H.R. El-Seedi et al. / Phytochemistry 52 (1999) 1739±1744
1743
3
.2. Plant material
Further puri®cation was achieved by dissolving the
combined fractions containing integerrenine in 0.1 N
hydrochloric acid, basi®cation with solid NaHCO and
The bark of Heisteria nitida (Engl.), was collected
3
by Dr Felipe Ghia in 1992 at the Reserva Biologica,
Jatun Sacha, Provincia del Napo, Ecuador. Voucher
specimens are deposited in the Herbario Economico,
Escuela Politecnica Nacional, EPN, Quito, Ecuador,
(G. F. 539) and in the Herbarium of the Department
of Systematic Botany, Uppsala University, Sweden.
extraction with chloroform followed by prep. TLC
using CHCl ±MeOH (99:1) as eluent, 240 mg, mp 2788
3
22
(acetonitrile), lit. [21] mp 2748, a � 2328C (CHCl ; c
D
3
+
0.2) HRFAB-MS, found: m/z [M+H] , 535.3286.
+
Cal. for [M+H] C H N4 O , 535.3284; EI-MS
31
43
4
1
and the H NMR data agreed with published data of
integerrenine (Tschesche et al., 1967; Medina &
Spiteller, 1981; Lagarias et al., 1979).
3.3. Extraction
Anorldianine 27-N oxide 2 was puri®ed by chroma-
The plant material was dried at 408 in the dark in a
tography on
a
CH Cl ±MeOH (1:1) as eluent. Further puri®cation
Sephadex LH-20 column using
ventilated hood and grounded. The material, 1.1 kg,
was extracted exhaustively at room temperature three
times with light petroleum (40±608) with occasional
stirring followed by three times with methanol for 8
days each time. The extracts were evaporated in vacuo
to give 8.3 and 86.5 g of a gelatinous and an oily ma-
terial respectively. The methanol extract was parti-
tioned between ethyl acetate and water to give 17.2 g
of an ethyl acetate soluble fraction. An insoluble resi-
due (2.8 g) was discarded. The water phase was freeze
dried to give 66.6 g of crude material which consisted
mainly of carbohydrates. It was not further investi-
gated.
2
2
was achieved by dissolving the combined fractions con-
taining 2a in 0.1 N hydrochloric acid, basi®cation with
solid NaHCO3 and extraction with chloroform. The
KBr
max
� 1
yield of 2a was 45 mg. IRn
cm 3343, 1675 (sec-
ondary amide), 2790 (N0Me), 1623 (conjugated
MeOH
C1C), 1241, 1115 (aryl ether). UVlmax (log E): 266
+
3.71), 320 (3.31). HRFAB-MS, found: [M+H] ,
(
+
501.3062, C H N O requires [M+H] , 501.3072;
27 41 4 5
EI-MS m/z (rel. int.): 500 [M] (<1), 439 (76), 328
+
(
(
(
25), 305 (78), 287 (8), 251 (19), 214 (19), 194 (30), 189
100), 166 (18), 135 (23), 114 (37), 97 (70), 89 (37), 70
1
99), 61 (60), 60 (71), 42 (74). The H and C NMR
13
data are listed in Table 1.
3.4. Isolation and puri®cation
Hydrolysis of anorldianine 27-N oxide 2a (ca 200 mg)
was hydrolyzed with 6 M HCl (100 ml, 1008, 16 h) in a
sealed glass tube. After evaporization, esteri®cation
was performed with 100 ml of 1 M methanolic HCl
The ethyl acetate fraction, 16 g, was subjected to
SEPARO column chromatography on silica gel 60
30 g) with gradient elution using hexane-CH Cl and
EtOAc-MeOH. At low solvent polarity stigmasterol,
(
2
2
(
1008, 30 min). For N-acetylation, MeOH±Ac O (4:1)
2
1
was added to the methyl ester in 100 ml H O at room
2
b-sitosterol and lupeol were eluted as identi®ed by H
temp. (45 min). The product in MeOH was analyzed
by GC-MS using an HP-5, 25 m fused silica WCOT
column, temp. programmed 1408 for 3 min±2308 for
NMR spectroscopy. The CH Cl -EtOAc fractions con-
2
2
tained impure integerrenine 1 mixed with fatty acid
material. Increased polarity gave small amounts of
impure anorldianine 27-N oxide 2. It was puri®ed by
chromatography on a Sephadex LH-20 column using
6
min, the MS of the peak at ret. time 8.2 min was
identical to an authentic sample of N-acetylproline
methyl ester.
gradient elution with CHCl ±MeOH, and EtOAc±
3
(
+)-Catechin was puri®ed by chromatography on a
Sephadex LH-20 column using CHCl ±MeOH (1:9) as
MeOH mixtures. At still higher eluent polarity (+)-
catechin, (� )-epicatechin and 4-hydroxy-3-methoxy-
benzoic acid were obtained.
3
2
2
eluent, amorphous solid, 46 mg, a +11.38 (Me CO;
D
2
1
13
c 0.2). The H and C NMR data agreed with lit.
data (Morimoto, Nonaka, Nishioka, Ezaki
Takizawa, 1985; Balde et al., 1991).
0)-Epicatechin was puri®ed by chromatography on
Stigmasterol was puri®ed by chromatography on
prep. TLC plates using CHCl ±MeOH (99:1) as eluent,
&
3
1
3 mg. It was identi®ed by its H NMR spectrum.
Â
6
(
b-Sitosterol was puri®ed by chromatography on
a Sephadex LH-20 column using MeOH as eluent,
22
prep. TLC plates using CHCl ±MeOH (98:2) as eluent,
8
3
1
7 mg. It was identi®ed by its H NMR spectrum.
amorphous solid, 35 mg, a � 12.28 (Me CO; c 0.2).
D
2
1
The H and C NMR data agreed with lit. data
13
Lupeol was puri®ed by chromatography on prep.
TLC plates using CHCl ±MeOH (95:5) as eluent,
(Morimoto et al., 1985; Balde et al., 1991).
Â
4-Hydroxy-2-methoxybenzoic acid, was puri®ed by
chromatography on Sephadex LH-20 using MeOH as
3
22
amorphous solid, 57 mg, a +27.38 (CHCl ; c 0.4).
D
3
1
13
H and C NMR data agreed with lit. data (Connolly
&
Hill, 1991).
Integerrenine 1 was puri®ed by chromatography on
eluent followed by prep. TLC using CHCl
H O (3 : 9 : 1) as eluent, 28 mg. It was identi®ed by its
2
3
±MeOH±
1
a Sephadex LH-20 column using CH Cl as eluent.
2
H NMR spectrum.
2