Journal of Medicinal Plant Research

Planta medica

Organ der Gesellschaft für Arzneipflanzen­forschung

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

Editorial Board H . P. T. Ammon, Tübingen W. Barz, Münster E . Reinhard, Tübingen

•O. Sticher, Zürich H . Wagner, München M . H . Zenk, München

Advisory Board N . Anand, Lucknow R. Anton, Strasbourg

A . Baerheim-Svendsen, Leiden

H . Böhm, Halle F. Bohlmann, Berlin A . Cave, Chatenay-Malabry P. Delaveau, Paris Ding Guang-sheng,

Shanghai C. -J. Estler, Erlangen N . Farnsworth, Chicago H . Floss, Columbus H . Friedrich, Münster D . Fritz, Weihenstephan A . G . Gonzalez, La Laguna O. R. Gottlieb, Sao Paulo E . Graf, Köln 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 Ch . 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 124

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 Moenjod-aramine from Buxus p a p i l o s a 126

B a l s e v i c h , J . , K u r z , W. G. W.: The Role of 9- and/or 10-oxygenated Derivatives of Geraniol, Geranial, Nerol, and Neral in the Biosynthesis of Loganin and Ajmalicine 79

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 complemen-taire de la cytotoxicite de la melinonine F, alcaloide derive de la ß-carboline (Complementary Study of Cytotoxic Activity of Melinonine F) 158

Becker, H . , C h a v a d e j , S., W e b e r l i n g , F . : Valepotriates in V a l e r i a n a t h a l i c t r o i d e s 64

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 Accu-mulation 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 ) 191

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. . . . 138

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 204

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.f 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 176

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 . : Ac-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 Ex-tract 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 ) 255

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 Amarylli-daceae Alkaloid, on Testicular Function in Rats 252

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 62

Dehaussy, H . , T i t s , M . , A n g e n o t , L . \ La 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 ) 25

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 9 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 . . . . 63

E n d o , K . , O s h i m a , Y.t 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 188

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 ) . . . 170

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 I s e r t i a h y p o l e u c a ) 244

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

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 ­b i s s a t i v a ) 250

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 Poly-acrylamide-Hydrazide. Monoterpene Reduction by En-trapped M e n t h a C e l l s 9

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. IV. Influence of Lycorine on Growth Pattern of Three Animal Viruses 109

I s h i g u r o , K . , Y a m a k i , M . y T a k a g i , S.: Studies on Iridoid-related Compounds; III: Gentiopicral, the Aglucone of Gentiopicroside 208

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, VI . Unter­suchungen zur antiphlogistischen Wirkung von Chama-zulen und Matricin (Pharmacological Investigations with Compounds of Chamomile, V I . Investigations on the Antiphlogistic Effects of Chamazulene and Matricine) 67

J o s h i , K . C , S i n g h , P . , T a n e j a , S.: A Sesquiterpenoid Naph-thol from K y d i a c a l y c i n a 127

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 ) 20

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 51

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 246 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 185 K i s o , Y., T o h k i n , M . , H i k i n o , H . : Assay Method for Anti­

hepatotoxic Activity Using Carbon Tetrachloride In-duced Cytotoxicity in Primary Cultured Hepatocytes . . 222

K r a m , G . , F r a n z , G.\ Untersuchungen über die Schleim-polysaccharide aus Lindenblüten (Analysis of Linden Flower Mucilage) 149

Krüger, D . , J u n i o r , P . , W i c h t l , M . \ Neue Cardenolidglyko-side aus D i g i t a l i s l a n a t a (New Cardiac Glycosides from D i g i t a l i s l a n a t a ) 74

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 . 181

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 X X X I V 36

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 55

Contents III

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 -s u l a 60

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. In-duction of Callus Tissues and Detection of the Agents in Cultured Cells 115

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 143

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 : Com-ponents of Root Bark of Morus australis. I. Structure of a New 2-Arylbenzofuran Derivative, Mulberrofuran D . 90

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 Con-ditions on D O P A Accumulation in a Callus Culture of S t i z o l o b i u m hassjoo 120

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 N M R Chemical Shift Values for Aromatic Protons in 2,3,9,10-and 2,3,10,11-tetrasubstituted Tetrahydroprotoberberine Alkaloids 162

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:Ter t ia ry Phenolic Alkaloids from C h a s m a n t h e r a dependens . . . 17

O j e w o l e , J . A . O., A d e s i n a , S. K . : Mechanism of the Hypo-tensive Effect of Scopoletin Isolated from the Fruit of T e t r a p l e u r a t e t r a p t e r a 46

O j e w o l e , J . A . O., A d e s i n a , S. K . \ Cardiovascular and Neuro-muscular Actions of Scopoletin from Fruit of T e t r a p l e u r a t e t r a p t e r a 99

O k o g u n , J . /., Adeboye, J . O., O k o r i e , D . A . \ Novel Struc-tures of two Chromone Alkaloids from Root-Bark of S c h u m a n n i o p h y t o n m a g n i f i c u m 95

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 128

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 Helio-trine, the Major Alkaloid of H e l i o t r o p i u m i n d i c u m Seeds" 254

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 Furano-flavone from Seeds of P o n g a m i a g l a b r a 61

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 28

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 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 t h y m b r a 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 O-Methyltransferase 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

Rueffer, M . , N a g a k u r a , N . , Zenk, M . H . : Partial Purification and Properties of S-Adenosylmethionine: (R), (S)-Nor-laudanosoline-6-O-Methyltransferase from A r g e m o n e p l a t y c e r a s Cell Cultures 131

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 43

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 ) 199

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 (An iso-Homoprotoberberin-Alkaloid from the Roots of B e r b e r i s a c t i n a c a n t h a ) . . . . 32

Shoyama, Y., H a t a n o , K . , N i s h i o k a , / . : Clonal Multiplica-tion of P i n e l l i a t e r n a t a by Tissue Culture 14

S t o i a n o v a - l v a n o v a , B . , B u d z i k i e w i c z , H . , K o u m a n o v a , 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 Pro-duct of Gentiopicrin (Gentiopicroside) 211

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 Zu­sammensetzung der Convolvulaceen Harze IV. Die Gly-kosidsäuren 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 IV. 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 offi-c 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.: Helenalin-und 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 -c o r e a c o l l e t t i i 38

Yu D e - Q u a n , D a s , B . C.: Structure of Hydroxymuscopyri-dine A and Hydroxymuscopyridine B , Two New Constit­uents ofMusk 183

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 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

Galactose 149 Genkwanin 128

L

Lasiocarpine 254 Laudanosoline 196

Subject Index V

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

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

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

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

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 fami-lies 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 y Caesalpini-oideae = C a e s a l p i n i a c e a e and Papilion-oideae = 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

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

D

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

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

Erythrina lithosperma 22 Eschscholtzia tenuifolia 133 Euphorbiaceae 199

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

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

M

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

Index of Names of Organisms Vl l

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

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

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

R

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

S

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

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

U

Ulmus americana 181 Uncaria sinensis 188 Uncaria species 188

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 Page

Central nervous System anticonvulsive Magnolia officinalis, Magnoliaceae 103 muscle relaxant Magnolia officinalis 103 sedative Magnolia officinalis 103

Peripheral nervous System muscle relaxant Tetrapleura tetraptera, Mimosaceae 99 Autonomous nervous System spasmolytic Tabernaemontana dichotoma, Apocynaceae 28 Cardiovascular System hypotensive Tetrapleura tetraptera, Mimosaceae 46

hypotensive Tetrapleura tetraptera, Mimosaceae 99 Hormonal System antifertility Hippadine, Amaryllidaceae 252 Infectious deseases antibacterial Baccharis crispa, Asteraceae 128

antiviral Cliviaminiata, Amaryllidaceae 109 Tumors tumor promotion Pimelea simplex, Thymelaeaceae 3 Inflammation antiinflammatoric Chamomillareculita, Asteraceae 67

antiinflammatoric Crassocephalum multicorymbosum 255

Journal of P lanta 1983, Vol. 49, pp. 131-137, ©Hippokrates Verlag GmbH Medicinal " ^ 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 Pharmazeut ische Biologie der Universität München, D-8000 München 2, Federal Republic of Germany

** Kobe W o m e n ' s Col lege of Pharmacy, Kobe 658, Japan

Received: August 7 , 1 9 8 3 ; accepted: August 3 1 , 1 9 8 3

Key Word Index:

Argemone platyceras; Papaveraceae; Cell Suspen­sion Cultures; Alkaloid Biosynthesis; S-Adenosyl­methionine: (R), (S)-Norlaudanosoline-6-OMethyl-transferase.

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-methylnor-laudanosoline, and, to a minor extent, 7-O-methyl-norlaudanosoline 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 M values for (S) and (R)-norlaudanosoline were 0.2 mM, and for SAM, 0.05 mM. The transfera-se shows high Substrate specificity for tetrahydro-benzylisoquinoline alkaloids. Simple orthophenols, like phenylpropane derivatives, coumarins or fla-vonoids, are not accepted as Substrates. The enzyme is widely distributed in benzylisoquinoline-contain-ing plant cell cultures and is present in differentiated plants like Papaver somniferum.

Introduction

(S)-NLS is now firmly established as the first inter-mediate in benzylisoquinoline alkaloid biosynthesis [1]. A recently discovered enzyme [2] catalyses its

Abbreviations: NLS = Norlaudanosoline; S A M = S-Adeno-syl-L-methionine; S A H = S-Adenosylhomocysteine, N L S - O M T = S-Adenosylmethionine: (R), (S)-Norlaudanosoline-6-0-Me-thyltransferase.

formation from dopamine and 3,4-dihydroxypheny-lacetaldehyde.

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, protoberbe-rines, proaporphines, cularines, dibenzopyrrocoli-nes etc. (e.g. 3]. This means that NLS must be trans-formed to reticuline by three methylation reactions, two O-methylations at positions 6 and 4' and one N-methylation (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-me-thylation for instance in opium alkaloids. O-Methy-lation of NLS is therefore an important reaction in the early steps of the biosynthesis of reticuline, the universal branch point intermediate. O-Methyltrans-ferases have been reported to mediate the transfer of methyl groups from SAM 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]. Speci-fic-O-methyltransferases derived from plants and ac-ting on tetrahydrobenzylisoquinolines have so far not been reported. Yet incubation of poppy latex with (R,S)-norlaudanosoline and 1 4 C-SAM has led to the formation of labelled opium alkaloids, thus demon-strating 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 in-volved in isoquinoline biosynthesis in plants, we have investigated the O-methylation of NLS. In this report, we present the partial purification and pro-perties of a new enzyme named norlaudanosoline-6-O-methyltransferase from A . platyceras cell Suspen­sion cultures. This enzyme catalyses the predominant transfer of the S-methyl group of SAM to the pheno-lic OH-group at position 6 of NLS, and to a lesser ex­tent, to the 7-O-position.

132 Rueffer, Nagakura, Zenk

R!0

R 2 0

R 3 0

R«0 3. R1 R 2 R3 R« R5

Norlaudanosoline H H H H H 6- •0- methylnorlaudanosoline CH 3 H H H H 7- •0- methylnorlaudanosoline H CH 3 H H H 5- •0- methylnorlaudanosoline H H CH 3 H H U- -0- methylnorlaudanosoline H H H CH 3 H

Laudanosoline H H H H CH 3

Norreticuline CH 3 H H CH 3 H Nororientaline CH 3 H CH 3 H H Laudanidine CH 3 CH 3 H CH 3 CH 3

1H = a = (S); 1H = p = (R)

Materials and Methods P l a n t M a t e r i a l

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 % inocul-um. The cells were harvested after 8 days, frozen in liquid nitrogen and stored at - 2 0 ° C. Cell fresh weight was determined after filte-ring 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.

C h e m i c a l s The following Compounds were obtained from the indicated

sources: S-adenosyl-L-methionine hydrogen sulphate, Combithek (calibration proteins), S-adenosylhomocysteine, all from Boehrin-ger, Mannheim; S-adenosyl-L-methyl-3H-methionine was prepa-red enzymatically from methionine -S-methyl- 3H, (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-microgranu-lar form from Whatman, and hydroxyapatite from Bio-Rad. A l l other materials were of reagent grade. Liquid scintillation count-ing was performed in a toluene mixture (Rotiszint 22, Roth).

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 prepara-tions, laudanosoline was substituted for its nor-derivative. The as­say mixture consisted of KPO42* -buffer, p H 7.5 (130 mM), ascor-bate (130 mM) R/S-norlaudanosoline (0.3 mM), 3 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 reac-tion was terminated by addition of 200 ul Na 2C0 3-buffer (1 M , pH 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

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 (Si-gel; solvent System: Chloroform : methanol: acetic acid : water = 18:6:3:0.3). The zone containing radioactivity was scraped off, pu-rified for a second time in the solvent System: Chloroform : n-pro-panol : methanol : water = 45:15:60:40 (CHCl 3-phase), and its mass spectrum was measured in a Finnigan M A T 44S instrument.

Enzyme p u r i f i c a t i o n Step 1:100gfrozen tissue wasallowed tothawin0.1 M K P 0 4

2 -buffer, p H 7.5, containing 20 mM 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 4

2*-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 KP0 4

2 -buffer , pH 7.5, 20 m M ß-mercaptoethanol. Frac-tions 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 4

2*-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 4

2"-buf-fer, p H 7.5, 20 m M ß-mercaptoethanol, and a gradient of 10-200 m M K P 0 4

2 , 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 frac-

Enzymes from Argemone Cell Cultures 133

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

Molecular Weight Determination The molecular weight determination of the purified O-methyl-

transferase was carried out by gel filtration on a calibrated G-100-superfine 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 KP0 4

2 -buf fer , 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.

Results

The presence of O-methyltransferases in crude en­zyme extracts of plant cell cultures of different taxo-nomic origin was investigated using (R, S)-laudano-soline and C 3 H 3 -SAM as Substrates. Laudanosoline was chosen as an initial methyl group acceptor to avoid interference with N-methyltransferases potenti-ally 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-me-thyltransferase enzymes showed positive results. By far the highest absolute amount of O-methyltransfe-rase per unit culture fluid was found in Argemone platyceras (Papaveraceae), a plant species known to contain isoquinoline alkaloids of the pavine, proto-berberine, and aporphine types [12]. It was there-fore decided to use cells of this species to purify and characterize the O-methyltransferase. (R, S)-norlau-danosoline 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 homo-genate of the Argemone cells. It could not be found in

Table I Survey of distribution of (R, S)- laudanosol ine-methyltransferase activity in species of different isoquinoline alkaloid containing famil ies ( laudanosoline served as Substrate)

Plant material Family Enzyme activity pkat/l pkat/mg

medium protein

Cell cultures: Argemone platyceras Papaveraceae 6350 13.8 Corydalis sempervirens „ 4620 12.2 Glauciumflavum 2855 27.7 Fumaria officinalis 1950 11.1 Corydalis pallida „ 1480 8.2 Argemone intermedia 1360 33.7 Adlumia fungosa „ 1070 12.5 Papaver somniferum 1020 9.3 Chelidonium majus „ 915 6.8 Eschscholtzia tenuifolia 590 4.2

Berberis henryana Berberidaceae 2050 38.7 Berberis wilsonae „ 1650 13.9 Berberis stolonifera 1260 18.9

Thalictrum tuberosum Ranunculaceae 2060 26.6 Thalictrum sparsiflorum » 570 2.4

Cissampelos mucronata Menispermaceae 940 3.7

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 chro-matography as described under "Materials and Me­thods". This procedure yielded a purification of ap-proximately 80-fold with a recovery of 13 %. The da­ta for a typical purification procedure are summari-zed 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 deter-

mine the catalytic properties. The activity of the

Table II Purification procedure for O-methyltransferase f rom Argemone platyceras (R, S-norlaudanosoline as Substrate)

Purification step Total activity Total protein Specific activity Recovery Purification (pkat) (mg) pkat/mg (%) - f o l d

Crude extract 3626 259.6 14 100 1 Ammoniumsul f ate-precipitat ion

( 0 - 7 0 % ) 3409 178.3 19 94 1.4 Gel filtration (ACA = 34) 2290 15.2 150 63.2 10 DEAE-cel lulose chromatography 1124 2.5 459 31.0 32 Chromatography on hydroxyapat i te 477 0.4 1173 13.2 83

134 Rueffer, Nagakura, Zenk

•160

Fraction

Fig. 1. Elution profi le o f N L S - O M T from a hydroxyapati te column.

pkdt

Fig, 2. pH profi le of the catalytic activity of purified NLS-OMT from A, platyceras cell cultures. Buffers used: A - A Na-maleate/ NaOH; • - • K 2 H P 0 4 / K H 2 P 0 4 ; • - • T r i s /HCI ; • - • Na-bora-te.

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 sulpha-tes to a final concentration of 5 mM, and the reaction started by the addition of the labelled Substrate: C u + + (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 ED-TA. Of the organic enzyme inhibitors tested only p-chloromercuribenzoate (25 mM, 70 %) and iodoben-zoic 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-adeno-syl-homocysteine in the presence of 3 H-SAM and NLS, as Substrates. A Ki of 10 \im was determined for 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 % gly-cerol Solution.

As shown in Table III, the purified enzyme is abso-lutely specific for the tetrahydrobenzylisoquinoline

Enzymes from Argemone Cell Cultures 1 3 5

T a b l e III Substrate specif ici ty of partlally purified NLS-OMT f rom A. platyceras cell cultures

Substrate Enzyme pkat /mg enzyme

activity %*

Reaction product

(S)-Nor laudanosol ine 313 100 6- O-methyl norlaudanosol ine: 7- O-methylnorlaudanosol ine = 8:2

(R)-Nor laudanosol ine 288 92 sameas above (R, S) 4 ' -0-methy lnor laudanoso l ine 107 34 Norprotosinomenine (R, S) 5 ' -0-methy lnor laudanosol ine 255 81 Nororientaline (R, S)-Laudanosol ine 247 79 6-O-methyl laudanosol ine (R, S)-Norlaudanosol ine-1 -carboxylic acid 15 5 nd (R, S)-Norret icul ine 3 1 nd (R, S)-Nororiental ine 0 0 -(R, S)-Laudanidine 0 0 -(S)-Scoulerine 3 1 Tetrahydrocolumbamine 2,3-Dihydroxy-9,10-dimethoxy

protoberberine 22 7 Jatrorrhizine (R, S)-2,3-Dihydroxy-9,10-dimethoxy

tetrahydroprotoberber ine 15 5 Tetrahydrojatrorrhizine Adrenal ine 0 0 -Aesculet ine 0 0 -Caffe icacid 0 0 -Catechol 0 0 -Dopamine 0 0 -Quercet in 0 0 -

relative to (S)-NLS; nd = not determined

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-func-tions, followed by mono-methyl-derivatives. Di-O-methylated alkaloids are very poor Substrates. K M

values for this enzyme were determined using the fol-lowing Substrates: K M (S)-norlaudanosoline0.2mM; (R)-noi iaudanosoline 0.2 mM; (R,S)-4'-0-methyl-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 tetrahy-dropapaverine.

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 1 4 C-SAM as Substrates was subse-quently investigated. HPLC-chromatography under the conditions given, showed that only two radioacti-ve products were formed, one Compound with a ret-ention 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 norlauda-nosolines indicated the minor Compound to be 7-0-methyl-norlaudanosoline and the major Compound to be 6-O-methyl-norlaudanosoline. Preparative iso-lation of the major Compound from a 15 ml incuba­

tion mixture containing (R, S)-laudanosoline as Sub­strate, mass spectroscopy of the purified major un-known product showed a clear fragment (3,4-dihy-dro-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-CH3, 30 %), and m/z 162 (177-CH3,4 %). The spectrum clearly indicated methylation at the A-ring 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 lo-garithmic 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.

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 invol-ved in benzylisoquinoline synthesis, it is our primary

1 3 6 Rueffer, Nagakura, Zenk

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-di-hydroxyphenylacetaldehyde to yield (S)-norlauda-nosoline [1,2]. The aldehyde, rather than the 3,4-di-hydroxyphenylpyruvate, 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 reco-gnized 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-reticuli-ne has been amply demonstrated by in vivo experi-ments 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 3 -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 3 H-SAM 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 cha-racterization of the NLS-O-methyltransferase enzy­me. The test used for the analysis of the methylated NLS formed involved differential extraction of the

labelled product at pH 9.5 with isoamylalcohol lea-ving residual 3 H-SAM 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-methyl-NLS. It is noteworthy that using the plant enzyme there was not much difference in the methylation rate using the stereoisomers (S)- or (R)-NLS. Further-more there was no difference in methylation pattern observed using the pure optical isomers as Substrates. In both cases 80 % of the product formed was 6-0-methyl-NLS, formed regardless of whether (R)- or (S)-NLS was used as Substrate. This demonstration is

Table IV Compar ison of Norlaudanosol ine-O-methyl transferases of plant and animal origin

Characterist ics Enzyme source

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 M - 1.3mM -S A M : K M 0.05 mM 6.2 mM 1.0 m M pH-opt imum 7.5 7 .7 -8 .0 7.5 Position of methylat ion 6 and 7 6 and 7 6 and 7 Ratio for (S)-NLS 80:20 79:14 53:47 Ratio for (R)-NLS 80:20 26:68 47:53 Caffeic acid methylated No N.d. Yes

* This investigation was carried out in our laboratory using a mouse liver preparation according to [17]

Enzymes from Argemone Cell Cultures 137

N-H H3C0

H C K ^ ^ HO'

80% 20% (R, S)- Norlaudanosoli ne 6-O-Methyl - 7-0 - Methyl -

(R.S)-Norlaudanosoline (R.S)-Norlaudanosoline

Fig. 4. Reaction sequence catalysed by S-Adenosyl-L-methionine: (R), (S)-Nor laudanosol ine-6-0-methyl t ransferase.

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-methyl-NLS (68 %). Thus in the animal System, the position of O-methylation (either at atom 6 or 7) is largely di-rected by the particular isomeric form of the Substra­te. 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 consid-erably higher affinity for the Substrates than the ani­mal enzymes, as demonstrated by the low K M value for NLS and SAM. The animal Systems which do not show, in contrast to the plant enzyme, a high Substra­te specificity clearly demonstrate that the stereoiso-meric form of the alkaloid Substrate dictates the site of enzymatic O-methylation. The plant enzyme pre-fers 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 car-boxylic acid were a natural intermediate in benzyliso­quinoline biosynthesis, decarboxylation would have to precede methylation of this molecule.

The Observation presented above that the first me­thylation 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, ob-tained from in vivo experiments, indicate that during the early steps in papaverine formation 6-O-methyla-tion must precede 7-O-methylation. This is also con-sistent with the sequence of O-methylation of phene-thylamine 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-3H]-6-0-methylnorlaudanosoline (0.12 %) in feeding experiments to Litsea glutinosa [19] may be within experimental error, but demonstrates that

the enzyme Systems introducing the second O-me-thyl-group are probably rather unspecific.

The search for methylating enzymes in the path-way 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-me-thyltransferase.

Acknowledgements This investigation was supported by SFB 145 of Deutsche For­

schungsgemeinschaft, 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.

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