segum d i c e n l o s i n d i o s j y a s i a i i r n a n que mandaban l o s r e y e s I n c a s d e l P e r u l l e v a r l a c o p i a de c e s t e m a n t e n i m i e n t o en sus e j e r c i t o s , p a r a que comiendo de e l l o s s o l d a d o s , se o l v i d a s e n de s u s m u j e r e s . " F a t h e r Cobo ( 1856 ) " d i c e n l o s i n d i o s que comida es c o n t r a r i a a l a p o t e n c i a g e n e r a t i v e . " I n c a G a r c i l a s o da l a Vega ( 1 9 6 0 ) A l t h o u g h m a g i c a l b e l i e f s accompany t h e s e a c c o u n t s , t h e use o f T« t u b e r o s u m i n a f f e c t i n g human r e p r o d u c t i v e p o t e n t i a l has c o n t i n u e d t c the p r e s e n t * Modern w r i t e r s i n v a r i a b l y c i t e Cobo and G a r c i l a s o de l a Vega ( S o u k u p , 197G; F e r n a n d e z , 1973; Y a c o v l e f f and H e r r e r a , 1 S 3 5 ). Hodge ( 1849, 1951 ), Gade ( 1975 ), M o n t a l d o (1977) and O b l i t a s P o b l e t e (1969) g i v e e v i d e n c e s u p p o r t i n g the c o n t i n u a t i o n of t h i s b e l i e f i n t o modern t i m e s i n s p e c i f i c r e g i o n s . In the D e partment o f A n c a s h i n C e n t r a l P e r u , V asquez V a r e l a ( 1952 ) r e c o r d s t h a t 'mashuas* a r e c o n s i d e r e d as f o o d f o r women. Men r e f u s e to e a t t h e s e t u b e r s b e c a u s e t h e y b e l i e v e t h a t to do so p r o d u c e s i m p o t e n c e and an i n c a p a c i t y t o have c h i l d r e n . A re p e r t i n the J a.u,£na.l o f t h e Hor t i c u l t u r a 1 S o c i e t y of io_ndcn from 1855 s t a t e d , i n r e f e r e n c e t o t h e use and p r e p a r a t i o n o f 1 • tuberosum , t h a t " t h e l a d i e s o f La Paz a r e a l l v e r y f o n d o f t h e Ysano, and i n the s e a s o n of t h e t a i a c h a s l a r g e q u a n t i t i e s a r e sopped i n m o l a s s e s , and t a k e n as r e f r e s h m e n t s 11 d u r i n g the heat of the day." In modern B o l i v i a T. tuberosum i s b e l i e v e d t o induce m e n s t r u a t i o n ( O b l i t a s P o b l e t e , 1969) and i n Cuzco, Peru Trpca^clum ma.ius and T. semjariili Buch. are employed i n p o p u l a r medicine as emmenagogues ( H e r r e r a , 1940). In f o l k m e d i c i n e g e n e r a l l y , m e n s t r u a t i o n i s seen as a s i g n of f e m i n i n i t y and f e r t i l i t y (Conway and Slocumb, 1979) and e f f o r t s a r e made u s i n g herbs and o t h e r means to in d u c e l a t e m e n s t r u a l p e r i o d s . The o n l y r e p o r t on the g l u c o s i n o l a t e s o f Tropaeolum tuberosum i s from seeds of w i l d p l a n t s c o l l e c t e d i n Per u . K j a e r et a l . . , ( 1978 ) r e p o r t taenzy I i so t h i ocyanat e and 2 — p r o p y l i s o t h i o c y a n a t e ( F i g u r e 2. ) as the major components upon enzyme h y d r o l y s i s , w i t h 2 — b u t y I i s o t h i o c y a n a t e o c c u r r i n g i n l e s s e r amounts-. Tropa eo lum ma Jus i s r e p o r t e d to c o n t a i n l a r g e p r o p o r t i o n s c f e r u c i c and e i c o s e n o i c a c i d s i n t h e seeds and f l o w e r p e t a l s (Radwan, 1976). A l t h o u g h e r u c i c a c i d i n C r u c i f e r o u s seed o i l s has been i m p l i c a t e d i n p r o d u c i n g l e s i o n s i n c a r d i a c t i s s u e (McCutcheon e t a_l. , 1976 ), t h e r e i s n o t h i n g t o suggest t h a t the presence o f e i t h e r of these f a t t y a c i d s i n T. tuberosum would c o n t r i b u t e t o the re c o r d e d m e d i c i n a l u s e s . F o l k b e l i e f s c o n c e r n i n g L e p i d i u m mey et> i i , the raaca, have a s t r i k i n g c o r r e s p o n d e n c e t o those of Troqaeolum tuberosum , a l t h o u g h r e f e r e n c e s are s c a r c e . Leon (1964) p r o v i d e s the most a c c e s s i b l e and r e c e n t overview of i t s b i o l o g y and ethn o b o t a n y . B e s i d e s b e i n g c u l t i v a t e d f o r i t s e d i b l e r o o t ( F i g u r e 3. ), the most common use f o r maca i s as a f e r t i l i t y m e d i c i n e . I t i s xz R-N=C=S 2 -p ropy l R=(CH 3) 2 - C H -2 - b u t y l R = C 2 H 5 - C H -C H 3 e t h y l R = C 2 H 5 -2-methyI p r o p y l R = C H 3 - C H - C H 2 -benzyI p-met hoxybenzyl i H 3 Figure 2. Isothiocyantes of the genus Tropaeolum. 13 r e p o r t e d by the c h r o n i q u i l i s t s t h a t i n t h e time o f t h e S p a n i s h c o n q u e s t the I n d i a n s recommended f e e d i n g maca t o d o m e s t i c a n i m a l s t o combat low r e p r o d u c t i v e r a t e s a t h i g h a l t i t u d e s , and L e p i d » u m me y e n / i Wolp. CENTIME I C R S 2 F i g u r e 3 . E d i b l e r o o t s of L e p i d i u m meyeni j t h a t t h e S p a n i s h n o t i c e d the p o s i t i v e e f f e c t s * Today maca i s c u l t i v a t e d o n l y i n a few a r e a s o f c e n t r a l P e r u . Leon r e p o r t s t h a t maca i s now e a t e n by I n d i a n and vthite women who want t o have c h i l d r e n . I t i s s o l d i n t h e m a r k e t s f o r t h i s p u r p o s e . M i c h a e l F. Erown (1979), d o c t o r a l c a n d i d a t e i n a n t h r o p o l o g y a t the U n i v e r s i t y o f M i c h i g a n , r e p o r t s t h a t i n 1973 when he worked i n J u n i n , b e l i e f i n t h e f e r t i l i t y e f f e c t s o f maca was w i d e s p r e a d . However, t h e f a c t t h a t the b e l i e f a p p l i e d p a r t i c u l a r l y t o male f e r t i l i t y seems i n c o n t r a d i c t i o n t o t h e o t h e r b e l i e f s m e n t i o n e d above. I^£J_dAiim «ev_en.i,i. has n o t been s t u d i e d p h y t o c h e m i c a l l y . 14 L e u i d i u m s p e c i e s , as members o f t h e £ r a s s i c a c e a e , a r e known t o c o n t a i n g l u c o s i n o l a t e s • S p e c i e s f r o m o t h e r a r e a s o f the w o r l d have been s t u d i e d and f o u n d t o c o n t a i n a v a r i e t y o f g l u c o s i n o l a t e s i n c l u d i n g b e n z y l i s o t h i o c y a n a t e r e p o r t e d above f r o m T. jtub_erp.sum . V a r i a t i o n c o m p r i s i n g a l k y l and a l k e n y l d e r i v a t i v e s o c c u r w i t h i n t h e genus a l t h o u g h a r o m a t i c g l u c o s i n o l a t e s , w i t h or w i t h o u t h y d r o x y and methoxy s u b s t i t u t i o n s i n m— and p— p o s i t i o n s , p r e v a i l ( K j a e r and W a g n i e r e , 19*71 )• 3 , 4 , 5— I r i me t hoxybenzy Ig l u c os i n o l a t e o c c u r s i n L.. s o r d I d a A. G r a y ( K j a e r and Wagniere, 1971) and L» h y s s o p i f c l i u m D esv. ( K j a e r ejt q.\, , 1971). The o n l y s p e c i e s s t u d i e d from S o u t h A m e r i c a , i« b o n a r i e n s e L . f r o m A r g e n t i n a , i s r e p o r t e d t o c o n t a i n p — h y d r o x y and p — m e t h o x y b e n z y l i s o t h i o c y a n a t e ( K j a e r and S c h u s t e r , 1968). L . b o n a r i e n s e c o l l e c t e d i n Q u e e n s l a n d , A u s t r a l i a has b een shown t o c o n t a i n s o l e l y t h e p — methoxy compound ( K j a e r e t aJL. , 1 9 7 1 ) . The p a r a l l e l s between T r o o a e o l u m tuberosum and Lepi,c(iuii| m e y e n i i a s a g e n t s a f f e c t i n g f e r t i l i t y may be c o i n c i d e n t a l o r r e l a t e t o c u l t u r a l and e n v i r o n m e n t a l f a c t o r s u n c o n n e c t e d w i t h t h e p h y t o c h e m i s t r y and p h y s i o l o g i c a l e f f e c t s o f t h e p l a n t s . R e p r o d u c t i v e r a t e s a r e i n d e e d l o w e r and a c o n c e r n a t h i g h a l t i t u d e s ( S o b r e v i l l a _g_t _ a l . , 1968; Buck e t a l . , 1968 ) and f o l k b e l i e f s a s s o c i a t e d w i t h t h i s a r e t o be e x p e c t e d . The a s s o c i a t i o n o f two g l u c o s 1 n o l a t e — c o n t a i n i n g ' r o o t ' c r o p s w i t h t h i s c o n c e r n may s i m p l y be a r e s u l t of t h e i r s t r o n g and d i s t i n c t f l a v o u r w h i c h t e n d s to draw a t t e n t i o n t o the p l a n t s as m e d i c i n a l a g e n t s . 15 Systemat jcs_and_Eij^sli.J:^_c»xigin .Sll I r o c a e o l u g t u b e r o s u m The b e s t b o t a n i c a l d e s c r i p t i o n a f the s p e c i e s Trouaeoluro  t uberosum i s g i v e n by S p a r r e ( 1 9 7 3 ) . Bodge (1951 ) and Leon F i g u r e 4. V e g e t a t i v e p o r t i o n s of T r o c a e o l u m t u b e r o s u m (1964) p r o v i d e d e t a i l s o f c u l t i v a t i o n and t u b e r c h a r a c t e r i s t i c s , w h i l e H e r r e r a (1941) and C a r d e n a s (184S) g i v e r e p o r t s on s p e c i f i c c u l t i v a t e d forms from a r o u n d C u z c o , P e r u and B o l i v i a r e s p e c t i v e l y . S p a r r e d i f f e r e n t i a t e s two s u b s p e c i e s ; T r o p a e o l u m t uberosum s u b s p . tub. exc.s_um » t h e t u b e r p r o d u c i n g c u l t i v a r , and s u h s p . s i l v e s t r e S p a r r e , a w i l d t y p e known t o o c c u r s y m p a t r i c a I l y . S p a r r e c o n s i d e r s t h a t s u b s p . s i I v e s t r e c a n be d i s t i n g u i s h e d by i t s g r a c i l e l i f e form and l a c k o f t u b e r s . He s t a t e s , however, t h a t some s p e c i m e n s must c o n t a i n t u b e r s , 16 otherwise the s p e c i e s would not have been s e l e c t e d f o r c u l t i v a t io n . Gibbs et a l . (1978) have e s t a b l i s h e d the chromosome number of subsp. tuberosum d e f i n i t i v e l y at 2n=52. The chromosome number of subsp. s i l y e s t r e has not been e s t a b l i s h e d , although Gibbs et a_l. , (1S7S) s p e c u l a t e that i t i s l i k e l y a d i p l o i d and that through au t o p o l y p I o i d y i t has given r i s e to the t e t r a p l o i d c u l t i v a r , subsp. tubej»csijm . A l t e r n a t i v e l y they suggested that the c u l t i v a r may have a r i s e n through a h y b r i d i z a t i o n of subsp. s i l v e s t r e and another r e l a t e d s p e c i e s , perhaps IropaeoIum cochabambe Euch., followed by a l l o p o l y p l o i d y . Huynh (1967) has r e p o r t e d a chromosome count of 2n=26 f o r T. cochabambe • Both T_. cochabambe and T. tube rosum have been placed i n the s e c t i o n Mucronata ( S p a r r e , 1973). A base number of x=13 has been suggested for t h i s s e c t i o n (Gibbs e_t a I . , 1978 ) i n a d d i t i o n to x= 7 p r e v i o u s l y e s t a b l i s h e d f o r Trppaeolum . The r e p o r t c i t e d above on g l u c o s i n o l a t e s present i n the s p e c i e s , r e f e r s presumably to seeds of T.« tubero sum subsp. s i l ves- t r e • G l u c o s i n o l a t e s of subsp. tuberosum have not been i d e n t i f i e d . Kjaer e_t _aJL. ( 197S ) r e p o r t the g l u c o s i n o l a t e s of s e v e r a l s p e c i e s i n the genus i n c l u d i n g Tropaeolum cochabambe • Seeds of T. c^cijataan_be. produce, upon enzymatic h y d r o l y s i s , 2 — p r o p y l , 2 — b u t y l , 2—me thyIpropy1, and b e n z y l i s o t h i o c y a n a t e s • Because 4-methcxybenzyI a l c o h o l and the corresponding aldehyde were d e t e c t e d , 4 —methoxybenzylisothiocyanate i s l i k e l y present as w e l l . In a d d i t i o n , e t h y l i s o t h i o c y a n a t e was detected i n another s p e c i e s , T.. peieprinum L . • I s o t h i o c y a n a t e s known from the genus T ^ _ o j 3 a.eol__u,n] are shown i n F i g u r e 2. 17 A n d r o g e n s and p l a n t e s t r o g e n s G l u c o s i n o l a t e s as a c l a s s have not been s t u d i e d d i r e c t l y i n r e l a t i o n to a n i m a l f e r t i l i t y , b u t s p e c i f i c g l u c o s i n o l a t e — c o n t a i n i n g p l a n t s have t e e n i n c l u d e d i n s u r v e y s o f e s t r o g e n i c a c t i v i t y ( C h a u d h e r , 1966; F a r n s w o r t h e._i a l . . , 1975ab). No c o n s i s t e n t p a t t e r n o f a c t i v i t y a p p e a r s * A g e n t s d i r e c t l y a f f e c t i n g r e p r o d u c t i o n a r e l i k e l y t o be s t e r o i d s o r t o a p p r o x i m a t e s t e r o i d hormones i n a c t i v i t y i n b i n d i n g to the r e c e p t o r s i t e s r e s p o n s i b l e f o r m e d i a t i n g s e x u a l and r e l a t e d p h y s i o l o g i c a l p r o c e s s e s * A l t h o u g h s u c h h o r m o n a l a c t i o n s , p a r t i c u l a r l y i n f e m a l e s , a r e a r e s u l t o f complex i n t e r a c t i o n s o f more t h a n one compound, i t can be s a i d g e n e r a l l y t h a t a n d r o g e n i c and e s t r o g e n i c a c t i v i t i e s a r e o p p o s i t e , t h a t i s a compound h a v i n g s t i m u l a t o r y e f f e c t i n a f e m a l e might be a n t a g o n i s t i c t o a n d r o g e n c o n t r o l l e d p r o c e s s e s i n a male* Thus the f o l k u s e s o f I r o n a e olum tuberosum and Lep i d ium me yen, j i as f e m a l e f e r t i l i t y a g e n t s and male a n t a g o n i s t s have a c e r t a i n l o g i c t o them* A g e n t s t h a t e x h i b i t e f f e c t s or< e s t r o g e n r e c e p t o r s I n t h e f e m a l e may be e s t r o g e n s t h e m s e l v e s * E s t r o g e n i c a c t i v i t y i s known f r o m many p l a n t s ( L i e n e r , 1969 ), and e s t r o n e i t s e l f i s a known p l a n t c o n s t i t u e n t ( B e n n e t t e t a_l • , 1966 ) The Tr o p a e o l a c eae has n o t been s t u d i e d f o r the o c c u r e n c e o f e s t r o g e n * I s o f l a v o n e s f r o m p l a n t s o u r c e s have been shown t o have e s t r o g e n i c a c t i v i t y i n c a t t l e and s h e e p (Pope and W r i g h t , 1954) and e x t e n s i v e s t u d i e s have been dene on the mechanism o f a c t i o n o f t h i s g r o u p o f compounds ( S h e m i s h e t a l . , 1972; S h u t t and Cox, 1972). I s o f l a v a n o i d s a r e known p r i m a r i l y f r o m t h e F a b a c e a e and have 18 n e v e r been r e p o r t e d from the T r o p a e o l a c e a e • T r o p a e o l u m ma.jus , t h e o n l y r e p r e s e n t a t i v e o f t h i s f a m i l y to be examined f o r f l a v a n o i d s t does not c o n t a i n i s o f l a v a n o i d s ( D e l a v e a u , 1 9 6 7 ) . The one s t u d y on s c r e e n i n g f o r the h e m o l y t i c a c t i v i t y c h a r a c t e r i s t i c o f s a p o n i n s r e p o r t s n e g a t i v e r e s u l t s f o r t h e s e v e r a l s p e c i e s t e s t e d ( B l c a r d i e_i a l . , 1 9 S 8 ) . A n t i — a n d r o g e n s a r e net known from p l a n t s o u r c e s . M a i n w a r i n g * s ( 1 9 7 7 ) r e v i e w o f t h e mechanism o f a c t i o n o f a n d r o g e n s m e n t i o n s the a n t i — a n d r o g e n i c e f f e c t o f e s t r o g e n , b u t t h e b u l k o f work i n t h i s f i e l d i s d i r e c t e d a t v a r i o u s s y n t h e t i c compounds s u c h as c y p r o t e r o n e a c e t a t e and BCWT ( F i g u r e 5. ) w h i c h have s t e r o i d — r e l a t e d s t r u c t u r e s . O t h e r compounds, p a r t i c u l a r l y f l u t a m i d e ( 4 ' — n i t r o — 3 * t r i f l u r o m e t h y I i s o b u t y l a n i l i d e ) ( F i g u r e 5. ), e x h i b i t s i m i l a r c o m p e t i t i v e n e s s f o r b i n d i n g s i t e s on t h e a n d r o g e n r e c e p t o r p r o t e i n i n r a t p r o s t r a t e and s e m i n a l v e s i c a l ( P e e t s et. a I . , 1974 ), but show no s t r u c t u r a l r e l a t i o n t o t h e s t e r o i d s . The p a p e r s c o m p r i s i n g A£-ta Eridqcr i no I o g i a 92( 1979 ) Supplementurn 229 c o n c e n t r a t e on the s t e r o i d a l a n t i — a n d r o g e n TSAA—291 and i l l u s t r a t e the m e t h o d o l o g y a v a i l a b l e to s t u d y compounds w i t h s u c h a c t i v i t y . The d e s i r e f o r a s a f e and e f f e c t i v e male o r a l c o n t r a c e p t i v e p r o v i d e s t h e c u r r e n t i m p e t u s i n t o s t u d y i n g a n t i — a n d r c g e n s • M u z t a r e t a\• ( 1979b) have shown t h a t a l l y l i s o t h i o c y a n a t e l o w e r s the amount o f 17—keto s t e r o i d s e x c r e t e d i n r a t u r i n e . 17— Keto s t e r o i d s a r e breakdown m e t a b o l i t e s o f a n d r o g e n s and a d r e n o c o r t i c o s t e r o i d s (Goodman and G i l m a n , 1975). T h e i r l o w e r l e v e l s may i n d i c a t e a d e c r e a s e i n t e s t o s t e r o n e p r o d u c t i o n and 19 Figure 5. Structures of some known antiandrogens. 20 b l o o d l e v e l s , a l t h o u g h t h i s has n o t been e s t a b l i s h e d i n t h e c a s e o f a 11 y I i s o t h i o c y ana te • In s t u d i e s o f b o t h e s t r o g e n i c and a n t i — a n d r o g e n i c a c t i v i t y i t has been shown t h a t s m a l l c h a n g e s i n s t r u c t u r e a l t e r a c t i v i t y . M e t a b o l i s m Ijq v i v o has been shown t o enhance t h e a c t i v i t y o f c e r t a i n compounds. K a t c h e n and Euxbaum, (1975) have d e m o n s t r a t e d t h a t f l u t a m i d e i s c o n v e r t e d t o t r i f l u o r o — 2 — m e t h y l — 4 • n i t r o — m — I a c t o l u i d i d e , w h i c h may be the a c t i v e p r i n c i p l e . N i l s s o n (1961) d e m o n s t r a t e d t h e d e m e t h y l a t i o n o f the i s o f l a v a n o l d , E i o c h a n i n A ( 4 ' m e t h y l g e n l s t e i n ) t o g e n i s t e i n , a s l i g h t l y more p o t e n t p h y t o e s t r o g e n , i n sheep and c a t t l e r u m i n a l f l u i d . I f i n f a c t T. t vi b e r o s um and L . m e v e n i i do a f f e c t r e p r o d u c t i v e p r o c e s s e s , a mechanism of a c t i o n i s n o t i m m e d i a t e l y o b v i o u s . I s o t h i o c y a n a t e s , b e c a u s e o f t h e i r p r e s e n c e i n t h e p l a n t and t h e i r e f f e c t on u r i n a r y 17—keto s t e r o i d s , a r e o b v i o u s c a n d i d a t e s f o r s t u d y . A l l e s t r o g e n i c and a n t i a n d r o g e n i c compounds known, s t e r o i d a l or o t h e r w i s e , a r e a r o m a t i c . S i g n i f i c a n t y , T. • tuberosum c o n t a i n s a r o m a t i c g l u c o s i n o l a t e s and L• m e v e n i i i s l i k e l y t c c o n t a i n them a l s o . I s o t h i o c y a n a t e s a r e known t o be r e a c t i v e compounds . T h e i r b i o l o g i c a l a c t i v i t i e s w i l l be d i s c u s s e d i n r e l a t i o n t o t h e i r c h e m i s t r y • 21 G l u c o s i n o l a t e s and t h e i r m e t a b o l i t e s as b i o l o g i c a l l y a c t i v e compound s The s t r u c t u r e o f the g l u c o s i n o l a t e a n i o n was c o r r e c t l y e l u c i d a t e d by E t t l i n g e r and Lundeen, ( l c f £ 6 ) * T h e s e s e c o n d a r y p r o d u c t s a r e d e r i v e d frcm amino a c i d s and o c c u r as p o t a s s i u m s a l t s i n p l a n t t i s s u e s * G r o t and M a t i l e (1979) p r e s e n t e v i d e n c e t o s u g g e s t t h a t g l u c o s i n o l a t e s and t h e i r a s s o c i a t e d enzymes, t h e m y r o s i n a s e s , a r e b o t h ccmpartmented In v a c u o l e s * V a n E t t e n je_t 8.JL* ( 1979) showed t h a t i n B r a s s i c a o l e r a c e a t h e g r e a t e s t j g l u c o s i n o l a t e c o n c e n t r a t i o n i s i n t h e r e g i o n o f t h e v a s c u l a r b u n d l e s * When the t i s s u e i s c r u s h e d or damaged the g l u c o s i n o l a t e i s h y d r o l y z e d , u s u a l l y t e r m i n g t h e c o r r e s p o n d i n g i s o t h i o c y a n a t e ( F i g u r e 6* )• M y r o s i n a s e s m e d i a t e t h i s p r o c e s s ( B j o r k m a n , 1976 ) and a r e b e l i e v e d t o f u n c t i o n a s t h i o g l u c o s i d e g l u c o h y d r o l a s e s ( E * C * 3*2*3*1* }• M y r o s i n a s e s a r e g e n e r a l l y n o n - s p e c i f i c w i t h r e s p e c t to the s i d e c h a i n o f t h e a g l y c o n e and t h e i r a c t i v i t y i s o f t e n e n h a n c e d by t h e a d d i t i o n o f a s c o r b i c a c i d * The a g l y c o n e s o f g l u c o s i n o l a t e s decompose n o n — e n z y m a t i c a l l y u s u a l l y to the i s o t h i o c y a n a t e s * However, u n d e r p a r t i c u l a r c o n d i t i o n s n i t r i l e s and t h i o c y a n a t e s ( M i l l e r , 1965) , and o c c a s i o n a l l y o x a z o l i d i n e — 2 — t h i c n e s and c y a n o — e p i t h i o a l k a n e s a r e known t o a p p e a r , e i t h e r s p o n t a n e o u s l y or by an e n z y m a t i c a l l y c o n t r o l l e d r e a c t i o n * Eenn (1977) r e v i e w s t h e c a t a b o l i s m o f g l u c o s i n o l a t e s and the b i o l o g i c a l e f i e c t s a s s o c i a t e d w i t h t h i s c a t a b o l i s m * I s o t h i o c y a n a t e s , o r m u s t a r d o i l s , a r e t h e compounds r e s p o n s i b l e f o r t h e s h a r p c h a r a c t e r i s t i c m u s t a r d t a s t e i n g l u c o s i n o l a t e c o n t a i n i n g p l a n t s * MacLeod (1876) r e v i e w s t h e r o l e 22 o f i s o t h i o c y a n a l e s i - t h l o c y a n a t e s a n d n i t r i t e s i n t h e f l a v o u r p a l a t a b i l i t y o f C r u c i f e r o u s v e g e t a b l e s . In the Andes t h e s h a r p t a s t e a s s o c i a t e d w i t h T • tuberosum i s removed f r o m t u b e r s by 2-R-C N-OSOo yy J \ G I u HoO yy N-OSOr s + C 6 H 1 2 0 6 —) R-NCS + SO4 i s o t h i o c y a n a t e —) R-CN + S + SO4 -n j t r i I e —^ [R+] + S C N - + SoJ" t h i o c y a n a t e F i g u r e 6. .Breakdown o f g l u c o s i n o l a t e s b o i l i n g b e f o r e t h e y a r e e a t e n . B e a t i n g d e s t r o y s m y r o s i n a s e a c t i v i t y t h u s p r e v e n t i n g i s o t h i o c y a n a t e r e l e a s e . The w e l l known g o i t r o g e n i c e f f e c t s o f c a b b a g e and o t h e r c r u c i f e r o u s v e g e t a b l e s i s due t o t h e o x a z o l i d i n e — 2 — t h i o n e s d e r i v e d from th e c y c l i z a t i o n o f 2 — h y d r o x y a l k y l i s o t h i o c y a n a t e s , and a l s o t o t h i o c y a n a t e i o n s . T h e s e a n t i t h y r o i d a g e n t s have been shown t o i n h i b i t e n z y m a t i c h a l o g e n a t i o n r e a c t i o n s ( M o r r i s and Hager, 1966; S i l v e r s t e l n and H a g e r . 1974) and i n t h i s way a r e t h o u g h t t o i n t e r f e r e w i t h t h e i o d i n a t i o n o f t y r o s i n e t o p r o d u c e t h y r o x i n . I s o t h i o c y a n a t e s as w e l l may be g o i t r o g e n i c . Benn (1977) s u g g e s t s t h a t s o l u b l e t h i o u r e a r e s u l t i n g f r o m t h e 23 r e a c t i o n of i s o t h i o c y a n a t e w i t h t a s i c amino a c i d s may be r e s p o n s i b l e t o r t h i s a c t i v i t y . Ejorkman (1973) demonstrated the r e a c t i o n of i s o t h i o c y a n a t e s w i t h s u l p h y d r y l and amino groups i n p r o t e i n s , thus p r o v i d i n g a mechanism to account f o r t h e i r t o x i c i t y towards m i c r o o r g a n i s m s and i n s e c t s . • E l b o c i o * , or g o i t r e has been known from the Andes s i n c e the conquest ( L a s t r e s , 1951). A l t h o u g h 2.7-4.8% o f the p o p u l a t i o n have e n l a r g e d t h y r o i d s i n a r e a s where T. tuberosum i s consumed (Buck e_t 1968), the i n c i d e n c e i s not h i g h e r than from o t h e r r e g i o n s i n Peru and t h e r e i s no e v i d e n c e to a s s o c i a t e t h i s c o n d i t i o n s p e c i f i c a l l y w i t h the use of T. tuberosum • G l u c o s i n o l a t e c o n t a i n i n g p l a n t s a r e known t o have an a n t i m e t a b o I i c e f f e c t on d o m e s t i c a n i m a l s ( V a n E t t e n and Tookey, 1978 ). Animals f e d more than 10% of rapeseed meal, Bra,ssica flajeus, s u f f e r e d weight l o s s w h i l e c o n t i n u i n g to consume normal amounts of f e e d . Nuztar _et .§A« (1979a) showed t h a t a l l y l i s o t h l o c y a n a t e i n c r e a s e d a c t i v i t y c f both s u c c i n a t e dehydrogenase and l a c t a t e dehydrogenase i n r a t l i v e r . They suggest t h a t the c a t a l y t i c e f f e c t on o x i d a t i v e r e a c t i o n s may be s i m i l a r to t h a t of t h y r o x i n e which i n c r e a s e s the m e t a b o l i c r a t e i n s u b — c e l l u l a r m i t o c h o n d r i a and microsomes. The s i g n i f i c a n c e o f t h i s i n f o r m a t i o n i n the o v e r a l l h o m e o s t a s i s o f the organism i s not known. The f a t e of i s o t h i o c y a n a t e s , n i t r i l e s and t h i o c y a n a t e s i n the t e s t tube or i n l i v i n g systems i s not w e l l s t u d i e d . Benn (1977) s u g g e s t s t h a t amines and a l d e h y d e s , c a r b o x y l i c a c i d s and t h i o l s r e s p e c t i v e l y are the l o g i c a l f i n a l p r o d u c t s . D e t a i l e d gas chromatography (GLC ) of the seed v o l a t l l e s of Tropaeolum p e r i e r i n u i c and T. cochatambe by K j a e r et _a_l. (1978 ) s u p p o r t s the breakdown of i s o t h i o c y a n a t e s to aldehydes and k e t o n e s . 24 E l M i g i r a b e t a l . ( 1977 ) have i s o l a t e d and d e s c r i b e d i s o t h i o c y a n a t e s , t h i o u r e a s and t h l o c a r b a m a t e s d e r i v e d from P ent ad i n l a n d r.a b r a z z e a n a B a i l l o n . , an A f r i c a n p l a n t i n t h e f a m i l y P e n t a d i p l a n d r a c e a e • A l t h o u g h the u n u s u a l n e s s o f t h i s CH 2 -NH-C-0-(\ />-CH 73 S N-(4-hydroxy,benzyl) methylthlocarbamate S 3 H C O - ^ ^ - C H 2 - N H - C - N H - C H 2 - ^ > -OCH 3 N,N,Di(methoxy,4-benzyl) thiourea F i g u r e 7.°Examples c i t h i o u r e a s and t h i o c a r b a m a t e s from g l u c o s i n o l a t e c o n t a i n i n g p l a n t s r e p o r t would s u g g e s t a l l b u t t h e i s o t h i o c y a n a t e s t o be a r t i f a c t s 25 o f t h e e x t r a c t i o n p r o c e d u r e , t h e a r o m a t i c s t r u c t u r e s o f t h e p a r t i c u l a r compounds and t h e i r p o s s i b l e r e l a t i o n to e s t r o g e n i c o r a n t i — a n d r o g e n i c compounds makes them p o t e n t i a l l y i n t e r e s t i n g i n t h i s i n v e s t i g a t i o n * T h i o u r e a s s p e c i f i c a l l y w i l l be c o n s i d e r e d as f e r t i l i t y a f i e c t i n g compounds* S c h e l i n e (1978) r e v i e w s t h e mammalian m e t a b o l i s m o f g l u c o s i n o l a t e s i n humans and a n i m a l s * i o v i v o m e t a b o l i s m o f g l u c o s i n o l a t e s i s c o m p l i c a t e d by t h e n e c e s s i t y f o r t h i o g l u c o s i d e h y d r o l y s i s b e f o r e a b s o r p t i o n t a k e s p l a c e * T h i o g l u c o s i d a s e a c t i v i t y a p p e a r s t o be p r e s e n t i n some human i n t e s t i n a l b a c t e r i a ( O g i n s k y e t a l . , 1965) and l e a d s t o the p r o d u c t i o n o f i s o t h i o c y a n a t e s and r e l a t e d compounds and t h e i r a b s o r p t i o n i n the i n t e s t i n a l lumen. T a n i e t a l . (1974) i s o l a t e d E n t e r o b a c t e r c l o a c a e as a b a c t e r i u m w i t h p a r t i c u l a r l y h i g h m y r o s i n a s e a c t i v i t y • When s i n a l b l n was a d m i n i s t e r e d t o r a t s by stomach t u b e ( G r i f f i t h s , 1969) i t underwent e s t e r h y d r o l y s i s . S i n a p i c a c i d , d i h y r o s i n a p i c a c i d and p — h y d r o x y b e n z o i c a c i d were e x c r e t e d i n t h e 24 h o u r u r i n e . 3 — H y d r o x y — 5 — m e t h o x y p h e n y l p r o p i o n i c a c i d was r e l e a s e d on t h e s e c o n d d a y . Work by H r u s e w i t z e t .&1* ( 1977 ) s u g g e s t s t h a t i s o t h i o c y a n a t e s may be c o n j u g a t e d w i t h g l u t a t h i o n e and e x c r e t e d i n t h e u r i n e as m e r c a p t u r i c a c i d s * A s e r i e s o f i n t e r m e d i a t e r e a c t i o n s were d e t e c t e d i n v i t ro when b e n z y l i s o t h i o c y a n a t e was i n c u b a t e d w i t h r a t l i v e r and k i d n e y homogenates* B e n z y l i s o t h i o c y a n a t e and t h e c o r r e s p o n d i n g m e r c a p t u r i c a c i d ( F i g u r e 8*) were d e t e c t e d i n t h e u r i n e o f r a t s a d m i n i s t e r e d lOmg/kg c r a l l y * A c c o r d i n g t o B r u s e w i t z e t al» (1977) t h e m e r c a p t u r i c a c i d o f b e n z y I i s o t h i o c y a n a t e has been d e t e c t e d i n t h e u r i n e s of r a t s , h a m s t e r s , dogs, p i g s and humans, 26 b u t not In the u r i n e s o f r a b b i t s and g u i n e a p i g s , s u g g e s t i n g d i f f e r e n t r o u t e s o f m e t a b o l i c breakdown i n t h e s e two g r o u p s o f a n i m a l s . ^ ^-CH 2-NH-C-S-CH 2-CH-C0 2H S NH-CO-CH3 F i g u r e 8. W e r c a p t u r i c a c i d o f b e n z y l i s o t h i o c y a n a t e S e v e n t y - t h r e e d i f f e r e n t g l u c o s i n o l a t e s were known a s o f 1977. R e v i e w s by E t t l i n g e r and K j a e r (1968), and K j a e r and O l s e n L a r s e n (1973, 1976, 1977) p r o v i d e a c u m m u l a t i v e summary o f o n g o i n g I n v e s t i g a t i o n s and new r e p o r t s . The d i s t r i b u t i o n o f t h e s e compounds t h r o u g h o u t the p l a n t kingdom i s d e a l t w i t h by E t t l i n g e r and K j a e r (1968) and K j a e r ( 1 9 7 3 ) . The c u r r e n t p r e f e r r e d method f o r g l u c c s i n o l a t e d e t e c t i o n and d e t e r m i n a t i o n i s GLC—Mass S p e c t r o s c o p y o f i s o t h i o c y a n a t e s r e l e a s e d by e n z y m a t i c h y d r o l y s i s . C o l e s ' s (1976) work on t h e B r a s s i c a c e a e p e r h a p s b e s t i l l u s t r a t e s t h i s method i n the s c r e e n i n g o f l a r g e numbers o f p l a n t s when s t a n d a r d s a r e a v a i l a b l e . K j a e r e_t a_l • (1978) combined the c l a s s i c method of p a p e r c h r o m a t o g r a p h y ( P C ) 27 of t h i o u r e a d e r i v a t i v e s o f i s o t h i o c y a n a t e s w i t h GLC—MS i n t h e o n l y c o m p r e h e n s i v e s t u d y on t h e f a m i l y T r o p a e o I a c e a e • P a p e r c h r o m a t r a p h y was u s e d t o d e t e r m i n e t h e i s o t h i o c y a n a t e s o f T r o p a e o l y m tube rosum as d e s c r i b e d below* 28 M a t e r i a l s and Methods E x p e r i m e n t s which f o l l o w were c a r r i e d o u t to d e t e r m i n e why I r o n a e o l u n tuberosum and L e p i d i u m meyeni i a r e u s e d f o r f o o d and m e d i c i n e i n r e l a t i o n t c t h e s p e c i f i c p o i n t s r a i s e d i n the above d i s c u s s i o n * O u t l i n e of R e s e a r c h 1. E t h n o b o t a n i c a l l i t e r a t u r e on g l u c o s i n o l a t e c o n t a i n i n g p l a n t s was s u r v e y e d and s t u d i e d s t a t i s t i c a l l y f o r p a t t e r n s w h i c h s u p p o r t t h e s p e c i f i c u s e s o f T. t u b e r o s u m and L • mey. e j 3 i i * 2* G l u c o s i n o l a t e s o f T* tube r c sum subsp* t u b e r o s u m and L. meyeni i were d e t e r m i n e d and r e l a t e d to the s i m i l a r m e d i c i n a l u s e s o f t h e s e p l a n t s * 3* C r u d e e x t r a c t s , g l u c o s i n o l a t e s and t h e i r breakdown m e t a b o l i t e s were a s s a y e d f o r e s t r o g e n i c and a n t i — a n d r o g e n i c a c t i v i t y t o d e t e r m i n e t h e b a s i s f o r the p r e v a l a n t b e l i e f s c o n c e r n i n g s u c h p r o p e r t i e s f o r t h e s e p l a n t s * 4* I s o t h i o c y a n a t e s and c r u d e e x t r a c t s were a s s a y e d b i o l o g i c a l l y i n r e l a t i o n t o the a n t i b i o t i c , a n t i v i r a l and n e r a a t o c i d a I u s e s o f t h e p l a n t s * 5* The g l u c o s i n o l a t e s of T* tuberosum subsp* s i l v e s t r e and T• tub er o sum s u b s p * tuberosum were compared and a r e d i s c u s s e d i n r e l a t i o n t o the d e r i v a t i o n o f t h e c u l t i v a t e d 2 8 p l a n t . The chromosome number o f T. tuberosum subsp. s i j V v e s t r e was d e t e r m i n e d . 6. F r e e amino a c i d s were a n a l y z e d to s c r e e n f o r any non-p r o t e i n b i o l o g i c a l l y a c t i v e members of t h i s c l a s s o f compound. S t a t i s t i c aI A n a l y s j s and Data C o l l e c t i o n I n f o r m a t i o n o b t a i n e d from the l i t e r a t u r e s o u r c e s l i s t e d i n Supplementary E i b l i o g r a p h i e s I and I I was c o m p i l e d onto c a r d s f i l e d by genus, and s u b s e q u e n t l y coded and s t o r e d i n a 2—D raw d a t a m a t r i x . Data were r e c o r d e d by c a s e , each case r e f e r r i n g t o a s p e c i f i c mention of a use o f a p a r t i c u l a r s p e c i e s , e i t h e r by an a u t h o r or i n a s p e c i f i c g e o g r a p h i c a l or c u l t u r a l r e g i o n . Care was taken t o e l i m i n a t e r e p e t i t i o n of c a s e s where i n f o r m a t i o n had been c o m p i l e d from or i n t e r c h a n g e d between l i t e r a t u r e s o u r c e s . Data were s t o r e d under v a r i o u s c a t e g o r y headings ( v a r i a b l e s ) and coded to r e c o r d the p o s s i b l e c o n d i t i o n s ( v a l u e s ) i n the s p e c i f i c c a s e . M e d i c i n a l uses which compose the b u l k of the d a t a were c a t e g o r i z e d b r o a d l y by f u n c t i o n a l system, g e n e r a l l y as o u t l i n e d by Lewis and Elvin—Le«is( 1977 ) • New c a t e g o r i e s were c r e a t e d t o c o v e r c a s e s t h a t d i d not f i t those o r i g i n a l l y d e f i n e d and the v a l u e s r e c o r d e d i n c r e a s e d and e v o l v e d to meet the i n c r e a s i n g scope o f the s t u d y . The way d a t a was coded does r e f l e c t the i n t e r p r e t a t i o n of f o l k m e d i c i n a l taxonomies by the a u t h o r . A l t h o u g h t h i s i n t e r p r e t a t i o n i s a c o n s t a n t f a c t o r i n the 30 i n v e s t i g a t i o n , i t s e f f e c t on s u b s e q u e n t r e s u l t s i s d i f f i c u l t t o a s c e r t a i n , b u t i t i s b e l i e v e d t o be m i n i m a l . The raw d a t a m a t r i x was s t r u c t u r e d t o meet t h e r e q u i r e m e n t s o f t h e S t a t i s t i c a l Package f o r t h e S o c i a l S c i e n c e s C SPSS) ( N i e e_t a l . , 1 9 7 5 ) . A l l a n a l y s e s were c a r r i e d out on t h e Amdahl 470V/6 Model I I computer r u n under t h e M i c h i g a n T e r m i n a l System (MTS ) a t t h e UBC Computing C e n t r e . D a t a r e t r i e v a l was c a r r i e d o u t on the SPSS s y s t e m s f i l e s as r e q u i r e d . The t r a n s f o r m a t i o n powers o f t h e p a c k a g e were us e d t c r e d e f i n e t h e d a t a m a t r i x , and the X F r e q u e n c y ( E i t a , 1978 ) and C r o s s t a b s programs were u s e d t o g e n e r a t e f r e q u e n c y d a t a . F o r t h e b u l k o f t h e a n a l y s i s on m e d i c i n a l and n u t r i t i o n a l u s e s , a l l i n f o r m a t i o n r e c o r d e d as v a l u e s and v a r i a b l e s was t r a n s f o r m e d i n t o 41 new y e s o r no c a t e g o r i e s r e f l e c t i n g t h e f a l l o w i n g c o n c e p t s : 31 1 I n s e c t i c i d e 2 D i s i n f e c t a n t 3 \ e r r r i f u g e 4 E m e t i c 5 D i g e s t i v e 6 P u r g a t i v e 7 Stomach ache 8 A n t i d y s e n t r y 9 Rheumatism 10 F e b r i f u g e 11 Headache 12 An e s t h e t i c 13 N e r v o u s l o t f c e r 14 Eye 15 E a r 16 Me t a b o l i e 17 C r a l p r o b l e m 18 T o o t h a c h e 19 P o i s o n : d e r m a t i t i s 20 E e p r o d u c t i v e : h o r m o n a l 21 R e p r o c u c t i v e : c t h e r 22 R e s p i r a t o r y : i n f e c t i o n 23 R e s p i r a t o r y : a n t i c o u g h 24 R e s p i r a t o r y : o t h e r 25 S k i n : d e r m a t i t i s 26 S k i n l u l c e r 27 S n a k e b i t e 28 Wound 29 I n f l a m a t i o n 30 S k l n : o t h e r 31 T o n i c 32 U r i n a r y s y s t e m 33 L i v e r 34 C i r c u l a t o r y s y s t e m 35 D i s e a s e( m i s c • ) 36 Condiment 37 Dye 38 E d i b l e and B e v e r a g e 39 I n t e r n a l b l e e d i n g 40 P o i s o n 41 M i s c e l l a n e o u s F r e q u e n c i e s of t h e s e v a r i a b l e s were o b t a i n e d and compared s t a t i s t i c a l l y t o a s i m i l a r a r r a y o f e t h n o b o t a n i c a l u s e s c o m p i l e d by t a b u l a t i n g t h e f r e q u e n c i e s o f use o f a l l the p l a n t s c o n t a i n e d i n A l t s c h u K 1978) • The d a t a I n t h i s volume i s t a k e n as r e p r e s e n t a t i v e c f a n ormal d i s t r i b u t i o n o f m e d i c i n a l and 32 n u t r i t i o n a l u s e s o f p l a n t s * P o s i t i v e d i f f e r e n c e s f r o m t h i s d i s t r i b u t i o n were c o n s i d e r e d t o i n d i c a t e a s i g n i f i c a n t s e l e c t i v i t y f o r p a r t i c u l a r u s e s * D a t a from b o t h s o u r c e s were t a b u l a t e o t as a t h r e e — w a y c o n t i n g e n c y t a b l e by f i t t i n g a h i e r a r c h i a l l o g — l i n e a r model t o t h e c e l l f r e q u e n c i e s u s i n g t h e program P3F i n t h e B i o m e d i c a l Computer P r o g r a m s P - s e r i e s ( E M D P ) ( D i x o n and Brown. 1979) a n d DBC A c t , b o t h a v a i l a b l e a t t h e CBC Computing C e n t r e . The model AC,AB,BC was t e s t e d and a l i k e l i h o o d r a t i o c h i - s q u a r e c a l c u l a t e d to t e s t f o r i n d e p e n d e n c e . To d e t e r m i n e the s p e c i f i c m e d i c i n a l use r e s p o n s i b l e f o r any s t a t i s t i c a l s i g n i f i c a n c e , u s e v a r i a b l e s were c o l l a p s e d and t h e m o d e l l i n g p r o c e d u r e r e p e a t e d . V a r i a b l e s were c o l l a p s e d i n t o t h e f o l l o w i n g s u r e r - c a t e g o r i e s ; A) A n t i o r g a n i s m a 1 E) I n t e r n a l .organs C) N e r v o u s s y s t e m D) B e p r o d u c t i v e s y s t e m E) R e s p i r a t o r y s y s t e m F ) S k i n P o s i t i v e l y d i f f e r i n g c a t e g o r i e s o f t h e o r i g i n a l 41 were t e s t e d i n t u r n i n a t a b l e c o n t a i n i n g t h e s i x s u p e r — c a t e g o r i e s . Cnce s i g n i f i c a n t c a t e g o r i e s were i d e n t i f i e d the f r e q u e n c y o f t h e o r i g i n a l u s e s from the raw d a t a m a t r i x was d e t e r m i n e d u s i n g t h e C r o s s t a b s p r o g r a m . Table I. . A n a l y s i s o f C o n t i n g e n c y T a b l e s Uses (&) I T T T-Sa mple (C) Presence (B) L i t . Sample A l t s c h u l Yes No Yes r No l r~ i T T 1 3 9 401 41 - f -1 T~ - J X J 34 P l a n t M a t e r i a l T u b e r s and s e e d s o f T r o r a e o l u m tuberosum were o b t a i n e d f r o m P e r u . The s o u r c e and g e o g r a p h i c a l o r i g i n o f the c o l l e c t i o n s u s e d i n t h i s i n v e s t i g a t i o n a r e l i s t e d i n T a b l e I I . P l a n t s o f a l l c o l l e c t i o n s were p r o p a g a t e d on t h e campus o f t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a d u r i n g May t h r o u g h November i n 1978 and 1979. V o u c h e r s o f o r i g i n a l c o l l e c t i o n s and p r o p a g a t e d s p e c i m e n s o f b o t h T. t u b e r q sum s u b s p . tube rqsv^m and s u b s p . s i l v e s t p e a r e d e p o s i t e d i n the UBC H e r b a r i u m . V e g e t a t i v e , f l o r a l and s e e d m a t e r i a l was c o l l e c t e d a t v a r i o u s t i m e s t h r o u g h o u t t h e g r o w i n g s e a s o n , and was u s e d f r e s h , o r f r o z e n a t — 7 0 ° C. T u b e r s were h a r v e s t e d i m m e d i a t e l y a f t e r f r o s t — k i l l on O c t o b e r 25, 1978 and November 6, 1979, and were e i t h e r s t o r e d a t 4° C o r f r e e z e d r i e d and s t o r e d at - 7 0 ° C. R o o t s o f L e p i d iurn, mev e n i i were c o l l e c t e d i n W a y r i , D e p a r t m e n t of J u n i n , P e r u on J u l y 15, 1973 by M i c h a e l F. Brown. They were s u b s e q u e n t l y p r e s e r v e d i n p — d i c h l o r o b e n z e n e and d e p o s i t e d ( C a t a l o g u e No. 26o23) a t room t e m p e r a t u r e i n t h e Museum of A n t h r o p o l o g y , U n i v e r s i t y o f M i c h i g a n . They were o b t a i n e d on r e q u e s t f r c m t h a t i n s t i t u t i o n on December 8, 1979. S i n a p i s a i b a , a s o u r c e o f s i n a l b i h and the enzyme m y r o s i n a s e , was d o n a t e d by the C a n a d i a n G r a i n C o m m i s s i o n , V a n c o u v e r , from samples t a k e n f r o m C a n a d i a n e x p o r t s h i p m e n t s . 3 5 T a b l e I I . C o l l e c t i o n s of Tro£aeolum tuberosum No.. Name Source-) — 1 P r o v e n i a n c e C o l l e c t o r Date T. tuberosum ss p . , s i l v e s t r e • k i p a i s a n o ' * I . tuberosum ssp.,tuberosum  1chaucha i s a n o ' s s p . tuberosum (seeds) ssp. tuberosum 'oke i s a n o ' ssp. tuberosum • i s a l a i s a n o ' ssp..tuberosum ' k e l l o i s a n o ' ssp. . tuberosum • y a n i nawi i s a n o ' ** ssp . , tuberosum ssp. _ tuberosum Cuyo-cuyo, Puno,Peru Cuyo-cuyo Cuzco-Peru Cuyo-cuyo Cuyo-cuyo Cuyo-cuyo Cuyo-cuyo Huancayo, Peru Huancayo Johns (505) Johns (466) C o r t e s Bravo, Camino Camino Camino Camino Ca mino Camino D e c 3 77 Nov30 77 Nov25 77 May 78 May 78 May 78 Apr 14 78 J u n l l 78 J until 78 * A l l c o l l e c t i o n s a r e t u b e r s u n l e s s o t h e r w i s e s t a t e d ; ** ' y a n i nawi i s a n o ' i s e q u i v a l e n t t o ' i s a l a i s a n o ' 36 C h e m i c a l s G l u c o s i n o l a t e s , i s o t h i o c y a n a t e s and t h e i r t h i o u r e a d e r i v a t i v e s were o b t a i n e d from v a r i o u s s o u r c e s and u s e d a s s t a n d a r d s o r i n b i o l o g i c a l t e s t i n g . G l u c o s i n o l a t e s : B e n z y l g l u c o s i n c l a t e and s i n a l b i n as t e t r a m e t h y l ammonium s a l t s , and s i n i g r i n were o b t a i n e d from D r s . E.W. U n d e r h i l l and L.R. W e t t e r of the N a t i o n a l R e s e a r c h C o u n c i l C anada, P r a i r i e R e g i o n a l L a b o r a t o r y . I s o t h i o c y a n a t e s : S p e c i m e n s o f b e n z y I i s o t h i o c y a n a t e were o b t a i n e d from D r . E.W. U n d e r h i l l , and from F l u k a AG. A l l y l i s o t h i o c y a n a t e , p h e n e t h y l i s o t h i o c y a n a t e and p h e n y I i s o t h i o c y a n a t e were p u r c h a s e d f r o m Eastman Kodak Co.. D r . A n d e r s K j a e r p r o v i d e d s a m p l e s o f 2— b u t y l and 2 — p r o p y l i s o t h i o c y a n a t e s upon r e q u e s t b u t t h e s e compounds u n f o r t u n a t e l y decomposed to e q u i c k l y t o be u s e f u l , p— M e t h o x y b e n z y I i s o t h i o c y a n a t e was o b t a i n e d f r o m T. t u b e r o s u m s u b s p . t u b e r o s u n u s i n g the HPLC methods d e s c r i b e d b e l o w . 37 T h i o u r e a s : The t h i o u r e a d e r i v a t i v e o f 2 - b u t y l i s o t h i o c y a n a t e was o b t a i n e d f r o m C r . E . W • U n d e r b i l l and p h e n y l t h i o u r e a was p u r c h a s e d from Eastman Kodak. T h i o u r e a d e r i v a t i v e s were p r e p a r e d f r o m b e n z y l and p — r a e t h o x y b e n z y l i s o t h i o c y a n a t e s by t h e methods d e s c r i b e d b e l o w . M a t e r i a l s used i n a s s a y s f o r e s t r o g e n i c and a n t i — a n d r o g e n i c ac t i v i t i es : 17)9—3 B - e s t r ad i o I was p u r c h a s e d from t h e Amersham C o r p o r a t i o n . G e n i s t e i n was p u r c h a s e d from Eastman Kodak Co.. Androgen l e v e l s i n r a t b l o o d were t e s t e d u s i n g a T e s t o s t e r o n e / D i h y d r o t e s t o s t e r o n e RIA k i t p r e p a r e d by Amersham C o r p o r a t i o n . P r e p a r a t i o n o f EJLaU-i e x t r a c t s -G l u c o s i n o l a t e s and I s o t h i o c y a n a t e s : Two p r o c e d u r e s were f o l l o w e d t o o b t a i n and t o i d e n t i f y t h e i s o t h i o c y a n a t e breakdown p r o d u c t s o f the g l u c o s i n o l a t e s . The f i r s t u s e s endogenous enzyme, t h e s e c o n d a m y r o s i n a s e p r e p a r a t i o n o b t a i n e d from t h e s e e d s o f Sjjxaj3J_g a l b a . 38 Use o f endogenous enzyme ( C o l e , 1976 )J The p l a n t m a t e r i a l , e i t h e r f r e s h o r d r i e d , was mashed o r g r o u n d t o a f i n e c o n s i s t e n c y . I f n e c e s s a r y , w a t e r was added t o g i v e a p a s t e — l i k e c o n s i s t e n c y . The m i x t u r e was i n c u b a t e d f o r 2 h o u r s a t 37° C, a f t e r w h i c h i t was s h a k e n and mixed w i t h s e v e r a l p o r t i o n s o f M e C l 2 t o e x t r a c t i s o t h i o c y a n a t e s . Use of m y r o s i n a s e p r e p a r a t i o n ! The p l a n t m a t e r i a l was g r o u n d i n a Waring b l e n d e r w i t h 10% h o t m e t h a n o l . S o l v e n t was added t o c o v e r and t h e p r o c e d u r e r e p e a t e d u n t i l pigment i n t h e e x t r a c t was n e g l i g i b l e . The b l e n d e d m i x t u r e was f i l t e r e d , and t h e e x t r a c t f i l t e r e d h o t t h r o u g h C e l i t e and c o n c e n t r a t e d Ljl v a c c u o to a volume o f l e s s t h a n 5ml. To t h e f l a s k were added 50ml o f c i t r a t e — p h o s p h a t e b u f f e r (Dawscn _e._t a_l. , 1972 ) pH 6.6, 0.1ml o f a s c o r b i c a c i d s o l u t i o n (Rodman, 1£7£) and 2ml o f the m y r o s i n a s e p r e p a r a t i o n , p r e p a r e d a s o u t l i n e d by Rodman( 1878 ). I n c u b a t i o n t o o k p l a c e a t room t e m p e r a t u r e o v e r n i g h t . R e l e a s e d i s o t h i o c y a n a t e s were e x t r a c t e d i n t o e i t h e r d i e t h y l e t h e r or l d e C l 2 » 39 F r e e amino a c i d s : One t o two gram p o r t i o n s o f t u b e r s o f X« t u b e r o s u m were e x t r a c t e d t w i c e w i t h 15ml o f 85% h o t e t h a n o l . f i l t e r e d , e v a p o r a t e d to d r y n e s s u n d e r vacuum and t a k e n up i n 1ml o f l i t h i u m c i t r a t e b u f f e r pH 2.2. B u f f e r was p r e p a r e d as s p e c i f i e d f o r use on the Eeckman Model 120C Amino A c i d A n a l y z e r . Samples were f r o z e n and s t o r e d u n t i l a n a l y z e d . B e t e c t i c i n and D e t e r m i n a t i o n o f C q p s t j t u e n t s G l u c o s i n o l a 1es : Matsuot197C ) has p r o v i d e d s e v e r a l s o l v e n t s y s t e m s f o r s e p a r a t i o n of g l u c o s i n o l a t e s on s i l i c a g e l t h i n l a y e r c h r o m a t o g r a p h y ( T L C ) . The f o l l o w i n g s y s t e m s were u s e d w i t h s u c c e s s : a c e t c n e — c h l o r o f o r m — e t h a n o l — w a t e r (6:3:3.4:3); b u t a n o l — b e n z e n e — e t h a n o l — a m m o n i a ( 4 : 1 : 2 : 3 ) ; b u t a n o l — p r o p a n o l — a c e t i c a c i d -w a t e r (3:1:1:2). P a p e r c h r o m a t o g r a p h y ( P C ) o f g l u c o s i n o l a t e s was c a r r i e d o u t on Whatman N o . l p a p e r u s i n g a s o l v e n t s y s t e m o f b u t a n o l — e t h a n o l — w a t e r ( 4 : 1 : 4 )( Gmel i n and K j a e r , l S 7 0 ) . S p o t s were d e t e c t e d on F o l y g r a m S i l G / u v 2 5 4 TLC p l a t e s u s i n g a s h o r t wave u l t r a v i o l e t lamp, and by the e x p o s u r e of chromatograms t o i o d i n e v a p o r . TLC p l a t e s and p a p e r chromatograms were a l s o s p r a y e d w i t h s i l v e r n i t r a t e — N a O H r e a g e n t ( G m e l i n and K j a e r , 1970). P h e n o l s were d e t e c t e d u s i n g a 0.1% p — n i t r o b e n z e n e d i a z o n i u m 40 t e t r a f l u o r o h o r a t e s p r a y r e a g e n t o v e r s p r a y e d w i t h 5% NaOH ( P h i l l i p s , p e r s . comm. ). Chromatograms can be s p r a y e d f o r p h e n o l s , l e f t t o d r y and s u b s e q u e n t l y s p r a y e d w i t h the AgN03~NaCH r e a g e n t * I s o t h i o c y a n a t e s: The d e t e r m i n a t i o n c f i s o t h i o c y a n a t e s from t h e e n z y m a t i c h y d r o l y s i s of g l u c o s i n o l a t e s p r o v i d e s a s u p e r i o r r o u t e t o t h e i d e n t i t y o f g l u c o s i n o l a t e s « F o r a rough d e t e r m i n a t i o n o f c o n s t i t u e n t s , e t h e r e x t r a c t s were d i v i d e d i n t o 3 p o r t i o n s and t h e method o f Eodman( 1978 ) was u s e d to t e s t f o r SCN —, c y c l i c o x o z o l i d i n e t f c e n e s and i s o t h i o c y a n a t e s * T h l o c y a n a t e ( SCN - ) i s p r o d u c e d i n a l k a l i n e medium f r o m p - h y d r o x y b e n z y l g l u c o s i n o l a t e ( s i n a l b i n ) and i n d o l i c g l u c o s i n o l a t e s and was a s s a y e d w i t h a f e r r i c n i t r a t e r e a g e n t . The c y c l i c d e r i v a t i v e s o f — h y d r o x y and y—hydroxy s u b s t i t u t e d g l u c o s i n o l a t e s can be d e t e c t e d by s p o t t i n g on p a p e r and s p r a y i n g w i t h G r o t e ' s r e a g e n t ( G r o t e , 1931) and p r o m p t l y s t e a m i n g . A b l u e c o l o u r i n d i c a t e s a p o s i t i v e r e s u l t . I s o t h i o c y a n a t e s were d e t e c t e d a s t h i o u r e a d e r i v a t i v e s . The e t h e r e x t r a c t was r e a c t e d w i t h an e x c e s s o f e t h a n o l i c ammonia ( o n e p a r t c o n c e n t r a t e d aqueous ammonium h y d r o x i d e and f i v e p a r t s o f e t h a n o l ) . A f t e r s e v e r a l h o u r s t h e sample was e v a p o r a t e d and s p o t t e d on p a p e r . G r o t e ' s r e a g e n t was u s e d as above to d e t e r m i n e p r e s e n c e . I f t h i o u r e a s were p r e s e n t t h e y were s e p a r a t e d by p a p e r c h r o m a t o g r a p h y o r on A v i c e l TLC p l a t e s . The c l a s s i c method u s i n g paper( K j a e r and R u b i n s t e i n , 1853) i s s u p e r i o r t o TLC i n p r o v i d i n g b e t t e r r e s o l u t i o n . S t a n d a r d t h i o u r e a s where a v a i l a b l e 41 were u s e d t o i d e n t i f y s p o t s e i t h e r t h r o u g h c o m p a r i s o n o f Rf v a l u e s o r c o—ch>r cma t og r ap hy • The most s u c c e s s f u l s o l v e n t s y s t e m s (Rodman,197S; K j a e r e_t a l . , 1978) f o r t h e p r e s e n t i n v e s t i g a t i o n were f o u n d t o be: b e n z e n e — e t h a n o l — w a t e r ( 5 : 1 : 2 ) ; c h l o r o f o r m -w a t e r ( 5 : 1 ) ; t o l u e n e — a c e t i c a c i d — w a t e r (3•75:1•5:3 )• Rph's were c a l c u l a t e d as t h e r a t i o o f the d i s t a n c e o f the s p o t / d i s t a n c e o f p h e n y l t h l o u r e a . A more c o n v e n i e n t a p p r o a c h to t h e d e t e r m i n a t i o n o f i s o t h i o c y a n a t e s was worked out u s i n g H i g h P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h y ( BPLC ) and u l t r a v i o l e t d e t e c t i o n a t 245nm. C o n s i d e r a b l e e f f o r t was a p p l i e d to f i n d i n g an HPLC s y s t e m t h a t would s e p a r a t e t h e compounds d e t e c t e d by PC o f t h i o u r e a s * A l t h o u g h i s o t h i o c y a n a t e s show no s t r o n g a b s o r p t i o n maxima t h e y do a b s o r b i n t h e range o f 245 nm, the e x t i n c t i o n c o e f f i c i e n t o f e t h y l i s o t h i o c y a n a t e b e i n g l , 2 0 0 ( S c o t t , 1964)* A r o m a t i c i s o t h i o c y a n a t e s have i n c r e a s e d a b s o r p t i o n i n t h i s r e g i o n * MeClg e x t r a c t s were c o n c e n t r a t e d and p a s s e d t h r o u g h a s m a l l ( 1 0 — 2 0 m l ) p r e — c o l u m n o f S i l i c a g e l . The MeCl2 e l u t a n t was a g a i n c o n c e n t r a t e d t o a few ml. Samples a p p l i e d t o a normal p h a s e column were i n j e c t e d i n MeClg* F o r r e v e r s e p h a s e c h r o m a t o g r a p h y the MeCl2 e l u t a n t was d r i e d , t a k e n up i n a c e t o n i t r i l e and b e f o r e i n j e c t i o n was f i l t e r e d t h r o u g h a f r i t t e d d i s c f u n n e l t o remove p r e c i p i t a t e d l i p i d s * • A V a r i a n Model 500 L i q u i d C h r o m a t o g r a p h w i t h a V a r i a n S e r i e s 634 v a r i a b l e w a v e l e n g t h d e t e c t o r was u s e d and c h r o m a t o g r a p h y was c a r r i e d o u t a t ambient t e m p e r a t u r e * F o r r e v e r s e p h a s e c h r o m a t o g r a p h y a M i c r o p a k m—CH—10 a n a l y t i c a l column w i t h a c e t o n i t r i l e and w a t e r as t h e m o t i l e p h a s e was u s e d * Samples were i n j e c t e d a t 35% C H 3 C N and run -at a f l o w r a t e o f l m l / m i n u t e o v e r a g r a d i e n t o f 10%/minute t o 70% CH 3CN. 42 P r e p a r a t i v e c h r c m a t o g r a p h y was c a r r i e d o u t by a s i m i l a r method u s i n g a m—CH—10 p r e p a r a t i v e column and a H o w r a t e o f 2ml/minute • F o r n o r m a l phase c h r o m a t o g r a p h y a M l c r o p a k NH2—10 column w i t h M e C l g • i s o o c t a n e ( 3 C : 7 0 ) as t h e m o b i l e phase p r o v i d e d t h e b e s t r e s u l t s . S t a n d a r d s were c o — c h r o m a t o g r a p h e d w i t h a l l s a m p l e s where p o s s i b l e . F o r p r e p a r a t i v e work f r a c t i o n s from r e p e a t e d r u n s were p o o l e d , e x t r a c t e d i n t o MeCl2 and c o n c e n t r a t e d under vacuum. I d e n t i t y of sa m p l e s p r e p a r e d i n t h i s way were c o n f i r m e d by the t h i o u r e a — P C raethcd d e s c r i b e d a b o v e , and by mass s p e c t r a l a n a l y s i s a n d / o r NMR. Mass S p e c t r a l A n a l y s i s : Mass s p e c t r o m e t r y was c a r r i e d o u t on an A t l a s MAT (Bremen) CH4—B Mass S p e c t r o m e t e r . N u c l e a r M a g n e t i c Resonance S p e c t r o m e t r y : The 1B—NMR s p e c t r u m o f p — m e t h o x y b e n z y I i s o t h i o c y a n a t e ( 20mg ) was r e c o r d e d on a B r u k e r WP-80 F o u r i e r t r a n s f o r m s p e c t r o m e t e r i n CDCI3 ( 0 . 5 m l ) w i t h TMS ( 1 0 * ) as an i n t e r n a l s t a n d a r d . 43 Amino A c i d A n a l y s i s : F r e e amino a c i d s were d e t e r m i n e d u s i n g a Beckman Model 120C A u t o m a t i c Amino A c i d A n a l y z e r . B a s i c amino a c i d s were a n a l y z e d a c c o r d i n g to m a n u f a c t u r e r s r e c o m m e n d a t i o n s (Beckman, 1966) on a 14 by 1 cm c o l u m n . A c i d i c and n e u t r a l amino a c i d s and amides were s e p a r a t e d u s i n g l i t h i u m s a l t s as d i r e c t e d by the m a n u f a c t u r e r ( B e c k m a n , 1£66 ) on a 56 by 1 cm c o l u m n . Amino a c i d s p r e s e n t i n t u b e r s amples were i d e n t i f i e d by c o m p a r i s o n o f r e t e n t i o n t i m e s t o t h o s e o f the s t a n d a r d c a l i b r a t i o n m i x t u r e o f p r o t e i n amino a c i d s and o f o t h e r known common amino a c i d s . D e t e r m i n a t i o n o f Chromosome Number T u b e r s o f t h e c o l l e c t i o n , ' k i p a i s a n o ' ( T a b l e I I ) , c o n f i r m e d i n i d e n t i f i c a t i o n as T r o c a e o l u m tuberosum s u b s p . s i l v e s t r e on t h e b a s i s o f the g l u c o s i n o l a t e s i t c o n t a i n s ( s e e b e l o w ) , were s p r o u t e d i n v e r m i c u l i t e • Foot t i p s were f i x e d i n C a r n o y ' s s o l u t i o n and s t o r e d a t 4° C. T i p s were s t a i n e d i n a c e t o — c a r m i n e and t h e chromosome numbers were d e t e r m i n e d by D r . G e r a l d B. S t r a l e y , V a n l u s e n B o t a n i c a l G a r d e n , V a n c o u v e r . F i g u r e 9. T u b e r s of Troc a e o l . um tube r c sum s u b s p . s i l v e s t r e 45 T e s t s f o r B i o l o s i c a l A c t i v i t y A n t i b i o t i c a c t i v i t y : T e s t s f o r a n t i b i o t i c and p h o t o t o x l c a c t i v i t y were c a r r i e d o u t u s i n g t h e method o f D a n i e l s ( 1 9 6 5 ) - A n t i b i o t i c t e s t s o f t u b e r m a t e r i a l o f b o t h s u b s p e c i e s o f T.. t u b e rosum , and o f p u r e b e n z y l i s o t h i o c y a n a t e and p — n e t h o x y b e n z y l i s o t h i o c y a n a t e , were c a r r i e d o u t a g a i n s t : C a n d i d a al__bJtcan.s, , E s c h e r i c h i a c o l i , Pseudomonas f l u o r e s c e n s , and g t a p h y l o c o c c u s a l b u s • C a n d i d a a l b i c a n s was grown on p l a t e s c o n t a i n i n g S a b o u r a u d D e x t r o s e A g a r w h i l e , E . c_oJ._i , P. f l u o r e s c e n s and S. a Ibu s, were c u l t u r e d on D i f c o B a c t c — A g a r . • P l a t e s were i n o c u l a t e d w i t h a lawn o f b a c t e r i a o r y e a s t . P a p e r d i s k s (7mm d i a m e t e r ) were i n o c u l a t e d w i t h l O u l o f e t h e r s o l u t i o n s o f p u r e compound i n c o n c e n t r a t i o n s o f 10 and lmg/ml (100 and 10 ug r e s p e c t i v e l y ) , a l l o w e d t o a i r d r y and p l a c e d on the i n o c u l a t e d p l a t e s . P l a n t m a t e r i a l was s u r f a c e s t e r i l i z e d i n e t h a n o l . D i s s e c t e d p i e c e s were p l a c e d on t h e p l a t e s . A l l p l a t e s were i n c u b a t e d a t 37° C. A f t e r 24 h o u r s p l a t e s were o b s e r v e d f o r t h e c l e a r z o n e s around the d i s k o r p l a n t m a t e r i a l which i n d i c a t e s a n t i b i o s i s . P h o t o t o x i c i t y of t u b e r and l e a f m a t e r i a l o f t h e two s u b s p e c i e s o f T.. t u b e r c s u m were t e s t e d a g a i n s t C a n d i d a a l b i c a n s • P l a t e s were p r e p a r e d as a b o v e , i n d u p l i c a t e . One s e t o f p l a t e s were e x p o s e d t o longwave u l t r a v i o l e t l i g h t f o r 5 h o u r s a t 30° C ( f o u r S y l v a n n i a b l a c k - l i g h t b l u e f l u o r e s c e n t lamps, F20T12-BLB. L i g h t i n t e n s i t y 0. 6m\»att/cm 2 ). C o n t r o l s were k e p t wrapped i n a l u m i n i u m f o i l . I n c u b a t i o n and o b s e r v a t i o n s were as above. 46 A n t i v i r a l a c t i v i t y : To t e s t the p o s s i b l e a n t i v i r a l e f f e c t o f Tropa,eoluro t u b e r o s u m s u b s p . tuberosum , an e t h e r e x t r a c t o f t h e t u b e r s , p r e p a r e d a s above, was t e s t e d i n d u p l i c a t e a g a i n s t a sample o f He r p e s t y p e I v i r u s u s i n g t h e method of Van Den B e r g e e t &JL« ( 1 9 7 8 ) . M o n o l a y e r s o f c o n f l u e n t VERC c e l l s m a i n t a i n e d by Mr. B r i a n J u d d , D e p a r t m e n t o f M e d i c a l M i c r o b i o l o g y , U . B . C , were grown i n L i n b r o m u l t i w e l l p l a t e s and i n c u b a t e d w i t h s e r i a l 10-f o l d d i l u t i o n s c f t h e v i r u s s t r a i n M2989, w i t h o r w i t h o u t t h e a d d i t i o n o f the maximum n o n t o x i c d i l u t i o n (MNTD ) o f t h e p l a n t e x t r a c t . S t r a i n M2S89 was i s o l a t e d by D r . D.M. McLean, Department o f M e d i c a l M i c r o b i o l o g y , U . B . C , and grown i n VERO c e l l s m a i n t a i n e d on D u l h e c c o ' s M o d i f i e d E a g l e Medium (MEM) c o n t a i n i n g ; 3 . 7 g / l sodium b i c a r b o n a t e w i t h 10% c f G i b c o F e t a l B o v i n C a l f Serum ( F C S ) o b t a i n e d from the A m e r i c a n Type C u l t u r e . The MKTD, a measure o f t h e c y t o t o x i c i t y o f the p l a n t e x t r a c t , was d e t e r m i n e d p r i o r t o t h e v i r u s i n h i b i t o r y a s s a y . S e r i a l 1 0 - f c l d d i l u t i o n s o f the e x t r a c t i n MEM and 10% FCS were added t o VEEC m o n o l a y e r s which were examined u n d e r 100X m a g n i f i c a t i o n a f t e r 2 d a y s o f i n c u b a t i o n a t 37° C. C y t o p a t h i c e f f e c t ( C P E ) was i n d i c a t e d by a r o u n d i n g o f c e l l s and a s u b s e q u e n t d e s t r u c t i o n c f the m o n o l a y e r . V i r u s c o n t r o l , t i s s u e c u l t u r e c o n t r o l , and s o l v e n t c o n t r o l s were i n c l u d e d i n t h e t e s t . To t e s t the p o s s i b l e p r o t e c t i v e e f f e c t o f the e x t r a c t upon t h e c e l l s , an a l i q u o t ( 0 . 5 m l ) o f the c o n c e n t r a t e d H e r p e s sample was i n c u b a t e d f o r 1 hour a t 37° C w i t h 0.5ml o f t h e MNTD and then s e r i a l l y d i l u t e d and added t o VERO c e l l s as a b o v e . V i r u s t i t r e s i n the p r e s e n c e and a b s e n c e o f t h e p l a n t 4 7 e x t r a c t were d e t e r m i n e d a f t e r 2 days o f i n c u b u a t i o n a t 37° C. CPE was d e t e r m i n e d as a b o v e . A n t i v i r a l a c t i v i t y was e x p r e s s e d as a r e d u c t i o n f a c t o r o f the v i r a l t i t r e . N e m a t o c i d a l a c t i v i t y : Nematodes ( C a e n o r h a b d i t i s e l e a a n s ) were o b t a i n e d f r o m D r . D.L. B a i l l i e , D e p artment o f B i o l o g i c a l S c i e n c e s , Simon F r a s e r U n i v e r s i t y , i n 35 by 10mm p e t r i p l a t e s s u p p o r t i n g E s c h e r i c h i a c o l i on t h e N e m a t o c i d a l G r o w i n g Medium (NGM ) u s e d i n t h a t l a b o r a t o r y . l u l amounts o f e t h a n o l i c s o l u t i o n s o f b e n z y l i s o t h i o c y a n a t e i n c o n c e n t r a t i o n s f r o m 0—2% were d r o p p e d o n t o t h e c e n t r e o f the p l a t e s . The p l a t e s were examined a f t e r one h a l f an h o u r to d e t e r m i n e t h e L L i o o o f the c c n p c u n d . E s t r o g e n i c a c t i v i t y : T e s t s f o r e s t r o g e n i c a c i t i v i t y were c a r r i e d o ut u s i n g t h e methods o f , and u n d e r the d i r e c t i o n o f , F r a n c e s Newsome, Department of A n i m a l S c i e n c e , UBC. B o t h _i_Q vj.vo and i r j v i t r q e x p e r i m e n t s were done t o t e s t t h e e s t r o g e n i c e f f e c t s of e x t r a c t s o f X« tuberosum and p u r e compounds. 4 8 E f f e c t s of Jrccaeolum tuberosum on the e s t r u s c y c l e o f t h e g u i n e a p i g : A s i m p l e t e s t u s i n g f e m a l e g u i n e a p i g s was u n d e r t a k e n t o t e s t t h e f e r t i l i t y r e l a t e d emmenagogic b e l i e f s s u r r o u n d i n g T. t u b e r o s u m . G u i n e a p i g s e x h i b i t a r e g u l a r 14—18(16.5 on t h e a v e r a g e ) day e s t r u s c y c l e ( C o o p e r and S c h i l l e r , 1 9 7 5 ) . The o n s e t o f e s t r u s c a n be r e a d i l y d e t e c t e d by the d i s i n t i g r a t i o n o f t h e v a g i n a l c l o s u r e membrane, a u n i q u e s t r u c t u r e i n t h e g u i n e a p i g , and t h e o p e n i n g o f the v a g i n a l o r i f i c e . These c h a r a c t e r i s t i c s make t h e g u i n e a p i g an i d e a l a n i m a l f o r t h i s t y p e o f s t u d y . E a r l y o n s e t o f e s t r u s would be i n d i c a t i v e o f an emmenagogic e f f e c t . T w elve r e c e n t l y weaned v i r g i n a l b i n o g u i n e a p i g s were o b t a i n e d f r o m t h e A n i m a l C a r e C e n t r e a t UBC and were m a i n t a i n e d u n d e r c o n t r o l l e d l i g h t and t e m p e r a t u r e c o n d i t i o n s i n i n d i v i d u a l 10.5" by 1 9 " by 6.2" c a g e s o v e r t h e c o u r s e o f t h e e x p e r i m e n t . A d i e t o f P u r i n a G u i n e a F i g Chow s u p p l e m e n t e d w i t h l e t t u c e , a p p l e and sweet p o t a t o was p r o v i d e d t h r o u g h 3 n o r m a l e s t r u s c y c l e s t o t e s t t h e r e g u l a r p e r i o d i c i t y o f the a n i m a l s • S i x c o n t r o l a n i m a l s were m a i n t a i n e d on t h e b a s a l d i e t . S i x e x p e r i m e n t a l a n i m a l s were f e d a d i e t o f g r o u n d p e l l e t s mixed w i t h a p p r o x i m a t e l y 20% by d r y w e i g h t o f f r e e z e d r i e d t u b e r s o f T. t u b e r o s u m s u b s p . t^bj=xo_suijj • C o n t r o l a n i m a l s d u r i n g t h i s s t a g e o f the e x p e r i m e n t consumed on t h e a v e r a g e 70g o f d r i e d p e l l e t s p e r d a y . E x p e r i m e n t a l a n i m a l s were g i v e n c o m p a r a b l e amounts. Body w e i g h t s were d e t e r m i n e d b e f o r e and d u r i n g t h e e x p e r i m e n t . The a n i m a l s were o b s e r v e d and t h e i r e s t r u s c y c l e s r e c o r d e d f o r 40 days a f e r the e x p e r i m e n t a l f e e d i n g began. 49 R a d i o a c t i v e c o m p e t i t i v e b i n d i n g a s s a y : E x t r a c t s o f t u b e r s o f T r o p a e o l u m tuberosu m . s e e d s o f S i n a p i s a l b a , s t a n d a r d g l u c o s i n o l a t e s and i s o t h i o c y a n a t e s , and f r a c t i o n s o b t a i n e d i i o i i BPLC were a s s a y e d f o r e s t r o g e n i c a c t i v i t y . Samples and 1 1 0 — 3 H — e s t r a d i o l were i n c u b a t e d w i t h a p r e p a r a t i o n o f e s t r o g e n r e c e p t o r f r o m c a l f u t e r u s . C o m p e t i t i v e i n h i b i t i o n was measured as a f a c t o r of t h e d r o p i n t h e l a b e l bound t o the r e c e p t o r . R e s u l t s were compared t o t h o s e o b t a i n e d u s i n g known c o n c e n t r a t i o n s o f t h e i s o f l a v o n e , g e n l s t e i n ( F o p e and W r i g h t , 1953'J, a well—known p h y t o - e s t r o g e n . P l a n t e x t r a c t s made u s i n g p e r o x i d e — f r e e and r e c e n t l y d i s t i l l e d d i e t h y l e t h e r were d i v i d e d and p l a c e d i n 5ml t e s t t u b e s . The a l i q u o t s were d r i e d and s t o r e d i n the f r e e z e r i n 1.0ml o f r e c e n t l y d i s t i l l e d e t h a n o l . S t a n d a r d compounds and HPLC f r a c t i o n s were d i s s o l v e d i n 1.0ml e t h a n o l , and s e r i a l l y d i v i d e d and made up to 1.0ml. P r i o r to the a s s a y a l l t u b e s were d r i e d u n d e r a s t r e a m o f n i t r o g e n . G e n i s t e i n s t a n d a r d s o f 10t 50 and 200 u l o f a 200ng/ml s o l u t i o n were p l a c e d i n t u b e s and d r i e d . The r e c e p t o r c o n t a i n i n g c y t i s o l f r a c t i o n was p r e p a r e d from c a l f u t e r u s and t e s t e d f o r b i n d i n g c a p a c i t y by F r a n c e s Newsome. lO O u l c f t r i s b u f f e r (pH 8) was added t o a l l t u b e s , v o r t e x e d and a l l o w e d t o s t a n d f o r 30 m i n u t e s a t 25° C. 5 0 u l ( 1 . 2 5 x 10-2 u C i ) c f 3 H - e s t r a d i o l and 50-100 u l o f t h e c y t i s o l f r a c t i o n * d e p e n d i n g on the t e s t f o r b i n d i n g c a p a c i t y ) were added and a f t e r 30 m i n u t e s a t room t e m p e r a t u r e 1ml o f d e x t r o s e c o a t e d c h a r c o a l ( 0 . 5 g N o r i t a c t i v a t e d c h a r c o a l and 5 mg d e x t r o s e T40 i n 200 u l o f t r i s b u f f e r (pH 8 ) ) was added to p r e c i p i t a t e unbound 3 H — e s t r a d i o I • Tubes were c e n t r i f u g e d a t 3000g f o r 10 m i n u t e s and 50 t h e s u p e r n a t a n t c o u n t e d f o r r a d i o a c t i v i t y i n 10ml o f Amersham's PSC • A c t i v i t y c f t e s t samples was compared t o a p l o t o f t h e g e n i s t e i n s t a n d a r d * D i x o n p l o t : To d e t e r m i n e w h e t h e r i n h i b i t i o n o f e s t r o g e n b i n d i n g was c o m p e t i t i v e or n o n — c o r a p e t i t i v e • t h e p r o c e d u r e o f D i x o n ( 1 9 5 3 ) f o r enzyme i n h i b i t i o n was a d a p t e d * C o n c e n t r a t i o n o f b e n z y l i s o t h i o c y a n a t e i n h i b i t o r was v a r i e d o v e r two d i f f e r e n t s u b s t r a t e ( S ) ( 3 B - e s t r a d i o l ) c o n c e n t r a t i o n s * I n h i b i t o r c o n c e n t r a t i o n s were u s e d i n the range o f 0*C7 t o 2*94 X 1 0 — 5 m o l e s / r e a c t i o n t u b e * S u b s t r a t e c o n c e n t r a t i o n s o f 25 and 5 0 u l / r e a c t i o n tube were u s e d * The r e a c t i o n r a t e ( v ) was measured as DFM o f e s t r a d i o l bound* To d e t e r m i n e t h e n a t u r e o f i n h i b i t i o n 1/v was p l o t t e d v e r s u s £ l ] f o r b o t h s u b s t r a t e c o n c e n t r a t i o n s * The i n t e r c e p t p o s i t i o n i n r e l a t i o n t o the x a x i s d e t e r m i n e s t h e n a t u r e o f t h e i n h i b i t i o n * A n t i — a n d r o g e n i c a c t i v i t y : To t e s t the a n t i — a p h r o d i s i a c b e l i e f s a s s o c i a t e d w i t h T r o p a e o l u m tub erqsum • t u b e r s o f subsp* tuberosum were f e d t o male r a t s * W i s t a r w h i t e r a t s were o b t a i n e d f r o m the Z o o l o g y R e s e a r c h S n a i l A n i m a l U n i t , UBC* Male r a t s w e i g h i n g 280 t o 330 grams a t 12 weeks o f age were hous e d under c o n t r o l l e d l i g h t and 51 t e m p e r a t u r e c o n d i t i o n s i n p a i r s i n 10-5" by 19" by 6.2" c a g e s . C o n t r o l a n i m a l s were f e d a d i e t of g r o u n d P u r i n a Rodent L a b o r a t o r y Chow e v e r the c o u r s e o f the e x p e r i m e n t . E x p e r i m e n t a l a n i m a l s were f e d a m i x t u r e o f g r o u n d chow and p u l v e r i z e d f r e e z e d r i e d t u b e r s . I n i t i a l l y the a n i m a l s were o f f e r e d a m i x t u r e o f 10% by w e i g h t o f t h e t u b e r s . A n i m a l s were weighed r e g u l a r l y and the c o m p o s i t i o n o f the d i e t was v a r i e d up to a maximum o f 25% t u b e r m a t e r i a l so as t o m a i n t a i n a c o n s t a n t body w e i g h t . A f t e r two weeks f i v e e x p e r i m e n t a l a n i m a l s and f i v e c o n t r o l s were s e p a r a t e d and e a c h c a g e d w i t h a v i r g i n f e m a l e r a t f o r a p e r i o d of one week ( e s t r u s c y c l e i s 5 d a y s ) . Female r a t s w e i g h e d 225—275 grams a t 12 weeks o f age. C o n t r o l and e x p e r i m e n t a l males were m a i n t a i n e d on t h e i r r e s p e c t i v e d i e t s . F e m a l e s were removed f r o m t h e c a g e f o r € h o u r s e a c h day and a l l o w e d to f e e d on n o r m a l p e l l e t s . A f t e r t h e ' b r e e d i n g ' p e r i o d f e m a l e s were m a i n t a i n e d f o r 22 d a y s . t h e i r n o r m a l maximum g e s t a t i o n p e r i o d , t o d e t e r m i n e i f t h e y were p r e g n a n t . A t h i r d g r o u p o f male r a t s was m a i n t a i n e d f o r 2 weeks on a d i e t t h a t l i m i t e d t h e i r w e i g h t g a i n t o a s c l o s e t o z e r o as p o s s i b l e . M a l e s from a l l t h r e e g r o u p s were a n e s t h e t i z e d u n d e r d i e t h y l e t h e r . H e p a r i n (120 u n i t s i n 0.1 ml o f p h y s i o l o g i c a l s a l i n e ) was i n j e c t e d i n t r a v e n o u s l y . The t e s t i c l e s were e x p o s e d s u r g i c a l l y and t h e t e s t i c u l a r v e i n was clamped and c u t . A p p r o x i m a t e l y 0.2ml o f t e s t i c u l a r venous b l o o d was c o l l e c t e d d r o p w i s e i n t o a t e s t t u b e ( Sudo ej_ a l . , 1 9 7 S ) . The a o r t a was s e v e r e d and an a r t e r i a l b l o o d s a m p l e o f a t l e a s t 1ml was c o l l e c t e d . Samples were i m m e d i a t e l y f r o z e n a t — 7 0 ° C and s t o r e d u n t i l a n a l y s i s . A n d r o g e n l e v e l s were d e t e r m i n e d u s i n g Amersham's T e s t o s t e r o n e / E i h y r o t e s t e s t e r o n e RIA k i t f o l l o w i n g t h e 52 m a n u f a c t u r e r ' s i n s t r u c t i o n s f o r T o t a K t e s t o s t e r o n e + DHT ) l e v e I s • 5 3 R e s u l t s S t a t i s t i c a l A n a l y s i s o l D a t a Bank o f G l u c q s i n o l a t e - c o n t a i n i n a P l a n t s The t o t a l d a t a hank o l g l u c o s i n c l a t e - c o n t a i n i n g p l a n t s was c o m p r i s e d o l 774 c a s e s * 15.2% o f w h i c h r e f e r r e d e i t h e r t o e d i b l e o r b e v e r a g e u s e s * T h i s I r e q u e n c y was c o n s i d e r a b l y l e s s t h an t h e 28.5% o f e d i b l e and m e d i c i n a l usage t a b u l a t e d f r o m the work o f A l t s c h u l * To t e s t t h e s i g n i f i c a n c e o f the v a r i o u s m e d i c i n a l c a t e g o r i e s t h e t a b l e was r e d u c e d t o 595 c a s e s r e f e r r i n g s p e c i f i c a l l y t o m e d i c i n a l u s e s . T a b l e I I I c o n t a i n s c a t e g o r i e s h a v i n g a p o s i t i v e f r e q u e n c y d i f f e r e n c e from the d i s t r i b u t i o n o l A l t s c h u l t h a t was c o n s i c e r e d g r e a t enough t o be w o r t h t e s t i n g . The p r o b a b i l i t i e s o f the l i k e l i h o o d r a t i o c h i — s q u a r e s p e r f o r m e d i n r e l a t i o n to t h e c o l l a p s e d d a t a base showed t h a t o n l y c a t e g o r i e s 3,16,20,24 and 32 were u s e d s i g n i f i c a n t l y more t h a n e x p e c t e d , w i t h i n e a c h o l t h e s e c a t e g o r i e s one s p e c i f i c use f r o m t h e raw d a t a m a t r i x c o n t r i b u t e d the g r e a t e s t number o f c a s e s . W i t h i n t h e c a t e g o r y v e r m i f u g e , a l l c a s e s were c o n c e r n e d w i t h t h e t r e a t m e n t o f i n t e s t i n a l worms* W i t h i n the c a t e g o r y m e t a b o l i c , 38 c a s e s o r 92*7% were a n t i s c o r b u t i c s * W i t h i n t h e c a t e g o r y r e p r o d u c t i v e : h o r m o n a l , 13 c a s e s o r 44.8% were emmenagogues; 78.5% were a s s o c i a t e d s p e c i f i c a l l y w i t h f e m a l e c o n d i t i o n s , and o n l y 14.3% w i t h male c o n c e r n s . W i t h i n t h e c a t e g o r y r e s p i r a t o r y : o t h e r , 13 c a s e s o r £4.2% were e x p e c t o r a n t s , w h i l e 54 T a b l e I I I Use c a t e g o r i e s a n a l y z e d l o r s i g n i f i c a n t m e d i c i n a l e f f e c t s C a t e g o r y C a s e s ^ F r e q u e n c y ^ F r e q u e n c y P r o b a b i l i t y ( S a m p l e ) ( A l t s c h u l ) P** 3 V e r m i f u g e 12 4 E m e t i c 8 13 N e r v o u s 5 o t h e r 28 16 M e t a b o l i c 41 20 R e p r o d u c t i v e : h o r m o n a l 29 24 R e s p i r a t o r y : o t h e r 24 30 S k i n : o t h e r 46 31 T o n i c 20 32 U r i n a r y s y s t e m 49 33 L i v e r 16 34 C i r c u l a t o r y s y s t e m 25 2.2 1.0 0.0416* 1.3 0.6 0.3807 4.7 2.5 0.0644 6.9 0.4 <0.0001* 4.9 2.4 0.0326* 4.0 1.5 0.0058* 7.7 5.7 0.1316 3.4 1.7 0.1491 8.2 3.4 <0.0001* 2.7 1.7 0.4461 4.2 2.3 0.2534 ** P r o b a b i l i t y P o f t h e c o l l a p s e d d a t a base was 0.7031. * P <0.05 20.8% were c o n c e r n e d w i t h t r e a t i n g a s t h m a . W i t h i n t h e c a t e g o r y u r i n a r y s y s t e m , 39 c a s e s o r 79. 591 were d i u r e t i c s . 55 D e t e c t i o n and D e t e r m i n a t i o n o l C o p s t l t u e n t s TLC o f g l u c o s i n o l a t e s o l T r o p a e o l u m t u b e r osum subsp* tuberosum and Lejaidj_,u._nj meyeni i : G l u c o s i n o l a t e s o c c u r n a t u r a l l y as p o t a s s i u m s a l t s . S t a n d a r d s as t e t r a m e t r . y l ammonium s a l t s were n o t u s e f u l f o r c h r o m a t o g r a p h y . In t h e a b s e n c e o l s t a n d a r d s , TLC was c a r r i e d o u t to d e t e r m i n e t h e number o l c o n s t i t u e n t s and to compare the g l u c o s i n o l a t e p r o f i l e s o f T r o p a e o l u m t u b erosum s u b s p . t u b e r o sum and L e p i d i u n mey e n i i • E x t r a c t s from b o t h s p e c i e s gave n e g a t i v e r e s u l t s when s p r a y e d f o r p h e n o l i c g l u c o s i n o l a t e s . P h e n o l s were p r e s e n t i n e x t r a c t s f r o m b o t h s p e c i e s b u t d i d not g i v e s p o t s t h a t c o r r e s p o n d e d t o t h o s e r e v e a l e d by t h e AgNC3—NaCH r e a g e n t . S f ' s o f s p o t s d e t e c t e d i n b o t h s p e c i e s a r e r e c o r d e d i n T a b l e IV. I n b o t h s o l v e n t s y s t e m s th e major c o n s t i t u e n t s c o r r e s p o n d i n E f . L e p i d i u m meyeni i has a t l e a s t one u n i q u e g l u c o s i n o l a t e , w h i l e b o t h s p e c i e s show a nrin<cr c o n s t i t u e n t i n common. P a p e r c h r o m a t o g r a p h y o f g l u c o s i n o l a t e s : E x t r a c t s o f T r o p a e o l u m t u b e r o s u m s u b s p . t uberosum and L e p i d i u m meyen i i were compared as w i t h T L C . R f ' s a r e r e c o r d e d i n T a b l e IV. The major compound (Rf=0.21) p r o d u c e d a s i n g l e s p o t when s a m p l e s of b o t h s p e c i e s were c c — c h r o m a t o g r a p h e d . Lep i d iuro m e v e n i i a l s o p r o d u c e d a m i n e r s p o t w i t h an Rf o f 0.38. No 56 p h e n o l i c c o n p c u n d s were d e t e c t e d -T a b l e IV T h i n l a y e r and p a p e r c h r o m a t o g r a p h y o f g l u c o s i n o l a t e s ( R±s ) C h r o m a t o g r a p h y S o l v e n t Samples system T . tuberosum L . m e y e n i i s u b s p • tuberosum T h i n l a y e r bu t a n o I benzene e t h a n o l amnion i a 0. 1S( m a j o r ) ( 0 . 4 0 ) 0. 1 8 ( m a j o r ) 0.25 (0.40 ) b u t a n o l 0 . 4 3 ( m a j o r ) p r o p a n e I 0.21 a c e t i c a c i d w a t er 0.43(major ) 0.21 Pape r b u t a n o I e t h a n o l 0. 2 1( m a j o r ) 0. 21( major ) 0.38 57 A n a l y s i s o f i s o t h i o c y a n a t e s : C h e m i c a l t e s t s and p a p e r c h r o m a t o g r a p h y o f t h i o u r e a s E x t r a c t s o f i s o t h i o c y a n a t e s l i b e r a t e d by endogenous and e xogenous ( f r o m s e e d s o f S i n a p i s a l t a ) m y r o s i n a s e s o f b o t h s u b s p e c i e s o f T.. tuberosum , and o f L. meyen i i were n e g a t i v e f o r t h i o c y a n a t e s and c y c l i c o x o z o I i d i n e t h e n e s • A l l s a m p l e s o f J> t u b e r o s u m t e s t e d d i d r e a c t w i t h e t h a n o l i c ammonium t o p r o d u c e t h i o u r e a s . The i n c u b a t i o n of g r o u n d r o o t o f L. meveni i w i t h w a t e r f a i l e d t o r e l e a s e d e t e c t a b l e i s o t h i o c y a n a t e s . I s o t h i o c y a n a t e s were l i b e r a t e d by an exogenous enzyme p r e p a r a t i o n and were d e t e c t e d as t h i o u r e a d e r i v a t i v e s . R e s u l t s o f t h e p a p e r c hxomatography o f t h i o u r e a s o f a l l t h r e e t a x a a r e t a b u l a t e d i n T a b l e V. T,. t u b e rosum s u b s p . tube ro sum a p p e a r s t o c o n t a i n one i s o t h i o c y a n a t e w h i c h was i d e n t i f i e d as p— m e t h o x y b e n z y I i s o t h i o c y a n a t e . T u b e r s o f t h e s u b s p e c i e s s i I v e s t r e ( • k i p a i s a n o ' ) a p p e a r t o c o n t a i n b e n z y l i s o t h i o c y a n a t e a s the m a j o r c o n s t i t u e n t and a m i n o r c o n s t i t u e n t s i m i l a r i n Rph t o p u b l i s h e d r e s u l t s f o r 2 — p r o p y l t h i o u r e a . T h i s s e c o n d a r y c o n s t i t u e n t was d e t e c t e d o n l y i n t h e b e n z e n e — e t h a n o l — w a t e r s o l v e n t s y s t e m , and t h e n n o t c o n s i s t e n t l y . Seeds o f T. t u b e r o s u m s u b s p . s i 1 v e s t r e c o n t a i n 2 — b u t y l and 2 — p r o p y l i s o t h i o c y a n a t e s as m a j o r c o n s t i t u e n t s and o n l y a t r a c e o f b e n z y l i s o t h i o c y a n a t e . L e p i d i u m m_ey.e_Q_i_l , i n t h e one s o l v e n t s y s t e m t e s t e d , p r o d u c e d one s p o t c o r r e s p o n d i n g to b e n z y l i s o t h i o c y a n a t e . 58 T a b l e V P a p e r c h r o m a t o g r a p h y of t h i o u r e a d e r i v a t i v e s (Rph*s ) S o l v e n t s y s t e m S t a n d a r d t h i o u r e a s Samples B e n z y l p-Met* s - B u t y l T . t . T. t . L.m. s • tub • s . s i l . Benzene e t h a n o l w a t e r 1.C5 1.10 0.80 1.07 t u b e r 1.0 0.32 s e e d ( 1.0 ) 0.77 0 .36 1.03 To Luene a c e t i c a c i d wa t e r 0.8 0.8 0.7 0 .77 0.8 C h l o r o f o rm w a t e r 0.92 1.0 0.85 1.0 t u b e r 0 .92 s e e d 0.86 0 .54 *p-Met = p - M e t h o x y b e n z y I i s o t h i o c y a n a t e 58 HPLC a n a l y s i s of i s o t h i o c y a n a t e s I s o t h i o c y a n a t e s r e s o l v e d by HPLC methods were i d e n t i f i e d by c o m p a r i s o n o f r e t e n t i o n v a l u e s and c o — c h r o m a t o g r a p h y w i t h s t a n d a r d compounds, and by p a p e r c h r o m a t o g r a p h y ( o f t h i o u r e a d e r i v a t i v e s ) , mass s p e c t r o m e t r y and NMR s p e c t r o m e t r y o f c o l l e c t e d f r a c t i o n s . D a t a f o r t h e HPLC a n a l y s i s i s r e c o r d e d i n T a b l e V I . Samples o f 2 grams o f f r e s h m a t e r i a l were s u f f i c i e n t T a b l e VI BFLC a n a l y s i s o f i s o t h i o c y a n a t e s Mode Compound t r ( m i n . ) a k 1 R e v e r s e phase p - m e t h o x y b e n z y l 1 1 . 3 4 . 7 i s o t h i o c y a n a t e ( A ) 0 b e n z y l 1 1 . 3 4 . 7 i s o t h i o c y a n a t e ( A ) 1 . 1 5 2 - p r o p y l 1 0 . 1 4 .1 i s o t h i o c y a n a t e ( B ) Normal phase p — m e t h o x y b e n z y l 3 . 8 0 . 5 2 i s o t h i o c y a n a t e ( D ) 1 . 6 5 b e n z y l 3 . 4 0 . 3 4 i s c t h i o c y a n a t e ( C ) 1 .80 2 - p r o p y l 3 . 1 0 . 1 9 i s o t h i o c y a n a t e ( B ) 60 f o r a n a l y s i s when s t a n d a r d s were a v a i l a b l e . R e t e n t i o n t i m e s ( t r ) g i v e n a r e mean v a l u e s . V a r i a b i l i t y i n r e t e n t i o n t i m e s n e c e s s i t a t e d t h e r e g u l a r use o f s t a n d a r d s , and t h e i d e n t i t y o f compounds i n sample e x t r a c t s was b a s e d p r i m a r i l y on c o m p a r i s o n w i t h s t a n d a r d compounds. Column s e l e c t i v i t y ( a ) ( J o h n s o n and S t e v e n s o n , ISIS1} i s measured by the r e l a t i v e s e p a r a t i o n o f peaks and i s d e f i n e d by the e q u a t i o n t r 2 - trm a= t r i - tra where: t r i and t r 2 = t h e r e t e n t i o n t i m e s o f components 1 and 2 r e s p e c t i v e l y . tm = r e t e n t i o n time o f u n r e t a i n e d compounds ( s o l v e n t f r o n t ) a i n d i c a t e s t h e r e l a t i v e s e p a r a t i o n c f components. The g r e a t e r t h e v a l u e t h e g r e a t e r t h e s e p a r a t i o n o f two s u c c e s s i v e p e a k s . The column c a p a c i t y f a c t o r , k', ( J o h n s o n and S t e v e n s o n , 1978) i s g i v e n by t h e e x p r e s s i o n 61 t r l - tm k» = -tm and i s a measure o f the r e t e n t i o n o f compounds r e l a t i v e to the s o l v e n t f r o n t . The r e v e r s e p h a s e s y s t e m p r o v i d e d t h e b e s t r e s o l u t i o n f o r i s o t h i o c y a n a t e s i n g e n e r a l ( F i g u r e 10) b u t f a i l e d t o s e p a r a t e p-m e t h o x y b e n z y l and b e n z y l i s o t h i o c y a n a c e s . The peak h a v i n g t h e same r e t e n t i o n t i m e as e i t h e r o f t h e s e compounds i s l a b e l l e d A. Normal phase c h r o m a t o g r a p h y p r o v i d e d a c o n v e n i e n t method f o r d i s t i n g u i s h i n g t h e s e two s i m i l a r compounds ( F i g u r e 1 1 ) . B e n z y l i s o t h i o c y a n a t e i s r e f e r r e d t o as C; p—methoxybenzy1 i s o t h i o c y a n a t e i s r e f e r r e d t o as D. The s m a l l k* v a l u e s i n t h e n o r m a l p h a s e s y s t e m i n d i c a t e t h e low r e s o l u t i o n o f t h i s s y s t e m . The p o s s i b i l i t y o f o t h e r compounds h a v i n g i d e n t i c a l r e t e n t i o n t i m e s and b e i n g m i s t a k e n l y I d e n t i f i e d i s h i g h under t h e s e c o n d i t i o n s and the u s e f u l n e s s o f t h e s y s t e m f o r t h e a n a l y s i s o f unknown samples i s l i m i t e d w i t h o u t c o m p a r i s o n w i t h r e s u l t s f r o m o t h e r methods o f a n a l y s i s . R e v e r s e p h a s e c h r o m a t o g r a p h y o f e x t r a c t s o f t u b e r s o f ' k i p a i s a n o ' ( X» t u b e r o s u m s u b s p . s i l y e s t r e ) r e v e a l e d 4 peaks ( F i g u r e 10) • P r e p a r a t i v e c h r o m a t o g r a p h y o f e x t r a c t s from a p p r o x i m a t e l y 40 grams o f f r e s h t u b e r s p r o v i d e d s u f f i c i e n t p u r e compounds f o r a p p l i c a t i o n to o t h e r methods o f a n a l y s i s . Two of t h e c o l l e c t e d peaks (A and P.) r e a c t e d w i t h e t h a n o l i c ammonia t o p r o d u c e t h i o u r e a s . P a p e r c h r o m a t o g r a p h y i n d i c a t e d t h a t compound A i s b e n z y l i s o t h i o c y a n a t e and B i s 2 — p r o p y l i s o t h i o c y a n a t e . Mass s p e c t r o m e t r y ( T a b l e V I I I c o n f i r m e d t h e i d e n t i t y o f A b u t Figure 10. Reverse phase analysis of Tropaeolum tuberosum subsp. s i l v e s t r e 0.02 AU B A J £ J o 1 5 • 10 i 15 MINUTES 63 Figure 11. Resolution of benzyl and p-methoxybenzyl isothiocyanates by normal phase HPLC. D B.E T 0.2 AU 0 1 5 9 MINUTES 64 f a i l e d t o g i v e a m e a n i n g f u l r e s u l t f o r B b e c a u s e o f t h e low-b o i l i n g p o i n t o f t h e sample ( B . P . 2 — p r o p y l i s o t h i o c y a n a t e i s 138° C ) . T a b l e V I I Mass s p e c t r o m e t r y o f HPLC f r a c t i o n s Compound M a s s — t o — C h a r g e R a t i o ( m / e ) B e n z y l i s o t h i o c y a n a t e ( C J 149( 5 1 ), 91( 100 ), 6 5( 21 ), 51 ( 6 ), 39( 9 ), 32(12 ),28( 50 ) P - m e t h o x y b e n z y l 179(20),121( 1 0 0 ) , 9 1 ( 4 ) , 7 7 ( 4 ) , 6 5 ( 2 ) , i s o t h i o c y a n a t e C E) 5 1 ( 2 ) , 2 2( 1 1 ) , 2 8 ( 4 4 ) N,N,Di( 4-methoxy, b e n z y l ) 316(1S3,282(2),195( 1 5 ) , 1 7 9 ( 2 ) , 1 5 3 ( 3 ) , t h i o u r e a 136(35 ),121( 100),109( 3),91( 5 ),77( 7 ) N-( 4-methoxy, b e n z y l ) 2 7 1( 3 ) , 164 ( 5 ) , 1 08( 100 ) , 77 ( 3 ), 51( 3 ) m e t h y I t h i o c a r b a m a t e R e v e r s e p h a s e and p r e p a r a t i v e c h r o m a t o g r a p h y o f t u b e r s o f T. t u b e r o s u m s u b s p . tuberosum p r o d u c e d one peak ( A ) t h a t c o u l d be i d e n t i f i e d as an i s o t h i o c y a n a t e ( F i g u r e 1 2 ) . P a p e r c h r o m a t o g r a p h y o f i t s t h i o u r e a s gave i t s i d e n t i t y as m e t h o x y b e n z y I i s o t h i o c y a n a t e . Mass s p e c t r a l a n a l y s i s ( T a b l e V I I ) c o n f i r m e d t h i s d e t e r m i n a t i o n . The p a r e n t i o n (m/e 179) and the f r a g m e n t a t i c n p a t t e r n g i v i n g a m a j o r f r a g m e n t a t m/e 121 (CH3CCfcH^CH2— ) c o r r e s p o n d s t o t h a t e x p e c t e d f o r m e t h o x y b e n z y I i s o t h i o c y a n a t e (M.W. 179.25). F i g u r e 13 shows th e H l —NMR sp e c t r u m f o r 23mg o f the compound. C h e m i c a l s h i f t s ( ) o f 7 . 0 5 ( m u l t i p l e t ), 4 . 6 0 ( s i n g l e t ) and 3 . 8 0 ( s i n g l e t ) c o r r e s p o n d w i t h t h e s h i f t s c f a r o m a t i c , a l i p h a t i c and methoxy p r o t o n s ;ure 12. Reverse phase HPLC of tubers of Tropaeolum tuberosum subsp. tuberosum. A T 0. 2 AU _ J _ _ 0 1 10 15 MINUTES 67 r e s p e c t i v e l y ( S l l v e r s t e i n e_t a l . » 1974 ). The I n t e g r a l s o f t h e p e a k s have t h e r a t i o o f 4:2:3 e x p e c t e d f o r m e t h o x y b e n z y I i s o t h i o c y a n a t e . The m u l t i p l e t h a s t h e s p l i t t i n g p a t t e r n o f an AE q u a r t e t , t y p i c a l o f an a r o m a t i c r i n g s u b s t i t u t e d i n the p a r a p o s i t i o n * From the above e v i d e n c e i t i s c o n c l u s i v e t h a t t h e s o l e i s o t h i o c y a n a t e l i b e r a t e d i n e x t r a c t s o f T* t u b e r o s u m s u b s p . t u b e r o s u m i s p — m e t h o x y b e n z y l i s o t h i o c y a n a t e . T a b l e V I I I NMR d a t a f o r p — m e t h o x y b e n z y I i s o t h i o c y a n a t e 1H—NMR (80MHz, C D C l 3 ) £ ( p p m ) 7.05(4H,m), 4 . 6 0 ( 2 H , s ) , 3 . 8 0 ( 3 H , s ) A peak h a v i n g a l a r g e r e t e n t i o n time on the normal phase s y s t e m ( 1 7 . 0 m i n u t e s ) was c o l l e c t e d p r e p a r a t i v e l y and a n a l y z e d by mass s p e c t r o s c o p y . The p a r e n t mass (m/e 316) ( T a b l e V I I ) c o i n c i d e s w i t h a f o r m u l a of C i 7 H 2 t ) C 2 N 2 S . The f r a g m e n t a t i o n p a t t e r n i s c o n s i s t e n t w i t h t h e s t r u c t u r e o f N ,N , DM methoxy , 4— b e n z y l ) t h i o u r e a ( F i g u r e 7 ) . The m a s s — t o — c h a r g e r a t i o s c o r r e s p o n d w i t h t h a t r e c o r d e d f o r t h i s compound by E l M i g i r a b , e t aj. • ( 1977 ). E x t r a c t s o f S i p a p j, s aj_ba p r o d u c e d a peak w i t h a r e t e n t i o n t i m e o f 7.9 m i n u t e s cn t h e r e v e r s e p h a s e c o l u m n . The mass s p e c t r u m o f t h i s compound ( T a b l e V I I ) s u g g e s t s t h a t i t i s N—(4— h y d r o x y , b e n z y 1 ) m e t h y I t h i o c a r b a m a t e ( F i g u r e 7 ) . The l a r g e s t f r a g ment of t h i s s p e c t r u m c o r r e s p o n d s to HC.C6H._. CB2 — • T h i s i s t h e e x p e c t e d f r a g m e n t i n the breakdown o f p — h y d r o x y b e n z y I i s o t h i o c y a n a t e to t h i o c y a n a t e . T u b e r s o f a l l t h e c o l l e c t i o n s i n T a b l e I I and v a r i o u s p l a n t p a r t s from e a c h o f the two s u b s p e c i e s o f T r o p a e o l u m tuberosum 6 8 were s c r e e n e d by n ormal phase HPLC. A l l t u b e r s a m p l e s o f T. t u b e ros um s u b s p . tuberosum and s a m p l e s o f s e e d s , f l o w e r s and l e a v e s of c o l l e c t i o n no•6 showed s i m i l a r HPLC p r o f i l e s to t h a t r e c o r d e d i n F i g u r e 14. An e x t r a c t o f t u b e r s t h a t had been c o o k e d by b o i l i n g p r o d u c e d p - m e t h o x y b e n z y l i s o t h i o c y a n a t e when i n c u b a t e d w i t h the exogenous enzyme p r e p a r a t i o n . T u b e r s , l e a v e s , s e e d s and f l o w e r s o f * k i p a i s a n o * ( T. t uberosum s u b s p . s i I v e s t r e ) a l l c o n t a i n e d b e n z y l i s o t h i o c y a n a t e and t h e peak i d e n t i f i e d as 2 —propyl i s o t h i o c y a n a t e ! F i g u r e 1 5 ) . However, t h e r e l a t i v e q u a n t i t i e s v a r i e d . T u b e r s c o n t a i n e d t h e g r e a t e s t r e l a t i v e amount o f b e n z y l i s o t h i o c y a n a t e and o n l y n e g l i g i b l e amounts o f o t h e r c o n s t i t u e n t s . S eeds c o n t a i n e d r e l a t i v e l y more o f 2 —propyl i s o t h i o c y a n a t e t han any o t h e r p l a n t p a r t , as w e l l as one unknown p e a k . Peak E from r e v e r s e phase c h r o m a t o g r a p h y c c c h r o m a t o g r a p h e d w i t h A i n t h e n o r m a l phase s y s t e m . E was s u s p e c t e d t o be 2 —butyl i s o t h i o c y a n a t e s b u t i t s i d e n t i t y was not c o n f i r m e d . I f t h i s were 2 —butyl i s o t h i o c y a n a t e then the 2 —propyl peak ( A ) i n t h e n o r m a l p h a s e s y s t e m i s a c o m b i n a t i o n o f 2 —butyl and 2 —propyl i s o t h i oc yan a t e s . An e x t r a c t o f 7 g o f g r o u n d r o o t s of L e o i d i u m meyeni i i n c u b a t e d w i t h an enzyme p r e p a r a t i o n f r o m S i n a p i s a l b a , p r o d u c e d i s o t h i o c y a n a t e s t h a t were d e t e c t a b l e by HPLC. R e v e r s e phase and n o r m a l phase p r o f i l e s a r e r e c o r d e d i n F i g u r e 16. The l a r g e s t peak i n b o t h systems c o r r e s p o n d e d t o b e n z y l o r p— m e t h o x y b e n z y l i s o t h i o c y a n a t e s . R e v e r s e phase c h r o m a t o g r a p h y p r o d u c e d one u n i d e n t i f i e d peak. By n o r m a l phase c h r o m a t o g r a p h y t h e sample was r e s o l v e d i n t o 4 p e a k s . The l a r g e s t o f t h e s e c o r r e s p o n d e d t o b e n z y l I s o t h i o c y a n a t e and a s m a l l e r one t o 69 Figure 14. Normal phase HPLC of tubers of Tropaeolum tuberosum subsp. tuberosum. 0 . 0 2 AU -1_ o 1 i — i — t • • - J — i i M l N U T E S 70 seeds 0 1 B 5 i i i MINUTES 0.2 AU 1 tubers Figure 15. Normal phase HPLC of Tropaeolum tuberosum subsp. s i l v e s t r e . 71 0.05 AU Normal phase J L Reverse phase A-0 1 5 10 15 t • . i • • • « i • i. i _« j i i , MINUTES Figure 16. HPLC of Lepidium meyenii 72 p — m e t h o x y b e n z y I i s o t h i o c y a n a t e . The a r e a of the ' b e n z y l * peak i n r e v e r s e phase was 63%, w h i l e t h e a r e a of t h e combined b e n z y l and p — m e t h o x y b e n z y l p e a k s i n n o r m a l phase was 65%. T h i s r o u g h measure s u p p o r t s the s u p p c s l t i o n t h a t the two peaks were r e s o l v e d f r o m t h e m a j o r peak i n t h e r e v e r s e phase s y s t e m . The i d e n t i t y o f the two o t h e r peaks r e m a i n s unknown. Amino a c i d a n a l y s i s : S i x c l o n e s o f t u b e rosum s u b s p . t u b e r o s u m were a n a l y z e d . The f o l l o w i n g f r e e amino a c i d s were d e t e c t e d i n a l l s a m p l e s : a s p a r t i c a c i d , t h r e o n i n e , s e r i n e , a s p a r a g i n e , g l u t a m i c a c i d , p r o l i n e , ( g l y c i n e , a l a n i n e , v a l i n e , c y s t i n e , m e t h i o n i n e ( v e r y l o w ) , i s o l e u c i n e , l e u c i n e , t y r o s i n e , p h e n y l a l a n i n e , a r g i n i n e , h i s t i d i n e , l y s i n e , o r n o t h i n e , t r y p t o p h a n • S m a l l amounts o f a n o t h e r n e u t r a l a n i n o a c i d were d e t e c t e d i n some s a m p l e s . T h i s compound had a r e t e n t i o n t i m e s l i g h t l y f a s t e r t h a n t h a t o f v a l i n e and i s p r o b a b l y T-amino b u t y r i c a c i d . A n o t h e r unknown a p p e a r e d i n the a n a l y s i s o f b a s i c amino a c i d s . T h i s compound was r e a d i l y d e t e c t a b l e i n a l l s a m p l e s and had a r e t e n t i o n t i m e s l i g h t l y f a s t e r t h a n ammonia. I t s i d e n t i t y r e m a i n s u n d e t e r m i n e d . Q u a n t i t a t i v e l y , t h e r e s u l t s show c o n s i d e r a b l e v a r i a t i o n . R e l a t i v e amounts o f amino a c i d s c h anged from sample t o s a m p l e . No p a t t e r n s t h a t might r e l a t e t o g l u c o s i n o l a t e b i o s y n t h e s i s were d i s c e r n i b l e . 13 D e t e r m i n a t i o n o f Chromosome Number Root s q u a s h e s o f t u b e r s o f ' k i p a i s a n o ' ( T. tuberosum s u b s p * s i l v e s t r e ) gave a chromosome c o u n t o f 2n>42* The s m a l l s i z e o f t h e chromosomes made i t d i f f i c u l t t o d e t e r m i n e t h e number more p r e c i s e l y * T e s t s f o r B i o l o g i c a l A c t i v i t y A n t i b i o t i c a c t i v i t y : T u b e r s of b o t h s u b s p e c i e s of T* t u b e r o s u m showed a n t i b i o t i c a c t i v i t y ( T a b l e I X) a g a i n s t C a n d i d a a l b i c a n s , w h i l e o n l y subsp* t u b e r o s u a was a n t i b i o t i c a g a i n s t E s c h e r i c h i a c o l i and S t a p h y l o c o c c u s a I b u s • N e i t h e r t a x a was a n t i o b i o t i c a g a i n s t Pseudomonas f l u o r e s c e r s . P u r e p — m e t h o x y b e n z y l and b e n z y l i s o t h i o c y a n a t e s , d e t e c t e d i n subsp * tuberosum and s u b s p * s i l v e s t r e r e s p e c t i v e l y , showed a s i m i l a r p a t t e r n t o t h e p l a n t m a t e r i a l , e x c e p t t h a t b e n z y l i s o t h i o c y a n a t e i n h i b i t e d the g r o w t h of £• £oJL_i and was a n t i b i o t i c t o w a r d s .§• a Ibus, • A n t i b i o s i s was dose dependent a g a i n s t some o r g a n i s m s i n the c o n c e n t r a t i o n d i f f e r e n c e t e s t e d * The y e a s t , C_. a l b j e a n s , was t h e most s e n s i t i v e t o b o t h compounds, w h i l e E . c o l 1 and S. alb,us were s e n s i t i v e o n l y t o lOOug o f e i t h e r p — m e t h o x y b e n z y I o r b e n z y l i s o t h i o c y a n a t e * P h o t o t o x i c i t y t e s t s o f c r u d e p l a n t m a t e r i a l o f b o t h 74 s u b s p e c i e s a g a i n s t C. jsj.b_ic.ajis showed an i d e n t i c a l r e s u l t t o t h a t r e c o r d e d above* The o r g a n i s m s were k i l l e d i n e i t h e r l i g h t o r d a r k i n d i c a t i n g a l a c k o f p h o t o t o x i c a f f e c t * T a b l e IX A n t i b i o t i c a c t i v i t y T e s t T r o p a e o l u m tuberosum I s o t h i o c y a n a t e s o r g a n i s m s s u b s p e c i e s p - M e t h o x y b e n z y l B e n z y l t u b e r o s u m s i l v e s t r e lOOug lOug lOOug lOug C . a l b i c a n s ++ + ++ ++ ++ ++ E . c o l i + - + - i -P • f l u o r e s c e n s — — — — — — S . a l b u s ++ - ++ - + -+ = a n t i b i o t i c i = i n h i b i t i v e — = n e g a t i v e A n t i v i r a l a c t i v i t y : An e t h e r e x t r a c t o f t u b e r s o f T r o n a e clum tuberosum subsp* _tuheroj=ur. showed no a n t i v i r a l a c t i v i t y a g a i n s t H e r p e s t y p e I v i r u s * The r e s u l t s of t h e a s s a y f o r t h e a n t i v i r a l a c t i v i t y a r e r e c o r d e d i n T a b l e X* The e x p e r i m e n t a l t i t r e showed no r e d u c t i o n i n CPE ( c y t o p a t h i c e f f e c t ) i n r e l a t i o n to the v i r a l 75 T a b l e X A n t i v i r a l a c t i v i t y E x p e r i m e n t and c o n t r o l s 1. E x p e r i m e n t MNTD + H e r p e s + VEEC 2. V i r u s c o n t r o l H e r p e s -+ VEEC 3. P r o t e c t i v e c o n t r o l C y t o p a t h l c e f f e c t ( C P E ) He r p e s d i l u t i o n 10- 1 1 0 - 2 1 0 - 2 1 0 - 4 1 0 - 5 1 0 - 6 1 0 - 7 4+ 4+ 4+ 4-f 3+ 2+ ?+ 4+ 4+ 4+ 4+ 3+ 2+ 4+ 4+ 4+ 2+ ?+ ?+ 0 0.5ml o f 1 0 - 1 d i l u t i o n o f H e r p e s and 0.5ml MNTD i n c u b a t e d 1 h o u r and t h e n t i t r e d . 4. S o l v e n t c o n t r c K v i r a l ) - - - 3 + 3 + + 0 0.5ml o f 1 0 - 4 d i l u t i o n o f e t h e r + VEEC S o l v e n t d i l u t i o n 10- 1 1 0 - 2 1 0 - 3 1 0 - 4 5. S o l v e n t c o n t r o l ( V E E C ) 0 0 0 0 6. T i s s u e c u l t u r e c o n t r o l CPE = 0 The maximum n o n t o x i c d i l u t i o n (MNTD) o f t h e p l a n t e x t r a c t was 1 0 - 4 . 76 t i t r e ( 2 . V i r u s c o n t r o l ) . S i m i l a r l y when the MNTD (maximum non -t o x i c d i l u t i o n ) was i n c u b a t e d f i r s t w i t h H e r pes ( 3 . P r o t e c t i v e c o n t r o l ) no s i g n i f i c a n t d r o p i n CPE was o b s e r v e d . S o l v e n t c o n t r o l s 4 and £ i n d i c a t e t h a t the s o l v e n t d i d n o t a f f e c t e i t h e r v i r a l a c t i v i t y i n t h e range where CPE would be o b s e r v a b l e , o r VERO c e l l s i n t h e c o n c e n t r a t i o n s u s e d i n the i n c u b a t i o n . N e m a t o c i d a l a c t i v i t y : B e n z y l i s o t h i o c y a n a t e had a LD??? a g a i n s t nematodes ( C a e n o r h a b d i t i s glejaaiis ) of a p p r o x i m a t e l y 0.4% a f t e r h a l f an h o u r . The amount of compound a p p l i e d t o t h e p l a t e was 4.5ug. A c o n c e n t r a t i o n of 0 . 1 % ( l . l u g ) k i l l e d some nematodes a t t h e c e n t r e o f t h e p l a t e b u t t h o s e n e a r t h e p e r i p h e r y were a b l e t o a v o i d t h e t o x i c e f f e c t s . A f t e r 6 and 24 h o u r s t h e r e s u l t s were the same as a f t e r h a l f an h o u r . E s t r o g e n i c a c t i v i t y : E f f e c t o f T r o p a e o l u m tuberosum on t h e e s t r u s c y c l e o f t h e g u i n e a p i g A d i e t c o n t a i n i n g 20% by w e i g h t o f J . tuberosum s u b s p . t u b e r o s u m f a i l e d to a f f e c t t h e r e g u l a r i t y o f t h e e s t r u s 77 c y c l e o f f e i t a l e g u i n e a p i g s o v e r a p e r i o d o f 40 d a y s (2 c y c l e s ) ( T a b l e XI )• E x p e r i m e n t a l a n i m a l s showed a d r o p i n w e i g h t o v e r the f i r s t 2 weeks o f t h e f e e d i n g , b u t as t h e y became a c c u s t o m e d t o t h e d i e t t h e y g a i n e d w e i g h t a t a r a t e c o m p a r a b l e t o c o n t r o l a n i m a l s * T h i s i n i t i a l w e i g h t d r o p was n o t r e f l e c t e d i n any change i n t h e e s t r u s c y c l e o f t h e g u i n e a p i g s * T a b l e X I E f f e c t o f T r o p a e o l u m t u b e r o s u m on e s t r u s c y c l e Group Number o f Mean l e n g t h o f P r o b a b i l i t y a n i m a l s c y c l e ( d a y s ) P E x p e r i m e n t a l 6 15.67 ± C.82 0.9000* C o n t r o l 6 15.60 ± 0.89 * P> 0.05 R a d i o a c t i v e c o m p e t i t i v e b i n d i n g a s s a y C r u d e e t h e r e x t r a c t s of T. t u b e r o s u m s u b s p . tuberosum and of S i n a p i s a l b a p r o d u c e d a q u a l i t a t i v e I n h i b i t i o n o f e s t r a d i o l b i n d i n g when compared to s o l v e n t c o n t r o l s . F u r e i s o t h i o c y a n a t e s a l s o p r o d u c e d i n h i b i t i o n . An u n q u a n t i f i e d p — m e t h o x y b e n z y I i s o t h i o c y a n a t e f r a c t i o n o b t a i n e d f r o m HPLC showed i n h i b i t i o n p r o p o r t i o n a l t o s e v e r a l d i l u t i o n s . I n h i b i t i o n by s t a n d a r d b e n z y l and p h e n e t h y l i s o t h i o c y a n a t e s i s t a b u l a t e d i n T a b l e X I I i n r e l a t i o n t o nanograms o f . g e n i s t e i n t h a t would p r o d u c e an e q u a l 78 T a b l e X I I E s t r o g e n i c a c t i v i t y Compound n g / t u b e E q u i v a l e n t g e n i s t e i n ( n g ) P h e n e t h y l i s o t h i o c y a n a t e 3.3 x 10 6 43 1.1 x 1 0 6 16 1.0 x 1 0 5 13 1.0 x 10* 3 B e n z y l i s o t h i o c y a n a t e 3.4 x 10 6 42 1.1 x 1 0 6 19 1.0 x 10 6 7 1.0 x 1 0 s 3 1.0 x 1 0 4 2 N , N , D i ( m e t h o x y , 4 - b e n z y l ) 10 38 t h i o u r e a 2 17 0.2 7 0.01 i n h i b i t i o n . B e n z y l g l u c o s i n o l a t e i n amounts up t o 0.25mg p r o d u c e d no m e a s u r a b l e i n h i b i t i o n i n e s t r a d i o l a c t i v i t y . The d i f f e r e n c e between t h e amount o f i s o t h i o c y a n a t e added and t h e e q u i v a l e n t w e i g h t o f g e n i s t e i n t h a t would p r o d u c e the same i n h i b i t i o n i s i n the o r d e r o f 1 0 5 . The m a g n i t u d e o f t h e d i f f e r e n c e s u g g e s t s t h a t the i n h i b i t i o n o b s e r v e d i s n o t 79 c o m p e t i t i v e . The D i x c n p l o t ( F i g u r e 17) c o n f i r m s t h a t the i n h i b i t i o n i s n o n — c o m p e t i t i v e i . e . t h a t t h e i s o t h i o c y a n a t e s a c t by a f f e c t i n g t h e e s t r o g e n r e c e p t o r o t h e r t h a n a t i t s a c t i v e s i t e • One p o r t i o n o f N,N,Di(4—methoxy• b e n z y l I t h i o u r e a , o b t a i n e d f r o m e x t r a c t s of T r o p a e o l u m t u b e r o s u m s u b s p . t u b e r o s u m by HPLC and i d e n t i f i e d by mass s p e c t r o m e t r y was t e s t e d q u a n t i t a t i v e l y f o r e s t r o g e n i c a c t i v i t y . These r e s u l t s s u g g e s t t h a t t h i s compound i n h i b i t s e s t r a d i o l b i n d i n g i n the same o r d e r o f m a g n i t u d e as g e n i s t e i n . A n t i — a n d r o g e n i c a c t i v i t y : S a t b r e e d i n g M a l e r a t s f e d s u b s p . t y b e x o s u a d i d i m p r e g n a t i n g f e m a l e male r a t s e a c h were ( T a b l e XII I ). a d i e t c o n t a i n i n g n o t show any d e c l i n e r a t s . E x p e r i m e n t a l 60% s u c c e s s f u l a t t u b e r s o f T. t u b e r o s u m i n t h e i r s u c c e s s a t and c o n t r o l g r o u p s o f i m p r e g n a t i n g f e m a l e s 80 1 2 3 4 5 moles x 1 0 - 5 [l] Figure 17. Dixon plot of i n h i b i t i o n of e s t r a d i o l binding by benzyl isothiocyanate. 81 T a b l e X I I I S a t b r e e d i n g e x p e r i m e n t Group Number o f Number ( a n d 96) o f P r o b a b i l i t y a n i m a l s p r e g n a n t f e m a l e s P E x p e r i m e n t a l 5 3 (60%) 1.0* C o n t r o l 5 3 ( 6 0 % ) * P>0.05 A n d r o g e n l e v e l s M ale r a t s f e d a d i e t c o n t a i n i n g t u b e r s o f T. t u b e r o s u m s u b s p . t u b e r o s u a showed a 45% d r o p i n t o t a l l e v e l s o f t e s t o s t e r o n e p l u s d i h j d r o t e s t o s t e r c n e . The a r t e r i a l b l o o d o f e x p e r i m e n t a l a n i m a l s and c o n t r o l a n i m a l s had mean a n d r o g e n l e v e l s of 145 and 264 ng/ml of b l o o d serum ( T a b l e XIV ), r e s p e c t i v e l y . A S t u d e n t ' s t — t e s t p e r f o r m e d cn t h e two s a m p l e s showed the r e s u l t t c be s i g n i f i c a n t (p<0.0£). However, i n c o m p a r i s o n to a n i m a l s s t a r v e d t o t h e p o i n t where t h e y g a i n e d o n l y 2.5%/week o f t h e i r t o d y w e i g h t f o r 2 weeks, the d r o p i n a n d r o g e n l e v e l s was i n s i g n i f i c a n t . S t a r v e d r a t s had mean a n d r o g e n l e v e l s o f 155ng/ml, a 41% d r o p compared t o c o n t r o l s ( P = 0•0127 )• The d r o p i n t e s t o s t e r o n e / d l h y r o t e s t o s t e r o n e i s r e l a t e d t o t h e d i e t and n o t to body w e i g h t . C o n t r o l a n i m a l s 82 w e i g h i n g f r o m 309 t o 396 grams had c o m p a r a b l e t e s t o s t e r o n e l e v e l s * C o n t r o l r a t s g a i n e d 6.6%/week of t h e i r body w e i g h t . E x p e r i m e n t a l r a t s g a i n e d no w e i g h t o v e r t h e c o u r s e o f t h e s t u d y . T e s t o s t e r o n e l e v e l s i n b l o o d c o l l e c t e d f r o m t e s t i c u l a r v e i n s showed too much v a r i a t i o n u n r e l a t e d t o the p a r t i c u l a r group o f a n i m a l s , t o be m e a n i n g f u l i n t h i s i n v e s t i g a t i o n . T a b l e XIV L e v e l s o f t e s t o s t e r o n e + d i h y d r o t e s t o s t e r o n e i n r a t b l o o d serum Group Number o f T e s t o s t e r o n e + P r o b a b i l i t y a n i m a l s d i h y d r o t e s t o s t e r o n e P ( n g / m l ) C o n t r o l 13*** 264 ± 99 0.0036* E x p e r i m e n t a l 9 145 ± 54 0.6773** S t a r v e d 7 155 ± 42 * P<0.05 ** P>0.05 *** C o n t r o l and e x p e r i m e n t a l a n i m a l s f e d f o r 2 o r 3 weeks showed no o b s e r v a b l e d i f f e r e n c e i n l e v e l s and a r e g r o u p e d t o g e t h e r • 83 E i s c u s s i o n G l u c o s i n o l a t e s g i Trop a eo Ium tuberosum The r e s u l t s o l p a p e r c h r o m a t o g r a p h y of t h i o u r e a d e r i v a t i v e s , HPLC on r e v e r s e phase and n o r m a l phase s y s t e m s , mass s p e c t r o s c o p y and NMR a l l c o n f i r m t h a t t h e s o l e i s o t h i o c y a n a t e o b t a i n e d by h y d r o l y s i s from e x t r a c t s o f t u b e r s o f T r o p a e o l um tuberosum sub sp« A_aber_gsuro i s p—me thoxyb enzy I i s o t h i o c y a n a t e . P a p e r c h r o m a t o g r a p h y of t h i o u r e a s and HPLC a n a l y s i s o f s e e d s , f l o w e r s and l e a v e s o f T_* tu b e r o s u m subsp* Aubexos.yr_ gave i d e n t i c a l r e s u l t s to t h o s e o b t a i n e d f r o m t u b e r s * P a p e r c h r o m a t o g r a p h y o f g l u c o s i n o l a t e s r e v e a l e d a s i n g l e compound a l t h o u g h TLC r e v e a l e d a s e c o n d s p o t i n two s o l v e n t s y s t e m s when s p r a y e d w i t h AgN03—NaOH* T h i s s p o t may i n d i c a t e a m i n o r g l u c o s i n o l a t e c o n s t i t u e n t i n t h e t u b e r s b u t , c o n s i d e r i n g t h e c o n s i s t e n c y o f the e v i d e n c e from o t h e r methods o f a n a l y s i s , i t i s p r o b a b l y a g l y c o s i d e o t h e r t h a n a g l u c o s i n o l a t e * The s p o t i s c o n s i d e r e d c f minor i m p o r t a n c e * The s o l e d e t e c t a b l e g l u c o s i n o l a t e i n T r j:£a e o J. um J_ub^rjos U_Q subsp* tuberosum i s t h e r e f o r e p - m e t h o x y b e n z y l g l u c o s i n o l a t e * O n l y I s o t h i o c y a n a t e s were a n a l y z e d i n * k i p a i s a n o ' , t h e w i l d c o l l e c t i o n i d e n t i f i e d t e n t a t i v e l y a s T* tuberosum subsp* s i l v e s t r e • K j a e r e t ajL* , ( 197S ) d e t e c t e d b e n z y l and 2 — p r o p y l t h i o u r e a s as t h e major I s o t h i o c y a n a t e d e r i v a t i v e s and 2— 84 b u t y l t h i o u r e a as a minor c o n s t i t u e n t from s e e d s o f w i l d X« t u b e r o s u m • The BFLC and p a p e r c h r o m a t o g r a p h y methods f o r t h i o u r e a s u s e d i n t h i s s t u d y c o n f i r m e d the p r e s e n c e o f b e n z y l and 2 — p r o p y l i s o t h i o c y a n a t e s i n t u b e r s , s e e d s , f l o w e r s and l e a v e s o f t h e one c o l l e c t i o n a v a i l a b l e o f w i l d m a t e r i a l * 2 — B u t y l i s o t h i o c y a n a t e was d e t e c t e d i n s e e d s b u t n o t i n t u b e r s . B e n z y l i s o t h i o c y a n a t e was t h e major c o n s t i t u e n t i n t u b e r s b u t a m i n o r c o n s t i t u e n t i n s e e d s . Mass s p e c t r a l a n a l y s i s o f t h e b e n z y l i s o t h i o c y a n a t e f r a c t i o n f r o m r e v e r s e phase c h r o m a t o g r a p h y c o n f i r m e d th e p r e s e n c e o f t h i s compound, p — M e t h o x y b e n z y I i s o t h i o c y a n a t e was n o t d e t e c t e d i n t h i s m a t e r i a l . From t h e s e r e s u l t s i t can be c o n c l u d e d t h a t t h e m a t e r i a l a n a l y z e d by K j a e r e t 3.1. ( 1 8 7 8 ) and ' k i p a i s a n o ' a r e d i s t i n c t from s u b s p . t u b e r o s u m . I t i s c o n c l u d e d t h a t t h e y a r e b o t h examples o f T. t u b e r o s u m s u b s p . s i l v e s t r e , and t h a t b e n z y l , 2 — p r o p y l and 2 — b u t y l i s o t h i o c y a n a t e s c h a r a c t e r i z e t h i s s u b s p e c i e s . G l u c o s i n o l a t e s , o f L e n i d i u m roeyenii B e c a u s e o f the l i m i t e d amount o f m a t e r i a l a v a i l a b l e d e t e r m i n a t i o n s c f t h e g l u c o s i n o l a t e s o f L e n i d i u m m e v e n i i a r e t e n t a t i v e . A n a l y s i s o f i s o t h i o c y a n a t e s l i b e r a t e d by an exogenous m y r o s i n a s e p r e p a r a t i o n i n d i c a t e t h a t t h e major c o n s t i t u e n t i s b e n z y l i s o t h i o c y a n a t e , w h i l e p — m e t h o x y b e n z l i s o t h i o c y a n a t e o c c u r s i n c o m p a r a t i v e l y s m a l l e r amounts. B o t h HPLC and p a p e r c h r o m a t o g r a p h y o f t h i o u r e a s s u p p o r t t h i s c o n c l u s i o n . A n a l y s i s o f 85 g l u c o s i n o l a t e s by p a p e r c h r o m a t o g r a p h y and TLC c o n s i s t e n t l y d e t e c t e d 2 c o n s t i t u e n t s . The m a j o r component c o — c h r o m a t o g r a p h e d w i t h t h e m a j o r ; g l u c o s i n c l a t e i n T. t u b e r o s u m s u b s p . t u b e r o s u m ( p — m e t h o x y b e n z y I ) • T h i s s p o t i s l i k e l y t o t e b e n z y l and p— m e t h o x y b e n z y I g l u c o s i n o l a t e s c o m b i n e d . The i d e n t i t y o f t h e o t h e r s p o t i s u n d e t e r m i n e d and may be a n o n — g l u c o s i n o l a t e g l y c o s i d e . A l t h o u g h no o t h e r t h i o u r e a d e r i v a t i v e s a p p e a r , o t h e r p eaks on t h e BPLC c o u l d p o s s i b l y c o r r e s p o n d t o a t h i r d g l u c o s i n o l a t e • However, u n t i l f u r t h e r work c a n be c a r r i e d out the summation o f t h e s e r e s u l t s i s t h a t b e n z y l and p — m e t h o x y b e n z y l g l u c o s i n o l a t e s have been d e t e c t e d i n r o o t s c f I., m g i e n i i • j o c a r b a m a t e s and t h i o u r e a s i n g l u e o s i n o l a t e - c o n t a i n i n g p l a n t s R e s u l t s o b t a i n e d from mass s p e c t r a l a n a l y s i s o f HPLC f r a c t i o n s i n d i c a t e t h e p r e s e n c e o f t h i o c a r b a m a t e s and t h i o u r e a s i n i s o t h i o c y a n a t e e x t r a c t s f r o m g l u c o s i n o l a t e — c o n t a i n i n g p l a n t s . I n b o t h c a s e s th e s t r u c t u r e o f t h e d e t e c t e d c o n s t i t u e n t s can e a s i l y be s e e n as a r e a c t i o n p r o d u c t o f two m o l e c u l e s o f t h e i s o t h i o c y a n a t e known from t h e p l a n t . N — ( 4 — h y d r o x y , b e n z y l ) m e t h y l t h i o c a r b a m a t e i s f o r m e d from p — h y d r o x y b e n z y I i s o t h i o c y a n a t e i n £i.r_aj=_is a l b a • One m o l e c u l e o f t h e OH—C^K^CBz— f r a g m e n t • f o r m e d by t h e l i b e r a t i o n o f t h i o c y a n a t e from t h e i s o t h i o c y a n a t e r e a c t s w i t h a m o l e c u l e o f t h e p a r e n t i s o t h i o c y a n a t e t o form the t h i o c a r b a m a t e • Two m o l e c u l e s o f p— m e t h o x y b e n z y I i s o t h i o c y a n a t e from T. t u b e r o s u m s u b s p . tuberosum 86 r e a c t to f orm N , N ,D I( me t hoxy . 4 - b e n z y l J t h i o u r e a . T h e s e two p r o d u c t s have not been w i d e l y r e p o r t e d from g l u c o s i n o l a t e — c o n t a i n i n g p l a n t s , but t h e i r d e t e c t i o n i n e x t r a c t s f r o m T.. t u b e r o s u m s u b s p * tuberosum and £>• a l b a c o n f i r m s t h e r e p o r t o f E l M i g i r a b e_t a l . ( 1 S 7 7 ) t h a t t h e y e x i s t . C o n v e n t i o n a l methods o f a n a l y s i s o f i s o t h i o c y a n a t e s u s i n g p a p e r c h r o m a t o g r a p h y o f t h i o u r e a s and GC—MS may n o t have d e t e c t e d t h e s e compounds even i f t h e y were p r e s e n t . T h e i r p r e s e n c e may have been o v e r l o o k e d b e c a u s e t h e y were n o t known to o c c u r . Whether t h e s e p r o d u c t s a r e n a t u r a l l y formed i n b i o l o g i c a l s y s t e m s , o r w h e t h e r t h e y a r e a r t i f a c t s o f t h e e x t r a c t i o n p r o c e d u r e c a n n o t be d e t e r m i n e d f r o m t h i s s t u d y . C h e m o t a x o n o m l c a l l y t h e i r p r e s e n c e i s n o t r e l e v a n t as t h e y g i v e no new i n f o r m a t i o n on t h e g l u c o s i n o l a t e s p r e s e n t . However, t h e i r b i o l o g i c a l a c t i v i t y i s l a r g e l y u n s t u d i e d . E l M i g i r a b et a l . ( 1 9 7 7 ) r e p o r t t h i o c a r t a m a t e s to be a n t i b i o t i c . I f t h e s e t h i o c a r b a m a t e s and t h i o u r e a s can be shown to be p r e s e n t i n l i v i n g s y s t e m s , t h e i r b i o l o g i c a l a c t i v i t y i n r e l a t i o n t o t h e r e p o r t e d e f f e c t s o f g l u c o s i n o l a t e — c o n t a i n i n g p l a n t s i s a r e l e v a n t c o n c e r n . E v a l u a t i o n , o f HPLC methods oj a n a l y s i s c f i s o t h i o c y a n a t e s The method of HPLC a n a l y s i s d e v e l o p e d i n t h i s s t u d y i s more c o n v e n i e n t and more s e n s i t i v e t h a n t h e c l a s s i c a p p r o a c h o f p a p e r c h r o m a t o g r a p h y of t h i o u r e a d e r i v a t i v e s . T h i s method was more s u c c e s s f u l i n c o n s i s t e n t l y d e t e c t i n g 2 — p r o p y l i s o t h i o c y a n a t e and 87 was d e c i d e d l y s u p e r i o r i n r e s o l v i n g p - m e t h o x y b e n z y l and b e n z y l i s o t h i o c y a n a t e s , the twc c o n s t i t u e n t s most i m p o r t a n t i n t h i s s t u d y . I t i s d i f f i c u l t to e v a l u a t e the method i n r e l a t i o n t o t h e GC—Mass S p e c t r a l method o f a n a l y s i s as no d i r e c t c o m p a r i s o n s were a t t e m p t e d . When s t a n d a r d s a r e a v a i l a b l e HPLC i s p r o b a b l y c o m p a r a b l e i n r e s o l u t i o n w i t h GC. The r e l a t i v e l y low e x t i n c t i o n c o e f f i c i e n t o f i s o t h i o c y a n a t e s s u g g e s t s t h a t HPLC i s l e s s s e n s i t i v e i n d e t e c t i n g t h e s e compounds. The mass s p e c t r o m e t r y f a c i l i t y o f a GC-MS u n i t would g e n e r a l l y make t h i s s y s t e m more u s e f u l . A l i a b l i t y o f HPLC, i s t h a t f o r f u r t h e r a n a l y s i s by mass s p e c t r o m e t r y , f r a c t i o n s must be l a b o r i o u s l y c o l l e c t e d and t h e n the s o l v e n t e v a p o r a t e d . W i t h low b o i l i n g p o i n t compounds s u c h as 2 — p r o p y l i s o t h i o c y a n a t e t h i s method would be u n s u c c e s s f u l . W i t h s t a b l e compounds, however, p r e p a r a t i v e HPLC p r o v i d e s a c o n v e n i e n t method f o r i s o l a t i o n of compounds o f h i g h p u r i t y . The p o s s i b i l i t y of u s i n g f u r t h e r a n a l y t i c a l t o o l s s u c h as NMR, and o f h a v i n g m a t e r i a l a v a i l a b l e f o r b i o l o g i c a l work i s t h e r e f o r e e n h a n c e d • S y s t e m a t i c s o f T r o p a e o l u m tuberosum A n a l y s i s o f t h e g l u c o s i n o l a t e s o f T r o p a e o l u m tufoe rosum s u p p o r t s t h e a s s e s s m e n t c f two d i s t i n g u i s h a b l e s u b s p e c i e s w i t h i n t h e s p e c i e s ( S p a r r e , 1 £ 7 3 ) . T h i s s t u d y has d e t e r m i n e d t h a t p-m e t h o x y b e n z y I g l u c o s i n o l a t e i s t h e c h a r a c t e r i s t i c c o n s t i t u e n t o f 88 s u b s p . ±vjbero.su.tji and c o n f i r m e d t h a t b e n z y l , 2 - p r o p y l and 2 - b u t y l g l u c o s i n o l a t e s c h a r a c t e r i z e s u b s p . _gi.Aves.txe as d e t e r m i n e d by K j a e r e t a.l« ( 1 9 7 8 ) . The chromosome number o f " k l p a i s a n o ' i s 2n>42. I f x=13, as i s t y p i c a l f o r the s e c t i o n M u c r o n a t a o f t h i s genus ( G i b b s _et a l . , 1978 ), t h e n t h i s c l o n e o f T_. t u b e r o s u m s u b s p . s i l v e s t r e i s p o s s i b l y a t e t r a p l o l d w i t h 2n e q u a l t o 52 a s f o r s u b s p . Jtujbexo^um • However, more t h o r o u g h i n v e s t i g a t i o n o f t h e chromosome number i n c l u d i n g t h e e x a m i n a t i o n o f m e i o t i c t i s s u e i s n e c e s s a r y to r u l e o u t t h e p o s s i b i l i t y t h a t * k i p a i s a n o * i s a t r i p l o i d ( 2 n = 3 9 ) . I f T. tuberosum s u b s p . s i I v e s t r e i s a t e t r a p l o l d or a t r i p l o i d , the p d o i d y l e v e l and t h e d i f f e r e n c e i n c h e m i s t r y r u l e o u t an a u t o t e t r a p l o i d o r i g i n o f s u b s p . t ub e ro sum w i t h s u b s p . s i l v e s t r e as t h e p r o g e n i t o r , p— M e t h o x y b e n z y l g l u c o s i n o l a t e i s a c o n s t i t u e n t of s e v e r a l members o f t h e genus J i o j o a e c l u m i n c l u d i n g "J,r opaeo lum c o c habambe ( K j a e r e_t a j , . , 1978 ), a s p e c i e s t h a t S p a r r e ( 1973 ) s u g g e s t s as h a v i n g t a x o n o m i c a f f i n i t y w i t h J . t u b e r o s u m • The s u g g e s t i o n t h a t subsp* t ub e r o s u n a r o s e as a h y b r i d from cochabambe ( o r a n o t h e r s p e c i e s ) and T. tuberosum s u b s p . s i l v e s t r e ( G i b b s e_t a l . , 1978) c a n be s u p p o r t e d by t h e s e r e s u l t s . However T. cochabambe has been r e p o r t e d t o have a chromosome c o u n t o f 2n=26 (Huynh, 1967), and t h e r e f o r e would not h y b r i d i z e w i t h a t e t r a p l o l d s u b s p e c i e s c i T ropae olum Jijbjgrosxjm t o f o r m t e t r a p l o l d p r o g e n y . I f t h e c o l l e c t i o n s t u d i e d i s n o t t y p i c a l and d - i p l o i d s t r a i n s o f s u b s p . s i I v e s t r e do e x i s t , an a l l o t e t r a p l o i d o r i g i n o f t h e c u l t i v a r w i t h s u b s p . s i l v e s t r e a n d / o r T• cochabambe a s p r o g e n i t o r s i s s t i l l a p o s s i b i l i t y . The l i m i t e d sample s i z e o f s u b s p . s l i v e s t r e makes i t i m p o s s i b l e t o r u l e o u t t h e e x i s t e n c e 89 o f p o p u l a t i o n s w i t h c h e m i c a l d i f f e r e n c e s . S t r a i n s which p r o d u c e p - m e t h o x y b e n z y I g l u c o s i n o l a t e c o u l d have g i v e n r i s e t o s u b s p . t u b e r o s u m t h r o u g h s e l e c t i o n . F u r t h e r s y s t e m a t i c s t u d i e s o f the chromosome numbers and t h e c r o s s i n g c a p a b i l i t i e s w i t h i n t h e genus Tj:ojiae_gl_.uj. would be n e c e s s a r y t o make more c o n c l u s i v e s t a t e m e n t s a b o u t t h e e x a c t o r i g i n o f t h e c u l t i v a t e d s u b s p e c i e s o f T. tube rosum . An o b v i o u s a s s u m p t i o n o f t h e above d i s c u s s i o n i s t h a t s u b s p . t u b erpsum a r o s e from s u b s p . s i I v e s t r e r a t h e r t h a n v i c e v e r s a . G e n e r a l l y i t i s assumed t h a t c u l t i v a t e d f o r m s a r i s e from w i l d f o r m s , b u t the p o s s i b i l i t y t h a t "the w i l d s u b s p e c i e s a r o s e f r o m t h e h y b r i d i z a t i o n o f s u b s p . tuberqsum w i t h a n o t h e r t a x o n c a n n o t be r u l e d o u t . The s o — c a l l e d w i l d s u b s p e c i e s may i n f a c t be p a r t o f a crop—weed complex ( H a r l a n d , 1 9 75). ' K i p a i s a n o ' does e x i s t i n Cuyo-cuyo i n r e l a t i v e l y c l o s e p r o x i m i t y to f i e l d s o f the c u l t i v a r and h y b r i d i z a t i o n between i t and c u l t i v a t e d p l a n t s may have o c c u r r e d i n the p a s t o r be o n g o i n g . A s t e r i l e t r i p l o i d would be t h e l i k e l y p r o d u c t o f a h y b r i d i z a t i o n between J_. cochabambe and J. • t u b e rosum • ' K i p a i s a n o ' s e t s s e e d s from the m a j o r i t y of i t s f l o w e r s when grown i n V a n c o u v e r ) a l t h o u g h t h e s e e d was not t e s t e d f o r v i a b i l i t y . D i f f e r e n c e s i n the t u b e r s o f T. t u b e rosum s u b s p . s i l v e s t r e ( ' k i p a i s a n o * ) and T. tuberosum s u b s p . tuberosum s u p p o r t t h e s e p a r a t i o n o f a w i l d and a c u l t i v a t e d s u b s p e c i e s . T u b e r s o f ' k i p a i s a n o * ( F i g u r e 9 ) a r e more e l o n g a t e d i n s h a p e , and were o b s e r v e d i n t h e f i e l d and i n c u l t i v a t i o n t o have l o n g e r s t o l o n s . T u b e r s o f s u b s p . s i I v e s t r e s p r o u t r e a d i l y even when k e p t i n c o l d s t o r a g e . S p r o u t i n g o c c u r s from t h e apex o f t h e t u b e r , and many o f t h e t u b e r s a p p e a r to have an apex t h a t i s c a p a b l e o f 90 c o n t i n u e d g r o w t h . The w i l d s u b s p e c i e s i s b e t t e r a d a p t e d f o r v e g e t a t i v e p r o p a g a t i o n and d i s p e r s a l t h a n i s the o b l i g a t e c u l t i v a r • The r o l e o f human i n t e r v e n t i o n i n t o the o r i g i n and s e l e c t i v i t y of the c u l t i v a t e d s u b s p e c i e s i s a f a c t o r t h a t i s d i f f i c u l t t o a s s e s s . S e l e c t i o n o f s h o r t s t o l o n l e n g t h and e n l a r g e d t u b e r s w i t h g r e a t e r dormancy i s e x p e c t e d . S e l e c t i o n f o r t h e c h e m i c a l c o n t e n t o f t h e c u l t i v a r i n r e l a t i o n t o c u l t u r a l c o n c e p t s o f consumers i s a p o s s i b i l i t y . However, p - m e t h o x y b e n z y I g l u c o s i n o l a t e i n s u b s p . t u b e r o s u m c o u l d n o t have been s e l e c t e d f o r i f p l a n t s w i t h g e n e t i c c a p a b i l t i y to p r o d u c e i t were unknown. I f s u b s p . tuherosum d i d a r i s e from s u b s p . s i l v e s t r e , i t must have done so t h r o u g h h y b r i d i z a t i o n o r f r o m v a r i e t i e s w h i c h p r o d u c e p - m e t h o x y b e n z y l g l u c o s i n o l a t e . E t h n o p h a r m a c o l o e y o f T r c p a e o l u m t u b e r o s u m arjd L e p i d i u m meyeni j. The e t h n o b o t a n i c a l u s e s o f T r o c a e o l u m tuberosum and L e p i d j u m m e y e n i 1 a r e c o n s i s t e n t w i t h the p a t t e r n s shown by t h e s u r v e y of a l l g l u c o s i n o l a t e — c o n t a i n i n g p l a n t s . The use o f b o t h o f t h e s e s p e c i e s t o a f f e c t human r e p r o d u c t i o n c o r r e s p o n d s t o a s i g n i f i c a n t use f o r g l u c o s i n a l a t e — c o n t a i n i n g p l a n t s i n ways t h a t r e f l e c t some e f f e c t cn r e p r o d u c t i v e h o r m o n a l p r o c e s s e s . T. t u b e r o s u m , i s used as w e l l to a f f e c t k i d n e y c o n d i t i o n s and as an e x p e c t o r a n t , b o t h m e d i c i n a l u s e s t h a t a r e s i g n i f i c a n t l y g r e a t e r i n the s u r v e y s ample. The use o f t u b e r s c f T. t u b e r o s u m 91 t o t r e a t s h i n c o n d i t i o n s p a r a l l e l s a p o s i t i v e ( a l t h o u g h n o t s i g n i f i c a n t ) s e l e c t i o n f o r g l u c o s i n o l a t e — c o n t a i n i n g p l a n t s t o t r e a t s u c h c o n d i t i o n s . T h i s s t a t i s t i c a l s u p p o r t f o r t h e m e d i c i n a l u s e s o f X* tuberosum and L• meyeni i has a p h a r m a c o l o g i c a l b a s i s i n some c a s e s * The s i m i l a r i t y i n the c h e m i s t r y of T* tube r o sum subsp* t u b e rosum and L . meyeni i i n d i c a t e s t h a t , a t l e a s t i n t h e c o n c e p t i o n o f Andean p e o p l e s , t h e r e i s a r e l a t i o n s h i p between t h e p r e s e n c e o f a r o m a t i c g l u c o s i n o l a t e s and human r e p r o d u c t i v e p r o c e s s e s * The d e c r e a s e i n t e s t c s t e r o n e / d i h y d r o t e s t o s t e r o n e l e v e l s i n r a t s f e d t i t e r s o f T« tub e rosum s u b s p * t u b e rosum s u p p o r t s t h e b e l i e f s a t t a c h e d t o T. tuberosum as an a n t i -r e p r o d u c t i v e a g e n t * The f a c t t h a t e x p e r i m e n t a l r a t s were as c a p a b l e and a p p a r e n t l y as i n c l i n e d t o w a r d s i m p r e g n a t i n g f e m a l e s as were c o n t r o l s c o n t r a d i c t s t h e use o f the p l a n t as an a n t i -a p h r o d i s i a c f o r males* V a r y i n g t e s t o s t e r o n e l e v e l s do n o t have an e f f e c t on s e x u a l b e h a v i o u r i n mammals below a t h r e s h o l d l e v e l c o n s i d e r a b l y l e s s than t h a t s e e n i n t h i s s t u d y ( G o r z a l k a and Mogenson, 1977)* T e s t o s t e r o n e l e v e l s do a f f e c t sperm c o u n t ( P u r v i s e_t aj_* » 197E), a l t h o u g h no o b v i o u s e f f e c t on s p e r m a t o g e n i s i s i s e v i d e n t from t h i s i n v e s t i g a t i o n . The l a c k o f a s i g n i f i c a n t d i f f e r e n c e i n t h e a n d r o g e n l e v e l s o f e x p e r i m e n t a l and s t a r v e d a n i m a l s i n d i c a t e s t h a t X* tuberosum e x e r t s i t s e f f e c t on b a s i c m e t a b o l i s m and n o t by d i r e c t l y a f f e c t i n g a n d r o g e n p r o d u c t i o n . The f a c t t h a t t h e e x p e r i m e n t a l r a t s d i d n o t g a i n w e i g h t and t h e d r o p i n t e s t o s t e r o n e a s s o c i a t e d w i t h t h i s i s l i k e l y a r e s u l t o f t h e known a n t i m e t a b o l i c and c o u n t e r — n u t r i t i o n a l a c t i v i t y o f i s o t h i o c y a n a t e s . The r a t e o f f o o d c o n s u m p t i o n by e x p e r i m e n t a l a n i m a l s , a l t h o u g h n o t 92 n o t i c e a b l y l e s s t h a n t h a t o f c o n t r o l s , was n o t d e t e r m i n e d and l a c k o f w e i g h t g a i n may t o some e x t e n t be r e l a t e d t o t h e a n i m a l s ' a v e r s i o n t o the I s o t h i o c y a n a t e s c o n t a i n e d i n the f o o d * C o o k i n g the t u b e r s d o e s n o t d e s t r o y t h e g l u c o s i n o l a t e s * W h i l e c o o k i n g i m p r o v e s t h e p a l a t a b i l l t y of t h e f o o d ( p a r t i c u l a r l y i f l a r g e q u a n t i t i e s a r e consumed) by p r e v e n t i n g e n z y m a t i c p r o d u c t i o n of i s o t h i o c y a n a t e s i n t h e mouth, s p o n t a n e o u s and b a c t e r i a l h y d r o l y s i s can s t i l l l i b e r a t e i s o t h i o c y a n a t e s and o t h e r breakdown p r o d u c t s . The d r o p i n 1 7 — k e t o s t e r o i d l e v e l s i n u r i n e o f r a t s t e d a l l y l i s o t h i o c y a n a t e ( l i u z t a r e t a l . , 1979b) may be a r e s u l t o f an e f f e c t on a n d r o g e n s s i m i l a r t o t h a t s e e n i n t h i s e x p e r i m e n t . M u z t a r e t a l . ( 1 9 7 9 b ) make no m e n t i o n o f the e f f e c t o f a l l y l i s o t h i o c y a n a t e on w e i g h t g a i n * The use o f a s t a r v a t i o n i n d u c i n g f a c t o r as an a n t i a p h r o d i s i a c seems r a t h e r d r a s t i c and c o u n t e r p r o d u c t i v e i n m a i n t a i n i n g an e f f e c t i v e army* R e p o r t s o f t h e u s e o f ' i s a n o * by th e I n c a s i n s u c h a way a r e u n s u b s t a n t i a t e d by t h i s s t u d y * However, t h e s e e x p e r i m e n t a l r e s u l t s do s u p p o r t t h e e m p i r i c a l b a s i s f o r Ixsjae OJLUJS tuberosum p r o d u c i n g t h e d e c r e a s e i n r e p r o d u c t i v e p o t e n t i a l r e p o r t e d by t h e c h r o n i q u i l i s t s and modern o b s e r v e r s * The i d e a l male c o n t r a c e p t i v e would d e c r e a s e s p e r m a t o g e n e s i s w h i l e m a i n t a i n i n g l i b i d o . The c u r r e n t c o n c e r n i n f i n d i n g a u s e a b l e male c o n t r a c e p t i v e would w a r r a n t f u r t h e r i n v e s t i g a t i o n i n t o t h e b e h a v i o u r a l and b i o c h e m i c a l a c t i o n s o f T r o p a e o l u m t u b e r o s u m and i s o t h i o c y a n a t e s * The g e n e r a l a n t i m e t a b o I i c and a n t i n u t r 1 t i o n a l a c t i v i t i e s o f i s o t h i o c y a n a t e s a r e n o t w e l l u n d e r s t o o d , and f u r t h e r s t u d y i n t h i s a r e a m i g h t l e a d t o g r e a t e r u n d e r s t a n d i n g o f s p e c i f i c b i o c h e m i c a l c h a n g e s , s u c h as t h e e f f e c t on t e s t o s t e r o n e l e v e l s * 93 The a n t i m e t a b o I i c e f f e c t s o f i s o t h i o c y a n a t e s a r e n o t sex s p e c i f i c and wo u l d a f f e c t f e m a l e s a d v e r s e l y as w e l l . I t i s not known whether t h e y a f f e c t f e m a l e hormonal l e v e l s . F e m a l e f e r t i l i t y i s o f t e n a s s o c i a t e d w i t h m e n s t r u a t i o n i n f o l k b e l i e f s (Conway and Slocumb, 1979) and, as shown i n t h e s t a t i s t i c a l s u r v e y , b e l i e f s o f enmenagogic e f f e c t s a r e the l a r g e s t c o n t r i b u t e r s t o t h e t s e of g I u c o s i n o l a t e — c o n t a i n i n g p l a n t s i n a f f e c t i n g human r e p r o d u c t i o n . T h i s s t u d y f o u n d no c o n c l u s i v e e x p e r i m e n t a l e v i d e n c e to s u p p o r t t h e s e b e l i e f s . A d i e t c o n t a i n i n g t u b e r s of T. tuberosum s u b s p . tub erosum d i d n o t a f f e c t t h e r e g u l a r p e r i o d i c i t y of t h e e s t r u s c y c l e i n t h e g u i n e a p i g . A l t h o u g h j_n v i t r o s t u d i e s on e x t r a c t s o f J.. t u b e r o s u m and S i n a p i s a l b a showed a q u a 1 1 t i t a t i v e i n h i b i t i o n o f e s t r a d i o l b i n d i n g , q u a n t i t a t i v e l y i s o t h i o c y a n a t e s d i d not c o m p e t i t i v e l y I n h i b i t e s t r a d i o l b i n d i n g . N,N,Di(methoxy, 4 — b e n z y l ) t h i o u r e a e x t r a c t e d f r o m t u b e r s o f T. tuberosum s u b s p . t u b e r o s u m does s i g n i f i c a n t l y i n h i b i t e s t r a d i o l b i n d i n g . However, the l a c k o f i n f o r m a t i o n on t h e i m p o r t a n c e , o r t h e e x i s t e n c e , of t h i s compound i n b i o l o g i c a l s y s t e m s ( e i t h e r p l a n t o r a n i m a l t i s s u e ) , and t h e one time n a t u r e o f the e x p e r i m e n t make i t i m p o s s i b l e t o draw m e a n i n g f u l c o n c l u s i o n s as t o the l i k e l i h o o d o f t h i s compound h a v i n g e s t r o g e n i c a c t i v i t y i n g l u c o s i n o l a t e — c o n t a i n i n g p i a n t s . G u i n e a p i g s a r e known t c m e t a b o l i z e i s o t h i o c y a n a t e s d i f f e r e n t l y t h a n r a t s o r man and i t i s p o s s i b l e t h a t the f e e d i n g e x p e r i m e n t r e p e a t e d w i t h a n o t h e r t e s t o r g a n i s m w o u l d p r o d u c e a p o s i t i v e r e s u l t . T h i s u n c e r t a i n t y , the a m b i g u i t y s u r r o u n d i n g N , N , D i (methcxy, 4 - b e n z y I D t h i o u r e a , and the s p e c i f i c n a t u r e o f t h e e x p e r i m e n t s do n o t r u l e o u t the p o s s i b i l i t y t h a t o t h e r 94 e x p e r i m e n t s m i g h t show r e s u l t s t h a t would s u p p o r t the f o l k b e l i e f s r e l a t i n g t o f e m a l e r e p r o d u c t i v e p r o c e s s e s * F o l k u s e s o f T. tuberosum t h a t c o u l d be p r o d u c e d by t o x i c i t y t o w a r d s l o w e r o r g a n i s m s a r e w e l l s u p p o r t e d p h a r m a c o l o g i c a l l y * The a n t i b i o t i c a c t i v i t y of p — m e t h o x y b e n z y I i s o t h i o c y a n a t e i s a s h i g h o r h i g h e r t han t h a t o f b e n z y l i s o t h i o c y a n a t e a g a i n s t s e v e r a l s p e c i e s o f y e a s t and b a c t e r i a * The t r e a t m e n t o f some s k i n a i l m e n t s and p o s i t i v e e f f e c t s on t h e u r i n a r y s y s t e m c o u l d be e x p l a i n e d by a n t i b i o t i c a c t i v i t y ( s e e L i t e r a t u r e R e v i e w ) * The n e m a t o c i d a l a c t i v i t y o f b e n z y l i s o t h i o c y a n a t e shown heire s u p p o r t s the r e p o r t s (Gommers, 1973 ) t h a t g l u c o s i n c l a t e — c o n t a i n i n g p l a n t s a r e t o x i c t o nematodes* A n t i b i o t i c and n e m a t o c i d a l a c t i v i t i e s c o u l d b o t h c o n t r i b u t e t o t h e r e p u t e d c r o p p r o t e c t i v e b e n e f i t s o f i n t e r p l a n t i n g w i t h T. t u b e r o s u m s u b s p * tu berosum • I n s e c t i c i d a l u s e s o f T. t u b e r o s u m a r e s u p p o r t e d by s t u d i e s on o t h e r g l u c o s i n o l a t e — c o n t a i n i n g p l a n t s ( B l a u _g_t _a_l. » 1978 )* The f o l k b e l i e f s cn t h e e f f e c t s o f T. t u b e r o s u m on t h e k i d n e y s c o r r e s p o n d s t o the h i g h u s e o f g l u c o s i n o l a t e — c o n t a i n i n g p l a n t s a s d i u r e t i c s * The d i u r e t i c e f f e c t o f a l l y l i s o t h i o c y a n a t e shown by M u z t a r e t aj.* ( 1979b) s u p p o r t s t h e s e f o l k u s e s . O b l i t a s P o b l e t e ( 1969 ) r e p o r t s t h e use o f T* tu b e rosum a s an e x p e c t o r a n t and g l u c o s i n o l a t e — c o n t a i n i n g p l a n t s i n g e n e r a l a r e u s e d f o r t h i s p u r p o s e . The p h a r m a c o l o g i c a l b a s i s f o r s u c h use i s u n d e t e r m i n e d . The use of m u s t a r d i n p l a s t e r s t o t r e a t c o l d s and c h e s t c o n d i t i o n s r e l a t e s t o the i r r i t a n t e f f e c t o f i s o t h i o c y a n a t e s on t h e s k i n . T h i s c o u n t e r — i r r i t a n t e f f e c t may c o n t r i b u t e t c t h e r e p u t e d e x p e c t o r a n t p r o p e r t i e s o f I s o t h i o c y a n a t e s • A l t h o u g h a n t i s c o r t u t i c u s e s o f T. tuberosum a r e n o t known. 95 •the i m p o r t a n t u s e o f g l u e os i n o l a t e — c o n t a i n i n g p l a n t s f o r s u c h a p u r p o s e i s s u b s t a n t i a t e d i n T. tuberosum by t h e h i g h l e v e l s o f a s c o r b i c a c i d r e p o r t e d from the t u b e r s . The use o f T. tuberosum f o r t h i s p u r p o s e would he w a r r a n t e d . The n u t r i t i o n a l b e n e f i t s o f t h e h i g h a s c o r b i c a c i d l e v e l s of t u b e r s o f T. tu b e r o s u m have been d i s c u s s e d . From t h e above d i s c u s s i o n i t c a n been s e e n t h a t the n u t r i t i o n a l and m e d i c i n a l u s e s o f T r o p a e o l u m tuberosum and L e p i d i u m meveni i by human b e i n g s l i v i n g i n the Andes m o u n t a i n s have i n most c a s e s some p h y s i c a l b a s i s . The c o n t r i b u t i o n o f t h e s e p l a n t s to t h e w e l l b e i n g o f t h e s e g r o u p s o f p e o p l e i s r e a l and t h e i r u t i l i z a t i o n by t h e s e p e o p l e s g e n e r a l l y r a t i o n a l , p a r t i c u l a r l y i f t a k e n i n the c o n t e x t o f the n a t i v e e t i o l o g y o f d i s e a s e ( C r t i z de M o n t e U a n o , 19*75). The d o m e s t i c a t i o n o f T. t u b e r o s u m s u b s p . tuberosum i n t h e Andes i s e x p l a i n a b l e by t h e I n d i a n s ' l o n g s t a n d i n g c o n c e r n f o r b o t h the n u t r i t i o n a l and m e d i c i n a l p r o p e r t i e s o f i t s t u b e r s . The s t r o n g c o r r e l a t i o n between t h e s t a t i s t i c a l l y s i g n i f i c a n t u s e s o f g l u c o s i n o l a t e — c o n t a i n i n g p l a n t s , and t h e p h a r m a c o l o g i c a l b a s i s f o r t h e s e u s e s as b o r n e o u t i n t h e s t u d y o f T r o p a e o l u m tut) erosum s u b s p . tuberosum and L e p i d i u m meveni i, , d e m o n s t r a t e s t h e v a l i d i t y and p o t e n t i a l o f a s y s t e m a t i c a p p r o a c h t o t h e e v a l u a t i o n o f e t h n o b o t a n i c a l r e p o r t s i n g e n e r a l . The t a b u l a t i o n and s t a t i s t i c a l a n a l y s i s o f e t h n o b o t a n i c a l d a t a i s g r e a t l y s i m p l i f i e d u s i n g modern c o m p u t i n g methods. I f s u b s e q u e n t p h y t o c h e m i c a l and p h a r m a c o l o g i c a l i n v e s t i g a t i o n s c o n c e n t r a t e on s i g n i f i c a n t l y p o s i t i v e c o r r e l a t i o n s , the c h a n c e s o f d e t e c t i n g a c t i v e c o n s t i t u e n t s c a n be i n c r e a s e d . The a s s o c i a t i o n o f f o l k u s e s , s t a t i s t i c a l p a t t e r n s and p h y t o c h e m i s t r y i n t h i s s t u d y 96 i n d i c a t e s t h a t p h a r m a c o l o g i c a l a c t i v i t y c f known p h y t o c h e m i c a I c o n s t i t u e n t s c c u l d be p r e d i c t e d by a n a l y s i s o f the r e l a t i o n s h i p between e t h n o b o t a n i c a l use and p h y t o c h e m i s t r y • A d a t a bank c o m p r i s i n g e t h n o b o t a n i c a l r e p o r t s as w e l l a s p h y t o c h e m i c a l , chenio taxoncm i c and p h a r m a c o l o g i c a l i n f o r m a t i o n would be a v a l u a b l e t o o l i n i n t e r p r e t i n g r e s e a r c h r e s u l t s and i n i n c r e a s i n g t h e p r e d i c a b i I i t y c f r e s u l t s whether one a p p r o a c h e s e t h n o p h a r m a c o l o g i c a I p r o b l e m s f r o m the d i r e c t i o n o f e t h n o b o t a n y , p h y t o c h e m i s t r y or p h a r m a c o l o g y . 97 l i t e r a t u r e C i t e d A c t a E n d o c r l n o l o g i a . 1S79. 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