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W. Herz, Tallahassee. K. Hostettmann, Lausanne. H. Inouye, Kyoto. M. A. Iyengar, Manipal. F. Kaiser, Mannheim. F. H. Kem
Planta Journal of Medicinal Plant Research medica Organ der Gesellschaft für Arzneipflanzenforschung

Editor-in-Chief E . Reinhard, Tübingen Pharmazeutisches Institut A u f der Morgenstelle 8 D-7400 Tübingen

A . Baerheim-Svendsen, Leiden H . Böhm, Halle F. Bohlmann, Berlin A . Cave, Chatenay-Malabry P. Delaveau, Paris Editorial Board Ding Guang-sheng, Shanghai H . P. T. Ammon, Tübingen C. -J. Estler, Erlangen W . Barz, Münster N . Farnsworth, Chicago E . Reinhard, Tübingen H . Floss, Columbus •O. Sticher, Zürich H . Friedrich, Münster H . Wagner, München D . Fritz, Weihenstephan M . H . Zenk, München A . G . Gonzalez, L a Laguna Advisory Board O. R. Gottlieb, Sao Paulo N . Anand, Lucknow E . Graf, Köln R . Anton, Strasbourg H . Haas, Mannheim

E . Hecker, Heidelberg R . Hegnauer, Leiden W . Herz, Tallahassee K . Hostettmann, Lausanne H . Inouye, Kyoto M . A . Iyengar, Manipal F. Kaiser, Mannheim F. H . Kemper, Münster W . R. Kukovetz, Graz J. Lemli, Leuven Liang Xiao-tian, Beijing M . Lounasmaa, Helsinki M . Luckner, Halle J. Lutomski, Poznan H . Menßen, Köln E . Noack, Düsseldorf J. D . Phillipson, London

J. M . Rowson, Mablethorpe M . v. Schantz, Helsinki K . F. Sewing, Hannover E . J. Shellard, London S. Shibata, Tokyo C h . Tamm, Basel W . S. Woo, Seoul Xiao Pei-gen, Beijing

Contents Volume 49,1983

(f) Hippokrates

ISSN 0032-0943 Hippokrates Verlag Stuttgart

II

Contents 49,1983 A t t a - u r - R a h m a n , B a s h i r , M . \ Isolation of New Alkaloids from C a t h a r a n t h u s roseus A t t a - u r - R a h m a n , N i s a , M . , F a r h i , S.: Isolation of Moenjodaramine from Buxus p a p i l o s a B a l s e v i c h , J . , K u r z , W. G. W.: The Role of 9- and/or 10oxygenated Derivatives of Geraniol, Geranial, Nerol, and Neral in the Biosynthesis of Loganin and Ajmalicine Bassleer, R., M a r n e t t e , J . - M . , W i l i q u e t , P h . , D e P a u w - G i l l e t , M . - C L , C a p r a s s e , M . , A n g e n o t , L . \ Etüde complementaire de la cytotoxicite de la melinonine F, alcaloide derive de la ß-carboline (Complementary Study of Cytotoxic Activity of Melinonine F) Becker, H . , C h a v a d e j , S., W e b e r l i n g , F . : Valepotriates in Valeriana thalictroides Becker, H . , H e r o l d , S.: RP-8 als Hilfsphase zur Akkumulation von Valepotriaten aus Zellsuspensionskulturen von V a l e r i a n a w a l l i c h i i (RP-8 Auxiliary Phase for the Accumulation of Valepotriates from Cell-Suspension-Culture of V a l e r i a n a w a l l i c h i i ) B o u n t h a n h , C, R i c h e r t , L . , Beck, J . P . , H a a g - B e r r u r i e r , M . , A n t o n , R.: The Action of Valepotriates on the Synthesis of D N A and Proteins of Cultured Hepatoma Cells. . . . B r i a n g o n - S c h e i d , F . , L o b s t e i n - G u t h , A . , A n t o n , R.: H P L C Separation and Quantitative Determination of Biflavones in Leaves from G i n k g o b i l o b a C a p u t o , O., D e l p r i n o , L . , V i o l a , F . , C a r a m i e l l o , R. B a l l i a n o , G.: Biosynthesis of Sterols and Triterpenoids in Tissue Cultures of C u c u r b i t a m a x i m a C h a g n o n , M . , N d i b w a m i , A . , D u b e , S., B u m a y a , A . : A c tivite Anti-Inflammatoire d'Extraits de C r a s s o c e p h a l u m m u l t i c o r y m b o s u m (Anti-inflammatory Action of an Extract from C r a s s o c e p h a l u m m u l t i c o r y m b o s u m ) C h a t t o p a d h y a y , S., C h a t t o p a d h y a y , U., M a t h u r , P . P . , S a i n i , K . S., G h o s a l , S.: Effects of Hippadine, an Amaryllidaceae Alkaloid, on Testicular Function in Rats Chen W e i m i n g , Yan Y a p i n g , L i a n g X i a o t i a n : Alkaloids from Roots of A l s t o n i a yunnanensis

124 126

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158 64

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176

252 62

25

63

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F o u r n i e r , G . , P a r i s , M . : Mise en Evidence de Cannabinoi'des Chez P h e l i p a e a r a m o s a , Orobanchacees, Parasitant le Chanvre, C a n n a b i s s a t i v a , Cannabinacees (Detection of

G a l u n , £., A v i v , D . , D a n t e s , A . , F r e e m a n , A . \ Biotransformation by Plant Cells Immobilized in Cross-Linked Polyacrylamide-Hydrazide. Monoterpene Reduction by Entrapped M e n t h a C e l l s G h o s a l , Sh., S i n g h , A . K . , B i s w a s , K . : New6-Aryl-2-pyrones from G e n t i a n a p e d i c e l l a t a

240

H a f e z , A . , A d o l f , W., H e c k e r , E . \ Active Principles of the Thymelaeaceae. III. Skin Irritant and Cocarcinogenic Factors from P i m e l e a s i m p l e x

3

I e v e n , M . , v a n den B e r g h e , D . A . , V l i e t i n c k , A . J . \ Plant Antiviral Agents. I V . Influence of Lycorine on Growth Pattern of Three Animal Viruses I s h i g u r o , K . , Y a m a k i , M . T a k a g i , S.: Studies on Iridoidrelated Compounds; III: Gentiopicral, the Aglucone of Gentiopicroside

9

109

J a k o v l e v , V., I s a a c , O., F l a s k a m p , E . : Pharmakologische Untersuchungen von Kamillen-Inhaltsstoffen, V I . Untersuchungen zur antiphlogistischen Wirkung von Chamazulen und Matricin (Pharmacological Investigations with Compounds of Chamomile, V I . Investigations on the Antiphlogistic Effects of Chamazulene and Matricine) J o s h i , K . C , S i n g h , P . , T a n e j a , S.: A Sesquiterpenoid Naphthol from K y d i a c a l y c i n a Jossang, A . , Lebceuf, M . , C a b a l i o n , P . , C a v e , A . : Alcaloides des Annonacees. X L V : Alcaloides de P o l y a l t h i a n i t i d i s s i m a (Alkaloids from Annonaceae. X L V : Alkaloids of P o l y a l t h i a n i t i d i s s i m a )

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67 127

20

255

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E n d o , K . , O s h i m a , Y. K i k u c h i , H . , K o s h i h a r a , Y., H i k i n o , H . : Hypotensive Principles of U n c a r i a H o o k s E n g e l s h o w e , R.: Dimere Proanthocyanidine als Gerbstoffvorstufen in J u n i p e r u s c o m m u n i s (Tannin Producing Dimeric Proanthocyanidins in J u n i p e r u s c o m m u n i s ) . . . E s t e r l , A . , G a b , S., B i e n i e k , D . \ Z u r Kenntnis der Inhaltsstoffe von I s e r t i a h y p o l e u c a (On The Constituents of Isertia hypoleuca)

250

y

f

Dehaussy, H . , T i t s , M . , A n g e n o t , L . \ L a guattegaumerine, nouvel alcaloide bisbenzylisoquinoleinique de G u a t t e r i a g a u m e r i (Guattegaumerine, New Bisbenzylisoquinoline Alkaloid from G u a t t e r i a g a u m e r i ) D o m i n g u e z , X . A . , F r a n c o , R., C a n o , G., M a . Consue l o G a r c i a , F . , X o r g e A . D o m i n g u e z , S. J r . , L e o n a r d o de la P e n a M . : Isolation of a New Furano-l,4-Naphthaquinone, Diodantunezone from L a n t h a n a a c h y r a n t h i f o l i a . . . .

Cannabinoids in P h e l i p a e a r a m o s a , a Parasite of C a n n a bis sativa)

K i m u r a , Y., O h m i n a m i , H . , O k u d a , H . , B a b a , K . , K o z a w a , M . , A r i c h i , S.: Effects of Stilbene Components of Roots of P o l y g o n u m ssp. on Liver Injury in Peroxidized Oil-fed Rats K i s i e l , W.: 8-Epidesacylcynaropicrin from C r e p i s c a p i l l a r i s K i s o , Y., Suzuki, Y., W a t a n a b e , N . , O s h i m a , Y., H i k i n o , H . : Antihepatotoxic Principles of C u r c u m a l o n g a Rhizomes K i s o , Y., T o h k i n , M . , H i k i n o , H . : Assay Method for Antihepatotoxic Activity Using Carbon Tetrachloride Induced Cytotoxicity in Primary Cultured Hepatocytes . . K r a m , G . , F r a n z , G.\ Untersuchungen über die Schleimpolysaccharide aus Lindenblüten (Analysis of Linden Flower Mucilage) Krüger, D . , J u n i o r , P . , W i c h t l , M . \ Neue Cardenolidglykoside aus D i g i t a l i s l a n a t a (New Cardiac Glycosides from Digitalis lanata)

51 246 185

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149

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L a n g h a m m e r , L . , Schulze, G., G u j e r , R., M a g n o l a t o , D . , H o r i s b e r g e r , M . \ Isolation and Structure of a Rarely Occurring Cyanidanol Glycoside from Cortex Betulae . L e m l i , J . , C u v e e l e , J . , V e r h a e r e n , E . \ Chemical Identification of Alexandrian and Tinnevelly Senna. Studies in the Field of Drugs Containing Anthracene Derivatives XXXIV L o n g - Z e L i n , W a g n e r , H . , S e l i g m a n n , ö.\ Thalifaberine, Thalifabine and Huangshanine, Three New Dimeric Aporphine-Benzylisoquinoline Alkaloids

181

36

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III

Contents M a n - P o W o n g , T e h - C h a n g C h i a n g , H s o n - M o u C h a n g : Chemical Studies on Dangshen, the Root of C o d o n o p s i s p i l o sula M i s a w a , M . , H a y a s h i , M . , T a k a y a m a , S.: Production of Antineoplastic Agents by Plant Tissue Cultures. I. Induction of Callus Tissues and Detection of the Agents in Cultured Cells N a h r s t e d t , A . , W r a y , V., G r o t j a h n , L . , F i k e n s c h e r , L . H . , H e g n a u e r , R.: New Acylated Cyanogenic Diglycosides from Fruits of A n t h e m i s c a i r i c a N o m u r a , T, F u k a i , T. S h i m a d a , T, I h - S h e n g C h e n : Components of Root Bark of Morus australis. I. Structure of a New 2-Arylbenzofuran Derivative, Mulberrofuran D .

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O b a t a - S a s a m o t o , H . , K o m a m i n e , A . : Effect of Culture Conditions on D O P A Accumulation in a Callus Culture of S t i z o l o b i u m hassjoo O h i r i , F. C, V e r p o o r t e , R., B a e r h e i m Svendsen, A r . H NMR Chemical Shift Values for Aromatic Protons in 2,3,9,10and 2,3,10,11-tetrasubstituted Tetrahydroprotoberberine Alkaloids O h i r i , F . C , V e r p o o r t e , R., B a e r h e i m Svendsen, A:Tertiary Phenolic Alkaloids from C h a s m a n t h e r a dependens . . . O j e w o l e , J . A . O., A d e s i n a , S. K . : Mechanism of the Hypotensive Effect of Scopoletin Isolated from the Fruit of Tetrapleura tetraptera O j e w o l e , J . A . O., A d e s i n a , S. K . \ Cardiovascular and Neuromuscular Actions of Scopoletin from Fruit of T e t r a p l e u r a tetraptera O k o g u n , J . /., Adeboye, J . O., O k o r i e , D . A . \ Novel Structures of two Chromone Alkaloids from Root-Bark of Schumanniophyton magnificum

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P a l a c i o s , P . , G u t k i n d , G . , R o n d i n a , R. V. D . , de T o r r e s , R., C o u s s i o , J . D . : G e n u s B a c c h a r i s . II. Antimicrobial Activity of B . c r i s p a and B . n o t o s e r g i l a Pandey, V. B . , S i n g h , J . P . , M a t t o c k s , A . R., B a i l e y , £ . : A Note on "Isolation and Pharmacological Action of Heliotrine, the Major Alkaloid of H e l i o t r o p i u m i n d i c u m Seeds" P a t h a k , V. P . , S a i n i , T. R., K h a n n a , R. N . : A New Furanoflavone from Seeds of P o n g a m i a g l a b r a P e r e r a , P . , S a n d b e r g , F . , v a n Beek, T. A . , V e r p o o r t e , R.\ Tertiary Indole Alkaloids of T a b e r n a e m o n t a n a d i c h o t o m a Seeds P e r e r a , P . , v a n Beek, T. A . , V e r p o o r t e , /?.: Dichomine, a Novel Type of Iboga Alkaloid

162 17

46

Rueffer, M . , N a g a k u r a , N . , Zenk, M . H . : Partial Purification and Properties of S-Adenosylmethionine: (R), (S)-Norlaudanosoline-6-O-Methyltransferase from A r g e m o n e p l a t y c e r a s Cell Cultures

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S a r i y a r , G.: Alkaloids from P a p a v e r t r i n i i f o l i u m of Turkish Origin Seip, E . H . , O t t , H . H . , H e c k e r , £ . : Skin Irritant and Tumor Promoting Diterpene Esters of the Tigliane Type from the Chinese Tallow Tree ( S a p i u m s e b i f e r u m ) Sepulveda-Boza, S., F r i e d r i c h s , £., Puff, H . , B r e i t m a i e r , £.: Ein iso-Homoprotoberberin-Alkaloid aus den Wurzeln von B e r b e r i s a c t i n a c a n t h a ( A n iso-HomoprotoberberinAlkaloid from the Roots of B e r b e r i s a c t i n a c a n t h a ) . . . . Shoyama, Y., H a t a n o , K . , N i s h i o k a , /.: Clonal Multiplication of P i n e l l i a t e r n a t a by Tissue Culture Stoianova-lvanova, B . , Budzikiewicz, H . , Koumanova, B . , T s o u t s o u l o v a , A . , M l a d e n o v a , K . , B r a u n e r , A . : Essential Oil of C h r y s a n t h e m u m i n d i c u m

236

T e h - C h a n g C h i a n g , H s o n - M o u C h a n g , M a k , T h . C. W.: New Oleanene-type Triterpenes from A b r u s p r e c a t o r i u s and X-ray Crystal Structure of Abrusgenic Acid-Methanol 1:1 Solvate

165

v a n d e r S l u i s , W. G., v a n der N a t , J . M . , Spek, A . L . , I k e s h i r o , Y., L a b a d i e , R. P . : Gentiogenal, aConversion Product of Gentiopicrin (Gentiopicroside)

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28 232

R a v i d , U., P u t i e v s k y , E . : Constituents of Essential Oils from M a j o r a n a s y r i a c a , C o r i d o t h y m u s c a p i t a t u s and S a t u r e j a thymbra 248 Röder, E . , W i e d e n f e l d , H . , H o n i g , A . : Pyrrolizidinalkaloide aus Senecio a u r e u s 57 Rueffer, M . , N a g a k u r a , N . , Zenk, M . H . \ A Highly Specific OMethyltransferase for Nororientaline Synthesis Isolated from A r g e m o n e p l a t y c e r a s Cell Cultures 196

W a g n e r , H . , S c h w a r t i n g , G., V a r l j e n , J . , B a u e r , R., H a m d a r d , M . £., E l - F a e r , M . Z . , B e a l , J . : Die chemische Z u sammensetzung der Convolvulaceen Harze IV. Die Glykosidsäuren von I p o m o e a q u a m o c l i t , I . l a c u n o s a , I . p a n d u r a t a und C o n v o l v u l u s a l - s i r e n s i s (Chemical Constituents of the Convolvulaceae-Resins I V . The Glycosidic Acids of I p o m o e a q u a m o c l i t , I . l a c u n o s a , I . p a n d u r a t a and C o n v o l v u l u s a l - s i r e n s i s ) 154 W a t a n a b e , K . , W a t a n a b e , H . , G o t o , Y., Y a m a g u c h i , M . , Y a m a m o t o , N . , H a g i n o , K . : Pharmacological Properties of Magnolol and Hönokiol Extracted from M a g n o l i a offic i n a l i s : Central Depressant Effects 103 W i l l u h n , G., Röttger, P . - M . , M a t t h i e s s e n , U.: Helenalinund 11,13-Dihydrohelenalinester aus Blüten von A r n i c a m o n t a n a (Helenalin- and 11,13-Dihydrohelenalinester from Flowers of A r n i c a m o n t a n a ) 226 W i t t e , L . , B e r l i n , J . , W r a y , V., Schubert, W., K o h l , W., Höfle, G., H a m m e r , J . : Mono- and Diterpenes from Cell Cultures of T h u j a o c c i d e n t a l i s 216 Y a n g M i n g h e , Chen Y a n y o n g : Steroidal Sapogenins in D i o s corea collettii Yu D e - Q u a n , D a s , B . C.: Structure of Hydroxymuscopyridine A and Hydroxymuscopyridine B , Two New Constituents ofMusk Yu D e - q u a n , Y., D a s , B . C: Alkaloids of A c o n i t u m b a r b a t u m

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183 85

IV

Subject Index 49,1983 A Abietane 220 Abrusgenic acid 165 Abruslactone 165 Acevaltrate 64,191 Adaptogenes 240 Alkaloids 17, 20, 25, 28, 32, 43, 55, 57, 62, 79, 85, 95,109,124,126,131,158, 162, 188,196, 232, 244, 252, 254 Amyrin 177 Antineoplastic agents 115 Antiviral agents 109 Apigenin 128 Aporphine alkaloids 20 Aporphine derivatives 43 Apparicine 232 Arabinose 149 Arylbenzofuran derivatives 90 Avenasterol 177

B Benzylisoquinoline alkaloids 131 Berberine alkaloids 32 Biflavones 204 Bilobetin 204 Biosynthesis of alkaloids 131, 196 Biosynthesis of ajmalicine 79 Biosynthesis of loganin 79 Biosynthesis of sterols 176 Biosynthesis of triterpenoids 176 Biotransformation 9 Bisabolol 67 Bisbenzylisoquinoline alkaloids 20, 25, 55 Bisnorargemonine 14 Borneol 236 Bornyl acetate 236

C Cadambine 188 Caffeic acid 186 Camphor 216 Cannabinoids 250 Cardenolides 74 Carvacrol 216 Catechin 181 Catechinxylopyranoside 181 Cell cultures 9, 14 79, 115, 120, 131, 176, 191,196,216 Chamazulene 67 Cholestanol 177 Chromone alkaloids 95 Chrysanthenone 236 Chrysosplenetin 63 Cinchonamine 244 Cinnamic acid 186 Clonal multiplication 14

Cocarcinogens 3 Convolvulaceae resins 154 Coreximine 14 Coronaridine 28 Coumaric acid 186 Coumarins 46, 99 Cucurbitacin 176 Curcumin 185 Curcuminoids 185 Cyanogenic glycosides 143 Cycloartenol 177 Cymenol 216 Cynarin 224 Cynaropicrin 246 Cytotoxic action 158

Gentiogenal 211 Gentiopicral 208 Gentiopicrin 211 Gentiopicroside 208, 211 Geranial 79 Geraniol 79 Ginkgetin 204 Glycosidic acids 154 Glycyrrhetinic acid 224 Glycyrrhizin 224 Govanine 17 Guajazulene 67 Guattegaumerine 25

H D Daphnane derivatives 3 Dauricine 20 Daurisoline 20 Dehydrohelenalinester 226 Deoxyphorbol 199 Dichomine 232 Didrovaltrate 64, 138, 191 Diginatigenin derivatives 74 Digoxigenin derivatives 74 Dihydrocadambine 188 Dihydroquinamine 244 Diodantunezone 63 Diosgenin 38 Diterpene esters 3, 199 Diterpenes 216 Diterpenoid alkaloids 85 Dopa 120

Enzymes 196 Epilumicin 143 Epoxyphorbol 199 Erythrocentaurin 212 Essential oils 9, 67, 216, 236, 248

Fenchone 220 Ferruginol 220 Ferulic acid 186 Flavonoids 61, 63, 90, 128, 204 Fluorocarpamine 124 Fridelin 60 Furoflavones 61

Helenalin 226 Helenalinester 226 Helenanolides 226 Heliotrine 254 Hepatoma cells 138 Hibiscone 127 Hibiscoquinone 127 Hinikione 220 Hinokiol 220 Hippadine 252 Homovaltrate 64, 191 Honokiol 103 Huangshanine 55 Hydroxymuscopyridine 183 Hydroxymusizin glycoside 36

Iboga alkaloids 232 Ibogamine 28 Iboxygaine 232 Immobilized cells 9 Indicine 254 Indole alkaloid glucoside 188 Indole alkaloids 28, 62, 79, 124. 158, 188, 232 Iridoids208,211 Isochinoline alkaloids 196 Isodihydrocadambine 188 Isoginkgetin 204 Isoquinoline alkaloids 32 Isothujone 220 Isovaltrate 64, 191

K Kuwanon 90

G

L

Galactose 149 Genkwanin 128

Lasiocarpine 254 Laudanosoline 196

Subject Index Levomenol 67 Liliolide 240 Lindolhamine 20 Linoleic acid 220 Liriodenine 20 Lumicin 143 Lycorine 109

M Magnoiol 103 Mandelonitril glycosides 143 Matricine 67 Melionine 158 Mentenediol 220 Menthone 9 Methionine 224 Methylabrusgenat 165 Methylnorlaudanosoline 196 Methyltransferase 131, 196 Moenjodaramine 126 Monoterpenes 216 Morphinane derivatives 43 Morusin 90 Mucilage 149 Myrtanol 216

N

Naphthalene glycosides 36 Naphthaquinones 63 Neolignane derivatives 103 Neomenthol 9 Neral 79 Nerol 79 Norlaudanosoline 131 Normacusine 62 Nororientaline 196 Norreticuline 131

O

Opium alkaloids 131 Orientaline 196

P Pallidine 14 Palmatic acid 220 Papaver alkaloids 43 Pedicellatin 240

V

Pedicellin 240 Penduletin 63 Perakine 62 Perivine 232 Phorbol 199 Phytosterols 60, 176 Piceid51 Picroside 224 Pimelea factors 3 Pleiocarpamine 124 Podophyllotoxin 224 Polysaccharides 149 Proanthocyanidins 170 Protoberberin derivatives 32 Protoberberine alkaloids 20 Protocatechuic acid 186 Pseudoguaianolides 226,246 Puberaconitidine 85 Puberaconitine 85 Puberanidine 85 Puberanine 85 Pulegone 9 Pyrrolizidine alkaloids 57

Resins 154 Resveratrol 51 Reticuline 20, 131 Rhamnose 149 Rhoeadine derivatives 43

S

Sabinene 220 Sapogenins 38 Saponins 38, 60 Sarpagine 62 Schumannificine 95 Sciadopitysin 204 Scopoletin 46, 99 Secoiridoids 208,211 Sesquiterpene lactones 226, 246 Sesquiterpenes 127 Silybin 224 Simplexin 3 Sinapic acid 186 Sitosterol 220 Skin irritants 199 Spinasterol 177 Stemmadenine 28 Stepholidine 20 Steroidal alkaloids 126

Steroidal sapogenins 38 Stigmastenol 177 Stilbene51 Sugiol 220 Swertiamarin 240

Tabersonine 28 Tacaman alkaloids 232 Tannins 181 Taraxerol 60 Terpineol 216 Tertiary phenolic alkaloids 14 Tetrahydroalstonine 62 Tetrahydroisochinoline alkaloids 196 Tetrahydroprotoberberine alkaloids 162 Thalifaberine 55 Thalifabine 55 Thujaplicin derivatives 216 Thujone 220 Tigliane derivatives 3 Tinnevellin glycoside 36 Tripdiolide 109 Triterpenoids 60, 165 Tropolone 220 Tumor promotors 199

U

Ursolic acid 240 Ushinsunine 20

V

Valepotriates 64, 138, 191 Valtrate 64, 138,191 Vellosimine 62 Vindolinine derivatives 124 Vinorine 62 Voacangine 28 Voacristine 232 Voaphylline 28, 232 Vobasine 232

Y Yamogenin 38

Z Zierinxyloside 143

VI

Index of Names of Organisms 49,1983 Please note that the following plant families are listed as indicated below: Alsinaceae sub C a r y o p h y l l a c e a e Apiaceae sub U m b e l l i f e r a e Arecaceae sub P a l m a e Asteraceae sub C o m p o s i t a e Brassicaceae sub Cruciferae Clusiaceae sub G u t t i f e r a e Fabaceae sub L e g u m i n o s a e (including M i mosoideae = M i m o s a c e a e Caesalpinioideae = C a e s a l p i n i a c e a e and Papilionoideae = P a p i l i o n a c e a e = Fabaceae sensu stricto) Hypericaceae sub G u t t i f e r a e Lamiaceae sub L a b i a t a e Oenotheraceae sub O n a g r a c e a e Poaceae sub G r a m i n e a e y

Abrus cantoniensis 165 Abrus precatorius 165 Aconitum barbatum 85 Adlumia fungosa 133 Albertisia papuana 22 Alstonia yunnanensis 62 Althaea officinalis 152 Amaryllidaceae 109, 252 Annonaceae 20, 25 Anthemis altissima 143 Anthemis cairica 143 Anthocephalus cadamba 189 Apocynaceae 28, 62,79,124, 232 Araceae 14 Argemone intermedia 133 Argemone platyceras 131, 196 Arnica montana 226 Artemisia caruthii 67

B Baccharis crispa 128 Baccharis megapotamica 116 Baccharis notosergila 128 Baliospermum montanum 200 Berberidaceae 32,133 Berberis actinacantha 32 Berberis henryana 133 Berberis stolonifera 133 Berberis wilsonae 133 Betula species 181 Betulaceae 181 Blackstonia perfoliata 211 Bombax malabaricum 127 Boraginaceae 254 Brucea antidysenterica 116 Bryonia dioica 176 Buxaceae 126 Buxus papulosa 126

Cacalia floridana 57 Caesalpinia gilliesii 116 Caesalpiniaceae 36 Campanulaceae 60 Cannabis sativa 250 Cassia angustifolia 36 Cassia senna 36 Catharanthus roseus 79,124 Celastraceae 115 Cephalotaxaceae 115 Cephalotaxus harringtonia 115 Chamomilla recutita 67 Chasmanthera dependens 17, 162 Chelidonium majus 133 Chlorella ellipsoidea 178 Chrysanthemum indicum 236 Cissampelos mucronata 133 Clivia miniata 109 Codonopsis pilosula 60 Colchicum speciosum 116 Compositae 57,67,128,143,226,236,246, 255 Convolvulaceae 154 Convolvulus al-sirensis 154 Convolvulus microphyllus 154 Coridothymus capitatus 248 Corydalis pallida 133 Corydalis sempervirens 133 Crassocephalum multicorymbosum 255 Crepis capillaris 246 Crepis virens 246 Crinum asiaticum 252 Crinum augustum 252 Crinum latifolium 252 Crinum pratens 252 Cucumis sativus 176 Cucurbita maxima 176 Cucurbita pepo 177 Cucurbitaceae 176 Cupressaceae 170, 216 Curcuma longa 185

Fagara zanthoxyloides 116 Fumaria officinalis 133

Gentiana pedicellata 240 Gentianaceae 208, 211, 240 Ginkgo biloba 204 Ginkgoaceae 204 Glaucium flavum 133 Guatteria gaumeri 25

H Heliotropium indicum 116,254 Heliotropium supinum 254 Hibiscus species 127 Holacantha emoryi 116 Hura crepitans 3

I Ipomoea lacunosa 154 Ipomoea operculata 154 Ipomoea pandurata 154 Ipomoea quamoclit 154 Ipomoea turpethum 154 Isertia hypoleuca 244

Juniperus communis 170

Kydia calycina 127

D Dictiostelium discoideum 177 Digitalis lanata 74 Digitalis schischkinii 74 Dioscorea collettii 38 Dioscoreaceae 38

Labiatae 9,248 Lantana achyranthifolia 63 Leguminosae 36,46, 61, 99, 120,165 Lindera oldhamii 22, 26 Loganiaceae 158

M

Erythrina lithosperma 22 Eschscholtzia tenuifolia 133 Euphorbiaceae 199

Magnolia obovata 103 Magnolia officinalis 103 Magnoliaceae 103 Majorana syriaca 248 Malvaceae 127

Index of Names of Organisms Maytenus bucchananii 116 Menispermaceae 17, 133, 162 Menispermum dauricum 22 Mentha species 9 Mimosaceae 46, 99 Moraceae 90 Morus alba 90 Morus australis 90 Moschus moschiferus .183 Mycobacterium species 10

N

Nicotiana tabacum 250

O

Ochrosia moorei 116 Origanum compactum 249 Origanum floribundum 249 Origanum hirtum 249 Origanum majorana 249 Origanum maru 248 Origanum smyrnaeum 249 Origanum syriacum 248 Orobanchaceae 250

Vll

Penicillium expansum 211 Phelipaea ramosa 250 Pimelea linifolia 7 Pimelea prostrata 3 Pimelea simplex 3 Pinellia ternata 14 Podophyllum hexandrum 223 Polyalthia beccarii 20 Polyalthia emarginata 20 Polyalthia nitidissima 20 Polyalthia oligosperma 20 Polyalthia oliveri 20 Polyalthia suaveolens 20 Polygonaceae 51 Polygonum cuspidatum 51 Polygonum multiflorum 51 Polypodium vulgare 181 Pongamia glabra 61 Putterlickia verrucosa 115

Strychnos melinoniana 158 Strychnos usambarensis 158

T

Tabernaemontana dichotoma 28, 232 Tabernaemontana eglandulosa 232 Taxus brevifolia 116 Tetrapleura tetraptera 46, 99 Thalictrum dasycarpum 116 Thalictrum faberi 55 Thalictrum sparsiflorum 133 Thalictrum tuberosum 133 Thuja occidentalis 216 Thymelaeaceae 3 Thymus capitatus 248 Tilia species 149 Tiliaceae 149 Tripterygium wilfordii 115

R Ranunculaceae 55, 85,133 Rhus coriaria 248 Rubiaceae 95,188,244

U

Ulmus americana 181 Uncaria sinensis 188 Uncaria species 188

S

P Panax ginseng 60 Panax pseudo-ginseng 60 Papaver armeniacum 43 Papaver cylindricum 43 Papaver fugax 43 Papaver Orientale 196 Papaver somniferum 131,196 Papaver tauricola 43 Papaver triniifolium 43 Papaveraceae 43,131, 196 Papilionaceae 120

Sapium sebiferum 199 Satureja capitata 248 Satureja peltieri 249 Satureja thymbra 248 Schistosoma japonicum 199 Schumanniophyton magnificum 95 Schumanniophyton problematicum 97 Scolytus multistriatus 181 Scolytus ratzeburgi 181 Scrophulariaceae 74 Senecio aureus 57 Stizolobium hassjoo 120 Streptomyces clavuligerus 10

V

Valeriana edulis ssp. procera 64 Valeriana kilimandscharica 64 Valeriana thalictroides 64 Valeriana wallichii 138,191 Valerianaceae 64, 138, 191 Verbenaceae 63

Z Zingiberaceae 185

VIII

Pharmacology Index 49,1983 Biological Systems Organs / Diseases

Pharmacological Effects / Effects on

Plant/Constituent

Central nervous System

anticonvulsive muscle relaxant sedative muscle relaxant spasmolytic hypotensive hypotensive antifertility antibacterial antiviral tumor promotion antiinflammatoric antiinflammatoric

Magnolia officinalis, Magnoliaceae Magnolia officinalis Magnolia officinalis Tetrapleura tetraptera, Mimosaceae Tabernaemontana dichotoma, Apocynaceae Tetrapleura tetraptera, Mimosaceae Tetrapleura tetraptera, Mimosaceae Hippadine, Amaryllidaceae Baccharis crispa, Asteraceae Cliviaminiata, Amaryllidaceae Pimelea simplex, Thymelaeaceae Chamomillareculita, Asteraceae Crassocephalum multicorymbosum

Peripheral nervous System Autonomous nervous System Cardiovascular System Hormonal System Infectious deseases Tumors Inflammation

Page

103 103 103 99 28 46 99 252 128 109 3 67 255

1983, Vol. 49, pp. 131-137, ©Hippokrates Verlag GmbH

.

Journal of Medicinal

Planta "^5"

—Plant Research lllGIlICa

Partial Purification and Properties of S-Adenosylmethionine: (R), (S)-Norlaudanosoline-6-0-Methyltransferase from Argemone platyceras Cell Cultures M. Rueffer*, N. Nagakura** and M. H. Zenk* * Lehrstuhl P h a r m a z e u t i s c h e Biologie d e r Universität M ü n c h e n , D-8000 M ü n c h e n 2, Federal Republic of G e r m a n y ** K o b e W o m e n ' s C o l l e g e of Pharmacy, K o b e 6 5 8 , J a p a n R e c e i v e d : August 7 , 1 9 8 3 ; a c c e p t e d : A u g u s t 3 1 , 1 9 8 3

Key Word Index: Argemone platyceras; Papaveraceae; Cell Suspension Cultures; Alkaloid Biosynthesis; S-Adenosylmethionine: (R), (S)-Norlaudanosoline-6-OMethyltransferase.

Abstract A new enzyme, S-adenosylmethionine: (R), (S)norlaudanosoline-6-O-methyltransferase, was isolated from the soluble protein extract of A . platyceras cell cultures and purified approximately 80-fold. This enzyme catalyses the formation of 6-O-methylnorlaudanosoline, and, to a minor extent, 7-O-methylnorlaudanosoline from SAM and (S), as well as (R), norlaudanosoline. The apparent molcular weight of the enzyme is 47000 Dalton. The pH-optimum of the enzyme is 7.5, the temperature Optimum, 35° C. Apparent K values for (S) and (R)-norlaudanosoline were 0.2 mM, and for SAM, 0.05 mM. The transferase shows high Substrate specificity for tetrahydrobenzylisoquinoline alkaloids. Simple orthophenols, like phenylpropane derivatives, coumarins or flavonoids, are not accepted as Substrates. The enzyme is widely distributed in benzylisoquinoline-containing plant cell cultures and is present in differentiated plants like Papaver somniferum. M

Introduction (S)-NLS is now firmly established as the first intermediate in benzylisoquinoline alkaloid biosynthesis [1]. A recently discovered enzyme [2] catalyses its Abbreviations: N L S = Norlaudanosoline; S A M = S-Adenosyl-L-methionine; S A H = S-Adenosylhomocysteine, N L S - O M T = S-Adenosylmethionine: (R), (S)-Norlaudanosoline-6-0-Methyltransferase.

formation from dopamine and 3,4-dihydroxyphenylacetaldehyde. Further down the pathway, reticuline is proven to be a branch point intermediate in the biosynthesis of a vast array of structure types of benzylisoquinoline alkaloids, as for instance: morphinanes, protoberberines, proaporphines, cularines, dibenzopyrrocolines etc. (e.g. 3]. This means that NLS must be transformed to reticuline by three methylation reactions, two O-methylations at positions 6 and 4' and one Nmethylation (at atom 2). Since nor-reticuline has been amply demonstrated by feeding experiments to be a precursor of reticuline in vivo [4, 5], one has to assume that O-methylation of NLS precedes N-methylation for instance in opium alkaloids. O-Methylation of NLS is therefore an important reaction in the early steps of the biosynthesis of reticuline, the universal branch point intermediate. O-Methyltransferases have been reported to mediate the transfer of methyl groups from S A M to phenylpropanoid and flavonoid Compounds mainly at the meta position of the aromatic System, though para-O-methylation is not uncommon [6 and literature cited therein]. Specific-O-methyltransferases derived from plants and acting on tetrahydrobenzylisoquinolines have so far not been reported. Yet incubation of poppy latex with (R,S)-norlaudanosoline and C - S A M has led to the formation of labelled opium alkaloids, thus demonstrating indirectly the presence of methyltransferase enzymes [7]. There are, however, several reports on NLS-O-methylation, by animal enzymes [e.g. 8]. In our attempt to elucidate the enzymatic steps involved in isoquinoline biosynthesis in plants, we have investigated the O-methylation of NLS. In this report, we present the partial purification and properties of a new enzyme named norlaudanosoline-6O-methyltransferase from A . platyceras cell Suspension cultures. This enzyme catalyses the predominant transfer of the S-methyl group of SAM to the phenolic OH-group at position 6 of NLS, and to a lesser extent, to the 7-O-position. 14

132

Rueffer, Nagakura, Zenk R!0 R0 2

R0 3

R«0

3.

Norlaudanosoline 6- •0- methylnorlaudanosoline 7- •0- methylnorlaudanosoline

R1

R

H

H

CH

2

R3



R5

H

H

H

3

H

H

H

H

H

CH

H

H

H

5- •0- methylnorlaudanosoline

H

H

CH

3

H

H

U- -0- methylnorlaudanosoline

H

H

H

CH

H

Laudanosoline

3

3

H

H

H

H

CH

Norreticuline

CH

H

H

CH

3

H

Nororientaline Laudanidine

CH CH

H CH

CH H

H CH

H CH

1H = a = (S);

3

3

3

3

3

3

3

3

1H = p = (R)

Materials and Methods Plant Material A . p l a t y c e r a s cell culture was initiated and maintained since 1975 on Linsmeyer and Skoog (LS) medium [9]. Batch cultures in 1 litre Erlenmeyer flasks containing 250 ml medium were agitated on a gyratofy shaker (100 rpm) in diffuse light (750 lux) at 24° C and were subcultured at weekly intervals using about 10 % inoculum. The cells were harvested after 8 days, frozen in liquid nitrogen and stored at - 2 0 ° C. Cell fresh weight was determined after filtering the cells through a fritted glass funnel using suction. Aliquots were used for dry-weight determination. A l l other cell cultures were from our culture collections and were grown under identical conditions as given above.

no enzyme or no Substrate (NLS) yielded a blank value of about 8 % radioactivity, a value which was subtracted from all incubation mixtures. For product identification, the products were separated by H P L C using a Nucleosil-SA-column (25 mm x 3.2 mm i.d.) and 0.5 ammonium phosphate : methanol (80:20) as a solvent System. Retention times of the potential products were: 5'-0-methyl-NLS, 6.45 min.; 7-O-methyl-NLS, 7.35 min.; 4'-0-methyl-NLS, 8.31 min.; 6-O-methyl-NLS, 9.18 min. Preparative isolations were done by using the above incubation mixture x 100. The reaction product was extracted by ethylacetate and subjected to T L C (Sigel; solvent System: Chloroform : methanol: acetic acid : water = 18:6:3:0.3). The zone containing radioactivity was scraped off, purified for a second time in the solvent System: Chloroform : n-propanol : methanol : water = 45:15:60:40 (CHCl -phase), and its mass spectrum was measured in a Finnigan M A T 44S instrument. 3

Chemicals The following Compounds were obtained from the indicated sources: S-adenosyl-L-methionine hydrogen sulphate, Combithek (calibration proteins), S-adenosylhomocysteine, all from Boehringer, Mannheim; S-adenosyl-L-methyl- H-methionine was prepared enzymatically from methionine -S-methyl- H, (Radiochemical Centre Amersham) using Standard methods. A l l benzylisoquinoline alkaloids were synthesized according to Standard procedures. A C A 34 was purchased from L K B , DEAE-cellulose-microgranular form from Whatman, and hydroxyapatite from Bio-Rad. A l l other materials were of reagent grade. Liquid scintillation counting was performed in a toluene mixture (Rotiszint 22, Roth). 3

3

Enzyme purification Step 1:100gfrozen tissue wasallowed tothawin0.1 M K P 0 buffer, p H 7.5, containing 20 m M ß-mercaptoethanol, stirred for 20 minutes, pressed through cheese cloth, and centrifuged at 48000 xg for 10 min. Ammoniumsulphate precipitation was done from 0-70 % Saturation, centrifuged again for 10 min. at 48000 xg. The pellet was taken up in 0.1 M KP0 *-buffer, p H 7.5, containing 20 m M ß-mercaptoethanol. Step 2: The crude extract from step 1 (15 ml) was put on an U l trogel A C A 34 column (1 = 90 cm, 0 = 2.5 cm), equilibrated with 10 m M K P 0 - b u f f e r , p H 7.5, 20 m M ß-mercaptoethanol. Fractions of 4 ml were collected at a flow rate of 8 ml/h. The fractions (45-61) containing the enzyme activity were pooled and subjected to the next step. Step 3: The fractions containing the enzyme (59 ml), were subjected to ionexchange chromatography on DEAE-cellulose (1 = 10 cm, 0 = 1.5 cm). The column was equilibrated with 10 m M KP0 *-buffer, p H 7.5, containing 20 mM ß-mercaptoethanol, and a gradient was applied from 0-300 mM K C l (8 hrs). 4 ml Fractions were collected at a flow rate of 40 ml/h. The enzyme was found in fractions 56-64. The fractions containing the enzyme were pooled and applied to the next step. Step 4: The protein Solution was added to a hydroxyapatite column (1 = 10 cm, 0 = 1 cm), equilibrated with 10 m M KP0 "-buffer, p H 7.5, 20 m M ß-mercaptoethanol, and a gradient of 10-200 m M K P 0 , p H 7.5, was applied (8 hrs). 2 ml Fractions were collected at a flow rate of 30 ml/h. Enzyme activity was found in frac2

4

2

4

2

4

O - M e t h y l t r a n s f e r a s e Assay a n d P r o d u c t I d e n t i f i c a t i o n Düring enzyme purification, O M T activity was assayed against R/S-norlaudanosoline, except that with crude enzyme preparations, laudanosoline was substituted for its nor-derivative. The assay mixture consisted of KPO4* -buffer, p H 7.5 (130 mM), ascorbate (130 mM) R/S-norlaudanosoline (0.3 m M ) , H - S A M (0.1 m M , 10000 cpm), and varying amounts of enzyme in a total volume of 150 ul. The mixture was incubated for 45 minutes at 35° C and the reaction was terminated by addition of 200 ul Na C0 -buffer (1 M , p H 9.5). The methylated products were extracted by adding 400 ul isoamylalcohol and shaking for 45 min. The turbid mixture was cleared by centrifugation in an Eppendorf centrifuge for 5 min. 200 ul of the organic phase were transferred to scintillation vials and counted for radioactivity. Recovery of the methylated products was 95 % under these conditions. Blank mixtures containing either 2

3

2

3

2

4

2

4

2

4

Enzymes from Argemone Cell Cultures

133

Table I

tions 43-54 with N L S as a Substrate. Soluble protein was determined as described previously [10] or in more highly purified samples, with an optical method [11].

Survey of distribution of (R, S)-laudanosoline-methyltransferase activity in species of different isoquinoline alkaloid containing families (laudanosoline served a s Substrate)

Molecular Weight Determination The molecular weight determination of the purified O-methyltransferase was carried out by gel filtration on a calibrated G-100superfine column. Although only the Stokes radii of the proteins can be determined by this method, it is often used for the determination of the molecular weight assuming globular shape for the proteins. The column, 168 ml (1 = 95 cm, 0 = 1.5 cm), equilibrated with 10 m M K P 0 - b u f f e r , p H 7.5, 20 m M ß-mercaptoethanol, was eluted at a flow rate of 15 ml/h in 100 fractions of 2 ml. The column was calibrated with the proteins of the Combithek. Ferritin ( M W : 450000) was used for the determination of the void volume of the column. The Standards were monitored by the absorbance at 280 nm. The results are given as Stokes radii.

Plant material

Enzyme pkat/l medium

activity pkat/mg protein

Papaveraceae

6350



4620

13.8 12.2

Glauciumflavum

2855

27.7

Fumaria

officinalis

1950

11.1

Corydalis

pallida

Cell cultures:

2

4

Argemone

platyceras

Corydalis

sempervirens

Argemone Adlumia

Results

fungosa

3



somniferum

Chelidonium

The presence of O-methyltransferases in crude enzyme extracts of plant cell cultures of different taxonomic origin was investigated using (R, S)-laudanosoline and C H - S A M as Substrates. Laudanosoline was chosen as an initial methyl group acceptor to avoid interference with N-methyltransferases potentially present in the assay. Using these Substrates in the Standard assay, O-methyltransferases were detected in all cell cultures containing isoquinoline alkaloids thus far tested (Table I). Cell cultures of four different plant families tested for the presence of O-methyltransferase enzymes showed positive results. By far the highest absolute amount of O-methyltransferase per unit culture fluid was found in Argemone platyceras (Papaveraceae), a plant species known to contain isoquinoline alkaloids of the pavine, protoberberine, and aporphine types [12]. It was therefore decided to use cells of this species to purify and characterize the O-methyltransferase. (R, S)-norlaudanosoline was used as a Substrate in the purification of the enzyme since it was possible to demonstrate that using this plant tissue, there was no interference with N-methyltransferases under the conditons chosen for the assay. All of the NLS-methylating emzyme activity was detected in the 100000 xg supernatant of the homogenate of the Argemone cells. It could not be found in



intermedia

Papaver

3

Family

Eschscholtzia

majus



tenuifolia

1480

8.2

1360

33.7

1070

12.5

1020

9.3

915

6.8

590

4.2

Berberis

henryana

Berberidaceae

2050

38.7

Berberis

wilsonae



1650

13.9

Berberis

stolonifera

1260

18.9

2060 570

26.6 2.4

940

3.7

Thalictrum

tuberosum

Thalictrum

sparsiflorum

Cissampelos

mucronata

Ranunculaceae »

Menispermaceae

Differentiated plant:

pkat/gdwt pkat/mg

Papaver somniferum

218

1.6

the culture filtrate. The enzyme was isolated and partly purified by ammonium sulphate precipitation, A C A 34, DEAE-cellulose, and hydroxyapatite chromatography as described under "Materials and Methods". This procedure yielded a purification of approximately 80-fold with a recovery of 13 %. The data for a typical purification procedure are summarized in Table II. The protein Solution at the stage of highest purification did not contain any other enzymes of the isoquinoline biosynthetic pathway thus far tested. A typical elution profile of a hydroxyapatite column is shown in Fig. 1. Properties ofthe O-Methyltransferase The 80-fold purified enzyme was used to determine the catalytic properties. The activity of the

Table II Purification procedure for O-methyltransferase f r o m Argemone Purification step

platyceras

(R, S-norlaudanosoline a s Substrate)

Total activity

Total protein

Specific activity

Recovery

Purification

(pkat)

(mg)

pkat/mg

(%)

-fold

3626

259.6

14

100

(0-70%) Gel filtration (ACA = 3 4 )

3409

178.3 15.2

19

94

150

63.2

10

DEAE-cellulose c h r o m a t o g r a p h y

1124

2.5 0.4

459

31.0 13.2

32

1173

C r u d e extract

1

A m m o n i u m s u l f ate-precipitation

C h r o m a t o g r a p h y o n hydroxyapatite

2290 477

1.4

83

Rueffer, Nagakura, Zenk

134 •160

Fraction Fig. 1. Elution profile o f N L S - O M T f r o m a hydroxyapatite c o l u m n .

enzyme was measured at a ränge of pH 5-9 with different buffers as shown in Fig. 2. The O-methyltransferase from Argemone shows a clear pH-optimum at pH 7.5. The enzyme exhibits a temperature optimum at 35° C. The molecular weight of the O-methyltransferase determined by gel filtration on Sephadex G 100 was 47000 daltons. The enzyme was inhibited by preincubation for 15 min. at 35° C with the following metal ions added as sulphates to a final concentration of 5 mM, and the reaction started by the addition of the labelled Substrate: Cu (100% Inhibition), M n (15%), Z n (100%), M g (0%), C o (81%), N i (100%), S n (55 %), H g (100 %), F e (90 %). No Inhibition was found after preincubation with 25 mM E D T A . Of the organic enzyme inhibitors tested only pchloromercuribenzoate (25 mM, 70 %) and iodobenzoic acid (10 mM, 73 %) resulted in substantial Inhibition of the enzyme. This indicates that there is an SH-group requirement for füll enzyme activity. A competitive inhibition was observed using S-adenosyl-homocysteine in the presence of H - S A M and

pkdt

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

3

NLS, as Substrates. A Ki of 10 \im was determined for

Fig, 2. p H profile of the catalytic activity of purified N L S - O M T f r o m A, platyceras cell cultures. Buffers u s e d : A - A N a - m a l e a t e / N a O H ; • - • K H P 0 / K H P 0 ; • - • T r i s / H C I ; • - • Na-borate. 2

4

2

4

SAH. The enzyme shows a half life of 8 hrs at 30° C. At 4° C after a 4 week storage period, 50 % of the initial activity of the enzyme was lost. All enzyme activity was lost when the enzyme was frozen in 30 % glycerol Solution. As shown in Table III, the purified enzyme is absolutely specific for the tetrahydrobenzylisoquinoline

Enzymes from Argemone Cell Cultures

135

T a b l e III Substrate specificity of partlally purified N L S - O M T f r o m A. platyceras Substrate

cell cultures

Enzyme

activity

pkat/mg enzyme

%*

(S)-Norlaudanosoline

313

100

(R)-Norlaudanosoline

288

92

s a m e a s above

(R, S) 4 ' - 0 - m e t h y l n o r l a u d a n o s o l i n e (R, S) 5 ' - 0 - m e t h y l n o r l a u d a n o s o l i n e

107

34

Norprotosinomenine

255

81

Nororientaline

(R, S ) - L a u d a n o s o l i n e

247

79

15

Reaction product

6- O-methyl norlaudanosoline: 7- O-methylnorlaudanosoline = 8:2

5

6-O-methyllaudanosoline nd

(R, S)-Norreticuline

3

1

nd

(R, S)-Nororientaline

0

0

(R, S)-Laudanidine

0

(S)-Scoulerine

3

0 1

--

22

7

Jatrorrhizine

15

5

Tetrahydrojatrorrhizine

Aesculetine

0 0

0 0

Caffeicacid

0

0

Catechol

0

0

Dopamine Quercetin

0

0

0

0

----

(R, S)-Norlaudanosoline-1 -carboxylic acid

2,3-Dihydroxy-9,10-dimethoxy protoberberine

Tetrahydrocolumbamine

(R, S)-2,3-Dihydroxy-9,10-dimethoxy tetrahydroprotoberberine Adrenaline

nd = not d e t e r m i n e d

relative t o ( S ) - N L S ;

nucleus. None of the phenylpropanoids, phenolics, biogenic amines, coumarins or flavonoids were methylated by action of this enzyme. The best Substrates were the ones with four free phenolic-hydroxy-functions, followed by mono-methyl-derivatives. Di-Omethylated alkaloids are very poor Substrates. K M

values for this enzyme were determined using the following Substrates: K (S)-norlaudanosoline0.2mM; (R)-noi iaudanosoline 0.2 mM; (R,S)-4'-0-methylM

norlaudanosoline 1.1 mM; (R, S)-laudanosoline 0.3

mM. SAM 0.05 mM (with (S)-NLS as Substrate). The 80-fold purified OMT is absolutely free of any N-methylating activity as demonstrated by its lack of activity on norreticuline, nororintaline or tetrahydropapaverine. The best enzyme Substrate of the Compounds tested so far proved to be (S)-norlaudanosoline. The product of the reaction catalysed by the transferase using (S)-NLS and C - S A M as Substrates was subsequently investigated. HPLC-chromatography under the conditions given, showed that only two radioactive products were formed, one Compound with a retention time of 7.2 min. (20 yield) and the major Compound with a retention time of 9.2 min. (80 % yield). The retention times and co-chromatography with all four possible authentic monomethylated norlaudanosolines indicated the minor Compound to be 7-0methyl-norlaudanosoline and the major Compound 14

to be 6-O-methyl-norlaudanosoline. Preparative isolation of the major Compound from a 15 ml incuba-

tion mixture containing (R, S)-laudanosoline as Substrate, mass spectroscopy of the purified major unknown product showed a clear fragment (3,4-dihydro-6-methoxy-7-hydroxy-N-methyl-isochinolinium cation) with m/z 192 (100%) containing one N and one O-methyl-group which further fragmented to ml z 177 (192-CH , 30 %), and m/z 162 (177-CH ,4 %). The spectrum clearly indicated methylation at the Aring and was identical in every respect with the mass spectrum of authentic 6-O-methyllaudanosoline. Thus the major product of the NLS-OMT reaction was proven to be 6-O-methylnorlaudanosoline. The time course of the enzyme formation in Suspension cultures of Argemone cells is shown in Fig. 3. The enzyme is present in the inoculum only in low amounts. The activity peaks at day 8 of cultivation exactly at the point when the culture is leaving the logarithmic growth phase. A 30-fold increase in activity can be seen as compared with only 10-fold increase in dry cell matter. Düring stationary phase there is a drastic decrease of total activity of the enzyme. 3

3

Discussion Isoquinoline alkaloids comprise the largest group of alkaloids in plant kingdom. Relatively little is known about their biosynthesis at the cell-free level. In an attempt to elucidate the enzymatic steps involved in benzylisoquinoline synthesis, it is our primary

Rueffer, Nagakura, Zenk

136

aim to isolate and characterize those enzymes which are involved in reticuline biosynthesis, the central and branch-point intermediate in benzylisoquinoline metabolism in plants [3]. In previous studies, we were able to demonstrate that the initial reaction in formation of the benzylisoquinoline skeleton is the stereospecific condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde to yield (S)-norlaudanosoline [1,2]. The aldehyde, rather than the 3,4-dihydroxyphenylpyruvate, is the Substrate for this condensation reaction. The previously postulated intermediate norlaudanosoline-1-carboxylic acid [13-15] is most probably an artefact. Between the now recognized first metabolite in the pathway norlaudanosoline and reticuline, three methylation steps are involved, two O-methylations at carbon atom 6 and 4' and one N-methylation at atom 2. Since nor-reticuline has been amply demonstrated by in vivo experiments to be the immediate precursor of reticuline [e.g. 4, 5], the question arises, which of the phenolic groups of the norlaudanosoline molecule is methylated first on its way to nor-reticuline, the one in 6 or the one in 4' position. A survey of plant cell cultures using (R,S)-laudanosoline and -H-SAM as Substrates (in order to prevent N-methylation) demonstrated that all isoquinoline containing species contained good methylating activity. No methyltransferase activity was observed using (R,S)-NLS and H - S A M as Substrate with for instance C a t h a r a n t h u s roseus (Apocynaceae), a species which is known notio contain any benzylisoquinoline alkaloids. The highest amount of methyltransferase per volume of medium was observed in A . platyceras cell cultures, and it was decided to use this species for the isolation and characterization of the NLS-O-methyltransferase enzyme. The test used for the analysis of the methylated NLS formed involved differential extraction of the 3

3

labelled product at pH 9.5 with isoamylalcohol leaving residual H - S A M in the aqueous Solution. By using this assay the methylating enzyme could be purified about 80-fold and the major (80 %) product of reaction could unequivocally be identified as 6-O-methylnorlaudanosoline by HPLC and mass spectroscopy and comparison with the other four authentic mono-methyl-NLS species. The minor reaction product (20%) was identified as 7-O-methylNLS. It is noteworthy that using the plant enzyme there was not much difference in the methylation rate using the stereoisomers (S)- or (R)-NLS. Furthermore there was no difference in methylation pattern observed using the pure optical isomers as Substrates. In both cases 80 % of the product formed was 6-0methyl-NLS, formed regardless of whether (R)- or (S)-NLS was used as Substrate. This demonstration is 3

Table IV C o m p a r i s o n of Norlaudanosoline-O-methyltransferases of plant a n d animal origin Characteristics

Enzyme s o u r c e A.

platyceras

Rat [17]

Mouse*

(S)-NLS:K

M

0.2 m M

_

0.8 m M

(R)-NLS:K

M

0.2 m M

-

2.2 m M

(R,S)-NLS:K SAM:K

M

M

pH-optimum Position of methylation

-

1.3mM

-

0.05 m M

6.2 m M

1.0 m M

7.5 6 and 7

7.7-8.0 6 and 7

7.5 6 and 7

Ratio for (S)-NLS

80:20

79:14

53:47

Ratio for (R)-NLS

80:20

26:68

47:53

No

N.d.

Yes

Caffeic acid methylated

* This investigation w a s carried out in our laboratory using a m o u s e liver preparation according to [17]

Enzymes from Argemone Cell Cultures

137

N-H

HCK^^ (R, S)- Norlaudanosoli ne

Fig.

3

HO'

80% 6-O-Methyl (R.S)-Norlaudanosoline

4. Reaction s e q u e n c e catalysed by S - A d e n o s y l - L - m e t h i o n i n e : (R),

at variance with observations using a purified O-methyltransferase from rat liver [8, 16, 17], where it could be clearly shown that methylation of (S)-NLS yielded predominantly (79%) 6-O-methyl-NLS while the (R)-NLS isomer gave rise to 7-O-methylNLS (68 %). Thus in the animal System, the position of O-methylation (either at atom 6 or 7) is largely di-

H C0

20% 7-0 - Methyl (R.S)-Norlaudanosoline

(S)-Norlaudanosoline-6-0-methyltransferase.

the enzyme Systems introducing the second O-methyl-group are probably rather unspecific. The search for methylating enzymes in the pathway leading to reticuline must now concentrate on an O-methyltransferase methylating the 4'-position in 6-O-methyl-NLS, as well as on a norreticuline-N-methyltransferase.

rected by the particular isomeric form of the Substra-

te. A comparison of NLS-O-methyltransferases of plant and animal origin are given in Table IV. There are distinct differences between the animal and plant enzyme. The plant enzyme shows a considerably higher affinity for the Substrates than the animal enzymes, as demonstrated by the low K value

Acknowledgements This investigation was supported by SFB 145 of Deutsche Forschungsgemeinschaft, Bonn, as well as by Fonds der Chemischen Industrie. The technical assistance of GABRIELE WEBER is gratefully acknowledged. We thank Dr. E . FANNING for kind linguistic help.

M

for NLS and SAM. The animal Systems which do not show, in contrast to the plant enzyme, a high Substra-

References

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meric form of the alkaloid Substrate dictates the site

of enzymatic O-methylation. The plant enzyme prefers the 6 positions for methylation regardless of the steric configuration of the NLS Substrate.

It is also noteworthy that the previously assumed intermediate, (R,S)-norlaudanosoline-l-carboxylic acid [13-15] shows only about 5 % of the activity of (S)-NLS. This fact would indicate that even if the carboxylic acid were a natural intermediate in benzylisoquinoline biosynthesis, decarboxylation would have to precede methylation of this molecule. The Observation presented above that the first me-

thylation reaction of NLS occurs at the 6-O-position (see Fig. 4) is in perfect agreement with a finding by BROCHMANN-HANSSEN et al. [5]. Their results, obtained from in vivo experiments, indicate that during the early steps in papaverine formation 6-O-methylation must precede 7-O-methylation. This is also consistent with the sequence of O-methylation of phenethylamine and tetrahydroisoquinolines in the biosynthesis of peyote alkaloids [18]. The Observation that (R,S)-[l-^H]-4'-0-methylnorlaudanosoline (0.18 %) is slightly better incorporated into reticuline than (R,S)-[l- H]-6-0-methylnorlaudanosoline (0.12 %) in feeding experiments to Litsea glutinosa [19] may be within experimental error, but demonstrates that 3

(10) Bensadoun, A . and D. Weinstein: Anal. Biochem. 70, 241 (1976). (11) Warburg, O. and W. Christians: Biochem. Z. 310, 384 (1941). (12) Santavy, F. and R. H . F. Manske (ed.): The alkaloids, Vol. XVII, 385, New York, San Francisco, London, 1979, Academic Press. (13) Wilson, M. L . and C. J. Coscia: J. Am. Chem. Soc. 97, 431 (1975). (14) Battersby, A . R., R. C. F. Jones and R. Kazlauskas: Tetrahedron Lett. 1975,1873. (15) Scott, A . I., S.-L. Lee and T. Hirata: Heterocycles 1 1 , 159 (1978). (16) Collins, A . C , J. L . Cashaw and V. E . Davis: Biochem. Pharmacol. 22,2337 (1973). (17) Meyerson, L . R., J. L . Cashaw, K. D. McMurtrey and V. E . Davis: Biochem. Pharmacol. 28, 1745 (1979). (18) Paul, A. G . : Lloydia 36, 36 (1973). (19) Tewari,S.,D.S. Bhakuniand R. S. Kapil: J. Chem. Soc. Chem. Commun. 1975, 554.

Address: Prof. D r . M . H . Zenk, Pharmazeutische Biologie, Karlstraße 29, D - 8 0 0 0 München 2, Feder a l Republic ofGermany