Phytochemistry,
Vol. 34,No. 4, pp. 11471151,1993
Printedin Great Britain.
ALKALOIDS
003l-9422/93
$6.00
+ 0.00
0 1993PergamonPressLtd zyxwvutsrqpo
OF zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
ERYTHROXYLUM
ZAMBESIACUM
STEM-BARK*
PHILIPPE CHRISTEN,tS MARGARET F. ROBERTS,~ J. DAVID PHILLIPSON$
and WILLIAM C. EVANS~~
TDepartment of Pharmacognosy, University of Geneva, Sciences II, 30, Quai E.-Ansennet, CH-1211 Geneva 4, Switzerland;
@epartment of Pharmacognosy, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC 1N lAX,
U.K.; IlDepartment of Pharmaceutical Sciences,University of Nottingham, Nottingham NG7 2RD, U.K.
(Received in revised fom 14 April 1993)
Key Word Index--Erythroxylum
zambesiacum; Erythroxylaceae;
stem-bark; tropane alkaloids; nico-
tine; GC; GC-MS; chemotaxonomy.
Abstract-Twenty-seven
alkaloids were identified from the stem-bark of Erythroxylum zambesiacum by GC and GCMS. The characteristic alkaloids were tropane derivatives, together with nicotine. In addition to known substances
previously isolated from the root-bark, a further 20 alkaloids were identified in the stem-bark. The principal base
was 3a-(3’,4’,5’-trimethoxybenzoyloxy)tropane.
Additionally, three new alkaloids were characterized as 6fibenzoyloxytropan-3-one
and, tentatively, 6-isovaleryloxytropan-3-01 and 3-(2-methylbutyryloxy)tropan-6,7-diol.
INTRODUCTION
Erythroxylum zambesiacum is a small tree mainly confined to the Zambesi valley near the Victoria Falls. It was
first described in 1962 [ 1J and is related to the W. African
species E. mannii. The alkaloid analysis of the root-bark
of E. zambesiacum has been reported previously [2].
However the stem-bark does not appear to have been
investigated for alkaloids. Knowledge of the complete
alkaloid pattern is of interest not only phytochemically,
but also in relation to aspects of alkaloid biogenesis and
metabolism. As a further contribution to our studies on
the chemotaxonomy of this medicinally important genus,
we now report a detailed phytochemical investigation of
the alkaloidal pattern of the stem-bark of E. zambesiacum.
RESULTS AND DISCUSSION
A preliminary TLC examination of petrol and diethyl
ether extracts of the powdered stem-bark revealed the
presence of five major alkaloids as well as several other
minor bases. During recent years, capillary GC has been
used as a powerful method for the analysis of tropane
alkaloids [3]. The combination of capillary GC with
mass apectrometry is a sensitive tool which has shown
that tropane alkaloid containing plants generally have a
large number of alkaloids which were not detected by
older, less sensitive methods [4].
A simple GC and GC-MS procedure has been developed for the identification of tropane alkaloids in
crude plant extracts [S]; this revealed the presence of 27
*Part 11 in the series ‘Alkaloids of the genus Erythroxylwm’.
For part 10 see. ref. [20].
IAuthor to whom correspondence should be addressed.
compounds in the stem-bark of E. zambesiacum representing 0.1% of the dried plant material (Table 1). Of these,
seven alkaloids have already been reported to occur in the
root-bark. Twenty bases were unknown as constituents of
E. zambesiacum. Furthermore, three novel alkaloids have
been identified.
The characteristic alkaloids are tropanol esters of a
range of acids. The amino alcohols are tropine, 3,6dihydroxytropane and 3,6,7_trihydroxytropane and the
acids involved in esterification are listed in Table 2.
The alkaloid mixture was dominated by 3a-(3’,4’,5’-trimethoxybenzoyloxy)tropane
(22), 3a-(3’,4’,5’-trimethoxycinnamoyloxy)tropane
(24), 3a-(3’,4’,5’-trimethoxybenzoyloxy)tropan-6/I-01
(23), 68-benzoyloxytropan-3a-ol
(19) and 3a-phenylacetoxytropane
(17). Furthermore,
one nor-derivative (15) and nicotine (7) were identified
The latter compound has been isolated previously
in cultivated coca, E. coca [6] as well as in the stem-bark
of E. cuneatum [7]; it also co-occurs with tropane
alkaloids in Wettsteins’s tribe Salpiglossideae of the
Solanaceae.
Intermediates and products of side-reactions of the
tropane alkaloid biosynthetic pathway, e.g. hygrine (l),
hygrolines (2,3), tropinone (4), tropine (5) and pseudotropine (6) were identified by GC-MS as constituents of
E. zambesiacum stem-bark. The hygrolines are found as
two diastereoisomen which have not been detected previously in this species, but their trivial derivation from
hygrine makes them wmmon side-products of the biosynthetic route leading to the tropane alkaloids.
The El mass spectrum of8 exhibited a [M]’ at m/z 155
(corresponding to the formula CsH,,NO,) and a peak at
m/z 111 [M-C(6)HOH-C(7)H,]+
indicating
a 6hydroxy-3-one derivative. Furthermore, a strong peak at
1147
P. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGF
CHRISTEN et al.
1148
Table 1. Alkaloids identified in stem-bark of zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON
Erythroxylum zambesiacum
Compound
no.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
CM1+
Reference*
material
Reference?
mass spectrum
+
-.
+
+
+
+
+
+
+
+
+
+
+
Previously
identifiedt in
the root-bark zyxwvutsrqpo
Alkaloid
zin)
m/z
Hygrine
Hygroline A
Hygroline B
Tropinone
Tropine
Pseudotropine
Nicotine
Tropan-68-ol-3-one
Tropan-3a,6/?diol
3-(2-Methylbutyryloxy)tropane
3cc-Isovaleryloxytropan-6/Z-o] (valeroidine)
C,,H,,NO,
6-Isovaleryloxytropan-3-o&
6/I-Tigloyloxytropan-3a-ol
3-Phenylacetoxynortropane
3-(2-Methylbutyryloxy)tropan-6,7-dials
3a-Phenylacetoxytropane
6/?-Benzoyloxytropan-3-o@
6/LBenzoyloxytropan-3a-ol
3a-(3’,4’-Dimethoxybenzoyloxy)tropane (convolamine)
3-Cinnamoyloxytropan-6-01
b-(3’,4’,5’-Trimethoxybenzoyloxy)tropane
3a-(3’,4’,5’-Trimethoxybenzoyloxy)tropan-6/?-o]
3a-(3’,4’,5’-Trimethoxycinnamoyloxy)tropane
3a-(3’,4’,5’-Trimethoxybenzoyloxy)tropan68,7B-diol
3a-(3’,4’,5’-Trimethoxycinnamoyloxy~6/3benzoyloxytropane
3a-(3’,4’,5’-Trimethoxycinnamoyloxy)-6/Ibenzoyloxy-7jGacetoxytropane
2.62
2.85
3.07
3.96
4.49
5.45
8.07
8.68
10.12
12.68
22.91
23.03
23.11
23.33
23.66
23.80
24.53
26.95
29.00
141
143
143
139
141
141
162
155
157
225
241
239
241
239
245
257
259
259
261
34.79
35.88
37.85
41.38
43.95
305
287
335
351
361
47.15
367
+
+
+
57.76
481
+
+
+
59.03
539
+
+
+
+
+
+
+
+
+
+
_
+
+
-I-
.+
+
+
c
*Sample available from W. C. Evans.
PMS available or in literature.
$Alkaloid present in root-bark.
$New alkaloid.
Table 2. Acids recorded as ester components of tropanols in
E. zambesiacum stem-bark
Acetic (1)
2-Methylbutyric (2)
Isovaleric (2)
Tight (1)
Benzoic (4)
Phenylacetic (2)
Cinnamic (1)
3,4-Dimethoxybenzoic (1)
3,4,5-Trimethoxybenzoic (3)
3,4,5-Trimethoxycinnamic (3)
Numbers in parenthesis indicate number of alkaloids found
involving this acid.
m/z 83 is due to further decomposition of the m/z 111 ion
fragment by loss of carbon monoxide. This expulsion is
diagnostic for 6- and 7-oxygenated tropinones [S]. Other
typical fragments at m/z 97,82,57 and 42 were consistent
with an alkaloid having the structure, tropan-6@-ol-3one. This compound had identical properties (TLC, GC,
mass spectrum) with those of the synthesized compound
run under the same conditions. This is the first report of
the natural occurrence of tropan-68-ol-3-one. Tropan3a,6/&diol (9) was also identified as a constituent of the
stem-bark. It has not been reported previously as a
natural alkaloid from the genus Erythroxylum although it
has been already isolated from species of Datura [9] and
Schizanth
[lo].
3-(2-Methylbutyryloxy)tropane
(10) is reported to occur in the genus Erythroxyhm for the first time. However,
in the absence of other spectroscopic data, it was not
possible to assign the configuration of the substituent at
C-3. This alkaloid was isolated from Duboisia kichhardtii
[ 1 l] and recently identified by GC-MS in Datura innoxia
c41.
The CM] + of alkaloid 12 at m/z 239, together with
prominent ions at m/z 156,139, 122,96 and 94 suggested
a disubstituted tropane nucleus of molecular formula
C13HZ1N0, with an esterifying acid C,H,O,. The base
peak at m/z 113 indicated that the free hydroxyl group is
at the C-3 position. In the absence of other spectroscopic
1149
Alkaloids from zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLK
Erythroxylum
The EI mass spectral fragmentation of 18 was condata, it was not possible to define which isomer (angeloyl,
tigloyl or senecioyl) was attached to C-6.
sistent with that for a benzoic acid ester (m/z 122, 105,77)
The mass spectra of zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
11 and 13 showed identical [M]’
of a hydroxytropinone (m/z 111,110,83). The occurrence
at m/z 241 corresponding to the elemental composition
of fragment ion peaks at m/z 154 [M-C6H$0]+
and
138 [M -C,H$Oz]+
confirmed the presence of benzoic
Ci3Hz,N03. The fragmentation patterns are characteracid as the esterifying acid. The position of the ester group
istic of monoesters of dihydroxytropane. The alkaloid
within the molecule was established from the mass spec11 shows fragment ion peaks at m/z 197 [M-C(6)
trum in which loss of the C-6 and C-7 atoms gave rise to
HOH-C(7)HJ +, 156 [M-CSH90]+
and 140 [M
the base peak at m/z 111 [M - C(7)H,C&)HOCOC,H,]‘.
-CSH902]+
with a base peak at m/z 94. This confirms
the attachment of the ester function at C-3 and a free Such cleavage has been shown to occur most readily
when the C-6 and/or C-7 atoms of the tropane ring bear a
hydroxyl group at C-6 of the tropane nucleus. On the
other hand, alkaloid 13 had a base peak at m/z 113 and a substituent [S]. The [Ml+ corresponded with the formula C,,Hi,NOs.
The strong peak at m/z 83 supports
prominent ion at m/z 96, indicating that the free hydroxyl
is at the C-3 position and the esterifying acid at C-6. The
the 3-one assignment due to elimination of the carbonyl
assignments of alkaloids 11 and 13 as isovaleryl esters. group [13]. Other typical fragments at m/z 96,95,94,82
could be deduced from the fragmentation pattern of and 42 are characteristic of the fragmentation of the
tropane nucleus. The structure of the new alkaloid was
the acid moieties. The presence of [M - 151’ and
[M -43]+ ions and the absence of a [M - 29]+ ion are
confirmed at 6/I-benzoyloxytropan-3-one
by comparison
(TLC, GC, mass spectrum) with the semi-synthetic comassigned to the loss of an isovaleryl moiety. Valeroidine
and
(11) was first isolated as a by-product in the manufacture of pound prepared from 6fl-hydroxytropan-3-one
benzoyl chloride [ 163.
cocaine from Peruvian coca leaves and from the rootbark of zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
E. dekindtii [12]. Compound
13, tentatively
6/I-Benzoyloxytropan-3a-ol(19)
was identified by comidentified as 6-isovaleryloxytropan-3-01
has not been
parison (TLC, GC, mass spectrum) with an authentic
sample isolated from the leaves of E. cuneatum [7]. This
previously recorded. However, lack of material precluded
alkaloid has also been isolated from E. zambesiacum rootthe making of any stereochemical observations.
bark [2].
6fi-Tigloyloxytropan-3a-ol(14),
was identified by comFrom a biogenetic point of view, it is of interest to point
parison (TLC, GC, mass spectrum) with an authentic
out for the Solanaceae that it is usually considered that
sample. The alkaloid was first isolated from Datura
hydroxylation and esterification of the tropane ring at Ccornigera
[14] and has also been detected in
E. cuneatum [7].
6 and C-7 take place after the reduction of tropinone to
tropine and Y-tropine. Biosynthetic experiments originAlkaloid 15 gave a mass spectrum consistent with that
ally indicated the reductions to be non-reversible [17],
predicted for 3-phenylacetoxynortropane,
the base peak
but recently Hashimoto et al. [18] have demonstrated, for
at m/z 110 being typical of a substituted nortropan-3-ok
the presence of m/z 91 (C,H,) and the absence of m/z 119 Hyoscyamus root cultures, the reversibility of the mediated tropinone to tropine conversion. The identification
(ArCO) suggested phenylacetic acid as esterifying acid.
of alkaloids S, 9,18 and 19 suggests that the biosynthesis
This alkaloid has been previously reported as a minor
of 6-hydroxytropane
esters, at least in Erythroxylum
constituent of the root-bark of E. hypericijiofolium[15]. The
N-methyl analogue (17) was unambiguously identified by species, takes place before the reduction of the ketone at
C-3. This hypothesis has no chemical or biochemical
comparison (TLC, GC, mass spectrum) with the base
precedence and leads us to propose the possible new
prepared from tropine and phenylacetyl
chloride.
Compound 17 has been previously isolated from E. biosynthetic relationships which are illustrated in Fig. 1.
Furthermore, it is noteworthy that no 6,7dihydroxytropandekindtii [ 121.
Compound 16 is a new alkaloid identified as 3-(2- 3-one esters have been detected in the alkaloid mixture
methylbutyryloxy)tropan-6,7-diol.
Its mass spectrum is indicating that esterification at C-7 occurs after the
stereospecific reduction of the ketone. This hypothesis
characteristic of a monoester of 3,6,7_trihydroxytropane.
requires experimental verification and at this stage we
The [M]’ at m/z 257 corresponds to the molecular
cannot rule out the possibility that tropane alkaloids
formula C,sHz3N0,. Further indication of the 1w, came
from the CI mass spectrum which displayed a [M + H] + formed in the roots are hydrolysed and oxidized in the
peak at m/z 258. Since the base peak was at m/z 94 it was transpiration stream from roots to aerial parts.
The EI mass spectrum of alkaloid 20 exhibited a [M] +
considered that the alkaloid had the ester function attachThe
ed to C-3. The occurrence of a prominent peak at m/z 197 at m/z 305 consistent with the formula C,,H,,NO,.
fragmentation pattern [m/z 124 (base peak), 140, 94, 83,
[M-60]+
confirmed the attachment of two hydroxyl
82, 67, 421 corresponded to that of a 3-substituted
groups to C-6 and C-7. Lack of material prevented the
tropane nucleus. Ions at m/z 124 and 140 are consistent
application of other analytical techniques to assign the
stereochemical orientation of the substituent attached to with the loss of a dimethoxybenzoic acid from the [M] +.
The presence of this acid is further indicated by ions at
C-3. The esterifying acid was identified as 2-methylbum/z 165 [(MeO),C,H,CO]+
and 182 [(MeO),
tyric acid by the presence of ions corresponding to [M
C,H,C02H] +. In the absence of other spectroscopic
-Me]+ and [M -CzHJ+.
The absence of an ion corresdata, it was not possible to establish the configuration of
ponding to [M - C(Me),] + is evidence for the 2-methylbutyryl ester and ruled out the possibility of other acids. the substituent at C-3 and to define the position of the
1150
P. CHRISTENet al. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON
EXPERIMENTAL
Stem-bark
of E. zambesiacum N.
Robson was collected in Zambia from the same plants as
examined in ref. [Z].
2,3
TLC. Carried out on silica gel F,,, with (i) Me&OH,O-conc.
NH, (80:15:2)
and (ii) CHCl,-Me,COcont. NH, (50: 50: l), and on aluminium oxide 60 F254
with (iii) Et,O-EtOH
(95: 1).
GC and GC- M S. Alkaloids were identified by GC on
the basis of the similarity of R,s with authentic compounds and by their GC-MS fragmentation
patterns. An
instrument equipped with two detectors (NPD and FID)
was used in the present study as described previously [S].
A 15m x 0.252mm
i.d. fused-silica
capillary column
coated with the methylsilicone phase DB-1 (film thickness
0.25 pm) and with He as carrier gas at 0.5 bar pressure
was used. The temp. prog. was, isothermal 80” for 1 min,
80-310” at 4” min-‘, isothermal 310” for 5 min. Both inj.
and det. temps were maintained
at 360”. GC-MS was
performed in the EI mode at 70 eV. Operating conditions
were (i) isothermal 35” for 2 min, 35-100” at 30” min-‘,
100-300” at 10” min-‘,
isothermal
300” for 5 min.
(ii) isothermal 35” for 2 min, 35-300” at 30” min-I, isoth0” zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDC
OH
OH
OR
ermal 300” for 5 min. CI mass spectra were recorded on
19
9
5
6
the same instrument using NH, as reagent gas.
Fig. 1. Suggested biosynthetic Inter-relationships
between
Extraction of alkaloids. Powdered stem-bark (64g) was
alkaloids of the 3.6-dihydroxytropane-type.
extracted with 250 ml n-hexane. The dried defatted plant
material was mixed with Ca(OH), (12 g) and H,O (50 ml),
allowed to stand overnight and then exhaustively exmtthoxy groups on the aromatic ring. However, TLC,
tracted with EtzO. The Et,0 extract was coned to a small
GC retention time and mass spectral data were consistent
vol., acidified with 0.1 M HCl (10 ml) and extracted with
with the structure of convolamine [3o+‘$dimethoxybenEt,0 (3 x 5 ml). The aq. layer, basified by adding 17%
zoyloxy)tropane].
This alkaloid has been isolated preNH, soln was repeatedly
extracted
with petrol (bp
viously from Conuoloulus [ 191 but has not been reported
as a component of Erythroxylum.
4&60”) and Et,O. The petrol and ether extracts were
Compound
21 has been tentatively
identified as 3dried (Na,SO,)
to afford 2 residues of 29.7 mg and
cinnamoyloxytropan-6-01:
m/z 287 [M] +; 243 [M
66.9 mg, respectively.
- C(7)H,C(6)HOH]
+; 156 [M - PhCH=CHCO]
+; 140
Identijication of alkaloids. Retention
data and mass
[M -PhCH=CHCO,]
+; 131 [PhCH=CHCO]
+; 148
spectra of known bases were compared with those of
[PhCH=CHCO,H]
+. The 3cr,6/?-isomer was isolated as
authentic
material or from lit. values (Table 1). The
the principal alkaloid from the leaves of E. hypericijiilium stereochemistry
of substituents at C-3, C-6 and C-7 could
[20] and as a minor constituent
from the root-bark of E.
only be determined by GC with alkaloids for which ref.
australe [7]. However, it has not been reported
in E.
material was available. The 3 new alkaloids have the
zambesiacum.
following mass spectra.
A characteristic feature of E. zambesiacum is the abund6- Isoualery loxy tropan- 3- 01 (13). EIMS, m/z (rel. int.):
ance of 3’,4’,5’-trimethoxybenzoic
(22,23,25) and 3’,4’,5’- 241 [M]’
(ascribable
to C,,H,,NO,)
(7), 226 [M
trimethoxycinnamic
(24,26,27) derivatives in the alkaloid
-Me]+
(I), 224 [M-OH]’
(l), 198 [M-C3H7]+
(I),
mixture (Table 2). The compounds identified in the stem156 [M-C,H,O]+
(4), 140 [M-C,H,O,]+
(15), 113
bark are typical of those isolated in the root-bark
[2].
[M - C(7)H,C(6)HOCOC,H,]
+ (lOO), 96 (46), 94 (26), 82
Compounds
22-27 were compared
(TLC, GC, mass
(16), 57 (24), 42 (27).
spectra) with authentic compounds
and were consistent
3- (2- M ethy lbuty ry loxy )tropan- 6,7- diol(l6).
EIMS, m/z
with the assigned structures. The principal alkaloid iden(rel. int.): 257 [M]’ (ascribable to C,,H,,NO,)
(I), 228
tified in the stem-bark
is 3c(-(3’,4’,5’-trimethoxybenz[M -CIHS]+
(l), 197 [M-C(6)HOHC(‘T)HOH]+
(3),
oyloxy)tropane
(22). However, the corresponding
nor172 CM-C,H,O]+
(l), 156 [M-C,H,OJ+
(9), 102
derivatives of 22 and 23, as well as 6#?-benzoyloxytropan[C,H&O,H]+
(l), 95 (65), 94 (lOO), 57 (17), 42 (20).
3c(,7/?-diol, 6/?-benzoyloxy-3a-(3’,4’,5’-trimethoxycinna6P- Benzoy loxy tropan- 3- one.
(18). EIMS,
m/z (rel.
moyloxy)tropan-78-01,
3a-phenylacetoxytropan-68-01
int.): 259 [M]’ (ascribable to C,SH,,NO,)
(9), 201 (5),
and 6/?-benzoyloxytropan-3a-ol
have not been detected
154 [M-C6HSCO]+
(27), 138 [M-C,H&O,-J+
in the stem-bark, but they have been reported from the
(18), 137 (15). 122 [C,H,CO,H]+
(3), 111 [M
root-bark 121.
-C(7)H,C(6)HOCOC,H,]
+ (lOO), 110 [C,H,NO]
+
Plant
material.
Alkaloidsfrom Erythroxylum
(39), 105 [C6HSCO]+ (52), 96 (20), 95 (28), 94 (74), 83 (94),
82 (42), 77 (56), 42 (93). The EIMS was identical with that
of the synthetic compound
conditions.
[16] run under the same
1151 zyxwvutsrqp
8. Blossey, E. C., Budzikiewicz, H., Ohashi, M., Fodor,
G. and Djerassi, C. (1964) Tetrahedron 20, 585.
9. Evans, W. C. and Than, M. P. (1962) J. Phurm.
Pharmac. 14, 147.
10. Gambaro,
V., Lab&, C. and Castillo, M. (1983)
authors are indebted to Dr G. J.
Phytochemistry 22, 1838.
Langley and Dr M. E. Harrison (ULIRS Mass Spectro11. Rosenblum, E. I. and Taylor, W. S. (1954) J. Pharm.
metry Laboratory at The School of Pharmacy) for perPharmac. 6,410.
12. Al-Yahya, M. A. I., Evans, W. C. and Grout, R. J.
forming the GC-MS analysis. This work was supported
(1979) J. Chem. Sot. Perkin Trans I 2130.
by a grant from the Royal Society of Great Britain under
the European Science Exchange Programme to P.C. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHG
13. Ethier, J. C. and Neville, G. A. (1986) Can. J. SpectroSC. 31, 81.
REFERENCES
14. Evans, W. C. and Griffin, W. J. (1963) J. Chem. Sot.
4348.
1. Robson, N. K. B. (1962) Bol. Sot. Brot. Ser. 2 36, 7.
15. Al-Said, M. S., Evans, W. C. and Grout, R. J. (1986)
2. El-Imam, Y. M. A., Evans, W. C., Grout, R. J. and
J. Chem. Sot. Perkin Trans I 957.
Ramsey, K. P. A. (1987) Phytochemisrry 26,2385.
3. Martinsen, A., Hiltunen, K. and Huhtikangas,
A. 16. Agar, J. T. H. and Evans, W. C. (1976) J. Chem. Sue.
Perkin Trans I 1550.
(1992) Phytochemical Analy sis 3,69.
17. Leete, E. (1990) Plunta M ed. 56, 339.
4. Witte, A., Miiller, K. and Arfmann, H.-A. (1987)
18. Hashimoto,
T., Nakajima, K., Ongena, G. and
Planta M ed. 53, 192.
Yamada, Y. (1992) Plant Physiol. 100, 836.
5. Christen, P., Roberts, M. F., Phillipson, J. D. and
19. Yagudaev, M. R. and Aripova, S. F. (1986) Khim Prir.
Evans, W. C. (1990) Plant Cell Reports 9, 101.
6. Fikenscher, L. H. (1958) Pharm. W eekbl. 93,932.
Soedin. 22, 80.
20. Al-Said, M. S., Evans, W. C. and Grout, R. J. (1989)
7. El-Imam, Y. M. A., Evans, W. C. and Grout, R. J.
Phytochemistry 28, 3211.
(1988) Phytochemistry 27, 2181.
Acknowledgements- The