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Physiological and chemical activities of parthenin and other sesquiterpene lactones Picman, Anna Krystina 1977

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PHYSIOLOGICAL AND CHEMICAL A C T I V I T I E S OF PARTHENIN AND OTHER SESQUITERPENE LACTONES by ANNA KRYSTINA PICMAN D i p l o m a , C h a r l e s * U n i v e r s i t y i n P r a g u e , C z e c h o s l o v a k i a , 1971 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department o f B o t a n y We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA O c t o b e r , 1977 © Anna K r y s t i n a Pieman In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced deg ree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Depar tment o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depar tment o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date Q O ^ h j t t i i ABSTRACT Parthenium hysterophorus i s an important weed i n the Caribe a n and, more r e c e n t l y , i n I n d i a and A u s t r a l i a . The importance of t h i s composite as a medical hazard and a g r i c u l t u r a l pest i s reviewed. The sesqui t e r p e n e l a c t o n e , p a r t h e n i n , which occurs i n the trichomes of l e a v e s and stems, i s r e s p o n s i b l e f o r an epidemie o f 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 i n I n d i a . My study i s concerned with c e r t a i n chemical and b i o l o g i c a l p r o p e r t i e s o f t h i s pseudoguaianolide as w e l l a s those of a number of other s e s q u i t e r p e n e l a c t o n e s . , Adducts o f p a r t h e n i n and c y s t e i n e were prepared and i d e n t i f i e d by i s and NMR spectrometry. Formation o f these adducts was a f f e c t e d by temperature and c y s t e i n e c o n c e n t r a t i o n s . I t was found t h a t both a c t i v e s i t e s of p a r t h e n i n are r e s p o n s i b l e f o r i t s b i o l o g i c a l a c t i v i t i e s . The cyclopentenone moiety appears t o be r e s p o n s i b l e f o r a n t i m i c r o b i a l a c t i v i t y while the cx -methylene- f - l a c t o n e moiety seems to be r e s p o n s i b l e f o r t o x i c i t y and a l l e r g e n i c i t y . Feeding experiments with a l a n t o l a c t o n e showed t h a t t h i s compound i s a fe e d i n g d e t e r r e n t and t h a t i t a l s o has d e t r i m e n t a l e f f e c t s on the f l o u r b e e t l e , T r i b o l i u m confusum-* The a p p l i c a t i o n of p a r t h e n i n on hearts of grasshoppers, Melajaoplus sanauinijges, r e s u l t e d i n i n h i b i t i o n of myocardic a c t i v i t i e s . These f i n d i n g s support the view t h a t p l a n t s have evolved the pr o d u c t i o n of i i i sesguiterpene lactones as a means of t h e i r defense against herbivorous predators. i v TABLE OF CONTENTS PAGE ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i i TABLE OF CONTENTS i v L I S T OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v i i L I S T OF FIGURES v i i i ACKNOWLEDGEMENT X INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . 2 I . C h e m i s t r y o f s e s g u i t e r p e n e l a c t o n e s . . . . . . . . . . . . . . . . . . . 2 I I . D i s t r i b u t i o n o f s e s g u i t e r p e n e l a c t o n e s . . . . . . . . . . . . . . . 2 I I I . B i o l o g i c a l a c t i v i t i e s o f s e s g u i t e r p e n e l a c t o n e s . .... 4 A. 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 ........................ 4 B. C y t o t o x i c and tumor i n h i b i t o r y a c t i v i t y ...........,6 C. A n t i m i c r o b i a l a c t i v i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 D. A n t i f u n g a l a c t i v i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 E. A n t i h e l m i n t h i c a c t i v i t y and c h e m o p r o p h y l a x i s i n s c h i s t o s o m i a s i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 F. I n s e c t f e e d i n g d e t e r r e n t s ........................12 G. T o x i c i t y t o v e r t e b r a t e s . . . . . . . . . . . . . . . . . . . . . . . . . . 12 H. A l l e l o p a t h y . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . . 14 I v * £3E.ik§aiaS kY.§ier0£horus, L . . . . . . . . . . . . . . . . . . . . . . . . . 17 CHEMICAL PART ..... ,19 I n t r o d u c t i o n 19 M a t e r i a l s and Methods 23 I . P l a n t m a t e r i a l 23 I I . I s o l a t i o n o f p a r t h e n i n . . . . . . . . . . . . . . . . . . . . . . . . . . 23 V TABLE OF CONTENTS (cont'd) PAGE I I I . M i c r o b i o l o g i c a l t e s t f o r p h o t o t o x i c i t y of compounds ........ .............................. ,24 IV. T h i n l a y e r chromatography ....................... 25 V. Column chromatography f o r the i s o l a t i o n of the parthenin - c y s t e i n e biadduct .................... 28 VI. .Chemicals and instruments ....................... 28 Experimental and R e s u l t s 30 I. Adducts of p a r t h e n i n and other s e s q u i t e r p e n e l a c t o n e s with c y s t e i n e and g l u t a t h i o n e ........... 30 A. The e f f e c t o f temperature, r a t i o s of r e a c t a n t s , r e a c t i o n time, and pH of medium ... 30 B. I s o l a t i o n o f the mono- and biad d u c t s of p a r t h e n i n and c y s t e i n e 32 C. Parthenin and h e l e n a l i n r e a c t i o n s with reduced g l u t a t h i o n e .......................... 46 D. Reaction o f other s e s q u i t e r p e n e l a c t o n e s with c y s t e i n e .. .......... ........ ...... ...... 47 I I . Chemistry of Parthenium hysterophorus 50 A. D e t e c t i o n of p a r t h e n i n at d i f f e r e n t stages of development of P.. histeroEhorus- ........... 50 B. D e t e c t i o n o f pa r t h e n i n and some phen o l i c a c i d s i n trichomes and stems of £i ky^terj>2k20§ ....-50 C. G l y c o s i d e o f parthenin ....................... 53 D. P o l y a c e t y l e n i c compounds i n P.. hysterophorus ...... .............. ........ . ... 56 D i s c u s s i o n ........ ...... ................ 57 v i TABLE OF CONTESTS (cont'd) PAGE PHYSIOLOGICAL PART .. . . . .... . 66 Introduction .................... .... ...................66 Materials and Methods 70 I. Experimental organisms ...........................,70 II. Chemicals ......... ............................... 70 I I I . Feeding experiments ............................ 71 IV. Heartbeat experiments 72 V. A n t i b i o t i c a c t i v i t y test ......................... 73 Experimental and Results 74 I. Feeding experiments .............................. 74 II . Heartbeat experiments ........................... 74 II I . A n t i b i o t i c a c t i v i t y test .......................89 Discussion ...... ... ... ... ... ........ .. ...... . . . . ........ 92 BIBLIOGRAPHY ..... .... ..... ..... ............. . . . . . . . 9 5 v i i L I ST OF TABLES TABLE PAGE I . E f f e c t o f t e m p e r a t u r e on r e a c t i o n o f p a r t h e n i n and c y s t e i n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1 I I . E f f e c t o f d i f f e r e n t p a r t h e n i n - c y s t e i n e r a t i o s on a d d u c t f o r m a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 33 I I I . S t r u c t u r e and b i o l o g i c a l a c t i v i t i e s of some s e s g u i t e r p e n e l a c t o n e s . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . . 61 IV . Summary o f d a t a o f e x p e r i m e n t on f o o d s e l e c t i o n by T r i b o l i u m confusum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 77 V. E f f e c t s o f p a r t h e n i n on t h e f r e g u e n c y of h e a r t b e a t ....... 79 V I . E f f e c t o f e g u i m o l a r s o l u t i o n s o f SH compounds and p a r t h e n i n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 V I I . A n t i b i o t i c a c t i v i t y o f p a r t h e n i n - c y s t e i n e a d d u c t s a g a i n s t S t a p h y l o c o c c u s a l b u s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 V I I I . A n t i b i o t i c a c t i v i t y o f some s e s g 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 t a p h y l o c o c c u s a l b u s .. . ... .... . . . . . . . . . . . . . . . . . . . . . . . . 90 v i i i L I S T OF FIGURES 1. P r o p o s e d mechanism o f r e a c t i o n o f p a r t h e n i n w i t h c y s t e i n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........ 21 2. TLC o f p a r t h e n i n and h e l e n a l i n a d d u c t s w i t h t h i o l s ....... 27 3. NMR s p e c t r u m o f p a r t h e n i n 35 4. NMR s p e c t r u m o f c y s t e i n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 5. ..NMR s p e c t r u m o f p a r t h e n i n - c y s t e i n e monoadduct ............. 37 6.,NMR s p e c t r u m o f p a r t h e n i n - c y s t e i n e b i a d d u c t . . . . . . . . . . . . . . 38 7. NMR s p e c t r u m o f c y s t i n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 8. IR s p e c t r u m o f p a r t h e n i n .................................... 40 9. IR s p e c t r u m o f c y s t e i n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 10. IR s p e c t r u m o f p a r t h e n i n - c y s t e i n e monoadduct . . . . . . . . . . . . 42 11. IR s p e c t r u m o f p a r t h e n i n - c y s t e i n e b i a d d u c t . . . . . . . . . . . . . . 43 12. IR s p e c t r u m o f c y s t i n e . . . 4 4 13. S e s q u i t e r p e n e l a c t o n e s s t u d i e d f o r r e l a t i o n s h i p s between t h e i r s t r u c t u r e s and b i o l o g i c a l a c t i v i t i e s 48 14. S e e d l i n g s o f P a r t h e n i u m h y . s t e r o e h o r u s 51 15. P a r t h e n i u m h y s t e r o p h o r u s : t r i c h o m e s on stems; s e e d s ..... 52 16. P h e n o l i c a c i d s i d e n t i f i e d i n b o t h t r i c h o s i e and stem e x t r a c t s 54 17. UV s p e c t r a o f t r i c h o m e e x t r a c t , c a f f e i c and c h l o r o g e n i c a c i d 55 18. P r o p o s e d mechanism o f p a r t h e n i n - c y s t e i n e a d d u c t f o r m a t i o n ........ . . . . . . . . . . . . . . . . . . . . . . . .... ............ 59 ix L I S T OF FIGURES ( c o n t ' d ) FIGURE PAGE 19. E f f e c t o f a l a n t o l a c t o n e on t h e s u r v i v a l o f T r i f e o l i u j confusum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 20. E f f e c t o f v a r i o u s c o n c e n t r a t i o n s o f a l a n t o l a c t o n e on c h o i c e o f f o o d by T r i b o l i u m 76 21. E f f e c t o f p a r t h e n i n on t h e d u r a t i o n o f h e a r t b e a t . . . . . . . . 8 0 22. E f f e c t o f g l u t a m i c a c i d and a c e t y l c h o l i n e on t h e f r e g u e n c y o f h e a r t b e a t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 23. E f f e c t o f c y s t e i n e and DTT on t h e f r e g u e n c y o f h e a r t b e a t 83 24. E f f e c t o f t h e monoadduct on t h e f r e q u e n c y o f h e a r t b e a t and c y s t e i n e a p p l i e d on p a r t h e n i n - a r r e s t e d h e a r t ........ 84 25. A n t i b i o t i c a c t i v i t y o f p a r t h e n i n - c y s t e i n e a d d u c t s and r e l a t e d compounds a g a i n s t 5^ a l b u s 88 26. A n t i b i o t i c a c t i v i t y o f some s e s g 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.. a l t j u s . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 91 X ACKNOWLEDGEMENT I an very grateful to Dr. G. H. N. Towers for introducing me into t h i s topic and given i n s p i r a t i o n as well as for his invaluable advice throughout my work and comments on t h i s thesis. I would l i k e to thank Dr., R. H. E l l i o t t for the provision of f a c i l i t i e s , h i s advice, and help f u l suggestions p a r t i c u l a r l y on the physiological part of t h i s study. The assistance and encouragement of Dr. C. K., Wat are greatly appreciated. To the members of my comittee I thank f o r t h e i r h e l p f u l comments on this manuscript. I give my thanks to Elizabeth Graham who kindly helped with microbiological tests and to Pat Jamieson and the Department of Chemistry, 0. B. C., for measurements and help with int e r p r e t a t i o n of the NMR spectra. Also, I would l i k e to thank my husband Jaroslav, for his encouragement and help throughout t h i s study. 1 INTRODUCTION Sesquiterpene lactones are a group of naturally occuring compounds of plant o r i g i n . . I t i s known that many of these substances are active i n a number of ways and these a c t i v i t i e s most l i k e l y result from t h e i r reaction with SH groups of proteins. The pr e v a i l i n g hypothesis concerning the role of sesquiterpene lactones suggests that plants have evolved production of these compounds as a means of t h e i r defense against herbivorous predators. ,The exact mechanism of action of sesquiterpene lactones, however, i s not known. Parthenium hysterophprus i s i n some parts of the world an important pest species that causes serious medical and a g r i c u l t u r a l hazards. These detrimental properties are almost e n t i r e l y caused by the presence of the sesquiterpene lactone, parthenin, i n t h i s plant species. The present study was designed to investigate the mechanism of reaction between parthenin and some other sesquiterpene lactones with two t h i o l s , L-cysteine and reduced glutathione. The second part of t h i s study i s concerned with possible food deterrent properties of selected sesquiterpene lactones and some of th e i r physiological e f f e c t s when absorbed by a herbivore. z L I T E R A T U R E R E V I E W 2o_ Chemistry of -sesqu 1terpene lac tones Sesquiterpene lactones form a group of terpenoids derived from three isoprenoid units attached together via head - to -t a i l condensation and subseguent c y c l i z a t i o n followed by oxidative modifications (Geissman 1973, Herz 1973).Most of the several hundred known sesguiterpene lactones are c l a s s i f i e d on the basis of the i r carbocyclic skeletons as follows: germacranolides (with a ten-membered ri n g ) , eudesmanolides (6/6 - b i c y c l i c compounds), guaianolides, and pseudoguaianolides (5/7 - b i c y c l i c compounds) (Geissman and Crout 1969, loshioka et a l . 1973). an important common feature of sesquiterpene lactones i s a five-membered c\-methylene - J - lactone r i n g which i s oriented i n a l l lactones of known stereochemistry. ,,The lactone r i n g i s c i s or trans fused to the C-6 - C-7 or the C-8 - C-7 position. Basic s k e l e t a l modifications involve the incorporation of an epoxide r i n g , hydroxyls or e s t e r i f i e d hydroxyls, and/or t i g l i c or angelic acid at a variety of positions (Yoshioka et a l 1973). Some sesguiterpene lactones also contain halogens (Siuda and DeBernardis 1973). I I . Distribution of sesquiterpene lactones During the l a s t two decades a rapidly increasing number of sesguiterpene lactones (over 600 up to date) have been is o l a t e d from various plant sources. Generally, an i n d i v i d u a l plant species produces only one ske l e t a l type with i t s variations 3 ( e s t e r i f i c a t i o n and oxidation of the skeleton) but genera with a wide geographical range may form several types of structures (Mabry 1970). The highest concentration of lactones i s generally found i n the flowering heads and leaves (up t o 8 %- of the dry weight) while the stems and roots have no or very l i t t l e sesquiterpene lactones (Rodriguez et a l . 1976 a, 1976 b). However, the i s o l a t i o n of two sesquiterpene lactones from the root bark of Liriodendron t u l i p i f e r a (Magnoliaceae) (Doskotch and El-Feraly 1969) and from the bark of numerous B r a z i l i a n species of Eremanthus (Compositae) (Baker et a l . 1972) have been reported. Most of a l l the known sesquiterpene lactones have been i s o l a t e d from species of the Compositae. Differences in s k e l e t a l types and quantities of sesquiterpene lactones i n different species of t h i s family have been u t i l i z e d in chemotaxonomic studies (Herz 1973, Herout 1973, Geissman and Irwin 1973). Sesquiterpene lactones sporadically occur also i n many other fa m i l i e s of Anqiosperms such as Umbelliferae, Maqnoliaceae, Lauraceae, Winteraceae, I l l i c i a c e a e , Aristolochiaceae, Benispermaceae, Cortiariaceae, and Acanthaceae (Yoshika et a l . 1973). sesquiterpene lactones have also been i s o l a t e d from the liverworts (Hepaticeae)> F r u l l a n i a d i l a t a t a and F A tamarisci (Knoche et a l . 1969), Diplophyllum albicans (Benesova et a l . 1975) , and Porella species {Asakawa et a l . 1976). Also some mushrooms, such as Lactarius vellereus and pergamenus (Maqnusson and Thoren 1973) and L. blennius (Vidari et a l . 1976) , have been reported to contain this type of compound. 4 I I I . B i o l o g i c a l a c t i v i t i e s of segguiterpene lactones &« A2i®E2iS contact dermatitis The present findings on a l l e r g i c contact dermatitis (caused by sesguiterpene lactones) and photodermatitis (caused by polyacetylenic compounds activated by OV light) are dealt with in d e t a i l by Towers et a l . (1977). Most of the sesguiterpene lactones causing a l l e r g i c contact dermatitis have been reported from species of the Compositae. Cases of a l l e r g i c contact dermatitis caused by F r u l l a n i a species (an epiphytic liverwort) have been reported i n fo r e s t workers (Mitchell et a l . 1969, 1970) and the major s e n s i t i z o r s were is o l a t e d and i d e n t i f i e d as sesquiterpene lactones (Knoche et a l . 1969, Perold et a l . 1972, Asakawa et a l . 1976). , Many species of Compositae, including common weeds, have been reported to cause a l l e r g i c contact dermatitis (Mitchell 1969). Chrysanthemum i s one of the most common causes of t h i s kind of dermatitis 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 (e.g. Hausen and Schulz 1973, 1976). Sesquiterpene lactones occuring in plants of t h i s qenus gave strong positive r e s u l t s in the patch t e s t s (Mitchell et a l . 1971 a, Bleumink et a l . 1976). Present findings of continuing research on dermatitis caused by Partheniiim hysterophorus are summarized by Towers et al . ( 1 9 7 7 ) . Parthenium dermatitis has been known in the southern United States for almost h a l f a century and i n India since the introduction of Parthenium there i n 1956. While urbanization and 5 mechanization of farming caused a decline in the occurence of t h i s dermatitis i n North America, i n India the freguency of cases of t h i s a l l e r g y i s increasing to-an-extent'which makes t h i s plant a dangerous medical hazard. Sesguiterpene lactones, mainly parthenin, which are the major allergens of th i s plant, are located i n trichomes (Rodriguez et a l . 1976 a). Patients with developed s e n s i t i v i t y to these allergens freguently evolve cross s e n s i t i v i t y to the lactones of other species of Corapositae (Mitchell 1975). Parthenium dermatitis occurs most freguently i n persons who are in d i r e c t and continuous contact with t h i s plant (e.g. weed p u l l e r s ) . However, airborne dust containing trichomes and dried plant material could also be the cause of dermatitis i n individuals who are not d i r e c t l y exposed to l i v i n g plants. Parthenium dermatitis has been reported much more frequently i n adult males than females and no cases are known i n children before puberty (Towers et a l . 1977). As a r e s u l t of the testing of many plant species and t h e i r sesguiterpene lactones i t has been established that the exocyclic methylene on the /"-lactone i s responsible f o r a l l e r g e n i c i t y (when hydrogenated this group losses i t s a c t i v i t y ) . However, i t appears that t h i s group alone i s not 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 (Mitchell et a l . 1970, 1971 a, 1971 b, Mitchell and Dupuis 1971). In order to study the r e a c t i v i t y of t h i s group Dupuis et al..(1974) car r i e d out the reaction of a sesguiterpene lactone - alantolactone - with some amino acids. It was shown that alantolactone formed adducts with 6 cysteine, h i s t i d i n e , tryptophan, and lys i n e and i n t h i s form i t did not exhibit a l l e r g e n i c i t y . This finding supports the hypothesis that t h i s moiety could conjugate with sulphydryl groups of proteins in skin c e l l s by a Michael - type addition to form complete allergens producing contact dermatitis reactions (Mitchell 1975). I t has also been suggested and l a t e r proved that the c\ ,/3 unsaturated ketone function i n some sesquiterpene lactones can also undergo a Michael - type addition reaction with sulphydryl groups (Lee et a l . 1971, 1977 a). Thus the presence of two active s i t e s (one on the lactone, the second one on the cyclopentenone) i n parthenin and ambrosin could be responsible for the high a l l e r g e n i c a c t i v i t i e s of these lactones (Rodriguez 1975 a) . . B. Cytotoxic and tumor i n h i b i t o r y a j c t i v i t j Sesquiterpene lactones have been of great i n t e r e s t (especially i n the l a s t decade) because many of them exhibit antileukemic and tumor i n h i b i t o r y a c t i v i t y . Sesquiterpene lactones exhibiting these a c t i v i t i e s are qenerally found in species of the Compositae although two active germacranolides ( t u l i p i n o l i d e and costunolide) originate i n Liriodendron t u l i p i f e r a of the flagnoliaceae (Doskotch and El-Feraly 1969). Every year increasing number of new sesquiterpene lactones of plant o r i g i n are found to be antineoplastic agents. These are reviewed by Hartwell and Abbott (1969) and by Rodriguez et a l . (1976 b). Parthenin has been reported to be active against 7 dif f e r e n t tumor systems {Herz 1967, in Hartwell and Abbott 1969). In th e i r review Hartweli and Abbott (1969) have concluded that a l l known active sesquiterpene lactones possess a lactone ring and a l l but one of these are <^ ,/3 unsaturated with the CK -ethylenic linkage exocyclic i n every case. Later i t has been established that the conjugated -methylene group on the y ~ lactone i s an e s s e n t i a l requisite for c y t o t o x i c i t y (Kupchan 1970, Kupchan et a l . 1970, 1971).Changes such as saturation or additions to t h i s methylene group res u l t in the l o s s of cy 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 et a l . 1974, Howie et a l . 1974) . , The presence of a conjugated ester, cyclopentenone, or CA - methylene- 6 -lactone in addition 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 immediate and dir e c t factor responsible for c y t o t o x i c i t y among the sesquiterpene lactones was the introduction of the 0=C-C=CH1 system, whether i t involves the lactone or cyclopentenone. Additional a l k y l a t i n g groups may enhance cyt 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 further studies Lee et a l . (1972, 1974, 1S77 a) and Hall et a l . (1977) concluded that the CX -methylene- f -lactone group i s les s important than the (X ,($ -unsaturated ketone moiety with respect to c y t o t o x i c i t y as well as antimicrobial a c t i v i t y (Lee et a l . 1977 b). In a recent study of Jamieson and coworkers (1976) of bakkenolides from Petasites species, however, bakkenolide-A, a ft-methylene- j-lactone which does not have the 0=C-C=CHi system, i s active against c e l l s derived from human carcinomas similar to that of sesguiterpene lactones possessing 8 the -methylene group. This suggests that probably other s t r u c t u r a l features have to be taken into account in predicting cytotoxic a c t i v i t i e s of sesguiterpene lactones. Chemical and b i o l o g i c a l studies support the view that sesquiterpene lactones i n h i b i t tumor growth by s e l e c t i v e alkyl a t i o n of growth - regulatory b i o l o g i c a l macromolecules such as key enzymes which control 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. generally for t h i o l s over other nucleophiles, b. e s p e c i a l l y f o r p a r t i c u l a r sulphydryl enzymes, and c. s p e c i f i c a l l y f o r p a r t i c u l a r sulphydryl groups within those enzymes (Kupchan op. c i t . ) . This hypothesis i s supported by the observed l o s s of a c t i v i t y of sulphydryl enzymes phosphofructokinase (Hanson et a l . 1970) and glycogen synthase (Smith et a l . 1972) a f t e r reaction with some sesquiterpene lactones known to be tumor i n h i b i t o r s . Recently i t has been reported that helenalin and tenulin s i g n i f i c a n t l y i n h i b i t e d nuclear DNS synthesis and DNA polymerase enzymatic a c t i v i t y in tumor c e l l s and also i n t e r f e r e d with g l y c o l y t i c and mitochondrial energy processes.,(Lee et a l . 1977 a, H a l l et a l . 1977). In v i t r o reactions of these and other cyclopentenone - bearing compounds showed that they do not alkylate purine bases of nucleic acids but undergo addition with sulphydryl groups (L- cysteine, glutathione, sulphydryl enzymes). Thus i t has been concluded 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 metabolism with these lactones i s by a Michael - type addition reaction with the a v a i l a b l e -SH groups (of the enzymes) i n the tumor c e l l s but the possible 9 reaction of sesquiterpene lactone metabolite<s) with purines i s not excluded. C. Antimicrobial, a c t i v i t y During the search for antimicrobial agents from higher plants i t was found that many plants possess t h i s a c t i v i t y and that i s caused by compounds of various structures (Hitcher 1972, 1975). Vicfakanova et a l . (1971) tested extracts of fourty-seven species of plants containing sesquiterpene lactones and also fourteen i s o l a t e d sesquiterpene lactones against seven d i f f e r e n t microorqanisms and found seven preparations strongly active. These preparations exhibited the hiqhest degree of a c t i v i t y against Entamoeba h i s t o l y t i c a and Trichomonas vaginali s and weaker a c t i v i t y against Hycobacterium tuberculosis , Staphylococcus aureus, and Escherichia £Oli. Cnicus benedietus exhibits antimicrobial a c t i v i t y because of the presence of an unsaturated sesquiterpene lactone, c n i c i n , and some polyacetylenes (Vanhaelen-Fastre 196 8, 1972). It has been observed that two germacranolides, mikanolide and dihydromikanolide from Mikania monagasensis- (Compositae) i n h i b i t qrowth of Staphylococcus aureus and Candida albicans (Harthur et a l . 1975). Two pseudoquaianolides , hymenovin and t e n u l i n , were tested for t h e i r e f f e c t on qrowth of Bacillus thurinqiensis and shown to have antimicrobial a c t i v i t y with tenulin being considerably 10 more active than hymenovin. An i n t e r e s t i n g r e s u l t from t h i s study was the finding of a mutagenic effect of hymenovin (but not tenulin) on the bacterium (Norman et a l . 1976). Investigations of the relationships between the structures of sesquiterpene lactones and t h e i r a c t i v i t y reveal that compounds with an CA , -tmsubstituted cyclopentenone r i n g exhibit cytotoxic as well as antimicrobial a c t i v i t y {Lee et a l . 1974)., Recently, Lee and coworkers (1977 b) tested t h i r t y - s i x sesguiterpene lactones and related compounds for t h e i r antimicrobial a c t i v i t y against six s t r a i n s of bacteria. The tested compounds were active exclusively against Gram positive bacteria (Staphylococcus aureus and B a c i l l u s s u b t i l i s ) . , a l l of them were i n a c t i v e against Gram negative bacteria and also (except for f i v e compounds) against C.albicans (yeast). I t was demonstrated that the cyclopentenone bearing sesguiterpene lactones react with sulphydryl bionucleophiles, e.g. cysteine, (Lee et a l . 1977 a, H a l l et a l . 1977) in v i t r o which may account for t h e i r antimicrobial action (Lee et a l . 1977 b). S i g n i f i c a n t antimicrobial a c t i v i t y was found to be independent of an C* -methylene -J- lactone moiety. E s t e r i f i c a t i o n and epoxidation of hydroxyl groups enhanced the a c t i v i t y . D• Antifungal a c t i v i t y Alantolactone and isoalantolactone have been reported to i n h i b i t growth of pathogenic fungi Trichophyton cjzsseum , T^ . acuminatum, Epidermophyton Wolf - Kaufmann - P r i e s t l y (Olechnowicz - Stepien and Stepien 1963). 11 The e f f e c t of parthenin (from P. h ys te r o ph orus-) on certain phases of fungal growth has been observed (Char and Shankarabhat 1975). Parthenin i n h i b i t e d sporangial germination and zoospore mo t i l i t y i n Sclerospora graminicola but did not exhibit 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 g i l l u s f l a y u s at the same or greater concentrations. E» MtikSil ia^lliS a c t i v i t y and chemoprophylaxis i n schistosomiasis It has been reported that alantolactone (eudesmanolide) which possesses a strong s e n s i t i z o r i n human a l l e r g i c contact dermatitis also possesses antihelminthic a c t i v i t y against I a s c i o i a hepatica (Kim et a l . 1961). I t has been used as a vermifuge (Dupuis et a l . 1974). The wood o i l s of the common b r a z i l i a n trees , Eremanthus elaeagnus , Vanillosmopsis erythropappa . and Hoguinea yelutjna i n h i b i t penetration of cercariae of the trematode Sehistisoma mansoni into the animal skin (Baker et a l . 1972). Active compounds in these o i l s were the sesquiterpene lactones eremanthin, costunolide, and <X-cyclocostunolide. More recently goyazenolide, from Eremanthus goyazensis . was also shown to have the same properties (Vichnewski et a l . , 1976). Because dihydro-(3-cyclocostunolide which possesses a saturated lactone function was i n a c t i v e , i t suggests that the exocyclic methylene on the Y'-lactone ring i s responsible for t h i s a c t i v i t y . I t i s believed that the i n h i b i t i o n either of the penetration enzymes or of an enzyme within the cercaria or newly formed schistosomulum may be involved., 12 Insggt feeding deterrents The function of secondary plant substances i n the s u r v i v a l of plant species was recognized and elucidated by Fraenkel (1959). Since that time many chemicals in plants have been shown to play a very important r o l e in highly s p e c i f i c plant-insect relationships. Evidence that sesquiterpene lactones provide resistance to insect feeding has been experimentally tested on three species of Vernonia (Compositae) (Burnett 1974, Burnett et a l . 1974). Feeding experiments were conducted with larvae of six species of phytophagous Lepidoptera using an agar medium containing the sesquiterpene lactone, glaucolide-A (major lactone of Vernonia species) and with a Sernonia leaf powder. Larval feeding was greatly reduced being inversely proportional to the concentrations of glaucolide-A i n the medium. Larvae were also feeding on a medium with Vernonia f l a c c i d i f o l i a (lacking glaucolide-A) in preference to the media with Vernonia species that contained t h i s sesquiterpene lactone. From these results and also from the observation that the ingestion of t h i s compound had a detrimental e f f e c t on the growth and development of the insect i t has been concluded that glaucolide-A i s an e f f e c t i v e defensive compound to many insect species. G- Toxicity, to vertebrates There are several reports in the l i t e r a t u r e on the poisonous action of some sesquiterpene lactones on mammals. Poisoning of c a t t l e by feeding on members of the Compositae has 13 been reported by Sperry et a l . (1964), Kingsbury (1964), and Schmuz et a l . (1968). I v i e et a l . (1975 a) showed that hymenovin, the major sesguiterpene lactone of Hymenoxys odorata , i s a chemical responsible for t o x i c i t y of t h i s plant to grazing li v e s t o c k (affecting mainly sheep and goats) causing high losses of animals in Texas. Also the extreme t o x i c i t y to c a t t l e , sheep, and goats of Helenium microcephalum has been found to be caused by high levels of the sesguiterpene lactone, helenalin, present i n t h i s species ( S i t z e l et a l . 1976, i n Towers et a l . 1977 ). Vermeerin, a sesguiterpene dilactone of the ph y s i o l o g i c a l l y active vermeeric acid, causes vomiting disease i n sheep grazing on Geigeria species i n South A f r i c a (Anderson et a l . 1967). This sesguiterpene dilactone has been also found i n Hymenoxys ri c h a r d s o n i i . another plant of the American Southwest, which i s poisonous 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 plants, which contain sesguiterpene lactones, are eaten by dairy c a t t l e , their milk tastes b i t t e r (Herzer 1942). It has been established (by o r a l administration to a l a c t a t i n g cow- Ivi e et a l . 1975 b) that tenulin (the major sesguiterpene lactone of Helenium amarum) i s the active ingredient producing the b i t t e r taste of the milk. When Parthenium hysterophorus was fed to c a t t l e i n India i t was found to cause i l l n e s s or death of the animals (Narasimhan, T. R. , Ananth, H., Babu, E. R., Mangala, A., and Subba Rao, P. V. - unpublished r e s u l t s ) . The main histopathological changes were i n the l i v e r , kidneys, and intestines and are s i m i l a r to 14 the changes caused by hymenovin (contained in Hyraenoxys odorata) reported by I v i e et a l . (1975 a). The symptoms are most probably caused by parthenin. I t s t i l l remains to be investigated whether P.hystgrophorus poses a hazard to l i v e s t o c k grazing i n India on waste lands and f i e l d s infested with t h i s weed and whether parthenin, ingested by these animals, enters th e i r milk and then becomes hazardous to humans (Towers et a l . 1977). I v i e et a l . , (1975 a) suggested that the t o x i c i t y of sesquiterpene lactones (based on the known antimicrobial a c t i v i t i e s of these compounds) i s a r e s u l t of alterations i n the microbial composition of the rumen of animals thus a f f e c t i n g v i t a l metabolic functions.. This needs further detailed investigation. H» Allelopathy Allelopathy i s a s p e c i f i c mechanism evolved by some plant species that produce various types of phytotoxic chemicals reducing development of competitors. Many s t r u c t u r a l l y d i f f e r e n t compounds (including sesquiterpene lactones) of plant o r i g i n have been found to have an a b i l i t y to regulate the growth of other plants (Gross 1975). Parthenium hysterophorus i s a hazardous, widespread weed that i n f e s t s a g r i c u l t u r a l lands i n certain parts of India where i t causes a serious reduction of crops of almost a l l economically important plants and thus poses an a g r i c u l t u r a l hazard. The growth and y i e l d of several crop plants were considerably affected when they were grown i n s o i l containing 15 dried root and l e a f materials of t h i s weed (Kanchan and Jayachandra 1976). The same authors observed that treatment with dried plant material or aqueous leachates from roots of P.hysterophorus caused a marked suppression i n the qrowth and colonization of Rhizobia in leguminous plants. , Parthenin and plant extracts of p£Mslero2hoias exhibited i n h i b i t i o n of seed germination and growth of seedlings of JEkaseoljis vulgaris (Garciduenas et a l . 1972) and of wheat and r a g i (gleusjne cpracana) (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 water extracts showed i n h i b i t o r y a c t i v i t y . On the bases of t h i s and other data i t has been suggested that P. h ysterophorus contains a complex of i n h i b i t o r s with parthenin and some phenolic acids as the prominent constituents (Kanchan 1975, Kanchan and Jayachandra 1976). This i s also supported by the higher a c t i v i t y of a methanol extract °f Pft feysterophorns versus parthenin alone in the work of Garciduenas et a l . (1972). P. hysterophorus also produces large quantities of pollen which, when carried away and deposited on f l o r a l parts, cause the i n h i b i t i o n of production of f r u i t s and grain. This has been observed under natural and experimental conditions (Kanchan and Jayachandra 1976) but the compounds responsible f o r t h i s action are not known. , Dalvi and coworkers (1971) demonstrated the phytotoxic action of alantolactone by i t s i n h i b i t o r y e f f e c t on seed germination, seedling growth, and a rate of r e s p i r a t i o n of Phaseolus mnngo. The authors suggested that alantolactone 16 i n h i b i t s the enzymes associated with the degradation of starch (amylases) and proteins (proteases) as well as with the synthesis of new proteins and nucleic acids. , Further, vernolepin (a tumor i n h i b i t o r from Vernonia hymenolepis) i n h i b i t s the extension growth of wheat c o l e o p t i l e sections (Sequiera et a l . 1968). Auxin has been found to reduce the i n h i b i t o r y e f f e c t of vernolepin but there i s no evidence available for competitive interactions between these two compounds. Retardation of germination and growth of three species of grasses grown in the greenhouse affected by l i t t e r of artemisia tridentata in the s o i l has been reported by Schlatterer and Tisdale (1969). Sesguiterpene lactones obtained from t h i s and other species of Artemisia (arbusculin-A, a c h i l l i n , desacetoxymatricarin, v i s c i d u l i n - B and -C) i n h i b i t e d the growth but stimulated the r e s p i r a t i o n of Cucumis sativus (McCahon et a l . 1973). The presence of physiologically active or phytotoxic chemicals i n Artemisia could explain the widespread d i s t r i b u t i o n of t h i s plant species. Sesguiterpenoids with an exomethylene group conjugated to the -lactone carbonyl (heliangine, helianginol, pyrethrosin, and cyclopyrethrosin acetate) found i n Helianthus tubergsus> i n h i b i t e d the elongation of Avena c o l e o p t i l e sections and promoted adventitious root formation of Phaseolus mango cuttings (Shibaoka et a l . 1967)., These unsaturated lactones formed adducts with cysteine (via -SH group and the exomethylene group) and the adducts were in a c t i v e as were derivatives of these 17 compounds in which the methylene function was reduced to a methyl group. This suggests that the exomethylene group cn the X-lactone may be responsible for regulatory a c t i v i t y of plant growth (Gross 1975) . IV. Parthen!um h y s t e r o p h o r u s L . A detailed study of the genus Parthenium has been conducted by Rollins {1950). The genus Parthenium, included in the family Compositae, the t r i b e Heliantheae, subtribe Ambrosiinae, has four sections or subgenera. The section ftrgyrochaetae with P.hysterophorns i s composed of nine herbaceous taxa distributed in western Texas, northern Mexico, and South America. Leaves of P.hysterophorus are c h a r a c t e r i s t i c a l l y divided and covered on both sides with trichomes. The herbaceous stem, which also has trichomes, persists usually for one growing season but the roots can p e r s i s t f o r a t l e a s t three years and produce new shoots i f the plants are continuously cut back. The flowering head i s composed of f i v e p i s t i l l a t e ray f l o r e t s (each with two s t e r i l e d i s k - f l o r e t s ) and about forty staainate f l o r e t s . About 2,000 mature f r u i t s (achenes) are produced per plant. P o l l i n a t i o n i s probably by wind although i n s e c t s v i s i t i n g the flowering heads have been observed. The chromosome number 2n=34 i s probably the same i n a l l populations (Towers et a l . 1977) . Present knowledge of the biology and chemistry of Parthenium p a r t i c u l a r l y with regard to i t s importance as an a g r i c u l t u r a l and a medical hazard in India has been reviewed by 18 Towers and coworkers (1977). P.hysterophorus i s an abundant weed of the Caribbean is l a n d s , the Southern U.S., Mexico, some parts of Argentina and B o l i v i a . Within the l a s t hundred years i t has been introduced also to A f r i c a , Australia, and Southeastern Asia with an alarming rate of spreading p a r t i c u l a r l y i n India in the l a s t twenty years., Chemical analysis of plants of t h i s species from d i f f e r e n t l o c a l i t i e s showed the presence of two d i f f e r e n t major sesquiterpene lactones: parthenin in plants from U.S., Mexico, Cuba, Trinidad, and India; hymenin i n plants from southern B o l i v i a , c e n t ral Argentina and some populations from Texas (Rodriguez 1975 b). The structure of parthenin* i s o l a t e d from P.hysterophorus i n 1959 (Herz and Watanabe) , was determined to be that of a pseudoguaianolide (Herz et a l . 1962). Hymenin, the C-1 hydroxystereomer of parthenin, 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 s a l s o l a (Toribio and Geissman 1968) and l a t e r from P.hysterophorus (Rodriguez 1975 b). B i o l o g i c a l a c t i v i t i e s of P.hysterophorus , mainly due to the presence of parthenin, are dealt with i n the section e n t i t l e d B i o l o g i c a l A c t i v i t i e s of Sesguitrpene Lactones. if C H E M I C A L P A R T 19& Introdaction Kupchan (1970) and Kupchan et a l . (1971) focused t h e i r attention on the importance of the conjugated tx. -methylene - y -lactone moiety for the b i o l o g i c a l a c t i v i t y (especially cytotoxicity) of sesquiterpene lactones. The 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 adducts of L-cysteine and sesquiterpene lactones (Kupchan et a l . 1970, Dupuis et a l . 1974) and the i n h i b i t i o n of a c t i v i t y of phosphofructokinase and glycogen synthase (sulfhydryl-bearing enzymes) (Hanson et a l . 1970, Smith et a l . 1972) demonstrated that compounds containing an exocyclic methylene group on a ^-lactone ring attack b i o l o g i c a l nucleophiles by rapid Michael-type addition. Also the c l i n i c a l t e s t i n g of a number of sesquiterpene lactones f o r a l l e r q i c contact dermatitis showed that a l l active lactones possess an CX-methylene group on a ^-lactone r i n g . Thus t h i s group seems to be necessary to cause a positive patch test response (Mitchell et a l . 1970, Mitchell and Dupuis 1971)., Studies of the r e l a t i o n s h i p s between the structure and cytotoxic a c t i v i t y of sesquiterpene lactones have established that one of the s t r u c t u r a l requirements for s i g n i f i c a n t cytotoxic antitumor a c t i v i t y i s that the 0=C-C=CH2 moiety be part of an ester or part of a ketone or lactone (Lee et a l . 1971). Lee et a l . (1972, 1974, 1977 a, 1977 b) and H a l l et a l . (1977) suggested that t h i s system i n a ketone unit such as a (b -unsubstituted cyclopentenone moiety might act as an e s s e n t i a l 20 a l k y l a t i n g center for the maintenance of antimicrobial a c t i v i t y against gram positive bacteria and for the maintenance of the high l e v e l of c y t o t o x i c i t y of these compounds. It has also been proposed recently that i n some sesquiterpene lactones both s i t e s (C-2 and C-13) may be responsible for b i o l o g i c a l a c t i v i t i e s of some sesquiterpene lactones. Rodriguez (1975 a) has suggested that the t o x i c i t y of parthenin and ambrosia both of which are very active allergens i s due to the presence of a C-13 exocyclic methylene as well as a C2 - C3 endocyclic double bond a l l y l i c to the C4 carbonyl group. Such active s i t e s may form a Michael-type addition at both C-13 and C-2 positions by the addition of a nucleophile (e.g. skin protein). The proposed mechanism of t h i s reaction i s shown on Fig. 1. The present study has been i n i t i a t e d to investigate the following problems: 1. Testing of the p o s s i b i l i t y of the presence of two active s i t e s in parthenin by i t s reaction with cysteine. 2. The 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 parthenin - cysteine adducts. 3. The investigation of the nature and c h a r a c t e r i s t i c s of the parthenin - cysteine reaction (effects of temperature, r a t i o of both reactants, and pH of reacting medium). 4. Study of relationships between some other sesguiterpene lactones and cysteine with regard to t h e i r structures. 5. Additional testing of the reaction of parthenin with a sulphydryl peptide, i . e . glutathione. Fig. 1. Proposed mechanism of reaction of parthenin with cysteine (from Rodriguez 1975 a). 22 Only a few studies have been conducted to investigate the synthesis and d i s t r i b u t i o n of parthenin and other p o t e n t i a l l y b i o l o g i c a l l y active chemical compounds i n Parthenium hysterophorus (Kanchan 1975, Rodriguez et a l . 1976 a). The present study i s intended to provide some additional information on; 1. the synthesis of parthenin i n plants of different ages. 2. the d i s t r i b u t i o n of parthenin and some phenolic compounds i n c e r t a i n parts of the plant. 3. the p o s s i b i l i t y of the presence of parthenin in a g l y c o s i d i c form that would reduce autotoxicity ( t o x i c i t y to other plants has been reported by e.g. Kanchan 1975). Further, since i t has been reported that many species of the Compositae family that contain sesquiterpene lactones responsible f o r a l l e r g i c contact dermatitis, also exhibit phototoxic a c t i v i t y which i s caused by polyacetylenic compounds (Towers 1977), the p o s s i b i l i t y of the presence of photoactive polyacetylenic compounds in Parthenium hysterophorus requires investigation. This study should also provide a test for such a p o s s i b i l i t y . , 23 Materials and Methods I. Plant material Plants used f o r t h i s study were grown from seeds obtained from a Texas population of Parthenium hysterophprus . To fin d the best method for germination seeds were placed i n shady or sunny locations in p e t r i dishes with s o i l , sand, cotton, and f i l t e r paper. The best re s u l t s were obtained when seeds were placed in p e t r i dishes with wet s o i l or sand i n a sunny place (no matter at what time of the year). Under these conditions almost a l l seeds (95-99$) germinated in the 7th-20th day af t e r they were seeded. When plants had formed some leaves they were transfered to pots and placed i n an incubator maintained at 30°C during the day and 20°C at night. The length of the l i g h t period was 16 hours. 1 1 • I s o l a t i o n of parthenin Parthenin was isola t e d from p u r i f i e d extracts of Vjt ll2siero£horus (Texas collection) by the method of Rodriguez (1975 a). Leaves of dried P^ hysterophorus were pulverized i n a blender and chloroform was added to cover the powder. The mixture was allowed to stand overnight. The chloroform extract was f i l t e r e d and the f i l t r a t e evaporated to dryness i n a rotatory evaporator in vacuo. The tar - l i k e syrup was dissolved 24 i n 958 ethanol, 4% aqueous lead acetate solution was added to pre c i p i t a t e fatty acids, flavonoids, and phenolic acids, and the solution f i l t e r e d through a c e l i t e pad. The f i l t r a t e was concentrated i n vacuo, extracted with chloroform and the extract dried over anhydrous magnesium su l f a t e . The chloroform solution was f i l t e r e d , evaporated to dryness, and t h i s residue dissolved in benzene : acetone (2:1) and p u r i f i e d on a s i l i c a gel column packed i n benzene. The column was eluted with benzene, followed by increasing amounts of acetone. A l l f r a c t i o n s were monitored by TLC. Fractions containing parthenin were combined and partly evaporated.,Parthenin c r y s t a l l i z e d after addition of isopropyl ether and cooling i n a r e f r i g e r a t o r overnight., Purity of parthenin was checked by TLC, and by OV, IH, and HMR spectroscopy, as well as by determination of a melting point. I I I . Microbiological t e s t for phototoxicity- of compounds (according to Daniels,1965) Candida albicans culture { D.B.C. #54) was spread over agar plates of Sabaroud's medium with s t e r i l e cotton swabs. The compounds, plant material, and extracts to be tested were placed on prepared piates using highly phototoxic xanthotoxin as a reference. The plates were incubated at 35° C under U7 l i g h t , duplicate plates being maintained i n the dark. The plates were examined a f t e r 24 hours. Phototoxic compounds (such as xanthotoxin) form a : clear area around the s i t e where they are placed by k i l l i n g C. albicans when incubated under UV l i g h t but not i n the dark.. Plant material used was cut into pieces of about 1 cm (stems) or 1 cm (leaves) . Usually washing with 25 ethanol i s used to s t e r i l i z e the plant material but because polyacetylenic compouds could be removed by ethanol, the tests sere c a r r i e d out with roots washed with water or ethanol, leaves and stems washed with ethanol, water or not washed. Plant material was gently crushed with a glass rod to l e t the chemicals i n the material come i n contact with the culture of Candida albicans. Leaf and flower extracts: Eight grams of fresh leaves were ground i n a mortar with 150 ml of methanol and about 100 ml of water was added. The methanolic extract was f i l t e r e d o f f and the f i l t r a t e , a f t e r reduction i n volume, extracted with 150 ml of petroleum ether. The ether phase was evaporated to approximately f i v e ml and tested for phototoxicity towards Candida. The remaining aqueous methanolic layer was also reduced in volume and tested. In the same way six t y flowers (0.5 g) , that were completely open and covered with pollen, were ground and extracted, and the petroleum ether and methanolic f r a c t i o n s concentrated and tested. For the phototoxicity tests extracts were transfered to discs of f i l t e r paper (5 mm) and these placed on the agar plates with C. albicans. IV. Thin layer \Chromatoara£hy 1. Parthenin detection: Eastman s i l i c a gel plates with solvent systems: (a) benzene : acetone (1:4) (b) benzene : ethyl acetate (7:3) Developed plates were fumed with iodine vapors. Parthenin forms 26 a brown spot with Rf~0.57 i n system (a) and Rf=0.28 i n system (b) . 2. Detection of phenolic compounds: Eastman c e l l u l o s e plates with fluorescent in d i c a t o r : (a) One d i r e c t i o n a l TLG with upper phase of butanol : acetic a c i d : water (4:1:5) i (b) Two d i r e c t i o n a l TLC with upper phase of benzene : ac e t i c acid : water (6:7:3) followed by 2% formic acid in the second d i r e c t i o n . , Developed plates were examined under DV l i g h t and sprayed with 1% f e r r i c chloride (ethanol). 3., Sesquiterpene lactone-cysteine (glutathione) adduct detection: Eastman c e l l u l o s e plates without fluorescent indicator with upper phase of butanol : a c e t i c acid : water (4:1:5) as solvent system. Developed plates were sprayed with 0.25% ninhydrin i n acetone, heated for several minutes at 80° C and then exposed to iodine vapors to i n t e n s i f y the spots and to detect parthenin. Sesquiterpene lactones give brown spots with the highest Rf values. The mono- and biadducts give p o s i t i v e ninhydrin spots (violet color) with Hf=0.55 and Rf-0.19, respectively, L-cysteine forms a brown-yellow spot and glutathione a v i o l e t spot, both with Rf=0. 36. The DL-cystine spot i s pi n k - v i o l e t with Rf=0.13 (Fig. 2). 27 Fig. 2. TLC of parthenin and helenalin adducts with thiols. 1- glutathione, 2- helenalin - glutathione (1 : 4), 3- parthenin - glutathione (1 : 1), 4- parthenin - glutathione (1 : 4), 5- parthenin - L-cysteine (1 : 2) a- cystine, b- biadduct, c- glutathione or cysteine, d- monoadduct 28 v« Column chromatography for the i s o l a t i o n of the parthenin-cysteine biadduct Column chromatography on Sephadex LH-20 or c e l l u l o s e powder with butanol : acetic acid : water (4:1:5) upper phase was found to be suitable for the separation of compounds from the reaction mixture of parthenin and cysteine. However, the evaporation of solvents from pooled f r a c t i o n s on a rotatory evaporator at 40 -50° C took several hours and ended with decomposition of the biadduct. Higher temperatures (70° C) increased decomposition. Cellulose standard powder packed and then eluted with the upper phase of the solvent system butanol : water (4:5) was used for the removal of fast moving compounds. Then the solvent system methanol : water (1:1) could be successfully used to elute the cysteine - parthenin biadduct. V I « Chemicals and instruments 1-cysteine, Dl-cystine, glutathione ( f -L-glutamyl-L-cysteine-glycine) (reduced form) , and xanthotoxin (8-methoxypsoralen) were obtained from Sigma, St. Louis, Ho. Emulsin (2.5 units/mg; 1 unit frees 1 mol of glucose at 37°C) was obtained from ICN Pharmaceuticals, Inc., Cleveland, Ohio. Standard phenolic acids used were obtained as follows: chlorogenic and f e r u l i c acid - Fluka AG, Buchs SG, Switzerland; c a f f e i c acid - Sigma, and p-coumaric acid - A l d r i c h , Chem. Comp., Inc. Pure alantolactone and isoalantolactone were previously separated in our laboratory by Dr. G. Dupuis from commercial "Helenin" ( Sigma) by the method described by Dupuis et a l . (1974). Helenalin, damsin, tenulin, isotenxilin, 29 tetraneurin-D, and hymenin sere provided by Dr. E. Rodriguez. Column chromatography was carried out on Sephadex 1H-20 (Pharmacia Fine Chemicals, Uppsala, Sweden) and on Hhatman ce l l u l o s e standard powder., Belting points were determined on a Thomas - Hoover c a p i l l a r y melting point apparatus. Infrared spectra were recorded i n KBr discs on a Unicam SP. 200 G infrared spectrophotometer. U l t r a v i o l e t spectra were recorded on a Unlearn SP. 800 u l t r a v i o l e t spectrophotometer. Nuclear magnetic resonance spectra were measured i n D2O with DSS (sodium 2,2-dimethyl-2-silapentane-5-sulfonate) as an i n t e r n a l reference (parthenin i n CDCI3 with TMS) on a varian XL-100 spectrometer i n the Department of Chemistry, U.B.C. 30 Experimental and Resnits I. Adducts of parthenin and some other sesguiterpene lactones with cysteine and glutathione A• The e f f e c t of temperature, r a t i o s of reactants, reaction time, and £H of medium a) Effect of temperature Eguimolar agueous solutions of parthenin and cysteine were mixed and allowed to react at di f f e r e n t temperatures: 22°C, 50°C and 80°C. The reactions were checked by TLC at various time i n t e r v a l s and the i n t e n s i t i e s of spots on chromatograms were compared v i s u a l l y (Table I.). The rate of formation of the monoadduct increased with increasing temperature. The amount of the biadduct formed increased with increasing temperature and time. However, at 22° C there appeared to be a decrease in the amount of the biadduct formed with time. These r e s u l t s are i n agreement with estimated quantity of free cysteine and parthenin. Thus when there are high quantities of the monoadduct and also of the biadduct there i s generally no free cysteine and parthenin. Aqueous solutions of cysteine are oxidized to cystine and the amount of t h i s compound also increased. Table 1; E f f e c t of temperature on reaction of parthenin and cysteine (parthenin : cysteine 1 :1) . 22°C 50°C 80°C Adduct Adduct Adduct Time Mono— B i - Cysteine Cystine Parth. Mono- B i - Cysteine Cystine Parth. Mono- B i - Cysteine Cystine Parth. lOmin 4+ 4+ ++++ + + -H-H- + + + +4-H- + - + -30min 4+ ++ +4+ + + • ++++ + + + 44++ + - + l h r ++ + ++ + + -H-H- + + + +44+ ++ - + 2hrs +++ + + + + -H-H- ++ - + +44+ +++ - + 3hrs +4+ + - + - ++++ 4+ - + +44+ +++ - + 4hrs ++++ + - + • - ++++ ++ - + ++++ +4+ - + 20hrs 4+4+ + - + - ++++ ++ - + 44+ +++ + ++ Note: + to 1111 indicates Intensity of spot on chromatogram + indicates barely perceptible spot - no spot 32 b) Effect of changing r a t i o s of cysteine to parthenin. Aqueous mixtures of parthenin and of L-cysteine at concentrations 0.5x,1x, 2x, 3x, and 4x that of parthenin were allowed to react at room temperature for 3, 6, and for 24 hours. The r e s u l t s , summarized in Table I I . , show dependence of adduct formation on the concentration of cysteine. The monoadduct was formed in the highest quantity when cysteine and parthenin were present i n equal amounts. With increasing quantities of cysteine, there was an increase i n the amount of the biadduct formed, and at the same time a decrease i n the amount monoadduct. c) Effect of pH on reaction mixtures. Parthenin and L-cysteine i n r a t i o s of 1:1, 1:2, 1:3, and 1:4 {parthenin:cysteine) were allowed to react in water, aqueous phosphate buffer (0.2H and 0.05M) pH 7.4 or T r i s buffer pH 7.8 at three d i f f e r e n t temperatures (22 , 50 , and 80°C). Ide n t i c a l results were obtained at the three pHs under the d i f f e r e n t sets of conditions. B. Isolation of the mono- and biad ducts of parthenin and cysteine in previous studies phosphate buffer (pH aprox. 7) was used as a medium for reactions of sesquiterpene lactones with t h i o l s { Kupchan et a l . 1970, Dupuis et a l . 1974, Hall et a l . 1977). In the present study the necessity for removal of buffer s a l t s from reaction mixtures i n order to purify the adducts formed, Table 2. Eff e c t of d i f f e r e n t parthenin : cysteine ratios on adduct formation (at 22°C). Ratio P : SH 1 : 0.5 1 : 1 1 : 2 1 : 3 1 : 4 Adduct Adduct Adduct Adduct Adduct Time Mono- B i - SH SS p Mono- B i - SH SS P Mono- B i - SH SS P Mono- B i - SH SS P Mono- B i - SH SS P 3hrs -H- + » + + +++ + + - ++ ++ + + ++ 4-H- ++ + - + +4-H- +++ + -6hrs -H-f+ + + - -H-+ +++ + + ++ +++ + + - + ++++ +++ + -24hrs ++++ + + ' - -t-H- ++f - + + ++++ + + - + +++ ++ + -Note: SH- cysteine SS- cystine p- parthenin 34 resulted i n heavy losses of the adducts and since reactions carried out i n water were not d i f f e r e n t as f a r as yi e l d s were concerned, d i s t i l l e d water medium was used. a) Monoadduct Eguimolar aqueous solutions of parthenin (5.84 mg, representing 0.02 mmol, in 0.58 ml) and L-cysteine (2.42 mg, representing 0.02 mmol, i n 0.24 ml) were mixed and allowed to stand at room temperature. After 6 hours when TLC showed only one fast moving spot with ninhydrin, which was believed to be the monoadduct, the solution was shaken with chloroform to remove remaining parthenin.. The agueous phase, when freeze-dried, yielded 8.2 mg of a snowy white powder which was very soluble i n water, methanol, ethanol, butanol, acetone but insoluble i n chloroform. I t turned brown at 210 - 212° C and decomposed at 247° c . I t was unstable at room temperature and after 48 hours traces of cysteine and cystine and some of the biadduct could be detected by TLC. The NMH spectra of parthenin, cysteine, and the monoadduct are shown i n Pig. 3-5. In the spectrum of the adduct the c h a r a c t e r i s t i c signals f o r H-13 (J=5.63 and & =6.28) of parthenin have disappeared but the signals of H-2 (cf-7.67) and of H-3 (c(=6.17) remain. New signals, not present on the spectrum of pure parthenin are from cysteine Ha (or=3.10) and Hb (cf=4.03). Other add i t i o n a l signals are from small amounts of cystine (NHR in F i g . 7). IB spectra of parthenin, cysteine, monoadduct, and cystine are shown i n F i g . 8-12. The monoadduct exhibits an absorption at 1715 cm"' that represents the cyclopentenone chromophore of parthenin F i g . 3. NMR s p e c t r u m o f p a r t h e n i n . Ln Fig. 4. NMR spectrum of cysteine. *» 1 ' 1 1 1 1 1 1 1 1 1 1 1 1 ' 1 1 1 1 1 1 1 ' • , . , , ' " " T " I I I I I i . i • • i i , i 3 ' i I i i i I ' I i i ' I ' I ' i i ' I ' I ' i' i ' i ' i ' i ' i i V T i i ' I ' I ' i i ' I ' I ' i ' i ' i Y i' i \ ' I ' I ' f+ ! I ••!  I 1 1 1 1 1 1 1 ' 1 1 1 ' 1 1 1 1 1 1 1 1 ' ' , 9 8 7 6 p p m ( ) 5 1 1 1 1 1 1 1 1 1 1 1 1 ' 1 ' I i . . i • , 4 1 2 I I I I I O J O S P i g . 5. NMR s p e c t r u m o f p a r t h e n i n - c y s t e i n e m o n o a d d u c t . F i g . 6. NMR spectrum of parthenin-cysteine biadduct. F i g . 7. NMR s p e c t r u m o f c y s t i n e . 40 Fig. 8. I R spectrum of parthenin. 4i Pig.9. IR spectrum of cysteine. 42 43 Fig.11. IR spectrum of parthenin-cysteine biadduct. 44 F i g .12. IR spectrum o f c y s t i n e . 45 (1718 cm"1 ) but the 1742 cm"1 absorbtion of the 7-lactone ring of parthenin i s found at 1755 c i ' i n the spectrum of the monoadduct. The two smaller peaks of the cyclopentenone chromophore {1595 ca"' ) and the lactone ring {1662 cm"' ) of parthenin that are absent from the monoadduct spectrum are replaced by a broader peak of the carboxyl group of cysteine (1650 - 1580 cm"' ) . The sharp 3420 cm"' hydroxyl peak of parthenin i s replaced by broadened absorption (3300 - 2900cm1 ) c h a r a c t e r i s t i c of an aminoacid residue. The 2560 cm"1 absorption of the SB group of cysteine disappeared from the monoadduct spectrum. b) Biadduct Solution of parthenin (17,5 mg, representing 0.06 mmol, i n 1.75 ml water) and L-cysteine (14.5 mg, representing 0.12 mmol, i n 1.45 ml water) were mixed and allowed to react at room temperature f o r s i x hours and freeze-dried. To 15 mg of the freeze-dried residue 1 ml of butanol and 1 ml of water were added and the mixture shaken f o r one minute..The two layers which separated were checked by TLC. The butanol phase contained most of the monoadduct. The aqueous phase contained some monoadduct, cysteine, cystine, and a compound believed to be the biadduct. The aqueous layer was chromatographed on a column (25x2 cm) as desribed i n Methods. The f r a c t i o n s containing the biadduct were pooled and freeze-dried., To avoid increasing temperature a rotatory evaporator was used to speed up the evaporation of solvents. The biadduct obtained i n t h i s manner was a s l i g h t l y yellow 46 powder, f r e e l y soluble i n water, methanol, and ethanol, but insoluble i n butanol and chloroform. I t decomposed at 220°C. I t was found to be unstable at room temperature, decomposing to the monoadduct, cysteine, and cystine. The MSB spectrum of t h i s rather unstable compound (Fig. 6) shows a disappearance of signals for H-2 ( £ =7.67) and H-3 ( 6 = 6.17), and also for both H-13 { 6 =5.63 and cf=6.28) of parthenin (Fig. 3) . The signals c5=3.10 and =4.03 of cysteine (Fig. 4) are found in areas where parthenin has no signals. Certain peaks are from cystine (Fig. 7) which i s formed by decomposition of t h i s product. The IB spectrum of the biadduct i s shown i n Fig. 11. The 7-lactone (1742 cm-' and 1662 cm' ) and the cyclopentenone moiety (1718 cm"' and 1595 cm"' ) absorptions of parthenin have disappeared and only a s i n g l e peak (1751 cuT1 ) in the biadduct spectrum remains. As i n the spectrum of the monoadduct, broadened absorptions of aminoacid residues (1650 - 1580 cm"' and 3300 - 2900 cm"1 ) are present on the biadduct spectrum. Again, the SH absorption (2560 cm-1 ) of cysteine i s not present on t h i s spectrum., C. Parthenin and helenalin reactions with reduced glutathione Parthenin and helenalin in agueous solutions were allowed to react with eguimolar and f o u r - f o l d guantities of glutathione at rooffl temperature.TLC checks after six hours showed the formation of the monoadducts when eguimolar guantities of glutathione and both sesguiterpene lactones were used. When four-fold guantities of glutathione were used, helenalin formed til a biadduct. Parthenin, however, foraed only a monoadduct during the course of s i x days of reaction time {Fig. 2). D« lS§£ii2S °f other sesquiterpene lactones with, cysteine Because of i n s u f f i c i e n t quantities of available sesquiterpene lactones (Fiq.13) only one reaction with cysteine was ca r r i e d out. Egual amounts of these sesquiterpene lactones were dissolved i n aqueous ethanol. alantolactone and isoalantolactone were dissolved in pure ethanol. L-cysteine, i n four times as much a molar quantity of sesquiterpene lactones, was added to each solution and allowed to react with them at room temperature. The reactions were checked by TLC {3) . A l l sesquiterpene lactones tested formed monoadducts (spots between cysteine and sesquiterpene lactones) and only parthenin, hymenin, and helenalin formed also biadducts (spots between cysteine and cystine)..The r e s u l t s obtained a f t e r four and twenty-four hours were i d e n t i c a l . 48 F i g . 13. Sesquiterpene lactones studied for r e l a t i o n s h i p s between t h e i r structures and b i o l o g i c a l a c t i v i t i e s . Class: Pseudoguaianolides h e l e n a l i n damsin tenulin i s o t e n u l i n 49 F i g . 13. Cont'd Class: Eudesmanolides alantolactone isoalantolactone 50 I I . Chemistry of Parthenium hysterophorus Detection of parthenin at d i f f e r e n t stages of development of P,t. hysterophorus Plant material used for parthenin detection were seeds {Fig. 15 b,c)> whole seedlings(3-5 day old) with cotyledons only (Fig 14 a), 10-15 day old seedlings with f i r s t leaves bearing trichomes (Fig.14 b), and one-month old plants with 3-4 leaves each with trichomes (Fig. 14 c) . Plant materials were extracted either i n t a c t or after crushing with chloroform for 24 hours and the presence or absence of parthenin i n extracts checked by TLC (1). Crushed or intact plant material gave i d e n t i c a l r e s u l t s . Parthenin was found i n seeds and also i n seedlings with at least one true leaf bearing trichomes. Younger seedlings with cotyledons only did not contain parthenin. 8 - D e t e c t i o n of parthenin and so me ph enolic a elds in trichgmes-and - stems of P.yhysterophorus From ten fresh stems (approx. 8 cm long) 10 ag of trichomes (Fig., 15 a) were removed with a spatula and transferred to d i s t i l l e d methanol (10 ml). The stems from which the trichomes had been removed (1 g) were cut in small pieces and s i m i l a r l y extracted with methanol (25 ml) overnight. Both methanolic extracts were concentrated to minimum ivoluaes and checked by TLC (1, 2) for presence of parthenin and other compounds. Parthenin F i g .14. S e e d l i n g s o f P a r t h e n i n u m h y s t e r o p h o r u s ; A - 3-5 day o l d (x4), B- 10-15 day o l d (x3), C - 1 m o n t h o l d (x2). 52 53 was found only i n extracts of trichomes. There were no traces of parthenin i n stems from which the trichomes had been removed. Phenolic compounds found to occur i n larger quantities i n both extracts were: t - c a f f e i c acid { UV-light blue, f e r r i c c hloride-gray-green, Rf=0.79 on TLC 2a) and chlorogenic acid ( OV-light blue, f e r r i c chloride - green-gray, Rf=0.65 on TLC 2a)(Fig. 16)* The presence of these two acids was confirmed also by t h e i r UV spectra (Fig. 17). Other phenolic acids occurring i n much lower quantities which were i d e n t i f i e d i n both extracts were: p-coumaric acid (UV -dark blue, f e r r i c chloride-orange, Rf=0.92 on TLC 2a) and f e r u l i c acid C UV-light blue, f e r r i c chloride -brown, Rf=0.87 on TLC 2a){Fig. 16). c« Glycoside of parthenin Fresh leaves and stems of P. hysterophorus were tested for the possible presence of parthenin i n a glyco s i d i c form using emulsin (fr-glucosidase). Ten grams of fresh leaves and stems were cut with s c i s s o r s to small pieces, covered with hot d i s t i l l e d methanol (150 ml) and heated for about 10 minutes. The extract was then f i l t e r e d o f f and evaporated to dryness on a rotatory evaporator. The residue was dissolved in water (100 ml) and f i l t e r e d . From t h i s aqueous solution parthenin was removed by shaking eight times with 50 ml of chloroform. Each chloroform extract, evaporated to minimum volume, was checked by TLC for the presence of parthenin. When no parthenin was l e f t in the aqueous solution, 40 mg (=100 units) of emulsin were allowed to react with 50 ml of t h i s solution in an incubator (37° C). Another 50 ml of the agueous solution were l e f t i n a 54 F i g . 16. Phenolic acids i d e n t i f i e d i n both trichome and stem extracts. COOH COOH c a f f e i c acid f e r u l i c a c i d p-coumaric ac i d chlorogenic acid Fig.17. UV spectra of: 1- trichome ext r a c t , 2- c a f f e i c acid, 3- chlorogenic a c i d . 56 r e f r i g e r a t o r as a co n t r o l . After 24 hours 200 ml (4x50 ml) of chloroform was shaken with each solution and chloroform parts, after concentrations, checked by TLC ( l a ) . No parthenin was found i n either extract. D. Polyacetylenic compounds i n Pj. hysterophorus Different parts of adult plants were extracted and extracts were tested for the presence of phototoxic polyacetylenic compounds using the microbiological test f o r phototoxicity of Daniels (1965) . Plant material used for the phototoxicity test were leaves (green: from the lowest, medium and the highest part of the plants, yellow: from the bases of the plants, and dry: s t i l l attached to the plants), stems, roots, and flowers. None of plant parts tested, t h e i r extracts nor parthenin i n a c r y s t a l l i n e form were found to have phototoxic properties when tested on C. albicans test. Also OV spectra of the extracts did not show any obvious relationships to spectra of polyacetylenic compounds ( Bohlmann et a l . 1973). Parthenium hysterophorus does not appear to contain phototoxic chemicals or detectable amounts of polyacetylenes. 57 Discussion This study has proved that parthenin forms at l e a s t two adducts with cysteine. These two adducts were i s o l a t e d and i d e n t i f i e d by NMR and IR spectroscopy. One of them, the monoadduct, contains a c y s t e i n y l moiety attached to parthenin through the C-13 exocyclic methylene on the f - l a c t o n e r i n g , while the other one possesses an addit i o n a l molecule of cysteine on the C-2 position. The parthenin-cysteine reaction, tested under d i f f e r e n t temperature conditions, indicated that the formation of the monoadduct i s accelerated by higher temperatures. Also i t appears that the amount of biadduct produced increases with time at 50 and 80°C. However, the reaction carried out at 22°C indicated that the amount of biadduct produced decreased with time. The investigation c a r r i e d out with d i f f e r e n t r a t i o s of parthenin to cysteine c l e a r l y indicated that the highest quantity of monoadduct was formed at equimolar quantities of both reactants. This fact and also the formation of much smaller amounts of the biadduct i n t h i s case suggests that the bond on ^-lactone r i n g of parthenin (C-13) has a much higher a f f i n i t y for cysteine than does the cyclopentenone r i n g of parthenin (C-2). The data further indicate that with increasing surplus of cysteine in the reaction mixture the amount of biadduct formed 58 increases while the quantity of monoadduct decreases. This suggests that after a l l C-13 methylene groups have been saturated {at eguimolar guantities of parthenin and cysteine) addition of free cysteine r e s u l t s in i t s reaction with the other active s i t e at the C-2 position. Hence the amount of biadduct formed increases while that of monoadduct decreases. This indicates that the available guantity of cysteine determines the proportion of formed mono- and biadducts. However, i t i s also l i k e l y that both adducts w i l l decompose i n time since their bonds are not stable and free cysteine tends to become oxidized to i t s more stable form (cystine) which i s not avail a b l e for further reaction. a l l i n d i c a t i o n s from t h i s study are that both parthenin-cysteine adducts are unstable. Support f o r t h i s comes from observations of the p a r t i a l decomposition of the isol a t e d biadduct into monoadduct, free cysteine, cystine, and parthenin. Also the i s o l a t e d monoadduct p a r t i a l l y transfered into biadduct, parthenin, and cysteine and cystine. The pH of the reaction mixture did not influence the reaction between parthenin and cysteine. This indicates that various pH conditions , for example within the digestive t r a c t of animals, should not a f f e c t the parthenin reaction with nucleophiles. The formation of the bond between cysteine and C-13 of parthenin i s evidently the f i r s t step i n parthenin-cysteine reaction (the a f f i n i t y of C-13 appears to be much higher and bonds through i t are more st a b l e ) . The attachment of cysteine on Fig. 18. Proposed 'mechanism of parthenin-cysteine adduct formation. c 60 C-2 position of parthenin seems to be the second step i n t h i s reaction {its a f f i n i t y appears to be lower and the bond le s s stable than that on f-lactone r i n g ) • The proposed mechanism of formation of both adducts i s shown on F i g . 18. The reactions of cysteine with six selected sesguiterpene lactones that contain either an (A -methylene- J - lactone moiety or an unsaturated cyclopentenone r i n g resulted i n the formation of monoadducts {most l i k e l y through C-2 or C-13). Three other lactones containing both active s i t e s formed monoadducts as well as biadducts (Fig. 13, Tab.,III.), according to spectroscopic analysis of the i s o l a t e d adducts of parthenin-cysteine , the monoadducts were formed through C-13 while biadducts through C-13 and C-2. This suggests that the other sesguiterpene lactones that contain the same active s i t e s form s i m i l a r adducts. Kupchan (1970) and Kupchan et a l . (1971) suggested that the (A-methylene- y-lactone moiety i s responsible for the b i o l o g i c a l a c t i v i t i e s ( p a r t i c u l a r l y cytotoxicity) of sesguiterpene lactones, also Mitchell and Dupuis (1971) found that t h i s group was present i n a l l sesguiterpene lactones reported to give a positive patch test (no compound was a l l e r g e n i c i n the absence of t h i s group) . Lee and coworkers (1977 a,b), on the other hand, proposed that the unsubstituted cyclopentenone ring contributes to antimicrobial a c t i v i t i e s of sesguiterpene lactones against gram posit i v e bacteria and that t h i s a c t i v i t y appears to be independent of the presence or absence of the methylene group on the Y ~ l a c t o n e ri n9« To elucidate the relationships between structures and Table 3. Structure and b i o l o g i c a l a c t i v i t i e s os some sesquiterpene lactones. Sesquiterpene a--tnethylene-Adducts of parthenin with * L-cysteine A n t i b i o t i c a c t i v i t y against Allergenicity (Mitchell and lactone - y -lactone cyclopentenone Mono C-13 Mono C-2 Bl c-13 + C-2 S. albus Tox i c i t y Dupuis, 1971) Parhtenin + + + - + + + + 2 Hymenin + + + - + + 0 2 Helenalin + + + - + 0 + Damsin + . - . + - + 0 + Tenulin - + - + - ±5 Isotenulin - + + ± 0 _ Tetraneurin-D + - + - 0 _ Hymenovin + - 0 0 0 + 5 0 Alantolactone + - + - - - +6,6' + Isoalantolactone + - + - - - + 7 Monoadduct P-SH - + + 1 0 Biadduct P-SH - _ 0 0 Footnote to Table 3. P- parthenin, SH- cysteine 0 not tested * monoadducts through C-13 and C-2 had the same Rf on TLC 1- e f f e c t on heartbeat of M. sanguinipes 2- Subba Rao, Towers, and Rodriguez - unpublished r e s u l t s 3- the most ac t i v e from a l l sesquiterpene lactones tested; also very a c t i v e against S_. aureus and B. s u b t i l i s (Lee et a l . 1977 b) 4- act i v e also against B a c i l l u s thuringiensis ( a c t i v i t y of t e n u l i n was twice as great as of hymenovin -Norman et a l . 1976) 4'- not tested against S_. albus 5- of t e n u l i n for hamsters i s 1200 mg/kg; °^ hymenovin f o r hamsters, white mice, and sheep i s 250 mg/kg, 150 mg/kg, and 100 - 150 mg/kg, r e s p e c t i v e l y ( I v i e et a l . 1975 a,b) 6- t o x i c i t y to Tribolium confusum 6'- feeding deterrent to Clethrionomys gapperi (Gapper's red-backed vole) and Schistocerca gregaria (desert locust) (Pieman, unpublished r e s u l t s ) 7- t o x i c i t y to T_. conf usum (Pieman, unpublished r e s u l t s ) 62 b i o l o g i c a l a c t i v i t i e s of sesguiterpene lactones, r e s u l t s from t h i s study (further d e t a i l s on antimicrobial tests and effects of some sesguiterpene lactones on insect are given i n the Physiological Part) and other studies are summarized i n Table I I I . From t h i s table i t appears that antimicrobial a c t i v i t y was exhibited mostly by sesguiterpene lactones possessing the unsaturated cyclopentenone ring which i s i n agreement with Lee's (1977 b) finding. This i s , i n addition, supported by results from the test of mono- and biadducts for their antimicrobial a c t i v i t y . The monoadduct with : a free double bond on the cyclopentenone r i n g exhibited antimicrobial a c t i v i t y , while the biadduct was inac t i v e . The weaker positive response to the monoadduct may be the re s u l t of i t s i n s t a b i l i t y i n solution. On the other hand, a l l e r g e n i c i t y and t o x i c i t y appear to be associated with the presence of the (A-methylene - 7" -lactone moiety. Thus d i f f e r e n t active s i t e s of these compounds may be responsible for d i f f e r e n t types of a c t i v i t i e s against various groups of organisms. These findings could have important medical implications since sesguiterpene lactones that exhibit antimicrobial a c t i v i t y but do not cause a l l e r g e n i c i t y and also are not toxic to humans may be used as a n t i b i o t i c s . There appear to be some exceptions to the proposed scheme, however, as indicated by r e s u l t s of Mi t c h e l l and Dupuis (1971). In t h e i r study some p o t e n t i a l l y active sesguiterpene lactones (possessing the CX-methylene moiety) gave negative re s u l t s i n the patch test f o r a l l e r g e n i c i t y . But, as explained by the authors, these results could had been influenced by the fact 63 that not a l l patients exhibit the same degree of s e n s i t i v i t y to the same lactones and because small numbers of patients (1-6) were tested. Thus i t i s possible that the po t e n t i a l l y active sesguiterpene lactones when tested on a larger group of patients would be found to be al l e r g e n i c to some of them. Individuals vary considerably i n t h e i r s e n s i t i v i t i e s to these compounds. The reasons for t h i s are unknown. Hymenin, the diastereomer of parthenin, contains the oc -methylene -"^-lactone and a double bond on the cyclopentenone r i n g . Like parthenin,it produces mono- and biadducts on reaction with cysteine. Of ten Indian patients e x h i b i t i n g strong p o s i t i v e patch test reactions with parthenin, however, none responded to hymenin (Subba Sao, Towers, Bodriguez, unpublished r e s u l t s ) . These results indicate that possibly i t i s not only the <X -methylene-^-lactone moiety or the unsaturated cyclopentenone r i n g that i s exclusively responsible for the a l l e r g e n i c i t y but also the stereochemistry of the hydroxyl group on C-1. group. I t i s noteworthy that parthenin formed mono- and biadducts with cysteine but only a monoadduct with glutathione. .Helenalin, on the other hand, formed mono- and biadducts with both -cysteine and glutathione (the same results on helenalin were reported by Lee et a l . 1977). At present there i s i n s u f f i c i e n t information to explain the above differences between these lactones and t h i s obviously requires further investigation. U n t i l t h i s study was carried out, there was no information on the presence of parthenin i n Parthenium hysterophorus plants of different ages. It was found that parthenin was not present 64 i n seedlings younger than 10 days and i t s presence i s evidently associated with the development of trichomes. The presence of parthenin exclusively i n trichomes was also demonstrated by the i d e n t i f i c a t i o n of t h i s lactone in trichome extracts and by i t s absence in extracts of stems devoid of trichomes. These r e s u l t s thus confirm conclusions reached by Rodriguez et a l . (1976). The fact that parthenin i s located i n trichomes also explains why the highest quantity of t h i s lactone has been found in leaves (Rodriguez 1975 b). Leaves have more trichomes than do other parts of the plant. The glycosidic form of parthenin was not detected i n methanolic extracts of Parthenium hysterophorus. Plants use glycosylation as a detoxication mechanism for compounds that could be autotoxic to a plant (in a free form - such as phenols) (Pridham 1965). Parthenium hysterophorus most l i k e l y must have developed another way to prevent the autotoxicity by parthenin. The storage of parthenin exclusively i n trichomes may be an e f f i c i e n t way of avoiding autotoxicity., The analysis of stem and trichome extracts provided some additional information on the presence of phenolic compounds. I t was found that c a f f e i c acid> already reported from agueous extracts of Parthenium hysterophorus (Kanchan 1975), was present in both trichomes and stems. Also chlorogenic acid was i d e n t i f i e d i n both extracts for the f i r s t time. F e r u l i c and p-coumaric acids, that had been found i n extracts of whole Parthenium hysterophorus plants (Towers et a l . 1977) , were i d e n t i f i e d i n both trichome and stem extracts i n the present 65 study. No polyacetylenic compounds (that might cause a photodermatitis) were i d e n t i f i e d in the extracts of leaves and flowers of Partheninum hysterophorus. The absence of phototoxic reactions from d i f f e r e n t parts of this species was also demonstrated. These re s u l t s thus support the view that Parthenium dermatitis i s an a l l e r g i c contact dermatitis caused by sesguiterpene lactones and not a photodermatitis. Introduction Endogenous compounds which play no apparent role i n essential metabolic processes of plants are termed secondary plant substances (Czapek 1925, in Mothes 1976). Many substances are known to be insect attractants or repellents or to have a n t i b i o t i c properties. &s early as 1959, Fraenkel suggested that the function of some of these compounds was to provide a defense mechanism against various herbivores such as insects. , Sesquiterpene lactones have not been extensively examined for t h e i r importance in the defense of the plant against herbivores or pathogens. So f a r only one sesguiterpene lactone, glaucolide -A, has been shown to be a feeding deterrent to several lepidopteran species (Burnett 1974, Burnett et a l . 1974). a part of t h i s section i s concerned with the e f f e c t s of a sesquiterpene lactone ~ alantolactone - on the confused f l o u r beetle, frj|>oliag-:-confa-sa§. The major objectives were to f i n d out whether alantolactone functions as a feeding deterrent and whether i t has detrimental e f f e c t s to t h i s insect when absorbed. A second part of t h i s section i s concerned with the physiological e f f e c t s of sesguiterpene lactones once they are absorbed by the herbivore. As outlined previously, the toxic effects of sesquiterpene lactones on various organisms have been attributed to the fact that they conjugate with sulphydryl 67 groups of proteins. In the grasshopper, Somalea s i c r o ^ t e r a , free sulphydryl groups are necessary f o r muscular contractions (Bratkowski et a l . 1972). When the sulphydryl i n h i b i t o r s , N-ethyl maleimide, iodoacetic acid, p- chloromercuribenzoic acid, or raersalyl, are applied to nerve - muscle preparations of the insect, muscular contractions normally induced by the neuro -transmitter, glutamic acid, are blocked. However, the blockage caused by p- chloromercuribenzoic acid i s reversed completely by post-perfusion with d i t h i o t h r e i t o l and the i n i t i a l a c t i v i t y i s restored. Also the prior mixture of sulphydryl i n h i b i t o r s with t h i o l reagents (L- cysteine, reduced glutathione, or DTT) r e s u l t s i n a loss of efficancy on subsequent perfusion (Bratkowski et a l . , op.cit.) The insect heart provides an i d e a l system fo r examining the e f f e c t s of drugs, s a l t s , tissue homogenates, etc. on neuro-muscular function. The semi-isolated or exposed heart preparations are generally used in such studies (Jones 1974). The c i r c u l a t o r y system of insects i s an open one and the c i r c u l a t i o n of haemolymph i s produced by the a c t i v i t y of a dorsal longitudinal vessel comprising a posterior heart and an anterior aorta. The heart i s usually separated from the body cavity by a dorsal diaphragm. Haemolymph i s pumped from the back of the heart forward and out through the aorta. During relaxation blood passes i n t o the heart through valved openings (incurrent ostia) (Chapman 1974). Although the exact mechanism c o n t r o l l i n g heartbeat i s not known, i t has been established that the myogenic contractions of 68 heart muscle are regulated by a neurogenic pacemaker (Krijgsman 1 9 5 2 , Chapman 1 9 7 4 ) . To-date, at least four d i f f e r e n t neuro-transmitters have been implicated: 1 . acetylcholine or acetyl c h o l i n e - l i k e substances; 2 . catecholamines; 3 . 5 -hydroxytryptamine; and 4 . , certai n amino acids (e.g..glutamic acid) (Pitman 1 9 7 1 , Jones 1 9 7 4 ) . acetylcholine has been reported to stimulate the heart i n several insect species (Davey 1 9 6 4 , Jones 1 9 7 4 ) , including Melanoplus d i f f e r e n t i a l l s (Hamilton 1 9 3 9 ) . a t present i t appears that L- glutamate i s the most l i k e l y candidate for the excitatory neuromuscular transmitter substance at somatic muscle junctions and i t may also be involved at some v i s c e r a l muscle junctions (Pitman 1 9 7 1 , McDonald 1975) . a part of t h i s study was designed to investigate the following problems in the grasshopper. Mela noplus sa nq uin ipes: (1) Effects of parthenin on the freguency and duration of heartbeat. (2 ) Effects of parthenin concentration on the heartbeat. ( 3 ) Location of the major s i t e of parthenin action. ( 4 ) R e v e r s i b i l i t y of the blockage of heart a c t i v i t i e s by certain sulphydryl agents. In addition, i t was considered of in t e r e s t to test a hypothesis presented e a r l i e r namely that c e r t a i n s t r u c t u r a l features i n sesguiterpene lactones e.g., cyclopentenone or If -lactone rings, may be responsible f o r s p e c i f i c b i o l o g i c a l a c t i v i t i e s . For example, the parthenin - cysteine monoadduct, with the (X.-methylene function involved, should exhibit 69 antimicrobial a c t i v i t y while the biadduct {with both active s i t e s blocked) should be inactive. I t was proposed to test t h i s using staphylococcus albus. 70 Material and Methods I•• -ES§£imental organisms The insect used i n the feeding experiment was the confused f l o u r beetle, Tribolinm confusum {Coleoptera: Tenebrionidae)., The beetles were maintained on wheat flou r in a glass container covered with f i l t e r paper which was moistened once a week. The insects used i n the heartbeat experiments were of the non-diapause s t r a i n of the migratory grasshopper, Melanoplus sanquinipes {Orthoptera: Acrididae). The grasshoppers were maintained on an a l f a l f a meal mixture ( a l f a l f a , bran, brewer's yeast, corn o i l ) and supplemented dai l y by fresh l e t t u c e . The insects were reared at room temperature and at a 12/12 photoperiod. Both species were obtained from Dr. R. H. E l l i o t t from the Department of Plant Science, U. B. C. Cultures of Escherichia c o l i (O.B.C. #219) , Staphylococcus albus- (O.B.C. #48), and Candida albicans (O.B.C. #54) were obtained from the Department of Microbiology, U.B.C. , I I . Chemicals The sesguiterpene lactones, L-cysteine, DL-cystine, reduced glutathione, and the monoadduct of parthenin and cysteine were obtained or prepared as described e a r l i e r . D i t h i o t h r e i t o l (DTT) (2,3-dihydroxy-1,4-dithiobutane), L-glutamic acid, L - c y s t e i c acid, and acetylcholine chloride were obtained from Sigma 71 Chemical Company. Salts for preparation of Locke's solution were obtained from Fischer, S c i e n t i f i c Co. and glucose from Mallinckrodt, Chem. Works. I I I . Feeding experiments approximately 0 . 3 ml of 95 % ethanolic solutions of alantolactone at d i f f e r e n t concentrations were added to 50 mg samples of f l o u r . after evaporation of the ethanol, the f l o u r was transferred to p e t r i dishes with the bottoms l i n e d with f i l t e r paper. Five beetles were placed i n each dish. A small s t r i p of moistened f i l e r paper was added weekly to maintain proper humidity. The condition of animals was checked every day during a 60 day period. A t o t a l of 60 individuals of Tribolium confusua were offered f l o u r containing alantolactone of diff e r e n t concentrations (0 .2 - 14.0 %) . Two controls, one with f l o u r without a lactone and the other one without any food were also included. To establish whether the beetles could recognize the presence of alantolactone i n t h e i r food, i n d i v i d u a l beetles were offered a choice between normal food and that containing various concentrations of alantolactone (two p i l e s of 50 mg of f l o u r -one with and one without alantolactone - were placed on opposite sides of the p e t r i dish). One animal was placed i n the dish and i t s l ocation with regard to the p i l e s of f l o u r was recorded. Four d i f f e r e n t concentrations of alantolactone (1 - 7 %) were offered simultaneously with normal food to Tribolium. Ten beetles were used for each concentration. The location of 72 beetles was checked every f i v e minutes during the f i r s t two and half hours. Thereafter i t was checked every 12th hour f o r two days. These checks involved examination of the f l o u r every f i v e minutes for one hour. The r e s u l t s were evaluated as the r a t i o of v i s i t s on food with alantolactone to v i s i t s on food without alantolactone. , IV. Heartbeat experiments Fourteen- to twenty- day-old adult male grasshoppers were anaesthetized with carbon dioxide. After the legs were removed, the insect was pinned ventral side up on a paraffin - bottomed pe t r i dish. The body cavity was opened via a longitudinal i n c i s i o n extending from the anus to prothorax and the c u t i c l e pulled aside and pinned to the paraffin. To expose ; the heart, the fat body, trachea and testes were c a r e f u l l y removed and the gut deflected with minute pins. The heart was immediately washed with Locke's solution (after Humason, 1967) and observed for f i v e to ten minutes under a binocular microscope. After a regular heartbeat was established, the freguency was recorded and d i f f e r e n t solutions were applied . Parthenin was dissolved in d i s t i l l e d water or Locke's solution. The remaining compounds (L-cysteine, reduced glutathione, DTT, L-glutamic acid, and acetylcholine) were dissolved i n Locke's solution. unless otherwise stated, 50 u l of each solution were applied to the heart. The e f f e c t s of each compound on the freguency and duration of heartbeat were recorded., 73 v• A n t i b i o t i c a c t i v i t y test The A l l Purpose B a c t e r i a l Medium was used f o r E . c o l i and S.albus and Sabaroud's medium for C.albicans cultures. Each culture was spread over the agar plates with the n u t r i t i o n a l medium with s t e r i l e cotton swabs. The compounds to be tested {in a c r y s t a l l i n e form - 1.5 mg) were placed d i r e c t l y on the plates prepared i n t h i s way. The plates, i n duplicate, were incubated at 37 PC in the dark and examined a f t e r 24 hours i n the case of C.albicans or 48 hours with S.albus and E^c.oli. A n t i b i o t i c a c t i v i t y i s indicated i f a clear area i s seen around the s i t e where the compound has been placed. 7 a Experimental and Results I. Feeding experiments The e f f e c t s of alantolactone on the sur v i v a l of Tribolium confusum are shown in Fig.,19. The lowest concentration (0.2 %) did not a f f e c t the sur v i v a l of the beetles. But higher concentrations of alantolactone s i g n i f i c a n t l y reduced the surv i v a l of the beetles. a l l the control animals that were offered f l o u r without alantolactone survived for the duration of the experimental period. In the second control, where no food was offered, the beetles died i n a period comparable to that of animals offered higher concentrations of alantolactone., Results of the choice experiment showed that i n the course of the f i r s t two and half hours there did not seem to be any consistent tendency of beetles to s e l e c t food without alantolactone (Fig. 20). However, observations from the following two days showed that beetles avoided the food with alantolactone (Fig. 20, Table I?.). From these data i t also appeared that s i g n i f i c a n t l y more beetles avoided the food with 3, 5, and 7,% of alantolactone than that with 1S alantolactone. I I . Heartbeat experiments In preliminary t r i a l s Locke's solution or d i s t i l l e d water were applied on the hearts and changes in the rate of heartbeat Fig. 19. Effect of alantolactone on the survival of Tribolium confusum (mean + standard deviation)• 8 0 H 7 0 A 6 0 in •g 5 0 0) E 40 ro > £ 3 0 -1/1 2 0 10 B fl B B B — r ~ A 5 — r -6 T —i—• 8 —r-9 e 10 11 i 12 B - i n f r -13 14 no food Per cent alantolactone in food Fig. 20. Effect of various concentrations of alantolactone on choice of food by Tribolium ( • observations within f i r s t 2.5 hours, o after 2.5 hours). .9 * ft co c ro I? .6 o o > o o ro .5 .4 .3 .2 .1 o o 0 —i— 0 2 —r— 5 1 2 3 4 Per cent alantolactone in food —r— 6 7 ON 77 Table 4. Summary of data of experiment on food selection by Tribolium confusum (observations were made after 2.5 hour period during which the beetles were adjusted). Concentration of Observed v i s i t s on food alantolactone (%) with alant. without alant. Chi-square 0 (control) 1 3 5 7 108 64 49 62 37 150 168 213 234 195 10.308 .005<p<.001 5.013 .05<p<.025 0.310 . p>.05 1.817 p>.05 78 were found to be within 10% of the i n i t i a l frequency (Table V.). Thus rate changes i n subsequent experiments that exceeded 10% of the i n i t i a l frequency were considered s i g n i f i c a n t . Various concentrations of parthenin (0.1 mg/ml - 15 mg/ml), dissolved i n Locke's solution were applied on the heart. The lowest parthenin concentration used (0.1 mg/ml) appeared to have no s i g n i f i c a n t e f f e c t but increasing concentrations exhibited an increasing i n h i b i t o r y e f f e c t on the rate of heartbeat (Table V.). The time reguired for complete i n h i b i t i o n of the heartbeat was negatively correlated with parthenin concentration (Fig. ,21). Parthenin i n Locke's solution appeared to have the same o v e r a l l e f f e c t s on the rate of heartbeat as a corresponding concentration i n d i s t i l l e d water (Table V.). The only obvious difference was a shorter time required to stop the heartbeat with aqueous solutions. In a l l cases when the hearts stopped as a re s u l t of an application of parthenin they were washed with Locke's solution., Time i n t e r v a l s between the i n h i b i t i o n of the heart a c t i v i t y and washing the heart were between f i v e and twenty minutes. No parthenin-arrested heart exhibited any a c t i v i t y when checked at f i v e to ten minute i n t e r v a l s a f t e r washing in the course of t h i r t y to s i x t y minutes. Neurotransmitters To f i n d out the major s i t e of parthenin action two suspected neurotransmitters, L-glutamic acid and acetylcholine, were used. The e f f e c t s of these neurotransmitters on heartbeat are shown in F i g . 22., T a b l e 5 . E f f e c t s o f p a r t h e n i n on t h e f r e q u e n c y o f h e a r t b e a t . P a r t h . I n Mean Mean d e v i a t i o n s f r o m i n i t i a l h e a r t b e a t (bpm) L o c k e ' s Amount I n i t i a l — Mean t i m e s o l u t i o n a p p l i e d h e a r t b e a t T ime (min) t o s t o p (mg/ml) ( u l ) (bpm) 1 5 10 20 30 40 50 60 70 80 90 100 (min ) L o c k e ' s s o l u t i o n W 9 9 . 0 ± 6 . 3 0 0 - 1 . 0 1 1 . 4 0 . 4 1 3 . 8 0 . 8 1 5 . 2 0 . 6 + 4 . 7 0 . 6 1 4 . 0 2 . 8 + 5 . 8 3.616.2 3 . 8 + 5 . 9 0 0 D l s t . H 2 0 20 1 0 2 . 3 1 1 1 . 8 8 . 7 1 2 . 5 0 6 . 0 1 3 . 0 4 . 3 + 2 . 1 1.313.2 0 . 8 1 2 . 6 0 0 0 0 0 0 0 . 1 50 1 0 4 . 7 * 3 . 1 6 . 7 1 4 . 6 6 . 0 1 5 . 3 8.715.0 7 . 3 1 3 . 1 7 . 3 1 5 . 0 4 . 7 1 4 . 2 2 . 7 1 4 . 6 8 . 0 1 2 . 0 5.313.1 6 . 0 1 4 . 0 8 . 0 1 2 . 0 9 . 3 1 2 , . 3 1 . 0 50 1 1 0 . 3 * 4 . 7 1 2 . 0 1 6 . 6 - 1 . 0 1 9 . 6 - 3 . 0 1 8 . 5 5 . 0 1 7 . 0 3 . 7 1 4 . 9 2 . 3 1 3 . 8 1 . 2 1 4 . 0 -110.314 .7 - - - - 6 3 . 3 2 . 0 50 1 0 6 . 6 1 1 4 . 7 - 1 . 0 1 2 . 6 0 . 0 + 1 2 . 2 - 3 . 0 1 7 . 8 - 4 . 7 1 1 2 . 1 - 9 . 3 + 2 1 . 2 - 1 6 . 7 + 2 6 . 0 - 1 0 6 . 6 1 1 4 . 7 - - - - - 4 8 . 3 3 . 0 50 1 0 8 . 7 1 7 . 6 - 0 . 7 1 1 1 . 0 5 . 3 1 1 1 . 0 4 . 0 1 1 2 . 5 • - 3 2 . 0 1 5 9 . 2 - 3 9 + 5 3 . 3 - 7 2 . 0 + 5 9 . 2 - 1 0 8 . 7 1 7 . 6 - - - - - 4 1 . 7 4 . 0 50 8 9 . 3 1 7 . 0 - 2 8 . 7 1 4 6 . 2 - 6 1 . 0 1 4 3 . 5 - 6 1 1 4 4 . 1 • - 8 9 . 3 1 7 . 0 - - ' - - - - - 1 3 . 3 5 . 0 50 7 8 . 0 1 1 2 . 2 - 6 . 0 1 1 5 . 9 - 5 2 . 0 + 3 9 . 4 - 5 4 1 3 6 . 6 • - 5 9 . 3 1 2 7 . 3 - 7 8 . 0 1 1 2 . 2 - - - - - - 16.7 6 . 0 50 9 7 . 9 1 1 7 . 3 -30.6137 .4 -48.7143 .7 - 5 7 . 2 1 4 1 . 5 - 8 1 1 3 2 . 9 - 9 7 . 9 1 1 7 . 3 - - - - - ' - 1 3 . 0 1 0 . 0 50 9 8 . 0 1 1 6 . 5 - 1 8 . 3 1 3 4 . 2 - 3 5 . 8 + 3 7 . 8 - 5 6 . 3 1 3 9 . 2 - 9 8 1 1 6 . 5 - - - - - - - 11.5 1 0 . 0 20 7 3 . 8 1 5 . 7 - 3 . 2 1 7 . 8 - 3 7 . 8 + 4 2 . 8 - 6 1 . 2 1 3 2 . 3 - 7 3 . 8 1 5 . 7 - - - - - - 1 2 . 8 1 0 . 0 * 20 9 3 . 7 + 1 8 . 0 - 9 3 . 7 1 1 8 . 0 - - - - - - - - - - 1 . 0 1 5 . 0 50 106.018.5 - 2 4 . 0 + 2 2 . 6 - 6 6 . 0 1 4 8 . 1 - 1 0 6 . 0 + 8 . 5 - - - - - - - - 7.5 Fig. 21. Effect of parthenin on the duration of heartbeat. ' < — — i " 1 — — i 1 1 1 1 , , ,— 2 3 4 5 6 7 8 9 . 1 0 11 12 1 3 14 1 5 Concent ra t ion of par thenin (mg/ml Locke's sol.) Fig. 22. Effect of glutamic acid and acetylcholine on the frequency of heartbeat ( o 10 mM and • 20.5 mM glutamic acid, + 1 mM and x 10 mM acetylcholine)-30-n) -30 82 L-glutamic acid at 10 mM and 2 0 . 5 mM concentrations was applied to the heart previously i n h i b i t e d by the application of 20 .5 mM parthenin. Ho recovery of the heart a c t i v i t y was found. However, the control application of L- glutamate alone, at these concentrations, had no stimulatory e f f e c t on the heart but rather i n i t i a l l y reduced the rate of heartbeat (Fig. 2 2 ) . Acetylcholine i n 10 mM concentration, applied to the parthenin-arrested hearts of three animals f i v e minutes after they were stopped, did not lead to a recovery i n the heart a c t i v i t y . Acetylcholine of two concentrations (1 mM and 10 mM) when applied on the heart during the control t r i a l s i n i t i a l l y increased the frequency of the heartbeat (Fig. 2 2 ) . Sulphydryl compounds L z S y s t e i n e applied on the heart at a concentration of 20.5 mM s l i g h t l y increased the frequency of the heartbeat (Fig. 2 3 ) . Parthenin solution (20.5 mM) was applied on the hearts of two other animals. When these hearts showed no detectable beats, a 20 .5 mM solution of cysteine was applied. The frequency of the beats returned almost to the i n i t i a l values (Fig. 2 4 ) . An equimolar mixture of parthenin and L-cysteine (20.5 mM concentration) was applied on the heart (Table 71.). Application of t h i s solution a f t e r f i v e minutes of i t s preparation resulted in the l o s s of a c t i v i t y of the heart within f i v e minutes. Application of the mixture 20 minutes or 48 hours a f t e r F i g . 23. E f f e c t of L-cysteine and DTT on the frequency of heartbeat ( + 20.5 mM L-cysteine, o 13.6 mM and • 20.5 mM DTT)• co Fig. 24. Effect of the monoadduct on the frequency of heartbeat and cysteine applied on parthenin - arrested heart (application indicated by arrow); • parthenin, o cysteine, + monoadduct 20 E Q. S 20 t 01 OJ | 60 z 20 + 60 —+ Time (min) \> q 80 100 id \ CO - p -Table 6. Effect of equimolar solutions of SH compounds and parthenin. Time of applic. Average i n i t i a l Average deviations from i n i t i a l heartbeat (bpm) after heartbeat Time (min) Thiol mixing (bpm) 1 5 10 15 20 25 30 35 40 45 50 55 60 L-cysteine 5min 84.0 -40 .0 -84. * ,0 20min 70.0 12 .0 -20. 0 2.0 4.0 4.0 2.0 d 2 days 86.5 11 .0 8. 0 3.0 -3.0 -6.0 --7.0 • -8.0 -11.0 -17.5 d Glutathione 5min 65.0 -65 * .0 20min 64.5 -28 .0 -28. 0 -29.5 -36.0 -29.0 - * -64.5 24hrs 71.5 -71 .5 -71. 5 -71.5 -71.5 -66.0 - * -71.5 DTT immediately 75.5 -50 .5 -75. * 5 5min 65.5 6 .5 7. 5 16.5 20.0 20.5 --27.5 4.0 3.0 -14.5 -13.0 -21.5 4hrs 72.5 1 .5 4. 5 4.5 2.0 8.0 1.0 -2.5 -6.0 * Note: no activity of the heart d the heart dried out 86 preparation greatly reduced the i n h i b i t o r y effect of parthenin. The i s o l a t e d monoadduct of parthenin and L-cysteine i n 20.5 mM concentration, applied on the hearts of three animals, did not cause any s i g n i f i c a n t changes i n a c t i v i t y (Fig. 24). Glutathione - i n an equimolar solution with parthenin (20.5 mM concentration) when applied f i v e minutes, 20 minutes, and 24 hours aft e r mixing, resulted i n a rapid loss of a c t i v i t y of the heart (Table VI.). A 20.5 mM solution of glutathione applied f i v e and 20 minutes a f t e r hearts had been stopped by parthenin, gave no recovery of the heart a c t i v i t y i n three animals. However, a control application of a 20.5 mM solution of glutathione carried out after these experiments with another animals stopped the heart within the f i r s t minute. D i t h i o t h r e i t o l solution (13.6 mM) was applied immediately, 5 minutes, or 20 minutes after hearts were stopped by parthenin (also 13.6 mM). In a l l three cases the hearts recovered t h e i r a c t i v i t i e s but only for a r e l a t i v e l y short time (for 5 to 25 minutes) and thereafter stopped again± Control t r i a l s with a 13.6 and 20.5 mM solution d i r e c t l y applied on hearts of other animals showed that DTT alone has a s l i g h t l y i n h i b i t o r y e f f e c t on the rate of heartbeat (Fig. 23). An equimolar mixture of parthenin and DTT (20.5mM) was applied on the hearts. The application, a f t e r immediate preparation of the solution, resulted i n blockage of the heart a c t i v i t y . When t h i s solution was applied f i v e minutes afte r mixing the e f f e c t of parthenin was s i g n i f i c a n t l y reduced. Two hours after mixing there was no ef f e c t (Table VI.). 87 Table 7. A n t i b i o t i c a c t i v i t y of parthenin - cysteine adducts against Staphylococcus albus. Compound Ant i m i c r o b i a l a c t i v i t y Parthenin + * Monoadduct + Biadduct -L-cysteine -DL-cysteine -L-cysteic acid + Note: i n h i b i t i o n only at place where compound was placed Fig. 25. Antibiotic activity of parthenin-cysteine adducts related compounds against Staphylococcus albus. 1- parthenin, 2- monoadduct, 3- biadduct, 4- L-cysteine, 5- DL-cystine, 6- L-cysteic acid 89 I I I . A n t i b i o t i c a c t i v i t y t e s t To investigate the antimicrobial a c t i v i t y of parthenin three s t r a i n s of bacteria were used. Because parthenin exhibited no a c t i v i t y against C.albicans, weak a c t i v i t y against E A s o l i but strong a c t i v i t y against S.albus. the l a s t bacterium was selected for further t e s t s . The antimicrobial a c t i v i t y of parthenin-cysteine adducts and other compounds that could r e s u l t from the decomposition of the adducts (L-cysteine, DL-cystine, and L-cysteic acid) was tested on S. albus. I t was found that the monoadduct exhibited weak antimicrobial a c t i v i t y but the biadduct was in a c t i v e . L-cysteine and DL-cystine did not exhibit any a c t i v i t y while L-cysteic acid was strongly active (Table VII., F i g . 25). Eight other sesguiterpene lactones were tested f o r their a n t i b i o t i c a c t i v i t y . The r e s u l t s are presented i n Table VIII., F i g . 26. From Table I I I . i t appears that lactones possessing the unsaturated cyclopentenone r i n g exhibit antimicrobial a c t i v i t y while those lacking t h i s s t r u c t u r a l feature are i n a c t i v e with the exception of damsin that exhibited weak a c t i v i t y . 90 Table 8. A n t i b i o t i c a c t i v i t y of some sesquiterpene lactones against Staphylococcus albus. Sesquiterpene lactone A n t i m i c r o b i a l a c t i v i t y Parthenin + Hymenin + Helenalin + Damsin + Tenulin + I s o t e n u l i n + Tetraneurin-D -Alantolactone -Isoalantolactone — 91 Fig. 26. Antibiotic activity of some sesquiterpene lactones against Staphylococcus albus. 1- parthenin, 2- hymenin, 3- helenalin, 4- tenulin, 5- isotenulin, 6- damsin, 7- tetraneurin-D 1- parthenin, 2- alantolactone, 3- isoalantolactone 92 Discussion Alantolactone present i n f l o u r i n concentrations higher than 0.2 % affected the s u r v i v a l of Tribolium beetles. However, i t i s not c e r t a i n whether the beetles died as a r e s u l t of ingested alantolactone or whether they avoided the food and thus died of starvation. In any case, t h i s experiment showed that alantolactone had detrimental e f f e c t s on the experimental animals., When given a choice between food with and without alantolactone, Tribolium avoided alantolactone treated food. However, i t seems that the beetles need a ce r t a i n time to learn to discriminate between the types of food offered. , This experiment demonstrated that alantolactone i s a feeding deterrent to Tribolium conf usum. The experiment with grasshoppers showed that parthenin has an i n h i b i t o r y e f f e c t on the oyocardic a c t i v i t y . There was a positive c o r r e l a t i o n between parthenin concentration and rate of heartbeat. Subsequent washing of the hearts with saline did not lead to recovery. In s p i t e of the fact that L- glutamic acid has been considered recently to be the most l i k e l y neuromuscular transmitter i n insects (McDonald 1975), I did not observe the excitatory e f f e c t s of t h i s compound on the semi - i s o l a t e d heart °f Melanoplus sanguinipes. Moreover, t h i s substance did not 93 a l l e v i a t e the blockage in a c t i v i t y caused by parthenin. Acetylcholine {another suspected neurotransmitter) caused a s i g n i f i c a n t increase i n the rate of heartbeat i n almost a l l cases., This r e s u l t i s i n accordance with the findings of Hamilton (1939) on Melanoplus d i f f e r e n t i a l i s . In spite of t h i s excitatory a c t i v i t y , however, the application of acetylcholine to hearts in h i b i t e d by parthenin did not lead to recovery of the heartbeat. This indicates that the s i t e of parthenin action most l i k e l y i s on the post - synaptic membrane. A recovery of the heart a c t i v i t y by acetylcholine would point to the pre -synaptic membrane as a s i t e of parthenin action. Similar conclusions have been made by Bratkowski e t a l . (1972) who used d i f f e r e n t sulphydryl agents and a di f f e r e n t neurotransmitter on sk e l e t a l muscle of Bomalea microptera. The application of t h i o l s (DTT and L- cysteine) on the heart previously stopped by parthenin resulted in a recovery of the heart a c t i v i t y in a l l t r i a l s . A possible explanation i s that parthenin - S - protein adducts that may have formed were dissociated i n the presence of SH groups of applied t h i o l s . This would mean that parthenin would p r e f e r e n t i a l l y form complexes with applied t h i o l s . I f t h i s i s so, thi s property should be considered i n the treatment of allergy dermatitis caused by parthenin or other sesquiterpene lactones. Dnharmful t h i o l s which would p r e f e r e n t i a l l y form complexes with sesquiterpene lactones could be used. The i n h i b i t o r y e f f e c t of a mixture of parthenin and cysteine or parthenin and DTT (prepared i n vitro) on the rate of 94 heartbeat evidently decreased with increasing reaction time of the mixture before i t was applied on the heart, sith time there i s an increasing amount of adducts formed and one of those, the monoadduct of parthenin and cysteine i s i n e r t in t h i s t e s t . The other i s even more l i k e l y to be so. Thus there i s a decrease i n the amounts of free parthenin (as described in the Chemical Part) with time so that the preparation aft e r 48 hours i s t o t a l l y i nactive. The finding that parthenin exhibits strong a c t i v i t y against S. j albus , but weak or no a c t i v i t y against E. c o l i and albicans, respectively, i s i n agreement with the r e s u l t s of Lee et a l . {1977 b). According to these authors pseudoguaianolides and related compounds exhibit a c t i v i t y exclusively against Gram positive bacteria. This suggests that these lactones attack certain enzymes (with SH groups) that are important for l i f e and reproduction of these bacteria., The antimicrobial tests support the view that sesguiterpene lactones possessing the cyclopentenone ring exhibit antimicrobial a c t i v i t y . A more detailed discussion of t h i s problem i s presented in the Chemical Part. The r e s u l t s of this study indicate that sesguiterpene lactones function as feeding deterrents and have detrimental effects on insect and b a c t e r i a l physiological processes. This finding lends some support to the view that plants may have evolved the production of sesguiterpene lactones as a means of defense against herbivorous predators, fungi, and bacteria. B I B L I O G 8 a P H Y 95 CK.. Anderson, L.A.P., W.T. de Kock, K.G.R. Pachler, 1967. The structure of Vermeerin. A sesquiterpene dilactone from Geiq.jgr.ia africana Gries. Tetrahedron 23: 4153. Asakawa, Y., M. Toyota, M. Uemoto, and T. Aratani. 1976. Sesquiterpenes of s i x Porella species (Hepaticae). Phytochemistry 15: 1929-1931. Baker, p. ,M., c . C. Fortes, E. G. Fortes, G. G a z z i n e l l i , B. Gil b e r t , 3.„• N.; C. Lopes, J. Pellegrino, T.C.B. Tomassini, and W. Vichnewski. 1972. Chemoprophylactic aqents i n schistosomoasis: eremanthine, costunolide, «-cyclocostunolide and bisabolol. Pharm. Pharmac. 24: 853-857. Benesova, V., Z. Samek, and S. Vasickova. 1975. S t r u c t u r e of the s e s q u i t e r p e n i c l a c t o n e d i p l o p h y l l o l i d e and other components from the l i v e r w o r t D i p l o p h v l l u m a l b i c a n s (L.) Dum. , C o l l . Czechoslov. Chem. Commun. 40: 1966-1970. , Bleumink, E., J . C. Mi t c h e l l , T. A. Geissman, and G. H. M. Towers. 1976. Contact hypersensitivity to sesquiterpene lactones i n Chrysanthemum dermatitis. Cont^ Derm. 2: 81-88. Bohlmann, F., T. Burkhardt, and C. Zdero. 1973. Naturally occurring Acetylenes. Academic Press, London. Bratkowski, T. A., T. J. McDonald, and B. D. O'Brien. 1972. Effects of Sulphydryl Heagents on an Insect Nerve-muscle Preparation. jJ . Ins. Physiol. 18: 1949-1960. Burnett, W. C. 1974. Sesquiterpene lactones- herbivore feeding deterrents i n Vernonia (Compositae). Ph.D. Thesis, Univ. of Georqia. Burnett, H. C., S. B. Jones, T. J. Mabry, and H. G. Padolina. 1974. Sesquiterpene lactones-insect feeding deterrents in Vernonia. Biochem System. Ecology 2: 25-29. Chapman, B. F. 1974. The Insects. The English Universities Press, Ltd., London. Char, M. B.,S. and S.,Shankarabhat. 1975. Parthenin: A growth Inhibitor Behaviour i n Different Organisms. Experientia 31: 1164-1165. Dalvi, R. B., B. Singh, and D. K. Salunkhe. 1971. A study on Phytotoxicity of Alantolactone. Chem., B i o l . Interactions 3: 13-18. Davey, K. G. 1964. The cont r o l of v i s c e r a l muscles i n Insect. Adv. Ins. Physiol. 2: 219-245. Dupuis, G*, J . C. M i t c h e l l , and G. H. N. Towers. 1974. Reaction 96 of Alantolactone, an Allergenic Sesguiterpene Lactone with some Amino Acids. Resultant Loss of Immunologic Reactivity. Can. J. Biochem. 52: 575-581. Doskotch, R. W. and F.S. El-Feraly. 1969. Antitumor agents I I . : T u l i p i n o l i d e , a new germacranolide sesguiterpene, and costunolide., Two cytotoxic Substances from Liriodendron t u l i p i f e r a L. J. Pharm. S c i . 58: 877-880. Fraenkel, G. S. 1959. The raison d'etre of secondary plant substances. Science 129: 1466-1470. Garciduenas, H./ 8., X. A.Dominguez, and J. F. y G. Alanis. 1972. Hew Growth Inhibitors from Parthenium hysterophorus . Rev. Latinoamer. Quim. 3: 52-53. Geissman, T. a. 1973. The biogenesis of Sesguiterpene Lactones in Compositae. ,In V. C. Runeckles and T. J. , Mabry (eds.). Terpenoids: Structure, Biogenesis and D i s t r i b u t i o n . Recent Advances i n Photochemistry, Vol. 6. Academic Press, New York. Geissman, T. A. and D. H. G. Crout. 1969. Organic Chemistry of Secondary Plant Metabolism. Freeman, Copper and Co., San Francisco. , Geissmann, T. A. and M. A. Irwin. 1973. Chemical Constitution and Botanical A f f i n i t y i n Artemisia. In G. Benz and J. Sandesson (eds.) Chemistry and Botanical C l a s s i f i c a t i o n . Nobel Symposia 25. Academic Press, New York. Gross, D. 1975. Review. Growth Regulating Substances Of Plant Origin. Phytochemistry 14: 2105-2112. H a l l , I. H. , K.-H. Lee, E. C. Mar, and C. 0. Starnes. 1977. Antitumor Agents. 21. A Proposed Mechanism for I n h i b i t i o n of Cancer Growth by Tenulin and Helenalin and Related Cyclopentenones. J . Med. Chem. 20: 333-337. Hamilton, H. L. 1939. The Action of Acetylcholine, atropine and nicotine on the heart of the Grasshopper (Melanoplus d i f f e r e n t i a l i s ) . J . C e l l . Comp. Physiol. 13: 91-103. Hanson, R. ,-L., H. A. Lardy, and S. M. Kupchan 1970. In h i b i t i o n of Phosphofructokinase by Quinone Methide and Methylene Lactone Tumor Inhibitors. Science 168: 378-380. Hartwell, J., L. and B. J . Abbott. 1969. Antineoplastic P r i n c i p l e s i n Plants: Recent Developments in the F i e l d . Adv. Pharm. Chemoth. 7: 117-209. Hausen, B.. M. and K. H. Schulz. 1973.,Chrysanthemen - A l l e r g i e (1. Mitteilung). Berufsdermatosen 21: 199-214. 9 7 Hansen, B. M. and K.,,H.Schulz. ,1976. Chrysanthemum Allergy I I I . I d e n t i f i c a t i o n of the allergenes. Arch. Der. Res. 255: 111-121. Herout, V. 1973. A chemical compound as a taxonomic character. In G. Benz and J. Sandesson (eds.) Chemistry i n Botanical C l a s s i f i c a t i o n . Nobel Symposia 25. Academic Press, New York. Herz, W. 1973. Pseudoguaianolides in Compositae. In G. Benz and J. Sandesson (eds.) Nobel Symposia 25. Academic Press. New York. Herz, W. and H.,Hatanabe. 1959. Partenin, a new guaianolide. J . Am. Chem. ,Soc. 81: 6088-6089. Herz, W., H. Watanabe, M. Miyazaki, and Y. Kishida. 1962. The Structures of Parthenin and Ambrosin. J. Am. Chem. Soc. 84: 2601-2610. Herz, W., K. Aota, M. Holub, and Z. Samek. 1970. Sesguiterpene Lactones and Lactone Glycosides from Hymenoxys Species. J. Org. Chem. 35: 2611-2624. Herzer, F. H. 1942. Ass. Southern Agr. Workers Proc. Ann. Convention 4 3: 112. Howie, G. A., P. E. Manni, and J. M. Cassady. 1974. Synthesis of al k y l substituted alpha, beta-unsaturated gamma-lactones as potential antitumor agents. J . Med. Chem. 17: 840-845. „ Humason, G. L. 1967. Animal Tissue Techniques. W. H. Freeman and Co., San Francisco. Ivie, G. f . , A. W. Donald, W. Herz, R. Kannan, J. 0. Norman, and J. A. Veech. 1975 a. Hymenovin. Major Toxic Constituent of Wester Bitterweed (Hymenpxys odorata D C ) . J. Agric. Food Chem. 23: 84 1-845. Ivie, G.W., D. A. Witzel, and D. D. Rushing. 1975 b. To x i c i t y and Milk B i t t e r i n g Properties of Tenulin, the Major Sesguiterpene Lactone Constituent of Helenium amarum (Bitter Sneezweed). J . Agric. Food Chem. 23: 845-849. Jamieson, G. R., E. H. Reid, B. P. Turner, and A. T. Jamieson 1976. Bakkenolide-A. I t s d i s t r i b u t i o n i n Petasites species and cytotoxic properties. Phytochemistry 15: 1713-1715. Jones, J. C. 1974, Factors a f f e c t i n g Heart Rates i n Insects. In M. Rockstein (ed.) The Physiology of Insecta, Vol.,5. Academic Press. Kanchan, S. ; D. 1975. Growth Inhibitors from Parthenium hysterophorus Linn. Curr. Sci. (Bangalore, India) 44: 98 358-359. Kanchan, S. D. and Jayachandra. 1976. Proc. of the seminar on "Parthenium - a po s i t i v e danger," published by BIECO and the University of A g r i c u l t u r a l Sciences, Bangalore, India. Kim, C. S., T... K. Suh, and J. Y. Park. 1961. The p a r a s i t i c i d a l action of Inula helenium and alantolactone on Fasciola fegsatica in v i t r o . J . Taegu Med.,Soc. 3: 171-175. Kingsbury, J.M. 1964. Poisonous Plants of the United States and Canada. Prentice-Hall, Inc., s. Jersey. Knoche, H. G., G. Ourisson, and G. W. Perold. 1969. Allerg e n i c Component of a Liverwort; A Sesguiterpene Lactone. Science 166: 239-240. Krijgsman, B., J. 1952. Con t r a c t i l e and pacemaker mechanisms of the heart of arthropods. B i o l . Rev. 27: 320-346. Kupchan, s. M. ,; 1970. Recent Advances in the chemistry of Tumor I n h i b i t i r s of Plant Origin. Transact. N. York Acad. Sci. 32: 85-106. ,, Kupchan, S. M. 1974. Novel Natural Products with Antitumor A c t i v i t y . Feder. Proceedings 33: 2288-2295. Kupchan, S. M., D. C. Fessler, B. A. Eakin, and T. J. Ciacobbe. 1970. Reactions of Alpha Methylene Lactone Tumor Inh i b i t o r s with Model B i o l o g i c a l Nucleophiles. Science 168: 376-377. Kupchan, S. M., M. A. Eakin, and A. ,M. Thomas. 1971. .Tumor Inhibit o r s . 69., Structure-Cytotoxicity Relationship among the Sesquiterpene Lactones. J. Med. Chem. 14: 1147-1152. Lee, K.-H«, E.-S. Huang, C. Piandosi, J., Pagano, and T. A. Geissman. 1971. Cytotoxicity of Sesquiterpene Lactones. Cancer Res. 31: 1649-1654. Lee, K.-H., H. Furukawa, and E.-S. Huang 1972. Antitumor Agents. 3. Synthesis and Cytotoxic A c t i v i t y of Helenalin Amine Adducts and Relate Derivatives. J. Med..,Chem. 15: 609-61 1. Lee, K.-H., R. Meek, C. , Piandosi, and E.-S. Huang. .1973. Antitumor Agents. 4. Cytotoxicity and i n vivo A c t i v i t y of Helenalin Esters and Related Derivatives. J . Med. Chem. 16: 299-301. Lee, K.-H., T. Ibuka, and R.-Y. Wu. 1974 Beta Unsubstituted Cyclopentenone, a Structural Requirement for Antimicrobial and Cytotoxic A c t i v i t i e s . Chem. Pharm. B u l l . 22: 2206-2208. 99 lee , K.-H., I... H. H a l l , E.-C. Mar, C. 0. Starnes, S. a . ElGebaly, T. G. Waddell, R. , I. Hadgraft, C. G. Ru.ff.ner, and I. Weidner. 1977 a. Sesguiterpene Antitumor Agents: Inhibitors of C e l l u l a r Metabolism. Science 196: 533-536. Lee, K.-H., T. Ibuka, R.-Y. Wu, and T. A. Geissman. 1977 b. Structure-Antimicrobial A c t i v i t y Relationship among the Sesguiterpene Lactones and Related Compounds. Phytochemistry 16: 1177-1181. Ma.bry, T. J. . 1970. I n f r a s p e c i f i c Variation of Sesguiterpene Lactones in Ambrosia {Compositae): Applications to Evolutionary Problems at the Populational Level. In J. B. Harborne {ed.) Photochemical Phylogeny. Academic Press. Magnusson, G. and S. Thoren. 1973. Fungal Extractives. Two Sesquiterpenes from Lactarius. Acta Chem. Scand. 27: 1573-1578. Mathur, S. B., P. G. T e l l o , M. Fermin, and V. Mora-Arellano. 1975. Terpenoids of Mikania monagasensis-and t h e i r b i o l o g i c a l a c t i v i t i e s . Rev. Latinoamer. Quim. 6: 201-205. M i t c h e l l , J . , C. 1969. A l l e r g i c Contact Dermatitis from Compositae. Trans. St. John's Hosp. Der. Soc. 55: 174-18 3., Mi t c h e l l , J. C. 1975. Contact Allergy from Plants. In V. C. Runeckles (ed.) Recent Advances i n Phytochemistry Vol.9. Plenum Press, New York. M i t c h e l l , J . C. and G. Dupuis. ,1971. A l l e r g i c contact dermatitis from sesquiterpenoids of the Compositae family of plants. Br. J . Der. ,84: 139. / Mitchell, J . C., B. Schofield, B. Singh, and G. H. N. Towers. 1969. Allergy to F r u l l a n i a . A l l e r g i c Contact Dermatitis occurring i n Forest Workers Caused by Exposure to Fru 1 l a nia nisqnal leg s i s -„• Arch. Derm. 100: 46-49. . Mitchell, J. C , B. F r i t i g , B. Singh, and G. H. N. Towers. 1970. A l l e r g i c contact dermatitis from F r u l l a n i a and Compositae. J. Invest. Derm. 54: 233-239. Mitc h e l l , J. C , T. A. Geissman, G. Dupuis, and G. H. N. Towers 1971 a., A l l e r g i c Contact Dermatitis caused by Artemisia and Chrysanthemum species. J. Invest. Derm. 56: 9 8-101. Mi t c h e l l , J.,C., A. K. Roy, G. Dupuis, and G.,H. N. Towers. 1971 b. a l l e r g i c Contact Dermatitis from Ragweeds (Ambrosia Species) . Arch. Derm. 104: 73-76. 100 Mitchell, J . C., G. Dupuis, and T. A. Geissman. 1972. a l l e r g i c Contact Dermatitis from Sesquiterpenoids of Plants. B r i t . J. Derm. 87: 235-240. Mitcher, L. A l . 1972. Antimicrobial Agents from Higher Plants., I. Introduction , Rationale, and Methodology. Lloydia 35: 157-166. Mitcher, L. A. 1975. Antimicrobial Agents from Higher Plants. In V. C. Runeckles (ed.) Recent Advances i n Phytochemistry, Vol. 9. .Plenum Press, New York. , Mothes, K. 1976. Secondary Plant Substances as Materials for Chemical High Quality Breeding i n Higher Plants. In J. W. Wallace and R. L. Mansell (eds.) Recent Advances i n Phytochemistry, Vol. 10. Plenum Press, New York and London. McCahon, C. B., R. G. Kelsey, R..P. Sheridan, and F. Shafizadeh. 1973. Physiological e f f e c t s of compounds extracted from sagebrush. B u l l . Torrey Botan. Club 100: 23-28. McDonald, T. J . 1975. Neuromuscular Pharmacology of i n s e c t s . Ann. Rev. Entom. 20: 151-166. Norman, J. 0., J. H. Johnson, H. H. Mollenhauer, and S. M. Meola. 1976. Effects of Sesguiterpene Lactones on the Growth of gacili<is-^h-j^inqiens-i.s.-..,< Antimicrob. Agents Chemoth. 9: 535-539. Olechnowicz-Stepien, W. and S. Stepien. 1963. In v i t r o and i n vivo studies on the a c t i v i t y of helenin and i t s components against some species of dermatophytes. Dissert. Pharm. 15: 17-22. Perold, G. W., J. C. Muller, and G. Ourisson. 1972. Structure d'une lactone al l e r g i s a n t e : l e f r u l l a n o l i d e - 1 . Tetrahedron 28: 5797-5803. P e t t i t , G. R. and G. M. Cragg. 1973. Antineoplastic Agents 32. The Pseudoguaianolide Helenalin. Experientia 29: 781. Pitman, R. M. 1971. Transmitter Substances in Insects: A Review. Comp. Gen. Pharmac. ,2: 347-371. Pridham, J. B. 1965. Low Molecular Weight Phenols i n Higher Plants. Ann. Rev. Plant Physiol. 16: 13-36. Rodriguez, E. 1975 a. Sesguiterpene Lactones: D i s t r i b u t i o n , B i o l o g i c a l A c t i v i t y and I s o l a t i o n . Phytochem. B u l l . 8: 7-13. Rodriguez, E. 1975 b. The Chemistry and D i s t r i b u t i o n of Sesquiterpene Lactones and Flavonoids i n Parthenium (Compositae): Systematic and Ecoloqical Implications. Ph.D. Thesis, Univ. of Texas, Austin. 101 Rodriguez, E., M. 0. D i l l o n , T. J. Mabry, J. C. M i t c h e l l , and G. H. N. Towers. 1976 a. Dermatologically Active Sesguiterpene Lactones in Trichomes of Parthenium hysterophorus -, L. Compositae. Experientia 32: 236-237. Rodriguez, E., G. H. N. Towers, and J. C. M i t c h e l l . 1976 b. B i o l o g i c a l A c t i v i t i e s of Sesguiterpene Lactones- a Review. Phytochemistry 15: 1573-1580. R o l l i n s , R. C. 1950. The guayule rubber plant and i t s r e l a t i v e s . Contrib. Gray Herbarium 172; 1-73. Schlatterer, E. F. and E. w. Tisdale. 1969. E f f e c t s of l i t t e r of Artemisia, Chrysothamnus. and Tor, tula-on germination and growth of three perennial grasses. Ecology 50: 869-873. Segueira, L., R. J. Hemingway, and M. Kupchan. 1968. Vernolepin: A New , Reversible Plant Growth In h i b i t o r . Science 161: 789-790. Shibaoka, H., M. Shimokoriyama, S. Iriuchijima, and S. Tamura. 1967. Promoting A c t i v i t y of Terpenic Lactones i n Phaseolus rooting and th e i r Reactivity toward Cysteine. Plant C e l l Physiol. 8: 297-305. Siuda, J. F. and J. F. DeBernardis. 1973. Naturally Occurring Halogenated Organic Compounds. Lloydia 35: 107-143. Smith, C. H., J. Larner, A. M. Thomas, and M. Kupchan. 1972. Inactivation of glycogen synthase by the tumor i n h i b i t o r Vernolepin. vBioch. Biophys. Acta 276: 94-104. Smutz, E. M., B. N. Freeman, and R.E. Reed. 1968. Livestock-Poisoning Plants of Arizona. University of Arizona Press, Tuscon. Sperry, 0. E., J. w. Dollahite, G. 0. Hoffman, and B. J . Camp. 1964. Texas Plants Poisonous to Livestock. Texas ASM University, College Station. Toribio, P. P. and T. A. Geissman. 1968. Sesguiterpene lactones. New lactones from Hymenoclea salsola- T. and G. , Phytochemistry 7: 16231630. Towers, G. H. N. 1977. Contact Hypersensitivity and Photodermatitis evoked by Compositae. (in press) Towers, G. H. N., J . C. M i t c h e l l , E. Rodriguez, P. V. Subba Rao, and F. D. Bennett. 1977. . Biology and Chemistry of Parthenium hysterophorus L., a problem weed i n India, (in press) Vanhaelen-Fastre, R. 1968. Cnicus benedictus. ,  Separation of 102 antimicrobial constituents. Plant. Med. Phytother. 2: 294. ¥anhaelen-Fastre, R. 1972. A n t i b i o t i c and cytotoxic a c t i v i t i e s od c n i c i n , i s o l a t e d from Cnicus benedlctus. J . Pharm. Belg. 27: 683. Vichkanova, S. A., M. A. Rubinchik, and V. V. Adqina. 1971. Antimicrobial A c t i v i t y of Sesquiterpene lactones from Compositae. Tr. Vses. Nauchn. I s s l e d . Inst. Lekarstv. Aromat. Rast. 14: 230-238. Vichnewski, W.,S. J . S a r t i , B. Gilbe r t , and B. Herz. 1976. Goyazensolide, a Schistosomicidal Heliangolide from Eremanthus qoyazensis. Phytochemistry 15: 191-193. V i d a r i , G . , M. DeBernardi, P. V i t a - F i n z i , and G. Fronza. 1976. Sesquiterpenes from L a c t a r i us blennius. Phytochemistry 15: 1953- 1955. ~ Yoshioka, H., T. J. Mabry, and B. N. Timmermann. 1973. Sesquiterpene Lactones: Chemistry, NMR, and Plant D i s t r i b u t i o n . University of Tokyo Press. 

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