Quinolizidine alkaloids from
Genista ephedroides
Luisa Pistelli
!
,
*
, Alessandra Bertoli
!
, Isa Giachi
!
, Ivano Morelli
!
,
Patrizia Rubiolo
"
, Carlo Bicchi
"
!Dipartimento di Chimica Bioorganica e Biofarmacia-Universita%degli Studi di Pisa,via Bonanno 33, 56126 Pisa, Italy
"Dipartimento di Scienza e Tecnologia del Farmaco-Universita% degli Studi di Torino,via P. Giuria 9, 10125 Torino, Italy
Received 4 February 1999; received in revised form 4 January 2000; accepted 22 March 2000
Abstract
Alkaloids retamine, anagyrine, lupanine, 17-oxoretamine, 12-a-hydroxylupanine were
detected, along with four others unidenti"ed compounds in the aerial parts ofGenista
ephedro-idesD.C. ( 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Genista ephedroides; Leguminosae; Quinolizidine alkaloids; Chemotaxonomy
1. Introduction
Genista ephedroides D.C. (Leguminosae), belonging to tribe Genisteae, is a
perennial endemic shrub in Sardinia. The plant, commonly known in Italy as `Ginestra del Gasparrinia, grows on rocky ground near the sea and#owers from April to May (Pignatti, 1982).
All phytochemical analysis on Genista ephedroides have revealed the presence of
#avonoids (Pistelli et al., 1998) and quinolizidine alkaloids. In particular, Bernasconi et al. (1965) had identi"ed, only on the basis of TLC analysis, sparteine, lupanine, anagyrine, cytisine,N-methylcytisine and retamine.
In this paper we report a reinvestigation of alkaloid composition of Genista
ephedroidesby modern chromatographic techniques as GC and GC}MS (Kinghorn
*Corresponding author. Tel.:#39-50-44074; fax:#39-50-43321. E-mail address:[email protected] (L. Pistelli).
and Balandrin, 1984; Majchrzak et al., 1984; Greinwald et al., 1991, 1992, 1994; Abdel-Halim et al., 1992; Wink and Witte, 1993).
2. Materials and methods
Plant material: The aerial parts ofGenista ephedroideswere collected in May 1995
at Capo Testa, Sardinia, Italy and a voucher specimen (voucher URB-167/94) is held in the Herbarium of the Istituto di Botanica ed Orto Botanico (Universita` di Urbino-URB).
Extraction procedure: The dried aerial parts ofGenista ephedroidesD.C. (680 g) were
extracted in a Soxhlet apparatus withn-hexane, CHCl
3and MeOH in turn, to obtain
the n-hexane fraction (7.35 g), the chloroform fraction (14.4 g) and the methanol
fraction (58.7 g), respectively. The basic components of the chloroform fraction and the methanol fraction were obtained by extraction with H
2SO4 0.5 M followed by
alkalinization with 25% NH
4OH. Two extracts were thus obtained: CC1 (8.6 mg)
from 2.3 g of the chloroform fraction and MC
1 (720 mg) from 32 g of the methanol
fraction.
A part of the chloroform fraction (11.45 g) was also fractionated by Sephadex LH-20 (MeOH}CHCl
3 9 : 1) to give eight fractions C1}C8. Alkaloids were present
only in the "rst three fractions : C
1 (7 mg), C2 (1.484 g) and C3 (2.565 g). C2 and
C
3were further puri"ed to separate the alkaloids from other non-alkaloid components
by#ash chromatography (CHCl
3}MeOH}H2O 6 : 4 : 1 and 7 : 3: 0.3, respectively).
GC}MS analysis:The GC}MS analysis was performed with HP 5890 series II plus
gas chromatograph equipped with a Duran 50 capillary column (30 m]0.25 mm) coated with a "lm of polydimethylsiloxane, 7% biphenyl, 1% vinyl ("lm thickness 0.25lm) and a HP5988 mass-selective detector.
Analytical conditions: Injector and detector temperatures 250 and 2803C,
respective-ly; oven temperature programmed from 1903C (1@) to 2803C at 53C/min, then 10@ isothermal conditions at 2803C; carrier gas helium at constant #ow of 1.5 ml/min; source 45 eV; injection volume:1ll; split ratio: 1/10.
Alkaloid identixcation: The alkaloids mixtures were analysed by GC and GC}MS
and identi"ed on the basis of their Kovats retention indices (KI) and mass spectral fragmentation literature data (Greinwald et al., 1991,1992,1994; Kinghorn and Bal-andrin, 1984; Majchrzak et al., 1984). Furthermore, the compound identities were con"rmed by comparing their KI and mass spectra with those in Dr. Ludger Witte's database. The results are summarised in Table 1.
3. Results
The alkaloid pattern ofGenista ephedroides, investigated by GC and GC}MS, has revealed"ve known quinolizidine alkaloids, namely, retamine, lupanine, anagyrine, 17-oxoretamine and 12-a-hydroxylupanine (see Fig. 1), along with four other
Table 1
GC}MS data of quinolizidine alkaloids inGenista ephedroides
Compound KI M` m/z(abundance%)
1 Retamine 2008 250 98 134 97 41 207 96 232 150 84 175 250 (100) (31) (26) (23) (15) (14) (12) (10) (9) (8) (3) 2 Lupanine 2190 248 136 55 149 98 110 84 248 219 191
(100) (46) (44) (28) (23) (16) (16) (5) (3) 3 Anagyrine 2405 244 98 41 244 146 136 160 229
(100) (23) (18) (14) (9) (8) (3)
4 17-Oxoretamine 2278 264 98 97 247 110 41 96 136 264 84 236 (100) (87) (63) (55) (47) (41) (34) (25) (19) (3) 5 12-a-hydroxylupanine 2380 264 41 246 55 134 112 58 94 207 148
(100) (69) (58) (55) (42) (38) (30) (24) (18) 6 Unidenti"ed 2748 312 98 136 41 312 68 277 214 148 249 (100) (87) (25) (13) (10) (10) (8) (7) (6) 7 Unidenti"ed 2341 322 98 249 41 84 279 231 137 206 322
(100) (18) (18) (10) (10) (8) (8) (7) (3)
8 Unidenti"ed 1939 194 97 96 83 55 41 98 194 136 112 151 179 (100) (73) (55) (55) (51) (44) (38) (30) (29) (13) (5) 9 Unidenti"ed 2323 294 96 98 97 55 41 84 136 294 208 150 264
(100) (97) (80) (59) (57) (41) (27) (8) (8) (5) (4)
Identi"cation of these compounds was based on their Kovats indices and mass spectrum, which showed distinctive peaks of sparteine-type, lupanine-type and a
-pyridone alkaloids (Greinwald et al., 1991,1992,1994; Kinghorn and Balandrin, 1984).
Only retamine (1) (C
15H26N2O), among these components, was isolated as pure
compound in MC
1fraction. In agreement with the literature (Bernasconi et al., 1965;
Savelli and Sparatore, 1975) retamine turned out to be one of the most abundant quinolizidine alkaloids inGenista ephedroides.
It is interesting to note that 17-oxoretamine (4) (C
12H24N2O2) and 12-a
-hy-droxylupanine (5) (C
12H24N2O2) have never been detected inGenista ephedroides
before the present study. The di!erent localization of carbonyl group in compound
4was established on the basis of the fragment ion atm/z98 (100%) [C
6H12N]`, the
fragment ion at m/z110 (10%) [C
7H12N]`and the fragment ion atm/z236 (3%)
[C
11H24N2O]`. These peaks are typical of quinolizidine alkaloids with carbonyl
group on C ring (Majchrzak et al., 1984).
Among unidenti"ed compounds6}9 it was possible to suggest an hypothetical structure only for8and9. The molecular peak atm/z194 and base peak atm/z97 in mass spectrum of 8 suggested an hydrogenated derivative of cytisine (Wink et al., 1995), while compound9is probably a lupanine-type alkaloid, due to the presence of peaks atm/z150, 136 and 96. The structure of these compounds cannot be elucidated unambiguously with the data obtained until now.
4. Discussion
The genus Genista is known to contain numerous #avonoids as well as quinolizidine alkaloids (Southon, 1994).
We have noticed that chromatographic separation by gel"ltration on Sephadex LH-20 allowed a more complete isolation of alkaloid fraction than the acid}base extraction.
GC and GC}MS analysis has detected a di!erent qualitative composition of alkaloids compared to that of Bernasconi et al.(1965). Most noteworthy is we have not found evidence for the presence of sparteine, cytisine, N-methylcytisine, but we have identi"ed 17-oxoretamine (4) and 12-a-hydroxylupanine (5), which were not
mentioned in the Bernasconi et al.(1965) study. These di!erent observations might be due to the ambiguity of structural assignment using rf comparison of TLC.
Other factors which might account for the di!erent results are the source and geographical area of used plant material, since Bernasconi et al.(1965) collected the samples at the Royal Botanical Gardens, Kew, not in natural habitat.
Acknowledgements
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