Phytochemistry 51 (1999) 1171±1176
b-Carboline glucoalkaloids from Strychnos mellodora
Viviane Brandt a, Monique Tits a, Arjan Geerlings b, Michel FreÂdeÂrich a,
Jacques Penelle a, CleÂment Delaude a, Robert Verpoorte b, Luc Angenot a,*
a
Service de Pharmacognosie, CHU, Institut de Pharmacie, Universite de LieÁge, Tour 4, Avenue de l'HoÃpital 1, 4000 Liege, Belgium
Leiden University, Leiden/Amsterdam Center for Drug Research, Division of Pharmacognosy, Gorlaeus Laboratories, P.O. Box 9502, Einsteinweg
55, 2300 RA Leiden, Netherlands
b
Received 25 November 1998; received in revised form 20 January 1999; accepted 8 February 1999
Abstract
Two new Nb-methylated b-carbolinium glucoalkaloids, 3,4,5,6-tetradehydropalicoside and 3,4,5,6-tetradehydrodolichantoside,
together with the known b-carboline compounds desoxycordifoline (b-carboline 3-carboxylate glucoalkaloid) and melinonine F
(Nb-methylated harmanium cation), were isolated from Strychnos mellodora stembark. The structures of the compounds were
elucidated on the basis of spectroscopic studies. # 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Strychnos mellodora; Loganiaceae; b-Carboline; Glucoalkaloids; Anhydronium alkaloids; 3,4,5,6-Tetradehydropalicoside; 3,4,5,6-Tetradehydrodolichantoside; Desoxycordifoline; Melinonine F; 2D-NMR
1. Introduction
Strychnos mellodora S. Moore is an East-African
endemic species. It is a tree, growing up to 35 m in
height and distributed in the mountain rainforests
(800±1200m) of Tanzania, Zimbabwe and Mozambique (Leeuwenberg, 1969). The only phytochemical
research on this plant was part of a large screening of
69 Strychnos African species carried out in 1971 by
Bisset and Phillipson, who showed the presence of
tertiary alkaloids (Bisset & Phillipson, 1971). A ®rst
investigation on this plant in our laboratory has led to
the isolation and identi®cation of three indolic glucoalkaloids: dolichantoside (6), palicoside (5) and
strictosidine (7) (Tits et al., 1996). In continuation of
our studies on the alkaloidal composition of S. mellodora stembark, we isolated three Nb-methylated b-carbolinium compounds (1, 2 and 4) and a b-carboline
glucoalkaloid (3), which were all characterized by a
blue ¯uorescence under UV light. This paper deals
with the isolation and structural elucidation of the new
compounds, 3,4,5,6-tetradehydropalicoside (1) and
* Corresponding author.
3,4,5,6-tetradehydrodolichantoside (2) (UV, IR, MS
and NMR), as well as with the isolation and the
identi®cation of the known compounds, desoxycordifoline (3) (UV, MS and NMR) and melinonine F (4)
(TLC, HPLC, UV and MS). We ®nally present an
HPLC method for the identi®cation of the main alkaloids of S. mellodora stembark.
2. Results and discussion
An EtOH extract of the dried powdered stembark of
S. mellodora was separated by MPLC using a gradient
of MeOH in Me2CO. The initial fractions contained
dolichantoside (6) and compound 3, while the latter
aorded a mixture of palicoside (5) and compounds 1,
2, 4. Subsequent chromatography was needed to separate these polar, fragile products.
Compound 1, positive ES-mass spectrum m/z 527
[M]+ corresponding to the molecular formula
C27H30N2O9, showed an intense blue ¯uorescence
under UV light. The wavelengths of its lmax in acidic
or neutral solutions suggested a b-carbolinium chromophore. The bathochromic shift in alkaline solution
0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 1 - 9 4 2 2 ( 9 9 ) 0 0 1 2 9 - 6
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V. Brandt et al. / Phytochemistry 51 (1999) 1171±1176
corresponded to the zwitterionic form (pH-dependent)
anhydronium base (Gribble, 1988). The presence of a
b-carboline derivative, as suggested by the UV spec-
trum, was con®rmed by characteristic mass fragments
at m/z 155, 167, 182 and 196 (Caprasse, Coune, &
Angenot, 1983). The peak at m/z 363 indicated the loss
of a sugar moiety. Cleavage with b-glucosidase established that the sugar moiety was b-D-glucose.
A more detailed understanding of the molecular
structure of 1 was gained from its NMR spectra
(Tables 1 and 2). In the 400 MHz 1H NMR spectrum,
the presence of a b-carbolinium structure was con®rmed by the presence of six aromatic protons, four of
which corresponded to an unsubstituted indole moiety
and two more deshielded doublets whose chemicals
shifts were in accordance with a pyridinium ring. The
NMR data showed the presence of a quaternary Nbmethyl group (1H: 4.35 (s), 13C: 45 ppm). The
deshielded values for the methyl protons are consistent
with those of melinonine F and normelinonine F (1H:
4.20, 13C: 43.1 and 1H: 4.55, 13C: 46.3 ppm, respectively) (Caprasse et al., 1983] or other similar molecules, like chrysotricine (1H: 4.42, 13C: 45.2 ppm)
(Peng, Feng, Zheng, & Liang, 1997). Several resonances suggested the presence of a seco-iridoid moiety
similar to secologanin (Stevens, 1994). There was a
singlet at d 7.44 corresponding to the ole®nic H17 and
a doublet at d 5.95 for the hemiacetalic H21. The vinyl
group was identi®ed by doublets at d 5.31 (J=10.8
Hz) and d 5.23 (J=17.9 Hz) corresponding to the
methylenic protons of C18, which are respectively cis
and trans coupled and by the unique hydrogen H19
(doublet of doublets at 5.97 ppm). A doublet at d 4.78
with a coupling constant of 7.9 Hz, indicating b-con®guration of the linkage, was consistent with the
anomeric proton of a b-glucose moiety (Agrawal,
1992). This was supported by a complex set of resonances in the range 3.15±3.92 ppm. Moreover, the 13CNMR spectrum showed the sugar to be in the b-D-glucopyranose form as in dolichantoside, palicoside and
strictosidine (Stevens, 1994; Tits et al., 1996)]. The 1HNMR spectrum revealed the absence of an O-methyl
group. Two bands in the IR spectrum at nmax 1635
and 1385 cmÿ1 suggested the presence of a b-alkoxyacrylate function. This was demonstrated by the fact
that methylation of 1 with diazomethane gave the
methyl ester 2. Furthermore, the results of the COSY
spectrum showed that in the aliphatic region a larger
structural fragment could be assembled. The H18a±
H18b±H19 vinylic protons aorded a convenient entry
point in this system. The connectivities provided the
means of assembling the multispin substructure related
with the hydrogens bonded to C18±C19±C20± (C15±
C14)±C21 which further con®rmed the vincosan-type
skeleton (no N4C17 or N4C21 bond). Thus, the structure of 1 was established as 3,4,5,6-tetradehydropalicoside. 2D NMR experiments (1H±1H COSY and
HMQC) were used to establish the 1H and 13C assignments, which con®rmed the proposed structure.
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V. Brandt et al. / Phytochemistry 51 (1999) 1171±1176
Table 1
1
H NMR spectral data of compounds 1, 2, 3 and 7 in CD3OD (d in ppm and J in Hz)
H
1
2
3
5a
5b
6a
6b
9
10
11
12
14a
14b
15
17
18trans
18cis
19
20
21
N+±CH3
COOH
O±CH3
1'
2'
3'
4'
5'
6'a
6'b
±
8.34 d
±
8.34 d
±
8.24 d (8.0)
7.33 t (8.1)
7.66
7.66
3.6 m
3.5 m
3.45 m
7.44 (1H) s
5.23 d (17.9)
5.31 d (10.8)
5.97 dd (10.8, 17.9)
2.72 d (8.9)
5.95 d (8.9)
4.35 (3H) s
n. d. in CD3OD
±
4.78 d (7.9)
3.15 d (7.9)
3.35 m
3.22 m
3.35 m
3.92 d (10.8)
3.60 d (10.8)
±
8.28
±
8.36
±
8.31
7.36
7.71
7.75
3.75
3.65
3.50
7.51
5.46
5.35
6.04
2.83
6.15
4.44
±
2.90
4.91
3.27
3.30
3.32
3.45
4.01
3.76
a
b
d (6.3)
d (6.3)
d (8.0)
t (8.0, 7.3)
t (7.3, 8.3)
d (8.3)
m
m
m (3.4)
(1H) s
d (17.1)
d (10.2)
ddd (10.2, 17.1, 8.0)
ddd (9.4, 8.0, 3.4)
d (9.4)
(3H) s
(3H) s
d (7.9)
d (7.9)
m
m
m
d (11.9)
d (11.9)
3
7a
±
±
±
8.69
±
8.19
7.28
7.56
7.59
3.43
3.27
3.67
7.59
4.67
4.93
5.67
2.62
5.86
±
11.5
3.54
4.78
3.19
3.39
3.22
3.39
3.99
3.67
3.96
3.29
2.93
2.80
2.69
7.35
6.94
7.01
7.23
2.04
1.96
3.03
7.67
5.30
5.20
5.84
2.67
5.82
±
±
3.74
4.78
3.23
3.40
3.24
3.35
3.94
3.64
(1H) s
d (7.8)
t (7.8, 7.3)
t (7.3, 7.7)
d (7.7)
m
m
m
(1H) s
d (17.3)
d (10.7)
ddd (10.7, 17.3, 7.2)
dd (7.3, 7.2)
d (7.3)
sb
(3H) s
d (7.8)
d (7.8)
m
m
m
d (10.7)
d (10.7)
dd
ddd
ddd
ddd
ddd
dd
ddd
ddd
dd
ddd
ddd
ddd (4.6)
(1H) s
dd (17.3)
dd (10.6)
ddd (10.6, 17.3, 7.6)
ddd (9.0, 7.6, 4.6)
d (9.0)
(3H) s
d (7.9)
dd (7.9, 8.8)
dd (8.8, 8.8)
dd (8.8, 8.8)
ddd (2.3, 6.7, 8.8)
dd (11.8, 2.3)
dd (11.8, 6.7)
From Stevens (1994).
Observed in DMSO-d6.
Compound 2 was separated from 1 by prep. TLC
on silicagel using MeOH±NH4NO3, 1 M±NH4OH, 2
M (7:2:1) as mobile phase. This product emitted the
same blue ¯uorescence under UV light and pH-dependent UV spectrum as 1. The m/z 182 and 196 fragments in the ES+ mass spectrum as well as the typical
non-substituted indole nucleus and 3-substituted pyridinium ring protons in the down-®eld region of the 1H
NMR spectrum (Table 1) aorded the evidence for the
presence of a b-carbolinium structure similar to 1.
Again we noted the presence of a deshielded singlet
signal for a quaternary Nb-methyl group (1H: 4.44,
13
C: 44.5 ppm). The m/z 541 [M]+ peak from ES+
mass spectrometry led to the molecular formula
C28H32N2O9. Thus, 2 was heavier than 1 by 14 mm,
which could be consistent with the methylation of the
carboxylic function of 1. Indeed, the 1H and 13C
NMR spectral data of compound 2 (Tables 1 and 2)
were analogous to those of compound 1, except for
the signal at 1H: 2.90 (13C: 51 ppm), attributable to a
carbomethoxy group. This chemical shift re¯ected a
shielding of the methyl protons, like in other indolic
alkaloids as 10-hydroxypericyclivine and derivatives
(Pinchon et al., 1990). The examination of the molecu-
lar stereomodels in the case of Nb-methyl-b-carbolinium and Nb-methyltetrahydro-b-carboline alkaloids
can partially explain the dierence in shifts between
the carbomethoxy protons in 2 and dolichantoside (6)
(1H: 3.70, 13C: 51.9) (Coune & Angenot, 1978). The
steric dimensions of the quaternary Nb-methyl group
as well as the planarity of the b-carbolinium nucleus
in¯uence the position of the methyl ester in such a way
that preponderance of its position in the shielding zone
of the aromatic ring current from the b-carbolinium
moiety becomes plausible. Furthermore, reaction of
diazomethane with 1 provided the methylester 2, which
allowed us to assign the structure of 2 as 3,4,5,6-tetradehydrodolichantoside. This is believed to be the ®rst
report on the isolation of Nb-methylated b-carbolinium
glucoalkaloids from nature.
The stereochemistry remained to be considered. The
similarities between the chemical shifts of C15, C20 and
C21 in the new compounds 1 and 2 and in strictosidine
(7) (Table 2) suggested the same con®guration for
these carbons in the two alkaloids. The weak deshielding eect observed for C15 and C20 can be attributed
to the quaternary character of the Nb-methyl group.
Moreover the coupling constants in 2 between H15,
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V. Brandt et al. / Phytochemistry 51 (1999) 1171±1176
Table 2
13
C NMR spectral data of compounds 1, 2, 3 and 7 in CD3OD (d in
ppm). n.d.=not detected
C
1
2
3
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
N+±CH3
O±CH3
COOH
1'
2'
3'
4'
5'
6'
a
b
2
135.9
117.1
133.5
116.0
124.3
123.1
132.8
114.1
124.0
121.8
132.0
115.0
32.0
36.0
31.5
35.5
155.0
120.3
135.6
45.0
97.2
155.0
119.5
n.d.
45.0
96.5
45.0
±
±
100.8
75.1
78.0
72.2
79.1
63.0
44.5
51.0
±
100.5
74.5
78.0
71.3
78.0
62.9
3a
7b
135.6
142.9
135.6
114.2
128.4
121.7
121.4
119.9
128.4
111.6
141.6
34.0
34.5
108.7
153.2
117.6
133.8
44.4
96.1
171.3
±
50.6
168.4
99.0
73.2
76.6
70.4
76.6
61.8
137.7 or 136.3
51.7
43.2
22.4
108.4
128.5
118.5
119.6
121.9
111.8
137.7 or 136.3
37.1
32.7
110.8
155.3
119.1
136.1
43.0
97.6
170.1
±
52.2
±
100.3
74.6
78.0
71.7
78.7
62.9
Quaternary carbons deduced from the APT spectrum.
From Stevens (1994).
H20 and H21 are consistent with the a-con®guration of
H15 and H20, and the b-con®guration of H21
(Levesque & Jacquesy, 1983). Thus, the proposed relative con®gurations of C15, C20 and C21 are those commonly accepted from the biogenetical hypothesis: 15a(S ), 20-a(R ) and 21-b(S ).
In the same fraction, compound 4 (m/z 197 [M]+),
which displayed a b-carbolinium UV spectrum, was
identi®ed as melinonine F after comparison by TLC
and diode-array HPLC with an authentic sample isolated in our laboratory from Strychnos usambarensis
roots (Caprasse et al., 1983).
The MPLC pooled fractions containing compound 3
and dolichantoside (6) were submitted to an HSCCC
process to yield pure products. The UV data of 3 in
basic, neutral and acidic conditions presented a bathochromic shift in acidic solution and were consistent
with a fully aromatic b-carboline structure. In the
1 1
H H COSY NMR spectrum, a system of four aromatic protons showed that the benzenoid ring of the
b-carboline was unsubstituted and a singlet at d 8.69
indicated a substitution of the 5-position of the pyridinic ring. All characteristic resonances of a seco-iridoid
moiety were present in the NMR spectra. In addition,
the ES+ mass spectrum providing a m/z 571 [M+H]+
peak, suggested the presence of desoxycordifoline, an
alkaloid previously isolated from Adina species
(Rubiaceae) (Merlini & Nasini, 1968; Brown &
Warambwa, 1978). The comparison of UV, MS and
1
H NMR spectra with those from literature con®rmed
this hypothesis. The 1H NMR data of 3 are listed in
Table 1. The 13C NMR data in Table 2 do not seem
to be available in previous literature. Moreover, this is
the ®rst time that a tryptophan-derived glucoalkaloid
has been found in a member of the family of
Loganiaceae.
Finally, a HPLC method for the analysis of alkaloids in the crude EtOH extract of S. mellodora stembark (1±7) was developed (see chromatogram in Fig.
1). The quanti®cation of 5 (0.41%), 6 (0.66%) and 7
(0.018%) in S. mellodora stembark was performed
using the corresponding references as external standards (Brandt, 1996). Using dolichantoside as external
standard, the amounts of 1, 2, 3 and 4, calculated with
reference to the dried drug, showed that these compounds represented ca. 0.9% of b-carboline alkaloids.
Adding up the previous results, we observe that the
total amount of glucoalkaloids in S. mellodora is particularly high (ca. 2%) and superior to levels of alkaloids usually found in plants.
The biological activity of both new anhydronium
bases glucosides 1 and 2, especially the cytotoxic and
antiparasitic properties, is now subject for further investigation. We have to consider those new products
as leads for the development of new drugs given the
encouraging results already obtained with b-carboline
compounds especially in the ®eld of cancerology, neurology and psychopharmacology (Rollema, Booth, &
Castagnoli, 1988; Funayama et al., 1996; Malgrange et
al., 1996; Picada, da Silva, Erdtmann, Henriques, &
Henriques, 1997).
We have isolated six glucoalkaloids from S. mellodora, including strictosidine, the recognized exclusive
precursor of all monoterpenoid indole and quinoline
alkaloids (Phililipson & Zenk, 1980; Massiot &
Delaude, 1988). This species, with signi®cant amounts
of those alkaloids, is a very primitive one from a phylogenetical point of view. It is a potential source of
glucoalkaloids useful for biotechnological experiments
and studies concerning the biosynthetic pathway of
those alkaloids (Stevens, 1994).
3. Experimental
3.1. Plant material
The stembark of S. mellodora S. Moore was collected in Chirinda Forest (Zimbabwe) by CD. Voucher
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V. Brandt et al. / Phytochemistry 51 (1999) 1171±1176
detection and elution by EtOH), followed by puri®cation on Sephadex LH 20 eluting with MeOH.
3.3. 3,4,5,6-Tetradehydropalicoside (1)
Obtained as an amorphous brownish-yellow powder.
UV (EtOH) lmax nm (log e ): 222 (3.97), 255 (3.06),
311 (2.85), 373 (2.31); (EtONa) lmax nm (log e ): 229
(3.88), 287 (3.29), 335 (2.70). IR (KBr) nmax cmÿ1:
3401, 2926, 1670 (sh), 1635, 1528, 1503, 1385, 1201,
1157, 1076, 944, 893, 825, 759. Positive ES±MS±MS
m/z: 527 [M]+, 364 [527-Glc]+, 249 [C17H17N2]+, 221
[C15H13N2]+, 196 [C13H12N2]+, 182 [196-CH2]+, 167
[182-CH3]+, 155. 1H and 13C NMR data, recorded on
a BRUKER 400 MHz spectrometer, are listed in
Tables 1 and 2.
3.4. 3,4,5,6-Tetradehydrodolichantoside (2)
Fig. 1. HPLC pro®le of an EtOH/HOAc (99:1) extract of Strychnos
mellodora stembark. See numbering in text.
specimens (L. Pauwels 7831) are kept in the
Herbarium of the Botanical Garden of Belgium at
Meise.
3.2. Extraction and isolation
Dried and powdered stembark (1 kg) was extracted
with EtOH and EtOH±HOAc (99:1) at room temp.
The combined extracts were conc. in vacuo to give 49
g residue. 4 g of this residue were fractionated by
MPLC on a silica gel 60 using a Me2CO/MeOH gradient. The Me2CO/MeOH (99:1) fraction (266 mg)
yielded 3 (10 mg) and 6 (109 mg) after puri®cation by
high speed counter current chromatography (HSCCC)
in a multilayer-coil separator±extractor, ®tted with a
2.6 mm i.d. coiled tubing and Me2CO±n-BuOH±H2O
(1:8:10) as solvent. The upper organic phase was used
as the stationary phase and the lower aq. phase as the
mobile phase (descending mode). The (80:20) to
(50:50) Me2CO/MeOH fractions (637 mg) containing
1, 2, 4 and 5 were ®rstly puri®ed by HSCCC using
CHCl3±MeOH±H2O (7:13:8) as solvent. The lower organic phase was used as stationary phase and the
upper aq. phase as mobile phase (ascending mode). As
5 (70 mg) was obtained pure in this HSCCC system,
compounds 1 (47 mg), 2 (67 mg) and 4 (13 mg) were
®nally separated by prep. TLC on silica gel (2 mm) in
MeOH±NH4NO3, 1 M±NH4OH, 2 M (7:2:1) (UV
Obtained as an amorphous brownish-yellow powder.
UV (EtOH) lmax nm (log e ): 209 (3.96), 254 (3.69),
311 (3.52), 375 (2.93); (EtONa) lmax nm (log e ): 213
(3.92), 288 (3.84), 336 (3.28), 438 (2.67). IR (KBr) nmax
cmÿ1: 3401, 2925, 2854, 1690 (sh), 1635, 1527, 1503,
1384, 1245, 1196, 1157, 1076, 943, 825, 761. Positive
ES±MS±MS m/z: 541 [M]+, 379 [541-Glc]+, 351, 347,
319, 221, 196, 182. 1H and 13C NMR data, recorded
on a BRUKER DMX 600 MHz spectrometer, are
listed in Tables 1 and 2.
3.5. Desoxycordifoline (3)
Obtained as a white powder. UV (EtOH) lmax nm
(log e ): 239 (4.58), 268 (4.54), 336 (3.70), 349 (3.71);
(EtONa) lmax nm (log e ): 240 (4.52), 260 (4.44), 268
(4.43), 285 (4.13), 337 (3.71), 352 (3.72); (EtOH+HCl)
lmax nm (log e ): 220 (4.46), 240 (4.46), 278 (4.51), 376
(3.69). Positive ES±MS±MS m/z: 593 [M+Na]+, 571
[M+H]+, 409 [M±Glc]+, 363, 359, 181, 167. 1H and
13
C NMR data, recorded on a BRUKER DMX 600
MHz spectrometer, are presented in Tables 1 and 2.
3.6. Melinonine F (4)
UV, MS, 1H NMR and
et al., 1983).
13
C NMR see lit (Caprasse
3.7. Palicoside (5)
UV, MS, IR, 1H NMR and
(Morita et al., 1989).
13
C NMR see lit
13
C NMR see lit
3.8. Dolichantoside (6)
UV, MS, IR, 1H NMR and
(Coune & Angenot, 1978).
1176
V. Brandt et al. / Phytochemistry 51 (1999) 1171±1176
3.12. Strictosidine (7)
UV, MS, IR, 1H NMR and
(Smith, 1968; Stevens, 1994).
13
C NMR see lit
the ``Fondation LeÂon Fredericq'' and by the Belgian
National Fund for Scienti®c Research (FNRS) (grant
No. 3451997).
3.10. Enzymatic hydrolysis of glucoalkaloids
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3.11. HPLC analysis
The stationary phase was a RP8 Lichrosorb SelectB
column (5 mm, 250 4 mm) and heptanesulfonic acid
(pH 3.7)/acetonitrile (75:25) at a ¯ow rate of 1 ml/min
was used as eluent. Alkaloids were detected at 260 nm
by a diode-array detector. They appear in the following order (elution time in min): 3 (5.0), 1 (7.2), 5 (9.6),
2 (14.5), 4 (17.9), unidenti®ed peak (20.4), 7 (25.3) and
6 (27.2).
3.12. Methylation of 1
A MeOH solution of 1 (0.5 mg) to which an excess
of CH2N2 in ether was added, was stirred for a few
min after which the solvent was evaporated. A TLC
system using MeOH±NH4NO3, 1 M±NH4OH, 2 M
(7:2:1) as mobile phase and UV detection showed that
the product of methylation was 2.
Acknowledgements
We thank Professor J. De Graeve and his collaborator J.C. Van Heugen for providing the mass spectra,
Dr. G. Llabres (CREMAN. Universite de LieÁge) for
taking the spectra of 1 and A.W.M. Lefeber (Gorleaus
Laboratories. University of Leiden) for measuring the
other NMR spectra. This research was supported by
Michel Frédérich