全 文 ：Received 27 Mar. 2003 Accepted 19 Sept. 2003
Supported by the National Natural Science Foundation of China (20272084) and the National Key Basic Research and Development Plan
Program (G1998-051120).
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Triterpenoids from Erythrophleum fordii
LI Nan, YU Fang, YU Shi-Shan*
(Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China)
Abstract: A phytochemical investigation of the bark of Erythrophleum fordii Oliv. furnished five compounds.
One is a new triterpenoid, namely 20S, 24S-epoxy-23S, 25-dihydroxy-dammarane-3-one (1), and four are
known, identified to be 20S , 25-epoxy-24R -hydroxydammarane-3-one (2), 20S, 24S -epoxydammarane-3b,
25-diol (3), betulinic acid (4), morolic acid acetate (5). All the known compounds were isolated from the
species for the first time. The structure of compound 1 was elucidated on the basis of spectroscopic
analyses.
Key words: Erythrophleum fordii ; triterpenoid; cytotoxic activities; KB cells; A2780 cells
Erythrophleum fordii Oliv. is used as Chinese tradi-
tional medicine with the main actions: invigoration and pro-
moting blood circulation. To date, no previous chemical
and pharmacological investigations have been reported on
it.
In the course of screening for cytotoxic compounds,
the 95% ethanol extract of the dry barks of E. fordii was
partitioned into EtOAc and H2O to afford an EtOAc-soluble
portion, which showed cytotoxic activities against KB cells
with the IC50 0.08 mg/mL, A2780 cells with the IC50 3.1
mg/mL. Phytochemical investigation on the EtOAc fraction
led to the isolation and identification of five compounds,
one is new, named 20S, 24S-epoxy-23S, 25-dihydroxy-
dammarane-3-one (1), and four are known (2-5), identified
to be 20S, 25-epoxy-24R-hydroxydammarane-3-one (2), 20S,
24S-epoxydammarane-3b,25-diol (3), betulinic acid (4),
morolic acid acetate (5). We report herein the isolation and
structure elucidation of the new compound. The structures
of the known compounds (2-5) were identified by the com-
parison of their 1H-NMR and 13C-NMR data with those
reported in literatures (Mochammad et al.,1980; Antonio et
al., 1981; Peter and Stephen, 1985; Carl et al., 1993).
Bioactive tests exhibited that all the compounds had no
cytotoxic activity against KB cells and A2780 cells.
1 Results and Discussion
Compound 1 was obtained as colorless crystal, mp 202-
203 ℃, [a]18D +45
。 (c 1.00, CHCl3). Libermann-Burchard
reaction was positive. The HR-FABMS gave a molecular
ion at m/z 475.382 4 ([M+H]+) corresponding to the molecu-
lar formula C30H50O4. The IR spectrum of compound 1
indicated the presence of hydroxyl groups (3 408, 1 072
cm-1 ) and a carbonyl group (1 707 cm-1). The 1H-NMR
spectrum (in CDCl3) of compound 1 showed eight methyl
group signals at d 1.38 (3H, s), 1.36 (3H, s),1.28 (3H, s), 1.08
(3H, s), 1.04 (3H, s), 1.00 (3H, s), 0.94 (3H, s), and 0.89 (3H,
s). The 13C-NMR spectrum of compound 1 and DEPT ex-
periments confirmed that the molecular contained 30
carbons, including eight methyl groups, nine methylenes,
six methines (two oxygenated methines), and seven qua-
ternary carbons. Compound 1 was assigned as a
triterpenoid which showed a close structural similarity to
cabraleone (20S, 24S-epoxy-25-hydroxy-dammarane-3-one)
( Peter and Stephen, 1985), except for additional signal of
an oxo-carbon (d 74.22) and differences in the chemical shifts
around C-22 (Table 1).
The EI-MS presented ion peaks at m/z 159, 141, 123,
presumably derived from the side chain, which could thus
include a hydroxylated tetrahydrofuryl isopropanol. The
proton at dH 3.44 (H-24) resonate as doublets with coupling
constant (J = 2.5 Hz), indicating the hydroxyl group at C-23
with a trans-proton. The structure of compound 1 was also
supported by HMBC (Fig.1).
The stereochemistry of C-20 should be S configuration,
since the C-20 epimer (R configuration) of compounds with
the tetrahydrofuran ring has been shown to be quite un-
stable and be converted easily to the corresponding tet-
rahydrofuran derivative. ( Masahiro et al., 1968 ) .
Information on the additional stereochemistry of com-
pound 1 was obtained by the use of selective NOE
irradiation. In this experiment, irradiation at dH 3.44 (H-24)
not enhance the signal at dH 1.38 (CH3-21), while irradiation
Acta Botanica Sinica
植 物 学 报 2004, 46 (3): 371-374
Acta Botanica Sinica 植物学报 Vol.46 No.3 2004372
at dH 4.43 (H-23) gave NOE on CH3-26 ( dH 1.36) and CH3-27
( dH 1.36). On the basis of above data, the structure of com-
pound 1 was established as 20S, 24S-epoxy-23S, 25-
dihydroxy-dammarane-3-one.
2 Experimental
2.1 General experimental procedures
Melting points were determined on an XT-4 micromelting
point apparatus and uncorrected. Optical rotations were
obtained using a Perkin-Elmer 241 polarimeter. IR spectra
were recorded using KBr disks on a Perkin-Elmer 683 FT
infrared spectrometer. The NMR spectra were taken with
TMS as internal standard on an Inova 500 FT-NMR
spectrometer. Mass spectra were measured on VG ZAB-2F
spectrometer. Silica GF254 for thin-layer chromatography
analysis and silica gel (160-200 mesh) for column chroma-
tography were produced by Qingdao Marine Chemical
Company, Qingdao, China. Solvents and chemicals were
analytical grade and purchased from Beijing Chemical
Company, Beijing, China.
2.2 Plant materials
Barks of Erythrophleum fordii Oliv. were collected from
Guangxi Province of China, and identified by Prof. Liu Shou-
Yang (Guangxi College of Chinese Traditional Medicine,
Guangxi Province, China) in August 2000. Barks were har-
vested and air-dried at room temperature in darkness. A
voucher specimen was deposited in Institute of Materia
Medica, Chinese Academy of Medical Sciences and Pe-
king Union Medical College, Beijing, China.
2.3 Extraction and isolation
The dried and chopped barks of E. fordii (15 kg) were
extracted three times with 95% hot EtOH .The combined
EtOH extract was evaporated under reduced pressure to
yield a dark brown syrup ( 2 910 g ), which was partitioned
between EtOAc and H2O. The EtOAc-soluble layer (400 g)
was directly chromatographed over silica gel by eluting
with a gradient of CH2Cl2-MeOH (20:1 to 1:1) to afford twelve
fractions. The fraction 3 (47.7 g) was chromatographed over
silica gel with gradient of Petro ether-EtoAc (9:1 to 1:1) to
yield compounds 1 (12 mg), 2 (10 mg), 3 (22 mg), 4 (47 mg),
5 (381 mg).
2.4 Identification
20S, 24S-epoxy-23S, 25-dihydroxy-dammarane-3-one
(1) Colorless crystal; mp 202-203 ℃; [a]18D +45°(c 1.00,
CHCl3); Libermann-Burchard reaction positive; IR (KBr) vmax
3 408, 1 707, 1 072 cm-1; EI-MS m/z 438 [M-2H2O]+ (2), 205
(23), 159 (18), 141 (50), 123 (100), 107 (44); HR-FABMS m/z
[M+H]+ 475.382 4 (calcd. for C30H51O4, 475.378 7). 1H-NMR
(500 MHz, CDCl3) d 4.43 (1H, br, H-23), 3.44 (1H, d, J=2.5 Hz,
H-24), 2.43-2.50 (2H, m, H-2), 1.40-1.50 (1H, m, H-17), 1.38,
1.36, 1.28, 1.08, 1.04, 1.00, 0.94, 0.89 (each 3H, s, 21, 26, 27,
28, 29, 19, 18, 30-Me); 13C-NMR (125 MHz, CDCl3), see
Table 1.
20S, 25-epoxy-24R-hydroxy-dammarane-3-one (2)
Colorless crystal (EtOAc); mp 188-189 ℃; [a]18D +37°(c
0.80, CHCl3); Libermann-Burchard reaction positive; IR
(KBr) vmax 3 485, 1 685, 1 082, 1 020 cm-1; EI-MS m/z 440
[M-H2O]+ (2), 205 (20), 143 (42), 125 (100), 107 (50); HR-
FABMS m/z [M+H]+ 459.386 2 (calcd. for C30H51O3, 459.383 8).
1H-NMR (500 MHz, CDCl3) d 3.38 (1H, dd, J = 9, 6.5 Hz),
2.39-2.53 (2H, m), 1.21, 1.20, 1.18, 1.08, 1.04, 1.02, 1.00, 0.94,
0.86 (each 3H, s); 13C-NMR (125 MHz, CDCl3), see Table 1.
20S, 24S-epoxy-dammarane-3b, 25-diol (3) Colorless
crystal (CH3OH); mp 158-159 ℃; [a]18D +24°(c 0.85,
CHCl3); Libermann-Burchard reaction positive; IR (KBr)
vmax 3 438, 1 045 cm-1; EI-MS m/z 424 (2) [M-H2O]+, 143
(100), 125 (50); 1H-NMR (500 MHz, CDCl3) d 3.64 (1H, dd,
J=15, 5 Hz, H-24), 3.20 (1H, dd, J=11.5, 4.5 Hz, aH-3), 1.19,
1.15, 1.11, 0.98, 0.98, 0.88, 0.85, 0.78 (each 3H, s, 27, 26, 21,
18, 28, 30, 19, 29-Me); 13C-NMR (125 MHz, CDCl3), see
Table 1.
Betulinic acid (4) Colorless crystal (CH3OH); mp 252-
253 ℃; [a]18D -36°(c 1.00, CHCl3); Libermann-Burchard
reaction positive; IR (KBr) vmax 3 454, 3 076, 1 689, 1 643,
1 043, 1 032, 984 cm-1; EI-MS m/z 456 (12) [M]+, 207 (50), 189
(100). 1H-NMR (500 MHz, C5D5N) d 4.76 (2H, s, H-30), 3.44
(1H, dd, J = 9, 7.5 Hz, aH-3), 1.78 (3H, s, Me-29), 1.21, 1.05,
1.04, 0.99, 0.81 (each 3H, s); 13C-NMR (125 MHz, C5D5N),
see Table 1.
Morolic acid acetate (5) Colorless crystal (CH3OH),
mp 263-265 ℃; [a]18D +20°(c 0.99, CHCl3); Libermann-
Burchard reaction positive; IR (KBr) vmax 3 446,1 734, 1 695
cm-1; EI-MS m/z 453 (2) [M-COOH]+, 203 (40), 189(100);
Fig.1. The selected HMBC correlations for compound 1, indi-
cated by arrows from 1H to 13C.
LI Nan et al.: Triterpenoids from Erythrophleum fordii 373
1H-NMR (500 MHz, CDCl3) d 5.18 (1H, s, H-19), 4.48 (1H, dd,
J=11, 6 Hz, H-3), 2.05 (3H, s, -OCOCH3), 1.00, 0.99, 0.98,
0.89, 0.85, 0.84, 0.79, 0.77 (each 3H, s); 13C-NMR (125 MHz,
CDCl3), see Table 1.
3 Bioassays
Cytotoxicity against human cancer cell lines for EtOAc-
soluble portion was measured in a five-day MTT test at
Department of Pharmacology, Institute of Materia Medica,
Chinese Academy of Medical Sciences and Peking Union
Medical College, using A2780 human epithelial carcinoma
cells and KB human epidermoid cancer cells (Dominic et
al., 1988). Cells were placed in a 96-well plate. The cells
were dissolved in 0.2 mL of DMSO, and the absorbance at
540 nm was measured in a microplate reader.
Acknowledgements: We are grateful to the department of
medicinal analysis of Institute of Materia Medica, Chinese
Academy of Medical Sciences and Peking Union Medical
College, for performing EI-MS, NMR and IR, and thank the
department of pharmacology in our institute, for cytotoxic-
ity testing.
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Table 1 13C-NMR (in CDCl3) data for compounds 1-5 and cabraleone
Position 1 Cabraleone 2 3 4 5
1 39.92 (2) 39.96 (2) 39.90 39.06 39.3 38.6
2 34.08 (2) 34.12 (2) 34.13 27.42 28.3 23.7
3 218.06 (0) 217.96 (0) 18.24 78.95 78.2 81.0
4 47.40 (0) 47.42 (0) 47.41 38.97 39.6 37.8
5 55.30 (1) 55.41 (1) 55.34 55.86 56.0 55.6
6 19.63 (2) 19.71 (2) 19.67 18.28 18.8 18.1
7 34.57 (2) 34.66 (2) 34.55 35.29 34.9 34.5
8 40.29 (0) 40.35 (0) 40.30 40.38 41.2 40.7
9 50.16 (1) 50.23 (1) 50.16 50.83 51.0 51.1
10 36.84 (0) 36.91 (0) 36.84 37.15 37.6 37.1
11 22.32 (2) 23.36 (2) 22.18 21.80 21.2 20.9
12 25.85 (2) 25.84 (2) 25.41 25.84 26.2 26.0
13 42.98 (1) 43.35 (1) 42.02 42.85 38.7 41.4
14 49.99 (0) 50.04 (0) 50.27 50.03 42.9 42.6
15 31.50 (2) 31.46 (2) 30.18 31.44 31.3 29.4
16 27.08 (2) 27.80 (2) 27.66 27.42 32.9 33.5
17 50.66 (1) 49.84 (1) 51.55 49.84 56.7 47.9
18 15.17 (3) 15.23 (3) 15.13 15.48 47.8 136.7
19 16.09 (3) 16.08 (3) 16.06 16.23 49.8 133.3
20 86.73 (0) 86.40 (0) 74.56 86.54 151.4 32.1
21 27.95 (3) 24.11 (3) 21.85 24.05 30.3 33.4
22 44.03 (2) 34.91 (2) 31.85 34.77 37.6 33.3
23 74.22 (1) 27.05 (2) 25.11 26.36 28.7 27.9
24 85.87 (1) 86.50 (1) 75.58 86.30 16.4 16.5
25 72.09 (0) 70.27 (0) 76.10 70.25 16.5 16.0
26 24.06 (3) 26.40 (3) 26.72 27.14 16.5 16.7
27 29.27 (3) 26.78 (3) 25.50 27.81 14.9 14.9
28 26.73 (3) 27.15 (3) 29.95 27.99 178.9 181.1
29 20.97 (3) 21.02 (3) 20.98 15.35 19.5 30.3
30 16.31 (3) 16.34 (3) 16.50 16.40 110.0 29.1
Spectra determined in CDCl3; data reported in ppm. Carbon types determined by a DEPT experiment and reported as 0 (quaternary), 1 (CH),
2 (CH2), or 3 (CH3).
Acta Botanica Sinica 植物学报 Vol.46 No.3 2004374
Peter G W, Stephen A. 1985. Dammarane triterpenes from the
stem bark of Commiphora dulzielii. Phytochemistry, 24:2925-
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