Journal of Medicinal Plant Research Planta medica Organ der Gesellschaft für Arzneipflanzen- forschung 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 (§)• Hippokrates ISSN 0032-0943 Hippokrates Verlag Stuttgart
11
Embed
Planta Journal of medica - uni- · PDF fileBiosynthesis of triterpenoids 176 Biotransformation 9 Bisabolol 67 Bisbenzylisoquinoline alkaloids 20, 25, 55 Bisnorargemonine 14
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
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
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 ofCultured Hepatoma Cells. . . . 138
B r i a n q 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., 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 r . 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 I 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 , 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., 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 DimericProanthocyanidins 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 . \ Zur 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 Cannabinoides 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
Cannabinoi'ds 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 . y 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 . y 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
l 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 , A/., T a k a g i , 5.: 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 . , Leboeuf, 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 F r a n z , G.: Untersuchungen über die Schleim-polysaccharide aus Lindenblüten (Analysis of Linden Flower Mucilage) 149
Krüger, £>., 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 , /., 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 . t 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 . : ! 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, /i.:Tertiary 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 . L , 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 . : Genus 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 , E . \ 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 , R.\ 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 Oilsfrom 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 , E . : 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 , E . \ 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 , I . : 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 - I 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 der 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, a Conversion 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 , B a u e r , R., H a m -d a r d , M . E . , 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 , 1. 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
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 , 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
A highly specific new enzyme, S-adenosyl-L-me-thionine: (6-O-methyl-norlaudanosoline)-5; -O-me-thyltransferase which catalyses the formation of nororientaline from 6-O-methyl-norlaudanosoline and SAM was discovered, partially purified, and charac-terized. Argemone platyceras cell Suspension cultures served as enzyme source.
Introduction
Tetrahydroisoquinoline biosynthesis in plants in-volves a number of O- and N-methylation Steps [e.g. 1] of norlaudanosoline, the first precursor in the pathway. In a previous communication, we have described the isolation of a first specific O-methyltrans-ferase. In Argemone platyceras cell Suspension cultures this enzyme methylates predominantly the 6-0-position of (S)- as well as (R)-norlaudanosoline, me-diating the transfer of a methyl-group from S-adeno-syl-L-methionine to NLS [2]. Düring the purification of this enzyme, a second enzymatic activity was discovered which utilises 6-O-methyl-NLS to introduce a second methyl group in the 5;-position of the mole-cule to yield nororientaline. Again SAM serves as a methyl group donator. Nororientaline is a principal immediate precursor of papaverine in Papaver somniferum [1] and is also undoubtedly converted to orientaline by N-methylation. Orientaline itself has
been isolated from opium poppy [3] and is metaboli-zed in Papaver Orientale via orientalinone to isothe-baine [4]. Therefore nororientaline can be consid-ered as a specific precursor of a number of isoquinoli-ne alkaloids in higher plants. This report describes the partial purification and characterization of a new enzyme which was designated S-adenosyl-L-methio-nine: (6-0-methyl-norlaudanosoline)-5'-0-methyl-transferase.
Material and Methods A r g e m o n e p l a t y c e r a s cells were cultivated as described pre
viously [2]. Chemicals used were essentially those described in the preceeding communication [2]. The new enzyme, described here, was purified through Steps 1-4 as described previously [2]. How-ever, the enzyme was eiuted from hydroxyapatite columns at frac-tions 56-65 in contrast to the previously described NLS-6-O-me-thyltransferase which was eiuted between fractions 43 and 54. Enzyme assays were carried out as follows: (R, S)-6-0-methyl-nor-laudanosoline (0.3 mM), S A M - 3 H (10000 cpm, 0.1 mM), K P 0 4
2 -buffer, p H 7.5 (130 mM) were incubated in a total volume of 150 ul at 35° C for 45 min. The reaction was terminated by the addition of 200 ul Na 2C0 3-buffer (1 M , p H 9.5). The methylated product was extracted by adding 400 ul isoamylacohol and shaking for 45 min. After centrifugation for 5 min. in an Eppendorf centrifuge, 200 ul of the organic phase were transferred to scintillation vials and counted. A blank value of about 10 % (obtained by assay mixtures containing either no enzyme or no Substrate), had to be subtracted from all incubation mixtures. Recovery of thje methylated product was 92 % under these conditions.
The products were separated and identified by H P L C using a Nucleosil-SA-column (25 mm x 3.2 mm i.d.) and 0.5 M ammo-nium phosphate : methanol (70:30) as a solvent System. Retention time of the potential products were: norisoorientaline, 7.14 min.; nororientaline, 8.20 min.; norprotosinomenine, 8.84 min.; norre-ticuline, 10.94 min.
Preparative isoiations were done using the above incubation mixture x 100. The reaction product was extracted by ethylacetate and purified twice by thin layer chromatography (Si-gel; solvent Systems: 1) Chloroform : methanol : acetic acid : water = 18:6:3:0.3; 2) acetone : Chloroform : diethylamine = 5:4:1). Mass spectra were determined in a Finnigan M A T 44S instrument.
Results and Discussion
Abbreviations: NLS = Norlaudanosoline; S A M = S-Adeno-syl-L-methionine; S A H = S-Adenosylhomocysteine.
Düring the purification of the 6-O-methyltransfe-rase evidence was obtained that its reaction product
O-Methyltransferase for Nororientaline Synthesis 1 9 7
Table I Purification protocol for the ö ' -O-methyl t ransferase f rom A. platyceras cell Suspension cultures (125 g fwt)
Purification step Volume Total activity Protein Spec. activity Yield Purification
(ml) (pkat) (mg/ml) (pkat/mg) factor
Crude extract 236 4647 1.10 17.97 1
( N H 4 ) 2 S 0 4 precipitation 23 5733 10.02 24.9 100 1.4
GelTiltration 69 5703 0.22 375.5 99.48 20.9
DEAE chromatography 35 1127 0.07 506.5 19.65 28.2
Hydroxyapati te 12 327.7 0.038 718.8 5.7 40.0
Table II Some characterist ics of (6-0-methy lnor laudanoso l ine) -5 ' -0-
methyltransferase f rom A. platyceras cell cultures
Characterist ics 5 ' -0-methy l t ransferase
Molecular weight 47.000
pH-opt imum 7.5
Temperature Optimum 35° C
K M for 6-O-methylnor laudano-
soline 0.4 m M
K M for SAM 0.05 m M
Kj for SAH 0.034 m M
Table III Substrate specifity of 40-fold purified 5 ' -0-methy l t ransferase
using S A M as methyl group donator
Substrate pkat/mg % Product of
reaction
(R, S) -6-0-methy l -
norlaudanosol ine 314 100 Nororientaline
(R)-Norlaudanosol ine 0 0 -(S)-Norlaudanosol ine 0 0 -(R, S)-Laudanosol ine 0 0 -
is transformed by the introduction of a second methyl group in the presence of excess SAM and crude enzyme preparations. The two enzymes involved are clearly separated by chromatography on hydroxyapatite. The enzyme was partially purified by using (R,S)-6-0-methyl-norlaudanosoline as Substrate. As shown in Table I the enzyme could be recovered with 6 % yield. Purification was about 40-fold.
The characteristic properties of the enzyme are li-sted in Table II. The enzyme has a half life of 33 hrs at 30° C and retains füll enzymatic activity in the presence of 0.05 % NaN 3 at 4° C after 4 weeks. The enzyme is, however, completely inactivated by freezing even in the presence of 30 % glycerol. The enzyme is inhibited completely by Fe 3 + and H g 2 + ions at 5 mM concentration and is strongly inhibited by 5 mM p-chloromercuribenzoate (84 %), N-ethylmaleimide (82 %), and 10mM jodobenzoic acid (79 %). This in-dicates that SH-groups are present at the active Center of the enzyme. Similar findings have been repor-
+ SAM
6-0-Methylnorlaudanosoline
ted for the unspecific meta and p a r a directing cate-chol-O-methyltransferases from plant origin [5]. The general properties of the enzyme, described here, are very similar to the previously described norlaudano-soline-6-O-methy ltransferase.
The Substrate specifity of the 5'-0-methyltransfe-rase was of considerable interest. 18 Substrates were tested for their ability to accept methyl groups from SAM catalysed by S-adenosyl-L-methionine: (6-0-methyl-norlaudanosoline)-5'-0-methyltransferase. None of the phenols, phenylpropanoids, flavonoids, coumarins, and biogenic amines tested, served as Substrates. Out of 12 different isoquinoline alkaloids tested [2] only (R,S)-6-0-methyl-norlaudanosoline was methylated. Surprisingly neither (R) or (S)-nor-laudanosoline nor (R, S)-laudanosoline served as a Substrate (Table III) which shows a hitherto unique Substrate specifity of an O-methyltransferase in plant kingdom [6]. Since the 6-O-methyl-norlaudanosoline used was a racemate, it is not known whether either
+ SAH
Nororientaline
1 9 8 Rueffer, Nagakura, Zenk
Hippokrates
Kompendium der Phytotherapie Von S. C H R U B A S I K und J. C H R U B A S I K , Freiburg
1983. 156 Seiten, 15,5x23 cm, kartoniert D M 34,— ISBN 3-7773-0618-5
Der Trend zur Naturheilkunde und da besonders zur Phytotherapie nimmt zu. Doch um die Heilkräuter therapeutisch nutzen zu können, bedarf es eines intensiven Studiums. Dieses Kompendium soll Studierenden und Ärzten helfen, die interne Applikation pflanzlicher Drogen zu erleichtern und dadurch zur Gesunderhaltung wie zur Behandlung von Krankheiten beizutragen. Das Buch ist für alle Ärzte, die sich für die Phytotherapie interessieren oder diese anwenden.
Ich bestelle aus dem Hippokrates Verlag Stuttgart durch die Buchhandlung:
Expl. C H R U B A S I K / C H R U B A S I K , Kompendium der Phytotherapie. D M 34 — ISBN 3-7773-0618-5
Unterschrift
Name (möglichst Stempel)
Straße
Ort
Datum (Preisänderung vorbehalten)
Hippokrates Verlag Stuttgart 57
of the two stereoisomers is preferentially methylated. As shown by HPLC (retention time 8.20 min.) and mass spectroscopy ([M + l ] + 316, 100%, meas-ured with dci), the reaction product of the enzyme was unequivocally nororientaline. As shown in Fig. 1, the new enzyme therefore exclusively methylates the 5'-position of 6-O-methyl-norlaudanosoline.
Using 1 4 CH 3 -SAM as one Substrate and this novel enzyme as catalyst it was possible to synthesize 5'-14CH3-nororientaline with excellent yields. Using callus of A . platyceras, feeding experiments with the labelled Substrate showed that nororientaline was ex-tensively metabolized by Argemone, yielding several alkaioids which are now being investigated. This finding demonstrates that the new enzyme is of phy-siological importance in the plant tissue.
Our results confirm previous findings [1, 2] that 6-O-methylation clearly precedes methylation at C-7 and methylation at ring C within the reticuline/orientaline pathway.
In addition the results obtained on the specific NLS-6-O-methyltransferase [2] and especially the 5'-O-methyltransferase described here clearly demon-strate that O-methylation precedes N-methylation.
Acknowledgements
Our thanks are due to Miss GABRIELE WEBER for excellent tech-nical assistance and Dr. H . IBELGAUFTS for his kind linguistic help. This investigation was supported by SFB 145 of Deutsche Forschungsgemeinschaft, Bonn, including a Finnigan M A T 44S in-strument and by Fonds der Chemischen Industrie (literature Provision).
References
(1) Brochmann-Hanssen, E . , C -H. Chen, R. Chen, H.-C. Chiang, A. Leung and K. McMurtrey: J. Chem. Soc. Perkin 1,1975, 1531. (2) Rueffer, M . , N. Nagakura and M. H. Zenk: Planta Med., in press (1983). (3) Brochmann-Hanssen, E . , C -H. Chen, H.-C. Chiang, C.-C. Fu and H. Nemoto: J. Pharm. Sei. 62, 1291 (1973).
(4) Batterby, A. R. in Taylor, W. I. and A. R. Battersby (eds.): Oxi-dative coupling of phenols, p. 119, New York 1967, Marcel Dekker. (5) Tsang, Y.-F. and R. K. Ibrahim: Z. Naturforsch. 34c, 46 (1979). (6) Poulton, J. E . , Stumpf, P. K. and E. E. Conn (eds.): The Bioche-mistry of Plants, Vol. 7, p. 667, New York 1981, Academic Press.
Address: P h a r m a z e u t i s c h e B i o l o g i e , Karlstraße 2 9 ,
D - 8 0 0 0 München 2, F e d e r a l R e p u b l i k o f G e r m a n y