Planta medica

Journal of Medicinal Plant Research

Editor - in - Chief Editoriai Board Hippokrates Verlag E. Reinhard, Univ. Tübingen H.P.T. Ammon, Tübingen Stuttgart Pharmazeutisches Institut W. Barz, Münster a

Auf der Morgenstelle E. Reinhard, Tübingen 7400 Tübingen O. Sticher, Zürich

H. Wagner, München M. H. Zenk, Bochum

Contens Vol. 34/1978

© Printed in Germany

3

Index of Plant Names

A c a l y p h a i n d l c a 410 A c r O n y c h i a p o r t e r i 130 Aesculus i n d i c a 3 3 7 A f r a m o m u m m e l e g u e t a 7 A l a n g i a c e a e 381 A l a n g i u m l a m a r c k i i 381 A l o e 113 A m m i m a j u s 1 6 7 A n a p h a l i s c o n t o r t a 94 A n d r a c h n e c o l c h i c a 4 0 8 y 410 - d e c a i s n e i 4 0 9 A n g e l i c a a r c h a n g e l i c a 1 6 7 A p i u m g r a v e o l e n s 1 6 7 A p o c y n a c e a e 37, 390 A r n i c a l o n g i f o l i a 2 9 9 A s t e r a c e a e 2 9 9 , 328 A t a l a n t i a r o x b u r g h i a n a 130

B a c c h a r i s c r i s p a 328 B e y e r i a l a e s c h e n a u l t i i 410 B i g n o n i a c e a e 2 1 9 B r i d e l i a e x a l t a t a 408 - m o n o i c a 413 - o v a t a 408 - t o m e n t o s a 408 B t t p l e u m m f a l c a t u m 1 6 0 , 2 7 5 , 2 9 4 B u r k i l l a n t h u s m a l a c c e n s i s 130

C a c t a c e a e 2 0 7 C a m p a n u l a r o t u n d i f o l i a 10 C a p s i c u m spec. 7 C a s s i a a n g u s t i f o l i a 430 - l a e v i g a t a 3 1 9 C a t h a r a n t h u s 37, 2 5 9 C e n t r a n t h u s r u b e r 203 C e p h a e l i s 381 C h r y s a n t h e m u m p a r t h e n i u m 80 - v u l g a r e 80 C i n c h o n a l e d g e r i a n a 1 , 16 - s u c c i r u b r a 18 C i n n a m o m u m c e y l a n i c u m 2 9 4 C l a u s e n a e x c a v a t a 130 C n i c u s b e n e d i c t u s 80 C n i d o s c o l u s s t i m u l o s u s 410 - t e x a n u s 410 C o l l i g u a j a i n t e g e r r i m a 410 C o m p o s i t a e 5, 7 9 , 328 C o n v o l v u l u s m i c r o p h y l l u s 222

C r a t a e g u s o x y a c a n t h a 226 C r a t a e v a r e l i g i o s a 223 C r o c u s s a t i v u s 9 C y n a r a c a n n a b i n u m 80 - s c o l y m u s 80

D i g i t a l i s l a n a t a 4 4 , 9 3 , 2 2 5 , 443 - p u r p u r ea 122 D r y o p t e r i s spec. 1 4 4 , 3 9 7 — c a r t h u s i a n a 3 9 7 - filix-mas 398

E l a t e r i o s p e r m u m t a p o s 4 0 9 E p h e d r a c e a e 291 E p h e d r a spec. 291 E r y t h r o x y l u m coca 1 4 , 1 9 E s c h e r i c h i a c o l i 375 E u p a t o r i u m c a n n a b i n u m 80 E u p h o r b i a c e a e 1 8 3 , 408 E u p h o r b i a b o o p h t h o n a 4 0 9 - c l u t i o i d e s 4 0 9 - d r u m m o n d i i 4 0 9 - m a r g i n a t a 183 ~ p e p l u s 4 0 9 E u o d i a e u n e u r a 130 - g l a b r a 130 - m a c r o c a r p a 130 - r o x b u r g h i a n a 130 E u o n y m u s p e n d u l u s 211 E u r y c o m a l o n g i f o l i a 3 3 9

F a b a c e a e 4 1 4 F u s a r i u m 232

G e n t i a n a l u t e a 1 1 3 , 1 1 4 - p a n n o n i c a 1 1 4 - p e d i c e l l a t a 442 - p n e u m o n a n t h e 176 - p u n c t a t a 1 1 4 - p u r p u r e a 1 1 4 G l y c o s m i s c a l c i c o l a 130 G l y c y r r h i z a 2 9 4 G r o s s h e i m i a m a c r o c e p h a l a 80 G y m n a n t h e s l u c i d a 410 G y m n o s p o r i a m o n t a n a 211

H a g e n i a a b y s s i n i c a 1 4 4 , 153 H a r p a g o p h y t u m procumbens 94

4

H e v e a b r a s i l i e n s i s 4 0 9 - s p r u c e a n a 410 H o l a r r h e n a f l o r i b u n d a 4 7

I n d i g o f e r a s u f f r u c t i o s a 172 I n u l a h e l e n i u m 80 I p e c a c u a n h a (— U r a g o g a ) 3 8 1 , 430 I p o m o e a m u r i c a t a 93 I r i s spec. 9

J a t r o p h a a n g u s t i d e n s 410 - capensis 410 J u r i n e a a l a t a 80

K n i g k t i a d e p l a n c h e i 66

L a t h y r u s h i r s u t u s 420 - o d o r a t u s 420 - s i l v e s t r i s 420 - t i n g i t a n u s 420 L a w s o n i a i n e r m i s 9 L e g u m i n o s a e 1 7 2 , 335 L i l i a c e a e 216 L i m o n i u m g m e l i n i i 218 - p e r e z i i 218 L o b e l i a 430 L o g a n i a c e a e 5 3 , 57, 6 2 , 2 6 4 L y g o s r a e t u m ( = G e n i s t a r . ) 335

M a l l o t u s 1 4 4 M a n i h o t a i p a 4 0 9 - c a r t h a g i n e n s i s 4 0 9 - g l a z i o v i i 4 0 9 - u t i l i s s i m a 4 0 9 M a r a n t a c e a e 323 M a r k h a m i a s t i p u l a t a 2 1 9 M a t r i c a r i a c h a m o m i l l a 113 M e n i s p e r m a c e a e 2 7 4 M e r c u r i a l i s a n n u a 410 M e r e n d e r a c a u c a s i c a 216 M e r i l l i a c a l o x y l o n 130 M e r o p e a n g u l a t a 130 M e s e m b r y a n t h e m u m e d u l e 226 M i t r a g y n a p a r v i f o l i a 2 5 9 - speciosa 2 6 , 253 M o m o r d i c a c h a r a n t i a 275 M y r t a c e a e 5

N e l i a m e y e r i 226 N i c o t i a n a g l a u c a 73 - t a b a c u m 73 N o t o c a c t u s c o n c i n n u s 2 0 7

Index of Plant Names

O p u n t i a a u r a n t i a c a 2 0 7 — b r a s i l i e n s i s 2 0 7

P a e o n i a 2 9 4 P a n a x ginseng 2 9 4 P a p a v e r b r a c t e a t u m 135 - O r i e n t a l e 135 - s o m n i f e r u m 1 4 , 135 P a r a m i g n y a l o b a t a 130 P e t r o s e l i n u m s a t i v u m 1 6 7 P h a r n a c e u m c o r y m b o s u m 4 0 9 - s u f f r u t i c o s u m 4 0 9 P h y l l a n t h u s g a s s t r o e m i i 4 0 9 - l a c u n a r i u s 4 0 9 - n i r u r i 4 0 9 - speciosus 4 0 9 P h y t o l a c c a a m e r i c a n a 8 7 P i n e l l i a t e r n a t a 2 9 4 P i p e r n i g r u m 7 P l u m b a g i n a c e a e 218 P o r a n t h e r a c o r y m b o s a 408 - m i c r o p h y l l a 408 P s e u d o m o n a s a u r e o f a c i e n s 3 5 4 P s o r a l e a c o r y l i f o l i a 168 P y c n a r r h e n a m a n i l l e n s i s 2 7 4 - n o v o g u i n e e n s i s 2 7 4

R a n u n c u l u s l a n u g i n o s u s 10 R a u w o l f i a c u m m i n s i i 390 - i v o r e n s i s 390 - l i b e r i e n s i s 390 - m o m b a s i a n a 395 - v o m i t o r i a 395 R h a m n u s f m n g u l a 311 R o s a c e a e 153 R u b i a c e a e 2 6 , 2 5 3 , 3 8 1 R u t a c e a e 1 2 9 , 338 R u t a g r a v e o l e n s 168

Scute I l a r i a 2 9 4 S e c u r i n e g a r a m i f l o r a 4 0 9 - s u f f r u t i c o s a 408 S i m a r u b a c e a e 3 3 9 S k i m m i a l a u r e o l a 338 S m y r n i u m c o n n a t u m 215 S t a p h y l o c o c c u s a u r e u s 346 S t r e p t o m y c e s a n t i b i o t i c u s 363 - a r e n a e 370 - griseus 346 - v i o l a c e o r u b e r 372 S t r y c h n o s c a e s p i t o s a 53 - d a l e 2 6 4 - d o l i c h o t h y r s a 62

Index of Plant Names 5

- e l a e o c a r p a 2 6 4 - procumbens 1 0 9 - g o s s w e i l e r i $3 T r i g l o c h i n m a r i t i m a 410 - u r c e o l a t a 62 T r i g o n e l l a f o e n u m - g r a e c u m 4 1 4 - u s a m b a r e n s i s 57 T r i p h a s i a t r i f o l i a 130 - v a r i a b i l i s 5 7 S t y r a x officinalis 403 U r a g o g a 3 8 1 , 430 - t o n k i n e n s e 9

V a l e r i a n a m e x i c a n a 305 T e t r a c t o m i a r o x b u r g h i i 130 - officinalis 1 1 3 , 1 2 0 - t e t r a n d r a 130 T h a p s i a v i l l o s a 218 W i g g i n s i a a r e c h a v a l e t a i 2 0 7 T h a u m a t o c o c c H S d a n i e l l i i 323 T i l i a a r g e n t e a 93 Z a n t h o x y l u m m y r i a c a n t h u m 130 T r i b u l u s t e r r e s t r i s 9 4 , 1 8 8 Z i z y p h u s 2 9 4 T r i c h o l e p s i s a n g u s t i f o l i a 1 0 9 Z i n g i b e r o f f i c i n a l e 7, 2 9 4 - g l a b e r r i m a 1 0 9 Z y g o p h y l l a c e a e 188

6 Subject Index

Subject Index Acevaltrat 120 Actinorhodin 370 Acylglycosides 93 Aescin 337 Ajamalicinic acid 38 Ajmalan diacetate 392 Ajmalicine 27, 31, 37, 40, 254, 394 19-epi Ajmalicine 40 Ajmalicinine 394 Ajmaline 195,394 Akagerine 264 Akuammigine 35,254 Alangiside 381 Alatolide 80 Allergie contact dermatitis 299 Aloin 121 Aloinosid 121 Alstonine 40 Amarogentin 115 Amaropanin 115 Amaroswerin 115 Anethol 5 Anguidine 231 Anhydronium bases 390 Anthocyanins 323 Anthroneglycosides 311 Antibiotics 345 Antiphlogistic activity 97 Antitumor survey 129 Apigenin 226,335 Arecolin 195 Aricine 394 Arnifolin 300 Atropin 195

Benzoic acid 9 Benzoisochromane quinones 373 Berbamine 274 Betainyl-anguidine 231 Bisabolol 116 Bisbenzylisoquinoline alkaloids 274 Bis Indolealkaloids 57, 62

Campesterol 215,339 Capsaicin 6 Carvon 5 Corynoxine B 31 Cassia senna 311

Catharanthinc 40 Cell Suspension cultures 73 Cephaelin 195,381 Cerotic acid 211 Chamazulen 116 Chavicine 6 Chinchonin 195 Chichonidin 195 Chinidin 195 Chinin 195 Chloroperoxidase 352 Chlorothricin 345, 362 Ciliaphylline 27, 31,253 Cinnamic acid 222 Cinchonidine 14 Cinnamyl cinnamate 9 Cinnamylcocaine 14 Citronellol 426 Cnicin 80 Cocaine 14 Codeine 14,195 Colchicin 195 Colchicine alkaloids 216 Concanavaline A 233,423 Conessine 47, 49 Coniferyl benzoate 9 Conan 195 Contact allergens 299 Convulsant 264 Corynantheal 394 Corynantheidine 26,31,254 Corynantheol 394 Corynoxeine 27,31 Corynoxine 27,31 Coumarins 338 p-Coumaryl- benzoate 9 Crocin 9 Cuscohygrine 14 Cupreine 14 Cyanogenic glucosides 408 Cyanogenesis 408 Cycloheximide 232 Cycloartenediol 109 Cynaropicrin 80 Cytosine arabinoside 232, 239

Daidzein 335 Demethylation 73 N-Demethylindolmycin 348

Subject Index 7

Deoxydiaboline 58 Desacetylretuline 58 Desmethoxycentaureidin 225 Diacetylajmaline 394 Dianthroneglycosides 311 Dia-Valtrate 203 Didrovaltrat 120, 306 Digoxin 44 Dihydroindoles 390 Dihydronorpurpeline 394 Dihydrophenylalanine 345,357 Dimeric indole alkaloids 62 Dioscin 188 Diosgenin 188, 223,414 D N A synthesis 231 Dolichantoside 53 Dulcitol 211

Emetin 195, 391,430 Endolobine 394 En-In-Dicycloäther 116 Ephedrin 195, 291 Epipicraphylline 394 Epifriedelinol 211 Esdragol 5 Eugenol 5 Eupatoriopicrin 80

Fanchinoline 275 Flavones 176 Flavonoids 225, 319, 323, 328, 335 Flavonol glycoside 323 Frangularoside 311 Frenolicin 370 Friedelin 211 Furocoumarins 167 Furostanolbisglycoside 188

Geissospermine 40 Gentiopikrosid 115 Gingerols 6 Gitogenin 415 Glycoflavones 323 Granaticin 345, 369, 372 Griseusin 370 Grossheimin

Haemoiysis Harpagosid HeLa cells Helenalin

Helenalinacetate 299 Helenalinmethacrylate 299 Heteroyohimbine 390 Hevea brasiliensis 1 Hispidol 335 Hispidulin 444 Holadienine 47 Holarrheline 47 Holarrhesine 47 Homoacevaltrat 120 Homobaldrinal 305, 306 Homodidrovaltrat 120 Homovaltrat 120 Hydroxyurea 232 Hydroxykynurenic acid 218 Hygrine 14 Hypertensive activity 291

Immunosuppressiv activity 233 Indole alkaloids 37, 264, 390 Indolepyruvic acid 348 Indolmycenic acid 348 Indolmycin 345,346, 348 Ionones 9 Ipecoside 381 Ipomine 93 Isocorynantheidine 253,254 Isodidrovaltrat 120 Isofangchinoline 274 Isomitrafoline 26,31 Isomitraphylline 26,31,254 Isoorientine 442 Isopayantheine 253,254 Isoquinoline alkaloids 381 Isorhynchophylline 31,254 Isoscoparine 176 Isospeciofoline 26,31 Isostrychnobiline 57 Isotetrandrine 275 Isovaltrat 120, 306 Isovincoside 259 Isovitexine 176

Jaceosid 444 Javaphylline 254

80 Kaempferol 222 Kalafungin 370

160 Kribine 264 97

235 Lapachol 219 299 Lapachone 219

8 Subject Index

Laticifers 183 Lawson 9 Lectins 420 Leucocristinesulfat 40 Leucosidinesulfat 40 Leucosinesulfat 40 Lignan 403 Limacine 274 Linalool 5 Linamarin 409 Lobeline 430 Lochnerine 40 Louisfieserone 172 Luteolin 444 Lymphocytes 231

Manihotoxin 409 Maokonine 291 Marmesin 167 Matricin 116 Menthol 5 Methionine 346 ß-Methylindolepyruvic acid 348 O-Methylpsychotrin 195 Mitraciliatine 31,253,254 Mitrafoline 26,31 Mitragynine 26, 27,31,254 - oxindole B 31 Mitrajavine 254 Mitraphylline 26, 27,31,254 Monoterpenes 426 Morphin 14,195 Mouse mastocytoma cells 233 Mucilages 207 Muscle-relaxant effects 264 Mycotoxic activity 232

Nanaomycin 370 Naphtocyclinone 345, 369, 370 Narcotin 195 Naringenin 335 Neogitogenin 415 Nepetrin 444 Nicotine 73,195 Nordihydrotoxiferine 57 Normacusine 394 Normitoridine 394 Nornicotine 73 2-N-norobamegine 275 Norpurpeline 394 Norseredamine 394 Nortetraphyllicine 394

Obamegine 275 Ovalicin 231 Oxindole alkaloids 26, 259

Palmitone 219 Papaverin 195 Paradols 6 Parthenolide 80 Paulownin 219 Payantheine 26, 31, 254 Payanthine 26 Pectolinarigenin 225 Pentadecanolide 5 Pericyclivine 391,394 Phaeantine 274 Phaseolunatin 409 Phloroglucinol derivatives 144, 153, 397 Phyllanthin 409 Phytohormones 73 Phytolaccosides 87 Pinitol 172 Piperetine 6 Piperine 6 Procyanidin 226 Protein synthesis 231 Protodioscin 188 Pseudoquaianolide 299 Psoralen 167 Psychotrin 195 Puromycin 232 Purpeline 394 Pycnamine 274 Pyrrolnitrin 345,351

Quercetin 335 Quercetrin 337 Quinidine 14 Quinine 14 Quinoline 218

Radioimmunoassay 37 Repanduline 275 Rescinnamine 40, 394 Reserpine 40,195, 394 Reticulocytes 233 Retuline 58 Rifampicin 233 Rhynchociline 27, 31, 253 Rhynchophylline 27,31,254

Subject Index 9

Rosmarinic acid 5 Rutaretin 167

Safranal 9 Saiko-Keishi-To 294 Saikosaponin 160,275 Sandwicine 40 Sapogenins 414 Saponarine 176 Saponines 339 Sarpagan 390 Sarpagine 40,394 Scopolamin 195 Sennoside 311,430 Seredamine 394 Seredamine-17-O-trimethoxybenzoate 394 Serpentine 37, 40, 394 Serpentinic acid 38 Serum proteins 420 Sesquiterpene lactones 79, 299 Sesquiterpenes 231 Shogaols 6 Siaresinolic acid 9 Sitosterol 172, 211, 215, 219, 222, 223, 335,

337,339 Specifoline Speciociliatine Speciofoline Speciogynine Specionoxeine Speciophylline Spectinomycin Spleen lymphocytes Steroidal alkaloids Sterols Stigmasterol Strictosidine Strychnobiline Styraxin

31 26,31,254

27 26,31,254

26 26,31

345,360 233

47 339

215,339 259

57 403

Tanacetin Taxiphyllin Tectol Testicular function Tetrahydroalstonine Tetrandine Tetraphyllicine Thebain Thymol Tiliroside Tribuloside Trichophyton Tricontane Tricontanol Triglochinin Triterpenoidsaponins Tropacocaine Tropane alkaloids Tropanol Tryptophan

Vaccinia virus Valepotriate Valepotriates Valtrat Valtrathydrine Vanilline Verrucarin Vinblastine Vinblastinesulfat Vincamin Vincoside Vincristine Vindoline Vindolinine Vomalidine

Yohimbine

80 409 219 275

35, 40 275 394 195

5 93

93, 94 351 223 223

408, 410 87 14 66

195 346

233 305 203

120, 306 120

9 231 195 40

40, 195 259 195 40 40

394

40,195, 390, 394

10

Contents

A B E , H . , M . S A K A G U C H I , H . KONISHI, T. T A N I , S. A R I C H I , The Effects of Saikosaponins on

Biological Membranes. I. The Relationship between the Structures of Saikosaponins and Haemolytic Activity. 160

A B E , H . , S. ODASHIMA, S. A R I C H I , The Effects of Saikosaponins on Biological Membranes. II. Changes in Electron Spin Resonance Spectra from Spin Labelled Erythrocyte and Erytrocyte Ghost Membranes 287

A D E S I N A , S. K., J. B. H A R B O R N E , The Occurrence and Identification of Flavonoids in T h a u -m a t o c o c c u s d a n i e l l i B E N T H 323

A R E N S , H . , J. STÖCKIGT, E. E. WEILER, M . H . Z E N K , Radioimmunoassays for the Determina­

tion of the Indole Alkaloids Ajmalicine and Serpentine in Plants 37 ÄYRÄS, P. and C.-J. W I D E N , N M R Spectroscopy of Naturally Occuring Phloroglucinol

Derivatives. Part I: The Carbon-13 Spectral Properties of Some Model Compounds 144 B A N D O N I , A. L., J. E. M E D I N A , R. V . D. R O N D I N A , J. D . COUSSIO, Genus Baccharis L.,

I: Phytochemical Analysis of a non Polar Fraction from B . c r i s p a SPRENGEL 328 B A R E , C . E., V . K. T O O L E , W . A . G E N T N E R , Temperature and Light Effects on Germination

of P a p a v e r b r a c t e a t u m L I N D L . , P . O r i e n t a l e L., and P . s o m n i f e r u m L. 135 B A R Z , W . , M . K E T T N E R , W . H Ü S E M A N N , On the Degradation of Nicotine in Nicotiana Cell

Suspension Cultures 73 B H A T I A , R. K . , D . R. L O H A R , D . D . C H A W A N , S. P. G A R G , Chemical Control of Yield of

Sennosides in S e n n a Leaves 437 BISHT, N . P. S. and R. SINGH, Chemical Studies of C o n v o l v u l u s m i c r o p h y l l u s SIEB. 222 B L O S Z Y K , E., B. GEPPERT, B. D R O Z D Z , Quantitative Determination of Sesquiterpene Lactones

in Plant Material by Infrared Spectroscopy 79 B O W E N , I A N , H . and J O H N R. LEWIS, Rutaceous Constituents, Part 10: A Phytochemical

and Antitumor Survey of Malayan Rutaceous Plants 129 B U R R E T , E., A . J. C H U L I A , A. M . DEBELMAS, K . H O S T E T T M A N N , Presence d'Isoscoparine,

d'Isoscoparine -7-0- Glucoside et de Saponarine dans G e n t i a n a p n e u m o n a t h e L. (Presence of Isoscoparine, Isoscoparine -7-0- Glucoside and Saponarine in G e n t i a n a p n e u m o n a n t h e L.) 176

C H A R I , U . M . , M . J O R D A N , H . W A G N E R , Structure Elucidation and Synthesis of Naturally Occuring Acylglycosides; Structure of Tiliroside, Tribuloside and Ipomine 93

C H A W L A , A. S., V . K . K A P O O R , P. K . S A N G A L , Cycloart - 23 ene - 3ßy 25 - diol from

T r i c b o l e p s i s g l a b e r r i m a 109 C H U L I A , A. J., K . H O S T E T T M A N N , M . L. B O U I L L A N T , A . M . M A R I O T T E , Contribution a

l'Etude du Genre G e n t i a n a . Un 3'-0-Glucoside d'Isoorientin chez G e n t i a n a p e d i c e l l a t a (Contribution to the Study of the Genus G e n t i a n a . A 3'-0-GIucoside of Isooriendine in G e n t i a n a p e d i c e l l a t a ) 442

C O U N E , C , L. A N G E N O T , Le Dolichantoside, un Alcaloide Nouveau du S t r y c h n o s goss-w e i l e r i E X C E L L (Dolychantoside, a New Alkaloid from S t r y c h n o s g o s s w e i l e r i ) 53

D A L L ' O L I O , G . , B. TOSI, A . BRUNI, A New Rapid Fluorescent Labelling Method for the Detection of the Latex Tissues in Euphorbiaceae Plants 183

D I X I T , V . P., P. K H A N N A , S. K . B H A R G A V A , Effects of M o m o r d i c a c h a r a n t i a L. Fruit Extract on the Testicular Function of Dog 280

D O M I N Q U E Z , X . A. , C. M A R T I N E Z , A. C A L E R O , X . A . D O M I N Q U E Z jr., M . H I N O J O S A , A. Z A -

MUDIO, W . W A T S O N , V . Z A B E L , Mexican Medicinal Plants, X X X I : Chemical Components from „Jiquelite", I n d i g o f e r a s u f f r u t i c o s a , M I L L . 172

E L S A Y E D , M . A . , A B D E L S A L A M , M . A. , A B D E L S A L A M , N . A. , M O H A M M E D , Y. A. , Spectro-

photometric Determination of Emetine and Lobeline by Charge Transfer Complexation 430

Contents 11

E L SHERBAINI, A . E. A. , H . I. E L SISSI, M . A . M . N A W W A R , M . A . E L A N S A R I , The Flavonoids

of the Seeds of L y g o s r a e t u m 335 E R D Ö S , A. , R. F O N T A I N E , H . F R I E H E , R. D U R A N D , T H . PÖPPINGHAUS, Beitrag zur Pharma­

kologie und Toxikologie verschiedener Extrakte, sowie des Harpagosids aus H a r p a g o ­p h y t u m p r o c u m h e n s D . C . (Contributions to the Pharmacology and Toxicology of difTerent Extracts as well as the Harpagosid from H a r p a g o p h y t u m p r o c u m h e n s ) 97

FLOSS, H . G. , C . - J . C H A N G , O. M A S C A R E T T I , K . SHIMADA, Studies on the Biosynthesis of

Antibiotics 345 F R A N K E , W . , B. BRUMMER, Flüchtige Stoffwechselprodukte aus A s c o i d e a h y l e c o e t i (Volatile

Substances from A s c o i d e a h y l e c o e t i ) 332 F R A N K E , W . und B. BRUMMER, Terpene aus A s c o i d e a h y l e c o e t i (Terpenes from A s c o i d e a

h y l e c o e t i ) 426 H A N D J I E V A , N . and v. G . Z A I K I N , DIA-Valtrate a New Valepotriate from C e n t r a n t h u s

r u b e r (L.) D C . 203 H A R D M A N N , R. and R. G . S T E V E N S , The Influence of N A A and 2,4-D on the Steroidal

Fractions of T r i g o n e l l a f o e n u m - g r a e c u m Static Cultures 414 H A R T M A N N , G . R., H . R I C H T E R , E. M . W E I N E R , W . Z I M M E R M A N N , O n the Mechanism of

Action of the Cytostatic Drug Anguidine and of the Immunosuppressive Agent Ovalicin, two Sesquiterpenes from Fungi 231

H E G N A U E R , R., Arzneipflanzen, Gestern, Heute und Morgen (Medicinal plants, in the past, today and tomorrow) l

H E R R M A N N , H . D. , G . W I L L U H N , B. M . H A U S E N , Helenalinmethacrylate, an new Pseudo-

guaianolide from the Flowers of A r n i c a m o n t a n a L. and the Sensitizing Capacity of their Sesquiterpene Lactones 299

H I E R M A N N , A. und T H . K A R T N I G , Flavonoide in den Blättern von D i g i t a l i s l a n a t a 2 . Mit­teilung (Flavonoids in the Leaves of D i g i t a l i s l a n a t a , 2nd Communication) 225

H I E R M A N N , A. , Flavonoide in den Blättern von D i g i t a l i s l a n a t a , III. (Flavonoids in the Leaves of D i g i t a l i s l a n a t a , III) 443

H O Y E R , G . A., A . H U T H , J . N I T S C H K E , C H . v. SZCZEPANSKI, Holarrhesin, ein neues Steroid-

alkaloid aus H o l a r r h e n a floribunda (Holarrhesin, a new Steroidal Alkaloid from H o l a r -r h e n a floribunda) 47

H U H T I K A N G A S , A. , A . H U U R R E , M . L O U N A S M A A , Studies on the Biosynthetic Origin of the

Acyl Side Chains of D r y o p t e r i s Fern Constituents 397 IKRAM, M . , M . ISRAR K H A N , N . K A W A N O , Chemical Investigation of Aesculus indica 337

INNOCENTI, G. , F. D A L L ' A C Q U A , P. RODIGHIERO, G . C A P O R A L E , Biosynthesis of O-Alkyl -

furocoumarins in A n g e l i c a a r c h a n g e l i c a 167 Iwu, M . M . and W . E. C O U R T , The Alkaloids of R a u w o l f i a c u m m i n s i i 390 JOSHI, K. C , R. K. B A N S A L and R. P A T N I , Chemical Constituents of G y m n o s p o r i a m o n t a n a

and E u o n y m u s p e n d u l u s 211 JOSHI, K. C , P. SINGH, R. T. PARDASANI, Chemical Constituents of the Stern Heartwood

of M a r k h a m i a s t i p u l a t a 219 K O L O D Z I E J , H . und H . FRIEDRICH, Konstitutionsermittlung eines Procyanidins in N e l i a

m e y e r i 226 LEMLI , J., J . C U V E E L E , Les transformations des heterosides anthroniques pendant le sechage

des feuilles de C a s s i a senna et de R h a m n u s f r a n g u l a , X X X I (Transformation of anthron-glycosides by drying of the leaves of C a s s i a senna and R h a m n u s f r a n g u l a . Investigations on anthraquinone drugs, X X X I ) 311

L O U N A S M A , M . , G . MASSIOT, * H Nuclear Magnetic Resonance Spectral Analysis of Some K n i g h t i a d e p l a n c h e i Alkaloids 66

L O U N A S M A A , M . et P. V A R E N N E , Derive's Phloroglucinoliques d * H a g e n i a a b y s s i n i c a . V : Spectrometrie de Masse en Ionisation Chimique de la Kosotoxine, de la Protokosine et de l'a-Kosine. (Phloroglucinol Derivatives of H a g e n i a a b y s s i n i c a . V : Chemical Ionisation Mass Spectrometry of Kosotoxin, Protokosin and a-Kosin) 153

12 Contents

L O U N A S M A A , M . and H . P U H A K K A , Stereoselective Reduction of l-Acetyl-3, 4, 6, 7, 12, 12b - hexahydroindole (2,3- a) quinolizine to the Corresponding 1 - (1-Hydroxyethyl) Derivative 180

M E N D E Z , J . , 6-Hydroxykynurenic Acid from Leaves of L i m o n i u m Species 218 M O Y N A , P. and J . D . D i F A B I O , Composition of Cactaceae Mucilages 207 N A K A G U R A , N . , G . H Ö F L E , D . C O G G I O L A , M . Z E N K , The Biosynthesis of the Ipecac Alkaloids

and of Ipecoside and Alangiside 381 O E I - K O C H , A . L J . K R A U S , Inhaltsstoffe von E u r y c o m a l o n g i f o l i a , I: Sterole, Saponine

(Constituents of E u r y c o m a l o n g i f o l i a I: Sterols, Saponines) 339 P E R E , D. , P. R O U G E , S. LASCOMBES, Precipitation des Glycoproteines du Serum Humain

Normale par les Lectines de quelques Especes de L a t h y r u s (Precipitation of Human Serum Glycoproteins by the Lectins from Various L a t h y r u s Species) 420

R O L F S E N , W., L. B O H L I N , S. K. Y E B O A H , M . G E E V A R A T N E , R. V E R P O O R T E , New Indole

Alkaloids of S t r y c h n o s d a l e and S t r y c h n o s e l a e o c a r p a 264 SETHI , V . K. , M . P. J A I N and R. S. T H A K U R , Chemical Constituents of C r a t a e v a r e l i g i o s a 223 S H E L L A R D , E . J., P. J . H O U G H T O N , M A S E C H A B A R E S H A , The Mitragyna Species of Asia,

Part X X X I : The Alkaloids of M i t r a g y n a speciosa K O R T H from Thailand 26 S H E L L A R D , E. J., P. J . H O U G H T O N , M A S E C H A B A R E S H A . The Mitragyna Species of Asia,

Part X X X I I The Distribution of Alkaloids in Young Plants of M i t r a g y n a speciosa K O R T H grown from Seed obtained from Thailand 253

S O O D , S., B. O . G U P T A , S. K . BANERJEE, Constituents of Skimmia laureola 338

S T A H L , E . und E. W I L L I N G , Extraktion von Alkaloiden mit überkritischen Gasen in direkter Koppelung mit der Dünnschicht-Chromatographie (Extraction of Alkaloids with Super-critical Gases in Direct Coupling with Thin - Layer Chromatography) 192

S U G A Y A , A . , T . T S U D A , E . S U G A Y A , M . T A K A T O , K. T A K A M U R A , Effect of Chinese Medicine

„Saiko-Keishi-To" on the Abnormal Bursting Activity of Snail Neurons 294 T A M A D A , M . , K. E N D O , H . H I K I N O , Maokinine, Hypertensive Principle of E p h e d r a Roots 291 TITS, M . J . G . , L. A N G E N O T , Nouveau Alcaloides bis - Indoliques dans L'Ecorce des

Racines du S t r y c h n o s v a r i a h i l i s (New Bis - Indole Alkaloids in the Bark of S t r y c h n o s v a r i a h i l i s Root) 57

T I T T E L , G . , V . M . C H A R I , H . W A G N E R , HPLC-Analyse von V a l e r i a n a m e x i c a n a Extrakten (HPLC-Analysis of V a l e r i a n a m e x i c a n a Extracts) 305

TLWARI, R. D . , J . SING, Phytochemical Investigation of C a s s i a l a e v i g a t a Pods, Part I: Isola­tion and Structural Studies of two new Flavonol Glycosides 319

T O M O W A , M . P. und R. G J U L E M E T O W A , Steroidsaponine und Steroidsapogenine. V I : Furo-stanolbisglykosid aus T r i b u l u s t e r r e s t r i s L. (Steroidsaponines and Steroidsapogenines. V I : Furostanolbisglycoside from T r i b u l u s t e r r e s t r i s L.) 188

U L U B E L E N , A . and A. A T E S , Steroidal Compounds of S m y r n i u m c o n n a t u m 215 U L U B E L E N , A . and M . T A N K E R , Alkaloids of M e r e n d e r a c a u c a s i c a 216 U L U B E L E N , A. , Y . SAIKI, H . L O T T E R , V . M . C H A R I , H . W A G N E R , Chemical Components of

S t y r a x o f f i c i n a l i s IV. The Structure of a New Lignan Styraxin 403 V A N V A L E N , F.: Contribution to the Knowledge of Cyanogenesis in Angiosperms 408 V E R P O O R T E , R., E. W. K O D D E , A. B A E R H E I M - S V E N D S E N , A Chromatographie Comparison

of S t r y c h n o s d o l i c h o t h y r s a and S t r y c h n o s u r c e o l a t a 62 V E R P O O R T E , R., A . H . M . V A N RIJZEN, J . SIWON, A . B A E R H E I M - S V E N D S E N , Tertiary alkaloids

of P y c n a r r h e n a n o v o g u i n e e n s i s . Studies on Indonesian Medicinal Plants I 274 W I C H T L , M . , Drogenanalytik und Arzneibuch, kritisch betrachtet (Drug Analysis and Phar-

makopoea, a Critical Survey) 113 Woo, W. S., S. S. K A N G , H . W A G N E R , O . SELIGMANN, V . M . C H A R I , Triterpenoid Saponins

from the Roots of P h y t o l a c c a a m e r i c a n a 87

Planta medica

Journal of Medicinal Plant Research

Editor-in-Chief Editorial Board E. Reinhard, Univ. Tübingen H.P.T.Ammon, Tübingen Pharmazeutisches Institut W. Barz, Münster Auf der Morgenstelle E. Reinhard, Tübingen 7400 Tübingen O. Sticher, Zürich

November 1978 Vol. 34

Hippokrates Verlag Stuttgart

N o . 3 H. Wagner, München M. H. Zenk, Bochum

On the Mechanism of Action of the Cytostatic Drug Anguidine and of the Immunosuppressive Agent Ovalicin, two Sesquiterpenes from Fungi Guido R. Hartmann, Hartmut Richter, Erika M. Weiner and Wolfgang Zimmermann

Institut für Biochemie der Ludwig-Maximilians-Universität München, Bundesrepublik Deutschland.

Key Word Index: Anguidine; Verrucarin; Ovalicin; Fungal Sesquiterpenes; Lymphocytes; DNA Synthesis, Protein Synthesis.

Abstract A at the low concentration of 10 8 mol/1 . t . , . j . inhibit protein synthesis in lymphocytes

The sesquiterpene betainyl-anguidine r J • J I r J J -

* Dedicated to Professor A D O L F B U T E N A N D T i s blocked at similarly low concentra-

and the structurally related verrucarin from mouse spieen rapidly after addi-tion to the cell culture. D N A synthesis

on the occasion of his 75th birthday. tions whereas R N A synthesis is much

232 Hartmann et al.

less reduced. The following observa-tions support the notion that D N A syn­thesis is blocked via inhibition of pro­tein synthesis: (i) D N A synthesis in a cell-free System is not reduced by the drug; (ii) in cell culture protein synthe­sis is inhibited more rapidly and at lower concentrations than D N A syn­thesis; (iii) inhibitors of protein synthe­sis such as cycloheximide or puromycin are similar to betainyl-anguidine in their action on lymphocytes whereas specific inhibitors of D N A synthesis such as cytosine arabinoside or hydroxy-urea only partially reduce protein syn­thesis in lymphocytes.

The sesquiterpene ovalicin at a con-centration of 10 1 0 mol/1 acts as a very potent inhibitor of D N A synthesis in proliferating lymphocytes and in lym-phoma cells. R N A and protein synthe­sis are only weakly affected. The fol­lowing observations support the con-clusion that D N A synthesis is blocked only indirectly: (i) D N A synthesis in a cell-free System is not reduced by ova­licin; (ii) a cell-free System for D N A synthesis prepared from ovalicin-trea-ted lymphocytes shows an impaired synthetic activity; (iii) in cell culture the action of ovalicin is not immediate and requires a 8-15 hour period of in-cubation.

Introduction

Fungi produce a large number of chemical Compounds which are highly toxic either for bacteria or for animals and plants. They are toxic because they inhibit biochemical reactions essential for sustaining life. Their unusual speci-

ficity for either animals and plants or bacteria is based on fundamental dif-ferences existing between molecular constituents of the prokaryotic and eukaryotic cell. If the receptor for the target of such a toxic Compound occurs only in a eukaryotic cell exclusively this type of cell will be affected. The specificity of action is particularly pro-nounced if such a Compound possesses a very high affinity for its target. Then very small concentrations are sufficient for the inhibitory action to be observed. If higher concentrations (10-6 - 10 3

mol/1) are required the high specificity of action may be lost because weak for-ces such as hydrophobic interactions may provoke unspecific binding of these Compounds also to other cellular constituents with concomitant impair-ment of their biological functions. Therefore Compounds which elicit bio­logical effects at very low concentration command higher interest.

In recent years the effort to suppress the uncontrolled growth of tumor cells and other rapidly proliferating cells such as committed lymphocytes has stimulated a general interest in Com­pounds which act cytostatic for euka­ryotic cells.

In this report the mode of action of two diff erent Compounds will be discus-sed which exhibit a high cytostatic activity at very low concentration and act exclusively against eukaryotic cells.

Chemically both Compounds are distantly related since they possess the basic structure of a sesquiterpene.

/. A n g u i d i n e a n d r e l a t e d C o m p o u n d s Anguidine is produced by various

species of F u s a r i a and shows mycotoxic

Anguidine, Ovalicin 233

anguidine: trichothec-9-ene-3,4,15-triol,12,13-epoxy, 4,15-diacetate, (3a, Aß):

O

R t = O H ; R 2 = R 3 = O - C - C H 3 betainyl-anguidine (chloride):

O O II + II

Rj = 0 - C - C H 2 - N ( C H 3 ) 3 ; R 2 = R 3 = 0 - C - C H 3

ci-verrucarin A : R, = H ; R 2 , R 3 = dicarbonic acid ester of:

O OII C H 3 O

II I I II - 0 - C - C H - C H - C H 2 - C H 2 - 0 - C - C H = C H - C H

O

II - C H - C - O -

activity. Chemically it is closely related with the group of verrucarins [1]. Cy­tostatic activity of anguidine is obser-ved at a concentration of 0.5 X 10-8

mol/1 in mouse mastocytoma cells as well as in human tumor cells (KB cells). At a similarly low concentration the propagation of the D N A containing vac-cinia virus and its cyptopathogenic eff ect is suppressed [2], With respect to the molecular target of anguidine it has been observed previously that protein synthesis in a cell culture is inhibited at a concentration of 3 X 10"7 mol/1 in eukaryotic cells such as rabbit reticu-locytes or mouse mastocytoma cells. At the same concentration, however, D N A synthesis is equally blocked [2]. Synthe­sis of proteins and of D N A is achieved

by biochemically very different proces-ses. Therefore the question arises if an­guidine acts on such unlike biosynthetic pathways by two different independent modes of action. This ist not a far-fet-ched question. For example, it has been observed that the antibiotic rifampicin specifically blocks R N A synthesis in bacteria [3]. On the other hand it inhi-bits the multiplication of vaccinia virus in cell cultures by a completely different mechanism [4]. The simultaneous Inhi­bition of two different biochemical reactions, however, may also be explai-ned without the assumption of two dif­ferent modes of action if both reactions are tightly coupled i n v i v o .

In view of the observed possibly im-munosuppressive activity [2] we have studied the molecular mode of action of anguidine in spieen lymphocytes from mice. In the absence of an antigenic or mitogenic Stimulus lymphocytes are metabolically very inactive. Lympho­cytes may be induced to proliferate by mitogens such as concanavalin A as is indicated by a strongly increased syn­thesis of R N A and proteins followed by a rapid synthesis of D N A after 25 h of incubation. In most of our experi-ments we have used the chemically modified derivative betainyl-anguidine which exhibits the same biological ef-fects as anguidine but is much better soluble in water. If betainyl-anguidine is added simultaneously with mitogen and radioactively labelled leucine to lymphocytes in cell culture the incor-poration of the radioactively labelled amino acid into proteins, determined after a 20 h incubation period, is redu­ced by more than the half in the pre­sence of only 1 X 1 0 8 mol/1 inhibitor.

234 Hartmann et al.

T a b l e I I n h i b i t i o n of p r o t e i n s y n t h e s i s i n l y m p h o c y t e s by b e t a i n y l - a n g u i d i n e o r v e r r u c a r i n A . 2 fA.% concanavalin A , 10 nmol mercaptoethanol, 5 [AC\ (3H)leucine (resulting specific radioactivity in the medium 13 Ci/mol) and inhibitor (as indicated) were added to 106 B6D2 mouse lympho­cytes from spieen (preincubated at 37° C for 10-20 h) in Eagle's M E M containing 5% fetal calf serum (38) (total volume 1.0 ml). After incubation for 20 h at 37° C incorporation of radioactive iabel into acid insoluble material was determined (39) (control in the absence of betainyl-angui­dine: 4731 ± 1 4 9 counts X min" 1; control in the absence of verrucarin A : 8531 ± 9 1 4 counts X min"1).

inhibitor concentration Inhibition of protein (M) synthesis in cell culture (%>)

betainylanguidine 4 X 10*' 20 1 X 10"8 79

verrucarin A 4 X 10"9 30 1 X 10"8 83

T a b l e I I I n f l u e n c e of t h e c e l l c y c l e o n t h e i n h i b i t o r y a c t i o n of v e r r u c a r i n A . The experiments were performed essentially as described in table I except that 2 X 10"8 mol/l verrucarin A and (3H)leucine were added to the cultures at the time indicated after addition of concanavalin A .

verrucarin A pulse of incorporation incorporation added at [3H]leucine in absence of drug (h) (h) (cpm) (cpm)

7. 8 - 1 0 69 412 15. 16 - 18 81 1079 39. 40 - 42 45 447

The same effect is observed x with ver­rucarin A, a Compound closely related to anguidine (table I). Cellular pro­tein synthesis is blocked rather rapid­ly. Incubation for only 3 h with 2X10"8

mol/1 verrucarin Ais sufficient for a very strong inhibitory effect to be observed. This is observed no matter whether toxin is added 7,15 on 39 hours after the mito­gen (table II). To determine whether the synthesis of all cellular proteins is redu­ced to the same extent the following ex-periment was performed: 2X10"8 mol/1

inhibitor was added to stimulated lym­phocytes which was followed by radio­actively labelled methionine one hour later. After two more hours the cellu­lar proteins were separated by Polya­crylamide gel electrophoresis in pre­sence of dodecylsulfate. Except for a single protein zone migrating with the relative electrophoretic mobility of 0.52 the synthesis of all other radioactively labelled proteins is uniformly reduced (Fig 1). This is also true for the biosyn­thesis of histones as will be discussed

Anguidine, Ovalicin 235

8 6 4 m i g r a t i o n [cm]

<

2

e F i g . 1 . G e l - e l e c t r o p h o r e t i c a n a l y s i s of r a d i o a c t i v e p r o t e i n s s y n t h e s i z e d d u r i n g i n c u b a t i o n of s t i m u -l a t e d l y m p h o c y t e s w i t h (^S) m e t h i o n i n e i n t h e presence o r absence of b e t a i n y l - a n g u i d i n e . 5X10 7 lymphocytes stimulated as described in table III, were incubated for 39 h at 37° C . 1 h after addition of 2X10" 8 M betainyl-anguidine (BAC) 0.25 mCi (^S) methionine (specific radioactivity 20 Ci/mol) were added and the incubation continued for 2 h. Subsequently the cells from 4.5 ml of the incubation mixture were isolated by centrifugation. After washing witfc cold 0.85% N a C l half of the cells were lysed for 10 min at room temperature by suspending them in about two volumes 60 m M tris-HCl p H 6.8 containing 10% glycerol and 2.7% dodecylsulfate (total volume 50 fi\). 20 /u\ of this mixture (equivalent to 10e cells) were applied to a slab gel containing a linear gradient of 5-15% acrylamide with 0.1 % dodecylsulfate. Electrophoresis was carried out at 60 V overnight [43]. Autoradiography was performed with the dried slab gel for 6 days. The developed film was scanned with a densitometer.

below. Histones are proteins tightly associated with D N A in eukaryotic cells.

These observations support the con-clusion that in lymphocytes anguidine and related Compounds act in general at very low concentration on protein

synthesis. Furthermore, the action of anguidine and its derivatives is not restricted to a certain type of cell such as lymphocytes or to a certain species of animal as follows from experiments with rabbit reticulocytes [5] and HeLa cells [6]. Since they are effective at

236 Hartmann et al.

lower concentrations than other inhibi­tors of protein synthesis such as puro-mycin or cycloheximide (table VI and VIII and unpublished experiments) their general application in biochemical and biological experiments is suggested.

On the other hand, derivatives of anguidine also act as very potent inhi­bitors of D N A synthesis as is shown by the following experiment: inhibitor is added together with mitogen to a cell culture of stimulated lymphocytes.

When D N A synthesis is determined by incubation with radioactively labelled thymidine a 50% inhibition is observed in the presence of only 0.7 X 10 8 mol/1 of the drug, similar to the inhibition of protein synthesis. Doubling of the con­centration of the inhibitor leads to a complete inhibition. Verrucarin A is even more active. D N A synthesis is in-hibited at a 10 fold lower concentra­tion than protein synthesis (table III). Obviously anguidine is a very potent

T a b l e I I I I n h i b i t i o n of D N A s y n t h e s i s i n l y m p h o c y t e s by b e t a i n y l - a n g u i d i n e , v e r r u c a r i n A o r o v a l i c i n s t u d i e d i n c e l l c u l t u r e a n d i n a c e l l - f r e e S y s t e m . 100 concanavalin A, 0.5 ^amol mercaptoethanol and betainyl-anguidine were added to 5X107

lymphocytes in medium (see table I) (total volume 45 ml). After 38 h incubation at 37° C 0.9 ml aliquots of the cell Suspension were transferred to test tubes containing 1 ^uCi (3H)thymidine (specific radioactivity 6.7 Ci/mmol). Incorporation of radioactive label into acid insoluble material was determined after 8 h incubation at 37° C . Control without betainyl-anguidine: 26817±1351 counts X min"1. The experiments with verrucarin A in cell culture were performed essentially as described in table I except that (3H)leucine was omitted. After 36 h incubation 0.1 ml medium containing 1 ^wCi ( 3H) thymidine (specific radioactivity 2 Ci/mmol) and 10 nmol mercaptoethanol was added. 18 h later incubation was terminated. Control wihout verrucarin A : 9016±1784 counts X min"1. Experiments with ovalicin in cell culture: 1 ,wg concanavalin A and inhibitor was added to 106 lymphocytes preincubated for 12 h. After 38 h 1 ^ C i (3H)thymidine (specific radioactivity 6.7 Ci/mmol) was added and incubation continued for 3 h. Control without ovalicin: 61843 ±2313 counts X min' 1 . Synthesis of D N A in a cell-free System was measured essentially as described [7]. Nuclei were from lymphocytes stimulated for 46 h with concanavalin A in presence of 10 ftM mercaptoethanol in the experiment with betainyl-anguidine (as in table I). In the experiment with ovalicin mercaptoethanol was left out. Incubation of the cell-free System was 45 min at 37° C . Control without betainylanguidine: 1890 counts X min"1 X 10'6 nuclei; control without ovalicin: 436 counts X min"1 X 10"6 nuclei.

inhibitor concentration of drug inhibition of D N A synthesis (°/o) (M) in cell culture in a cell-free System

betainyl-anguidine 4 X 10"9 12 1 X 10"8 98 2 X 10"6 - 7

verrucarin A 4 X 10"9 33 1 X 10"9 96

ovalicin 2 X 10"10 50 4 X 10"10 73 3 X 10"6 75 0

Anguidine, Ovalicin 237

inhibitor of the synthesis of proteins as well as of D N A in lymphocytes. This f act has to be taken into consideration when discussing the molecular mode of action of this drug. In the case of verrucarin A D N A synthesis is even more sensitive than protein synthesis. Consequently we have to investigate the question if anguidine derivatives are double-hea-ded inhibitors or if they block a pro-cess common to both protein and D N A synthesis.

Such a process would be the produc-tion of energy in the form of A T P required for both biosynthetic reactions. However this is not the case as is shown by the following argument. Biosynthe­sis of R N A also requires energy in the form of A T P . However, R N A synthesis in lymphocytes is much less reduced by a concentration of the inhibitor which inhibits protein synthesis distinctly wit-

hin 3 h. Even 100 fold higher concentra­tions of the drug reduce R N A synthesis by only 50% (table IV). Such a relati-vely weak effect on R N A synthesis has been previously observed in mouse mastocytoma cells [2]. Of course, there are many other biochemical pathways which precede both D N A and protein synthesis. Any of them could be the target of anguidine. Instead of discuss­ing them separately we would like to mention several arguments which sup­port the notion that anguidine and its derivatives block D N A synthesis via the inhibition of protein synthesis.

(i) D N A synthesis in a cell-free System ist not inhibited by derivatives of anguidine. This process can be stu-died with nuclei obtained from stimu­lated lymphocytes [7]. During incuba­tion of this System the activated precur-sors of D N A , the four deoxynucleoside

T a b l e I V C o m p a r i s o n of i n h i b i t o r y a c t i o n of b e t a i n y l - a n g u i d i n e o n R N A - a n d p r o t e i n s y n t h e s i s . Inhibition of protein synthesis (3 h incubation): similar to experiments described in Fig 1 except that after 40 h incubation 0.9 ml aliquots of the cell Suspension was added to 0.1 ml Hank's Solution containing inhibitor. After 1 h (8H)leucine was added and incorporation determined after another 2 h at 3 7 ° C (control without drug: 1 6 2 2 9 ± 3 0 1 0 counts X min"1); 20 h incubation: essentially as described in table I (control without drug 4731 ± 1 4 9 counts X min"1). Inhibition of R N A synthesis: 3 h incubation with drug: drug was added to 106 lymphocytes (table 1), 18 h after concanavalin A . 2 hours later 50 nCi (1 4C)uridine (specific radioactivity 415 (Ci/mol) was added and incorporation into acid insoluble material determined after 1 h (control without drug 693 ± 2 1 counts X min"1). 12 h incubation with drug: concanavalin A, (1 4C)uridine and drug were added together to the lymphocytes. Incorporation was determined after 12 h incubation (control without drug: 5 0 9 4 ± 4 0 9 counts X min"1).

percent inhibition of incorporation of betainyl-anguidine (M) leucine after uridine after

3 h 20 h 3 h 12 h incubation with drug incubation with drug

2 X 10"8 62 95 28 41 1 X 10"7 96 100 - 50 2 X 10"6 - 52 69

238 Hartmann et al.

T a b l e V Dependence of i n h i b i t o r y a c t i o n of b e t a i n y l - a n g u i d i n e o n p r o t e i n o r D N A s y n t h e s i s o n t h e t i m e of i n c u b a t i o n . Stimulated lymphocytes as described for the experiments in cell culture with betainyl-anguidine in table III were used. Short incubation with drug: 40-43 h after addition of concanavalin A ; long incubation with drug: 0-40 h after addition of concanavalin A ; protein synthesis was measured by incubation with (3H)leucine for 2 h; D N A synthesis was determined by incubation with (3H)thymidine for 2 h.

incubation period with molar concentration of drug required for betainyl-anguidine 50 percent inhibition of

protein synthesis D N A synthesis

short 1,5 X 10'8 5,0 X 10"8

long 0,5 X 10'8 0,5 X IQ'8

triphosphates dATP, dTTP, dGTP and dCTP, are incorporated into D N A Even in the presence of 2X10"6 mol/1 betain-ylanguidine this reaction is only slightly reduced (table III).

(ii) In a cell culture protein synthesis is inhibited more rapidly and at lower concentration of the drug than D N A synthesis. Comparing the inhibitory effects of betainyl-anguidine observed after a long or a short incubation period it is immediately obvious that the inhi­bition of protein synthesis decreases much less than that of D N A synthesis upon shortening the incubation time. The concentration of the inhibitor has to be increased by a factor of ten to in-hibit D N A synthesis to the same extent as protein synthesis during a short expo-sure to the drug (table V).

(iii) Specific inhibitors of protein synthesis such as puromycin or cyclo-heximide inhibit D N A synthesis in lymphocytes similarly to the derivati­ves of anguidine. This conclusion is derived from the Observation that puro­mycin or cycloheximide inhibit D N A synthesis in stimulated lymphocytes at

the same concentration as protein syn­thesis. Similar to betainyl-anguidine the effect of cycloheximide on D N A syn­thesis increases with the length of the exposure time. Smaller concentrations of the inhibitor are sufficient at a long incubation time (table VI).

All these observations are consistent with the hypothesis that protein bio­synthesis is the primary target of angui­dine and its derivatives. Subsequently D N A synthesis ceases. This effect is due to the tight coupling of D N A synthesis to protein synthesis in eukaryotic cells [8] which has been demonstrated with a large number of inhibitors of protein synthesis and by use of amino acid ana-logues in many different types of cells [9-15]. Probably the tight coupling is mediated by the process of histone bio­synthesis which is absolutely required for the formation of chromatin [16-18]. D N A biosynthesis can not be the pri­mary target of anguidine. Otherwise any blocking of D N A synthesis by spe­cific inhibitors should lead to a general termination of protein biosynthesis. This question was studied in the f ollowing ex-

Anguidine, Ovalicin 239

T a b l e V I I n f l u e n c e of i n h i b i t o r s of p r o t e i n s y n t h e s i s o n t h e D N A s y n t h e s i s i n l y m p h o c y t e s . The effect of puromycin on protein synthesis was measured under conditions similar to those described in table I except that puromycin was added 39 h after concanavalin A. 1 h later the cells were isolated by centrifugation and resuspended in medium containing, in addition to mitogen, mercaptoethanol and puromycin, 5 ,«Ci (3H)leucine (specific radioactivity 50 Ci/mol). Incorporation was determined after 1 h. Control without puromycin: 4800 counts X min"1. For measuring D N A synthesis the isolated cells were resuspended in medium as above except that (3H)leucine was replaced by 1 fxQX (3H)thymidine. Incorporation was determined after 1 h. Control without puromycin: 6800 count X min" 1. Experiments with a 6 h incubation of drug: similar to those described in table III with betainyl-anguidine except that betainyl-anguidine or cycloheximide were added 38 h after concanavalin A . 4 h later 1 p C i (3H)thymidine or 5 / *Ci (3H)leucine were added and incorporation determined after 2 h. Controls without drug: incor­poration of (3H)leucine 15850 counts X min" 1; incorporation of (3H)thymidine 20705 counts X min"1. Experiments with cycloheximide or betainyl-anguidine (incubation with drug for 2 or 12 h resp.): similar to table III except that drug was added either 29 or 39 h after concanavalin A . 1 /aCi(3H)thymidine was added 40 h after mitogen and the incubation terminated after 1 h. Control without cycloheximide (2 h pulse): 4642 counts X min" 1; (12 h pulse): 11242 counts X min" 1; control without betainyl-anguidine (2 h pulse): 3972 counts X min' 1 ; (12 h pulse): 10886 counts X min" 1.

inhibitor concentration period percent inhibition of incorporation (M) of incubation of

with drug (h) leucine thymidine

puromycin 1,0 X 10"4 2 93 87 cycloheximide 7,2 X 10"7 6 86 70 betainyl-anguidine 0,2 X 10"7 6 92 83

cycloheximide 1,8 X 10"7 2 - 4 12 - 67

betainyl-anguidine 0,2 X 10"7 2 - 7 12 - 85

rapidly and more efficiently than D N A synthesis during a short incubation (table V). Therefore D N A synthesis is hardly the primary target of this drug.

If protein synthesis i n v i v o is the pri­mary target of anguidine one would expect that this drug is also a very potent inhibitor of protein synthesis in a cell-free ribosomal System. Indeed, it has been demonstrated previously by many investigators that this classs of Compounds inhibits initiation as well as elongation of protein synthesis on eukaryotic ribosomes in a cell-free

periments: Cytosine arabinoside2 inhi­bits D N A synthesis in stimulated lym­phocytes. Simultaneously biosynthesis of histones but not of other proteins de-creases strongly (Fig. 2). Similarly hy-droxyurea3 inhibits D N A synthesis in lymphocytes much more strongly than protein synthesis (table VII). Contrary to these inhibitors of D N A synthesis anguidine blocks protein synthesis more

2 an antimetabolite of biosynthesis of dCTP. 3 a potent inhibitor of ribonucleoside di-

phosphate reductase, of an enzyme required for the synthesis of D N A precursors.

240 Hartmann et al.

H2o H3

12 10 8 6 4 2 0 m i g r o t i o n [ cm )

© e F i g . 2 . G e l - e l e c t r o p h o r e t i c a n a l y s i s of r a d i o a c t i v e l a b e l l e d n u c l e a r p r o t e i n s s y n t h e s i z e d d u r i n g i n c u b a t i o n of s t i m u l a t e d l y m p h o c y t e s w i t h ( 1 4 C ) l e u c i n e i n t h e presence o r absence c y t o s i n e a r a b i n o s i d e . Experiments were performed similar to Fig. 1 except that 20 ^wCi (14C)leucine (specific radio­activity 309 Ci/mol) and 4.5 X10" 5 M cytosine arabinoside were added. After incubation nuclei were prepared as described [7] and dissolved in dodecylsulfate as in Fig. 1. To resolve the histones the gel System described [44] was used. Nuclear proteins [7] from 2X10 6 lymphocytes were applied to the gel.

Table V I I I n f l u e n c e of i n h i b i t o r s of D N A s y n t h e s i s o n p r o t e i n s y n t h e s i s i n l y m p h o c y t e s . Experiments were performed similarly to those described in table III except that the drug in 0.1 ml medium was added to 0.9 ml aliquots of stimulated lymphocytes 39 hours after mitogen. One hour later (3H)thymidine or (3H)leucine were added and incorporation into acid insoluble material measured after another 2 hours of incubation; controls without drug for D N A syn­thesis: 1) 14381 ± 1 0 4 6 counts X min"1 (experiments with hydroxyurea); 2) 12240 ± 7 5 5 counts X min"1 (experiments with cytosine arabinoside). Controls without drug for protein synthesis: 1) 8 9 1 7 ± 2 2 3 (experiments with hydroxyurea); 2) 6240 counts X min"1 (experiments with cytosine arabinoside).

inhibitor concentration percent inhibition of incorporation of (M) thymidine leucine

hydroxyurea 1,3 X 10"4 77 10 13,0 X 10"4 96 36

cytosine 4,1 X 10"7 65 0 arabinoside 41,0 X 10"7 91 10

Anguidine, Ovalicin 241

T a b l e V I I I I n f l u e n c e of i n h i b i t o r s of p r o t e i n s y n t h e s i s i n c e l l c u l t u r e o n t h e r i b o s o m a l s y n t h e s i s of p r o t e i n s i n a c e l l - f r e e S y s t e m . Experiments in cell culture: Essentially as described in table IV except that verrucarin A or betainyl-anguidine were added 39.5 h after concanavalin A and (3H)leucine 30 min later. Incubation was terminated 1 h later. Experiments with cycloheximide were carried out similarly to the experiments in cell culture with betainyl-anguidine described in table III except that the drug was added 38 h after concanavalin A. 2 h later (3H)leucine was added and the incubation continued for 2 more hours. I n v i t r o experiments: essentially as described [40] with rat liver supernatant [41]. Ribosomes were prepared from lymphocytes stimulated for 42 h with concan­avalin A (table III). Experiments with added m R N A : 50 ,«g poly rU and 1 /uCl (3H)phenylalanin (specific radioactivity 9 Ci/mmol); time of incubation 40 min except for cycloheximide (20 min); control without drug: about 4100 counts X min"1 in both experiments. In the experiments using endogenous m R N A 1 ^ C i (3H)leucine was used as radioactive precursor; time of incubation: 20 min. Control without drug: 5195 counts X min - 1 .

inhibitor molar concentration required for 50 percent inhibition of protein synthesis

in ce 11 culture in a cell-free System poly U endogenous m R N A

no effect at verrucarin A 2 X 10"8 2 X 10'5 2 X 10"5

betainyl-anguidine 2 X 10*8 2 X 10'5 > 2 X 10"4

cycloheximide 4 X 10'7 4 X 10*4 1 X 10'3

system [5, 6, 19-25]. It is rather strik-ing, however, that usually more than 10"6 mol/1 anguidine or verrucarin A are required for inhibition under these conditions. We have confirmed these Observation for the inhibition of protein synthesis in a cell-free system with ribo­somes from stimulated lymphocytes using either polyribouridylic acid or endogeneous R N A as messenger. To obtain 50% inhibition 2 X 10"5 mol/1 of the toxin is required (table VIII). Obviously in this system protein syn­thesis is also much less sensitive to betai­nyl-anguidine than in cell culture. This unexpected discrepancy between the effect in cell culture and in a cell-free system has been observed with other inhibitors of protein synthesis such as cycloheximide [20, 26-28] and emetine

[12, 28] in various eukaryotic cells and cellfree Systems. We have confirmed the Observation with cycloheximide in sti­mulated lymphocytes (table VIII). It may be explained by assuming the pre­sence or absence of a soluble protein factor which determines the sensitivity to the inhibitors. On the basis of this assumption the concentration of such a soluble factor would control the inhibi­tory activity of the drugs. Indeed, such a soluble protein has been isolated from the supernatant of a yeast lysate which renders sensitivity towards cy­cloheximide to the cell-free system from yeast [29].

//. O v a l i c i n Ovalicin has been isolated from the

culture medium of the ascomycete Pseud-

242 Hartmann et al.

Ovalicin

eurotium ovalis STOLK by SIGG and

WEBER . The same authors have deter­mined its chemical structure [30]. Its im-munosuppressive activity i n v i v o deser-ves particular attention: the number of antibody producing cells in the spieen of mice immunized with sheep red blood cells decreases to less than one per cent if 600 mg ovalicin per kg mou­se is injected one day after immunisa-tion. With later injection the inhibitory effect declines rapidly. Similarly the graft versus host reaction is distinctly delayed by ovalicin. The drug reduces the number of mitoses in spieen cells of immunised mice whereas the mitotic index in the jejunum is unchanged [31, 32]. These observations suggested a study of the action of ovalicin on cells of the lymphatic system. Following our proposal SCHIMPL and WECKER have

investigated the effect of the drug on the induction of antibody production in cultures of mouse spieen cells [33]. Sur-prisingly the activity of ovalicin in this system turned out to be much higher than in the animal. Even at a concen­tration of 4 X 1 0 9 mol/1 a distinct in­hibition was observed [34]. One reason for the apparently lower activity in the animal may be the much higher meta-bolic turnover rate of the drug.

We decided to analyse the mode of action of ovalicin by measuring a more

convenient reaction in a lympocyte cell culture system. For this purpose we have chosen thymidine incorporation into D N A as an indicator of cell proli-feration in lymphocytes stimulated by mitogen. If ovalicin is added together with concanavalin A to murine splenic lymphocytes incorporation of radio­actively labelled thymidine into D N A during the S-phase is strongly reduced. Even at a concentration of 2 X 1 0 1 0

mol/1 ovalicin a 5 0 % decrease of thy­midine incorporation is observed (table III). However, at much higher concen­trations of the drug inhibition of D N A synthesis is by no means complete. The extent of this ovalicin resistant thymi­dine incorporation depends on many factors such as the concentration of the mitogen, cell density, presence of mer­captoethanol or time of measurement of D N A synthesis.

Ovalicin is not only active on D N A synthesis induced artificially by plant lec-tins such as concanavalin A, purified phy-tohaemagglutinin or poke weed mitogen (data not shown). In a mixed lympho-cyte culture proliferation is induced by the presence of several different anti-genic determinants on the cell surface of the allogeneic lymphocytes. In this system ovalicin also is strongly inhibi­tory to D N A synthesis (table IX).

Al l these observations demonstrate that ovalicin is one of the most potent low-molecular inhibitors of prolifera­tion of lymphocytes.

A Suspension of spieen cells contains populations of many different cells, among them lymphocytes of B and T type. Therefore the question arises if ovalicin inhibits proliferation only in certain types of cells. B and T cells may

Anguidine, Ovalicin 243

T a b l e I X I n f l u e n c e of o v a l i c i n o n t h e t h y m i d i n e i n c o r p o r a t i o n i n t o p r o l i f e r a t i n g l y m p h o c y t e s . M i x e d l y m p h o c y t e c u l t u r e : A mixture of 5 X10 5 spieen lymphocytes each from C 57 Bl/6 and DBA/2 mice were incubated at 3 7 ° C with or without drug inEagle's M E M containing 5°/o fetal calfserum [38] (total volume 1 ml). After 48 h / * C i (3H)thymidine (specific radioactivity 10 Ci/mmol) were added and the incubation continued for 6 h. L y m p h o h l a s t o m a c e l l s : Exponentially growing cells were cultured at 37° C in Dulbecco's modified Eagle's medium (without nonessential amino acids) containing 10% fetal calf serum (total volume 15 ml) with or without drug. After 48 h 0.9 ml aliquots were transferred to test tubes, 1 f t C i (3H)thymidine (specific radioactivity 0.5 Ci/mmol) in 0.1 ml was added and the incubation continued for 1 h. Spleen c e l l s ( a t h y m i c n u l n u m o u s e ) : 106 cells were incubated in the presence of 2 (A,% lipopolysac-charide and ovalicin for 36 h (total volume 1 ml). Subsequently 1 ^ C i (3H)thymidine was added and the incubation continued for 24 h. H u m a n p e r i p h e r a l b l o o d l y m p h o c y t e s : lymphocytes from fresh human blood (42) were treated as described under spieen cells except that 5 ^ug/ml Concanavalin A was used as mitogen.

type of cell ovalicin (mol/1) incorporation of [3H] thymidine (counts X min'1) inhibition

+ ovalicin — ovalicin (°/o)

mixed mouse lymphocytes 0,3 X 10' 1 1 2538 ± 917 6471 ± 1305 61 (C 57 Bl/6 + D B A 2)

S 49.1 lymphoblastoma 1,0 X 10'9 35410 ± 760 70820 ± 340 50 spieen cells

(athymic nu/nu mouse) 3,0 X 10-7 4609 ± 343 13557 ± 1213 65 peripheral human lymphocytes 2,0 X 10-7 1971 ± 260 3131 ± 226 37

be stimulated separately by various mitogens such as concanavalin A which induces only T cells whereas lipopoly-sacdiaride from the outer membrane of gram-negative bacteria induces only B cells [35], In each case the proliferation induced by these type-specific mitogens is inhibited by very small concentra­tions of ovalicin (table IX). This is also true for the mitogen induced prolifera­tion of human peripheral lymphocytes (table IX). Similarly, thymidine incor­poration in transformed lymphocytes such as monoclonal S 49.1 mouse lym-phoma cells [36] is strongly reduced after 48 h incubation with the toxin (table IX). This Observation demonstra-tes the direct action of ovalicin on

lymphocytes. Its action is not mediated by cell-cell interaction, for example via macrophages.

The inhibitory action of ovalicin on cells different from those of the lym-phatic system has also been studied. Thymidine incorporation in 3T6 mouse fibroblasts or HeLa cells is distinctly less inhibited by ovalicin. Incubation with 2 X l O 7 mol/1 drug for two days results in only 30-40 % inhibition whereas at this concentration inhibition in S 49.1 lymphoma cells is 80% at this concentration. However, the smaller in­hibitory effect on 3T6 or HeLa cells is already detectable at the low drug con­centration of 2 X l O 9 mol/1 (data not shown). These observations suggest

244 Hartmann et al.

that lymphocytes are more susceptible to the action of ovalicin than other cells, in agreement with observations on the eff ects of the drug on the various animal tissues [31].

In the experiments reported so far incorporation of the nucleoside thymi­dine into D N A was used as analytical tool to demonstrate the activity of ova­licin. If D N A synthesis is directly affec­ted the drug should be able to block the incorporation of deoxyribonucleoside triphosphates into D N A in a cell free system from lymphocytes [7]. Howe­ver, even at concentration of 3 X 10*6

mol/1 ovalicin does not act inhibitory in this system (table III). Obviously the polycondensation of precursors to D N A is not the target of the drug. On the other hand D N A synthesis is clearly reduced in a cell-free system prepared from lymphocytes which had been preincubated with ovalicin compared with the activity of a system from untreated cells (table X). This Observa­tion supports the notion that ovalicin inhibits the formation of an essential

element of the D N A synthesizing machinery.

If this notion is correct it is be expec-ted that the time of addition of the in­hibitor to the cell culture should clearly influence the extent of inhibition. In-deed, this notion has been confirmed. If ovalicin is added to a spieen cell cul­ture at various times after mitogenic Stimulation maximal inhibition is ob­served only when the drug is added until the 6th to the 8th hour after mito­gen. If added 16-20 h after mitogen no effect is measured on the D N A synthesis measured 14 h later (Fig. 3). Obvious­ly the early phase of lymphocyte Stimu­lation is particularly sensitive to the inhibitor.

On the other hand, characteristic reactions occurring immediately after addition of the mitogen are not at all reduced by the drug. This conclusion may be derived from the Observation that ovalicin added 6 h after the mito­gen shows its füll inhibitory activity (Fig. 3). The same conclusion is derived from the investigation of the lipid

T a b l e X I n f l u e n c e of o v a l i c i n o n t h e i n c o r p o r a t i o n of ( * H ) d T T P i n t o D N A of n u c l e i of l y m p h o c y t e s . The experiment was performed as described in table III except that nuclei were prepared from lymphocytes (2X10 6 cells/ml) stimulated for 46 h with 1 ,ug/ml concanavalin A in the absence of mercaptoethanol; concentration of ovalicin: 7X10" 7 mol/1.

pretreatment of [3H] d T T P incorporation into nuclei lymphocytes (counts X min"1 X 10*6 nuclei)

+ Con A 115 4- ovalicin

+ Con A 231 — ovalicin

— Con A 31 — ovalicin

Anguidine, Ovalicin 245

• i i i i i i i

1 1 • 0 4 8 12 16

addition of ovalicin after mitogen (h)

F i g . 3. I n f l u e n c e o f t h e t i m e of a d d i t i o n of o v a l i c i n o n ( s H ) t h y m i d i n e i n c o r p o r a t i o n i n t o D N A of s t i m u l a t e d l y m p h o c y t e s . 1 /ig/ml concanavalin A was added to 106 lymphocytes (preincubated for 12 h at 37° C). 2X10* 7 M ovalicin were added after the time indicated on the abscissa. 30 h after mitogen (3H)thymidine was added and the incubation continued for 6 h. Controls without ovalicin: 2 1 8 3 5 ± 1 0 9 4 countsX m i n 1 = 100%.

turnover which, in lymphocyte mem­branes, is significantly increased very soon after addition of mitogen [37]. The lipid turnover is increased also in presence of ovalicin (table XI).

These observations may lead to the conclusion that ovalicin acts on stimu­lated lymphocytes only during a very short phase of the cell cycle (6-12 h after addition of mitogen). However, this conclusion could not be confirmed. Rather a 14-16 h incubation period of the drug is required for maximum in­hibition of stimulated spieen lympho­cytes from mouse as is shown by the

following experiments: in the first ex­periment the drug was added to the cell culture various times after the mitogen. 20 h after addition of the mitogen the inhibitor was removed from all samp-les by repeated washing (Fig. 4 (•)). The strongest inhibition of D N A syn­thesis was observed when ovalicin had acted on the cells for at least 16 h. In the second experiment the drug was added uniformly to all samples 6 h after mitogen. It was then removed from the samples after 2-14 h incuba­tion (Fig. 4 ( ± ) ) . As in the previous experiment an incubation period of at

246 Hartmann et al.

T a b l e X I I n f l u e n c e of o v a l i c i n o n t h e i n c o r p o r a t i o n of ( u C ) a c e t a t e i n t o l e c i t h i n f r o m l y m p h o c y t e s . Lymphocytes from spieen (2X10 6 cells/ml, preincubated for 15 h) were incubated in Eagle's M E M containing 2.5% calf serum, 1 ag/ml concanavalin A and 10 umol/1 mercaptoethanol. Incorpora­tion of ( 1 4C) acetate (56 Ci/mol) into lecithin was measured essentially as described [37]. Concen­tration of ovalicin: 2X10" 7 mol/l (added with Con A).

additions incorporation of [14C]acetate into lecithin from lymphocytes between 0-4 h after addition of Con A (counts X min"1)

lecithin content (in % of total lipids)

+ Ccn A 904 15.6 + ovalicin

+ Con A 803 14.3 — ovalicin

— Con A 567 13.5 — ovalicin

least 14 h is required to obtain the strongest inhibitory effect. This long incubation period may be required for several reasons: existence of various populations of cells in the culture, a very slow permeation of the drug into the cells or a metabolic activation of ovalicin.

However we were unable to detect any metabolic products in the medium of the cell culture after a long incuba­tion with the drug which would inhibit stimulated lymphocytes with higher activity (data not shown).

The preceding experiments show that the cell cultures have to be incubated with ovalicin for a rather long period of time to obtain a maximal inhibitory effect. But at which time can the first effect of the drug be observed? If the rate of D N A synthesis is measured using short pulses of radioactively labelled thymidine and starting imme-diately after addition of mitogen, an

incorporation of thymidine into D N A is observed as soon as 7-8 h after mito­gen (Fig. 5). Although exceedingly small, it is clearly higher than that ob­served in a cell culture not having received mitogen. If 2 X 10"7 mol/1 ovalicin are added together with the mitogen even this very early incorpora­tion is distinctly inhibited (Fig. 5). Again the effect of ovalicin is not obser­ved immediately after addition but requires an incubation period of several hours (data not shown).

Al l these observations are at variance with D N A synthesis as a direct target of the drug. Therefore the action of the toxin on the biosynthesis of other mac-romolecules has also been investigated.

Incorporation of radioactively la­belled uridine into the acid insoluble material of cell lysate was used to assay for the biosynthesis of R N A . Incorpo­ration of uridine into R N A is signifi-cant even in resting lymphocytes. It is

Anguidine, Ovalicin 247

time (h) of addition of drug {•)

16 12 8 i* 0

0 U 8 12 16

time(h) of removal of drug W —* F i g . 4 . I n f l u e n c e of t h e l e n g t h of i n c u b a t i o n p e r i o d of l y m p h o c y t e s w i t h o v a l i c i n o n t h e i n h i b i t i o n of D N A s y n t h e s i s . 1 4«g ConA was added to 106 lymphocytes (preincubated for 12 h at 37° C) in 1 ml medium. In one series of experiments (•) 3X10" 9 M ovalicin were added after addition of ConA at the time indicated on the upper abscissa and the drug removed 20 h after addition of mitogen by sedimen-ting of the cells and resuspending in fresh medium. This washing procedure was repeated twice. Controls were treated similarly. 25 h after addition of mitogen 1 ^uCi (3H)thymidine was added and the incubation continued for 11 h. In a second series of experiments (A) 3X10" 8 M ovalicin were added 6 h after mitogen and removed by repeated washings after the times indicated on the lower abscissa. Controls without drug: 29198 ± 2 6 8 9 counts X min' 1 = 100%.

not reduced even by 3 X 10"7 mol/1 ovalicin. In lymphocytes stimulated with concanavalin A R N A synthesis is only weakly inhibited after 20 h incu­bation with the drug (Table XII). Ob-viously, R N A synthesis as followed by total incorporation of a precursor, is not the target of the drug.

Furthermore, this Observation also indicates that the production of energy in lymphocytes is not the target of ova­licin because R N A synthesis is strongly dependent on this process.

In eukaryotic cells, D N A synthesis is tightly coupled to protein synthesis as has been discussed in the section on anguidine. Therefore the influence of ovalicin on protein synthesis has been investigated. The 'incorporation of radioactively labelled leucine into the acid insoluble material of cell lysates was used to assay for protein biosyn­thesis. 14 h incubation with 2 X 10'7

mol/1 ovalicin leads to a 20 % reduction of protein synthesis whereas the small extent of D N A synthesis is already redu-

248 Hartmann et al.

10 12 14 16

incubation (h) after addition of mitogen

F i g . ß. I n f l u e n c e of o v a l i c i n o n D N A s y n t h e s i s i n l y m p h o c y t e s observed s h o r t l y a f t e r a d d i t i o n of m i t o g e n . Lymphocytes were stimulated as described in table XI . Incorporation of (3H)thymidine (0.5 Ci / mmol, 1 ^aCi/ml) during a one hour pulse was measured; concentration of ovalicin used: 2X 10"7 mol/1.

ced by 60-70% at this early phase of the cell cycle (table XIII). The inhibi­tion of protein synthesis is increased to 40% when the incubation period with the drug is extendedto40h (table XIII). This increased extent of inhibition may

be the consequence of the blocked bio­synthesis of D N A as has been discussed above. In this context we wish to men-tion the Observation that the elongation process of protein biosynthesis as mea­sured with ribosomes from stimulated

Anguidine, Ovalicin 249

T a b l e X I I I n f l u e n c e of o v a l i c i n o n t h e i n c o r p o r a t i o n of ( i 4 C ) u r i d i n e i n t o R N A of s t i m u l a t e d l y m p h o c y t e s . 1 ag concanavalin A/ml and 3X10* 7 mol/1 ovalicin were added to 106 lymphocytes/ml (preincu-bated for 12 h at 3 7 ° C). After 20 h 80 nCi ( 1 4C)uridine (specific radioactivity 414 Ci/mol) were added and the incubation continued for 1 h.

additions incorporation of [ 1 4C] uridine during a one hour pulse (counts X min"1)

+ Con A 2651 + ovalicin

+ Con A 3419 — ovalicin

— Con A 1170 + ovalicin

— Con A 1186 — ovalicin

T a b l e X I I I I n h i b i t i o n by o v a l i c i n of t h e i n c o r p o r a t i o n of ( * H ) l e u c i n e o r ( s H ) t h y m i d i n e i n t o a c i d i n s o l u b l e m a t e r i a l of s t i m u l a t e d l y m p h o c y t e s . Preincubated lymphocytes were stimulated essentially as described in table XI . Protein synthesis was followed by incorporation of (3H)Ieucine (specific radioactivity 57 Ci/mol when added 12 h after mitogen, 13 Ci/mol when added 36 h after mitogen) during a 2 h pulse essentially as described in table I. Incorporation of (3H)thymidine (specific radioactivity 0.5 Ci/mol) during a 2 h pulse was determined essentially as described in table III. Concentration of ovalicin: 2X10' 7 mol/1 (added with Con A) .

additions time of addition of radioactive precursor

incorporation of [3H] leucine (counts X min*1)

incorporation of [ 3H] thymidine (counts X min"1)

+ ovalicin 12 5700 ± 139 443 ± 30 — ovalicin 12 7741 ± 27 1094 ± 28 + ovalicin 36 4391 ± 62 13250 ± 934

— ovalicin 36 8565 ± 308 40594 ± 1664

lymphocytes in a cell-free system is not inhibited by 3 X 10*7 mol/1 ovalicin (table XIV).

If the inhibition of protein biosyn­thesis by the toxin is the direct conse-quence of the inhibition of D N A syn­thesis the formation of the histones should be affected particularly as has been shown above for cytosine arabino­

side by electrophoretic analysis (Fig. 2). However, this is not found. In the pre­sence of ovalicin the formation of all proteins is uniformly reduced (data not shown).

Summarizing the numerous observa­tions reported we have to conclude that neither D N A nor protein synthesis is a direct target of ovalicin. This notion is

250 Hartmann et al.

T a b l e X I V Effect of o v a l i c i n o n t h e p o l y r U d i r e c t e d s y n t h e s i s of p o l y p h e n y l a l a n i n e i n a c e l l - f r e e s y s t e m w i t h r i b o s o m e s f r o m s t i m u l a t e d l y m p h o c y t e s . Ribosomes were prepared from lymphocytes stimulated as described in table XI except that mercaptoethanol was added 23 h after addition of mitogen. The experiments with the cell-free system were performed as in table VIII except that the time of incubation was 15 min.

additions incorporation of [3H] Phenylalanine (counts X min"1)

no 2630 ± 494 + 0.7 X10' 6 mol/1 ovalicin 2707 ± 310 + 1X10" 5 mol/1 ovalicin 2545 ± 293 without ribosomes 1040 ± 271

supported by a comparison of the action of ovalicin with that of cyclo­heximide or cytosine arabinoside on the transformation of small resting lym­phocytes into large blast cells. After addition of the mitogen to a cell culture the average diameter of the cells increa-ses up to the 48th hour of incubation as measured in the microscope. If cyclo­heximide is added together with the mitogen the average diameter of the lymphocytes remains unchanged as in the absence of mitogen. Obviously con-tinuous protein synthesis is required for blast cell formation. In the presence of cytosine arabinoside the average cell diameter increases up to the 24th hour almost as much as in the absence of this specific inhibitor of D N A synthesis. Düring the following day no further in-crease is observed. The effect of ovali­cin is very different from that of the drugs mentioned above. Here the for­mation of blast cells continues up to two days of incubation although it is distinctly slower than in the absence of

ovalicin (Fig. 6). In contrast D N A syn­thesis, as measured simultaneously by incorporation of thymidine into the acid insoluble material of the cells, is inhibited very extensively (Fig. 6). These oberservations indicate that the mechanism of action of ovalicin is very different from that of specific inhibi­tors of protein and D N A synthesis. Further investigations to pinpoint the target will be required. They promise to be rewarding since they will reveal the reason for the exceptionally high sensi­tivity of lymphocytes to the toxin as well as a key reaction crucial for lym-phocyte proliferation, since otherwise 10"10 mol/1 ovalicin would not be ex-pected to act inhibitory.

Acknowledgements

These investigations have been supported in part by the Sonderforschungsbereich 105 (Cyto-Iogische Grundlagen der experimentellen Biolo­gie) and by the Fonds der Chemischen Industrie. The toxins used have been kindly provided by Drs. H . - P . SIGG and H . STAEHELIN, Sandoz A G ,

Anguidine, Ovalicin 251

'c

>>

s

>

o

E g X3 <D cn o > o

I I I I

+ con A

(89%]

•con A + cytosine-arabinoside

(74%)

con A+cycloheximide

2U 36 48

incubation (hours) F i g . 6. I n h i b i t i o n of t h e t r a n s f o r m a t i o n of s m a l l l y m p h o c y t e s i n t o b l a s t c e l l s by o v a l i c i n , c y t o s t n e a r a b i n o s i d e o r c y c l o h e x i m i d e . Lymphocytes were stimulated as described in table X L Drugs were added togehter with the mitogen: 2X10" 7 M ovalicin, 1X10" 6 M cycloheximide or 1X10" 5 M cytosine arabinoside, respec-tively. About 5X10 5 washed cells (in 0.25 ml) were mixed with 0.5 ml warm 0.5% agarose in phosphate-buffered saline, and applied to slides.The coated slides were treated with 0.5% glutaral-dehyde in phosphate-buffered saline, washed three times for 5 min in destilled water, air-dried and Giemsastained [45]. The diameter of the stained cells was estimated at a magnification of 1:1250 in arbitrary units and the average diameter of 250 cells calculated. D N A synthesis (values in per cent are given in brackets) at 24, 36 and 48 h of incubation after addition of concanavalin A was determined by an one hour (3H)thymidine pulse (1 ^aCi/ml; 0.5 Ci/mmol) in 0.9 ml aliquots of the cell culture. 100% of D N A synthesis at 24, 36 and 48 h respectively: 19957 ± 8 4 , 50285 ± 3286, 116805 + 222 counts X min' 1 , respectively. In the presence of cycloheximide or cytosine arabinoside the incorporation of thymidine into the acid-insoluble material of the cells is neglegible.

and by Dr. C . T A M M , Institut für Organische Chemie, University of Basel. The first investi­gations on the mode of action of betainyl-an­guidine were performed by Dr. T . N E U D E C K E R in our laboratory. We wish to express our deep gratitude to Drs. A . SCHIMPL, E . W E C K E R and

C. J U N G W I R T H , Institut für Virologie und Im­munbiologie, University of Würzburg, for ad-vice, suggestions and many helpful discussions

and for providing us with informations about unpublished experiments. We are obliged to Dr. U . G E H R I N G , Institut für Biologische Chemie, University of Heidelberg, for providing us with lymphoblastoma cells and for advice to grow them in cell culture and to Dr. E.-L. W I N N A K -KER and his group, Institut für Biochemie, Uni­versity of München, for help in the experiments with HeLa and 3T6 cells.

252 Hartmann et al.

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