Ankara Ecz. Fak. Der. j. Fak. Pharm. Ankara 15, 9 (1985) 15, 9 (1985)
A l k a l o i d s o f G e n i s t a a c a n t h o c l a d a D C . Genista a c a n t h o c l a d a D C . ' n ı n Alkaloitleri
Fatma TOSUN* Mekin TANKER** Tuncel ÖZDEN*** Ali TOSUN*
S U M M A R Y
In this research, six q u i n o l i z i d i n e alkaloids w e r e isolated f r o m the aerial parts of Genista acanthoclada a n d their picrates w e r e p r e p a r e d . T h e y were identified as sparteine, r e t a m i n e , l u p a n i n e , anagyrine, N-methylcytisine a n d cytisine b y T L C , m e l t i n g points, I R spectra a n d e l e m e n t a r y analysis. T h e total alkaloid c o n t e n t o f aerial parts of Genista acanthoclada was f o u n d to be 0 . 1 2 % by using titrimetric m e t -h o d ( 1 ) .
ÖZET
Bu çalışmada Genista acanthoclada'nın toprak üstü kısımlarından altı kinolizidin alkaloidi izole edilmiş ve pikrat tuzları hazırlanmıştır. B u bileşikler, İ T K , e r g i m e dereceleri, I R spektrumları v e elementel analizleri ile spartein, retamin, anagirin, N-metilsitisin ve sitisin ola rak teşhis edilmiştir. Titrimetrik y ö n t e m l e yapılan miktar tayininde Genista acanthoclada'nın t o p r a k üstü kısımlarının total alkaloit miktarı % 0 . 1 2 olarak bulunmuştur.
Redaksiyona verildiği tarih: 5.12.1984
*Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Etiler Ankara, Turkey.
** Department of Pharmacognosy, Faculty of Pharmacy, University of Ankara, ***Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ankara.
K e y W o r d I n d e x :
Genista acanthoclada; F a b a c e a e ; Q u i n o l i z i d i n e alkaloids.
T h e presence of quinolizidine alkaloids in Genista species has been k n o w n for a l o n g time. A l t h o u g h , there are a large n u m b e r of publi cations dealing with the alkaloids of Genista species, there is o n l y o n e paper dealing with Genista acanthoclada D C . (Fabaceae) ( 2 ) .
In this study we isolated sparteine, retamine, lupanine, anagyrine, N-methylcytisine and cytisine from the aerial parts of Genista acant-hoclada collected in I z m i r ( T u r k e y ) .
E X P E R I M E N T A L
T h e plant material was collected in M a y 1980 from Zeytinalan, Çeşme ( I z m i r ) and identified by us. A v o u c h e r specimen, A E F N o . 12701, is deposited in the H e r b a r i u m on the Faculty of Pharmacy, University of Ankara.
Elementary analysis were m a d e with Perkin Elmer M o d e l 240 Elemental Analyser. IR spectra were r e c o r d e d in K B r discs with a Pye U n i c a m S P 1025 I R Spectrophotometer. M e l t i n g points were determined on a Buchi 510 M e l t i n g Point Apparatus and n o t correc ted.
E x t r a c t i o n of total alkaloids
T h e dried and p o w d e r e d aerial parts of Genista acanthoclada D C . (1 kg) were extracted in a Soxhlet apparatus with methanol. T h e extract was evaporated under v a c u u m to dryness. T h e residue was extracted with 5 % aqueous H2S 04. T h e c o m b i n e d aqueous acidic solutions were extracted with c h l o r o f o r m to r e m o v e the nonbasic substances, and this was discarded. T h e aqueous solution was then m a d e basic ( p H = 1 2 ) with 33 % aqueous N a O H and extracted with c h l o r o f o r m . T h e c o m b i n e d c h l o r o f o r m extracts were dried with anhydrous N a2S O4, filtered and evaporated under v a c u u m to dryness, 1.14 g of a crude alkaloidal mixture was obtained. T h e crude alkalo-idal mixture was dissolved in methanol and e x a m i n e d by thin-layer c h r o m a t o g r a p h y . For the T L C , silica gel G F2 5 4 a n d the developers S1 ( c y c l o h e x a n e : diethylamine = 7 : 3 ) , S2 ( c y c l o h e x a n e : diethyla-m i n e = 9:1) and S3 ( c h l o r o f o r m : m e t h a n o l : 25 % a m m o n i u m
hydro-Alkaloids of Genista acanthoclada D C . 11
x i d e = 8 5 : 1 5 : 1 ) w e r e used. After d r y i n g for o n e h o u r a t 1 2 0 ° C the c h r o m a t o g r a m s w e r e e x a m i n e d u n d e r u l t r a v i o l e t l i g h t 2 5 4 n m a n d then s p r a y e d w i t h D r a g e n d o r f f reagent, six alkaloids a p p e a r e d as o r a n g e - r e d spots w i t h the f o l l o w i n g Rf values. S1 ( 0 . 0 7 , 0 . 3 0 , 0 . 3 5 . , 0 . 6 1 , 0 . 7 9 , 0 . 9 3 ) , S2 ( 0 . 0 3 4 , 0 . 0 7 , 0 . 0 9 , 0 . 2 4 , 0 . 5 0 , 0 . 8 3 ) , S3
( 0 . 0 7 , 0 . 1 8 , 0 . 4 2 , 0 . 6 1 , 0 . 6 6 , 0 . 6 9 ) .
I s o l a t i o n o f t h e a l k a l o i d s f r o m t h e c r u d e e x t r a c t
P r e p a r a t i v e T L C w a s used for the s e p a r a t i o n o f the c r u d e alkal o i d a alkal extract. T h e c r u d e a alkal k a alkal o i d a alkal m i x t u r e w a s d i s s o alkal v e d i n m e t h a -n o l a -n d c h r o m a t o g r a p h e d o -n P L C plates o f silica gel G F2 5 4. Fl, F2 a n d F3 w e r e o b t a i n e d f r o m the c h r o m a t o g r a m w i t h S2 s o l v e n t system ( C h r o m a t o g r a m - I ) . After the e x t r a c t i o n o f Fl, F2 a n d F3 f r o m the c h r o m a t o g r a m , the area b e t w e e n start a n d the s p o t w i t h the Rf= 0 . 2 4 was eluted w i t h c h l o r o f o r m : m e t h a n o l ( 7 : 3 ) a n d e v a p o r a t e d u n d e r v a c u u m t o dryness. T h e residue was dissolved i n m e t h a n o l a n d a p p l i e d t o P L C plates o f silica gel G F2 5 4. F4, F5 a n d F6 w e r e o b t a i n e d f r o m the c h r o m a t o g r a m w i t h S3 s o l v e n t system ( C h r o m a t o g r a m - I I ) .
Q u a n t i t a t i v e a n a l y s i s o f t o t a l a l k a l o i d s
A c r u d e alkaloidal m i x t u r e w a s o b t a i n e d f r o m the d r i e d a n d p o w -d e r e -d aerial parts o f G e n i s t a a c a n t h o c l a -d a ( 2 0 g ) w i t h the m e t h o -d m e n t i o n e d a b o v e . T h e c r u d e alkaloidal m i x t u r e was treated w i t h excess o f 0 . 0 1 N H C 1 s o l u t i o n a n d b a c k titration was m a d e with 0 . 0 1 N N a O H s o l u t i o n using m e t h y l red a s i n d i c a t o r ( 1 ) . T h i s m e t h o d was c a r r i e d o u t in the s a m e w a y for 5 times and a v e r a g e result was f o u n d as 0 . 1 2 % .
RESULTS and DISCUSSION
In this research, the a l k a l o i d s of the aerial parts of Genista acant-hoclada w e r e isolated a n d their structures w e r e i d e n t i f i e d b y I R spect-r o s c o p y a n d e l e m e n t a spect-r y analysis.
F A U G E R A S a n d P A R I S studied o n Genista acanthoclada c o l l e c ted i n G r e e c e i n 1967. T h e y isolated r e t a m i n e f r o m stems a n d N -m e t h y l c y t i s i n e fro-m f l o w e r s o f this plant, a n d o n l y i d e n t i f i e d l u p a n i n e , a n a g y r i n e a n d cytisine b y gas c h r o m a t o g r a p h y . T h e s e workers m e n -t i o n e d -tha-t spar-teine is n o -t presen-t in -the aerial par-ts of -the plan-t.
In this study, we h a v e f o u n d the total alkaloid c o n t e n t as 0 . 1 2 % a n d isolated six q u i n o l i z i d i n e alkaloids. After p r e p a r i n g their picrate salts we identified t h e m as sparteine, retamine, lupanine, anagyrine, N-methylctisine and cytisine.
F1 ( S p a r t e i n e )
T h e u p p e r b a n d ( Rf = 0 . 8 3 ) o f the c h r o m a t o g r a m - I was eluted w i t h c h l o r o f o r m : m e t h a n o l ( 7 : 3 ) a n d e v a p o r a t e d u n d e r v a c u u m . An o i l y residue r e m a i n e d . Its picrate was p r e p a r e d . T h e picrate salt o b t a i n e d was 6 8 m g , y e l l o w needles, m . p . 2 0 8 ° C ( 3 ) , Rf ( S1 0 . 9 3 , S2 0 . 8 3 , S3 0 . 0 7 ) .
Analysis o f the c o m p o u n d gave the following results: C , 4 6 . 8 7 ; H , 4 . 6 7 ; N , 1 6 . 1 0 ; 0 , 3 2 . 3 6 % .
A c c o r d i n g to these results the m o l e c u l a r formula was calculated t o b e C2 7H3 2N8O1 4.
E l e m e n t a r y analysis, T L C , m . p . a n d I R spectra c o m p a r i s o n o f the salt w i t h the standart sample s h o w e d that F1 was sparteine. Fi-gure-1 shows the I R spectra o f the c o m p o u n d F1 a n d sparteine d i p i c -rate ( p r e p a r e d f r o m M e r c k sparteine sulphate).
Figure-1. IR spectra of sparteine dipicrate and F, A = Sparteine dipicrate, B = F1
F2 ( R e t a m i n e )
T h e s e c o n d b a n d ( Rf = 0 . 5 0 ) was eluted w i t h c h l o r o f o r m : met h a n o l ( 7 : 3 ) , c o n c e n t r a t e d u n d e r v a c u u m to a small v o l u m e and
refri-Alkaloids of Genista acanthoclada D C . 13
g e r a t e d for 4 8 h o u r s . T h e y e l l o w crystals w h i c h settled o u t w e r e r e c r -ystallized f r o m a c e t o n e a n d the crystals o b t a i n e d w e r e w a s h e d w i t h c o l d a c e t o n e t o g i v e 108 m g colorless r e g u l a r prisms w i t h m . p . 1 6 4 ° C ( 4 ) , Rf ( S1 0 . 7 9 , S2 0 . 5 0 , S3 0 . 1 8 ) .
Analysis o f the c o m p o u n d g a v e the f o l l o w i n g results: C , 7 1 . 9 0 ; H , 1 0 . 5 0 ; N , 1 1 . 1 8 ; 0 , 6 . 4 2 % .
T h e m o l e c u l a r f o r m u l a w a s c a l c u l a t e d t o b e C1 5H2 6N2O . E l e m e n t a r y analysis, T L C , m . p . a n d I R spectra ( 5 ) c o m p a r i s o n o f the c o m p o u n d s h o w e d that F2 w a s r e t a m i n e .
T h e T L C , m . p . ( 1 6 8 ° C ) a n d I R spectra (6) c o m p a r i s o n o f the d i p i c r a t e salt o f the c o m p o u n d also p r o v e d its i d e n t i t y .
F3 ( L u p a n i n e )
T h e third b a n d ( Rf= 0 . 2 4 ) was eluted w i t h c h l o r o f o r m : m e t h a -n o l ( 7 : 3 ) a -n d c o -n c e -n t r a t e d u -n d e r v a c u u m . Its p i c r a t e salt p r e p a r e d was 8 2 m g , m . p . 1 7 9 ° C ( 7 ) , Rf ( S1 0 . 6 1 , S2 0 . 2 4 , S3 0 . 6 9 ) .
Analysis o f the c o m p o u n d g a v e the f o l l o w i n g results: C , 5 2 . 7 5 ; H , 5 . 7 1 ; N , 1 4 . 6 0 ; 0 , 2 6 . 9 4 % .
A c c o r d i n g t o the results the m o l e c u l a r f o r m u l a w a s c a l c u l a t e d t o b e C2 1H2 7N508.
E l e m e n t a r y analysis, T L C , m . p . a n d I R spectra ( 6 ) c o m p a r i s o n o f the salt s h o w e d that F3 w a s l u p a n i n e .
F4 ( A n a g y r i n e )
T h e u p p e r b a n d ( Rf = 0 . 6 6 ) o f the c h r o m a t o g r a m - I I w a s eluted w i t h c h l o r o f o r m : m e t h a n o l ( 7 : 3 ) a n d c o n c e n t r a t e d u n d e r v a c u u m t o g i v e a l i g h t y e l l o w o i l y l i q u i d . It w a s crystallized as p i c r a t e . Its p i c r a t e i n needles was 9 6 m g , m . p . 2 4 3 ° C , Rf ( S1 0 . 3 5 , S2 0 . 0 9 , S3 0 . 6 6 ) .
A n a l y s i s o f the c o m p o u n d g a v e the f o l l o w i n g results: C , 5 3 . 1 8 ; H , 4 . 9 0 ; N , 1 4 . 9 2 ; 0 , 2 7 . 0 0 % .
T h e m o l e c u l a r f o r m u l a w a s c a l c u l a t e d t o b e C2 1H2 3N5O8. E l e m e n t a r y analysis, T L C , m . p . a n d I R spectra ( 8 ) c o m p a r i s o n o f the salt s h o w e d that F4 w a s a n a g y r i n e .
F5 ( N - M e t h y l c y t i s i n e )
T h e s e c o n d b a n d ( Rf = 0 . 6 1 ) w a s eluted w i t h c h l o r o f o r m : m e t -h a n o l ( 7 : 3 ) a n d c o n c e n t r a t e d u n d e r v a c u u m t o dryness. T -h e residue w a s dissolved i n m e t h a n o l a n d p r e c i p i t a t e d a s F5 p i c r a t e . After crystal-l i z a t i o n , F5 p i c r a t e w a s o b t a i n e d 118 m g , l o n g y e l l o w needles, m . p . 2 2 9 ° C ( 9 ) , Rf ( S1 0 . 3 0 , S2 0 . 0 7 , S3 0 . 6 1 ) .
Analysis o f the c o m p o u n d g a v e the f o l l o w i n g results: C , 5 0 . 0 1 ; H , 4 . 4 2 ; N , 1 6 . 1 4 ; O , 2 9 . 4 3 % .
A c c o r d i n g t o these results the m o l e c u l a r f o r m u l a was c a l c u l a t e d t o b e C1 8H1 9N5O8.
E l e m e n t a r y analysis, T L C , m . p . a n d I R spectra (7) c o m p a r i s o n o f the salt s h o w e d that F5 was N - m e t h y l c y t i s i n e .
F6 ( C y t i s i n e ) T h e third b a n d ( Rf= 0 . 4 2 ) w a s e l u t e d w i t h c h l o r o f o r m : m e t h a -n o l ( 7 : 3 ) a -n d e v a p o r a t e d u -n d e r v a c u u m t o dry-ness. T h e residue w a s dissolved i n m e t h a n o l a n d p r e c i p i t a t e d a s F6 p i c r a t e . T h e p i c r a t e salt crystallized w a s 187 m g , m . p . 287 C , Rf (Sl 0 . 0 7 , S2 0 . 0 3 4 , S3 0 . 4 2 ) .
Analysis o f the c o m p o u n d g a v e the f o l l o w i n g results: C , 4 8 . 6 8 ; H , 4 . 1 2 ; N , 1 6 . 7 1 ; O , 3 0 . 4 9 % .
T h e m o l e c u l a r f o r m u l a was c a l c u l a t e d t o b e C1 7H1 7N508. E l e m e n t a r y analysis, T L C , m . p . a n d I R spectra ( 1 0 ) c o m p a r i -s o n o f the -salt -s h o w e d that F6 was cytisine.
A c k n o w l e d g e m e n t
T h e a u t h o r s are grateful t o P r o f . D r . A . U L U B E L E N f o r her k i n d g r a n t o f r e t a m i n e , l u p a n i n e , a n a g y r i n e p e r c h l o r a t e a n d N -m e t h y l c y t i s i n e p i c r a t e , used as standart c o -m p o u n d s in this research.
Alkaloids of Genista acanthoclada D C . 15
REFERENCES
1- Paech, K. and Tracey, M . V . , Moderne Methoden der Pflanzenanalyse, 4, 372, Springer, Berlin-Göttingen-Heidelberg (1955).
2- Faugeras, G. and Paris, M . , "Sur les alcaloides du Genista acanthoclada D C : repartition, isolement de la retamine", Compt. Rend. Acad. Sc., Paris, Ser. D, 264 (10), 1290 (1967).
3- Paech, K. and Tracey, M. V . , Moderne Methoden der Pflanzenanalyse, 4, 461, Springer, Berlin-Göttingen-Heidelberg ( 1955).
4- Mollov, N. M . , Ivanov, I. H. and Panov, P.P., "Alkaloids of Representatives of the Family Leguminosae, Common in Bulgaria", Compt. Rend. Acad. Bulgare des Sc., 24 (12), 1657 (1971).
5- Sandberg, F., "Phytochemical and Pharmacological Studies on Some Alkaloidal Plants of Egypt", Pakistan J. Sci. Ind. 4, 280 (1961).
6- Ulubelen, A. and Doguc, T., Phytochemical Studies on the Flowers of Genista lydia Boiss
(Leguminosae), Doctorate Thesis, Faculty of Pharmacy, University of Istanbul, Istanbul
(1974).
7- Galinovsky, F. in Zechmeister, L. (Ed,). Progress in the Chemistry of Organic Natural
Products, 8, 245, Springer, Wien (1951).
8- Faugeras, G., "Alcaloides et polyphenols des Legumineuses. X X I I . -Les alcaloides du Genista cinerea D C " , Ann. Pharm, Franç., 29 (4), 241 (1971).
9- Faugeras, G. and Paris, M . , "Alcaloides et polyphenols des Legumineuses. X V . - S u r les alcaloides du Genista equisetiformis Spach; Isolement de la N-methylcytisine et de la cytisine", Ibid., 27 (4), 269 (1969).
10- Faugeras, G., Paris, R. and Meyruey, M . H . , "Sur les alcaloides du Retama raetam
