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UBC Theses and Dissertations

Analytical and biological studies of sesquiterpene lactones 1981

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ANALYTICAL AND BIOLOGICAL STUDIES OF SESQUITERPENE LACTONES by ANNA KRYSTINA PICMAN Dip loma, C h a r l e s ' U n i v e r s i t y in Prague, C z e c h o s l o v a k i a , 1971 M.Sc . , U n i v e r s i t y of B r i t i s h Co lumbia , 1977 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Botany We accept t h i s t h e s i s as conforming to the r e q u i r e d s tandard THE UNIVERSITY OF BRITISH COLUMBIA January 1981 (c) Anna K r y s t i n a Pieman In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library 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 reference and study. I further agree that permission for extensive copying of t h i s thesis for s c h o l a r l y purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or pu b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date T^dUut^t, 4/ /9<f/ ABSTRACT Three r e l a t e d a n a l y t i c a l and b i o l o g i c a l s t u d i e s of s e s q u i t e r p e n e l a c t o n e s main ly from Compositae were conduc ted . In the f i r s t one I examined v a r i o u s t h i n - l a y e r chromatography (TLC) t e c h n i q u e s for i d e n t i f i c a t i o n of s e s q u i t e r p e n e l a c t o n e s and p o l . y a c e t y l e n e s . V a n i l l i n and p-d imethy laminobenza ldehyde were found to be most u s e f u l of s e v e r a l benzaldehyde and benzo ic a c i d d e r i v a t i v e s fo r the d e t e c t i o n of these compounds. C o l o r s produced wi th these two reagents and with 78 s e s q u i t e r p e n e l a c t o n e s and 25 p o l y a c e t y l e n e s were of a l l shades of the spectrum and were s p e c i f i c for i n d i v i d u a l compounds. T h e r e f o r e , these reagents can be used as an a i d in the i d e n t i f i c a t i o n of these compounds by TLC. These spray reagents are the most s e n s i t i v e of a l l reagents which have so f a r been used fo r the d e t e c t i o n of s e s q u i t e r p e n e l a c t o n e s or p o l y a c e t y l e n e s us ing TLC. • The v a n i l l i n spray reagent was used in the second study for the d e t e c t i o n and p r e l i m i n a r y i d e n t i f i c a t i o n of major s e s q u i t e r p e n e l a c t o n e s in crude e x t r a c t s o f ' 29 samples of Parthen ium hys te rophorus r e p r e s e n t i n g specimens from v a r i o u s areas of i t s world d i s t r i b u t i o n . Some s e s q u i t e r p e n e l a c t o n e s d e t e c t e d by t h i s techn ique were subsequent ly i s o l a t e d and a l s o i d e n t i f i e d by NMR a n a l y s e s . Three s e s q u i t e r p e n e l a c t o n e s , c o r o n o p i l i n , h y s t e r i n , and d i h y d r o i s o p a r t h e n i n , were shown for the f i r s t time to occur in t h i s s p e c i e s . . i i i The chemica l a n a l y s e s of 10 s e l e c t e d compounds from 29 samples i n d i c a t e d the e x i s t e n c e of 11 chemica l types of P. h y s t e r o p h o r u s . The most widespread i s the North American type which i s a l s o p resent in B e l i z e , A u s t r a l i a , and I n d i a . However, the South American samples d i f f e r g r e a t l y in t h e i r s e s q u i t e r p e n e l a c t o n e compos i t i on from the North American p l a n t s . In a d d i t i o n , the comparison of samples from v a r i o u s South American p o p u l a t i o n s showed a h igh degree of d i v e r s i t y in c h e m i s t r y . These r e s u l t s suggest the p o s s i b i l i t y of the e x i s t e n c e of e i t h e r s e v e r a l forms, or s u b s p e c i e s , or perhaps even s e v e r a l s p e c i e s of t h i s p l a n t . In the t h i r d study I examined the r e l a t i o n s h i p between v a r i o u s b i o l o g i c a l a c t i v i t i e s of s e s q u i t e r p e n e l a c t o n e s and t h e i r chemica l s t r u c t u r e s . A n t i m i c r o b i a l s c r e e n i n g t e s t s were performed with. 57 s e s q u i t e r p e n e - l a c t o n e s a g a i n s t 6 b a c t e r i a and 45 s e s q u i t e r p e n e l a c t o n e s a g a i n s t 3 f u n g i . The presence of c e r t a i n s t r u c t u r a l m o i e t i e s in the compounds seems to be necessary for a n t i m i c r o b i a l a c t i v i t i e s . However, d i f f e r e n t f u n c t i o n a l groups appear to be r e q u i r e d fo r the d i f f e r e n t s k e l e t a l c l a s s e s of s e s q u i t e r p e n e l a c t o n e s and fo r d i f f e r e n t types of m ic roorgan i sms . In a d d i t i o n , some other f u n c t i o n a l groups appear to enhance a c t i v i t y whereas o thers reduce a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s . F i n a l l y , the c o n f i g u r a t i o n of some f u n c t i o n a l groups a lone may s i g n i f i c a n t l y i n f l u e n c e the a c t i v i t y of c e r t a i n s e s q u i t e r p e n e l a c t o n e s . These r e s u l t s i n d i c a t e that there i s no s imple g e n e r a l r e l a t i o n s h i p between the a n t i m i c r o b i a l a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s and t h e i r chemica l s t r u c t u r e . i v Four s t r u c t u r a l l y s i m i l a r s e s q u i t e r p e n e l a c t o n e s were t e s t e d for t h e i r e f f e c t s on s u r v i v a l of f l o u r b e e t l e s . Three compounds c o n t a i n i n g the e x o c y c l i c methylene group on the l a c t o n e r i n g ( c o r o n o p i 1 i n , h e l e n a l i n , and p a r t h e n i n ) s i g n i f i c a n t l y reduced s u r v i v a l of b e e t l e s , whereas t e n u l i n , l a c k i n g t h i s moiety , had no e f f e c t s . T h e r e f o r e , the e x o c y c l i c methylene i s most l i k e l y r e s p o n s i b l e fo r the d e t r i m e n t a l p r o p e r t i e s of these s e s q u i t e r p e n e l a c t o n e s on s u r v i v a l of the b e e t l e s . Many s e s q u i t e r p e n e l a c t o n e s are p o t e n t i a l a l l e r g e n s . In the l a s t study c r o s s - r e a c t i v i t y to 6 s t r u c t u r a l l y s i m i l a r s e s q u i t e r p e n e l a c t o n e s of p a r t h e n i n s e n s i t i z e d guinea p i g s was examined. The presence of the exomethylene on the l a c t o n e r i n g was found to be necessary fo r c r o s s - r e a c t i v i t y . However, compounds which were p r o g r e s s i v e l y s t r u c t u r a l l y more d i f f e r e n t than p a r t h e n i n , gave weaker a l l e r g e n i c r e s p o n s e s . V TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES ix LIST OF FIGURES . . . x i i i ACKNOWLEDGEMENTS xv PREFACE 1 1. Chemistry of s e s q u i t e r p e n e l a c t o n e s • ••• 1 2. D i s t r i b u t i o n of s e s q u i t e r p e n e l a c t o n e s 3 SECTION I . V i s u a l i z a t i o n reagents for s e s q u i t e r p e n e l a c t o n e s and p o l y a c e t y l e n e s on t h i n l a y e r chromatograms 7 I n t r o d u c t i o n 7 Exper imenta l 9 (1) Chemica ls 9 (2) Spray reagents 10 (3) TLC p l a t e s 10 (4) Reagent s e n s i t i v i t y t e s t 11 (5) Test of v a r i o u s reagents for t h e i r r e a c t i o n s wi th s e s q u i t e r p e n e l a c t o n e s 12 R e s u l t s and D i s c u s s i o n 13 SECTION I I . Comparat ive study of the major s e s q u i t e r p e n e l a c t o n e s and other s e l e c t e d compounds in v a r i o u s p o p u l a t i o n s of P. hys te rophorus 31 I n t r o d u c t i o n 31 E x p e r i m e n t a l 35 v i (1) P l a n t m a t e r i a l 35 (2) Chemica ls and Instruments 35 (3) Th in l a y e r chromatography 36 (4) Chromatographic i d e n t i f i c a t i o n of p a r t h e n i n and hymenin 36 (5) I s o l a t i o n and i d e n t i f i c a t i o n of s e s q u i t e r p e n e l a c t o n e s from p l a n t m a t e r i a l . . . 36 (a) P a r t h e n i n and c o r o n o p i l i n 36 (b) Hymenin 38 (c) H y s t e r i n 39 (6) P r e p a r a t i o n of d i h y d r o i s o p a r t h e n i n and t e t r a h y d r o p a r t h e n i n 39 (7) P h o t o l y s i s of p a r t h e n i n and c o r o n o p i l i n 40 (8) D e t e c t i o n of s e s q u i t e r p e n e l a c t o n e s in p l a n t m a t e r i a l 41 R e s u l t s and D i s c u s s i o n 43 C o n c l u s i o n s 52 SECTION III- B i o l o g i c a l a c t i v i t i e s of s e s q u i t e r p e n e l a c t o n e s 68 I n t r o d u c t i o n 68 (1) A n t i m i c r o b i a l a c t i v i t y 68 (2) A l l e l o p a t h i c a c t i v i t y 69 (3) A n t i h e l m i n t h i c a c t i v i t y and chemoprophy lax is in s c h i s t o s o m i a s i s 71 (4) E f f e c t s on i n s e c t s 72 (5) E f f e c t s on mammals 72 (6) A l l e r g i c c o n t a c t d e r m a t i t i s 74 (7) C y t o t o x i c a c t i v i t y 76 (8) Mutagenic a c t i v i t y 78 (9) A n t i - i n f l a m m a t o r y a c t i v i t y 78 (10) A n a l g e s i c a c t i v i t y 79 Chapter 1. A n t i b a c t e r i a l a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s 81 I n t r o d u c t i o n 81 Exper imenta l 83 (1) A n t i m i c r o b i a l a c t i v i t y s c r e e n i n g t e s t 83 R e s u l t s and D i s c u s s i o n 84 (1) A c t i v i t y a g a i n s t i n d i v i d u a l b a c t e r i a 84 (2) R e l a t i o n s h i p between chemica l s t r u c t u r e and a n t i m i c r o b i a l a c t i v i t y 86 (a) Germacranol ides 88 (b) G u a i a n o l i d e s 90 (c) Eudesmanol ides 91 (d) P s e u d o g u a i a n o l i d e s 92 Cone lus i ons 97 Chapter 2. A n t i f u n g a l a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s . . . 120 I n t r o d u c t i o n 120 Exper imenta l 122 (1) A n t i f u n g a l a c t i v i t y s c r e e n i n g t e s t 122 R e s u l t s and D i s c u s s i o n 124 (1) A n t i f u n g a l a c t i v i t y a g a i n s t i n d i v i d u a l fung i 124 (2) R e l a t i o n s h i p between chemica l s t r u c t u r e and a n t i f u n g a l a c t i v i t y 125 (a) Germac r a n o l ides 127 v i i i (b) G u a i a n o l i d e s 128 (c) Eudesmanol ides ' 129 (d) P s e u d o g u a i a n o l i d e s 130 C o n c l u s i o n s 132 Chapter 3. E f f e c t s of s e l e c t e d p s e u d o g u a i a n o l i d e s on s u r v i v a l of the f l o u r b e e t l e 151 I n t r o d u c t i o n 151 E x p e r i m e n t a l 153 (1) Feed ing experiment 153 R e s u l t s and D i s c u s s i o n 154 Chapter 4. C r o s s - r e a c t i v i t y between s e s q u i t e r p e n e l a c t o n e s r e l a t e d to p a r t h e n i n in p a r t h e n i n s e n s i t i z e d guinea p i g s 164 I n t r o d u c t i o n 164 E x p e r i m e n t a l 167 (1) Guinea p i g s e n s i t i z a t i o n 167 (2) C r o s s - r e a c t i v i t y t e s t s 168 R e s u l t s and D i s c u s s i o n 170 REFERENCES 178 APPENDIX. Chemica l s t r u c t u r e s of s e s q u i t e r p e n e l a c t o n e s examined in t h i s study 191 i x LIST OF TABLES Tab le 4 T a b l e '5. Tab le 1. Co lo r r e a c t i o n s of s e s q u i t e r p e n e l a c t o n e s with v a n i l l i n or p-d imethy laminobenza ldehyde spray reagent 17 Tab le 2. Co lo r r e a c t i o n s of p o l y a c e t y l e n i c compounds wi th v a n i l l i n or p-d imethy laminobenza ldehyde spray r e a g e n t s 24 Tab le 3. Minimum q u a n t i t i e s (ug) of s e l e c t e d s e s q u i t e r p e n e l a c t o n e s and p o l y a c e t y l e n e s d e t e c t a b l e wi th v a r i o u s reagents 27 Co lo r r e a c t i o n s of some n a t u r a l l y o c c u r i n g compounds wi th v a n i l l i n spray reagent 28 Summary of chemica l a n a l y s e s of P. hys te rophorus from v a r i o u s l o c a l i t i e s by two- d i r e c t i o n a l TLC and NMR 60 Tab le 6. Types of P^ hys te rophorus as determined by chemica l a n a l y s e s (NMR, TLC) of samples from v a r i o u s l o c a l i t i e s 65 Tab le 7. Degree of d i s i m i l a r i t y between i n d i v i d u a l chemica l types of Pj_ h y s t e r o p h o r u s . 66 Tab le 8. Sc reen ing t e s t for a n t i m i c r o b i a l a c t i v i t y of s e l e c t e d s e s q u i t e r p e n e l a c t o n e s a g a i n s t s i x b a c t e r i a 99 Tab le 9. Summary of a n t i m i c r o b i a l a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s from i n d i v i d u a l X s k e l e t a l c l a s s e s a g a i n s t s i x b a c t e r i a 104 Tab le 10. Summary of a n t i m i c r o b i a l a c t i v i t y of s e l e c t e d s e s q u i t e r p e n e l a c t o n e s a g a i n s t i n d i v i d u a l bac te r i a 105 Tab le 11. Summary of a n t i b a c t e r i a l a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s as r e l a t e d to the presence or absence of the exomethylene on the Y - l a c t o n e r i n g 106 Tab le 12. A n t i m i c r o b i a l a c t i v i t y of ge rmacrano l ides as r e l a t e d to the presence or absence of the methylene group on the Y - lac tone, r i ng 107 Tab le 13. A n t i m i c r o b i a l a c t i v i t y of ge rmacrano l ides a g a i n s t a lbus as r e l a t e d to the presence or absence of v a r i o u s f u n c t i o n a l group-s 108 Tab le 14. A n t i m i c r o b i a l - a c t i v i t y of gua i a n a l ides as r e l a t e d to the presence or absence of the methylene group on the f - l a c t o n e r i n g 110 Tab le 15. A n t i m i c r o b i a l a c t i v i t y of g u a i a n o l i d e s a g a i n s t S^ a lbus as r e l a t e d to the presence or absence of v a r i o u s f u n c t i o n a l groups I l l Tab le 16. A n t i m i c r o b i a l a c t i v i t y of eudesmanol ides as r e l a t e d to the presence or absence of the methylene group on the Y - l a c t o n e r i n g 113 Tab le 17. A n t i m i c r o b i a l a c t i v i t y of eudesmanol ides a g a i n s t a lbus as r e l a t e d to the presence or absence of v a r i o u s f u n c t i o n a l groups 114 Tab le 18. A n t i m i c r o n b i a l a c t i v i t y of p s e u d o g u a i a n o l i d e s as r e l a t e d to the presence or absence of the x i methylene group on the - l a c t o n e and the u n s u b s t i t u t e d cyc lopentenone r i n g '. 116 Tab le 19. A n t i m i c r o b i a l a c t i v i t y of p s e u d o g u a i a n o l i d e s a g a i n s t a lbus as r e l a t e d to the presence or absence of v a r i o u s f u n c t i o n a l groups. 118 Tab le 20. Sc reen ing t e s t fo r a n t i m i c r o b i a l a c t i v i t y . o f s e l e c t e d s e s q u i t e r p e n e l a c t o n e s a g a i n s t three fungi 135 Tab le 21. Summary of a n t i m i c r o b i a l a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s from i n d i v i d u a l s k e l e t a l c l a s s e s a g a i n s t three fung i 138 Tab le 22. Summary of a n t i m i c r o b i a l a c t i v i t y of s e l e c t e d s e s q u i t e r p e n e l a c t o n e s a g a i n s t i n d i v i d u a l fung i 139 A c t i v i t y of s e l e c t e d s e s q u i t e r p e n e l a c t o n e s a g a i n s t two s p e c i e s of y e a s t s 140 Summary of a n t i f u n g a l a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s as r e l a t e d to the presence or absence of the exomethylene on the y - l a c t o n e r i n g 141 Tab le 25. A n t i f u n g a l a c t i v i t y . of g e r m a c r a n o l i d e s , g u a i a n o l i d e s , and eudesmanol ides as r e l a t e d to the presence or absence of the methylene on the - l a c t o n e r i n g 142 Tab le 26. A n t i m i c r o b i a l a c t i v i t y of ge rmacrano l i des a g a i n s t T\ mentaqrophytes as r e l a t e d to the presence or absence of v a r i o u s f u n c t i o n a l groups . . .' 144 Tab le 23 Tab le 24 x i i Tab le 27. A n t i m i c r o b i a l a c t i v i t y of g u a i a n o l i d e s a g a i n s t T\_ mentagrophytes as r e l a t e d to the presence or absence of v a r i o u s f u n c t i o n a l groups . . . 146 Tab le 28. A n t i m i c r o b i a l a c t i v i t y of eudesmanol ides a g a i n s t T^ mentagrophytes as r e l a t e d to the presence or absence of v a r i o u s f u n c t i o n a l groups 147 Tab le 29. A n t i f u n g a l a c t i v i t y of pseudog.ua i a n o l i des as r e l a t e d to the presence or absence of the methylene group on the f - l a c t o n e and the unsubst i t u t e d eye lopentenone r i n g 148 Tab le 30. A n t i m i c r o b i a l a c t i v i t y of p s e u d o g u a i a n o l i d e s a g a i n s t T^ mentagrophyte.s as r e l a t e d to the presence or absence of v a r i o u s f u n c t i o n a l groups 149 Tab le 31. F u n c t i o n a l groups of p s e u d o g u a i a n o l i d e s which were s t u d i e d fo r t h e i r e f f e c t s on s u r v i a v a l of f l o u r b e e t l e s . • 163 Tab le 32. C r o s s - r e a c t i v i t y between s e s q u i t e r p e n e l a c t o n e s r e l a t e d to p a r t h e n i n in p a r t h e n i n s e n s i t i z e d guinea p i g s 176 Tab le 33. R e l a t i o n s h i p between c r o s s - r e a c t i v i t y in p a r t h e n i n - s e n s i t i z e d guinea p i g s to p a r t h e n i n and r e l a t e d s e s q u i t e r p e n e l a c t o n e s and t h e i r chemica l s t r u c t u r e 177 x i i i LIST OF FIGURES F i g u r e 1. Basic- s k e l e t a l c l a s s e s of s e s q u i t e r p e n e l a c t o n e s 2 F i g u r e 2. hys te rophorus grown in a greenhouse from achenes c o l l e c t e d in A u s t i n , Texas 54 F i g u r e 3. D i s t r i b u t i o n of P^ hys te rophorus and l o c a l i t i e s from which p l a n t samples fo r chemica l a n a l y s e s were ob ta ined 55 F i g u r e 4. Chemical s t r u c t u r e s of s e s q u i t e r p e n e l a c t o n e s examined in t h i s S e c t i o n 56 F i g u r e 5. NMR spectrum of crude c h l o r o f o r m e x t r a c t of P. hys te rophorus from Texas 57 F i g u r e 6. NMR spectrum of' crude c h l o r o f o r m extr-act of P. hys te rophorus from A r g e n t i n a 58 F i g u r e 7. Major spots d e t e c t e d by v a n i l l i n spray reagent on p l a t e s deve loped by two- d i r e c t i o n a l TLC of crude c h l o r o f o r m e x t r a c t s of F\_ hys te rophorus 59 F i g u r e 8. A n t i f u n g a l a c t i v i t y of some s e s q u i t e r p e n e l a c t o n e s a g a i n s t (a) Microsporum cooke i and (b) Tr i chophyton mentagrophytes 134 F i g u r e 9. Chemical s t r u c t u r e s of s e l e c t e d p s e u d o g u a i a n o l i d e s t e s t e d fo r t h e i r e f f e c t s on s u r v i v a l of f l o u r b e e t l e s 158 F i g u r e 10. E f f e c t of h e l e n a l i n of v a r i o u s c o n c e n t r a t i o n s on s u r v i v a l of T r i b o l i u m confusum 159 x i v F i g u r e 11. E f f e c t of c o r o n o p i l i n of v a r i o u s c o n c e n t r a t i o n s on s u r v i v a l of Tr i b o l i u m confusum 160 F i g u r e 12. E f f e c t of p a r t h e n i n of v a r i o u s c o n c e n t r a t i o n s on s u r v i v a l of . Tr i b o l ium conf usum . . ., 161 F i g u r e 13. E f f e c t of t e n u l i n of v a r i o u s c o n c e n t r a t i o n s on s u r v i v a l of Tr i b o l i u m confusum 162 F i g u r e 14. Chemical s t r u c t u r e s of s e l e c t e d p s e u d o g u a i a n o l i d e s used for c r o s s - r e a c t i v i t y t e s t s 175 XV ACKNOWLEDGEMENTS I would l i k e to thank my s u p e r v i s o r , P r o f e s s o r G. H. N. Towers, for i n v a l u a b l e d i s c u s s i o n s and a d v i c e throughout t h i s study as w e l l as for h e l p f u l comments on t h i s t h e s i s . I a l s o thank members of my r e s e a r c h committee, Dr . A. D. M. G l a s s , Dr . D. G. K i l b u r n , Dr . J . C. M i t c h e l l , and Dr . I . E. P. T a y l o r , fo r h e l p f u l s u g g e s t i o n s on t h i s m a n u s c r i p t . I am g r a t e f u l to many i n d i v i d u a l s l i s t e d in the S e c t i o n s I and II who k i n d l y p r o v i d e d samples of s e s q u i t e r p e n e l a c t o n e s , p o l y a c e t y l e n e s , and Parthenium- h y s t e r o p h o r u s . The study would not have been p o s s i b l e without t h e i r h e l p . I thank Z. Abramowski, F. B a l z a , Dr . .1. P a n f i l , and Dr . C . -K . Wat for h e l p f u l d i s c u s s i o n s . F. Ba l za a l s o a s s i s t e d wi th the hydrogenat ion of p a r t h e n i n , performed at the Department of Chemis t ry , U . B . C . , and with the i d e n t i f i c a t i o n of h y s t e r i n . I thank M. M. Tracey of the Department of Chemis t ry , U . B . C . , f o r measurements of many NMR s p e c t r a . I am g r a t e f u l to Dr . R. H. E l l i o t t who k i n d l y p r o v i d e d the f l o u r b e e t l e s for my exper iements and R. A. Norton fo r a s l i d e of P_;_ hy s t e r o p h o r u s . F i n a l l y , I would l i k e to express my s p e c i a l thanks to my husband, Dr . J . Pieman, fo r h i s encouragement and i n v a l u a b l e h e l p in a l l a spec t s of my work. 1 PREFACE 1. Chemi st ry of sesqu i terpene l a c t o n e s Sesqu i te rpene l a c t o n e s are a group of t e r p e n o i d s d e r i v e d from i s o p r e n o i d u n i t s a t t a c h e d together v i a h e a d - t o - t a i l condensa t ion and subsequent c y c l i z a t i o n fo l l owed by o x i d a t i v e m o d i f i c a t i o n s (Geissman 1973, Herz 1973). Most of the known n a t u r a l l y o c c u r r i n g s e s q u i t e r p e n e l a c t o n e s are c l a s s i f i e d a c c o r d i n g to t h e i r c a r b o c y c l i c s k e l e t o n s i n t o four b a s i c c l a s s e s : ge rmacrano l ides (with a ten-membered r i n g ) , eudesmanol ides. (6/6 b i c y c l i c . compounds), g u a i a n o l i d e s and p s e u d o g u a i a n o l i d e s (5/7 b i c y c l i c compounds) (Geissman and Crout 1969, Yoshioka et a l . 1973; F i g . 1 ) . An important common f e a t u r e of s e s q u i t e r p e n e l a c t o n e s i s the presence of a V - l a c t o n e r i n g wi th an (7v-methy lene in many of them. The l a c t o n e r i n g i s / i - o r i e n t e d in a l l l a c t o n e s of known s t e r e o c h e m i s t r y . I t i s c i s or t r a n s fused to the C6,C7 or the C8,C7 p o s i t i o n . Bas i c s k e l e t a l m o d i f i c a t i o n s i n v o l v e the i n c o r p o r a t i o n of an epoxide r i n g , h y d r o x y l s or e s t e r i f i e d h y d r o x y l s , and/or t i g l i c or a n g e l i c a c i d at a v a r i e t y of p o s i t i o n s (Yoshioka et a l . 1973) . A few s e s q u i t e r p e n e l a c t o n e s occur in g l y c o s i d i c form ( e . g . Herz et a l . 1970, 1978) and some c o n t a i n ha logens (Siuda and DeBernard i s 1973) . F i g . 1. Basic skeletal classes of sesquiterpene lactones. 3 2. D i s t r i b u t i o n of s e s q u i t e r p e n e l a c t o n e s Approx imate ly 900- s e s q u i t e r p e n e l a c t o n e s are known at p resent (Mabry and G i l l 1979) but the number of these compounds i s r a p i d l y i n c r e a s i n g . A great m a j o r i t y of s e s q u i t e r p e n e l a c t o n e s occur in members of the Composi tae , and these compounds toge ther wi th p o l y a c e t y l e n e s c h a r a c t e r i z e t h i s l a r g e fami ly of p l a n t s (Mabry and Bohlmann 1977). D i f f e r e n c e s in s k e l e t a l types and q u a n t i t i e s of s e s q u i t e r p e n e l a c t o n e s in d i f f e r e n t genera and s p e c i e s of t h i s f a m i l y have been u t i l i z e d in chemotaxonomic s t u d i e s ( e . g . Mabry 1970, Herz 1973, Herout 1973, Geissman and Irwin 1973, Ke lsey and S h a f i z a d e h 1979) . G e n e r a l l y , an i n d i v i d u a l p l a n t w i l l produce s e s q u i t e r p e n e l a c t o n e s of .one s k e l e t a l type most ly with o x i d a t i v e and e s t e r i f i c a t i o n v a r i a t i o n s on that s k e l e t o n . However, d i f f e r e n t s p e c i e s of genera wi th a wide g e o g r a p h i c a l range or sometimes even d i f f e r e n t p o p u l a t i o n s of a s i n g l e s p e c i e s may form d i f f e r e n t s k e l e t a l t y p e s , as i s known, fo r example, for d i f f e r e n t s p e c i e s of the genus Ambros ia (Mabry 1970). In many compos i tes s e s q u i t e r p e n e l a c t o n e s are l o c a t e d in t r i chomes which cover a e r i a l p a r t s of the p l a n t s (Rodr iguez et a l . 1976a, Pieman 1977, Pieman et a l . 1979, Pieman and Towers, u n p u b l i s h e d r e s u l t s ) . In Parthenium hys te rophorus s e s q u i t e r p e n e l a c t o n e s are found a l s o in p o l l e n (Pieman et a l . 1980), achenes , and s e e d l i n g s ( in the f i r s t t rue l e a f which bears t r i c h o m e s ) . Younger s e e d l i n g s , wi th c o t y l e d o n s o n l y , l ack t r i chomes and do not c o n t a i n d e t e c t a b l e amounts of s e s q u i t e r p e n e l a c t o n e s (Pieman 1977., Pieman et a l . 1979) . The h i g h e s t c o n c e n t r a t i o n of 4 s e s q u i t e r p e n e l a c t o n e s (up to 8% of the dry weight of p l a n t s ) i s u s u a l l y found in the f l o w e r i n g heads and. l e a v e s . Stems have smal l q u a n t i t i e s of these compounds (Rodr iguez et a l . 1976a), presumably because of a r e l a t i v e l y sma l le r s u r f a c e area covered with t r i chomes in stems than in l eaves (Pieman 1977) . Roots u s u a l l y have no or very smal l q u a n t i t i e s of s e s q u i t e r p e n e l a c t o n e s (Rodr iguez et a l . 1976a,b , Pieman 1977, Pieman et a l . 1980), however, the i s o l a t i o n of two s e s q u i t e r p e n e l a c t o n e s from the root bark of L i r iodendron t u l i p i f e r a (Magnol iaceae) has been r e p o r t e d (Doskotch and E l - F e r a l y 1969). A l though most of a l l known s e s q u i t e r p e n e l a c t o n e s have been i s o l a t e d from s p e c i e s of the Composi tae , these compounds occur s p o r a d i c a l l y a l s o in other f a m i l i e s of p l a n t s . In Ang i ospe rms,. they have been r e p o r t e d from U m b e l l i f e r a e , Magno l i aceae , Lauraceaeae , Win te raceae , I l l i c i a c e a e , A r i s t o l o c h i a c e a e , Menispermaceae, C o r t i a r i a c e a e , and Acanthaceae (Yoshioka et a l . 1973, and r e f e r e n c e s t h e r e i n ) . A few s e s q u i t e r p e n e l a c t o n e s have a l s o been i s o l a t e d from l i v e r w o r t s ( H e p a t i c e a e ) , such as F r u l l a n ia d i l a t a t a and F^ tamar i sc i (Knoche et a l . 1969), D i p l o p h y l l u m a l b i c a n s (Benesova et a l . 1975, Asakawa et a l . 1979), and P o r e l l a s p e c i e s (Asakawa et a l . 1976). The fungus, A s p e r g i l l u s te r reus ( R a n i e r i and C a l t o n 1978 ), and some mushrooms, for example L a c t a r i u s v e l l e r e u s , L. pergamenus (Magnusson and Thoren 1973), and L^ b l e n n i u s ( V i d a r i et a l . 1976), have been found to c o n t a i n t h i s type of compounds. Sesqu i te rpene l a c t o n e s a long with many other groups of compounds such as a l k a l o i d s , p h e n o l i c compounds, and g l y c o s i d e s 5 are p roduc ts of secondary metabol ism whose f u n c t i o n s in the pr imary metabol ism of p l a n t s are not known (Czapek 1925, in Mothes 1976). I t has been suggested (F raenke l 1959) that these compounds evo lved in p l a n t s as a means of defense a g a i n s t h e r b i v o r o u s p r e d a t o r s such as i n s e c t s . They may a l s o p lay a r o l e in reduc ing c o m p e t i t i o n wi th other p l a n t s ( D a l v i et a l . 1971') and in the defense of p l a n t s a g a i n s t v a r i o u s pathogenic organisms (Wal lace and M a n s e l l 1976) . T h i s t h e s i s r e p o r t s s t u d i e s on c e r t a i n b i o l o g i c a l and chemica l a s p e c t s of s e s q u i t e r p e n e l a c t o n e s . In the f i r s t s e c t i o n , I d e s c r i b e a new t h i n l a y e r chromatography (TLC) v i s u a l i z a t i o n techn ique for i d e n t i f i c a t i o n of s e s q u i t e r p e n e l a c t o n e s . In the s e c t i o n I I , I r epor t the use of t h i s new TLC techn ique in a chemotaxonomic study of major s e s q u i t e r p e n e l a c t o n e s p resent in v a r i o u s p o p u l a t i o n s ©f h y s t e r o p h o r u s . The purpose of t h i s study was to e s t a b l i s h the r e l a t i o n s h i p s between v a r i o u s p o p u l a t i o n s of t h i s common weed throughout i t s recent range of d i s t r i b u t i o n . F i n a l l y , in the S e c t i o n I I I , I r epor t v a r i o u s b i o l o g i c a l a c t i v i t i e s of s e s q u i t e r p e n e l a c t o n e s in r e l a t i o n to t h e i r chemica l s t r u c t u r e s . T h i s study was des igned to e l u c i d a t e the r o l e of v a r i o u s f u n c t i o n a l groups in d e t e r m i n i n g the a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s a g a i n s t v a r i o u s types of o rgan isms . 6 S E C T I O N I 7 V i s u a l i zat ion reagents for s e s q u i t e r p e n e l a c t o n e s and p o l y a c e t y l e n e s on t h i n l a y e r chromatograms Int roduct i on The Compositae f a m i l y c h a r a c t e r i s t i c a l l y c o n t a i n s two b i o l o g i c a l l y a c t i v e types of secondary m e t a b o l i t e s , namely s e s q u i t e r p e n e l a c t o n e s and p o l y a c e t y l e n e s (Heywood et a l . 1977) . In s p i t e of an i n c r e a s i n g i n t e r e s t in the c y t o t o x i c , a n t i b i o t i c , p h o t o t o x i c , and a n t i n e o p l a s t i c a c t i v i t i e s of these compounds (Rodr iguez et a l . 1976b, Towers and Wat 1978) s a t i s f a c t o r y reagents for t h e i r v i s u a l i z a t i o n on 'thin l a y e r chromatograms have not been r e p o r t e d . The techn iques g e n e r a l l y used fo r the d e t e c t i o n of s e s q u i t e r p e n e l a c t o n e s by t h i n l a y e r chromatography (TLC) or paper chromatography (PC) are UV l i g h t ( I v i e et a l . 1 9 7 5 a , b ) , . exposure to i o d i n e vapours (Mabry 1970, Rodr iguez 1975a), s p r a y i n g wi th a s o l u t i o n of KMnO^(Rodriguez 1975a), or wi th c o n c e n t r a t e d UzSO^ (Geissman and G r i f f i n 1971), f o l l o w e d by h e a t i n g ( H a l l et a l . 1977). These methods are g e n e r a l l y u n s p e c i f i c g i v i n g the same c o l o r r e a c t i o n wi th almost a l l u n s a t u r a t e d compounds. A c i d c a t a l y z e d c o l o r r e a c t i o n s of c e r t a i n s e s q u i t e r p e n e l a c t o n e s have been d e s c r i b e d (Geissman and G r i f f i n 1971, G r i f f i n et a l . 1971), but the techn ique i s r e s t r i c t e d to those l a c t o n e s which form c o l o r complexes in a c i d i c s o l u t i o n . P o l y a c e t y l e n i c compounds are o f t e n i d e n t i f i e d by t h e i r h igh ly , c h a r a c t e r i s t i c UV s p e c t r a (Bohlman et a l . 1973) but , with the 8 e x c e p t i o n , of th iophenes ( C u r t i s and P h i l l i p s 1962), no s imple r e l i a b l e method for the v i s u a l i z a t i o n of these compounds by TLC or PC has been d e s c r i b e d . In t h i s s e c t i o n I d e s c r i b e a new techn ique for the s e p a r a t i o n , v i s u a l i z a t i o n , and i d e n t i f i c a t i o n of s e s q u i t e r p e n e l a c t o n e s and p o l y a c e t y l e n e s . T h i s techn ique i s based on the use of s e v e r a l a c i d i c reagents c o n t a i n i n g e i t h e r v a n i l l i n or r e l a t e d benzaldehyde or benzo ic a c i d d e r i v a t i v e s which are h i g h l y s e n s i t i v e for these compounds and which g i ve c h a r a c t e r i s t i c c o l o r s wi th i n d i v i d u a l s e s q u i t e r p e n e l a c t o n e s or p o l y a c e t y l e n e s a f t e r TLC on s i l i c a g e l . Spray reagents c o n t a i n i n g v a n i l l i n in m i n e r a l a c i d s are commonly used in the d e t e c t i o n of s t e r o i d s ( S t a h l 1969, Ba ja j and Ahuja 1979), h igher a l c o h o l s , p h e n o l s , e s s e n t i a l o i l s ( S t a h l 1969), and c a n n a b i n o i d s (Tewari and Sharma 1979). An a c i d i c s o l u t i o n of p-d imethy laminobenza ldehyde i s a known spray reagent fo r a z u l e n e s , i n d o l e d e r i v a t i v e s , urea and u r e i d e s , and n i t r o compounds (Merck) . Other s t r u c t u r a l l y s i m i l a r benzaldehydes have a l s o been r e p o r t e d for use as spray reagents a l though most ly for s t e r o i d and a l k a l o i d d e t e c t i o n (Merck) . 9 Exper imenta l (1) Chemica ls Sesqu i te rpene l a c t o n e s m e l c a n t h i n - B , c o n f e r t i f l o r i n , c i n e r e n i n , melampodin-A and -B , e n h y d r i n , and melampodinin were k i n d l y p r o v i d e d by D r . N . H. F i s h e r ( L o u i s i a n a S ta te U n i v e r s i t y , Baton Rouge, L a . , U . S . A . ) , h e l e n a l i n , hymenin, h y s t e r i n , c o n c h o s i n - A , cumanin, e u p a t o r i o p i c r i n , and santamarine by Dr . A. Romo de V i v a r ( U n i v e r s i d a d N a c i o n a l Autonoma de Mexico , M e x i c o ) , ambros in , a x i v a l i n , i v a x i l l a r i n , damsin, t e n u l i n , i s o - t e n u l i n , t e t r a n e u r i n - A , - B , -D, and - E , c o n c h o s i n - B , and hymenin by Dr . E. Rodr iguez ( U n i v e r s i t y of C a l i f o r n i a , I r v i n e , C a l i f . , U.S.A-.-), g l a u c o l i d e - A , -'B, -D, - E , - F , and -G and m a r g i n a t i n by D r s . T. J . Mabry and M. Be tkousk i ( U n i v e r s i t y of Texas , A u s t i n , T x . , U . S . A . ) , p a r t h e n o l i d e -9 -$-hydroxy1 and e l e p h a n t o p i n by Dr . J . M. Cassady (Purdue U n i v e r s i t y , West L a f a y e t t e , I n d . , U . S . A . ) , quadrone by Dr . R. L. R a n i e r i (W. R. Grace &' C o . , Co lumbia , Md. , U . S . A . ) , f r u l l a n i a l a c t o n e by Dr . G. O u r i s s o n ( U n i v e r s i t y of S t r a s b o u r g , F r a n c e ) . A l a n t o l a c t o n e and i s o - a l a n t o l a c t o n e (as a mixture "He len in") were purchased from Sigma, Chem. Co. and (X- san ton in from F l u k a , S w i t z e r l a n d . A l l Other s e s q u i t e r p e n e l a c t o n e s were p r o v i d e d by the l a t e P r o f e s s o r T. A. Geissman ( U n i v e r s i t y of C a l i f o r n i a , Los A n g e l e s , C a l i f . , U . S . A . ) , were p r e v i o u s l y i s o l a t e d in our l a b o r a t o r y from v a r i o u s p l a n t s o u r c e s , 'or were prepared as d e s c r i b e d in the S e c t i o n I I . P o l y a c e t y l e n e s number 24 and 25 (Tab le 2) were i s o l a t e d by K. 10 Downum and number 20 (Table 2) by R. A. Norton in our l a b o r a t o r y from v a r i o u s p l a n t s o u r c e s . Methyl 2 - t h i e n y l ketone was purchased from Eastman Kodak Co. and th iophene • from I ON P h a r m a c e u t i c a l s , Inc . A l l other p o l y a c e t y l e n e s were k i n d l y p r o v i d e d by Dr . J . Lam ( U n i v e r s i t y of Aarhus , Denmark). A l l s e s q u i t e r p e n e l a c t o n e s and p o l y a c e t y l e n e s were used without f u r t h e r p u r i f i c a t i o n . Other c h e m i c a l s which were used as s tandards or in spray s o l u t i o n s were ob ta ined from common commercia l sources and were used without f u r t h e r p u r i f i c a t i o n . (2) Spray reagents One of the f o l l o w i n g c h e m i c a l s , v a n i l l i n , p- d imethy laminobenza ldehyde , p -hydroxybenza ldehyde , s a l i c y l a ldehyde , m-an isa ldehyde , c innamaldehyde, p -hydroxybenzo ic a c i d , or v a n i l l i c a c i d .(0.5g) was d i s s o l v e d in a s o l u t i o n c o n s i s t i n g of 9 ml of e thano l (95%), 0.5 ml of c o n c e n t r a t e d s u l p h u r i c a c i d , and 3 drops of a c e t i c a c i d . T h i s reagent shou ld be f r e s h l y p repared be fo re use . (3) TLC p l a t e s S i l i c a ge l p l a t e s , without gypsum and with f l u o r e s c e n t i n d i c a t o r (Polygram, Brinkmann Ins t ruments , I n c . ) , were spo t ted wi th 5-10 ug of each compound. Sesqu i te rpene l a c t o n e s were d i s s o l v e d in c h l o r o f o r m or ace tone , p o l y a c e t y l e n e s in e t h a n o l , o ther compounds (Table 4) in v a r i o u s s o l v e n t s . The p l a t e s were deve loped in a s tandard chamber (without chamber s a t u r a t i o n ) wi th a s o l v e n t system of c h l o r o f o r m - acetone (6 : 1 ) . A f t e r 11 chromatography the p l a t e s were a i r d r i e d , sprayed wi th the reagent s o l u t i o n and d i r e c t l y p l a c e d on a h o t . p l a t e (TekPro H e a t S t i r 36, S c i e n t i f i c P roducts) and s lowly heated (temperature on 70°C). When v a n i l l i n was used as a spray reagent the background become y e l l o w i s h and wi th c o n t i n u e d h e a t i n g s l i g h t l y v i o l e t . C o l o r s formed by r e a c t i o n with the compounds t e s t e d were recorded at 10 minutes a f t e r t h i s t reatment (Table 4) and a l s o 24 hours l a t e r (Tab le 1 ) . Some p o l y a c e t y l e n e s changed c o l o r s a f t e r 48 hours (Table 2) where there was no change of c o l o r a f t e r 24 hours . The c o l o r s formed are u n s t a b l e and change on s t a n d i n g . Some p r e s e r v a t i o n was ach ieved by wrapping p l a t e s in p l a s t i c f i l m (Saran Wrap) and s t o r i n g p l a t e s in da rk . The c o l o r s were recorded us ing the Handbook of Co lour (Kornerup and Wauscher 1967) . (4) Reagent s e n s i t i v i t y t e s t Each of f i v e s e s q u i t e r p e n e l a c t o n e s (Table 3 ) , in acetone s o l u t i o n , was a p p l i e d in q u a n t i t i e s rang ing from 0.05 to 20.0 yg on TLC p l a t e s and deve loped as d e s c r i b e d above. Developed p l a t e s were v i s u a l i z e d under UV l i g h t or exposed to i o d i n e vapours or sprayed wi th a s o l u t i o n of e i t h e r KMnO^, v a n i l l i n or p- dimethylamonobenzaldehyde reagent f o l l o w e d by h e a t i n g . The minimum q u a n t i t i e s of s e s q u i t e r p e n e l a c t o n e s forming v i s i b l e spots on the chromatograms were r e c o r d e d . In the same way the s e n s i t i v i t i e s of v a n i l l i n and p-d imethy laminobenza ldehyde were t e s t e d wi th two n a t u r a l p o l y a c e t y l e n e s , p h e n y l h e p t a t r i y n e (No. 20 in Tab le 2) and the th iophene d e r i v a t i v e , fl-terthienyl (No. 25 in Tab le 2 ) . . The s e n s i t i v i t y of i s a t i n was t e s t e d on the 12 l a t t e r compound. The minimum q u a n t i t i e s of both p o l y a c e t y l e n e s ( in e t h a n o l ) s t i l l showing d i s t i n c t c h a r a c t e r i s t i c curves of t h e i r UV s p e c t r a were recorded (Unicam Sp. 800) . (5) Test of var i ous reagents for the i r reac t ions with s e s q u i t e r p e n e l a c t o n e s F i v e to ten ug of each of 12 s e l e c t e d s e s q u i t e r p e n e l a c t o n e s , t a m a u l i p i n - A , a l a n t o l a c t o n e , i s o a l a n t o l a c t o n e , p u l c h e l l i n - C , p a r t h e n i n , c o r o n o p i l i n , h e l e n a l i n , d e s a c e t o x y m a t r i c a r i n , cumambrin-B a c e t a t e , t e n u l i n , i s o - t e n u l i n , and x a n t h i n i n , in c h l o r o f o r m or acetone s o l u t i o n were spo t ted on 13 s i l i c a ge l p l a t e s . Each p l a t e was d i r e c t l y (without d e v e l o p i n g ) sprayed wi th one of the spray reagents l i s t e d above or 5% (w/v) aqueous s o l u t i o n of KMnOij , concenta ted H^SO^, or 95% e t h a n o l - h y d r o c h l o r i c a c i d (1 : 1) f o l l o w e d by h e a t i n g to o b t a i n the most i n t e n s i v e c o l o r s or was exposed to i o d i n e vapours . C o l o r s were recorded d u r i n g f i r s t 10 minutes and a f t e r 24 h o u r s . 13 R e s u l t s and P i scuss i on Of the reagents t e s t e d fo r TLC v i s u a l i z a t i o n of s e s q u i t e r p e n e l a c t o n e s in a c i d i c s o l u t i o n s only benzaIdehydes and v a n i l l i c a c i d produced d i s t i n c t c o l o r s with i n d i v i d u a l l y t e s t e d compounds. In c o n t r a s t to t h i s other reagents ( i o d i n e vapours , KMnO^ s o l u t i o n , and c o n c e n t r a t e d R^SO^) produced on ly g r e y i s h to brownish c o l o r s wi th s e s q u i t e r p e n e l a c t o n e s . The e t h a n o l - h y d r o c h l o r i c a c i d spray gave d i s t i n c t c o l o r s with some s e s q u i t e r p e n e l a c t o n e s but d i d not g i ve any c o l o r wi th o t h e r s . Sesqu i te rpene l a c t o n e s r e p r e s e n t i n g a l l the b a s i c s k e l e t a l c l a s s e s ( F i g . l ) and a few m o d i f i e d s e s q u i t e r p e n e l a c t o n e s ( a l t o g e t h e r 78; Tab le 1) and- 25 p o l y a c e t y l e n e s (Tab le 2) were t e s t e d wi th the v a n i l l i n and p-d imethy laminobenza ldehyde r e a g e n t s . V a n i l l i n was chosen because i t produces a great range of c o l o r s and p-d imethy laminobenza ldehyde because i t g i v e s a d i f f e r e n t spectrum of c o l o r s when compared wi th v a n i l l i n . V a n i l l i n was t e s t e d wi th a few r e p r e s e n t a t i v e s of o ther c l a s s e s of compounds (Table 4 ) . Monoterpenes, s t e r o i d s , and c a r o t e n o i d s gave very b r i g h t c o l o r s , f a t t y a c i d s gave g r e y i s h c o l o r s , and some aromat ic a c i d s d i d not g ive any c o l o r . F l a v o n o i d s g e n e r a l l y produced ye l l ow c o l o r s . The s m a l l e s t q u a n t i t i e s of s e s q u i t e r p e n e l a c t o n e s d e t e c t a b l e by TLC by v a r i o u s reagents are shown in Tab le 3. The most s e n s i t i v e were v a n i l l i n and p-d imethy laminobenza ldehyde which can be used to d e t e c t 0.05 ug of c e r t a i n s e s q u i t e r p e n e 14 l a c t o n e s . D imethy laminobenza ldehyde , in some c a s e s , was even more s e n s i t i v e than v a n i l l i n . The s m a l l e s t q u a n t i t y of a s e s q u i t e r p e n e l a c t o n e which can be d e t e c t e d depends.on the b r i g h t n e s s and i n t e n s i t y of the c o l o r s deve loped on chromatograms. Thus t e n u l i n (No. 63 in Appendix) which produces b r i g h t orange red c o l o r s wi th v a n i l l i n and p- d imethy laminobenza ldehyde i s d e t e c t a b l e on chromatograms at 0.05 ug whi le h e l e n a l i n (No. 66 in Appendix) which g i v e s a weaker r e d d i s h orange c o l o r with v a n i l l i n and a g r e y i s h ye l low c o l o r wi th p-d imethy laminobenza ldehyde i s on ly d e t e c t a b l e at 0.5 and 0.1 pg. P a r t h e n i n (No. 47 in Appendix) which forms a b l u i s h green c o l o r wi th v a n i l l i n can be d e t e c t e d at 0.5 ug but the golden ye l low c o l o r with p-d imethy laminobenza ldehyde i s v i s i b l e at a c o n c e n t r a t i o n lOx lower . A l p h a - t e r t h i e n y l (No. 25 in Tab le 2) which g i v e s a very b r i g h t c o l o r with. v a n i l l i n , p- dimethy. laminobenzaldehyde, and i s a t i n has a lower l i m i t of d e t e c t i o n of 0.05 ug. P h e n y l h e p t a t r i y n e (No. 20 in Tab le 2) ( forming the l e a s t i n t e n s i v e c o l o r of a l l p o l y a c e t y l e n e s I t e s t e d ) can be d e t e c t e d on TLC p l a t e s wi th v a n i l l i n or p- d imethy laminobenza ldehyde reagent only at the l e v e l of 0.5 ug which , however, i s s t i l l lOOOx lower than the minimum amount needed fo r i t s d e t e c t i o n by UV spec t rophotomet ry . Some of the reagents which I have t e s t e d , p a r t i c u l a r l y a c i d i c s o l u t i o n s of v a n i l l i n and p-d imethy laminobenza ldehyde , are the most s u i t a b l e fo r the i d e n t i f i c a t i o n of s e s q u i t e r p e n e l a c t o n e s and of p o l y a c e t y l e n i c compounds. These reagents produce a v a r i e t y of co lo rs , wi th s t r u c t u r a l l y very d i f f e r e n t compounds. The d i f f e r e n c e s in c o l o r s produced by v a r i o u s s e s q u i t e r p e n e 15 l a c t o n e s and other compounds a l l o w s for the ready d e t e c t i o n of i m p u r i t i e s in c r y s t a l l i n e p r e p a r a t i o n s . The TLC method can be made h i g h l y s p e c i f i c by : (1) the use of s e v e r a l reagents that form d i f f e r e n t c o l o r s or shades wi th the same compound, (2) the use of s tandard compounds,, and (3) e v a l u a t i n g Rf v a l u e s . The wide range of c o l o r s produced with a l l types of s e s q u i t e r p e n e l a c t o n e s and p o l y a c e t y l e n e s makes i t d i f f i c u l t to a s s i g n the format ion of a p a r t i c u l a r c o l o r to a chemica l s t r u c t u r e . T h i s i s supported by the f a c t that the s t r u c t u r a l l y s i m i l a r s e s q u i t e r p e n e l a c t o n e s , such as damsin (No. 54 in A p p e n d i x ) , c o r o n o p i l i n (No. 53 in Append ix) , and p a r t h e n i n (No. 47 in A p p e n d i x ) , produce q u i t e d i f f e r e n t c o l o r s wi th the v a n i l l i n reagent (Table 1) whi le d i a s t e r e o i s o m e r s produce i d e n t i c a l c o l o r s as i s the case with p a r t h e n i n and hymenin (numbers 47 and 51 in A p p e n d i x ) . Hydroxybenzoic a c i d d i d not produce d i s t i n c t c o l o r s with s e s q u i t e r p e n e l a c t o n e s but p-hydroxybenzaldehyde formed a l a r g e v a r i e t y of c o l o r s . Both v a n i l l i c a c i d and v a n i l l i n , on the other hand, gave a good range of c o l o r s . A l though the most d i s t i n c t i v e and v a r i a b l e c o l o r s were produced wi th p-hydroxybenzaldehyde and v a n i l l i n , c e r t a i n s e s q u i t e r p e n e l a c t o n e s might be more c o n v e n i e n t l y d e t e c t e d wi th a d i f f e r e n t spray reagent (e . g . , c innamaldehyde forms a very b r i g h t s t a b l e c o l o r wi th h e l e n a l i n ) . To c o n c l u d e , I recommend t h i s TLC techn ique as a s i m p l e , f a s t , s e n s i t i v e , and s p e c i f i c method that can be used in a p r e l i m i n a r y search fo r s e s q u i t e r p e n e l a c t o n e s and p o l y a c e t y l e n e s in crude ( p l a n t ) e x t r a c t s of the Compositae and a l s o in the i d e n t i f i c a t i o n of i n d i v i d u a l compounds. I used t h i s techn ique the study of chemis t ry of major s e s q u i t e r p e n e l a c t o n e s Parthen i um hys te rophorus which i s d e s c r i b e d in the S e c t i o n I I . 17 Table 1* Color reactions of sesquiterpene lactones with v a n i l l i n or p-dimethylaminobenzaldehyde spray reagent. Sesq. lactone No. i n Appendix Name Reagent Rf v a n i l l i n p-dimethylamino- (CHCl^: Structure benzaldehyde Me^CO, 6:1) (reference) Germacranolides: 1 Parthenolide d u l l blue d u l l v i o l e t greyish ruby l i g h t brown 0.83 2 Pyrethrosin d u l l green l i g h t brown brownish red orange 0.80 3 Mifcanolide, dihydro- b l u i s h grey grey brownish orange brownish orange 0.52 4 Chamissonin, di a c e t y l blue greyish yellow brownish grey brownish grey 0.83 5 Tamaulipin-A greyish v i o l e t greenish grey dark brown dark brown 0.52 6 Tamaulipin-B grey grey brown l i g h t brown 0.57 7 Eupatoriopicrin d u l l green dark brown greyish brown dark brown 0.15 8 Melampodin-A d u l l blue b l u i s h grey greyish brown ol i v e brown 0.43 9 Melampodin-B greyish green d u l l blue brownish orange brownish orange 0.60 10 Enhydrin v i o l e t pastel v i o l e t l i g h t brown greyish green 0.77 18 Table 1. Continued. 11 Cinerenin grey grey brownish orange brownish orange 0.19 3 12 Melampodinin greyish blue grey greyish brown greyish brown 0.50 2 13 Melcanthin-B d u l l blue reddish grey greyish brown greyish brown 0.07 4 14 Glaucolide-A o l i v e grey greyish v i o l e t purple l i g h t brown brownish grey 0.75 5 15 Glaucolide-B o l i v e grey greyish v i o l e t l i g h t brown brownish grey 0.68 5 16 Marginatin dark ruby v i o l e t brown ol i v e brown ol i v e brown 0.86 6 17 Glaucolide-D dark v i o l e t dark v i o l e t l i g h t brown l i g h t brown 0.70 7 18 Glaucolide-E dark v i o l e t dark v i o l e t l i g h t brown l i g h t brown 0.80 7 19 Glaucolide-F o l i v e grey greyish v i o l e t purple l i g h t brown brownish grey 0.74 8 20 Glaucolide-G dark ruby v i o l e t brown ol i v e brown ol i v e brown 0.87 8 21 Elephantopin grey grey yellowish grey greyish yellow 0.34 9 22 Parthenolide, 9-0i-OH dark blue d u l l v i o l e t v i o l e t l i g h t brown 0.37 l i g h t brown 9 19 Table 1. Continued. Guaianolides: 23 Cumambrin-A dark blue d u l l green reddish brown l i g h t brown 0.59 24 Cumambrin-B greyish turquoise l i g h t brown d u l l green l i g h t brown 0.42 25 Cumamb rin-B, dihydro- dark blue d u l l green reddish brown l i g h t brown 0.32 10 26 Cumambrin-B, tetrahydro- dark blue dark purple brownish red reddish brown 27 Cumambrin-B, greyish turquoise reddish brown acetate d u l l green l i g h t brown 0.14 0.61 10 10 2 8 Cumamb rin-B, formyl greyish turquoise l i g h t brown d u l l green l i g h t brown 0.55 10 29 Matricarin purplish red greyish red greyish yellow greyish yellow 0.84 30 Matricarin, desacetoxy- greyish ruby purplish pink yellow yellow 0.83 31 Grossheimin greyish yellow greyish yellow greyish yellow greyish yellow 0.43 32 I v a l i n , pseudo- v i v i d v i o l e t -*• dark blue reddish brown 0.43, 0.35 11 o l i v e reddish brown 20 Table 1. Continued. Eudesmanolides: 33 Alantolactone d u l l blue grey v i o l e t brown greyish Magenta 0.91 1 34 Alantolactone, tetrahydro- d u l l blue blue v i o l e t brown greyish Magenta 0.91 1 35 Alantolactone, i s o - d u l l blue bluish grey v i o l e t brown greyish Magenta 0,91 1 36 I v a l i n purple greenish grey dark brown purplish grey 0.51 1 37 Ivasperin v i o l e t blue greenish grey l i g h t brown l i g h t brown 0.15 1 : 38 P i n n a t i f i d i n b l u i s h red -»-red yellowish orange v i v i d yellow v i v i d yellow 0.65 1 39 Pulchellin-C brownish red greyish ruby greyish orange greyish orange 0.14 1 40 0(~Santonin greyish brown d u l l green greyish orange brownish orange brownish orange 0.81 1 41 Ludovicin-A b l u i s h grey brownish grey dark brown brownish grey 0.58 1 42 Ludovicin-B bl u i s h grey brownish grey dark brown brownish grey 0.41 1 43 Ludovicin-C dark blue brownish grey greyish ruby greyish ruby 0.89 1 Table 1. Continued. 44 Reynosin d u l l blue v i o l e t grey l i g h t brown I l i g h t brown 0.58, 0.49* 1 45 F r u l l a n i a lactone dark green ol i v e greyish magenta purplish grey 0.91 1 46 Santamarine dark blue dark v i o l e t l i g h t brown l i g h t brown 0.63 1 Pseudoguaianolides: 47 Parthenin b l u i s h green greyish yellow golden yellow golden yellow 0.44 1 48 Parthenin, dihydroiso- v i v i d orange l i g h t yellow l i g h t yellow greyish yellow 0.45 12 49 Parthenin, tetrahydro- d u l l blue d u l l blue orange -* l i g h t brown brownish red 0.49 12 50 Parthenin, photolytic product v i v i d v i o l e t yellowish grey yellowish grey yellowish grey 0.76 13 51 Hymenin bluish green greyish yellow golden yellow golden yellow 0.44 1 52 Ambrosin yellow -* orange red greenish yellow l i g h t orange l i g h t orange 0.67 1 53 Coronopilin blue greyish magenta yellow -> orange red red 0.46 1 54 Damsin v i v i d yellow yellowish white golden yellow golden yellow 0.73 1 55 Hysterin grey grey greyish brown greyish brown 0.19 1 56 Tetraneurin-A greyish green greyish green brownish yellow brownish yellow 0.42 1 22 Table 1. Continued. 57 Tetraneurin-B blue bluish v i o l e t brownish red brownish red 0.44 1 58 Tetraneurin-D d u l l green olive brown brownish orange l i g h t brown 0.09 1 59 Tetraneurin-E d u l l green o l i v e brown greyish orange greyish orange 0.09 1 60 Conchosin-A l i l a c greyish blue pink>-pastel blue pastel blue 0.05 1 61 Conchosin-B yellowish green l i g h t yellow golden golden 0.42 1 62 C o n f e r t i f l o r i n , desacetyl greyish yellow greyish yellow greyish yellow greyish yellow 0.25 1 63 Tenulin orange red brownish orange reddish orange reddish orange 0.44 1 64 Tenulin, i.so- greyish yellow greyish yellow yellows-greyish . orange greyish orange 0.71 1 65 G a i l l a r d i l i n brownish grey-> -> d u l l green d u l l green greyish yellow greyish yellow 0.59 1 66 Helenalin yellow-Kreddish orange greyish yellow greyish yellow greyish yellow 0.42 1 67 Flexuosin-B v i o l e t red-v greyish orange pale yellow yellow-Hreddish golden reddish golden 0.53 1 68 Spathulin black golden brown 0.07 1 brown golden brown 69 B a l d u i l i n o l i v e yellow greyish yellow yellow yellow 0.63 1 23 Table 1. Continued. 70 Cumanin brownish yellow-* -•greyish brown greyish brown Other sesquiterpene lactones: 71 Xanthinin .brownish red ruby 72 Psilostachyin d u l l green blue 73 Psilostachyin-C d u l l green greenish grey 74 Vernolepin 75 Vernomenin 76 Quadrone 77 A x i v a l l i n 78 I v a x i l l a r i n grey greyish brown l i g h t brown grey deep yellow-* -KLight yellow l i g h t yellow v i o l e t v i o l e t grey orange red l i g h t orange brownish yellow-*- 0.21 -•greyish brown l i g h t brown v i v i d yellow 0.82 orange yellow grey 0.59 grey blue+grey 0.50 grey dark yellow 0.28 dark yellow dark yellow 0.29 dark yellow v i v i d yellow 0.84 yellow b l u i s h v i o l e t 0.61 greyish v i o l e t greyish v i o l e t 0.43 yellow 14 •*> =change during heating the plate * =two spots of i d e n t i c a l color and inte n s i t y References: 1 Yoshioka et a l . (19 73) 2 Fisher et a l . (1976) 3 Perry and Fisher (1975) 4 Fisher et a l . (19 78) 5 Padolina et a l . (1974a) 6 Padolina et a l . (1974b) F i r s t color recorded after 10 min; second after 24 hours. 7 Betkouski et a l . (1975) 8 M. Betkouski (pers. comm.) 9 J . M. Cassady (pers. comm.) 10 T. A. Geissman (pers. comm.) 11 Devon and Scott (1972) 12 Herz et a l . (1962) 13 Kagan et a l . (1971) 14 Ranieri and Calton (1978) Table 2. Color reactions of polyacetylenic compounds with v a n i l l i n or p-dimethylaminobenzaldehyde spray reagents. Reagent Compound v a n i l l i n p-dimethylamino- benzaldehyde Rf (CHC1 : Me cof 6:1) t t (1) CH -(C=C) -(CH=CH) -CH-CH -CH -OH • OH (2) CH3-(C=C)3-CH=CH-ko^ (3) CH=C-C-CH=C<^3-C5Hn (4) CH3-CH2-CH2-C=C-CH= Q=0 (5) ^~^)-(C=C) 2-CH=CH-CHO (6) <^"^)-(C£C) 2-CH=CH-CH2OH (7) CH -CH=CH-(C=C)-(CH=CH) -CH-CH CH -OH OH (8) CH3-(CH=CH)2-C=C-(CH=CH)2-(CH2)5-OAc (9) CH3-CH=CH-(C=C)2-(CH2)4-CH=CH2 (10) CH3-CH=CH- (C=C) 2-CH2-CH=CH- ( CH^ 5~CH=CH2 d u l l blue greyish brown deep orange bl u i s h v i o l e t blue dark yellow brownish beige brownish beige o l i v e brown grey reddish grey o l i v e yellow brownish orange reddish orange greyish Magenta * v i o l e t o l i v e yellow greyish yellow o l i v e yellow o l i v e brown yellowish brown l i g h t brown 0.16 0.71 0.87 0.83 0.91 0.63 0.17 0.92 0.93 0.93 Table 2. Continued. Compound Reagent v a n i l l i n p-dimethylamino- benzaldehyde Rf (CHC1- : Me2CO; 6:1) (11) CH -CH=CH-(C=C)2-(CH=CH) ̂ (CH^-OAc greyish brown yellowish brown 0.91 (12) CH -CH=CH-(C=C) -CH=CH-CH-CH-CH=CH J OH OH (13) CH -(C=C) -(CH=CH) -CH-CH -CH J J Z OAc * OAc (14) CH 3-(C=C) 2-CHSQ^ (15) CH3-(C=C) 2 - C H ^ o A (16) CH -CH=CH-(C=C) -CH=CH-CH-CH OH J J OH (17) CH -CH=CH-(C=C) -CH=CH-CH-CH OH 3 Cl (18) CH3-(C=C) 3-CH=CH^) dull green olive brown and deep Magenta olive brown deep violet * violet greyish brown olive greenish grey yellowish brown olive and greyish ruby olive brown- greyish violet olive * greyish brown greyish brown greenish grey 0.36 0.89, 0.73 0.90 ** 0.79 0.20 0.79 0.92 Table 2. Continued. Compound Reagent v a n i l l i n p-dimethylamino- benzaldehyde Rf (CHC1 : Me2C0f 6:1) (19) CH =CH-CH-(C=C) -CH-CH=CH-C H 6 H • 6 H ' L^ (20) \ /> (^C) 3-CH 3 (21) CH 3-(CH 2) 2-(C=C) 2-CH=CH-COOCH 3 (22) Q (23) (24) (25) ^VQ-CC-CH-CH greyish brown dark blue * blue dark blue olive-* - K l u l l blue v i v i d red greenish blue bluish green dark brown * o l i v e brown greyish yellow grey d u l l blue orange red v i o l e t grey v i v i d blue 0.58 0.95 0.98 0.95 0.88 0.90 0.90 ** =change of color after 48 hrs k =two spots of i d e n t i c a l i n t e n s i t y "change during heating the plate Table 3. Minimum quantities (yg) of selected sesquiterpene lactones and polyacetylenes detectable with, various reagents. iodine 1 KMnO. 4 p-dimethylamino- i s a t i n UV Compound UV l i g h t vapours (5%, H20) v a n i l l i n benzaldehyde (0.4%, cone. H_S0.) 2 4 spectrophotometry Parthenin 1.0 1.0 0.5 0.5 0.05 NT NT Coronopilin 5.0 1.0 0.5 0.05 0.05 NT NT Tenulin 1.0 1.0 0.5 0.05 0.05 NT NT Helenalin 1.0 1.0 0.5 0.5 0.1 NT NT Alantolactone 5.0 1.0 0.5 0.1 0.1 NT NT a-terthienyl NT NT NT 0.05 0.05 0.05 2.5 mg/ml Phenylhep t a - t riyne (PHT) NT NT NT 0.5 0.5 NT 0.5 mg/ml NT =not tested 28 Table 4. Color reactions of some naturally occurring compounds with vanill in spray reagent. Compound Color Rf (CHCl3:Me2CO, 6:1) Monoterpenes: d-limonene citronellol citronellal menthol acetate Steroids: cholesterol ergosterol Carotenoids: 3-carotene Fatty acids: stearic oleic Flavonoids: quercetin kaemferol myricetin-3-0-Arab kaemferol-3-0-Glu quercetin-3-0-Glu Aromatic acids: anisic p-hydroxybenzoic vanil l ic caffeic ferulic p-coumaric p-aminobenzoic dark blue dark blue dark blue dark blue purple purple dark blue grey greyish blue yellow-> orange yellow-*- orange golden yellow golden yellow golden yellow 0.94 0.76 0.86 0.90 0.76 0.76 0.93 0.79 0.79 0.18 0.37 0.00 0.00 0.00 pastel violet pastel blue vivid yellow 0.49 0.47 0.42 29 Table 4. Continued. Compound Color Rf (CHCl3:Me2CO, 6:1) Miscellaneous compounds: catechol resorcinol hydroquinone coumarin hydroxycoumarin bluish red vivid red deep magenta 0.46 0.29 0.29 - =no color reaction -»• =change during heating the plate 30 S E C T I O N I I 31 A compa ra t i ve study•of the ma j or sesqu i terpene l a c t o n e s and other s e l e c t e d compounds in v a r i o u s p o p u l a t i o n s of PARTHENIUM HYSTEROPHORUS I n t r o d u c t ion Pa r then i um hys te rophorus L. i s i n c l u d e d in the t r i b e H e l i a n t h e a e , s u b t r i b e Ambros i inae of the Compositae (Asteraceae) f a m i l y ( R o l l i n s 1950). T h i s s p e c i e s i s a r e p r e s e n t a t i v e of the s e c t i o n Argyrochaetae which i s composed of n ine herbaceous taxa ( R o l l i n s , op. c i t . ) . The p l a n t ( F i g . 2) i s e r e c t , branched, l e a f y , wi th a r i g i d herbaceous stem which p e r s i s t s u s u a l l y for one growing season . The r o o t s , however, can p e r s i s t fo r at l e a s t three years and produce new s h o o t s . Leaves are a l t e r n a t e , h i g h l y d i v i d e d and covered on both s i d e s wi th t r i chomes which are a l s o present on the stems. The c a p i t u l u m ( f l o w e r i n g head) i s heterogamous, numerous, and composed of f i v e f e r t i l e p i s t i l l a t e ray f l o r e t s , and about f o r t y f e r t i l e s taminate d i s k - f l o r e t s . The mature f r u i t s (achenes) with two d i s k - f l o r e t s a t t a c h e d at the base and the subtend ing brack of female f l o r e t f a l l o f f together as a u n i t c a l l e d an achene complex. The achenes a lone are obovate , b l a c k , crowned by the p e r s i s t e n t remnants of c o r o l l a , appendages i and s t y l e ( R o l l i n s 1950) . About 2000 mature achenes are produced per p l a n t . P o l l i n a t i o n i s p robab ly by wind, a l though i n s e c t s might p lay a r o l e . The chromosome number (2n=34) i s p robab ly the 32 same in a l l p o p u l a t i o n s (Towers et a l . 1977b). Today P_;_ hys terophor us i s d i s t r i b u t e d throughout the t r o p i c s ( F i g . 3 ) , o c c u r r i n g p r i m a r i l y in a reas d i s t u r b e d by man (Towers et a l . 1977b). A c c o r d i n g to R o l l i n s (1950), the s p e c i e s i s n a t i v e to the r e g i o n around the Gu l f of Mex ico , i n c l u d i n g the West I n d i e s , arid p o s s i b l y a l s o to c e n t r a l Argen t i na .. Wi th i n the l a s t hundred years t h i s weed was i n t r o d u c e d to A f r i c a , A u s t r a l i a , and s o u t h - e a s t e r n A s i a . In A u s t r a l i a i t was f i r s t recorded in Queensland in 1955, then i t was e r a d i c a t e d but a c c i d e n t a l l y r e - i n t r o d u c e d aga in in 1958 (Hase ler 1976). In 1956 P. hys te rophorus was a l s o recorded in Ind ia (Rao 1956) . I t s spread in the f o l l o w i n g years r e s u l t e d in s e r i o u s a g r i c u l t u r a l proSlems in both c o u n t r i e s and i t has become a . s e r i o u s med ica l hazard in c e r t a i n p a r t s of I n d i a , be ing a source of a l l e r g i c c o n t a c t d e r m a t i t i s (Towers et a l . 1977b). The d e t r i m e n t a l p r o p e r t i e s of P^ hys te rophorus have been a t t r i b u t e d main ly to the presence of a p a r t i c u l a r s e s q u i t e r p e n e l a c t o n e , p a r t h e n i n ( F i g . 4 ) , whose content in t h i s p l a n t may be as h igh as 8% of the dry weight (Rodr iguez et a l . 1976a) . P a r t h e n i n i s not on ly the major a l l e r g e n r e s p o n s i b l e for a l l e r g i c c o n t a c t d e r m a t i t i s in Ind ia (Lonkar et a l . 1976) but , in a d i t i o n , i t appears to be r e s p o n s i b l e fo r the a l l e l o p a t h i c p r o p e r t i e s of P^ hys te rophorus (Kanchan 1975) . Pa r then in was i s o l a t e d from P^ hys te rophorus by Herz and Watanabe (1959) and shown to be a p s e u d o g u a i a n o l i d e (Herz et a l . 1962) . Hymenin ( F i g . 4 ) , the C - l hydroxys te reo i somer of p a r t h e n i n , was f i r s t i s o l a t e d and i d e n t i f i e d from Hymenoclea 33 s a l s o l a ( T o r i b i o and Geissman 1968) and l a t e r a l s o from P. hys te rophorus (Rodr iguez 1975b). Whi le p a r t h e n i n was found to. be the major c o n s t i t u e n t of P^ hys te rophorus from U . S . A . , Mex ico , West I n d i e s , and I n d i a , hymenin was found in p o p u l a t i o n s from southern B o l i v i a and c e n t r a l A r g e n t i n a and a l s o in one p o p u l a t i o n in Texas (Rodr iguez 1975b, Towers et a l . 1977b). Ambrosin (No. 52 in Appendix) has a l s o been r e p o r t e d to occur together with p a r t h e n i n in p l a n t s from one p o p u l a t i o n from Texas (Rodr iguez et a l . 1976a). More r e c e n t l y Ind ian workers (Sohi et a l . 1979) have i d e n t i f i e d t e t r a n e u r i n - A ( F i g . 4 ) , known from P^ alp inum v a r . t e t r a n e u r i s (Ruesch and Mabry 1969), and a new s e s q u i t e r p e n e l a c t o n e which they named h y s t e r o p h o r i n in e x t r a c t s of P. h y s t e r o p h o r u s . A l l the s t r u c t u r e s g iven by Sohi et a l . (1979) are i n c o r r e c t . On the b a s i s of d i f f e r e n c e s in the occurence of p a r t h e n i n and hymenin in v a r i o u s p o p u l a t i o n s of P^ hys te rophorus Rodr iguez (1975b) d i v i d e d t h i s s p e c i e s i n t o the " p a r t h e n i n race" and "hymenin r a c e " . In a d d i t i o n to d i f f e r e n c e s in the chemis t ry of the s e s q u i t e r p e n e l a c t o n e s , P^ hys te rophorus a l s o d i s p l a y s a number of m o r p h o l o g i c a l l y d i v e r s e forms. In South Amer i ca , p a r t i c u l a r l y , there i s a great degree of v a r i a t i o n in the s i z e and other m o r p h o l o g i c a l c h a r a c t e r s of t h i s s p e c i e s ( R o l l i n s 1950) . In t h i s study I have examined the major s e s q u i t e r p e n e l a c t o n e s and other s e l e c t e d c o n s t i t u e n t s of hys te rophorus from v a r i o u s l o c a l i t i e s us ing a s e n s i t i v e v i s u a l i z a t i o n TLC 34 method d e s c r i b e d in the S e c t i o n I . Us ing these r e s u l t s , I e s t a b l i s h e d : • (1) r e l a t i o n s h i p s between v a r i o u s p o p u l a t i o n s from the Amer i cas ; (2) the p robab le o r i g i n of hys terophorus which has been i n t r o d u c e d i n t o A u s t r a l i a and I n d i a . 35 Exper imenta l (1) P lan t m a t e r i a l P l a n t s were c o l l e c t e d by var ious , persons (see Tab le 5) in 1975-1980, a i r d r i e d and kept as herbar ium specimens or in paper e n v e l o p e s . One sample (No. 15 in Tab le 5) c o n s i s t e d of achenes o n l y . Vouchers have been d e p o s i t e d in the U n i v e r s i t y of B r i t i s h Columbia Herbar ium. When f r e s h p l a n t s were used in t h i s s tudy , they were grown from achenes c o l l e c t e d in B e l i z e , A u s t r a l i a , or Texas ( F i g . 2 ) . Achenes were l e f t to germinate in wet s o i l in covered p e t r i d i s h e s . When c o t y l e d o n s and the f i r s t l eaves appeared s e e d l i n g s were t r a n s f e r e d to pots and p l a c e d in a greenhouse. (2) Chemica ls and ins t ruments Standards of s e s q u i t e r p e n e l a c t o n e s were o b t a i n e d as d e s c r i b e d in the S e c t i o n I . The m e l t i n g p o i n t s were determined on a Thomas-Hoover c a p i l l a r y m e l t i n g p o i n t a p p a r a t u s . Nuc lear magnetic resonance s p e c t r a were measured in CDCl^with TMS as an i n t e r n a l s tandard on V a r i a n XL-100 (100 MHz) or Bruker S p e c t r o s p i n (80 MHz) spec t rometers in the -Department of C h e m i s t r y , U . B . C . The Rayonet Photochemica l Reactor used was p r o v i d e d • by the Department of Chemis t ry , U .B .C . and the hydrogenat ion of p a r t h e n i n was c a r r i e d out in the Department of Chemis t ry , 'U .B .C . 36 (3) Th in l a y e r chromatography O n e - d i r e c t i o n a l TLC with a so l ven t system ^chloroform- aceton.e (6:1) was c a r r i e d out as d e s c r i b e d in the S e c t i o n I . T w o - d i r e c t i o n a l TLC: p l a t e s 20x20 cm were deve loped f i r s t l y in c h l o r o f o r m - a c e t o n e ( 6 : 1 ) , a i r d r i e d , and then run in a second d i r e c t i o n in a s o l v e n t system w a t e r - e t h a n o l ( 5 : 1 ) . A l l p l a t e s were v i s u a l i z e d by the v a n i l l i n spray reagent as d e s c r i b e d in the S e c t i o n I . (4) Chromatographic ident i f i c a t ion of p a r t h e n i n and hymenin The s e p a r a t i o n of s t e r e o i s o m e r s by TLC may sometimes be a c h i e v e d by m u l t i p l e development of p l a t e s in the same s o l v e n t system (Dr. I . P a n f i l , p e r s . communicat ion) . Two s t e r e o i s o m e r s , p a r t h e n i n and hymenin, in crude p l a n t . e x t r a c t s were i d e n t i f i e d by TLC us ing t h i s method. The TLC p l a t e s 20x20 cm were f i r s t l y deve loped in c h l o r o f o r m - a c e t o n e ( 6 : 1 ) , a i r d r i e d , and then deve loped in a second d i r e c t i o n in h e p t a n e - e t h e r - e t h y l a c e t a t e ( 3 0 : 6 5 : 5 ) . Chromatography in the second s o l v e n t was repeated ten t i m e s , with d r y i n g o f f the p l a t e s in between the runs . P a r t h e n i n moves ahead of hymenin under these c o n d i t i o n s . (5) I s o l a t ion and i d e n t i f i c a t i o n of sesqu i terpene l a c t o n e s f rom p l a n t m a t e r i a l (a) P a r t h e n i n and c o r o n o p i l i n Shoots of d r i e d P. hys te rophorus . (Texas or B e l i z e c o l l e c t i o n ) were p u l v e r i z e d in a b l e n d e r , covered wi th 37 c h l o r o f o r m • and a l lowed to stand o v e r n i g h t . The c h l o r o f o r m e x t r a c t was f i l t e r e d and the f i l t r a t e evaporated to dryness on a r o t a t o r y evapora tor i_n vacuo. The syrup was. d i s s o l v e d in 95% e t h a n o l , 4% aqueous l ead a c e t a t e s o l u t i o n was added to p r e c i p i t a t e f a t t y a c i d s , f l a v o n o i d s , and p h e n o l i c acids. , and the s o l u t i o n was f i l t e r e d through c e l i t e ( a n a l y t i c a l f i l t e r a i d ) . The f i l t r a t e was c o n c e n t r a t e d j_n vacuo, e x t r a c t e d with c h l o r o f o r m and the e x t r a c t d r i e d over anhydrous magnesium s u l f a t e . The c h l o r o f o r m s o l u t i o n was f i l t e r e d , evaporated to d r y n e s s , and the r e s i d u e d i s s o l v e d in benzene-acetone (2:1) and a p p l i e d to a s i l i c a ge l column packed in benzene. The column was e l u t e d wi th benzene f i r s t , f o l l o w e d by i n c r e a s i n g amounts of acetone (benzene:acetone 1 0 : 1 , 10 :5 , 5:5, 5:10, 1:10, and acetone o n l y ) . F r a c t i o n s c o n t a i n i n g p a r t h e n i n (checked by TLC wi th benzene-acetone , 1:4, and b e n z e n e - e t h y l a c e t a t e , 7 :3; i o d i n e vapors) were combined and p a r t l y e v a p o r a t e d . A f t e r a d d i t i o n of i s o p r o p y l e ther and c o o l i n g in a r e f r i g e r a t o r white c r y s t a l s appeared . However, us ing v a r i o u s s o l v e n t systems for TLC development and the v a n i l l i n spray reagent I found that the p a r t h e n i n , i s o l a t e d in t h i s way, was a mixture of two compounds. S i x t y mg of t h i s mixture was chromatographed on a s i l i c a g e l column and e l u t e d with c h l o r o f o r m - a c e t o n e , 6 :1 . E a r l y f r a c t i o n s gave a b lue spot (Rf=0.46) on TLC p l a t e s . The combined f r a c t i o n s y i e l d e d white c r y s t a l s , m.p.. l77°C, NMR (CDC1 3 wi th TMS): 6 6.29 ( d, H-13b), 5.61 ( d , H-13a), 4.95 ( d, H - 6 ) , 1.17 ( s, C -10- Me), 1.10 ( s, C-5-Me).. The NMR and m.p. co r respond to the r e p o r t e d v a l u e s of c o r o n o p i l i n (see F i g . 4) (Yoshioka et a l . 1973). and the Rf and c o l o r on TLC p l a t e s were i d e n t i c a l wi th 38 those of an a u t h e n t i c sample of c o r o n o p i l i n . ( P r o f . T. A. Ge issman) . L a t e r f r a c t i o n s y i e l d e d pure p a r t h e n i n (F-ig. 4 ) , g i v i n g a b l u i s h - g r e e n c o l o r with v a n i l l i n (Rf=0.44, m.p. 167°C, NMR ( C D C I 3 w i th TMS) : c f7 .50 ( d , H-2) , 6.33 ( d, H-13b), 6.25 ( d, H-3) , 5.62 ( d , H-13a) , 5.02 ( d , H-6) , 1.30 ( s, C-5-Me), 1.14 ( d , C-10-Me).. The pa r then i n-cor onopi 1 i n mixture be fore chromatography had m.p. 164°C and a NMR spectrum i d e n t i c a l with that g iven fo r p a r t h e n i n (Yoshioka et a l . 1973). (b) Hymenin D r i e d shoots of A r g e n t i n i a n P_;_ hys te rophorus (No. 19 in Tab le 5) (only 8.5g were a v a i l a b l e ) were cut wi th s c i s s o r s and e x t r a c t e d wi th c h l o r o f o r m o v e r n i g h t . The e x t r a c t was f i l t e r e d , evaporated to a minimum volume and a p p l i e d to a column ( s i l i c a g e l ) * p a c k e d wi th c h l o r o f o r m - a c e t o n e , 6 :1 . The column was e l u t e d wi th the same s o l v e n t system and a l l f r a c t i o n s were checked by TLC. F r a c t i o n s g i v i n g a b l u i s h - g r e e n spot (Rf=0.44) were combined and p a r t l y e v a p o r a t e d . White c r y s t a l s appeared a f t e r t reatment with i s o p r o p y l e t h e r . The NMR spectrum of t h i s compound ( C D C l 3 w i t h TMS):cf7.50 ( d , H-2) , 6.25 ( d, H-13b), 6.18 ( d , H-3) , 5.55 ( d, H-13a) , 4.88 ( d , H-6) , 1.10 ( d , C-10-Me), 1.05 ( s, C-5-Me) . T h i s NMR spectrum cor responds to the r e p o r t e d v a l u e s for hymenin ( F i g . 4) (Yoshioka et a l . 1973). The c o l o r and' Rf on TLC p l a t e s were i d e n t i c a l wi th those of an a u t h e n t i c sample of hymenin (Dr. A. Romo de V i v a r ) . 3 9 (c ) H y s t e r i n D r i e d leaves of an A r g e n t i n i a n sample (No. 20 in Tab le 5) (on ly 8g were a v a i l a b l e ) were cut wi th s c i s s o r s , e x t r a c t e d , and chromatographed as d e s c r i b e d above. E a r l i e r f r a c t i o n s c o n t a i n e d hymenin (TLC, NMR), l a t e r f r a c t i o n s gave a grey spot (Rf=0.19) a f t e r TLC and the NMR ( C D C I 3 wi th TMS): cf 6.20 ( d , H-13b), 5.45 ( d , H-13a) , 4.50 ( d , H-6) , 3.85 ( s, C-10-CH 0 ) , 2.08 ( s, a c e t y l - M e ) , 0.80 ( s, C-5-Me) . These NMR va lues co r respond to those r e p o r t e d for h y s t e r i n ( F i g . 4) (Yoshioka et a l . 1973). The c o l o r and Rf on chromatograms were i d e n t i c a l wi th those of an a u t h e n t i c sample of h y s t e r i n (Dr. A. Romo de V i v a r ) . (6). P repara t ion of d i h y d r o i s o p a r t h e n i n and t e t r a h y d r o p a r t h e n i n The procedure of hydrogenat ion of p a r t h e n i n d e s c r i b e d by Herz et a l . (1962) was f o l l o w e d . A s o l u t i o n of 100 mg of p a r t h e n i n in 10 ml of e t h a n o l (95%) was hydrogenated at room temperature and atmospher ic p r e s s u r e with 10 mg of 10% p a l l a d i u m on c h a r c o a l ( N u t r i t i o n a l B i o c h e m i c a l s , C o r p . ) . A f t e r 5 hours of r e a c t i o n the s o l u t i o n c o n t a i n e d two new compounds but no p a r t h e n i n (TLC, NMR). The s o l u t i o n was f i l t e r e d and comp le te l y evaporated _i_n vacuo. The r e s i d u e was d i s s o l v e d in acetone and c r y s t a l s (need les) were formed a f t e r the a d d i t i o n of petro leum e t h e r . Complete s e p a r a t i o n of these two compounds was ach ieved by us ing p r e p a r a t i v e TLC ( s i l i c a ge l ) w i th the s o l v e n t system c h l o r o f o r m - a c e t o n e , 6 :1 . P u r i t y of these compounds was checked by TLC and NMR. A f t e r v a n i l l i n spray and h e a t i n g the p l a t e , one of these compounds (Rf=0.45) gave a v i v i d orange spot f ad ing 40 r a p i d l y on c o o l i n g the p l a t e g i v i n g l i g h t ye l low c o l o r . The NMR spectrum (CDC1 3 wi th TMS) : cf 5.42 (s b road , H-6) , 0.83 ( s, C-5- Me), 1.12 ( d , C-10-Me) cor responds to the NMR va lues of d i h y d r o i s o p a r t h e n i n (see F i g . 4) (Herz et a l . 1962). The second compound (Rf=0.49) gave d u l l b lue c o l o r wi th the same reagent and the NMR spectrum ( C D C I 3 w i th TMS) : c f4 .77 ( d, H-6) , 1.2 ( s, C-5-Me) c o r r e s p o n d i n g to that of t e t r a h y d r o p a r t h e n i n (see F i g . 4) (Herz et a l , . 1962) . The approximate r a t i o of d i h y d r o - and t e t r a h y d r o p a r t h e n i n formed from p a r t h e n i n was 2:1 (NMR). (7) P h o t o l y s i s of par then i n and co ronop i1 in The p rocedures of Kagan et a l . (1971) and Romo de V i v a r et a l . (1978) were f o l l o w e d . A s o l u t i o n of p a r t h e n i n (100 mg) in e t h y l a c e t a t e (100 ml) under a n i t r o g e n atmosphere was d i s t r i b u t e d e q u a l l y in four Pyrex tubes and these were p l a c e d in the Rayonet Photochemica l Reactor (25°C, 350 nm) for 4 h o u r s . Contents of a l l tubes were combined and evaporated on a r o t a t o r y e v a p o r a t o r , y i e l d i n g h o n e y - l i k e m a t e r i a l wi th some white c r y s t a l s , presumably p a r t h e n i n . The mixture was d i s s o l v e d in e t h y l a c e t a t e - h e x a n e , 1:1, and a p p l i e d to a column of s i l i c a ge l packed and e l u t e d wi th the same s o l v e n t system. The f r a c t i o n s g i v i n g a v i v i d v i o l e t spot (R'f = 0 . 8 0 ) , presumably of the p h o t o l y t i c product of p a r t h e n i n , were poo led and evaporated on a r o t a t o r y e v a p o r a t o r . The compound was checked immediate ly by NMR (CDC13 w i th TMS) : cf 6.34 ( d , H-13b) , 5.60 ( d , H-13a ), 4.48 (d , H-6) , 3.60 (complex) , 2 .90-3 .90 (complex) , 1 .80-2.40 (complex) , 1.27 ( s, C-5-Me) , 1.03 ( ,d, C-10-Me) . These v a l u e s cor respond to the NMR v a l u e s g iven for the p h o t o l y t i c product of p a r t h e n i n 41 (Kagan et a l . 1971) . To examine the r a t e of the p h o t o r e a c t i o n , one Pyrex tube with 10 ml of e t h y l a c e t a t e with 10 mg of p a r t h e n i n (under n i t r o g e n ) was p l a c e d in the Rayonet Photochemica l Reactor and the s o l u t i o n checked by TLC at 20 minute i n t e r v a l s . The format ion of the photoproducts of p a r t h e n i n and c o r o n o p i l i n in v a r i o u s s o l v e n t s and in a c r y s t a l l i n e form were a l s o examined. P a r t h e n i n or c o r o n o p i l i n e i t h e r in s o l u t i o n s (5 mg in 5 ml of benzene, e t h y l a c e t a t e or water) or in a c r y s t a l l i n e form in Pyrex tubes under n i t r o g e n were p l a c e d h o r i z o n t a l l y under a UV lamp ( S y l v a n i a B l a c k l i t e ; l i g h t output at 320-380 nm) for 24 hours . The s o l u t i o n s were then c o n c e n t r a t e d on a r o t a t o r y evapora tor and checked by TLC fo r the presence of new compounds. (8) Detec t i on of s e s q u i t e r p e n e l a c t o n e s i n p l a n t mater i a l One gram of each P^ hys te rophorus sample (only p a r t s of shoots were used) was cut with s c i s s o r s i n t o sma l l p i e c e s and covered wi th 30 ml of c h l o r o f o r m and o c c a s i o n a l l y shaken by hand d u r i n g 2 days of e x t r a c t i o n . Each e x t r a c t was f i l t e r e d through f i l t e r paper and the remain ing p l a n t m a t e r i a l washed twice wi th 30 ml of c h l o r o f o r m . The combined f i l t r a t e s were evaporated to d ryness _in vacuo -on a r o t a t o r y evapora tor and l e f t in a d e s i c c a t o r j_n vacuo for one hour to remove remain ing c h l o r o f o r m . Each sample was then d i s s o l v e d in a minimum amount of CDCI3 and used for NMR a n a l y s i s (80 MHz). The NMR s p e c t r a (with the lower f i e l d peaks maximized and -the methyl peaks min imized) of the 42 crude c h l o r o f o r m e x t r a c t s ( F i g . 5,6) were compared with lower f i e l d peaks of the NMR s p e c t r a of the a u t h e n t i c samples of the f o l l o w i n g s e s q u i t e r p e n e l a c t o n e s : P a r t h e n i n : H-2(d) , H-3(d) , H-13b(d) , H-13a(d) , H - 6 ( d ) ; C o r o n o p i l i n : <6 4.9 of H-6(d) was used for c o r o n o p i l i n i d e n t i f i c a t i o n in e x t r a c t s c o n t a i n i n g p a r t h e n i n (H-13a,b o v e r l a p with those of p a r t h e n i n ) ; c o r o n o p i l i n c o u l d not be i d e n t i f i e d in e x t r a c t s c o n t a i n i n g hymenin because the d o u b l e t s of H-6 o v e r l a p ; Hymenin: H -2(d) , H~3(d), H-13b(d) , H-13a(d) , H - 6 ( d ) ; H y s t e r i n : H-6(d) o n l y ; peaks of H-13a,b o v e r l a p with those of hymen i n; D i h y d r o i s o p a r t h e n i n : H-6(s_ b r o a d ) . The crude c h l o r o f o r m e x t r a c t s of a l l P^ hys te rophorus samples were chromatographed by o n e - d i r e c t i o n a l and* two- d i r e c t i o n a l TLC ( F i g . 7 ) . A l l major spots' were recorded 10 minutes and 24 hours l a t e r . The presence of the f o l l o w i n g s e s q u i t e r p e n e l a c t o n e s was checked by comparison t h e i r p o s i t i o n and c o l o r on TLC p l a t e s with a u t h e n t i c samples of p a r t h e n i n or hymenin (both have i d e n t i c a l c o l o r and p o s i t i o n on chromatograms), c o r o n o p i l i n , h y s t e r i n , t e t r a n e u r i n - A , t e t r a h y d r o p a r t h e n i n , d i h y d r o i s o p a r t h e n i n , the p h o t o l y t i c product of p a r t h e n i n , and t e t r a n e u r i n - E and -D. 43 R e s u l t s and P i s c u s s i o n P a r t h e n i n was i d e n t i f i e d by NMR in most of the samples (Table 5) as the major s e s q u i t e r p e n e l a c t o n e . , Hymenin was present in a l l A r g e n t i n i a n samples and in two samples from Jamaica (No. 14 and 15; Tab le 5 ) . I t was i d e n t i f i e d by NMR and by t w o - d i r e c t i o n a l TLC with m u l t i p l e development . C o r o n o p i l i n ( F i g . 4; i d e n t i f i e d by NMR, m. p . , Rf and c o l o r on TLC p l a t e s ) was i s o l a t e d from e x t r a c t s of d r i e d P. hys te rophorus c o l l e c t e d in B e l i z e and Texas . I t was f i r s t i s o l a t e d and i t s s t r u c t u r e determined from Ambrosia p s i l o s t a c h y a (Herz and Hognauer 1961) and l a t e r r e p o r t e d from numerous s p e c i e s of Ambrosia and Parthenium (Rodr iguez 1975b), but not from P^ h y s t e r o p h o r u s . C o r o n o p i l i n was not d e t e c t e d in t h i s s p e c i e s e a r l i e r because i t i s not separa ted and d i s t i n g u i s h e d from p a r t h e n i n by the c o n v e n t i o n a l methods of TLC a n a l y s e s of s e s q u i t e r p e n e l a c t o n e s ( e . g . UV l i g h t , i o d i n e v a p o r s , or aqueous KMnO^), and because i t s NMR spectrum o v e r l a p s to some extent with that of p a r t h e n i n . C o r o n o p i l i n was d e t e c t e d (TLC and NMR) in a l l samples of P_;_ hys te rophorus in which p a r t h e n i n ( F i g . 4) was found to be the major s e s q u i t e r p e n e l a c t o n e (Table 5 ) . The r a t i o of p a r t h e n i n to c o r o n o p i l i n was always approx imate ly 10:1 (determined by NMR). C o r o n o p i l i n was a l s o d e t e c t e d by TLC in two A r g e n t i n i a n samples (No. 22, 23; see Tab le 5) c o n t a i n i n g hymenin ( F i g . 4) as the major s e s q u i t e r p e n e l a c t o n e , and by TLC and NMR in the B o l i v i a n sample, which c o n t a i n s n e i t h e r p a r t h e n i n nor hymenin (sample No. 18; Tab le 5 ) . 44 C o r o n o p i l i n from p l a n t s c o n t a i n i n g hymenin, might , in f a c t be the d i a s t e r e o i s o m e r of c o r o n o p i l i n , but because of a s c a r c i t y of A r g e n t i n i a n p l a n t m a t e r i a l t h i s c o u l d not be v e r i f i e d by NMR. T h e r e f o r e , both the A r g e n t i n i a n and one B o l i v i a n samples were compared wi th the B e l i z i a n sample ( c o n t a i n i n g p a r t h e n i n ) by two- d i r e c t i o n a l TLC us ing m u l t i p l e development of p l a t e s in s o l v e n t s by which hymenin and p a r t h e n i n were c l e a r l y s e p a r a t e d . C o r o n o p i l i n was present in a l l examined samples and there was no ev idence for the presence of i t s d i a s t e r e o i s o m e r . H y s t e r i n ( F i g . 4) was i s o l a t e d and i d e n t i f i e d by NMR, Rf , and c o l o r on TLC p l a t e s from the A r g e n t i n i a n sample (No. 20; Tab le 5 ) . T h i s l a c t o n e was f i r s t i s o l a t e d and i t s s t r u c t u r e determined by Romo de V i v a r et a l . (1966) from p l a n t m a t e r i a l which was l a t e r e s t a b l i s h e d to be b i p i n n a t i f idum (Herz 1968, Rodr iguez et a l . 1971) . H y s t e r i n was d e t e c t e d by TLC and/or NMR in three A r g e n t i n i a n samples (No. 20, 21, 22; Tab le 5) and in one sample from Jamaica (No. 14; Tab le 5 ) . In a l l these samples hymenin was the major s e s q u i t e r p e n e l a c t o n e (Table 5 ) . T e t r a n e u r i n - A ( F i g . 4) was d e t e c t e d by TLC in a l l samples c o n t a i n i n g p a r t h e n i n , w i th the e x c e p t i o n of B r a z i l i a n p l a n t s . I t was never found together wi th hymenin (Table 5 ) . Two d e r i v a t i v e s of hydrogenated p a r t h e n i n were checked fo r t h e i r presence in Pj_ h y s t e r o p h o r u s . D i h y d r o i sopar then in was d e t e c t e d by TLC and NMR on ly in one A r g e n t i n i a n sample (No. 23; Tab le 5 ) , but t e t r a h y d r o p a r t h e n i n was not d e t e c t e d in any sample. 45 Compound No.. 1 (see F i g . 7) was present in crude e x t r a c t s of both B r a z i l i a n and B o l i v i a n samples and a l s o in two samples from A r g e n t i n a (No. 22, 23; Tab le 5 ) . Compound No. 6 (see F i g . 7) was t y p i c a l of a l l A r g e n t i n i a n samples and o c c u r r e d a l s o in the samples from Jamaica (No. 14; Tab le 5) which was i d e n t i c a l w i th A r g e n t i n i a n sample No. 20 (Table 5 ) . Compound No. 8 ( F i g . 7) was d e t e c t e d on ly in two samples from A r g e n t i n a (No. 21, 22; Tab le 5 ) . Compound No. 9 (see F i g . 7) had i d e n t i c a l c o l o r and Rf on TLC as t e t r a n e u r i n - D or - E . However, these two s e s q u i t e r p e n e l a c t o n e s were not i d e n t i f i e d in crude e x t r a c t s of P. hys te rophorus samples by NMR. The compound number 9 was p resent in most samples examined (Table 5 ) . . Compound No. 10 (see F i g . 7 ) ; three, samples from A r g e n t i n a .(No. 9, 20, 22; Tab le 5) and one from Jamaica (No. 14; Table 5) c o n t a i n e d a r e l a t i v e l y l a r g e q u a n t i t y of t h i s compound, whi,ch on o n e - d i r e c t i o n a l TLC comp le te ly covered hymenin and c o r o n o p i l i n s p o t s , thereby p r e v e n t i n g d e t e c t i o n of these s e s q u i t e r p e n e l a c t o n e s . T h i s compound p r e c i p i t a t e s from crude c h l o r o f o r m e x t r a c t s on s tand ing for s e v e r a l days . I t i s not s o l u b l e in most s o l v e n t s . The p h o t o l y t i c product of hymenin, c o n f e r d i o l i d e , has been found to occur n a t u r a l l y together wi th hymenin in P^ confertum v a r . l y ra tum (Romo de V i v a r et a l . 1978) . The f a c t that P. hys te rophorus c o n t a i n s a l a r g e q u a n t i t y of p a r t h e n i n or hymenin, 46 and c o r o n o p i l i n suggests the p o s s i b i l i t y that t h i s s p e c i e s might a l s o c o n t a i n the p h o t o l y t i c p roduc ts of these s e s q u i t e r p e n e l a c t o n e s . I t was found that the p h o t o l y t i c product of p a r t h e n i n ( F i g . . 4) was formed a f t e r 20 minutes of UV i r r a d i a t i o n _in_ v i t r o and i t s amount was i n c r e a s i n g s t e a d i l y for about three and h a l f hours when the r e a c t i o n mixture c o n s i s t e d of approx imate ly 80% of t h i s compound. There was no change in the amount of t h i s product wi th f u r t h e r i r r a d i a t i o n . The i s o l a t e d product was uns tab le in c r y s t a l l i n e form at room temperature ( a f t e r 7 days s e v e r a l spots bes ide that of the main compound were d e t e c t e d ) . The p h o t o r e a c t i o n of p a r t h e n i n o c c u r r e d when p a r t h e n i n was d i s s o l v e d in e t h y l a c e t a t e , benzene, or water , but p a r t h e n i n in a c r y s t a l l i n e form d i d not change a f t e r 24 hours of UV i r r a d i a t i on .• C o r o n o p i l i n underwent the p h o t o l y t i c r e a c t i o n on ly when d i s s o l v e d in e t h y l a c e t a t e or benzene but not in water or when i t was in a c r y s t a l l i n e form. When benzene was used, two new s p o t s , bes ide c o r o n o p i l i n .(Rf = 0.46) were d e t e c t e d ; one spot of i d e n t i c a l Rf (0.80) and c o l o r ( v i v i d v i o l e t ) wi th the photoproduct of p a r t h e n i n and a second, very i n t e n s i v e p ink spot (R f=0 .63) . C o r o n o p i l i n in e t h y l a c e t a t e formed the same produc ts and, in a d d i t i o n , s e v e r a l new spots were d e t e c t e d . The compounds were not i d e n t i f i e d . The p h o t o l y t i c p roduc ts of p a r t h e n i n and c o r o n o p i l i n were not d e t e c t e d by TLC in crude e x t r a c t s of any of the 29 d r i e d samples of P. hys te rophorus or in f r e s h l eaves of p l a n t s grown from achenes from B e l i z e and A u s t r a l i a . Ambrosin (No. 52 in Appendix) was not d e t e c t e d in any p l a n t 47 sample, in s p i t e of the e a r l i e r r epor t on i t s occur rence in t r i chomes of hys te rophorus (Rodr iguez et a l . 1976). R e l a t i o n s h i p s between the chemis t ry of P^ hys te rophorus from v a r i o u s l o c a l i t i e s The twenty n ine samples of h y s t e r o p h o r u s , examined in t h i s s tudy , c o u l d be d i v i d e d i n t o 11 chemica l t y p e s , a c c o r d i n g to the presence or absence of compounds which were examined by the TLC method used and/or NMR of t h e i r crude e x t r a c t s (Table 6 ) . In North America on ly types I and II are p r e s e n t ; these d i f f e r on ly with respec t to the compound No. 9 (Tab les 6 and 7 ) ; p a r t h e n i n i s the major s e s q u i t e r p e n e l a c t o n e . In C e n t r a l America type I i s present and p a r t h e n i n i s the major s e s q u i t e r p e n e l a c t o n e . The West I n d i e s samples are of types I I , V I I , and X I ; type II i s the North American type with p a r t h e n i n as the major s e s q u i t e r p e n e l a c t o n e , whereas types VII and XI c o n t a i n hymenin ( l o c a l i t y No. 14 i s i d e n t i c a l wi th the A r g e n t i n i a n sample No. 20; see Tab le 6 ) . The South American samples can be d i s t i n g u i s h e d as f o l l o w s : B r a z i l : the present types d i f f e r on ly in the compound No. 9 (Tab les 6, 7 ) ; p a r t h e n i n i s the major s e s q u i t e r p e n e l a c t o n e . B o l i v i a : the present type V i s c l o s e s t to type IV but d i f f e r s by the absence of p a r t h e n i n ; c o r o n o p i l i n i s the major s e s q u i t e r p e n e lactone. . Argent i n a : types VI-X are present (Table 6 ) ; types VI and VII d i f f e r e n t on ly wi th respec t to the presence of 48 h y s t e r i n ; hymenin i s the major s e s q u i t e r p e n e l a c t o n e . Type VII i s most s i m i l a r to type VIII., from which i t d i f f e r s by the presence of the compound No. 8 and absence of the compound No. 10. Type IX d i f f e r s g r e a t l y from a l l o ther t ypes , wi th the g r e a t e s t s i m i l a r i t y to type VII and VIII from which i t d i f f e r s in three compounds (Tab les 6, 7 ) . Type X r e p r e s e n t s the most d i s t i n c t p o p u l a t i o n of £_;_ h y s t e r o p h o r u s , d i f f e r i n g from a l l other types by at l e a s t four compounds (Tab les 6, 7 ) . Only type I i s p resent in samples from I n d i a and A u s t r a l i a ; p a r t h e n i n i s the major s e s q u i t e r p e n e l a c t o n e . A l l samples from North Amer i ca , B e l i z e , I n d i a , A u s t r a l i a , and one sample from Ja-maica be long to type I and I I , which are almost i d e n t i c a l , d i f f e r i n g on ly in one compound (Tab les 6 , 7 ) . These r e s u l t s suggest that the p o p u l a t i o n s examined are c l o s e l y r e l a t e d and may be of the same o r i g i n . Because samples of P. h y s t e r o p h o r u s from Ind ia and A u s t r a l i a d i f f e r g r e a t l y from a l l South Ameriacan samples, i t seems reasonab le to conc lude that these p o p u l a t i o n s o r i g i n a t e d in North America.. T h i s i s c o n s i s t e n t wi th an e a r l i e r s u g g e s t i o n that P_;_ hys te rophorus was i n t r o d u c e d a long with g r a i n imported from the U. S. A. to Ind ia (Vartak 1968) and A u s t r a l i a (Hase ler 1976). R e s u l t s of my chemica l a n a l y s e s exc lude an a l t e r n a t i v e e x p l a n a t i o n that Ind ian p o p u l a t i o n s of P^ hys te rophorus o r i g i n a t e in A r g e n t i n a (Lonkar and Jog 1972) . 49 Compared to North American samples, South American p o p u l a t i o n s of hys te rophorus are extremely d i v e r s e . In f a c t , each sample examined r e p r e s e n t s a s p e c i a l chemica l type (Tab le 6) d i f f e r i n g in 1-8 compounds from a l l o ther South American samples (Table 7 ) . A h i g h . d e g r e e of d i v e r s i t y in c h e m i s t r y and the e a r l i e r r e p o r t e d m o r p h o l o g i c a l d i f f e r e n c e s between v a r i o u s South American p o p u l a t i o n s of hys te rophorus ( R o l l i n s 1950) suggest the e x i s t e n c e of s e v e r a l d i f f e r e n t forms on t h i s c o n t i n e n t . These d i f f e r e n c e s in South American p o p u l a t i o n s c o u l d be a consequence of three s i t u a t i o n s . F i r s t , h y b r i d i z a t i o n with c l o s e l y r e l a t e d s p e c i e s might promote a h igher degree of d i v e r s i t y among hys te rophorus p o p u l a t i o n s in South Amer i ca . T h i s seems to be supported by o b s e r v a t i o n s of R o l l i n s (1950) that .many m o r p h o l o g i c a l c h a r a c t e r s of South American P^ hys te rophorus appear to d e r i v e from both , P^ conf ertum and P_;_ b i p i n n a t i f idum, . T h i s view appears to be a l s o supported by the f a c t that hymenin, which i s p resent in P^ confer tum and hymenin and h y s t e r i n in P. b i p i n n a t i f idum (Rodr iguez 1975b, Romo de V i v a r et a l . 1978) i s a l s o p resent in South American p o p u l a t i o n s of P^ hys te rophorus (Table 5 ) . On the other hand, the h y p o t h e s i s i n v o k i n g h y b r i d i z a t i o n wi th P^ b i p i n n a t i f idum and P^ con fer tum, as a cause of a h igher degree of d i v e r s i t y in South American p o p u l a t i o n s of P^ hys te rophorus i s u n l i k e l y because, a c c o r d i n g to R o l l i n s (1950), P^ b i p i n n a t i f idum and P_;_ conf ertum are not known south of Mex ico . If h y b r i d i z a t i o n wi th these s p e c i e s was 50 an important source of new forms, then we would expect a h igher degree of d i v e r s i t y among samples c o l l e c t e d in North Amer i ca . However, h y b r i d i z a t i o n wi th other c l o s e l y r e l a t e d s p e c i e s n a t i v e to South America might be an important source of a g r e a t e r degree of d i v e r s i t y among South American p o p u l a t i o n s of P. h y s t e r o p h o r u s . T h i s p o s s i b i l i t y r e q u i r e s a d e t a i l e d comparat ive study of morphology, anatomy, g e n e t i c s , and chemis t ry of P. hys te rophorus and other c l o s e l y r e l a t e d s p e c i e s . Second ly , i t has been suggested that p o l y p l o i d y has p layed a r o l e in p roduc ing the d i v e r s i t y of forms of P_̂  hys te rophorus in c e n t r a l South America ( R o l l i n s 1950). The a v a i l a b l e d a t a , however, show that chromosome number (2n=34) i s un i fo rm among v a r i o u s p o p u l a t i o n s , i n c l u d i n g one from Cordoba, A r g e n t i n a ( R o l l i n s 1950, Towers et a l . 1977b). T h i r d l y , d i f f e r e n t chemis t ry and d i f f e r e n c e s in morphology among South American p o p u l a t i o n s of P^ hys te rophorus may r e p r e s e n t v a r i o u s a d a p t a t i o n s of p l a n t s to l o c a l c o n d i t i o n s , such as the degree of h u m i d i t y , p redator p r e s s u r e s , pathogens, c o m p e t i t i v e r e l a t i o n s h i p s with other p l a n t s , e t c . T h i s p o s s i b i l i t y r e q u i r e s the study of p h y s i o l o g y and eco logy of these probab ly l o c a l l y adapted p o p u l a t i o n s . Assuming that Pj_ hys te rophorus o r i g i n a t e s in an area around the Gu l f of Mexico ( R o l l i n s 1950), from my chemica l a n a l y s e s p o s s i b l e r e l a t i o n s h i p s between the examined p o p u l a t i o n s of P. h y s t e r o p h o r u s can be r e c o n s t r u c t e d . From Tab les 6, 7 i t can be seen that the two B r a z i l i a n samples are most s i m i l a r to those from North Amer i ca . These and the B o l i v i a n ' sample, which i s 51 c h e m i c a l l y s i m i l a r to the B r a z i l i a n p o p u l a t i o n s (Tab les 6, 7 ) , thus rep resen t forms of hys te rophorus i n t e r m e d i a t e between North American types and the extremely d i v e r g e n t A r g e n t i n i a n types (Tab le 7 ) . T h i s view i s supported a l s o by the f a c t that the A r g e n t i n i a n p o p u l a t i o n s , which are most s p e c i a l i z e d as i n d i c a t e d by the h i g h e s t number of compounds examined (Table 6 ) , e x h i b i t a h i g h ' d e g r e e of d i v e r s i t y among themselves ( i n d i v i d u a l samples d i f f e r from one another in 1-5 compounds; Tab le 7 ) . The samples of hys te rophorus from Jamaica i n d i c a t e a c l o s e r e l a t i o n s h i p wi th both North and South American t y p e s . One sample was i d e n t i c a l with No. 20 from A r g e n t i n a , whereas the second sample was i d e n t i c a l wi th North American type II (Table 5 ) . The t h i r d sample (type XI) d i f f e r e d from a l l other samples (Table 5) but because of i n s u f f i c i e n t i n f o r m a t i o n on three compounds, i«ts r e l a t i o n s h i p s to other types c o u l d not be d e t e r m i n e d . The presence of hymenin in t h i s sample, however, i n d i c a t e s the p robab le o r i g i n of t h i s P_;_ hys te rophorus p o p u l a t i o n in South Amer i ca . 52 Conelus i ons The use of v a n i l l i n as a TLC spray reagent fo r p r e l i m i n a r y i d e n t i f i c a t i o n of major s e s q u i t e r p e n e l a c t o n e s in P. hys te rophorus r e s u l t e d in f i n d i n g three compounds, c o r o n o p i l i n , h y s t e r i n , and d i h y d r o i s o p a r t h e n i n , which were shown for the f i r s t time to occur in t h i s s p e c i e s . The presence of these sesqu i te rpene , l a c t o n e s was con f i rmed by NMR a n a l y s e s . The chemica l a n a l y s e s of s e l e c t e d compounds from v a r i o u s samples of P^ hys te rophorus i n d i c a t e d the e x i s t e n c e of 11 chemica l types of t h i s s p e c i e s . However, i t has to be c o n s i d e r e d that the number of samples was sma l l and sampl ing d i d not cover the whole range of r e c e n t l y d e s c r i b e d P^ hys te rophorus d i s t r i b u t i o n . T h e r e f o r e , i t i s l i k e l y that the number of chemica l types i s an u n d e r e s t i m a t i o n . A more e x t e n s i v e survey i s necessary for a b e t t e r unders tand ing of the d i v e r s i t y and r e l a t i o n s h i p s between v a r i o u s p o p u l a t i o n s , e s p e c i a l l y those in South Amer i ca . In any c a s e , d i f f e r e n c e s in the c h e m i s t r y of v a r i o u s p o p u l a t i o n s of hys te rophorus shown by t h i s study suggest the p o s s i b i l i t y of the e x i s t e n c e of s e v e r a l forms, s u b s p e c i e s , or perhaps even s p e c i e s . T h i s has a l r e a d y been i n d i c a t e d by R o l l i n s (1950) on the b a s i s of h i s study of v a r i o u s m o r p h o l o g i c a l f e a t u r e s of P^ h y s t e r o p h o r u s . However, the i m p l i c a t i o n s of d i f f e r e n c e s in chemis t ry between v a r i o u s p o p u l a t i o n s of P. h y s t e r o p h o r u s shown by t h i s study w i l l r e q u i r e a more d e t a i l e d taxonomic s t u d y . 54 Fig. 2. Parthenium hysterophorus grown i n a greenhouse (U.B.C.) from achenes collected i n Austin, Texas. Fig. 3. D i s t r i b u t i o n of P_. hysterophorus (according to R o l l i n s (1950) and Towers et a l . (1977)) and l o c a l i t i e s from which plant samples for chemical analyses were obtained. 56 Fig. 4. Chemical structures of sesquiterpene lactones examined in this section. Coronopilin Tetrahydroparthenin Dihydroisoparthenin Parthenin, photolytic product Fig. 5. NMR spectrum of crude chloroform extract of P_. hysterophorus from Texas ( l o c a l i t y 10). This represents the most widespread chemical type I containing parthenin and coronopilin as major sesquiterpene lactones. Fig. 6. NMR spectrum of crude chloroform extract of P_. hysterophorus from Argentina ( l o c a l i t y 20). This represents the chemical type VII containing hymenin and hysterin. Fig. 7. Major spots detected by v a n i l l i n spray reagent on plates developed by two-directional TLC of crude chloroform extracts of P_. hysterophorus. 1 o - — 2 10 o 0 7' 8 O 9 >. 2nd water - ethanol, 5 • ] Spot No. Color a f t e r 10 min (after 24 hours) Rf values Compound 1 Bluish red (purple) 0.56; 0.46 unidentified 2 Blue (greyish magenta) 0.46; 0.54 coronopilin 3 Bluish green (greyish yellow) 0.44; 0.59 parthenin, hymenin 4 V i v i d orange ( l i g h t yellow) 0.45; 0.54 dihydroisoparthenin 5 Greyish green (greyish green) 0.42; 0.65 tetraneurin-A 6 Greenish blue (greenish blue) 0.32; 0.65 unidentified 7 Grey (grey) 0.19; 0.58 hysterin 8 V i o l e t -*- greyish green (greyish green) 0.19; 0.58 unidentified 9 D u l l green (ol i v e brown) 0.09; 0.71 unidentified 10 Yellow (yellow) 0.44; 0.18 unidentified Table 5. Summary of chemical analyses of P_. hysterophorus from various l o c a l i t i e s by two-directional TLC and NMR ( D compound detected, • compound not detected by TLC; A compound detected, A compound not detected by NMR). Numbers of in d i v i d u a l l o c a l i t i e s correspond to those i n Fi g . 3. No. 3 L o c a l i t y ; N o x ^ q 2 Par. N o > 4 N q > 5 N o # 6 N o > 7 N o < 8 N o < 9 No.10 date of c o l l e c t i o n ; or No. c o l l e c t o r Unk. Cor. Hym. Par. Hym. DHP T.-A Unk. Hys. Unk. Unk. Unk. 1 F l o r i d a : • B A B A A • A B • • A • B • Hwy 90-Quincy; October 19 75; G. H. N. Towers 2 F l o r i d a : D B A B A A D A B • • A • B • Hwy 90-Marianna; October 1975; G. H. N. Towers 3 Alabama: D B A B A A • A B • • A • • • Montgomery; October 1975; G. H. N. Towers 4 Alabama: • B A B A A • A B • • A • • • Selma; October 1975; G. H. N. Towers 5 Alabama: D B A B A A D A B • • A • • • Hwy 80W-Uniontown; October 1975; G. H. N. Towers Table 5. (continued) 6 Texas: Forney; October 1975; G. H. N. Towers • • • • A • A • • A • • 7 Texas: Dalas; October 1975; G. H. N. Towers i B A A A • A • • A • • 8 Texas: Hwy 84-Mexia; October 1975; G. H. N. Towers • • • • A • A • • A • • 9 Texas: Hwy 35-Waco; October 1975; G. H. N. Towers • a A • A • A • D A D • • 10 Texas: Austin 1975 D. R. DiFeo • a a A A DA • • D A • • 11 Mexico: Mexico C i t y ; November 1975; G. H. N. Towers • • A A A • A • • A • • 12 B e l i z e : Indian Church; May 1979; T. Arnason • B A A A DA D DA D D Table 5. (continued) 13 Jamaica: • D A B A A D A B • • A • • • Montego Bay; December 1975; G. H. N. Towers 14 Jamaica: • D A B A A • A • B . B A • B B Kingston; December 1975; G. H. N. Towers 15 Jamaica: • • B A A * • ? • ? • ? • St. Mary; June 1980; K. Stuart 16 B r a z i l : n n B B A B A A D A D d • A • B • Corumba 19 S 57 30'W 1979 I. Dale 17 B r a z i l : B B A B A A • A • • • A • • • Londrina 23°30'S 51°30'W; 1979; I. Dale 18 . B o l i v i a : B B A D A A • A • • • A • . • • Santa Cruz 17°47'S 63°10'W; 1979; I. Dale 19 Argentina: Jujuy, • • A Q ^ A • A • B • A • B B San Salvador de Jujuy; February 1976; G. H. N. Towers Table 5. (continued) 20 Argentina: Salta, Pamp Blanca 24°30'S 64°30'W 1979; I. Dale 21 Argentina: Tucuman, 26 30'S 65°W; 1979; I. Dale 22 Argentina: La Rioja 29 S 67°W; 1979; I. Dale 23 Argentina: Cordoba, Arroyo l a Pampa; February 1976; G. H. N. Towers 24 India: Bangalore; P. V. Subba Rao 25 A u s t r a l i a : Queensland, Charter's Towers; March 1980; I. Dale 26 A u s t r a l i a : Queensland, C o l l i n s v i l l e ; February 1980; I. Dale A A • A • • A • A • A • ** A • • A A • A • ** A • A A B A • • A • B • A A • A • • A • • A A • A B • • A • • A A • A B • • A • • ON Co Table 5. (continued) 27 A u s t r a l i a : Queensland, • D A D A A • A • • D A • Mt. Douglas; March 1980; I. Dale 28 A u s t r a l i a : Queensland, • D A B A A • A • • D A • Elgen Downs; March 1980; I. Dale 29 A u s t r a l i a : Queensland, • D A D A A D A • • D A • Clermont; 1979; I. Dale ? Analyses incomplete because of i n s u f f i c i e n t sample; * Presence of hymenin was determined by two-directional TLC with multiple development (see Experimental for further explanation); ** Hysterin was undetectable by TLC because of being covered by compound No. 8; *** Coronopilin was undetectable on NMR because i t s peaks overlapped with those of hymenin. Note: Cor.=coronopilin; Par.=parthenin; Hym.=hymenin; DHP=dihydroisoparthenin; T.-A=tetraneurin-A; Hys.=hysterin; Unk.=unknown compound. Table 6 . Types of P_. hysterophorus as determined by chemical analyses (NMR, TLC) of samples from various l o c a l i t i e s . Chemical Compounds examined type Par. Hym. Cor. Hys. T.-A DHP No.l No. 6 No. 8 No.9 No. 10 L o c a l i t y I + - + - + _ - - - + - Fl o r i d a (1,2), Texas (6-8,10), Mexico (11), Belize (12), India (24), A u s t r a l i a (25-29) I I + - + - + - - - - - Alabama (3-5), Texas (9), Jamaica (13) I I I • '• + - + - - - + - - + - B r a z i l (16) IV + . . - + - _ _ + - - - - B r a z i l (17) V _ _ + _ _ _ + _ _ _ _ B o l i v i a (18) VI - + - - _ . _ _ . + - + + Argentina (19) VII - + - + _ _ _ + - + + Argentina (20), Jamaica (14) VIII _ + _ + - _ _ + + + _ Argentina (21) IX - + + + _ _ + + + + + Argentina (22) X - + + - _ + + + _ + _ Argentina (23) XI - + - ? ? - - - - ? - Jamaica (15) Note: Par.=parthenin; Hym.=hymenin; Cor.=coronopilin; Hys.=hysterin; T.-A=tetraneurin-A; DHP=dihydroisoparthenin; No.l, 6, 8, 9, 10=unknown compounds; + =compound present; - =compound absent. Table 7. Degree of d i s s i m i l a r i t y between in d i v i d u a l chemical types of P_ . hysterophorus (type numbers correspond with those given i n Table 6 ). Table gives t o t a l number of compounds i n which in d i v i d u a l types d i f f e r from a l l others . Type XI i s not included because of i n s u f f i c i e n t data. Chemical type Chemical type I I I I I I IV V VI VII VIII IX X I - •1 2 3 4 6 7 7 8 6 I I 1 - 3 2 3 7 8 8 9 7 I I I 2 3 - 1 2 6 7 7 6 4 IV 3 2 1 - 1 7 8 8 7 5 V 4 3 2 1 - 6 7 7 6 4 VI 6 7 6 7 6 - 1 3 4 4 VII 7 8 7 8 7 1 - 2 3 5 VIII 7 8 7 8 7 3 2 - 3 5 IX 8 9 6 7 6 4 3 3 - 4 X 6 7 4 5 4 4 5 5 4 - 67 S E C T I O N I I I 68 B i o l o g i c a l a c t i v i t i e s of sesqu i terpene l a c t o n e s Q I n t r o d u c t i o n Sesqu i te rpene l a c t o n e s e x h i b i t a v a r i e t y of a c t i v i t i e s a g a i n s t d i f f e r e n t types of o rgan isms . In the f o l l o w i n g pa r t I w i l l summarize the major f i n d i n g s on i n d i v i d u a l types of b i o l o g i c a l a c t i v i t i e s of these compounds. 1. A n t i m i c r o b i a l a c t i v i t y Some s e s q u i t e r p e n e l a c t o n e s have been r e p o r t e d to i n h i b i t , growth of b a c t e r i a and/or fung i ( e . g . O lechmnowicz-Step ien and S t e p i e n 1963, V ichkanova et a l . 1971, V a n h a e l e n - F a s t r e 1968, 1972, Mathur et a l . 1975, Char and Shankarabhat 1975, Norman et a l . 1976, Lee et a l . 1977b, Towers et a l . 1977a) . I t has been suggested (Lee et a l . 1974, 1977b) that the u n s u b s t i t u t e d eye lopentenone r i n g i s a p r e r e q u i s i t e for a n t i m i c r o b i a l a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s , and that i t i s independent on the presence or absence of the W-methylene-V- l a c t o n e mo ie ty . 69 2. A l l e l o p a t h i c a c t i v i t y Parthenium h y s t e r o p h o r u s , a hazardous , widespread weed that has i n f e s t e d a g r i c u l t u r a l lands in many p a r t s of Ind ia and A u s t r a l i a , causes a s e r i o u s r e d u c t i o n of many e c o n o m i c a l l y important c rop s p e c i e s . The growth and y i e l d of s e v e r a l c r o p s p e c i e s were c o n s i d e r a b l y a f f e c t e d when they were grown in s o i l c o n t a i n i n g d r i e d root and l e a f m a t e r i a l s of t h i s weed (Kanchan and Jaychandra 1976). These authors a l s o observed that d r i e d p l a n t m a t e r i a l or aqueous e x t r a c t s from roo ts of P. h y s t e r o p h o r u s caused a s u p p r e s s i o n in the growth and c o l o n i z a t i o n of R h i z o b i a in leguminous p l a n t s . Sarma et a l . (1976) found that the seed germinat ion of three c rop p l a n t s (Arach i s hypogea, C r o t a l a r i a juncea , and Phaseolus mungo) was not a f f e c t e d by aqueous e x t r a c t s of h y s t e r o p h o r u s , however, the e x t r a c t s had a pro found i n h i b i t o r y e f f e c t on r a d i c l e gr.owth and dry weight of the p l a n t s examined. P a r t h e n i n and water e x t r a c t s of E\_ hys te rophorus i n h i b i t e d seed germina t ion and growth of s e e d l i n g s of Phaseo lus v u l g a r i s (Garc iduenas et a l . 1972), wheat, and r a g i ( E l e u s i n e coracana) (Kanchan 1975). In the l a t t e r work almost a l l f r a c t i o n s of e x t r a c t s showed i n h i b i t o r y a c t i v i t y . On the b a s i s of these and other data i t has been suggested that P^ hys te rophorus c o n t a i n s a complex of i n h i b i t o r s with p a r t h e n i n and some p h e n o l i c a c i d s as the prominent c o n s t i t u e n t s (Kanchan 1975, Kanchan and Jayachandra 1976). P. hys te rophorus produces l a r g e q u a n t i t i e s of p o l l e n w h i c h , ' when c a r r i e d away a-nd d e p o s i t e d on f l o r a l p a r t s of o ther p l a n t s , 70 i n h i b i t the ge rmina t i on o f . p o l l e n and the growth of p o l l e n tube of other s p e c i e s . T h i s has been observed under both exper imenta l and n a t u r a l c o n d i t i o n s but the compounds r e s p o n s i b l e for t h i s a c t i o n have not been i d e n t i f i e d (Kanchan and Jayachandra 1976, 1980). The f a c t that s e s q u i t e r p e n e l a c t o n e s have been found in p o l l e n of hys te rophorus (Pieman et a l . 1980) suggests that these compounds c o u l d be r e s p o n s i b l e for p o l l e n a l l e l o p a t h y of t h i s s p e c i e s . D a l v i et a l . (1971) demonstrated the p h y t o t o x i c a c t i o n of a l a n t o l a c t o n e by i t s i n h i b i t o r y e f f e c t s on seed g e r m i n a t i o n , s e e d l i n g growth, and ra te of r e s p i r a t i o n of Phaseo lus mungo. The authors suggested that a l a n t o l a c t o n e i n h i b i t s amylases and p r o t e a s e s and enzymes i n v o l v e d i n . t h e s y n t h e s i s of new p r o t e i n s and n u c l e i c a c i d s . Sesqu i te rpene l a c t o n e s from s p e c i e s of Artemi s ia i n h i b i t e d the growth but s t i m u l a t e d the r e s p i r a t i o n of Cucumi s sat ivus (McCahon et a l . 1973 ). L i t t e r of Artemi s ia t r i d e n t a t a i n h i b i t e d ge rmina t i on and growth of three s p e c i e s of g r a s s e s ( S c h l a t t e r e r and T i s d a l e 1969) p o s s i b l y due to s e s q u i t e r p e n e l a c t o n e s p resent in t h i s s p e c i e s . S e s q u i t e r p e n e l a c t o n e s from K e l i a n t h u s tuberosus i n h i b i t e d the e l o n g a t i o n of Avena c o l e o p t i l e s e c t i o n s and promoted a d v e n t i t i o u s root fo rmat ion of Phaseolus mungo c u t t i n g s (Shibaoka et a l . 1967 a . b ) . Only l a c t o n e s with the exomethylene on the l a c t o n e r i n g e x h i b i t e d t h i s a c t i v i t y . Reduct ion of the l a c t o n e s i n v o l v i n g the methylene group l e d to i n a c t i v e compounds. A l s o the adducts formed with c y s t e i n e ( v i a the 71 exomethylene group) were i n a c t i v e . Some other s e s q u i t e r p e n e l a c t o n e s have been r e p o r t e d to possess growth r e g u l a t i n g a c t i v i t y (Gross 1975) . A number of such compounds have been i s o l a t e d in the search for ant i tumor agents of p l a n t o r i g i n . For example, v e r n o l e p i n from Vernon ia hymenolepi s i n h i b i t s the ex tens ion growth of wheat c o l e o p t i l e s e c t i o n s (Sequ iera et a l . 1968). 3. A n t i h e l m i n t h i c a c t i v i t y and chemoprophylax i s i n , s c h i s t o s o m i a s i s It has been r e p o r t e d that a l a n t o l a c t o n e , which i s a very s t ron g s e n s i t i z o r in human a l l e r g i c c o n t a c t d e r m a t i t i s , has been used as a vermifuge (Dupuis et a l . • 1974) . I t possesses a n t i h e l m i n t h i c a c t i v i t y a g a i n s t Fa sc i o l a hepat i c a (Kim et a l . 1961) . A l p h a - s a n t o n i n and i t s d e r i v a t i v e s are a l s o w e l l known as imporatant a n t i h e l m i n t h i c and a s c a r i c i d a l agents (Haynes 1948) . E remanth in , c o s t u n o l i d e , (i - c y c l o c o s t u n o l i d e , and g o y a z e n o l i d e from the wood o i l s of the common b r a z i l i a n t r e e s (Eremanthus e l a e a q n u s , E. goyazens i s, Van i1 losmops i s e r y t h r o p a p p a , and Moguin ia v e l u t ina) i n h i b i t p e n e t r a t i o n of c e r c a r i a e of the trematode Sch i stosoma man son i i n t o an imal s k i n (Baker et a l . 1972, V ichnewski et a l . 1976). I t has been suggested that the e x o c y c l i c methylene on the l a c t o n e r i n g of these s e s q u i t e r p e n e l a c t o n e s i s r e s p o n s i b l e fo r t h i s a c t i v i t y by i n h i b i t i n g e i t h e r the p e n e t r a t i o n enzymes or enzymes w i t h i n the c e r c a r i a . 72 4. Ef f e c t s on i n s e c t s It has been e s t a b l i s h e d that the s e s q u i t e r p e n e l a c t o n e g l a u c o l i d e - A from Vernon ia s p e c i e s p r o v i d e s r e s i s t a n c e to i n s e c t f eed ing and that the i n g e s t i o n of t h i s compound has a d e t r i m e n t a l e f f e c t on the growth and development of L e p i d o p t e r a n l a r v a e (Burnet t et a l . 1974). Another s e s q u i t e r p e n e l a c t o n e , a l a n t o l a c t o n e , i s a f eed ing d e t e r r e n t to T r i b o l i u m con fusum and a l s o a f f e c t s the s u r v i v a l of b e e t l e s (Pieman et a l . 1978). P a r t h e n i n from P^ hys te rophorus i n h i b i t s hear tbeat of g rasshoppers most l i k e l y by b l o c k i n g t h i o l c o n t a i n i n g compounds important f o r normal heart a c t i v i t y . T h i s i s i n d i c a t e d by the f a c t that the a c t i v i t y of p a r t h e n i n - a r r e s t e d h e a r t s can be r e s t o r e d by t h i o l a d d i t i o n (Pieman et a l . 1981). 5. E f f e c t s on mamma 1s The s e s q u i t e r p e n e l a c t o n e , g l a u c o l i d e - A , has been r e p o r t e d to act as a f eed ing d e t e r r e n t to r a b b i t s and deer (Burnet t et a l . 1977, Mabry and G i l l 1979) . In a d d i t i o n , there are s e v e r a l r e p o r t s on the po isonous a c t i o n of many s p e c i e s of the Compositae to l i v e s t o c k g r a z i n g on them ( e . g . Sperry et a l . 1964, K ingsbury 1964, Schmuz et a l . 1968). I v i e et a l . (1975a) showed that hymenovin, the major s e s q u i t e r p e n e l a c t o n e of Hymenoxys o d o r a t a , i s r e s p o n s i b l e fo r t o x i c i t y of t h i s p l a n t to g r a z i n g sheep and g o a t s . A l s o the extreme t o x i c i t y of Helen ium microcephalum to c a t t l e , sheep, and goats has been found to be caused by a h igh l e v e l of the s e s q u i t e r p e n e l a c t o n e , h e l e n a l i n , 73 present in t h i s s p e c i e s (Wi t ze l et a l . 1976; in Towers et a l . 1977b). V e r m e e r i n , a s e s q u i t e r p e n e d i l a c t o n e of the p h y s i o l o g i c a l l y a c t i v e vermeer ic a c i d , causes vomi t ing d i s e a s e in sheep g r a z i n g on Ge iger ia s p e c i e s in South A f r i c a (Anderson et a l . . 1 9 6 7 ) . T h i s d i l a c t o n e i s a l s o present in Hymenoxys r icha rdson i i , an American p l a n t po isonous to l i v e s t o c k (Herz et a l . 1970). I t has been known for many years that when p l a n t s , which c o n t a i n s e s q u i t e r p e n e l a c t o n e s , are consumed by d a i r y c a t t l e , t h e i r mi lk t a s t e s b i t t e r (Herzer 1942) . I t has been e s t a b l i s h e d that t e n u l i n from Helen i um amarum i s the a c t i v e i n g r e d i e n t r e s p o n s i b l e for the b i t t e r t a s t e of the m i l k . Pa r then i um h y s t e r o p h o r u s , when fed to c a t t l e and b u f f a l o e s in I n d i a , was found to cause i l l n e s s or death of the a n i m a l s , most l i k e l y because of p a r t h e n i n p resent in t h i s s p e c i e s (Narasimhan et a l . 1977). The . e x t r a c t of P^ hys te rophorus from which p a r t h e n i n and other s e s q u i t e r e p e n e l a c t o n e s were removed c o u l d be used as a p r o t e i n r i c h fodder (Savangihar and J o s h i 1978), c l e a r l y showing that s e s q u i t e r p e n e l a c t o n e s are r e s p o n s i b l e for the p o i s o n i n g a c t i o n of t h i s p l a n t . I v i e e-t a l . (1975a) suggested that the p o i s o n i n g a c t i o n of s e s q u i t e r p e n e l a c t o n e s such as hymenovin on g r a z i n g mammals might be a consequence o f : (1) t h e i r r e a c t i o n wi th s u l p h y d r y l groups of key enzymes (see a l s o Hanson et a l . 1970); and (2) the e f f e c t s of s e s q u i t e r p e n e l a c t o n e s on the m i c r o b i a l compos i t i on of the rumen. In a d d i t i o n , Seth and Bhat ia (1978) demonstrated tha t p a r t h e n i n i s a d i r e c t c a r d i a c depressant in dogs and r a t s . 74 6. A l l e r g i c con tac t dermat i t i s In fo rmat ion on a l l e r g i c c o n t a c t d e r m a t i t i s caused by s e s q u i t e r p e n e l a c t o n e s and p h o t o d e r m a t i t i s caused by p o l y a c e t y l e n i c compounds a c t i v a t e d by UV l i g h t was reviewed by Towers (1979) . Most of the s e s q u i t e r p e n e l a c t o n e s c a u s i n g a l l e r g i c c o n t a c t d e r m a t i t i s have been r e p o r t e d from s p e c i e s of the Compositae but there are a l s o examples from other f a m i l i e s such as the Lauraceae and Magnol iaceae ( M i t c h e l l 1969, M i t c h e l l et a l . 1970, 1971a,b, 1972, Bleumink et a l . 1976, Lonkar et a l . 1974, Evans and Schmidt 1980). Cases of a l l e r g i c c o n t a c t d e r m a t i t i s evoked by F r u l l a n ia s p e c i e s (an e p i p h y t i c l i v e r w o r t ) in f o r e s t workers have a l s o been r e p o r t e d ( M i t c h e l l et a l . 1969) . • The major s e n s i t i z e r s from F r u l l a n i a s p e c i e s were i s o l a t e d and i d e n t i f i e d as s e s q u i t e r p e n e l a c t o n e s (Knoche et a l . 1969, M i t c h e l l et a l . 1970, Pexold et a l . 1972, Asakawa et a l . 1976) . The recent f i n d i n g s of c o n t i n u i n g r e s e a r c h on d e r m a t i t i s caused by hys te rophorus were summarized by Towers et a l . (1977b). A l l e r g i c con tac t d e r m a t i t i s caused by t h i s s p e c i e s was r e p o r t e d from the southern U n i t e d S ta tes 50 years ago (French 1930) but mechan iza t ion of farming caused a d e c l i n e in i t s i n c i d e n c e . On the c o n t r a r y , in I n d i a , where the s p e c i e s was a c c i d e n t a l l y i n t r o d u c e d in 1956, the f requency of cases of Parthenium d e r m a t i t i s has been i n c r e a s i n g s i n c e i t was f i r s t r e c o g n i z e d in 1965 (Lonkar and Jog 1972). A l l e r g i c c o n t a c t d e r m a t i t i s due to Parthenium deve lops from repeated c o n t a c t s w i th t h i s p l a n t or p o s s i b l y with i t s d i s s e m i n a t e d t r i chomes and 75 d r i e d p l a n t p a r t s (Lonkar et a l . 1974). T y p i c a l i n i t i a l p a t i e n t s were farmers and weed p u l l e r s but more r e c e n t l y some persons l i v i n g in c i t i e s , who were not d i r e c t l y exposed to the l i v i n g p l a n t s , a l s o deve loped t h i s a l l e r g y . The weed has become an important d e r m a t o l o g i c a l and h e a l t h problem (Towers et a l . 1977b, and r e f e r e n c e s t h e r e i n ) . Pa r then i um d e r m a t i t i s has been r e p o r t e d much more f r e q u e n t l y in a d u l t males than in females and no cases are known in c h i l d r e n be fo re puberty (Towers et a l . 1977b). Sesqu i te rpene l a c t o n e s , main ly p a r t h e n i n , are c o n s i d e r e d to be the a l l e r g e n s of t h i s p l a n t s p e c i e s (Lonkar et a l . 1976) . The genus Chrysanthemum (with i t s many s p e c i e s and v a r i e t i e s ) i s one of the most common causes of a l l e r g i c c o n t a c t d e r m a t i t i s among f l o r i s t s and h o r t i c u l t u r i s t s . Sesqu i te rpene l a c t o n e s present in ' these p l a n t s are r e s p o n s i b l e fo r the d e r m a t i t i s ( e . g . Hausen and Schu lz 1973, .1975, 1976). Other common weeds, h o r t i c u l t u r a l p l a n t s , or v e g e t a b l e s from the Compositae such as Ambros i a , Artemi s i a , A s t e r , C i c h o r ium, Cosmos, D a h l i a , H e l i a n t h u s , Helen i um, M a t r i c a r i a , S o l i d a g o , and many o t h e r s have been r e p o r t e d to cause a l l e r g i c c o n t a c t d e r m a t i t i s ( e . g . M i t c h e l l 1969, Evans and Schmidt 1980) . I t has been e s t a b l i s h e d that the exomethylene on the l a c t o n e r i n g i s r e s p o n s i b l e for a 1 l e r g e n i c i t y of s e s q u i t e r p e n e l a c t o n e s , however, t h i s group a lone • i s not always immuno log i ca l l y s u f f i c i e n t ( M i t c h e l l et a l . 1970, 1971a,b; M i t c h e l l and Dupuis 1971, E p s t e i n et a l . 1980) . S ince p a r t h e n i n undergoes a r e a c t i o n wi th c y s t e i n e v i a the exomethylene on the l a c t o n e r i n g as w e l l as v i a the C2-C3 double bond (Pieman et a l . 76 1979) the presence of these two a c t i v e s i t e s in a molecu le of ' pa r then in c o u l d be r e s p o n s i b l e fo r i t s s t rong a l l e r g e n i c i t y . 7. C y t o t o x i c a c t i v i t y Sesqu i te rpene l a c t o n e s are of great i n t e r e s t in cancer r e s e a r c h because many of these compounds e x h i b i t a n t i l e u k e m i c , c y t o t o x i c , and/or tumor i n h i b i t o r y a c t i v i t y . The r e l a t i o n s h i p between chemica l s t r u c t u r e of s e s q u i t e r p e n e l a c t o n e s and t h e i r c y t o t o x i c a c t i v i t y was i n v e s t i g a t e d by many r e s e a r c h e r s . In t h e i r review of a n t i n e o p l a s t i c agents of p l a n t o r i g i n , H a r t w e l l and Abbott (1969) conc luded that a l l known a c t i v e s e s q u i t e r p e n e l a c t o n e s possess an Of, ̂ - u n s a t u r a t e d . l a c t o n e r i n g . L a t e r i t was e s t a b l i s h e d that the con jugated exomethylene group on the l a c t o n e i s an e s s e n t i a l r e q u i s i t e for c y t o t o x i c i t y • (Kupchan 1970, Kupchan et a l . 1970, 1971) . Changes such as s a t u r a t i o n or a d d i t i o n s to t h i s methylene group r e s u l t e d in the l o s s of c y t o t o x i c i t y and tumor i n h i b i t i o n ( e . g . Kupchan et a l . 1971, P e t t i t and Cragg 1973, Howie et a l . 1974). The presence of a con jugated e s t e r , eye lopentenone , or exomethylene on the l a c t o n e in a d d i t i o n appear to enhance c y t o t o x i c i t y . However, Lee et a l . (1971) demonstrated that the most d i r e c t f a c t o r r e s p o n s i b l e fo r c y t o t o x i c i t y among s e s q u i t e r p e n e l a c t o n e s i s the 0=C-C = CH2. system, whether i t i n v o l v e s the l a c t o n e or c y c l o p e n t e n o n e . A d d i t i o n a l a l k y l a t i n g groups may enhance c y t o t o x i c i t y s i g n i f i c a n t l y (Lee et a l . 1973). In t h e i r f u r t h e r s t u d i e s Lee et 77 a l . (1972, 1974, 1977a) and H a l l et a l . (1977) conc luded that the CX-methylene-V- lactone moiety i s l e s s important than the u n s a t u r a t e d ketone moiety wi th respec t to c y t o t o x i c i t y as w e l l as a n t i m i c r o b i a l a c t i v i t y (Lee et a l . 1977b). However, bakkenol i d e - A , which i s a /3-methylene-V- lactone that does not have the 0=C-C=CH2 system, i s c y t o t o x i c (Jamieson et a l . 1976) . T h i s suggests that other s t r u c t u r a l f e a t u r e s of s e s q u i t e r p e n e l a c t o n e s a l s o p lay an important r o l e in c y t o t o x i c a c t i v i t y of these compounds. Schlewer et a l . (1979) s t u d i e d the c y t o t o x i c a c t i v i t y of 20 s y n t h e t i c methy lene- - b u t y r o l a c t o n e s . Many of these compounds were c y t o t o x i c , however, because c h e m i c a l l y c l o s e compounds d i f f e r e d g r e a t l y in t h e i r a c t i v i t y , these au thors conc luded that there was no c l e a r s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p . Chemical and b i o l o g i c a l s t u d i e s support the view that s e s q u i t e r p e n e l a c t o n e s i n h i b i t tumor growth by s e l e c t i v e a l k y l a t i o n of g r o w t h - r e g u l a t o r y b i o l o g i c a l macromolecules such as key enzymes which c o n t r o l c e l l d i v i s i o n (Kupchan 1974) . These tumor i n h i b i t o r s may have a t r i p l e s e l e c t i v i t y : (a) for t h i o l s over other n u c l e o p h i l e s such as amines; (b) for p a r t i c u l a r s u l p h y d r y l enzymes; and (c) for p a r t i c u l a r s u l p h y d r y l groups w i t h i n those enzymes (Kupchan 1974). T h i s h y p o t h e s i s i s supported by the observed l o s s of a c t i v i t y of s u l p h y d r y l enzymes p h o s p h o f r u c t o k i n a s e (Hanson et a l . 1970) and g lycogen synthase (Smith et a l . 1972) a f t e r t h e i r r e a c t i o n wi th some s e s q u i t e r p e n e l a c t o n e s known to act as tumor i n h i b i t o r s . I_n v i v o s t u d i e s (Lee et a l . 1977a, H a l l et a l . 1977) showed that s e s q u i t e r p e n e 78 l a c t o n e s i n h i b i t e d nuc lea r DNA s y n t h e s i s and DNA polymerase enzymatic a c t i v i t y in tumor c e l l s and i n t e r f e r e d with g l y c o l y t i c and m i t o c h o n d r i a l energy p r o c e s s e s . These au thors conc luded that the i n h i b i t i o n of c e l l u l a r enzyme a c t i v i t i e s and metabol ism with these l a c t o n e s i s by t h e i r r e a c t i o n with the a v a i l a b l e t h i o l groups of the enzymes in the tumor c e l l s . 8. Mutagen i c a c t i v i t y P a r t h e n i n , a s e s q u i t e r p e n e l a c t o n e from P^ h y s t e r o p h o r u s , has been r e p o r t e d to have the a b i l i t y to break human l e u c o c y t e chromosomes rn v i t r o and to induce m i c r o n u c l e i f o rmat ion in % the po lychromic e r y t h r o c y t e s of mice _i_n v i v o (Vaidya et a l . 1978 ). The exact mechanism u n d e r l y i n g the observed c y t o g e n e t i c damage caused by p a r t h e n i n i s not known. Bac i 1 l u s t h u r i n g i e n s i s which i s e x t e n s i v e l y used as an i n s e c t i c i d e can mutate to B^ anthrac i s , a pathogenic mic roorgan ism for man and other a n i m a l s . In t h e i r s t u d i e s on e f f e c t s of s e s q u i t e r p e n e l a c t o n e s on B^ t h u r i n g i e n s i s Norman et a l . (1976) found that hymenovin but not t e n u l i n has t h i s mutagenic e f f e c t . 9. Ant i - i n f l a m m a t o r y . act i v i t y H a l l et a l . (1979) t e s t e d some s e s q u i t e r p e n e l a c t o n e s fo r a n t i - i n f l a m m a t o r y a c t i v i t y in r o d e n t s . In the carageenan in f lammat ion s c r e e n i n g t e s t s and in the t e s t s fo r i n h i b i t i o n of 79 the w r i t h i n g r e f l e x , the exomethylene on the l a c t o n e r i n g of the s e s q u i t e r p e n e l a c t o n e s was found to be r e q u i r e d fo r po tency . The s e s q u i t e r p e n e l a c t o n e s were on ly m a r g i n a l l y e f f e c t i v e a g a i n s t induced p l e u r i s y . In the a n t i - a r t h r i t i c t e s t s compounds with the methylene on the l a c t o n e r i n g , the u n s u b s t i t u t e d eye lopentenone r i n g , and. the epoxy eye lopentenone were s i g n i f i c a n t l y a c t i v e at r e l a t i v e l y low doses . 10. A n a l g e s i c act i v i t y A crude e x t r a c t of Helen i um amarum was found to have a n a l g e s i c a c t i v i t y and i n h i b i t e d the w r i t h i n g syndrome in mice p r e v i o u s l y induced by i n j e c t i o n of a c e t i c a c i d . A m a r i l i n (a p s e u d o g u a i a n o l i d e ) i s o l a t e d from t h i s p l a n t e x t r a c t was found to be r e s p o n s i b l e fo r the major pa r t of the a n a l g e s i c a c t i o n . A l s o some other s e s q u i t e r p e n e l a c t o n e s , h e l e n a l i n , t e n u l i n , i s o t e n u l i n , and t h e i r d e r i v a t i v e s , showed a n a l g e s i c a c t i v i t y which was, however, weaker than that of a m a r i l i n (Lucas et a l . 1964) . The above review of v a r i o u s b i o l o g i c a l a c t i v i t i e s of s e s q u i t e r p e n e l a c t o n e s i n d i c a t e s that these n a t u r a l l y o c c u r r i n g compounds i n f l u e n c e d i f f e r e n t p h y s i o l o g i c a l p r o c e s s e s of many types of o rgan isms . However, these e a r l i e r s t u d i e s do not u s u a l l y p r o v i d e s u f f i c i e n t d a t a ' f o r e x p l a i n i n g the mode of a c t i o n of s e s q u i t e r p e n e l a c t o n e s and hence the r e l a t i o n s h i p between chemica l s t r u c t u r e s and v a r i o u s b i o l o g i c a l a c t i v i t i e s of these compounds. T h e r e f o r e , in t h i s study T examined a c t i v i t i e s 80 of s e l e c t e d s e s q u i t e r p e n e l a c t o n e s a g a i n s t v a r i o u s types of o rgan isms, in p a r t i c u l a r a g a i n s t b a c t e r i a (Chapter 1 ) , fung i (Chapter 2 ) , i n s e c t s (Chapter 3 ) , and mammals (Chapter 4 ) . The main purpose of my study was: (1) to r e l a t e b i o l o g i c a l a c t i v i t i e s of s e s q u i t e r p e n e l a c t o n e s to t h e i r - chemica l s t r u c t u r e ; and (2) to e s t a b l i s h whether there i s any c l e a r p a t t e r n between chemica l s t r u c t u r e s and a c t i v i t i e s of s e s q u i t e r p e n e l a c t o n e s a g a i n s t v a r i o u s types of o rgan isms. 81 CHAPTER 1 Ant i b a c t e r i a l a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s I n t r o d u c t ion Many p l a n t s e x h i b i t a n t i m i c r o b i a l a c t i v i t y caused by compounds of v a r i o u s s t r u c t u r e s ( e . g . V ichkanova et a l . 1971, M i t s c h e r et a l . 1972, M i t cher 1975, Towers et a l . 1977a, Ieven et a l . 1978) . Sesqu i te rpene l a c t o n e s have been r e p o r t e d to e x h i b i t a n t i b a c t e r i a l a c t i v i t y , fo r example x a n t h a t i n from Xanthium pennsyIvan icum ( L i t t l e et a l . 1950), c n i c i n from Cn i cus b e n e d i c t u s (Vanhae len -Fas t re 1968, 1972), a c r o p t i l i n from Ac ropt i Ion repens , mibu lac tone from A r t e m i s i a taur i c a , l a c t o c i n from C i chor i um i n tybus , and s a u r i n from Saussurea p u l c h e l l a (Vichkanova et a l . 1971), and m i k a n o l i d e and d i h y d r o m i k a n o l i d e from Mi kan i a monaganen s i s (Mathur et a l . 1975). Lee et a l . ( 1 9 7 7 b ) t e s t e d 36 n a t u r a l s e s q u i t e r p e n e l a c t o n e s or t h e i r d e r i v a t i v e s a g a i n s t two Gram p o s i t i v e and three Gram n e g a t i v e b a c t e r i a and found 19 of them to be a c t i v e . These s t u d i e s showed that some s e s q u i t e r p e n e l a c t o n e s i n h i b i t growth of b a c t e r i a and that t h i s a c t i v i t y i s a s s o c i a t e d wi th the e x o c y c l i c methylene group on the l a c t o n e ( V a n h a e l e n - F a s t r e 1972 ), or with an (X,/3- u n s u b s t i t u t e d cyc lopentenone r i n g (Lee et a l . 1964, 1977b). S ince on ly a sma l l number of n a t u r a l l y o c c u r i n g s e s q u i t e r p e n e s were examined, there i s a need f o r a l a r g e r s c a l e i n v e s t i g a t i o n . 82 T h e r e f o r e , I screened 57 s e s q u i t e r p e n e l a c t o n e s fo r t h e i r a c t i v i t y a g a i n s t 6 b a c t e r i a . The purpose of t h i s i n v e s t i g a t i o n was: (1) to examine more thorough ly which s e s q u i t e r p e n e l a c t o n e s e x h i b i t a n t i b a c t e r i a l a c t i v i t i e s ; (2) to compare a c t i v i t i e s of i n d i v i d u a l s e s q u i t e r p e n e l a c t o n e s a g a i n s t v a r i o u s types of b a c t e r i a ; (3) to eva lua te a n t i b a c t e r i a l a c t i v i t i e s of s e s q u i t e r p e n e l a c t o n e s in r e l a t i o n to t h e i r chemica l s t r u c t u r e . 83 Exper imenta l C u l t u r e s of. S taphy lococcus a lbus ( U . B . C . #48), Bac i l l u s s u b t i l i s ( U . B . C . #221), S t r e p t o c o c c u s f a e c a l i s ( U . B . C . #197), Escher i c h i a c o l i ( U . B . C . #219), Proteus v u l g a r i s ( U . B . C . #15), Proteus m i r a b i l i s ( U . B . C . #5) and Pseudomonas. f l u o r e s c e n s ( U . B . C . #9) were o b t a i n e d from the Department of M i c r o b i o l o g y , U . B . C . The s e s q u i t e r p e n e l a c t o n e s were i s o l a t e d , p r e p a r e d , or o b t a i n e d as d e s c r i b e d in the S e c t i o n s I and I I . (1) Ant i m i c r o b i a l act i v i ty s c r e e n i n g t e s t Each c u l t u r e was spread over an agar p l a t e c o n t a i n i n g the n u t r i t i o n a l medium (Bacto N u t r i e n t B r o t h , D i f c o , L a b . , M ich igan) us ing s t e r i l e c o t t o n swabs. C r y s t a l s • ( a p p r o x i m a t e l y 1.5 mg) of the s e s q u i t e r p e n e l a c t o n e s to be t e s t e d were p l a c e d d i r e c t l y on the p l a t e s prepared in t h i s way. The p l a t e s , in d u p l i c a t e , were incubated at 37°C in the dark and examined a f t e r 24 hours . Lac tones which caused a c l e a r area with complete b a c t e r i a l growth i n h i b i t i o n were c o n s i d e r e d s t r o n g l y a c t i v e . Those in which some c o l o n i e s were present in the o therwise c l e a r area were c o n s i d e r e d weakly a c t i v e , and those which d i d not i n h i b i t growth of b a c t e r i a were c o n s i d e r e d to be i n a c t i v e (Table 8 ) . Some s e s q u i t e r p e n e l a c t o n e s ob ta ined l a t e r du r ing my study were t e s t e d on P^ mi r a b i l i s i n s t e a d of on P_̂_ v u l g a r i s which was no longer a v a i l a b l e . 84 R e s u l t s and P i s c u s s i o n R e s u l t s of the a n t i b a c t e r i a l s c r e e n i n g t e s t s (Table 8) show that the m a j o r i t y of s e s q u i t e r p e n e l a c t o n e s t e s t e d are a n t i m i c r o b i a l a g e n t s . Out of 309 t e s t s performed with 57 s e s q u i t e r p e n e l a c t o n e s a g a i n s t 6 b a c t e r i a (some l a c t o n e s were t e s t e d a g a i n s t fewer b a c t e r i a ) 88 (29%) were s t r o n g l y p o s i t i v e and 48 (16%) were weakly p o s i t i v e . Out of 57 s e s q u i t e r p e n e l a c t o n e s examined, 38 (67%) s t r o n g l y i n h i b i t e d the growth of at l e a s t one bac te r ium, 15 (26%) were only weakly a c t i v e , and on ly 4 (7%) had no ev ident e f f e c t on t h e i r growth (Table 9 ) . While I examined approx imate ly only 6% of a l l p r e s e n t l y known s e s q u i t e r p e n e l a c t o n e s , my s t u d i e s show that when these compounds are c a t e g o r i z e d a c c o r d i n g to t h e i r s k e l e t a l c l a s s e s , there are between 56 and 82% 'of s t r o n g l y a c t i v e compounds w i t h i n i n d i v i d u a l c l a s s e s (Table 9 ) . Eudesmanol ides appear to have the l a r g e s t p r o p o r t i o n of a c t i v e compounds, whereas g u a i a n o l i d e s seem to be the l e a s t f r e q u e n t l y a c t i v e a g a i n s t b a c t e r i a (Table 9) . (1) Act iv i ty a g a i n s t i n d i v i d u a l b a c t e r i a Most of the s e s q u i t e r p e n e l a c t o n e s examined were a c t i v e a g a i n s t s u b t i 1 i s and S_. a lbus (Table 1 0 ) . However, the p r o p o r t i o n of l a c t o n e s d i s p l a y i n g a c t i v i t y a g a i n s t i n d i v i d u a l b a c t e r i a l s p e c i e s decreased in the f o l l o w i n g o r d e r : Pj_ v u l g a r i s (or mi r a b i 1 i s ) , f a e c a l i s, E . c o l i , and f l u o r e s c e n s (Table 1 0 ) . Great d i f f e r e n c e s in the p r o p o r t i o n of compounds 85 i n h i b i t i n g the growth of i n d i v i d u a l b a c t e r i a i n d i c a t e that the a n t i b a c t e r i a l a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s must be p a r t i a l l y determined by d i f f e r e n c e s in b i o c h e m i s t r y and p h y s i o l o g y of v a r i o u s types of b a c t e r i a . G e n e r a l l y more l a c t o n e s i n h i b i t e d Gram p o s i t i v e than Gram n e g a t i v e b a c t e r i a (Table 1 0 ) . Approx imate ly 64% of the s e s q u i t e r p e n e l a c t o n e s t e s t e d i n h i b i t e d the growth of Gram p o s i t i v e b a c t e r i a ( t h i s i n c l u d e s compounds e x h i b i t i n g both , s t r o n g and weak i n h i b i t i o n ) , whereas on ly 24% of the compounds were a c t i v e a g a i n s t Gram negat ive b a c t e r i a . I t i s p o s s i b l e that the weaker a n t i m i c r o b i a l a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s a g a i n s t Gram negat ive b a c t e r i a i s a r e s u l t of the more complex outer l a y e r s of Gram negat i ve c e l l s , which probab ly reduce t h e i r p e r m e a b i l i t y -to drugs (see F r a n k l i n and Snow 1975), such as s e s q u i t e r p e n e l a c t o n e s . In a d d i t i o n , the presence of c y s t e i n e r e s i d u e s in the p r o t e i n - 1 i p o p o l y s a c c h a r i d e outermost l a y e r , as known from c o l i ( F r a n k l i n and Snow 1975), might prevent p e n e t r a t i o n of these compounds through the outer membrane by r e a c t i n g with l a c t o n e s . The s t ronger a n t i b a c t e r i a l a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s a g a i n s t Gram p o s i t i v e than Gram n e g a t i v e b a c t e r i a i s s i m i l a r to that r e p o r t e d by Lee et a l . (1977b) who examined a c t i v i t y of s e l e c t e d s e s q u i t e r p e n e l a c t o n e s and t h e i r d e r i v a t i v e s a g a i n s t 5 b a c t e r i a . They c o n c l u d e d , however, tha t the compounds they s t u d i e d were a c t i v e on ly a g a i n s t Gram p o s i t i v e b a c t e r i a , such as S taphy lococcus aureus and subt i 1 i s , but were i n a c t i v e a g a i n s t Gram negat ive s p e c i e s , such as 86 c o l i , Sa lmone l l a e n t e r i t i d i s , and K l e b s i e l l a pneumon i a e . Us ing the s c r e e n i n g t e s t I found that s e s q u i t e r p e n e l a c t o n e s i n h i b i t e d growth of Gram n e g a t i v e b a c t e r i a s t r o n g l y in 11% and weakly in 13% out of 150 t e s t s (Table 1 0 ) . The gram n e g a t i v e b a c t e r i a , P. v u l g a r i s and P^ mi r a b i l i s , were i n h i b i t e d even more f r e q u e n t l y than the Gram p o s i t i v e S^ f a e c a l i s. These d i f f e r e n c e s between the r e s u l t s of Lee et a l . (1977b) and my study are l i k e l y a consequence o f : (1) the use of a l a r g e r number of s e s q u i t e r p e n e l a c t o n e s by me, (2) the f a c t that d i f f e r e n t s e s q u i t e r p e n e l a c t o n e s were t e s t e d in these two s t u d i e s , (3) the use of d i f f e r e n t microorgan isms in the t e s t s , and f i n a l l y (4) the use of d i f f e r e n t techn iques for t e s t i n g the a n t i m i c r o b i a l a c t i v i t y by Lee et a l . (1977b) and in my s t u d y . (2) R e l a t i o n s h i p between chemica l s t r u c t u r e and a n t i b a c t e r i a l a c t i v i t y It has been suggested that the mechanism of a n t i b a c t e r i a l a c t i o n of compounds 'with u n s a t u r a t e d ketone and l a c t o n e f u n c t i o n s i s by r e a c t i o n of these groups with t h i o l groups of enzymes ( C a v a l l i t o and H a s k e l l 1945) . More r e c e n t l y , in_ v i t r o r e a c t i o n s between the u n s u b s t i t u t e d eye lopentenone and/or e x o c y c l i c methylene on the l a c t o n e r i n g of s e s q u i t e r p e n e l a c t o n e s and t h i o l a g e n t s , such as c y s t e i n e , have been demonstrated (Kupchan et a l . 1970, 1971, Lee et a l . 1977a, H a l l et a l . 1977, Pieman et a l . 1979) . T h i s ev idence suggests that the a n t i b a c t e r i a l a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s might be through t h e i r r e a c t i o n with t h i o l c o n t a i n i n g compounds e . g . 87 enzymes. Lee e t . a l . (1977a) conc luded that one of the s t r u c t u r a l requ i rements for c y t o t o x i c ant i tumor as w e l l as a n t i m i c r o b i a l a c t i o n of s e s q u i t e r p e n e l a c t o n e s i s the presence of the (5 - u n s u b s t i t u t e d c y c l o p e n t e n o n e . They a l s o suggested that s i g n i f i c a n t a n t i m i c r o b i a l a c t i v i t y i s independent of the presence or absence of an CX-methy l e n e - V - l a c t o n e mo ie ty . However, these c o n c l u s i o n s were reached on the b a s i s of t e s t s of on ly 13 s e l e c t e d s e s q u i t e r p e n e l a c t o n e s and some of t h e i r d e r i v a t i v e s . T h e r e f o r e , in the f o l l o w i n g s e c t i o n I examined the r e l a t i o n s h i p between a n t i b a c t e r i a l a c t i v i t y and the presence of v a r i o u s f u n c t i o n a l groups of a l a r g e r sample of main ly n a t u r a l l y o c c u r i n g s e s q u i t e r p e n e l a c t o n e s . The exomethylene on the l a c t o n e r e p r e s e n t s one of the a c t i v e s i t e s of s e s q u i t e r p e n e l a c t o n e s as demonstrated by the r e a c t i o n of some of them with c y s t e i n e ( e . g . Pieman et a l . 1979). To examine a p o s s i b l e r o l e of t h i s moiety in the a n t i b a c t e r i a l a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s , I d i v i d e d a l l l a c t o n e s i n t o two groups based on the presence or absence of t h i s moiety (Table 1 1 ) . Approx imate ly 50% of t e s t s wi th s e s q u i t e r p e n e l a c t o n e s p o s s e s s i n g the exomethylene on the l a c t o n e were p o s i t i v e and 36% of t e s t s i n c l u d i n g l a c t o n e s l a c k i n g t h i s moiety gave p o s i t i v e r e s u l t s . The f a c t that some l a c t o n e s without the exomethylene were a c t i v e ( s e v e r a l of them s t r o n g l y ) suggests that t h i s group i s not necessary fo r a n t i b a c t e r i a l a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s . In a d d i t i o n , t h i s moiety i s not always s u f f i c i e n t for the a c t i v i t y because many s e s q u i t e r p e n e l a c t o n e s p o s s e s s i n g the C13-methylene gave n e g a t i v e r e s u l t s . 88 To e l i m i n a t e p o s s i b l e d i f f e r e n c e s in a c t i v i t y between major groups of s e s q u i t e r p e n e l a c t o n e s due to t h e i r d i f f e r e n t b a s i c s k e l e t o n s , I e v a l u a t e d the s k e l e t a l c l a s s e s i n d i v i d u a l l y . I n d i v i d u a l c l a s s e s were grouped a c c o r d i n g to the presence or absence of the e x o c y c l i c methylene on the l a c t o n e r i n g ; p s e u d o g u a i a n o l i d e s were a l s o d i v i d e d a c c o r d i n g to the presence or absence of another p o s s i b l y a c t i v e s i t e , the C2,C3 double bond. Fur thermore , I a l s o examined other f u n c t i o n a l groups for t h e i r p o s s i b l e importance in the a n t i b a c t e r i a l a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s . S ince the l a r g e s t number of t e s t s were u s u a l l y performed with S^ a lbus whose growth was f r e q u e n t l y i n h i b i t e d by t e s t e d l a c t o n e s (Table 10) , I examined the r e l a t i o n s h i p between the presence of f u n c t i o n a l groups in s e s q u i t e r p e n e l a c t o n e s and t h e i r i n h i b i t i o n of growth of t h i s 4 s p e c i e s . (a) Germacrano l ides Out of 32 t e s t s performed wi th a t o t a l of 12 ge rmacrano l i des on three Gram p o s i t i v e b a c t e r i a , 19 t e s t s i n c l u d e d compounds p o s s e s s i n g the C13-exomethylene on the l a c t o n e r i n g . Of these 84% were p o s i t i v e (most ly s t r o n g l y ; see Tab le 1 2 ) . Of a l l 13 t e s t s i n c l u d i n g l a c t o n e s without the C13- methylene f u n c t i o n , 77% were p o s i t i v e (Table 1 2 ) . T h i s c l e a r l y suggests that in ge rmacrano l ides the methylene on the l a c t o n e i s not s o l e l y r e s p o n s i b l e for a n t i b a c t e r i a l a c t i v i t y of these compounds, a l though t h i s moiety might c o n t r i b u t e to the observed a c t i v i t y in some c a s e s . 89 Germacrano l ides were g e n e r a l l y l e s s a c t i v e a g a i n s t Gram n e g a t i v e b a c t e r i a . S ince t h i s l ack of a c t i v i t y was s i m i l a r for compounds p o s s e s s i n g or l a c k i n g the exomethylene (Table 12) , these r e s u l t s support the c o n c l u s i o n reached above for Gram p o s i t i v e b a c t e r i a that t h i s moiety does not p l a y an important r o l e in a n t i m i c r o b i a l a c t i v i t y . Tab le 13 g i v e s i n f o r m a t i o n on other f u n c t i o n a l groups of ge rmacrano l i des t e s t e d in r e l a t i o n to t h e i r a n t i b a c t e r i a l a c t i v i t y a g a i n s t a l b u s . I t appears that a n t i b a c t e r i a l a c t i v i t y of s e l e c t e d compounds or the lack of i t cannot be s imply e x p l a i n e d e i t h e r by the presence or absence of any g iven f u n c t i o n a l group. Some of these groups are p resent in both c a t e g o r i e s of l a c t o n e s wi th and without a c t i v i t y . These i n d i v i d u a l groups e i t h e r do not a f f e c t the a c t i v i t y of g e r m a c r a n o l i d e s or t h e i r a c t i v i t y might become expressed on ly in the presence (or absence) of some other g r o u p ( s ) . On the other hand, f u n c t i o n a l groups p resent on ly in l a c t o n e s wi th or without a n t i b a c t e r i a l a c t i v i t y might enhance or h inder t h e i r a c t i v i t y . For example, a l l g l a u c o l i d e s ( A , D , F , and G) and m a r g i n a t i n , which have s i m i l a r s t r u c t u r e s but l ack the exomethylene on the l a c t o n e r i n g , are a c t i v e ( s t r o n g l y and weakly) as w e l l as i n a c t i v e (Table 13) . M a r g i n a t i n and g l a u c o l i d e - G which possess the double bonds between C1-C10 and C7-C11 and R2 on C8 are s t r o n g l y a c t i v e . G l a u c o l i d e - F , which does not possess the C1-C10 double bond but has the C7-C11 double bond as w e l l as R2, i s on ly weakly a c t i v e . T h i s i n d i c a t e s that the C7-C11 double bond i s an important a c t i v e s i t e but that the C1-C10 double bond enhances the a c t i v i t y of m a r g i n a t i n and g l a u c o l i d e - G . 90 G l a u c o l i d e - D , m i s s i n g the C7-C11 double bond but p o s s e s s i n g the C1-C10 double bond, i s i n a c t i v e . It i s p o s s i b l e t h a t : , (a) the C1-C10 double bond i t s e l f i s not an i m p o r t a n t , a c t i v e s i t e of g l a u c o l i d e s , or (b) that the C1-C10 i s an a c t i v e s i t e but i t s a c t i v i t y i s i n h i b i t e d in g l a u c o l i d e - D by the a c e t y l group present on C2. T h i s group, which i s not p r e s e n t ' on o ther g l a u c o l i d e s or m a r g i n a t i n , might reduce the a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s (see a l s o p s e u d o g u a i a n o l i d e s , t h i s c h a p t e r ) . A l s o the epoxy group which sometimes reduces or i n c r e s a s e s . the a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s (Lee et a l . 1977b) in R4 on C8 might reduce the a c t i v i t y of g l a u c o l i d e - D . (b) Gua i a n o l i d e s Out of 18 t e s t s wi th g u a i a n o l i d e s p o s s e s s i n g the exomethylene 11 were p o s i t i v e (-9 s t r o n g l y ) a g a i n s t three Gram p o s i t i v e b a c t e r i a (Table 1 4 ) . However, of a l l t e s t s performed wi th l a c t o n e s l a c k i n g t h i s group on ly 4 (out of 9) were p o s i t i v e (only w e a k l y ) . These r e s u l t s i n d i c a t e that the e x o c y c l i c methylene c o u l d p lay a r o l e in de te rmin ing the a n t i b a c t e r i a l a c t i v i t y of the s e l e c t e d g u a i a n o l i d e s . The sample s i z e i s , however, too smal l for making a d e f i n i t e c o n c l u s i o n . In c o n t r a s t to Gram p o s i t i v e b a c t e r i a the exomethylene on the l a c t o n e does not seem to p lay any r o l e in the a c t i v i t y a g a i n s t Gram n e g a t i v e b a c t e r i a where both groups of g u a i a n o l i d e s wi th and without the C13-methylene showed very l i t t l e a c t i v i t y (Tab le 1 4 ) . As can be seen from Tab le 15 there i s no c l e a r r e l a t i o n s h i p 91 between the presence or absence of v a r i o u s f u n c t i o n a l groups on the s k e l e t o n of d i f f e r e n t g u a i a n o l i d e s and the a c t i v i t y a g a i n s t S. a l b u s . I t i s c l e a r that c e r t a i n p o t e n t i a l l y a c t i v e groups ( e . g . C3-C4 and C1"C10 double bonds in d e s a c e t o x y m a t r i c a r i n ) are not by themselves s u f f i c i e n t fo r the i n h i b i t i o n of growth of t h i s as w e l l as other t e s t e d b a c t e r i a (Tab les 8 , 1 5 ) . But i t i s p o s s i b l e that (1) these groups might enhance or reduce the a c t i v i t y of some other g r o u p ( s ) , or (2) the a c t i v i t y of such groups c o u l d become expressed on ly in the presence of another g r o u p ( s ) . For example, cumambrin-A p o s s e s s i n g a 'C3-C4 double bond i s i n a c t i v e whereas cumambrin-B a c e t a t e which l a c k s t h i s double bond ( t h i s i s the only d i f f e r e n c e between these two compounds) i s a c t i v e . Hence the C3-C4 double bond in t h i s i n s t a n c e reduces a c t i v i t y of another f u n c t i o n a l group(s) present in both cumambrins. I t i s a l s o p o s s i b l e that the a c t i v i t y of any g iven p o t e n t i a l l y a c t i v e group might be i n f l u e n c e d by i t s c o n f i g u r a t i o n on a s e s q u i t e r p e n e l a c t o n e m o l e c u l e . Thus, in c e r t a i n c o n f i g u r a t i o n s , the a c t i v i t y of these presumably a c t i v e f u n c t i o n a l groups might be reduced or even i n h i b i t e d . (c) Eudesmanol ides Out of 27 t e s t s wi th eudesmanol ides p o s s e s s i n g the C13- methylene 67% were p o s i t i v e (most ly s t r o n g l y ) a g a i n s t Gram p o s i t i v e b a c t e r i a (Table 16) . T h i s suggests that t h i s group i s at l e a s t p a r t i a l l y r e s p o n s i b l e fo r i n h i b i t i n g p r o p e r t i e s of eudesmanol ides a g a i n s t the b a c t e r i a examined. T h i s i s supported by a complete l o s s of a c t i v i t y of oc-santonin which does not possess the e x o c y c l i c methy lene . However, the presence of the 92 m e t h y l e n e - Y - l a c t o n e moiety i s not s u f f i c i e n t for a c t i v i t y of these compounds s i n c e 9 out of 27 eudesmanol ides p o s s e s s i n g t h i s group were i n a c t i v e (Table 1 6 ) . T e s t s c a r r i e d out on Gram negat i ve b a c t e r i a were most ly n e g a t i v e for both groups of l a c t o n e s with the exomethylene present or absent (Table 1 6 ) . Tab le 17 summarizes other f u n c t i o n a l groups present in v a r i o u s eudesmanol ides t e s t e d a g a i n s t a l b u s . A l l eudesmanol ides s t r o n g l y a c t i v e a g a i n s t t h i s bac ter ium possess .the ft-OH group (except p i n n a t i f i d i n which possesses a ketone group i n s t e a d ) on C l or C2. T h i s suggests that t h i s group might enhance the a n t i b a c t e r i a l a c t i v i t y of eudesmano l ides . Thus a l a n t o l a c t o n e might be a c t i v e as a r e s u l t of the presence of the C13-exomethylene (and p robab ly C5-C6 double bond) but i t i s weaker because i t l a c k s the h y d r o x y l groups . A l s o the lack of the a c t i v i t y of CX-santonin, compared with the s t r o n g l y a c t i v e , s t r u c t u r a l l y s i m i l a r l u d o v i c i n - C , might r e s u l t not on ly from the absence of the e x o c y c l i c methylene but a l s o from the lack of the h y d r o x y l groups on C l and C2. (d) Pseudogua i a n o l i d e s It has been p r e v i o u s l y suggested that p s e u d o g u a i a n o l i d e s may possess two a c t i v e s i t e s (the exomethylene on the l a c t o n e r i n g and the C2,C3 double bond) which are r e s p o n s i b l e fo r t h e i r v a r i o u s b i o l o g i c a l a c t i v i t i e s (Lee et a l . 1 9 7 4 , 1977a,b, H a l l et a l . 1977, Pieman et a l . 1979) . Tab le 18 summarizes r e s u l t s of a n t i b a c t e r i a l t e s t s of s e l e c t e d p s e u d o g u a i a n o l i d e s p o s s e s s i n g 93 bo th , e i t h e r , or n e i t h e r of these f u n c t i o n a l g roups . Compounds p o s s e s s i n g the e x o c y c l i c methylene a lone were s t r o n g l y a c t i v e a g a i n s t Gram p o s i t i v e b a c t e r i a in 32% of the t e s t s (but none a g a i n s t S_;_ f a e c a l i s) . However, the f a c t that 45% of a l l t e s t s were n e g a t i v e suggests that the presence of the CX-methylene-T- l a c t o n e moiety a lone does not guarantee a n t i b a c t e r i a l a c t i v i t y of p s e u d o g u a i a n o l i d e s p o s s e s s i n g t h i s group. T h i s moiety a p p a r e n t l y p l a y s an even l e s s important r o l e in a n t i m i c r o b i a l a c t i v i t y a g a i n s t Gram negat ive b a c t e r i a s i n c e 86% of the t e s t s were nega t i ve (Table 1 8 ) . Out of 22 p s e u d o g u a i a n i o l i d e s examined on ly two l a c t o n e s ( t e n u l i n and i s o t e n u l i n ) lack the e x o c y c l i c methylene but possess the (A , f i - cyc lopentenone r i n g . Both l a c t o n e s were a c t i v e a g a i n s t a l l t h r e e Gram p o s i t i v e b a c t e r i a . However, these l a c t o n e s were i n a c t i v e when t e s t e d a g a i n s t Gram n e g a t i v e b a c t e r i a , wi th the e x c e p t i o n ' o f t e n u l i n which weakly i n h i b i t e d growth of v u l g a r i s (Tab les 8, 1 8 ) . T h i s i n d i c a t e s that the presence of a C2-C3 double bond in p s e u d o g u a i a n o l i d e s may be a s s o c i a t e d wi th the a c t i v i t y a g a i n s t Gram p o s i t i v e but not a g a i n s t Gram negat i ve b a c t e r i a . However, 55% of t e s t s c a r r i e d out on Gram p o s i t i v e b a c t e r i a us ing l a c t o n e s l a c k i n g t h i s moiety (but p o s s e s s i n g an exomethylene on the l a c t o n e ) were p o s i t i v e . T h i s f a c t i n d i c a t e s that the presence of the ^ ,/5-unsubst i tu ted eye lopentenone i s not always a requirement for the a n t i b a c t e r i a l a c t i v i t y of p s e u d o g u a i a n o l i d e s . Tab le 18 shows that when both a c t i v e s i t e s (the eye lopentenone r i n g and the exomethylene on the l a c t o n e r i n g ) 94 are p r e s e n t , p s e u d o g u a i a n o l i d e s e x h i b i t e d a s t r o n g a n t i b a c t e r i a l a c t i v i t y a g a i n s t a lbus and s u b t i 1 i s . T h i s i n d i c a t e s that the presence of both m o i e t i e s enhances the a c t i v i t y of p s e u d o g u i a i a n o l i d e s a g a i n s t these two b a c t e r i a . However, there was no c l e a r r e l a t i o n s h i p between the presence of the two m o i e t i e s and the a c t i v i t y of those p s e u d o g u a i a n o l i d e s a g a i n s t S. f a e c a l i s and three Gram negat i ve b a c t e r i a (Table 18) . These d i f f e r e n t r e s u l t s c o u l d p o s s i b l y be e x p l a i n e d by d i f f e r e n t c h e m i s t r i e s of d i f f e r e n t b a c t e r i a i n v o l v e d . The three p s e u d o g u a i a n o l i d e s c o n t a i n i n g l a c t o n e s l a c k i n g both m o i e t i e s (the e x o c y c l i c methylene and the e n d o c y c l i c C2,C3 double bond) were i n a c t i v e a g a i n s t f i v e b a c t e r i a t e s t e d (Table 1 8 ) . However, two of these compounds ( d i h y d r o i s o p a r t h e n i n and t e t r a h y d r o p a r t h e n i n ) weakly i n h i b i t e d growth of subt i 1 i s . These r e s u l t s aire c o n s i s t e n t wi th the view that the two m o i e t i e s p l a y the most important r o l e but other f u n c t i o n a l groups might a l s o i n f l u e n c e the a c t i v i t y of some p s e u d o g u a i a n o l i d e s a g a i n s t b a c t e r i a . To e s t a b l i s h whether the a c t i v i t y of the e x o c y c l i c methylene and /3-unsubst i tu ted eye lopent'enone c o u l d be i n f l u e n c e d by t h e i r i n t e r a c t i o n s with other p o t e n t i a l l y a c t i v e groups p resent in p s e u d o g u a i a n o l i d e s , I examined the r e l a t i o n s h i p between a n t i m i c r o b i a l a c t i v i t y of 22 s e l e c t e d p s e u d o g u a i a n o l i d e s a g a i n s t a lbus and the presence of v a r i o u s f u n c t i o n a l groups (Table 1 9 ) . Two major c o n c l u s i o n s can be made from the r e s u l t s . F i r s t , the e x o c y c l i c methylene group i t s e l f appears to be important but i s not s u f f i c i e n t to c o m p l e t e l y i n h i b i t the growth 95 of b a c t e r i a . Another a c t i v e group must be present on a s e s q u i t e r p e n e l a c t o n e molecule to make the p s e u d o g u a i a n o l i d e an a c t i v e compound. The group might for example be the C2,C3 double bond whose presence a lone i s s u f f i c i e n t fo r a c t i v i t y a g a i n s t Gram p o s i t i v e b a c t e r i a . Th i s group augments the a c t i v i t y of the exomethylene on the l a c t o n e (or v i c e v e r s a ) , l e a d i n g to growth i n h i b i t i o n of at l e a s t two Gram p o s i t i v e b a c t e r i a (Table 1 8 ) . S e c o n d l y , as conc luded e a r l i e r , t h e - p r e s e n c e of a methylene f u n c t i o n on C13 i s u s u a l l y a s s o c i a t e d with a n t i m i c r o b i a l a c t i v i t y . However, there are a l s o four compounds p o s s e s s i n g t h i s moiety which had no v i s i b l e i n h i b i t o r y e f f e c t s on growth of S. a lbus (Table 1 9 ) . S ince the monoadduct of p a r t h e n i n wi th L- c y s t e i n e v i a exomethylene on the l a c t o n e only s l i g h t l y i n h i b i t e d growth of S^ a l b u s , whereas p a r t h e n i n had s t r o n g i n h i b i t o r y e f f e c t s (Pieman 1977) , the CX-methylene- V - l a c t o n e moiety must be p a r t i a l l y r e s p o n s i b l e for the a n t i m i c r o b i a l a c t i v i t y of p a r t h e n i n and probab ly a l s o of other p s e u d o g u i a n o l i d e s . T h e r e f o r e , the lack of a c t i v i t y of four p s e u d o g u i a n o l i d e s p o s s e s s i n g t h i s moiety c o u l d most l i k e l y be e x p l a i n e d by the presence of other group(s) which c o n t e r a c t the e f f e c t s of the exomethylene group. T h i s idea i s supported by the f a c t that a l l four i n a c t i v e p s e u d o g u a i a n o l i d e s p o s s e s s i n g the exomethylene on the l a c t o n e a l s o have a c e t y l g roup(s) on C4 or C14 (Table 1 9 ) . The sugges t i on that these a c e t y l groups might exer t i n h i b i t o r y e f f e c t s on the a c t i v i t y of the C13~methylene i s fur thermore supported by ev idence on a c t i v i t y of s t r u c t u r a l l y s i m i l a r l a c t o n e s which d i f f e r on ly in the presence or absence of the a c e t y l g roups . For example, c o r o n o p i l i n i s s t r o n g l y a c t i v e but 96 t e t r a n e u r i n - A , which d i f f e r s from c o r o n o p i l i n on ly by the presence of a C 1 5 - a c e t y l group, i s weakly a c t i v e , and t e t r a n e u r i n - B , which d i f f e r s from c o r o n o p i l i n only by the presence of a C 1 4 - a c e t y l group, i s comp le te l y i n a c t i v e (Table 1 9 ) . H a l l et a l . (1979) reached s i m i l a r c o n c l u s i o n s in t h e i r study on a n t i - i n f l a m m a t o r y a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s . These authors conc luded that the h y d r o x y l group may p lay a s i g n i f i c a n t r o l e in r e c e p t o r b i n d i n g s i n c e e s t e r i f i c a t i o n (as OCOCH^) or e l i m i n a t i o n of the h y d r o x y l group d r a s t i c a l l y reduced the a n t i - i n f l a m m a t o r y a c t i v i t y of the compounds s t u d i e d . However, in c o n t r a s t to these s u g g e s t i o n s , there are three p s e u d o g u a i a n o l i d e s which e x h i b i t e d s t rong a n t i b a c t e r i a l a c t i v i t y (Table 19) , and which a l s o possess a C13-methylene as w e l l as an a c e t y l g r o u p ( s ) . The a c t i v i t y of these compounds might be e x p l a i n e d by the presence of a d d i t i o n a l a c t i v e s i t e s which f u r t h e r enhance the a c t i v i t y of the e x o c y c l i c methy lene . Thus, c o n c h o s i n - B i s a (3-unsubst i t u t e d eye lopentenone , g a i l l a r d i l i n has an epoxy group, and s p a t h u l i n has two h y d r o x y l g roups . In a d d i t i o n , the p o s i t i o n of a c e t y l s on the s k e l e t o n c o u l d p l a y an important r o l e and thereby i n f l u e n c e the a c t i v i t y of p s e u d o g u i a n o l i d e s . A l l i n a c t i v e compounds have a c e t y l groups on C4 or C14. In c o n t r a s t , the s t r o n g l y a c t i v e compounds have the a c e t y l group on C6, C9, or C15. (Table 1 9 ) . The importance of the p o s i t i o n of the a c e t y l group can f u r t h e r . b e seen from the f a c t that t e t r a n e u r i n - A which has an a c e t y l on C15 i s on ly weakly a c t i v e , whereas t e t r a n e u r i n - B which has a c e t y l on C14 ( t h i s i s the only d i f f e r e n c e in chemica l s t r u c t u r e between the 97 two compounds) i s i n a c t i v e (Table 1 9 ) . C o n c l u s i o n s There i s no s imple g e n e r a l r e l a t i o n s h i p between the a n t i b a c t e r i a l a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s and t h e i r chemica l s t r u c t u r e . My r e s u l t s suggest that the a n t i b a c t e r i a l a c t i v i t y of these compounds c o u l d be determined by any combinat ion of the f o l l o w i n g f a c t o r s : (1) The presence or absence of f u n c t i o n a l groups such as an e x o c y c l i c methylene on the l a c t o n e r i n g in some l a c t o n e s , or an ( i -unsubst i t u t e d eye lopentenone r i n g in p s e u d o g u i a n o l i d e s , or a C7-C11 double bond in g e r m a c r a n o l i d e s ; (2) The presence or absence of v a r i o u s a d d i t i o n a l groups which enhance ( e . g . ^ - h y d r o x y l in eudesmanol ides) or reduce ( e . g . a c e t y l on C4 or C14 in p a s e u d o g u a i a n o l i d e s ) the a n t i b a c t e r i a l a c t i v i t y of a g iven l a c t o n e ; (3) The p o s i t i o n of i n d i v i d u a l a d d i t i o n a l g roups , which i n c r e a s e or reduce the a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s , on a s k e l e t o n ( e . g . the a c e t y l group on C4 and C14 a p p a r e n t l y reduces the a c t i v i t y of some p s e u d o g u i a i a n o l i d e s but in p o s i t i o n s on C6, C9, and C15 i t does not seem to p lay an important r o l e ) ; (4) The c o n f i g u r a t i o n of f u n c t i o n a l groups on a s k e l e t o n ( e . g . the (X- or Pi- p o s i t i o n of a h y d r o x y l f u n c t i o n in eudesmano l ides) ; (5) The a v a i l a b i l i t y and a c c e s s i b i 1 i t y 'of v i t a l t h i o l compounds 98 present in b a c t e r i a and t h e i r chemica l a f f i n i t y for s e s q u i t e r p e n e l a c t o n e s . For example, a l though p a r t h e n i n _i_n v i t r o forms a monoadduct wi th L - c y s t e i n e through the C13 e x o c y c l i c methylene (Pieman et a l . 1979), i t forms a monoadduct _in_ v i t r o with t h i o p h e n o l through i t s unsubst i tu ted cyclope.ntenone r i n g ( P a n f i l and Towers; u n p u b l i s h e d ) . F u r t h e r , when L - c y s t e i n e i s in excess , i t forms two adducts with p a r t h e n i n . In c o n t r a s t , g l u t a t h i o n e always forms with p a r t h e n i n on ly a s i n g l e adduct (Pieman et a l . 1979). Table 8. Screening test for antimicrobial a c t i v i t y of selected sesquiterpene lactones against s i x bacteria. Sesquiterpene lactone Response to Number i n Appendix Name S_. albus B. s u b t i l i s S. f a e c a l i s E. c o l i P. vulgaris P. fluorescens Germacranolides 1 Parthenolide + NT - + NT NT 2 Pyrethrosin + + + - - + 4 Chamissonin, acetyl + + - - - 5 Tamaulipin-A + - - - - - 7 Eupatoriopicrin + + + - + - 14 Glaucolide-A + + - - - NT 16 Marginatin + + NT - + NT 17 Glaucolide-D - - + - - . 19 Glaucolide-F + + NT - NT NT 20 Glaucolide-G + + + - + + 21 Elephantopin - + + - * - 22 Parthenolide, it 9-fl-OH + + + + + + Table 8. Continued. Guaianolides 23 Cumambrin-A - - - * - 24 Cumambrin-B - + - - * + - 25 Cumambrin-B, dihydro- + + + + + — 26 Cumambrih-B, tetrahydro- + - - - - - 27 Cumambrin-B, acetate + + + + - • — 28 Cumambrin-B, formyl + - - - — 30 Matricar i n , desacetoxy- - - - - — 31 Grossheimin + + - - * + - 32 I v a l i n , pseudo- Eudesmanolides + + + + + 33 Alantolactone + + - - - - 35 Alantolactone, i s o - - + — — - + 36 I v a l i n + + - - - - Table 8. Continued. 37 Ivasperin + + - - + - 38 P i n n a t i f i d i n + + - - + - 39 Pulchellin-C + + + - - 40 c<-Santonin - - - - + - 41 Ludovicin-A + NT - + NT NT 42 Ludovicin-B + NT NT - NT NT 43 Ludovicin-C + + + - - - 46 Santamarine - + - - Pseudoguaianolides 47 Parthenin + + + + + - 48 Parthenin, dihydroiso- - + - - - - 49 Parthenin, tetrahydro- - + - ' - - - 51 Hymenin + + + • + + - 52 Ambrosin + + NT + NT NT 53 Coronopilin + + - + + - 54 Damsin + + + - - - Table 8. Continued. 55 Hysterin - + - - - - 56 Tetraneurin-A + + - - * - 57 Tetraneurin-B + NT - * NT 58 Tetraneurin-D - + - - - - 59 Tetraneurin-E - + - ' - * - 60 Conchosin-A + - - - + - 61 Conchosin-B + NT - - NT - 63 Tenulin + + + - + - 64 Tenulin, iso- • + + + - - - 65 G a i l l a r d i l i n + NT - NT NT NT 66 Helenalin + + - + * + - 67 Flexuosin-B - - - - ' - - • 68 Spathulin + + - - - - 69 Balduilin + + + - - - 70 Cumanin + + - - + - Table 8. Continued. Other sesquiterpene lactones 76 Quadrone - - NT NT NT NT 77 A x i v a l i n + + - _ . _ * • . _ 78 I v a x i l l a r i n + + NT + NT - NT= not tested * = tested on P_. m i r a b i l i s . += complete i n h i b i t i o n += some colonies present -= no i n h i b i t i o n 104 Table 9. Summary of antimicrobial activity of sesquiterpene lactones from individual skeletal classes against six bacteria. No. (%) of sesq. lactones active against at least one bacterium Number (%) inactive Class strongly weakly sesq. lact. Total Germacranolides 8 4 0 12 Guaianolides 5 2 2 9 Eudesmanolides 9 2 0 11 Pseudo- guaianolides 14 7 1 22 Other sesq. lactones 2 0 1 3 Total 38 (6.7) 15 (26) 4 (7.) 57 105 Table 10. Summary of antimicrobial activity of selected sesquiterpene lactones against individual bacteria. No . (%) pos. responses Number (%) negative responses Total No. Bacterium Strong Weak tests S. albus 31 (54) 10 (18) 16 (28) 57 B. subtilis 33 (64) 10 (19) 9 (17) 52 S. faecalis 8 (16) 10 (20) 32 (64) 50 Total Gram positive 72 (45) 30 (19) 57 (36) 159 E. c o l i 7 (13) 6 (11) 42 (76) 55 P. vulgaris/ P. mirabilis 8 (17) 11 (23) 29 (60) 48 P. fluo- rescens 1 (2) 3 (6) 43 (92) 47 Total Gram negative 16 (11) 20 (13) 114 (76) 150 Table 11. Summary of an t i b a c t e r i a l a c t i v i t y of sesquiterpene lactones as related to the presence or absence of the exomethylene on the Y-lactone ri n g . No. sesq. l a c t . with No. (%) responses No. (%) responses Positive No. sesq. l a c t . without P o s i t i v e Bacterium . C13=CH2 .Strong Weak Negative C13=CH2 Strong Weak Negative S. albus 42 28 5 9 15 3 5 7 B. s u b t i l i s 37 29 5 3 15 4 5 6 S. fa e c a l i s 38 6 7 25 12 2 3 7 E. c o l i 41 7 5 29 14 0 1 13 P. vu l g a r i s / P. m i r a b i l i s 35 8 6 21 13 0 5 8 P. fluorescens 36 1 2 33 11 0 1 10 Total 229 (100) 79 (35) 30 (13) 120 (52) 80 (100) 9 (11) 20 (25) 51 (64) Table 12* Antimicrobial a c t i v i t y of germacranolides as related to the presence or absence of the methylene group on the Y-lactone r i n g . No. (%) responses Bacterium No. sesq. l a c t . with C13=CH„ No. (%) responses Positive No. sesq. l a c t . without Positive Strong Weak Negative C13=CH„ Strong Weak Negative S. albus 7 B. s u b t i l i s 6 S. f a e c a l i s 6 Total Gram positive E. c o l i P_. v u l g a r i s / P_. m i r a b i l i s 6 P_. fluorescens 6 Total Gram negative 19 (100) 19 (100) 14 (74) 1 0 1 0 1 2 (10) 1 1 2 1 1 1 3 (16) 5 4 4 2 (11) 4 (21) 13 (68) 5 5 3 13 (100) 5 4 2 11 (100) 2 2 1 0 0 5 ( 39 ) 5 ( 39 ) 3 ( 23) 3 (27) 8 (73) Table 13. Antimicrobial a c t i v i t y of germacranolides against S. albus as related to the presence or absence of various functional groups. Presence or absence of functional groups <D CD Sesquiterpene lactone o o o o o S ° ^ < Appendix Name • 3 V Q -.5 S 3 V 3 o o o » » >- o o o u o o » Strongly active: 1 Parthenolide + - - - - - + - - + - _ - - - - - + 2 Pyrethrosin - + _ - - - + + - + _ - - - - - + 4 Chamissonin, diacetate + _ - - + - + + - + _ _ - - - - + • 7 Eupatoriopicrin + _ - - - - + - + _ _ + - - - - + 16 Marginatin + _ _ - - + - - + _ + _ + - - + - 20 Glaucolide-G + - + - - + - + - + - - + - 22 Parthenolide, _ 9-«-0H + _ - - - + - - + - - - - - - Weakly active: 5 Tamaulipin-A + _ _ * - - + - + _ _ _ - - - + ^ o 14 Glaucolide-A - - + - - + - ' - + ~ + - - + - + - Table 13. Continued. 19 Glaucolide-F Inactive: 17 Glaucolide-D 21 Elephantopin + + + + + + + * OH 0—c=o ** 1 I C2 CIO 0 OH R = _ 0 -c-C-CH O 1 " 2 CH CH„ 6 H 2 V -0-C-C-CH„ CH„ 0 V -O-C-C-CH J CH J -O-C-C-CH. CH, Table 14. Antimicrobial a c t i v i t y of guaianolides as related to the presence or absence of the methylene group on the r-lactone r i n g . No. (%) responses No. (%) responses No. sesq. l a c t . with Positive No. sesq. l a c t . without P o s i t i v e Bacterium C13=CH2 Strong Weak Negative C13=CH2 Strong Weak Negative S. albus 6 4 0 2 3 0 2 1 B. s u b t i l i s 6 5 0 1 3 0 1 2 S. f a e c a l i s 6 0 2 4 3 0 1 2 Total Gram positive 18 (100) 9 (50) 2 (11) 7 (39) 9 (100) 0 4 (44) 5 (56) E. c o l i 6 0 2 4 3 0 1 2 P. v u l g a r i s / P. m i r a b i l i s 6 1 2 3 3 0 1 2 P. fluorescens 6 1 0 5 3 0 0 3 Total Gram negative 18 (100) 2 (11) 4 (22) 12 (67) 9 (100) 0 2 (22) 7 (78) Table 15. Antimicrobial a c t i v i t y of guaianolides against S_. albus as related to the presence or absence of various functional groups. Presence or absence of functional groups Sesquiterpene lactone No. i n Appendix Name cu cu c c o o M CM CM o 4-) CJ 33 33 33 o oo o S3 <! o O c_> U c_> o o CJ O nj u ca O O U-t 1 II II II II 11 ii rH •> rH 1 1 1 O o CO i— i CM CO CO 1 l oo oo oo i—( t—i f—1 o u u CJ O >- O >- u u u U u o Strongly active 27 Cumambrin-B, acetate 28 Cumambrin-B, formyl 31 Grossheimin 32 I v a l i n , pseudo + + + + - + - - + + - - + Weakly active 25 26 Cumambrin-B, dihydro Cumambrin-B, tetrahydro Table 15. Continued. Inactive: 23 Cumambrin-A - - - + . + - • - + - + -• + 24 Cumambrin-B - . - - + + - + - - + - + 30 Matricarin, desacetoxy + + - + + - _ _ _ _ _ _ Table 16. Antimicrobial a c t i v i t y of eudesmanolides as related to the presence or absence of the methylene group on the Y-lactone ri n g . No. (%) responses No. (%) responses No. sesq. l a c t . with C13=CH2 Positive No. sesq. l a c t . without C13=CH2 P o s i t i v e Bacterium Strong Weak . Negative Strong Weak Negative S. albus 10 7 1 2 1 0 0 1 B. s u b t i l i s 8 6 2 0 1 0 0 1 S. f a e c a l i s 9 0 2 7 1 0 0 1 Total Gram positive 27 (100) 13 (48) 5 (19) 9 (33) 3 (100) 0 0 3 (100) E. c o l i 10 1 1 8 1 0 0 1 P. vu l g a r i s / P. m i r a b i l i s 8 2 1 5 1 0 1 0 P. fluorescens i 8 0 0 8 1 0 0 1 Total Gram negative 26 (100) 3 (12) 2 (8) 21 (81) 3 (100) 0 1 (33) 2 (67) Table 17. Antimicrobial a c t i v i t y of eudesmanolides against _S. albus as related to the presence or absence of various functional groups. Presence or absence of functional groups Sesquiterpene lactone No. i n Appendix Name PC re a O 1 o i CM o 1 IS 1 C2. 1 o II s 1 t—i — < CM o cj u C J EC O * k I CO u (D 0) EG c C o o o CM 4-1 4-1 w 1 m vD o oo o C J C2 O u cj C J U cd C J oa II 1 II II II II t-H .» r-( CO CO CO in 1 1 1—( u u C J C J u C J >- C J >- C J Strongly active: 36 I v a l i n 37 Ivasperin 38 P i n n a t i f i d i n 39 Pulchellin-C 41 Ludovicin-A 42 Ludovicin-B 43 Ludovicin-C + + + + - - - + + - - - + * - - + - + - + - + — rt* ** _ — - + - - + * * + + + + + + + + + + + + + + Weakly active: 33 Alantolactone Table 17. Continued. Inactive: 35 Alantolactone, i s o - - - - - - - - + - - - + + AO tX-santonin - - + * * - - + - + - 46 Santamarine - + - - - - + - - - + - + * =0 ** -CH-CH- v 0 ' Table 18. Antimicrobial a c t i v i t y of pseudoguaianolides as related to the presence or absence of the methylene group on the Y-lactone and the unsubstituted cyclopentenone r i n g . Bacterium No. (%) responses No. sesq. l a c t . with Positive C13=CH? only Strong Weak Negative No. sesq. l a c t . with C2=C3 only No. (%) responses P o s i t i v e Strong Weak Negative _S. albus 11 13. s u b t i l i s 10 S. fa e c a l i s 10 4 6 0 3 3 1 4 1 9 1 0 1 0 0 0 Total Gram posi t i v e 31 (100) 10 (32) 7 (23) 14 (45) 6 (100) 4 (67) 2 (33) E. c o l i 10 P_. v u l g a r i s / P. m i r a b i l i s 10 P. fluorescens 10 1 0 2 0 7 10 2 2 0 0 1 0 Total Gram negative 30 (100) 2 (7) 2 (7) 26 (86) 6 (100) 1 (17) 5 (83) Table 18. Continued. No. (%) responses No. (%) responses No. sesq. l a c t . with C13=CH2 Positive No. sesq. l a c t . without C13=CH2 P o s i t i v e Bacterium and C2=C3 Strong Weak Negative and C2=C3 Strong Weak Negative S. albus 6 6 0 0 3 0 0 3 B. s u b t i l i s 5 5 0 0 3 0 2 1 S. fa e c a l i s 5 1 2 2 3 0 0 3 Total Gram pos i t i v e 16 (100) 12 (75) 2 (13) 2 (13) 9 (100) 0 2 (22) •7 (78) E. c o l i 6 3 1 2 3 0 0 3 P. v u l g a r i s / P. m i r a b i l i s 4 3 0 1 3 0 0 3 P. fluorescens ; 5 0 0 5 3 0 0 3 Total Gram negative 15 (100) 6 (40) 1 (7) 8 (53) 9 (100) 0 0 9 (100) Table 19. Antimicrobial a c t i v i t y of pseudoguaianolides against S_. albus as related to the presence or absence of various functional groups. Presence or absence of functional groups Sesquiterpene lactone pa o 101 16 bo ne  r) r 1 CM Pd u n CO i—i CJ o C1 5- 0A C No. i n Appendi: x. Name i r-( cj C2 -0 H C2 =C 3 C3 -0 H C4 -0 H o ii -* c_> C6 .C 7 Y- la c1  00 u O (0 « iH I o >- C6 -0 A<  C9 -0 A<  C1 4- 0i  C1 5- 0A C Strongly active: 47 Parthenin •+ - + - - + + - - - + - - 51 Hymenin +<A) - + - - + + - - - + - 52 Ambrosin - - + - - + + - - + - • - 53 Coronopilin + - - - - + + - - - + - - 54 Damsin - - - - - + + - - - + - - 61 Conchosin-B + - + - + + - - - + - + 63 Tenulin - - + - - + - + a - - - - 65 G a i l l a r d i l i n - - b - - - + + - + - - 66 Helenalin - - + - - + - + c - + - . - 68 Spathulin - + - - + - • - + + + + - - 69 B a l d u i l i n - - + - - + - + + - + - - Table 19. Continued. Weakly active: 56 Tetraneurin-A 60 Conchosin-A 64 Tenulin, iso- 70 Cumanin + + + • + + +((3) +(/3) - + + + + + Inactive: 48 Parthenin, dihydroiso- 49 Parthenin, tetrahydro- 55 Hysterin 57 Tetraneurin-B 58 Tetraneurin-D 59 Tetraneurin-E 67 Flexuosin-B + + + +(<x) + +((3) - e + + + + + •+ + + + a= C6—C7 OH b= C3-C4 ^0' -OH d= C2 C15 N 0 ' / C H 3 e= -OAc f= C6-0-C-CH=C 0 \CH„ 120 CHAPTER 2 Ant i f unga1 a c t i v i t y of sesqu i terpene l a c t o n e s Int roduct ion S e v e r a l s t u d i e s of h igher p l a n t s , t h e i r e x t r a c t s , or chemica l s i s o l a t e d from them show that some s p e c i e s c o n t a i n a n t i f u n g a l compounds ( e . g . V ichkanova et a l . 1971, Camm et a l . 1975, Towers et a l . 1977a, Ieven et a l . 1978, Muir 1979, and r e f e r e n c e s t h e r e i n ) . C e r t a i n s e s q u i t e r p e n e l a c t o n e s have been r e p o r t e d to possess a n t i f u n g a l p r o p e r t i e s . For example, x a n t h a t i n from Xanthium pennsy lvan icum s t r o n g l y i n h i b i t e d the growth of T r i chophy ton mentaqrophytes and Candida a l b i c a n s and s l i g h t l y i n h i b i t e d growth of s i x other fungi ( L i t t l e et a l . 1950). A l a n t o l a c t o n e and i s o a l a n t o l a c t o n e have been r e p o r t e d to i n h i b i t the growth of T^ mentagrophytes , T. acuminatum, and Epidermophyton sp . ( O l e c h n o w i c z - S t e p i e n and S t e p i e n 1963). The e f f e c t of p a r t h e n i n on c e r t a i n phases of funga l growth has been r e p o r t e d . T h i s l a c t o n e i n h i b i t e d s p o r a n g i a l ge rmina t ion and zoospore m o t i l i t y in S c l e r o s p o r a qramin i c o l a but d i d not e x h i b i t any a c t i v i t y in the c o n i d i a l development of A s p e r q i 1 l u s f l a v u s at the same or g r e a t e r c o n c e n t r a t i o n s (Char and Shankarabhat 1975) . Mathur et a l . (1975) found m ikano l i de and d i h y d r o m i k a n o l i d e , from Mi kan ia monagasens is , to be a c t i v e a g a i n s t Candida a l b i c a n s . A l a r g e s c a l e study by Towers et a l . 121 (1977a) showed that out of 65 s e s q u i t e r p e n e l a c t o n e s t e s t e d a g a i n s t a l b i c a n s on ly 3 compounds, m i k a n o l i d e , g l a u c o l i d e - B , and p s e u d o i v a l i n were a n t i b i o t i c . Lee et a l . (1977b) who examined the. a c t i v i t y of 36 s e s q u i t e r p e n e l a c t o n e s and t h e i r d e r i v a t i v e s a g a i n s t C_;_ a l b i c a n s a l s o conc luded that only 5 l a c t o n e s i n h i b i t e d growth of t h i s y e a s t . These s t u d i e s t h e r e f o r e suggest that s e s q u i t e r p e n e l a c t o n e s r a r e l y e x h i b i t the a c t i v i t y a g a i n s t a l b i c a n s . In t h i s study I t e s t e d 45 s e s q u i t e r p e n e l a c t o n e s for t h e i r a n t i f u n g a l a c t i v i t i e s a g a i n s t 3 fung i and 7 l a c t o n e s a g a i n s t two s p e c i e s of y e a s t s . The purpose of t h i s study was t o : (1) examine which s e s q u i t e r p e n e l a c t o n e s e x h i b i t a n t i f u n g a l a c t i v i t i e s , (2) compare the a c t i v i t i e s of i n d i v i d u a l s e s q u i t e r p e n e l a c t o n e s a g a i n s t v a r i o u s types of f u n g i , and (3) to r e l a t e a n t i f u n g a l a c t i v i t i e s of. s e s q u i t e r p e n e l a c t o n e s to t h e i r chemica l s t r u c t u r e . 122 Exper imenta l C u l t u r e s of Microsporum cooke i ( U . B . C . #86), T r i c h o p h y t o n mentagrophytes ( U . B . C . #132), Fusar ium sp . ( U . B . C . #77), Saccharomyces c e r e v i s i a e ( U . B . C . #140), and Candida a l b i c a n s ( U . B . C . #54) were ob ta ined from the Department of M i c r o b i o l o g y , U . B . C . The s e s q u i t e r p e n e lactone's were i s o l a t e d , p r e p a r e d , or ob ta ined as d e s c r i b e d in the S e c t i o n I I . (1) Ant i funga l a c t i v i t y s c r e e n i n g t e s t M y c e l i a of M̂  cooke i , T. mentagrophytes , and Fusar ium sp . were scraped from the c u l t u r e s , suspended in s t e r i l e water , and w e l l mixed with c o t t o n swabs to' form a f i n e s u s p e n s i o n . Each of the m y c e l i a l suspens ions or c u l t u r e s of yeast was even ly spread wi th s t e r i l e c o t t o n swabs over agar p l a t e s c o n t a i n i n g the n u t r i t i o n a l medium (Bacto Sabouraud Dextrose Agar , D i f c o L a b . , M i c h i g a n ) . C r y s t a l s (approx imate ly 1.5 mg) of the s e s q u i t e r p e n e l a c t o n e s to be t e s t e d were p l a c e d d i r e c t l y on the agar p l a t e s . M. c o o k e i , T. mentagrophytes, and Fusar ium s p . , in d u p l i c a t e , were grown at room temperature in the dark and checked a f t e r 5 and 20 days of growth ( F i g . 8 ) . The p l a t e s with C^ a l b i c a n s and S. c e r e v i s i a e , in d u p l i c a t e , were incubated at 37°C in the dark and examined 24 hours l a t e r . Lac tones which caused a comp le te l y c l e a r area of funga l growth i n h i b i t i o n around the s i t e where they were p l a c e d were c o n s i d e r e d s t r o n g l y a c t i v e , whereas those i n h i b i t i n g growth on ly w i t h i n an area where c r y s t a l s were p l a c e d were c o n s i d e r e d weakly a c t i v e . I n a c t i v e compounds were those 123 which had no o b s e r v a b l e e f f e c t s on the growth of f u n g i . 124 R e s u l t s and P i scuss ion R e s u l t s of the a n t i f u n g a l a c t i v i t y s c r e e n i n g t e s t s on M. cooke i , T. mentagrophytes , and Fusar i um sp . (Table 20) show that a m a j o r i t y of s e s q u i t e r p e n e l a c t o n e s examined possess at l e a s t weak a n t i f u n g a l p r o p e r t i e s . Out of 130 t e s t s with 45 s e s q u i t e r e p e n e l a c t o n e s 24 (19%) were s t r o n g l y p o s i t i v e , 29 (22%) were weakly p o s i t i v e , and 77 (59%) were n e g a t i v e . Out of 45 s e s q u i t e r p e n e l a c t o n e s examined 13 (29%) s t r o n g l y i n h i b i t e d growth of at l e a s t one of the three f u n g i , 15 (33%) were only weakly a c t i v e . Only 17 (38%) s e s q u i t e r p e n e l a c t o n e s d i d not have any apparent e f f e c t on the growth of the three fung i examined (Table 2 1 ) . Tab le 21 summarizes the a n t i f u n g a l a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s based on s k e l e t a l c l a s s e s . Eudesmanol ides have the h i g h e s t p r o p o r t i o n of s t r o n g l y a c t i v e l a c t o n e s , whereas g e r m a c r a n o l i d e s have the h i g h e s t p r o p o r t i o n of i n a c t i v e compounds. However, sample s i z e s w i t h i n i n d i v i d u a l s k e l e t a l c l a s s e s of s e s q u i t e r p e n e l a c t o n e s are too smal l for making any d e f i n i t e c o n c l u s i o n s on the importance of the b a s i c s k e l e t a l s t r u c t u r e in de te rmin ing the a n t i f u n g a l a c t i v i t i e s of these compounds. (1) Ant i funga l a c t i v i t y aga i n s t i n d i v i d u a l fung i O v e r a l l a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s examined was approx imate ly the same for both dermatophytes , M_;_ cooke i , and T. mentagrophytes (Table 22) ; out of 45 s e s q u i t e r p e n e l a c t o n e s 125 t e s t e d a g a i n s t these two s p e c i e s approx imate ly one h a l f i n h i b i t e d growth ( s t r o n g l y or weakly) of these f u n g i . On the c o n t r a r y , Fusar ium sp. was i n h i b i t e d (only weakly) in 13% out of 40 t e s t s . These marked d i f f e r e n c e s in the s e n s i t i v i t y of the s k i n fungi and Fusar ium to s e s q u i t e r p e n e l a c t o n e s suggests that these fung i must d i f f e r g r e a t l y in the t h e i r p h y s i o l o g y and chemi st r y . Four, out of 14 t e s t s of 7 s e s q u i t e r p e n e l a c t o n e s a g a i n s t two y e a s t s (§_;_ c e r e v i s i a e and a l b i c a n s ) were p o s i t i v e (Table 23) . P s e u d o i v a l i n was a c t i v e a g a i n s t both y e a s t s but p a r t h e n i n and h e l e n a l i n i n h i b i t e d growth of c e r e v i s i a e o n l y . T h i s i n d i c a t e s that i n d i v i d u a l s p e c i e s of yeas t s may g r e a t l y d i f f e r in t h e i r s e n s i t i v i t y to the same s e s q u i t e r p e n e l a c t o n e s . (2) Re la t i onsh ip between chem i ca1 s t r u c t u r e and a n t i f u n g a l a c t i v i t y To my knowledge, no study has yet been conducted that would examine the mechanism of a n t i f u n g a l a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s . I_n v i t r o r e a c t i o n s between the e x o c y c l i c methylene on the l a c t o n e r i n g or the u n s u b s t i t u t e d eye lopentenone wi th v a r i o u s t h i o l s have been demonstrated ( e . g . H a l l et a l . 1977, Lee et a l . 1977a, Pieman et a l . 1979). These r e s u l t s suggest t h a t , i_n v i v o , s e s q u i t e r p e n e l a c t o n e s c o u l d r e a c t through these a c t i v e s i t e s wi th v a r i o u s v i t a l l y important t h i o l s such as enzymes and consequent ly might have v a r i o u s d e t r i m e n t a l e f f e c t s on a g iven organ ism. In t h i s way s e s q u i t e r p e n e l a c t o n e s might 126 a l s o i n t e r f e r e with v a r i o u s funga l p h y s i o l g i c a l p r o c e s s e s , c o n s e q u e n t l y d e s t r o y i n g the c e l l s or s lowing down t h e i r growth r a t e . In the f o l l o w i n g p a r t , I examine the r e l a t i o n s h i p between the presence of v a r i o u s f u n c t i o n a l groups of s e s q u i t e r p e n e l a c t o n e s and t h e i r a n t i f u n g a l a c t i v i t y . A study of adduct fo rmat ion wi th t h i o l s through the exomethylene on the l a c t o n e r i n g ( e . g . Pieman et a l . 1979) suggests that t h i s moiety p r e s e n t s a p o s s i b l e a c t i v e s i t e of s e s q u i t e r p e n e l a c t o n e s which might be r e s p o n s i b l e . f o r t h e i r v a r i o u s b i o l o g i c a l a c t i v i t i e s . Tab le 24 summarizes the a n t i f u n g a l a c t i v i t y of a l l s e l e c t e d s e s q u i t e r p e n e l a c t o n e s a c c o r d i n g to the presence or absence of the methylene group on the l a c t o n e . About 50% of t e s t s i n c l u d i n g s e s q u i t e r p e n e l a c t o n e s p o s s e s s i n g t h i s group were p o s i t i v e and 50% were n e g a t i v e . Out of 39 t e s t s wi th l a c t o n e s l a c k i n g the C l3 -methy lene only 6 were weakly p o s i t i v e , ' whereas the r e s t were n e g a t i v e . These r e s u l t s suggest that a l though the e x o c y c l i c methylene .probably may p lay a r o l e in the a n t i f u n g a l a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s , some other f u n c t i o n a l i t i e s a l s o c o n t r i b u t e . Thus the presence or absence of the exomethylene on the l a c t o n e r i n g a lone cannot f u l l y e x p l a i n the a n t i f u n g a l a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s . To e l i m i n a t e d i f f e r e n c e s in a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s due to d i f f e r e n c e s in t h e i r b a s i c s k e l e t a l s t r u c t u r e , I a n a l y z e d the i n d i v i d u a l s k e l e t a l c l a s s e s s e p a r a t e l y . F i r s t l y , I d i v i d e d s e s q u i t e r p e n e l a c t o n e s a c c o r d i n g to the presence or absence of the C13-methylene. P s e u d o g u a i a n o l i d e s were d i v i d e d 127 a l s o a c c o r d i n g to the presence or absence of the C2-C3 double bond which i s another p o t e n t i a l l y a c t i v e s i t e . Fur thermore , I a l s o examined the r o l e of other f u n c t i o n a l groups of s e s q u i t e r p e n e l a c t o n e s which might i n f l u e n c e the a n t i f u n g a l a c t i v i t y of these compounds. S ince the s t r o n g e s t p o s i t i v e responses were ob ta ined on T\_ mentagrophytes on which a l s o a l l a v a i l a b l e s e s q u i t e r p e n e l a c t o n e s were t e s t e d , I chose t h i s fungus for a c l o s e r study of the r e l a t i o n s h i p between the f u n c t i o n a l groups of s e s q u i t e r p e n e l a c t o n e s and t h e i r a b i l i t y to i n h i b i t growth of f u n g i . In the f o l l o w i n g pa r t I w i l l d i s c u s s r e s u l t s o b t a i n e d for i n d i v i d u a l c l a s s e s of s e s q u i t e r p e n e l a c t o n e s . (a) Germac r a n o l i d e s Out of 13 t e s t s a g a i n s t three f u n g i , i n c l u d i n g ge rmacrano l i des which possess the e x o c y c l i c methy lene, 54% were e i t h e r s t r o n g l y or weakly p o s i t i v e (Table 2 5 ) . On the other hand, a l l 15 t e s t s i n c l u d i n g germacrano l ides without the e x o c y c l i c methylene were n e g a t i v e . Thus the presence of the e x o c y c l i c methylene i s p robab ly necessary but not always s u f f i c i e n t fo r a n t i f u n g a l a c t i v i t y of t h i s group of l a c t o n e s . The presence or absence of v a r i o u s f u n c t i o n a l groups in ge rmacrano l i des in r e l a t i o n to t h e i r a c t i v i t y a g a i n s t T. mentagrophytes i s summarized in Tab le 26. E l e p h a n t o p i n i s the on ly l a c t o n e p o s s e s s i n g the e x o c y c l i c methylene on the l a c t o n e r i n g which d i d not i n h i b i t growth of t h i s fungus. The lack of 128 a c t i v i t y in t h i s compound might be a r e s u l t of the presence of two epoxy groups on a molecu le (Table 26) which probab ly c o u n t e r a c t the e f f e c t s of the e x o c y c l i c methy lene . The presence of at l e a s t one epoxy group i s a l s o common to a l l o ther i n a c t i v e germacrano l ides but s ince a l l of these compounds a l s o lack the e x o c y c l i c methylene, i t i s i m p o s s i b l e to dec ide on the r e l a t i v e importance of these m o i e t i e s . In a d d i t i o n , there are other f u n c t i o n a l groups which might p l a y a r o l e in de te rmin ing a n t i f u n g a l a c t i v i t i e s of g e r m a c r a n o l i d e s . For example, the C4,C5 double bond i s p resent in a c t i v e ge rmacrano l i des but i s absent from a l l i n a c t i v e compounds (Table 2 6 ) . T h i s f u n c t i o n a l i t y thus appears to enhance the a n t i f u n g a l a c t i v i t y of g e r m a c r a n o l i d e s . However, i t w i l l be necessary to examine a l a r g e r number of compounds more s i m i l a r in s t r u c t u r e be fo re any c o n c l u s i o n s can be drawn. (b) G u a i a n o l i d e s There i s no c l e a r r e l a t i o n s h i p between the presence of the exomethylene on the l a c t o n e r i n g and the a n t i f u n g a l a c t i v i t y of g u a i a n o l i d e s (Table 25) . Because one compound without the exomethylene ( d e s a c e t o x y m a t r i c a r i n ) weakly i n h i b i t e d growth of both dermatophytes , some other group(s) must be r e s p o n s i b l e for the a n t i f u n g a l a c t i v i t y of t h i s and p o s s i b l y other r e l a t e d compounds. The a c t i v i t y of d e s a c e t o x y m a t r i c a r i n a g a i n s t T. mentagophytes might be e x p l a i n e d by the presence of C1,C10 129 double bond which i s a l s o p resent in a s t r o n g l y a c t i v e p s e u d o i v a l i n (Table 2 7 ) . Data are i n s u f f i c i e n t to f u r t h e r examine the importance of other f u n c t i o n a l g roups . (c) Eudesmanol ides Out of 21 t e s t s i n c l u d i n g 7 eudesmanol ides p o s s e s s i n g the exomethylene, 67% were p o s i t i v e whi le a l l t e s t s i n c l u d i n g a l a c t o n e l a c k i n g t h i s group (0(-santonin) were n e g a t i v e (Table 2 5 ) . Thus i t appears that the presence of the C13-methylene may be a s s o c i a t e d with the a n t i f u n g a l a c t i v i t y of t h i s c l a s s of l a c t o n e s . However, s i n c e some l a c t o n e s wi th t h i s group d i d not e x h i b i t any a c t i v i t y in the t e s t s , the presence of t h i s moiety i s not always s u f f i c i e n t to cause the a n t i f u n g a l a c t i v i t y perhaps because some other f u n c t i o n a l i t y may negate i t . T h i s p o s s i b i l i t y i s supported by data on three s t r u c t u r a l l y s i m i l a r eudesmano l ides . I v a s p e r i n , which d i f f e r s from the s t r o n g l y a c t i v e i v a l i n on ly by the presence of a h y d r o x y l group on C l , d i d not have any v i s i b l e e f f e c t s on growth of T. metaqrophytes (Table 2 8 ) . S i m i l a r l y , p u l c h e 1 1 i n - C , which d i f f e r s from i v a l i n only by the presence of a h y d r o x y l group on C3, was a l s o i n a c t i v e . Hence the lack of a c t i v i t y of i v a s p e r i n and p u l c h e l l i n - C can be e x p l a i n e d on ly by the a d d i t i o n a l h y d r o x y l group which must have c o u n t e r a c t e d the e f f e c t s of the e x o c y c l i c methy lene . Other f u n c t i o n a l groups (see Tab le 28) might a l s o p l a y a r o l e , however, I do not have s u f f i c i e n t data on s i m i l a r compounds to examine t h i s p o s s i b i l i t y . 130 (d) Pseudoqua i a n o l i d e s Tab le 29 summarizes r e s u l t s on a c t i v i t y of p s e u d o g u a i a n o l i d e s in r e l a t i o n to the presence or absence of the e x o c y c l i c methylene and/or C2,.C3 double bond. The e x o c y c l i c methylene a lone does not appear to p lay an important r o l e in a c t i v i t y of p s e u d o g u a i a n o l i d e s a g a i n s t fungi because a m a j o r i t y of the compounds p o s s e s s i n g t h i s moiety d i d not e x h i b i t a c t i v i t y a g a i n s t t e s t e d fungi (Table 29) . A l s o the C2,C3 double bond does not appear to p lay a r o l e in a n t i f u n g a l a c t i v i t y because a l l 6 t e s t s ( i n c l u d i n g 2 l a c t o n e s ) gave nega t i ve r e s u l t s (Table 29) . There thus appears to be no r e l a t i o n s h i p between the presence or absence of both m o i e t i e s and the a c t i v i t y of p s e u d o g u a i a n o l i d e s a g a i n s t the fungi , examined (Table 29) . T h e r e f o r e I conc lude that e i t h e r other f u n c t i o n a l groups are r e s p o n s i b l e for a c t i v i t y of these s e s q u i t e r p e n e l a c t o n e s or these m o i e t i e s are important but t h e i r e f f e c t s are f r e q u e n t l y c o u n t e r a c t e d by other f u n c t i o n a l g roups . To examine the two p o s s i b l e e x p l a n a t i o n s , in Tab le 30, I summarized v a r i o u s f u n c t i o n a l groups of p s e u d o g u a i a n o l i d e s in r e l a t i o n to t h e i r a c t i v i t y a g a i n s t T^ mentagrophytes . P a r t h e n i n , which possesses a hydroxy l group on C l in ( X - p o s i t i o n , i s s t r o n g l y a c t i v e but hymenin, which d i f f e r e s from p a r t h e n i n on ly by /3 -pos i t i on of t h i s hydroxy l group i s i n a c t i v e . T h i s i n d i c a t e s that the c o n f i g u r a t i o n of the hydroxy l on C l a lone i s an important f a c t o r de te rmin ing a c t i v i t y of these compounds. T h i s h y d r o x y l group p o s s i b l y i n f l u e n c e s the a c t i v i t y of p s e u d o g u a i a n o l i d e s through i t s i n t e r a c t i o n s wi th the 131 exomethylene and cyc lopentenone r i n g . T h i s view i s supported by the f a c t that ambrosin which d i f f e r s from p a r t h e n i n and hymenin on ly by the absence of the h y d r o x y l group on C l i s s t r o n g l y a c t i v e (Table 30) . T h i s shows the importance of the exomethylene and/or the C2,C3 double bond. Damsin which a l s o l a c k s t h i s h y d r o x y l group and the C2,C3 double bond i s s t i 1 1 ' s t r o n g l y a c t i v e . T h i s suggests that the C13-methylene a lone might be r e s p o n s i b l e ( i f the hydroxy l group on C l i s absent) for s t rong a n t i f u n g a l a c t i v i t y . But the presence of the C l hydroxy l in c o r o n o p i l i n , which i s o therwise i d e n t i c a l w i th damsin (Table 30) , r e s u l t s in a complete l o s s of a c t i v i t y . The importance of the C2,C3 double bond i s apparent from the i n a c t i v i t y of c o r o n o p i l i n which l a c k s t h i s group but i s o therwise i d e n t i c a l to the s t r o n g l y a c t i v e p a r t h e n i n (Table 30) . Thus s i n c e the lack of the C2,C3 double bond r e s u l t s in a complete l o s s of - a c t i v i t y , t h i s moiety must a l s o p lay an important r o l e . These comparisons t h e r e f o r e suggest t h a t : (1) both the exomethylene and the C2,C3 double bond are important c o n s t i t u e n t s r e s p o n s i b l e for a n t i f u n g a l a c t i v i t i e s , of p s e u d o g u a i a n o l i d e s ; and (2) the h y d r o x y l group on C l ( p a r t i c u l a r l y in / 3 - p o s i t i o n ) reduces the e f f e c t s of the two m o i e t i e s . In a d d i t i o n , a comparison of o ther i n a c t i v e p s e u d o g u a i a n o l i d e s (shown in Tab le 30) shows that a common f e a t u r e of a l l but one compound i s the presence of e i t h e r an e s t e r f u n c t i o n ( u s u a l l y a c e t y l ) or other g roup ing ( in t e n u l i n ) , and in many cases a l s o a h y d r o x y l group on e i t h e r C l , C2, C3, C4, or C15. These groups might have reduced the a c t i v i t y of e i t h e r the exomethylene, the C2,C3 double bond, or both 132 m o i e t i e s . On the b a s i s of these r e s u l t s I conc lude that the exomethylene on the l a c t o n e r i n g and the C2,C3 double bond are p robab ly the major c o n s t i t u e n t s r e s p o n s i b l e for a n t i f u n g a l a c t i v i t y of p s e u d o g u a i a n o l i d e s but that t h e i r e f f e c t s are f r e q u e n t l y c o u n t e r a c t e d by other f u n c t i o n a l g roups . C o n c l u s i o n s The a n t i f u n g a l a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s appear to have a m u l t i p l e c a u s a t i o n , as wi th b a c t e r i a . The f o l l o w i n g s t r u c t u r a l f e a t u r e s of s e s q u i t e r p e n e l a c t o n e s may p lay a r o l e e i t h e r i n d i v i d u a l l y or in combinat ion wi th o t h e r s in p roduc ing the a n t i f u n g a l e f f e c t s . (1) The presence of the exomethylene the l a c t o n e r i n g appears to be necessary for a n t i f u n g a l a c t i v i t y in ge rmacrano l i des and eudesmano l ides . However, the presence of t h i s group i s not always s u f f i c i e n t for a c t i v i t y . (2) The presence of the C1,C10 double bond in g u a i a n o l i d e s or the C2,C3 double bond in p s e u d o g u a i a n o l i d e s may be r e s p o n s i b l e fo r the a c t i v i t y of some compounds, or these group ings might enhance the a c t i v i t y of o ther f u n c t i o n a l g r o u p ( s ) . (3) ' Some other f u n c t i o n a l groups appear to i n f l u e n c e the a c t i v i t y p o s s i b l y through t h e i r i n t e r a c t i o n s wi th the main a c t i v e f u n c t i o n a l i t i e s . For example, the C4,C5 double bond enhances the a c t i v i t y in g e r m a c r a n o l i d e s , whereas the h y d r o x y l groups in eudesmanol ides and 133 p s e u d o g u a i a n o l i d e s , the epoxy group(s) in g e r m a c r a n o l i d e s , and the a c e t y l groups in p s e u d o g u a i a n o l i d e s reduce the a n t i f u n g a l a c t i v i t i e s of some s e s q u i t e r p e n e l a c t o n e s . (4) The c o n f i g u r a t i o n of the h y d r o x y l group on C l in some p s e u d o g u a i a n o l i d e s i n f l u e n c e s t h e i r a n t i f u n g a l act i v i t y . The a c t i v i t y of s e s q u i t e r p e n e l a c t o n e s a g a i n s t fung i must a l s o be determined by d i f f e r e n c e s in the chemis t ry and p h y s i o l o g y of i n d i v i d u a l types of f u n g i . The p e r m e a b i l i t y of the v a r i o u s fung i examined here to the s e s q u i t e r p e n e l a c t o n e s used c o u l d be an important f a c t o r which was not examined. S e l e c t i v e uptake of o rgan i c compounds by c e l l s i s a wel l-known phenomenon. In a d d i t i o n , the s e s q u i t e r p e n e l a c t o n e s used in t h i s study d i f f e r in t h e i r s o l u b i l i t i e s and t h i s i s undoubtedly a l s o very impor tan t . These f a c t o r s c o u l d e x p l a i n d i f f e r e n t s e n s i t i v i t y of dermatophytes and other types of fung i t e s t e d in t h i s study a g a i n s t the same s e s q u i t e r p e n e l a c t o n e s . 134 Fig. 8. Antifungal activity of some sesquiterpene lactones (1- alanto- lactone, 2- isoalantolactone, 3- i v a l i n , 4- ivasperin, 5- pinnatifidin) against (a) Microsporum cookei and (b) Tricho- phyton mentagrophytes. 135 Table 20. Screening test for antimicrobial activity of selected sesquiterpene lactones against three fungi. The activity of individual sesquiterpene lactones was examined after 5 and 20 days of growth. If the activity after 20 days of growth differed from that after 5 days, the result is shown in parentheses • Sesquiterpene lactone Response to Number in Appendix Name M. cookei T. mentagrophytes Fusarium sp. Germacranolides 2 Pyrethrosin + + - 4 Chamissonin, diacetyl ± ± 7 Eupatoriopicrin ± ± 14 Glaucolide-A - - 16 Marginatin - - 17 Glaucolide-D - - 19 Glaucolide-F - 20 Glaucolide-G - - 21 Elephantopin - - NT 22 Parthenolide, 9-cn-OH Guaianolides + NT 23 Cumambrin-A ± ± 24 Cumambrin-B ± ± 26 Cumambrin-B, tetrahydro- - - 27 Cumambrin-B, acetate ± ± 136 Table 20. Continued. 30 Matricarin, desacetoxy- + + 31 Grossheimin + + - 32 Ivalin, pseudo- Eudesmanolides + + + 33 Alantolactone + + + 35 Alantolactone, iso- + + + 36 Ivalin + + - 37 Ivasperin ± ( - ) - - 38 Pinnatifidin + + - 39 Pulchellin-C ± (-) - - 40 & -Santonin - • - - 46 Santamarine Pseudoguaianolides + + 47 Parthenin + + - 48 Parthenin, dihydroiso- + + NT 49 Parthenin, tetrahydro- + ± NT 51 Hymenin + - - 52 Ambrosin + + + 53 Coronopilin ± (-)• - - 54 Damsin + + - 55 Hysterin - - 137 Table 20. Continued. 56 Tetraneurin-A - — _ 57 Tetraneurin-B - - - 58 Tetraneurin-D - - - 59 Tetraneurin-E - - - 63 Tenulin - - - 64 Tenulin, iso- - - - 66 Helenalin + + + 67 Flexuosin-B - - - 69 Balduilin - - - 70 Cumanin Other sesquiterpene + lactones — 77 Axivalin - ± (") - 78 Ivax i l l a r i n - + <±) NT NT= not tested += complete inhibition += weak inhibition -= no inhibition 138 Table 21. Summary of antimicrobial a c t i v i t y of sesquiterpene lactones from i n d i v i d u a l s k e l e t a l classes against three fungi. No. (%) of sesq. lactones active against at least one fungus Number (%) inactive sesq. l a c t . Class strongly weakly Total Germacranolides 2 2 6 10 Guaianolides 0 6 1 7 Eudesmanolides 5 2 1 8 Pseudo- guaianolides 4 5 9 18 Other sesq. lactones 1 1 0 2 Total 13 (29) 15 (33) 17 (38) 45 139 Table 22. Summary of antimicrobial a c t i v i t y of selected sesquiterpene lactones against i n d i v i d u a l fungi. No. (%) positive responses Fungus Strong Weak Number (%) negative Total No. responses tests M. cookei 11 (24) T. mentagrophytes 13 (29) Fusarium sp. 0 14 (31) 10 (22) 5 (13) 20 (45) 22 (49) 35 (88) 45 45 40 Total 24 (19) 29 (22) 77 (59) 130 140 Table 23. A c t i v i t y of selected sesquiterpene lactones against two species of yeasts. Sesquiterpene lactone Response to Number i n Appendix Name S. cerevisiae C. albicans 32 I v a l i n , pseudo- + + 33 Alantolactone - - 35 Alantolactone, i s o - - - 47 Parthenin + - 51 Hymenin - - 63 Tenulin - - 66 Helenalin + - Table 24. Summary of antifungal a c t i v i t y of sesquiterpene lactones as related to the presence or absence of the exomethylene on the Y-lactone ri n g . No. (%) responses No. (%) responses No. sesq. l a c t . with Positive No. sesq. l a c t . without Po s i t i v e Fungus C13=CH2 Strong Weak Negative C13=CH2 Strong Weak Negative M. cookei 32 11 11 10 13 0 3 10 T. men t agrophyt es 32 13 7 12 13 0 3 10 Fusarium sp. 29 0 5 24 11 0 0 11 S. cerevisiae 6 2 1 3 1 0 0 1 C. albicans 6 1 0 5 1 0 0 1 Total 105 (100) 27 (26) 24 (23) 54 (51) 39 (100) 0 6 (15) 33 (85) Table 25. Antifungal a c t i v i t y of germacranolides, guaianolides, and eudesmanolides as related to the presence or absence of the methylene group on the Y-lactone r i n g . No. (%) responses No. (%) responses Fungus No. sesq. l a c t . with C13=CH2 Positive Negative No. sesq. l a c t . without C13=CH2 P o s i t i v e Negative Strong Weak Strong Weak Germacranolides: M. cookei . 5 1 2 2 5 0 0 5 T. mentagrophytes 5 2 2 1 5 0 0 5 Fusarium sp. 3 0 0 3 5 0 0 5 Total 13 (100) 3 (23) 4 (31) 6 (46) 15 (100) 0 0 15 (100) Guaianolides: M. cookei 5 1 4 0 2 0 1 1 T. mentagrophytes 5 1 4 0 2 0 1 1 Fusarium sp. 5 0 1 4 2 0 0 2 Total 15 (100) 2 (13) 9 (60) 4 (27) 6 (100) 0 2 (33) 4 (67) K Table 25. Continued. Eudesmanolides: M. cookei 7 , 5 2 T_. mentagrophytes 7 5 0 Fusarium sp. 7 0 2 Total 21 (100) 10 (48) 4 (19) 0 1 0 0 1 2 1 0 0 1 5 1 0 0 1 7 (33) 3 (100) 0 0 3 (100) Table 26. Antimicrobial a c t i v i t y of germacranolides against _T. mentagrophytes as related to the presence or absence of various functional groups. Presence or absence of functional groups Sesquiterpene lactone o o o CM o o a CJ o u u r-i < J B C >-t , < < 3 < J m m - ^ r ^ c J o o O f - i i - i c M r o s t o c j X U II O I II I | O I II I « . r H - r H II I I I I CO CO A n n p n r H •* N a m e —' \-' CM c o \ < f < r v o v o i l o o o o o o o o . - i . - < A p p e i l U i X Mctlltt: c _ ) o c j c j c j U u c j u > - U > - c j u c j u c j c j c j Strongly active: 2 Pyrethrosin - + _ _ - _ + + _ + _ _ _ _ _ _ + 22 Parthenolide, 9-tt-OH + - - - - + - + - - - - - - + Weakly active: 4 Chamissonin, diacetate + - - - + - + + - + _ — _ _ - _ + 7 Eupatoriopicrin + - - - - - + - + - - + - - - - + Inactive: 14 Glaucolide-A - - + - - .+ - - + - + - - . + - + - 16 Marginatin + - - - _ + - - + + - + - - + - 17 Glaucolide-D + - - + - + - + - - - - _ + + _ 19 Glaucolide-F - - + - - + - - + - + - + - - + - Table 26. Continued. 20 Glaucolide-G + - - - - + - - + - + - + - - + - 21 Elephantopin + - - * _ + _ _ + _ - - - + - - + c T ^ i o V 0 OH -0-C-C-CH CH tn OH V 9 -0-C-C-CHo CH 3 CH3 0 R = -O-C-C-CH, CH„ " V -0-C-C-CH. 1 > CH2 Table 27. Antimicrobial a c t i v i t y of guaianolides against T. mentagrophytes as related to the presence or absence of various functional groups. Presence or absence of functional groups CD 0) Sesquiterpene lactone . No. i n ii r-l Appendix Name . C J Strongly active: 32 I v a l i n , pseudo + - - - + - ' + - - - . - + Weakly active: 23 Cumambrin-A - - - 24 Cumambrin-B 27 Cumambrin-B, acetate - 30 Matrica r i n , desacetoxy + + - 31 Grossheimin - - + o II CM C J O II CO C J C J II CO O PC o I o •u r~. CJ C J cd v£> I O CO CJ c j cd C J S 3 o I co C J < o I CO C J 33 o I o I—I C J CM 33 C J II o t—I C J CM 33 C J II CO I—I C J + - + - - + + - + + - + - + - + - + + - - + + - + + - + - - + + Inactive: 26 Cumambrin-B, tetrahydro Table 28. Antimicrobial a c t i v i t y of eudesmanolides against T_. mentagrophytes as related to the presence or absence of various functional groups. Presence or absence of functional groups Sesquiterpene lactone No. i n Appendix Name 35 <N 33 33 <r O • u II O o o C J i rH II 1—1 • CM II CO 1 CO H CO C J C_) C J C J C J C J CM 33 C J II <J" C J m C J II C J vO C J II m C J cu C o 4-1 c j cd •> rH VO I C J V - C3 O CM 4J EE c o o C J u c d II ~ rH CO I"-- I r H C J >- o Strongly active: 33 Alantolactone 35 Alantolactone, i s o - 36 I v a l i n 38 P i n n a t i f i d i n 46 Santamarine + + + + + + + + + + + Inactive: 37 Ivasperin 39 Pulchellin-C 40 rt-Santonin + + + A + + + + + + Table 29. Antifungal a c t i v i t y of pseudoguaianolides as related to the presence or absence of the methylene group on the Y-lactone and the unsubstituted cyclopentenone r i n g . No. sesq. l a c t . with No. (%) responses No. sesq. l a c t . with No. (% ) responses Positive P o s i t i v e Fungus C13=CH2 only Strong Weak Negative C2=C3 only Strong Weak Negative M. cookei 8 1 2 5 2 0 0 2 T. mentagrophyt es 8 1 0 7 2 0 0 2 Fusarium sp. 8 0 0 8 2 0 0 2 Total 24 (100) 2 (8) 2 (8) 20 (80) 6 (100) 0 0 6 (100) No. sesq. l a c t . No. sesq. l a c t . with C13=CH2 without C13=CH2 and C2=C3 and C2=C3 M. cookei 5 3 1 1 3 0 2 1 T. mentagrophyti es 5 3 0 2 3 0 2 1 Fusarium sp. 5 0 2 3 1 0 0 1 Total 15 (100) 6 (40) 3 (20) 6 (40) 7 (100) 0 4 (57) 3 (43) Table 30. Antimicrobial a c t i v i t y of pseudoguaianolides against T. mentagrophytes as related to the presence or absence of various functional groups. Presence or absence of functional group s Sesquiterpene lactone 35 O i I 33 O i 1 33 33 :on e :on e •O Ac  CM 33 O 1 No. i n C2 =C 3 0 o 1 1 1 1 C4 =0  * J u to | 1 oo a u <o •O Ac  C7 =C 11  C1 3= CH  < o i Appendix Name u CM u C2 =C 3 co ~* u c_> C4 =0  \o  i • U C7 =C 11  C1 3= CH  f—1 o m i — i u Strongly active: 47 Parthenin + - + - + + • - - - + - - 52 Ambrosin - - + - + • + - - - + - - 54 Damsin - - - - + + - - - - - 66 Helenalin - - + - + - • + a - + - - Weakly active: 48 Parthenin, dihydroiso- + - - - + + - - + - - • - 49 Parthenin, tetrahydro- + — — — + + — — — — - Table 30. Continued. Inactive: 51 Hymenin +(0) - + - - + + _ . _ _ + _ . _ 53 Coronopilin + - - - - + + - _ _ • + ' . _ . _ 55 Hysterin _ _ _ b - + - - + _ + 56 Tetraneurin-A + - - - - + + - - - + - b 57 Tetraneurin-B + - - - - + + - _ _ + + _ 58 Tetraneurin-D . + - - - + - + - - _ + + 59 Tetraneurin-E + - - - b - + - _ + _ + 63 Tenulin - - + _ _ + _ + c 64 Tenulin, i s o - - - + - - + - + + _ _ _ _ 67 Flexuosin-B - + - - - + - + d - - - 69 B a l d u i l i n - - + - - + - + + _ + _ _ 70 Cumanin - - - + + - - + _ _ + _ _ C6—C7 yCYi3 a= -OH a= -OAc c= d = c 6 _0- c _CH=C OH X C H , 151 CHAPTER 3 E f f e c t s of s e l e c t e d pseudogua i a n o l i d e s on s u r v i v a l of the f l o u r b e e t l e , TRIBOLIUM CONFUSUM Int roduct i on Secondary p l a n t substances that do not have any apparent f u n c t i o n in e s s e n t i a l metabo l i c p r o c e s s e s of p l a n t s are b e l i e v e d to p lay an important r o l e in defense of p l a n t s a g a i n s t h e r b i v o r e s , p a r t i c u l a r l y i n s e c t s ( F r a e n k e l 1959, Wal lace and M a n s e l l 1976). Sesqu i te rpene l a c t o n e s have not been e x t e n s i v e l y examined for t h i s p r o t e c t i v e f u n c t i o n . To date on ly g l a u c o l i d e - A (Burnet t et a l . 1974) and a l a n t o l a c t o n e (Pieman et a l . 1978) have been shown to be i n s e c t f e e d i n g d e t e r r e n t s ; g l a u c o l i d e - A i n f l u e n c e s the growth r a t e and s u r v i v a l of l e p i d o p t e r o u s l a r v a e (Burnet t et a l . 1974) and p a r t h e n i n e x h i b i t s c a r d i a c - i n h i b i t o r y p r o p e r t i e s in grasshoppers (Pieman et a l . 1981). I-n t h i s study I examined the e f f e c t s of s e v e r a l s e s q u i t e r p e n e l a c t o n e s in r e l a t i o n to t h e i r chemica l s t r u c t u r e on s u r v i v a l of the confused f l o u r b e e t l e , Tr i b o l i um confusum ( C o l e o p t e r a : T e n e b r i o n i d a e ) . T h i s s p e c i e s i s f r e q u e n t l y used to a s c e r t a i n the t o x i c p r o p e r t i e s of chemica l s on i n s e c t s . A l l of the s e l e c t e d s e s q u i t e r p e n e l a c t o n e s are p s e u d o g u a i a n o l i d e s 152 d i f f e r i n g main ly in the presence of two p o t e n t i a l l y a c t i v e s i t e s , «-methylene-f-lactone and fl,/j-unsaturated ketone mo ie ty . 153 Exper imenta l The b e e t l e s , o b t a i n e d from Dr . R. H. E l l i o t t , P lan t Sc ience Department, U . B . C , were ma in ta ined on wheat f l o u r in a g l a s s c o n t a i n e r covered with weekly moistened f i l t e r paper . C o r o n o p i l i n , h e l e n a l i n , p a r t h e n i n , and t e n u l i n ( F i g . 9) were i s o l a t e d or ob ta ined as d e s c r i b e d in the S e c t i o n I . (1) Feed ing exper iment D i f f e r e n t c o n c e n t r a t i o n s (0.2-10.0%) of s e s q u i t e r p e n e l a c t o n e s were prepared in 95% e t h a n o l . Approx imate ly 0.3 ml of each e t h a n o l i c s o l u t i o n was added to 50mg samples of f l o u r . A f t e r e v a p o r a t i o n of the e t h a n o l , the f l o u r was t r a n s f e r r e d to p e t r i d i s h e s which were l i n e d wi th f i l t e r paper . F i v e b e e t l e s were p l a c e d in each d i s h . A sma l l s t r i p of moistened f i l t e r paper was added weekly to ma in ta in proper h u m i d i t y . The b e e t l e s were checked every morning throughout a 60 day study p e r i o d . The t e s t was run in d u p l i c a t e s for a l l c o n c e n t r a t i o n s of p a r t h e n i n and c o r o n o p i l i n . Because of the s c a r c i t y of t e n u l i n only 0 .2 , 4, and 8% c o n c e n t r a t i o n s were d u p l i c a t e d , and for the same reason the t e s t wi th h e l e n a l i n was run on ly once. Two c o n t r o l s , one with f l o u r on ly and the other one without any food , were a l s o run in d u p l i c a t e . Data on s u r v i v a l r a t e s of the b e e t l e s were a n a l y z e d by Duncan's M u l t i p l e Range T e s t . Reference to ' s i g n i f i c a n c e ' in the tex t means a s i g n i f i c a n t d i f f e r e n c e s at the 0.05 l e v e l . 154 R e s u l t s and P i s c u s s i o n In a c o n t r o l t r i a l , ten b e e t l e s which were o f f e r e d f l o u r wi thout any l a c t o n e s u r v i v e d for an average of 58.9 +3.5 (SP) days . The e f f e c t s of food c o n t a i n i n g v a r i o u s s e s q u i t e r p e n e l a c t o n e s are shown in F i g u r e s 10-13. H e l e n a l i n ( F i g . 9) s i g n i f i c a n t l y reduced s u r v i v a l of the b e e t l e s ( F i g . 1 0 ) . I t s e f f e c t s i n c r e a s e d wi th i n c r e a s i n g c o n c e n t r a t i o n of t h i s compound up to 4%, but f u r t h e r i n c r e a s e in c o n c e n t r a t i o n had no a d d i t i o n a l s i g n i f i c a n t e f f f e c t s . The s u r v i v a l time of b e e t l e s o f f e r e d food wi th 4% or h igher c o n c e n t r a t i o n s of h e l e n a l i n was s i m i l a r to that of c o n t r o l b e e t l e s without food ( F i g . 10) . C o r o n o p i l i n ( F i g . 9 ) , in c o n c e n t r a t i o n s h igher than 3%, a l s o had a s i g n i f i c a n t e f f e c t on s u r v i v a l of Tr i b o l i um. The s t r o n g e s t e f f e c t , comparable to that of the c o n t r o l without f o o d , was recorded when food c o n t a i n e d 6% or more c o r o n o p i l i n ( F i g . 1 1 ) . P a r t h e n i n ( F i g . 9) a l s o s i g n i f i c a n t l y reduced s u r v i v a l of b e e t l e s , p a r t i c u l a r l y in the two h i g h e s t c o n c e n t r a t i o n s ( F i g . 1 2 ) . However, there was a great degree of v a r i a t i o n in s u r v i v a l r a t e s o f i n d i v i d u a l b e e t l e s w i t h i n i n d i v i d u a l t r i a l s ( F i g . 1 3 ) . T e n u l i n ( F i g . 9) which was o f f e r e d in 6 d i f f e r e n t c o n c e n t r a t i o n s (0.2-8.0%) had no s i g n i f i c a n t e f f e c t s on s u r v i v a l of the b e e t l e s compared wi th that of c o n t r o l an imals g iven food 155 without any s e s q u i t e r p e n e l a c t o n e ( F i g . 13) . The three s e s q u i t e r p e n e l a c t o n e s which reduced s u r v i v a l of f l o u r b e e t l e s had no s i g n i f i c a n t e f f e c t s in lower c o n c e n t r a t i o n s ; i . e . in 0.2-2.0% (par then in and h e l e n a l i n ) or up to 3% ( c o r o n o p i l i n ) . On the other hand, in h igher c o n c e n t r a t i o n s (4-10%) a l l three l a c t o n e s had a s i m i l a r adverse e f f e c t on b e e t l e s ' s u r v i v a l . T h e r e f o r e , I conc lude that p a r t h e n i n , c o r o n o p i l i n , and h e l e n a l i n had s t a t i s t i c a l l y s i m i l a r e f f e c t s on the s u r v i v a l of T^ confusum. Because in h igher c o n c e n t r a t i o n s these l a c t o n e s had s i m i l a r e f f e c t s on b e e t l e s ' s u r v i v a l r a t e s as s t a r v a t i o n , i t i s d i f f i c u l t to e s t a b l i s h whether the b e e t l e s d i e d as a d i r e c t r e s u l t of i n g e s t i n g s e s q u i t e r p e n e l a c t o n e s , or whether they r e f u s e d to eat the food c o n t a i n i n g these compounds and consequent l y d i e d of s t a r v a t i o n . T h i s problem i s moreover c o m p l i c a t e d by the p o s s i b i l i t y that the i n g e s t i o n of l a c t o n e s produces the same e f f e c t s as s t a r v a t i o n . Sesqu i te rpene l a c t o n e s c o u l d decrease the e f f i c i e n c y of d i g e s t i o n by t h e i r r e a c t i o n with t h i o l - a c t i v a t e d d i g e s t i v e enzymes. The presence of such p r o t e o l y t i c enzymes i s known from i n s e c t s (e . g . Houseman 1978). In a d d i t i o n , s t a r v a t i o n - 1 i k e e f f e c t s might a l s o be produced i f a g iven s e s q u i t e r p e n e l a c t o n e had d e t r i m e n t a l e f f e c t s on microorgan isms important for d i g e s t i o n . The a v a i l a b l e ev idence i s not s u f f i c i e n t to d i s c r i m i n a t e between these s u g g e s t i o n s . E s t a b l i s h i n g the b i o l o g i c a l s i g n i f i c a n c e of e f f e c t s of s e l e c t e d s e s q u i t e r p e n e l a c t o n e s on the s u r v i v a l of the f l o u r b e e t l e s r e q u i r e s the e v a l u a t i o n of f u n c t i o n a l groups which are 156 r e s p o n s i b l e for b i o l o g i c a l a c t i v i t i e s of these compounds. The presence of two f u n c t i o n a l groups in the four l a c t o n e s are summarized in Tab le 1. The three l a c t o n e s which reduced s u r v i v a l of the b e e t l e s ( p a r t h e n i n , h e l e n a l i n , and c o r o n o p i l i n ) a l l possess the methylene on the l a c t o n e r i n g . A double bond between C2-C3, however, does not seem to be r e s p o n s i b l e fo r d e t r i m e n t a l p r o p e r t y because c o r o n o p i l i n which l a c k s t h i s group had a s i m i l a r e f f e c t to p a r t h e n i n and h e l e n a l i n which both possess i t (Table 3 1 ) . T h i s suggests that the exomethylene on the l a c t o n e r i n g a lone i s r e s p o n s i b l e for reduced s u r v i v a l r a t e s of the b e e t l e s . T h i s view i s f u r t h e r supported by r e s u l t s on t e n u l i n which had no e f f e c t on b e e t l e s ' s u r v i v a l . Th i s l a c t o n e l a c k s the e x o c y c l i c methylene but possesses the double bond between C2-C3 (Table 31) . Other s t u d i e s concerned wi th the e f f e c t s of t e n u l i n on mammals showed that t h i s s e s q u i t e r p e n e l a c t o n e i s r e l a t i v e l y low in t o x i c i t y when compared to the h i g h l y t o x i c s e s q u i t e r p e n e l a c t o n e , hymenovin, c o n t a i n i n g the e x o c y c l i c methylene ( I v i e et a l , . 1 9 7 5 a , b ) . These authors conc luded that the exomethylene on the l a c t o n e r i n g a c c o u n t s , at l e a s t in p a r t , for the mode of a c t i o n of the s e s q u i t e r p e n e l a c t o n e s . They a l s o sugges ted , however, that the t o x i c i t y of t e n u l i n , which was observed on ly at ext remely h igh c o n c e n t r a t i o n s of t h i s compound, may be p a r t l y a t t r i b u t a l to the Q,/j-unsaturated ketone mo ie ty . My r e s u l t s are c o n s i s t e n t w i th the view that the i7i- methy lene- l f - l a c tone moiety may be important for the v a r i o u s b i o l o g i c a l a c t i v i t i e s of s e s q u i t e r p e n e l a c t o n e s (Rodr iguez et 157 a l . 1976b).. However, these r e s u l t s seem to c o n t r a s t the e a r l i e r f i n d i n g s that p a r t h e n i n , h e l e n a l i n , and c o r o n o p i l i n form d i f f e r e n t adducts with c y s t e i n e or g l u t a t h i o n e (Pieman et a l . 1979). These s t u d i e s suggested that l a c t o n e s which have more than one a c t i v e s i t e ( p a r t h e n i n and h e l e n a l i n ; see Table 31) shou ld e x h i b i t a s t ronger a c t i v i t y . T h i s was not the c a s e , however, because c o r o n o p i l i n e x h i b i t e d an a c t i v i t y s i m i l a r to that of p a r t h e n i n and h e l e n a l i n . I t i s p o s s i b l e that the fo rmat ion of the adduct through C2 i s l i k e l y to occur on ly in case of t h i o l s of lower mo lecu la r weight such as c y s t e i n e . T h i s i s suppor ted by the f a c t that p a r t h e n i n forms two adducts wi th c y s t e i n e but on ly one adduct wi th the t r i p e p t i d e g l u t a t h i o n e (Pieman et a l . 1979) . I f the t h i o l . con ta in ing compounds i n s i d e the f l o u r b e e t l e s are l a r g e mo lecu les such as p r o t e i n s , the s e s q u i t e r p e n e l a c t o n e s may be bound on ly to the methylene f u n c t i o n of the l a c t o n e . Then t h i s would r e s u l t in a .. s i m i l a r e f f e c t of p a r t h e n i n , h e l e n a l i n , and c o r o n o p i l i n on s u r v i v a l r a t e s of the b e e t l e s . To c o n c l u d e , t h i s study suggests that the (^-methylene-V - l a c t o n e moiety i s p robab ly r e s p o n s i b l e fo r the d e t r i m e n t a l p r o p e r t i e s of s e l e c t e d p s e u d o g u a i a n o l i d e s on s u r v i v a l of f l o u r b e e t l e s . The ft ^ - u n s a t u r a t e d ketone moiety does not seem to c o n t r i b u t e to those p r o p e r t i e s . 158 Fig. 9. Chemical structures of selected pseudoguaianolides tested for thei r effects on survival of f l o u r beetles. Coronopilin Tenulin Fig. 10. Effect of helenalin of various concentrations on s u r v i v a l of Tribolium confusum (mean + SD). 60 50 o OJ E -L 6 + AO 30 > 3 in 20 10 0 Helenalin in food % 8 10 No food VO Effect of coronopilin of various cancentrations on s u r v i v a l of TriboHum confusum (mean + SD). 60 50 AO 30 20 10 0 0 I 1 1 1 1 " 1 1 1 1 i \ • 1 2 3 A 5 6 7 8 9 10 No food Coronopilin in food, % Fig. 12. Effect of parthenin of various concentrations on s u r v i v a l of Tribolium confusum (mean + SD). 60 50 AO h 30 20 10 _1 I I L 1 2 3 4 5 6 7 8 9 10 No food Parthenin in food, % F i g . 13. Effect of tenulin of various concentrations on s u r v i v a l of Tribolium confusum (mean + SD). 60 i 50 a -o AO E 30 a > > to 20 10 J I 1 L _l I I l_ 1 2 3 A 5 6 7 8 9 10 No food to Tenulin in food. % 163 Table 31. Functional groups of pseudoguaianolides which were studied for their effects on survival of f l o u r beetles. Sesquiterpene lactone Functional groups ot-methylene- y-lactone cyclopentenone Effect on beetles Parthenin Helenalin Coronopilin Tenulin + + + + + + + + 164 CHAPTER 4 C r o s s - r e a c t i v i t y between sesqu i terpene l a c t o n e s r e l a t e d to pa r then i n i n parthen in s e n s i t i z e d guinea p i g s Int roduc t i on A l l e r g i c con tac t d e r m a t i t i s , known as de layed h y p e r s e n s i t i v i t y or Type IV c e l l mediated h y p e r s e n s i t i v i t y ( R o i t t 1971), i s produced by c o n t a c t of the s k i n with low mo lecu la r weight c h e m i c a l s . The chemica l (hapten) r e a c t s in the s k i n wi th p r o t e i n ( c a r r i e r ) forming an an t igen which s e n s i t i z e s b lood lymphocytes . Rashes on the s k i n appear a f t e r re -exposure of the s e n s i t i z e d i n d i v i d u a l to the chemica l u s u a l l y w i t h i n 24 to 48 h o u r s . Many members of the Composi tae , i n c l u d i n g common weeds and c u l t i v a t e d p l a n t s , have been r e p o r t e d to cause a l l e r g i c c o n t a c t d e r m a t i t i s ( M i t c h e l l 1969, Evans and Schmidt 1980). Chemica ls r e s p o n s i b l e for t h i s d i s e a s e are s e s q u i t e r p e n e l a c t o n e s which are known to be potent a l l e r g e n s ( M i t c h e l l 1975a). Contact d e r m a t i t i s evoked by P^ hys te rophorus became a widespread d i s e a s e among people in I n d i a , a f t e r the i n t r o d u c t i o n of t h i s s p e c i e s in 1956. P a r t h e n i n i s c o n s i d e r e d to be the major a l l e r g e n of t h i s p l a n t (Lonkar et a l . 1976), but a l s o the r e c e n t l y d i s c o v e r e d t e t r a n e u r i n - A , h y s t e r o p h o r i n (Sohi et a l . 165 1979), and c o r o n o p i l i n (Pieman et a l . 1980) might p lay a r o l e in c a u s i n g Parthenium d e r m a t i t i s . C r o s s - r e a c t i o n s between v a r i o u s a l l e r g e n i c p l a n t s p e c i e s are common. P a t i e n t s who deve lop s e n s i t i v i t y to the l a c t o n e ( s ) of one s p e c i e s f r e q u e n t l y e x h i b i t c r o s s - s e n s i t i v i t y to c h e m i c a l l y r e l a t e d l a c t o n e s of o ther s p e c i e s of Compositae ( M i t c h e l l et a l . 1972, M i t c h e l l 1975a). H i g h l y s e n s i t i z e d i n d i v i d u a l s u s u a l l y show a wider spectrum of c r o s s - s e n s i t i v i t y than do weakly s e n s i t i z e d ones ( M i t c h e l l et a l . ' 1975a, Roed- Petersen and H j o r t h 1976). Only a few s t u d i e s have been conducted on t h i s complex phenomenon and the u n d e r l y i n g mechanism of c r o s s - s e n s i t i v i t y towards v a r i o u s s e s q u i t e r p e n e l a c t o n e s i s unknown. The study of c r o s s - s e n s i t i v i t y i s c o m p l i c a t e d by the f a c t that i n d v i d u a l s s e n s i t i z e d by i d e n t i c a l compound(s) deve lop t h e i r own c h a r a c t e r i s i c p a t t e r n s of c r o s s - sens i t i zat i on (Baer 1954) . On the b a s i s of t e s t s with many s e s q u i t e r p e n e l a c t o n e s , i t has been e s t a b l i s h e d that the presence of an (A -methylene group a t t a c h e d to the f - l a c t o n e r i n g i s a p r e r e q u i s i t e for a l l e r g e n i c a c t i v i t y ( M i t c h e l l and Dupuis 1971, M i t c h e l l et a l . 1975a), wi th the e x c e p t i o n of d e a c e t o x y m a t r i c a r i n where the p o s i t i v e response might have been caused by i m p u r i t i e s (Bleumink et a l . 1976). Hydrogenat ion of the methylene group r e s u l t s in a l o s s of a c t i v i t y ( M i t c h e l l et a l . 1970). Adducts i n v o l v i n g the methylene f u n c t i o n such as the a l a n t o l a c t o n e - a m i no a c i d adducts a l s o showed l o s s of a c t i v i t y (Dupuis et a l . 1974). However, i t appears that the exomethylene on the l a c t o n e r i n g a lone i s not 166 always s u f f i c i e n t to cause- a l l e r g e n i c i t y ( M i t c h e l l et a l . 1970, 1971a,b , M i t c h e l l and Dupuis 1971 , . E p s t e i n et a l . 1980). Negat ive r e s u l t s of pa tch t e s t s to compounds other than s e s q u i t e r p e n e l a c t o n e s which possess an u n s a t u r a t e d methylene group, i n c l u d i n g ^ - m e t h y l e n e - V - b u t y r o l a c t o n e , show, that a l a c t o n e r i n g together with a s e s q u i t e r p e n e a t t a c h e d to i t are immunolog ica l r e q u i s i t e s in a d d i t i o n to a methylene group ( M i t c h e l l et a l . 1972) . However, because a l l known a l l e r g e n s be long to a l l s k e l e t a l c l a s e s of s e s q u i t e r p e n e l a c t o n e s , s t r u c t u r a l d i f f e r e n c e s in a s k e l e t o n between c l a s s e s are not important ( M i t c h e l l et a l . 1970) . Comparison of o ther s t r u c t u r a l groups of s e s q u i t e r p e n e l a c t o n e s (hydroxy l group, a c e t y l a t e d or f o r m y l a t e d hydroxy l groups) and s t e r e o c h e m i s t r y at the l e v e l of attachment of the l a c t o n e r i n g i n d i c a t e tha t none of them i s i n v o l v e d in a c t i v i t y ( M i t c h e l l et a l . 1970). In t h i s study I i n v e s t i g a t e d the r e l a t i o n s h i p between- c r o s s - s e n s i t i v i t y and chemica l s t r u c t u r e of p a r t h e n i n and r e l a t e d s e s q u i t e r p e n e l a c t o n e s . The study was conducted on guinea p i g s s e n s i t i z e d to p a r t h e n i n . Guinea p i g s are e x t e n s i v e l y used in immunology and s t u d i e s of in f lammatory r e a t i o n s s i n c e the p h y s i o l o g i c a l c h a r a c t e r i s t i c s of their , s k i n are s i m i l a r to those of man (Wagner and Manning 1976). 167 Exper imenta l (1) Guinea p i g s e n s i t i z a t i o n The exper iments were c a r r i e d out on s i x a l b i n o female guinea p i g s which were fed wi th Guinea P ig Chow granu les ( P u r i n a , S t . L o u i s , Mo.) and water , supplemented with f r e s h l e t t u c e and c a r r o t . In the beg inn ing of the exper iments the an imals were 5 months o l d , each weighing between 615-933 g . Dur ing the f i r s t attempt at s e n s i t i z a t i o n , 50 p i of 5% p a r t h e n i n s o l u t i o n in 80% e thano l was a p p l i e d d a i l y for 18 days •to the same a r e a , approx imate ly 1.5 cm in d iameter , of the shaved f l ank of each a n i m a l . To ensure a proper a p p l i c a t i o n of the s o l u t i o n to the s k i n , newly grown h a i r s were shaved every 4 days wi th an e l e c t r i c shaver . The f i r s t erythema (+) o c c u r r e d on the 5 t h - 7 t h day of attempted i n d u c t i o n of s e n s i t i z a t i o n . On the 18th day • a l l an imals e x h i b i t e d a s t rong inf lammatory r e a c t i o n (++++). The a p p l i c a t i o n of p a r t h e n i n s o l u t i o n was stopped and the an imals were a l lowed to recover c o m p l e t e l y . A f t e r 22 days the o p p o s i t e f l a n k of a l l an imals were shaved and 5% p a r t h e n i n (80% e t h a n o l ) a p p l i e d . However, none of the an imals responded to p a r t h e n i n (checked a f t e r 24, 48, 72, and 96 h o u r s ) . To s e n s i t i z e the a n i m a l s , the procedure was repeated with p a r t h e n i n s o l u t i o n of a h igher c o n c e n t r a t i o n . To a v o i d the lengthy procedure of shav ing the a n i m a l s , p a r t h e n i n s o l u t i o n was a p p l i e d t h i s time 168 d i r e c t l y on the guinea p i g ears which are almost without h a i r s . F i f t y u l of 10% p a r t h e n i n s o l u t i o n ( i n 80% e t h a n o l ) was a p p l i e d da i l y - for 15 days (except on the 6th and 12th d a y ) . On the 3rd day some an imals deve loped s l i g h t erythema ( + ) at the p l a c e of a p p l i c a t i o n , and on the 6th day four of these an ima ls e x h i b i t e d a s t rong r e a c t i o n (++++). The s e n s i t i z a t i o n was then c o n t i n u e d by s o l u t i o n a p p l i c a t i o n on the o p p o s i t e ear of a i l an imals u n t i l a l l e x h i b i t e d the s t r o n g e s t r e a c t i o n (day 1 5 ) . E igh teen days l a t e r , a f t e r the an imals comp le te l y r e c o v e r e d , a 10% s o l u t i o n of p a r t h e n i n (as above) was a p p l i e d on t h e i r shaved f l a n k s . A l l guinea p i g s responded p o s i t i v e l y 24 or 48 hours a f t e r the a p p l i c a t i o n (Table 32) . The r e a c t i o n of s e n s i t i z e d an imals to p a r t h e n i n was checked aga in a f t e r c r o s s r e a c t i v i t y t e s t s with the same r e s u l t s . (2). Cross reac t i v i t y t e s t s Sesqu i te rpene l a c t o n e s used in t h i s study ( F i g . 14) were o b t a i n e d or prepared as d e s c r i b e d in S e c t i o n s I and I I . C h a l l e n g e s were performed wi th 50ul of 10% s o l u t i o n s of i n d i v i d u a l compounds which were a p p l i e d on the shaved f l a n k s of the a n i m a l s . The r e a c t i o n s were recorded every 24 hours for s e v e r a l days . The i n t e n s i t y of the r e a c t i o n s were e v a l u a t e d a c c o r d i n g to the f o l l o w i n g s c o r i n g symbols: 0 no r e a c t i o n ( + ) s l i g h t erythema + d i s t i n c t spo t ted erythema + + almost c o n f l u e n t erythema d i s t i n c t c o n f l u e n t erythema 169 ++++ in tense c o n f l u e n t erythema, e x u d a t i o n , spread ing i n t o nearby a r e a s . The presence of p o s i t i v e , r e a c t i o n s can induce other f a l s e p o s i t i v e . r e a c t i o n s , s o - c a l l e d "angry back" ( M i t c h e l l 1975b). To a v o i d t h i s "angry back syndrome", I a p p l i e d on ly one compound at a t ime . The next s e s q u i t e r p e n e l a c t o n e was a p p l i e d on the o p p o s i t e f l a n k of. the animal and on ly a f t e r at l e a s t 3 days a f t e r a complete recovery from the p r e v i o u s l y t e s t e d compound. If a s e s q u i t e r p e n e l a c t o n e d i d not cause a p o s i t i v e r e a c t i o n , a new l a c t o n e was a p p l i e d a l s o to the o p p o s i t e f l a n k at l e a s t 4 days l a t e r . A l l 6 an imals were t e s t e d with each compound except fo r hymenin and h y s t e r i n , where a l i m i t e d q u a n t i t y a l lowed for the t e s t i n g of 3 an imals o n l y . 170 R e s u l t s and D i s c u s s i o n The a p p l i c a t i o n of p a r t h e n i n s o l u t i o n a f t e r the s e n s i t i z a t i o n p e r i o d r e s u l t e d in a s t rong r e a c t i o n in a l l an imals (Table 32) . A l l an imals a l s o reac ted to c o r o n o p i l i n , and 2 out of 6 gave a p o s i t i v e r e a c t i o n to damsin (Table 3 2 ) . However, none of the an imals t e s t e d responded to hymenin, h y s t e r i n , d i h y d r o i s o p a r t h e n i n , and t e t r a h y d r o p a r t h e n i n (Table 32) . The r e l a t i o n s h i p between r e a c t i o n of an imals to t e s t e d l a c t o n e s and the presence of v a r i o u s f u n c t i o n a l groups i s summarized in Tab le 33. Hymenin ( F i g . 14) d i f f e r s from p a r t h e n i n on ly in the c o n f i g u r a t i o n of the hydroxy l group on C l . T h i s d i f f e r e n c e in s t e r e o c h e m i s t r y a lone r e s u l t e d in a complete l o s s of r e a c t i v i t y of guinea p igs to hymenin. C o r o n o p i l i n ( F i g . 14) , which d i f f e r s from p a r t h e n i n by the absence of a double bond between C2 and C3, gave p o s i t i v e responses in a l l 6 a n i m a l s , though g e n e r a l l y weaker than p a r t h e n i n (Table 32) . The f a c t that the 6 an imals g r e a t l y d i f f e r e d in t h e i r responses (Tab le 32) i n d i c a t e s tha t the absence of a double bond between C2 and C3 of p a r t h e n i n leads to d i f f e r i n g immunologica l r e a c t i o n s by d i f f e r e n t i n d i v i d u a l s . Damsin ( F i g . 1 4 ) , which d i f f e r s from p a r t h e n i n in two f u n c t i o n a l groups (Table 33) , gave a weak p o s i t i v e r e a c t i o n in 2 a n i m a l s . Thus, compared to c o r o n o p i l i n , the a d d i t i o n a l change on the p a r t h e n i n molecu le (the absence of a h y d r o x y l group on C l ) r e s u l t e d in a f u r t h e r decrease in s e n s i t i v i t y of p a r t h e n i n s e n s i t i z e d guinea p i g s . Another change in a s t r u c t u r e , as seen •171 in h y s t e r i n ( F i g . 14; T a b l e . 3 3 ) , r e s u l t e d in a complete l o s s of s e n s i t i v i t y in the examined a n i m a l s . D i h y d r o i s o p a r t h e n i n and t e t r a h y d r o p a r t h e n in ( F i g . 14) , two hydrogenated produc ts of p a r t h e n i n which c o n t a i n ft-methyl on the ^ - l a c t o n e r i n g , d i d not cause any r e a c t i o n (Table 32) . S ince the an imals d i d not g ive any response to d i h y d r o i s o p a r t h e n i n , w h i c h , possesses a double bond between C7 and C l l , t h i s f u n c t i o n a l group does not seem to p lay any r o l e in c r o s s - s e n s i t i v i t y in p a r t h e n i n - s e n s i t i z e d guinea p i g s . The above r e s u l t s i n d i c a t e that the presence of the (X - m e t h y l e n e - T - l a c t o n e moiety in s e s q u i t e r p e n e l a c t o n e s i s a p r e r e q u i s i t e for c r o s s - s e n s i t i v i t y of p a r t h e n i n s e n s i t i z e d guinea p i g s to the e x a m i n e d . p a r t h e n i n - r e l a t e d compounds. The presence- of t h i s group a lone,• however, i s not s u f f i c i e n t fo r c r o s s - s e n s i t i v i t y (see h y s t e r i n ; Tab les 3 2 , 3 3 ) . T h i s f i n d i n g i s c o n s i s t e n t with M i t c h e l l ' s (1975a) h y p o t h e s i s . o n the r o l e of the exomethylene on the l a c t o n e r i n g in a l l e r g i c c o n t a c t d e r m a t i t i s . On the b a s i s of t h e i r r e s u l t s , E p s t e i n et a l . (1980) conc luded that the presence of the methylene con jugated to the l a c t o n e r i n g does not a lone determine c r o s s - s e n s i t i v i t y or the lack of i t , but that i t i s r a t h e r the presence of v a r i o u s a d d i t i o n a l groups on a s k e l e t o n , which h inder g r e a t l y the r e a c t i v i t y of the e x o c y c l i c methy lene . My s tudy , however, shows that as the number of any s t r u c t u r a l changes ( e i t h e r through the a d d i t i o n , d e l e t i o n , or s h i f t s of groups) in a p a r t h e n i n molecu le was i n c r e a s e d ( p a r t h e n i n - c o r o n o p i l i n - damsin - h y s t e r i n ) , so the ' response of guinea p i g s became weaker (Table 32) . T h i s 172 o b s e r v a t i o n c o u l d be e x p l a i n e d in terms of the immunologica l p r o c e s s e s . I t i s known that i n d i v i d u a l organisms may form s e v e r a l d i f f e r e n t types of a n t i b o d i e s to a g iven an t igen ( R o i t t 1977). D i f f e r e n t types of a n t i b o d i e s present a copy of v a r i o u s p a r t s of a g iven ant igen and hence can f i t on ly these p a r t s ( R o i t t 1977) . T h e r e f o r e , as the number of s t r u c t u r a l changes in an a n t i g e n molecule i n c r e a s e s , the number of d i f f e r e n t types . of a n t i b o d i e s formed in response to a g iven a n t i g e n ( e . g . p a r t h e n i n - p r o t e i n complex) which can f i t a p r o g r e s s i v e l y more d i f f e r e n t a l l e r g e n should become s m a l l e r . Hence the s k i n r e a c t i o n of guinea p i g s becomes weaker. T h i s e x p l a n a t i o n i s a l s o suppor ted by r e s u l t s of Schlewer et a l . (1978) who examined c r o s s - s e n s i t i v i t y between s e s q u i t e r p e n e l a c t o n e s from a l i v e r w o r t ' F r u l l a n ia sp . and s e v e r a l s y n t h e t i c , s t r u c t u r a l l y s i m i l a r l a c t o n e s . . T h e s e authors r e p o r t e d that sk in r e a c t i o n of i n d i v i d u a l s t e s t e d to s y n t h e t i c compounds was weaker and one compound, the C(-methy l e n e - f - b u t y r o l a c t o n e , was u n r e a c t i v e . S ince the s y n t h e t i c compounds t e s t e d possessed the e x o c y c l i c methylene on the l a c t o n e r i n g , the o ther s t r u c t u r a l f e a t u r e s of these l a c t o n e s must have a l s o p layed a r o l e in c r o s s - s e n s i t i v i t y . Stampf et a l . (1978) ob ta ined s i m i l a r r e s u l t s from c r o s s - s e n s i t i v i t y t e s t s between a l a n t o l a c o n e and other s i m i l a r s e s q u i t e r p e n e l a c t o n e s . There are two other p o s s i b l e sources of v a r i a t i o n in response of an i n d i v i d u a l organism to. a g iven a l l e r g e n . F i r s t , s i n c e p a r t h e n i n forms two adducts with c e r t a i n t h i o l s such as c y s t e i n e (Pieman et a l . 1979) and t h e r e f o r e presumably d i f f e r e n t 173 types of a n t i g e n s , t h i s i s l i k e l y to f u r t h e r i n c r e a s e the number of d i f f e r e n t types of a n t i b o d i e s which p resent c o p i e s of v a r i o u s p a r t s of a hapten ( p a r t h e n i n ) - c a r r i e r ( t h i o l - p r o t e i n ) complex. If the c h e m i s t r y of i n d i v i d u a l s in terms of these t h i o l - p r o t e i n s d i f f e r , then t h i s i s l i k e l y to cause a great degree of v a r i a t i o n in a number of formed a n t i b o d i e s . Second, in a g iven i n d i v i d u a l , p a r t h e n i n might form a number of a n t i g e n s with d i f f e r e n t p r o t e i n s c o n t a i n i n g t h i o l g roups . The number of a n t i g e n s might f u r t h e r i n c r e a s e through v a r i o u s combinat ions of p a r t h e n i n wi th v a r i o u s c a r r i e r s in the case of a b i a d d u c t . These f a c t o r s r e q u i r e f u r t h e r i n v e s t i g a t i o n . A l s o changes in s t e r e o c h e m i s t r y of the p a r t h e n i n molecu le can l ead to a complete l o s s of c r o s s - r e a c t i v i t y , as in the case of hymenin (Table 3 2 ) . T h i s i s in agreement wi th r e s u l t s of t e s t s on p a r t h e n i n s e n s i t i z e d humans (Subba Rao et a l . 1978) and one i n s t a n c e of a p a t i e n t who responded p o s i t i v e l y to hymenin but not to p a r t h e n i n (Rodr iguez et a l . . 1977 ). T h i s d i f f e r e n t i a l immunolog ica l a c t i v i t y between two d i a s t e r e o i s o m e r s i s not u n u s u a l . I t has been a l s o documented for d - u s n i c a c i d which gave p o s i t i v e r e a c t i o n in 6 p a t i e n t s , whi le 1-usnic a c i d gave n e g a t i v e r e a c t i o n s in patch t e s t s fo r a l l e r g e n i c i t y ( M i t c h e l l 1966). The s t e r e o i s o m e r i c s p e c i f i c i t y in a l l e r g i c c o n t a c t d e r m a t i t i s can be e x p l a i n by the f a c t that whi le the exomethylene on the l a c t o n e r i n g appears to be necessary fo r combining wi th s k i n p r o t e i n s to produce a l l e r g e n s , the remain ing pa r t of the p a r t h e n i n molecu le may determine the s p e c i f i c 174 r e c o g n i t i o n by a n t i b o d i e s . Thus a l t e r a t i o n s in s t e r e o c h e m i s t r y may r e s u l t in a l o s s of a c t i v i t y to an o therwise c h e m i c a l l y i d e n t i c a l molecule (Subba Rao et a l . 1978) . To c o n c l u d e , r e s u l t s of t h i s study support the g e n e r a l idea of the importance of the # - m e t h y l e n e - f - l a c t o n e moiety in a l l e r g e n i c i t y . In a d d i t i o n , t h i s study a l s o suggests that any changes in the s t r u c t u r e of p a r t h e n i n appear to have a d d i t i v e e f f e c t s of weakening of the a l l e r g e n i c response , presumably because of a s m a l l e r number of d i f f e r e n t types of a n t i b o d i e s i n v o l v e d . 175 Fig. 14. Chemical structures of selected pseudoguaianolides used for cro s s - r e a c t i v i t y tests. Damsin CH 2OH AcO o Hysterin Dihydroisoparthenin Tetrahydroparthenin Table 32. Cross-reactivity between sesquiterpene lactones related to parthenin i n parthenin sensitized guinea pigs. F i f t y u l of 10% ethanolic solutions were applied. Only the strongest reactions observed during 72 hours after applications were recorded. Challenge with Animal Parthenin 'Hymenin Coronopilin Damsin Hysterin Dihydroiso- parthenin Tetrahydro- parthenin A +-H- 0 4+ (+) NT 0 0 B +++ NT (+) 0 0 0 0 C +++ 0 (+) 0 NT 0 o D +++ NT +++ (+) 0 0 0 E +++ 0 ++ 0 NT 0 0 F +++ NT (+) 0 0 0 0 * See text for scoring symbols used for evaluation of the i n t e n s i t y of reactions NT= not tested Table 33. Relationship between cross-reactivity i n parthenin sensitized guinea pigs to parthenin and related sesquiterpene lactones and thei r chemical structure. Functional groups Sesquiterpene a-methylene- cyclo- on C 1 double bond CĤ COO- No. p o s i t i v e lactone y-lactone pentenone ot-OH $-0H orH C7-C11 on C-4 reac t i o n s / t o t a l Parthenin + + + - - - - 6/6 Hymenin + + + - - 0/3 Coronopilin + - + - - - 6/6 Damsin + - - - + - - 2/6 Hysterin + - - - + - + 0/3 Dihydroiso- parthenin - - + - - + - 0/6 Tetrahydro- parthenin - - + - - - - 0/6 178 REFERENCES Anderson , L. A. P. , w. T. de Kock, K. G. R. P a c h l e r . 1967. 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L. M a n s e l l . 1976. B i o c h e m i c a l I n t e r a c t i o n s b e t ween P l a n t s and I n s e c t s . R e c e n t A d v a n c e s i n P h y t o c h e m i s t r y , V o l . 10. A c a d e m i c P r e s s , New Y o r k . Y o s h i o k a , H., T. J . M a b r y , and B. N. Timmermann. 1 9 7 3 . S e s q u i t e r p e n e L a c t o n e s : C h e m i s t r y , NMR, and P l a n t D i s t r i b u t i o n . U n i v e r s i t y o f T o k y o P r e s s . 191 APPENDIX. Chemical structures of sesquiterpene lactones examined i n this study. Germacranolides: 1 Parthenolide 2 Pyrethrosin 3 Mikanolide, dihydro- 4 Chamissonin, 5 Tamaulipin-A 6 Tamaulipin-B diacetyl 7 Eupatoriopicrin 8 Melampodin-A 9 Melampodin-B 192 C H 3 9°2 A x C H 3 »0 - C - C - C H OAcI 0 C H 3 O-CO 10 Enhydrin C 2 H 50 c 0 CHjOH °~CO 11 Cinerenin CH 3 992 ? H / C H 3 .o-c-c-c 0Ac| 0 CH 3 X 0Ac O-CO 12 Melampodinin o-c-c= ,CH3 0 CH3V1 CH20H o-co - 0 - C - C - C H Q II II J 0 CH2 o-co rfCvH3 PAc o-co 13 Melcanthin-B 14 Glaucolide-E 15 Glaucolide-B 0 - C - C = C H n 1 \ 0 CH 3 CH 3 OAc AcO AcO 0 C H 3 1  t 3 o - c - c 16 Marginatin 17 Glaucolide-D 18 Glaucolide-E C H 3 0-C-C=CH n 1 0 CH3 OAc 9H3 n 0 - C - C = C H if 1 0 CH, OAc O-CO 19 Glaucolide-F 20 Glaucolide-G 21 Elephantopin 193 OH I Parthenolide, 9-fl-OH Guaianolides: Cumambrin-A 24 Cumambrin-B Matricarin 30 Matricarin, desacetoxy- 25 Cumambrin-B, dihydro- 31 Grossheimin 194 I v a l i n , pseudo- Eudesmanolides: Alantolactone 34 Alantolactone, tetrahydro- Pulchellin-C 40 01 -Santonin 35 Alantolactone, i s o - 38 P i n n a t i f i d i n HO i Pseudoguaianolides: Parthenin, 51 Hymenin 52 Ambrosin photolytic product 196 CH 2OH 53 Coronopilin 54 Damsin AcO o 55 Hysterin CH 2OAc OAc U - C 0 HO C H ^ OAc 0 ^ c o 56 Tetraneurin-A 57 Tetraneurin-B 58 Tetraneurin-D CHjOH AcO 59 Tetraneurin-E 0- C B H 2 - OH " 60 Conchosin-A CH 2 0Ac 61 Conchosin-B OAc 62 Confertiflorin, desacetyl- 63 Tenulin 64 Tenulin, iso- 197 

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