New nortriterpenoid and ceramides from stems and leaves of cultivated Triumfetta cordifolia a rich (Tiliaceae)

Article abstract of DOI:10.1007/s11746-010-1584-z

To highlight the role of plants in traditional healing, the leaves and the stems of cultivated Triumfetta cordifolia were phytochemically studied yielding a new nor- ursane type (1), a new ceramide (2) and a new piperidinic ceramide derivative (3) named, respectively, 2α,19α-dihy-droxy-3-oxo-23-nor- urs-12-en-28-oic acid, (2R)-2-hydroxy- N-[(2S,3S,4R,26E)-1,3,4-trihydroxy-26- triaconten-2-yl] tetradecanamide and (2R,8Z)-2-hydroxy-{(2S,3R,5R,6S)- 3,5-dihydroxy-6-[(1E,5Z)-hexadeca-1,5-dienyl]-2-(β-D-glu- copyranosyloxy)methyl piperidine-1-yl} tetracos-8-enamide (3). These were obtained together with lupeol (4), stigmasterol (5), 3-O-β-D- glucopyranoside of β-sitosterol (6), tormentic acid (7) from stems and heptadecanoic acid (8), β-carotene (9), oleanolic acid (10), and 24-hydroxy- tormentic acid (11 ) from leaves. The structures were determined on the basis of NMR data (¹H-, ¹³C-, 2D-NMR analyses), mass spectrometry and confirmed by chemical transformations as well as comparison of spectral data with those reported in the literature. The FRAP method was used to evaluate the antioxidant activity of fractions collected from flash chromatography and isolated compounds. Among the fractions, four reduced Fe -TPTZ to Fe -TPTZ while isolated pure compounds showed no activity. AOCS 2010.

Full text of DOI:10.1007/s11746-010-1584-z

J Am Oil Chem Soc (2010) 87:1167–1177
DOI 10.1007/s11746-010-1584-z
ORIGINAL PAPER
New Nortriterpenoid and Ceramides From Stems and Leaves
of Cultivated Triumfetta cordifolia A Rich (Tiliaceae)
•
Louis Pergaud Sandjo Abdou Tchoukoua Hippolyte Nga Ntede Mehdi Yemloul
•
•
•
•
•
•
•
Enrico Perspicace Felix Keumedjio Franc¸ois Couty Gilbert Kirsch
Bonaventure Tchaleu Ngadjui
Received: 16 February 2010 / Revised: 23 March 2010 / Accepted: 24 March 2010 / Published online: 18 April 2010
Ó AOCS 2010
Abstract To highlight the role of plants in traditional
healing, the leaves and the stems of cultivated Triumfetta
cordifolia were phytochemically studied yielding a new nor-
ursane type (1), a new ceramide (2) and a new piperidinic
ceramide derivative (3) named, respectively, 2a,19a-dihy-
droxy-3-oxo-23-nor-urs-12-en-28-oic acid, (2R)-2-hydroxy-
N-[(2S,3S,4R,26E)-1,3,4-trihydroxy-26-triaconten-2-yl]
tetradecanamide and (2R,8Z)-2-hydroxy-{(2S,3R,5R,6S)-
3,5-dihydroxy-6-[(1E,5Z)-hexadeca-1,5-dienyl]-2-(b-D-glu-
copyranosyloxy)methyl piperidine-1-yl} tetracos-8-enamide
(3). These were obtained together with lupeol (4),
stigmasterol (5), 3-O-b-D-glucopyranoside of b-sitosterol
(6), tormentic acid (7) from stems and heptadecanoic acid
(8), b-carotene (9), oleanolic acid (10), and 24-hydroxy-
tormentic acid (11) from leaves. The structures were deter-
mined on the basis of NMR data (1H-, 13C-, 2D-NMR
analyses), mass spectrometry and confirmed by chemical
transformations as well as comparison of spectral data with
those reported in the literature. The FRAP method was used
to evaluate the antioxidant activity of fractions collected
from flash chromatography and isolated compounds.
Among the fractions, four reduced FeIII-TPTZ to FeII-TPTZ
while isolated pure compounds showed no activity.
L. P. Sandjo (&) ꢀ A. Tchoukoua ꢀ F. Keumedjio ꢀ
B. T. Ngadjui (&)
Department of Organic Chemistry, University of Yaounde I,
´
P.O. Box 812, Yaounde, Cameroon
e-mail: plsandjo@yahoo.fr
Keywords Cultivated Triumfetta cordifolia (Tiliaceae) ꢀ
Isolation ꢀ Structure elucidation ꢀ Antioxidant activity
´
Abbreviations
APT
B. T. Ngadjui
e-mail: ngadjuibt@yahoo.fr
Attached proton test
CC
Column chromatography
Correlation spectroscopy
Ferric reducing ability of plasma
Fourier transformed infra-red
Heteronuclear multiple bond
correlation
COSY
FRAP
FT-IR
HMBC
L. P. Sandjo ꢀ E. Perspicace ꢀ G. Kirsch
´
´
Laboratoire d’Ingenierie Moleculaire et Biochimie
Pharmacologique, Institut Jean Barriol,
University of Paul-Verlaine Metz, 1Bld Arago,
57070 Metz, France
H. N. Ntede
National Advanced School of Engineering,
´ ´
University of Yaounde I, P.O. Box 8390 Yaounde, Cameroon
HSQC
Heteronuclear single quantum
correlation
HR-ESI-MS
LCB
Higher resolution electrospray mass
spectrometry
M. Yemloul
Laboratoire de Methodologie RMN, Institut Jean Barriol,
´
Faculty of Science, University Henri Poincare, P.O. Box 239,
54506 Vandoeuvre-les-Nancy Cedex, France
Long chain base
´
MALDI-TOF-MS Matrix assisted laser desorption
ionisation-time of flight-mass
spectrometry
F. Couty
Institut Lavoisier de Versailles UMR 8086 University
of Versailles-St-Quentin-en-Yvelines, 45 Av. des Etats-Unis,
78035 Versailles Cedex, France
NMR
Nuclear magnetic resonance
NOESY
Nuclear overhauser effect spectroscopy
123

1168
J Am Oil Chem Soc (2010) 87:1167–1177
Introduction
C9H19
C15H31
9"'
5"'
2'"
4"
6'"
6"
Cultivated Triumfetta cordifolia A Rich (Tiliaceae) grows
as shrubs; there are similarities to the leaves, flowers and
height of the wild species. However, the stems of cultivated
T. cordifolia are bigger than those of wild species. The
people from West and North-west of Cameroon cultivate
and use it as foodstuff. The aqueous extract of freshly
stems is mixed with other ingredients and accompanied by
corn couscous. This meal is essentially eaten by women
during the first week after they gave given birth and by
convalescing patients. In previous studies, we reported new
compounds and other phytoconstituents from twigs and
leaves of the wild species. In the continuation of the
investigation of cultivated T. cordifolia, seven compounds
were isolated from the stems and among them, three
compounds identified as 2a-19a-dihydroxy-3-oxo-23-nor-
urs-12-en-28-oic acid (1), (2R)-2-hydroxy-N-[(2S,3S,4R,
26E)-1,3,4-trihydroxy-26-triaconten-2-yl] tetradecanamide
(2) and (2R,8Z)-2-hydroxy-{(2S,3R,5R,6S)-3,5-dihydroxy-
6-[(1E,5Z)-hexadeca-1,5-dienyl]-2-(b-D-glucopyranosyloxy)
methylpiperidine-1-yl}tetracos-8-enamide (3) are unre-
ported and four known compounds identified as lupeol (4)
[1], stigmasterol (5) [2], 3-O-b-D-glucopyranoside sitos-
terol (6) [3], tormentic acid (7) [4]. In addition, four known
secondary metabolites were also obtained from the leaves
and identified as heptadecanoic acid (8) [3], b-carotene (9)
[5], oleanolic acid (10) [1] and 24-hydroxytormentic acid
(11) [6]. Some of them are endowed with anti-inflamma-
tory, antimicrobial, analgesic and cytotoxic activities [7, 8].
These properties could identify these species as being good
health promoters.
4'"
3'"
3"
7"'
8"'
5"
HO
2"
1"
OH
6""
1"'
O
6
1
OH
4""
O
N
2
5
4
HO
O
HO
3
3""
1""
OH
1'
OH
Compound 3
This paper deals with the isolation and structural elu-
cidation of new compounds based on spectroscopic meth-
ods and some chemical transformations. The antioxidant
activity of some fractions obtained from flash chromatog-
raphy will also be reported.
Experimental Section
Plant Material
T. cordifolia A. Rich. was collected in December 2006
from the Nkongsamba Littoral region of Cameroon and a
specimen (N° 12830SRF Cam) has been deposited in the
´
National Herbarium of Yaounde, Cameroon.
General
IR spectra were recorded on a Perkin-Elmer (FT-IR system
spectrum BX spectrometer) using KBr disks. 1D- and 2D-
NMR spectra were carried out on a Bruker DRX-400 MHz.
Optical rotation was measured with a Perkin Elmer polar-
imeter model 341 at 589 nm. Melting points were mea-
sured in a Stuart Scientific Melting Point apparatus SMP3
and are uncorrected. The silica gel 60H (particle size
\45 lm), GF254 and 60A (size 70–200 lm) were used to
perform, respectively, flash chromatography, thin layer
chromatography and column chromatography. Iodine-silica
gel was employed as developer to visualize the spots on the
TLC plates. ESI-FT-ICR-MS and ESI-FT-ICR-MSn mea-
surements were conducted in the positive ion detection
mode on an Explorer High Resolution FT-ICR-MS (Var-
ian-IonSpec Corporation, Palo Alto, CA, USA) fitted with
a 9.4-T shielded superconducting magnet and a Micromass
Z-spray electrospray source. The used high voltage (HV)
was in the -4,000–3,600 V range. Nitrogen was used to
assist solvent evaporation. The temperatures of the source
and of the probe temperature were 90 and 100 °C,
respectively. Ions were trapped in the FT-ICR-MS cell with
30
HO
29
21
19
18
22OH
28
13
17
11
25
26
1
14
27
HO
O
2
16
O
9
10
3
5
7
4
23
Compound 1
3'
HO
O
14'
2'
1'
1
NH OH
3
26
5
30
4
2
27
OH OH
Compound 2
123

J Am Oil Chem Soc (2010) 87:1167–1177
1169
a 2V trapping potential. In these conditions, the average
mass measurement accuracy was typically better than
2 ppm, and the mass resolution close to 150,000 (m/Dm).
After calibration, the identification of the ions was ensured
by exact mass measurement and comparison with theoret-
ical isotopic pattern calculated by the ‘‘Omega 8 Exact
Mass Calculator’’ software (Varian-IonSpec Corporation,
Palo Alto, CA, USA). MS/MS experiments were conducted
in SORI–CID (sustained-off resonance irradiation collision
induced dissociation) mode. After isolation of parent ion by
ejection of other ions, sustained off-resonance irradiation
was performed during 60 ms with amplitude of 24 V bp.
Nitrogen was used as collision gas. The matrix used for the
MALDI-TOF-MS was a solution of 2,5-Dihydroxybenzoic
acid (2,5-DHB) 1 M in 50% acetonitrile 50% ultrapure
water, 0.1% trifluoroacetic acid (TFA); its measurements
were possible using a Bruker Reflex IV time-of-flight mass
spectrometer (TOF-MS) (Bruker-Daltonic, Bremen, Ger-
many) equipped with the SCOUT 384 probe ion source,
using a nitrogen pulsed laser (337 nm, model VSD-337ND,
Laser Science Inc., Boston, MA) with energy output of
400 lJ/pulse. LC-MS analyses were performed on an
HPLC system (LC pump P4000 and autosampler AS3000
from Thermo Separation Products) coupled to a LCQ Duo
Ion Trap detector (Thermo Electron, Zellik, Belgium)
equipped with an ESI interface run in the positive ion
mode. The separation of sample components was achieved
on an X-Terra MS C18 (5 lm particle size,
3.9 9 150 mm) (Waters, Overijse, Belgium), equipped
with an X-Terra MS C18 pre-column (5 lm particle size,
3.9 9 10 mm) and operated at 37 °C. Injection volume
was 15 lL. The mobile phase consisted of a mixture of
5 mM ammonium formate buffer at pH 3.8 (A) and ace-
tonitrile (B). Separation conditions for all compounds were
as follows: 0.0–0.50 min, A/B hold at 50/50, v/v; 0.50–
9.0 min, eluant B increase to 97%; 9.0–12.0 min, eluant B
hold at 97%, 12.0–12.5 min, eluant decrease to 50%, 12.5–
13.0 min, A/B hold at 50/50, Before each run the column
was equilibrated for 6 min at A/B 50/50. The flow rate for
column equilibration and analytical runs was 0.4 ml/min.
Ionization of the analytes was carried out as follows: sheath
gas flow rate (nitrogen), 47 arbitrary units; auxiliary gas
flow rate (helium), 18 arbitrary units; spray voltage,
5.0 kV; capillary temperature, 200 °C; capillary voltage,
36 V; Data acquisition was performed in a time segment
between 0.2 and 11.5 min after injection. The full MS-MS
spectrum of [M?H]? ions was monitored for all com-
pounds, isolation width was 2.5 m/z, normalized collision
energy was 28.0%. HR-ESI-MS and HR-ESI-MS/MS were
carried out using MicroTOF-Q 98 (Bruker-Daltonics,
Germany). The method used was tune low MS with source
type ESI and the acquisition parameters were: positive ion
polarity, set collision cell RF 150 Vpp, set capillary
6,000 V set dry heater 190 °C, set nebulizer 0.4 bar, set
dry gas 4.0 L/min.
Extraction and Isolation
Dried stems and leaves of T. cordifolia A. Rich. were pow-
dered to give, respectively, 5 and 1.8 kg of the powder. Both
powders were separately extracted by maceration at room
temperature with 5 (twigs) and 4 L (leaves) of methy-
lene:methanol (1:1). These were extracted three times and
each extraction lasted 48 h. The organic extracts were con-
centrated and yielded 248 and 184 g (for the stems and
leaves, respectively) of crude extract which were fractioned
by flash chromatography using fine silica gel 60H (particle
size\45 lm). The elution was performed with cyclohexane,
cyclohexane/EtOAc in order of increasing polarity (3:1 to
1:1 to 1:3), ethyl acetate, acetone and methanol. The frac-
tions collected were indexed A, B, C, D, E, F and G for the
twigs and A0, B0, C0, D0, E0, F0 and G0 for the leaves.
Chromatography of Stems
The fraction A (56 g) obtained from cyclohexane:ethyl
acetate (3:1, v/v) was purified by CC on silica gel 60A (size
70–200 lm) with different mixtures of cyclohexane:ethyl
acetate yielding 150 fractions: 25 mg of lupeol was
obtained from the fraction 30–45 eluted with cyclohex-
ane:ethyl acetate (95:5, v/v), 654 mg of sterol mixture
(stigmasterol and b-sitosterol) was isolated from the
fractions 48–52 eluted with cyclohexane:ethyl acetate
(82.5:7.5, v/v). The comparative thin layer chromatography
of D and E turned out to be identical and led us to mix
them. The purification of this mixture (36 mg) was done
with methylene chloride and the mixture methylene chlo-
ride:methanol in the order of increasing polarity yielding
150 fractions. This gave 6 mg of compound 2 in the frac-
tions 60–63 in the ratio 95:5 (v/v) of methylene chlo-
ride:methanol. In the same proportion of solvents, 475 mg
of 3-O-b-D-glucopyranoside of b-sitosterol were obtained
from the fractions 73–89. The fractions 54–57 collected
from the mixture methylene chloride:methanol 95:5 (v/v)
were purified with methylene chloride:ethyl acetate in the
order of increasing polarity (methylene chloride:ethyl
acetate 99:1, 98:2, 96:4, v/v) yielding 126 fractions. An
8-mg amount of compound 1 was isolated from those
numbered from 78 to 105 eluted in the ratio 49:1. The part E
(25 g) was purified by CC on silica gel 60 A (70–200 lm)
with methylene chloride:methanol in order of increasing
polarity yielding 83 fractions. Compound 3 (637 mg) was
obtained from the fractions 65–87 eluted with methylene
chloride:methanol 7:1. Tormentic acid (57 mg) was iso-
lated from G in the mixture methylene chloride:methanol
19:1 (v/v) from the fraction 1–5.
123

1170
J Am Oil Chem Soc (2010) 87:1167–1177
TOF-MS: 654 [C41H77O3N ? Na]?, 664 [C39H79O5N ?
Na]?, 668 [C42H79O3N ? Na]?, 682 [C43H81O3N ? Na]?.
Chromatography of Leaves
The cyclohexane fraction (4 mg) was eluted with petro-
leum ether:methylene chloride in the order of increasing
polarity yielding 98 fractions. Heptadecanoic acid (50 mg)
was isolated from the fraction 8–17 in the proportion of
mixture of solvents 98:2. Fractions 28–43 yielded 15 mg of
b-carotene in the ratio 97:3 (v/v). C0 (20 mg) was chro-
matographed with cyclohexane:ethyl acetate yielding 80
fractions. The ones eluted with cyclohexane:ethyl acetate
7:3 (v/v) gave 53 mg of oleanolic acid from the fractions
56–70. G0 was purified with methylene chloride:methanol
in the order of increasing polarity to give 147 fractions.
Among them, 122–128 eluted with methylene chlo-
ride:methanol (82.5:7.5, v/v) yielded 5 mg of 24-hy-
droxytormentic acid.
Methanolysis A 1-mg sample of 2 was refluxed (70 °C)
for 18 h in 3.5 ml of methanol containing 1.5 ml of 0.9 N
HCl under magnetic stirring. The medium was neutralized
with sodium carbonate and extracted with chloroform. The
organic phase was subjected to MALDI-TOF-MS analysis
and LCB was identified at m/z 484, [C30H61O3N ? H]?.
(2R,8Z)-2-Hydroxy-{(2S,3R,5R,6S)-3,5-dihydroxy-6-
[(1E,5Z)-hexadeca-1,5-dienyl]-2-(b-D-glucopyranosy-
loxy)methylpiperidine-1-yl}tetracos-8-enamide (3)
Brown solid; M.p. 201–203 °C; [a] ?3.89 (c 0.18; MeOH);
IR (KBr) mmax 3,336, 3,205, 2,918, 2,849, 1,622, 1,546,
1,469, 1,278, 1,068 cm-1; 1H and 13C NMR (Table 3); HR-
ESI-MS: 894.6938 [C52H95O10N ? H]?, 880.6717 [C51H93
O10N ? H]?, 866.6652 [C50H91O10N ? H]?, 852.6465
[C49H89O10N ? H]?, 838.6347 [C48H87O10N ? H]?,
754.5045 [C42H75O10N ? H]?, 736.5278 [C42H73
O9N ? H]?, 574.4852 [C36H63O4N ? H]?, 534.4463
[C26H47O10N ? H]?.
2a-19a-Dihydroxy-3-oxo-23-nor-urs-12-en-28-oic acid (1)
Colorless powder; M.p. 187–189 °C; [a] ?10.4 (c 0:06,
DMSO); IR (KBr) mmax 3,577, 3,446, 1,712, 1,687 cm-1
;
1H and 13C NMR (Table 1); HR-ESI-MS m/z: 495.3081
(calc. 495.3081) [C29H44O5 ? Na]?, Retro Diels-Alder
287.1620 [C16H24O ? Na]?.
Acetylation and Oxidative Cleavage of the Double Bond
A 11-mg sample (10.4 lmol) of compound 3 was dissolved
in 4 mL of pyridine and 4 mL of acetic anhydride was
added to this solution under magnetic stirring at room
temperature. The reaction was stopped after 1 h and the
acetylated product was obtained after evaporation under
vacuum. This product was dissolved in 4 mL of a mixture
of dioxane/water (3:1); two equivalents of pyridine, two
drops of 4% wt of an aqueous solution of osmium tetroxide
and four equivalents of sodium periodate were added. The
(2R)-2-Hydroxy-N-[(2S,3S,4R,26E)-1,3,4-trihydroxy-26-
triaconten-2-yl]tetradecanamide (2)
Brown solid; M.p. 136–138 °C; [a] -12.86 (c 0.14;
1
C5H5N); IR (KBr) mmax 3,334, 1,644, 1,624 cm-1; H and
13C NMR (Table 2); HR-ESI-MS m/z: 732.6447 [C44H87
O5N ? Na]?, 718.6283 [C43H85O5N ? Na]?, 704.6163
[C42H83O5N ? Na]? ESI-MS/MS: 340 [C17H35O4N ?
Na]?, 314 [C17H34O4N]?, 288 [C15H30O4N]?, MALDI-
Table 1 The 1H-NMR (400 MHz) and 13C-NMR (100 MHz) spectral data of compound 1 measured in DMSO-d6; d in ppm, J in Hz
Position
1
dH
dC (APT)
Position
11
dH
dC (APT)
Position
21
dH
dC
28.9
1.03 (m)
50.9
1.45 (m)
24.6
0.87 (m)
1.21 (m)
1.49 (m)
1.58 (m)
0.89 (d, 6.7)
–
2.20 (dd, 6.4, 12.8)
4.20 (m, 5.5, 6.5, 10.9)
2.00 (br d, 6.4)
5.16 (br s)
2
72.2
12
127.6
22
38.1
3
–
213.5
43.2
54.9
26.0
37.9
40.4
45.1
37.8
–
13
–
139.9
42.4
27.2
26.7
47.8
54.2
72.4
42.5
–
23
24
25
26
27
28
29
30
–
12.1
–
4
2.45 (m)
14
–
5
0.97 (m)
15
0.97 (m), 1.15 (m)
1.13 (s)
1.09 (s)
1.27 (s)
–
15.0
17.2
24.7
180.1
25.0
17.5
–
6
1.40 (m), 1.70 (m)
1.47 (m), 1.62 (m)
–
16
1.39 (m), 1.49 (m)
7
17
–
8
18
2.37 (s)
–
9
1.65 (m)
19
1.07 (s)
0.82 (d, 6.7)
–
10
–
20
2.44 (m)
3.72 (s)
OH-2
4.70 (d, 5.5)
OH-19
123

J Am Oil Chem Soc (2010) 87:1167–1177
1171
1
Table 2 The H NMR (400 MHz) and 13C (APT) NMR (100 MHz, C5D5N) spectral data of compound 2
Position
d(H) (m, J/Hz)
d(C) (APT)
COSY
HMBC
1a
4.38 (dd, 4.0, 10.8)
4.45 (dd, 8.0, 10.8)
5.10 (br s)
62.8
4.45
52.8, 78.1
52.8, 78.1
1b
62.8
4.38
2
52.8
4.30, 4.38, 4.45, 8.51
62.8, 78.1, 175.8
3
4.30 (dd, 4.8, 7.2)
4.22 (m)
78.1
4.22
34.5, 52.8, 62.8, 73.1
4
73.1
1.90, 2.20
1.20
5a and 5b
1.90 (m), 2.20 (m)
1.93 (m), 2.12 (m)
5.40 (m, 7.2, 16.0)
5.50 (m, 7.2, 16.0)
1.20 (br s)
34.5
130.4, 130.5
130.4, 130.5
33.0, 33.2
24, 27
33.0, 33.2
5.40, 5.50
1.93
25
130.5
26
6-23, 28, 29 40-140 (CH2)
130.6
2.12
33.0, 33.2
22.8, 23.1, 24.0, 26.0 30.0, 32.1
0.80
14.0, 22.8, 30.0, 32.1
52.8, 175.8
NH
10
20
30a and 30b
150, 30 (CH3)
8.51 (d, 8.0)
175.8
72.8
36.5
14.0
4.58 (dd, 3.6, 7.8)
1.97 (m), 2.15 (m)
0.80 (t, 8.0)
1.97, 2.15
1.20
26.0, 36.5, 175.8
22.8, 30.0, 32.1
1.20
reaction was left under magnetic stirring at room temper-
ature for 3 h. Quenching was done with 10 mL of an
aqueous solution of sodium thiosulfate and left under
magnetic stirring during 20 min about. The organic layer
was extracted three times with 15 mL of methylene chlo-
ride and concentrated under vacuum. The analysis by
MALDI–TOF of the organic residual phase gave important
peaks at m/z 241 [C16H33O ? H]?, 924 [M0 ? Na]? and
940 [M0 ? K]?.
sample-reagent mixing. The test was performed at 37 °C
and a 0–4 min reaction time window was used.
Results and Discussion
The crude organic extracts of stems and leaves were sepa-
rately subjected to repeated column chromatography to yield
seven and four compounds, respectively. Among those
obtained from stems there were a new nor-ursane derivative
1, a new ceramide 2 and a new piperidinic cerebroside 3.
Compound 1 was obtained as a white powder from a
mixture of methylene chloride/ethyl acetate (49:1, v/v). Its
positive mode HR-ESI-MS spectrum showed a peak at m/z
495.3081 (calc. 495.3081) corresponding to the molecular
formula [C29H44O5 ? Na]? accounting for eight double
bond equivalents. It responded to the Liebermann–Burchard
test of triterpenes. The reddish-purple color observed was
indicative of a triterpene type of triterpenoid. The IR spec-
trum showed intense absorption bands at 3,577 (free OH),
broad band at 3,446 (OH of carboxylic function), 1,712
Methanolysis A 2-mg sample of (3) was refluxed (70 °C)
for 18 h in 3.5 mL of methanol containing 1.5 mL of 0.9 N
hydrochloride acid under magnetic stirring. The medium
was neutralized with sodium carbonate and extracted with
chloroform. The organic phase was subjected to the LC-
ESI-MS analysis and the peak observed at m/z 424 corre-
sponding to retention time 10.96 and some fragment ion
led us to identify fatty acid methyl ester as methyl-2-
hydroxyhexacos-17-enoate.
FRAP Assay
(C=O of carbonyl function), 1,687 (C=C stretching) cm-1
.
The FRAP assay is one of the methods used today to
evaluate antioxidant power. It was developed by Benzie
and Strain [9]. Practically, it is based on a mixture of
1.5 mL of 10 volumes of 300 mmol/L acetate buffer, pH
3.6 ? 1 volume of 10 mmol/L 2,4,6-tripyridyl-S-triazine
(TPTZ) in 40 mmol/L hydrochloric acid ? 1 volume of
20 mmol/L (FeCl3) pre-warmed at 37 °C. Then 50 lL of
the sample and the standards were added to the mixture and
homogenized by vortex. The absorbance at 593 nm was
read against a reagent blank at a pre-determined time after
Two signals at dH/dC 5.19 (1H, br s)/127.9 and 139.9 cor-
responding to the trisubstituted double bond of D [12] ursene
1
type [1] were exhibited on the H- and 13C-NMR (APT)
spectra (Table 1) of 1. These spectra presented also six
intense signals of methyl groups in which, there were two at
dH/dC 0,89 (3H, d, J = 6.7 Hz)/12.1; 0.82 (3H, d,
J = 6.7 Hz)/17.1 and four singlets at dH/dC 1.07/25.0; 1.09/
17.2; 1.13/15.0; 1.27/24.2. This meant that, among the eight
Me groups of urs-12-enes, two are oxidized or missing but
on the APT spectrum, we had two signals of quaternary
123

1172
J Am Oil Chem Soc (2010) 87:1167–1177
1
Table 3 The H-NMR (400 MHz CD3OD) and 13C-NMR (APT) (100 MHz, CD3OD) spectral data of compound 3
Position
dH (m, J/Hz)
dC (APT)
COSY
HMBC
10a and 10b
3.82 (dd, 4.8, 10.2)
4.08 (dd, 9.2, 10.2)
4.25 (br d, 9.2)
69.7
4.08, 4.25, 4.27
3.82, 4.25, 4.27
3.82, 4.08, 3.61
1.55, 1.70, 4.25
3.61, 4.02
51.6, 104.7
75.5, 104.7
2
3
4
5
51.6
75.5
32.9
73.1
69.7, 75.5, 177.2
32.9, 51.6, 69.7
54.8, 75.5
3.61 (dd, 9.7, 10.0)
1.55 (m), 1.70 (m)
4.12 (br d, 4.8, 9.2,)
3.61, 4.02, 5.49,
5.75
32.9, 54.8, 132.0, 134.4
6
4.02 (m)
54.8
1.55, 4.25, 3.61,
4.12
51.6, 73.1, 177.2
100
200
5.49 (dd, 9.2, 14.6)
5.75 (br d, 14.6)
2.01 (br d, 4.8)
132.0
134.4
33.7
2.01, 2.08, 5.75
2.01, 2.08, 5.49
33.7, 73.1, 134.4.
33.7, 132.0
300a and 300b
4,12, 5.49, 5.75,
2.01
73.1, 132.0
2.08 (br d, 9.2)
400a and 400b
1.98 (br s), 2.00 (br s)
33.4
5.38, 5.39, 5.41,
5.75
131.2
2.05
1000
2000
–
177.2
73.0
35.7
–
–
4.03 (dd, 4.8, 9.2)
1.65 (m), 1.75 (m)
1.65, 1.75
1.30, 4.02
1.94, 2.00, 2.08
35.7, 177.2
3000a and 3000b
500, 600, 8000, 9000
26.1, 30.5, 30.7, 73.0
26.1, 28.4, 33.7,
5.38 (dd, 4.8, 9.2), 5.39 (dd, 4.8, 9.2), 5.41 130.7, 130.8
130.9, 131.2
(br d, 5), 5.42 (br s)
700, 7000, 10000
2.00 (m), 2.10 (m)
28.4
1.30, 5.38, 5.39,
5.41, 5.42
23.7, 30.7, 26.1, 30.5 130.7,
130.8, 130.9, 131.2
800-1500, 4000-6000, 11000- 1.30 (br s)
23000(CH2)n
23.7, 26.1, 30.7, 0.90, 1.94, 2.00
30.8
28.4, 35.7, 14.5
1600, 240002x (CH3)
0.90 (t, 6.9)
14.5
1.30
23.7, 26.1, 30.5, 30.7
Glucosyl
10000
20000
30000
40000
50000
60000a and 60000
4.27 (d, 9.0)
104.7
75.0
77.9
71.6
78.0
62.6
3.19, 3.37, 4.25
3.37, 4.27
3.30
69.7, 75.0
77.9, 104.7
71.6
3.19 (dd, 9.0, 9.2)
3.37 (dd, 9.0, 9.2)
3.30 (br d, 8.5)
3.29, 3.37
3.68, 3.88
3.29
78.0
3.29 (ddd, 4.8, 8.5, 9.2)
3.88 (br d, 12.1)
3.68 (dd, 4.8, 12.1)
62.6,
b
71.6, 78.0
carbons at dC 180.1 corresponding to the carboxylic function
C-28 [10] and at dC 213.5 attributable to the ketone at C-3
[10]. From the above-mentioned data, it was deduced that
one of the Me groups is missing and the other is oxidized.
Considering some characteristic signals of triterpenes of
urs-12-ene type [10] such as the ones observed at dH/dC
0.97 (H-5, m)/54.9, 1.65 (H-9, m)/45.1 and 2.37 (H-18, s)/
54.2, we have observed interesting long range correlations
on the COSY and HMBC spectra (Fig. 1). In fact, the
proton at dH 0.97 showed correlations with those at dH
0.89, 1.40, 1.70 and 2.45 as well as their carbons at dC 12.1,
26.0 and 43.2, respectively. The multiplicity of signals at
dH 0.89/12.1 appearing as a doublet and correlating with
the proton at dH 2.45 confirmed that only one Me group
was present at C-4. Additional interactions supporting this
structure were noted from both protons at dH 0.89 and 2.45
to the carbons at dC 54.9 and 213.5. The signal at dH 1.65
showed correlation with the proton at dH 2.00 and the
carbons at dC 15.0, 25.0 and 37.8. The protons (dH 1.13) of
carbon at dC 15.0 showed interactions with the carbons at
dc 37.8 and 50.9. Both protons at dH 1.03 and 2.20 linked to
the carbons at dc 37.8 and 50.9, respectively, interacted
with the carbon at dC 15.0, 37.8, 54.8, 72.2 and 213.5
showing that the ring A has an acyloin system. The signal
at dH 2.37 correlated with the carbons at dC 25.0, 42.5,
47.8, 72.9, 127.6, 139.9 and 180.1 corresponding to the
interactions observed in the derivatives of 19a-hydroxyurs-
12-en-28-oic acid [4]. The spatial geometry of hydroxyl
and methyl groups in position C-2 and C-4, respectively,
were determined using the NOESY spectrum (Fig. 2).
123

J Am Oil Chem Soc (2010) 87:1167–1177
1173
Fig. 3 NOESY interactions of
compound 2
H
O
H
H
HO
H
C10H21
H
O
H
NH
H
H
O
OH
H
OH
H
C25H49
O
H
H
OHHO
H
O
H
HMBC
COSY
confirming that this ceramide of dihydrosphingolipid type
has linked to a fatty acid having acyloin system [11]. The
trans-configuration of the double bond was evident from
the coupling constant between the olefinic protons
(J = 16.0 Hz) and the chemical shifts of the allylic carbons
at dc 33.0 and 33.2, which were expected at 26.5 ppm in
case of cis-configuration [12]. In the COSY spectrum of 2
(Table 2), the proton signal of the azomethine (dH 5.10)
was correlated with the hydroxymethylene (dH 4.38 and
4.45) and two oxygenated methines (dH 4.22 and 4.30)
suggesting the location of two hydroxyl groups at C-3 and
C-4. The third hydroxyl group was located at C-20 as result
of the correlations observed in the HMBC spectrum
(Table 2) from the oxymethine group (dH 4.58) and amine
group (dH 8.50) to the carbonyl ester (dC 175.8). The
methylene C-30 (dH 1.97 and 2.15) showed correlation with
the broad singlet at 30.0. The absolute configuration of
asymmetric carbons in phytosphingolipids is 20R, 2S, 3S
and 4R [11], and NOESY spectrum showed interactions
between the protons geminated to the oxygen, confirming
that 2 has the same absolute configurations (Fig. 3). By
application of the fragmentation mechanism developed by
Fong-Fu et al. [13], the peak observed at m/z 664 gave
information about the position of the double bond
(Scheme 1) by loss of C5H8. Its position in the long chain
base was confirmed by methanolysis. In fact, this reaction
(0.9 N hydrochloric acid:methanol/70 °C/18 h) gave the
fatty acid methyl ester and the long chain base which were
carefully characterized on the basis of mass spectrometry
as methyl-2-hydroxytetradecanoate and 2-amino-25-tria-
contene-1,3,4-triol (at m/z 484, [C30H61O3N ? H]?) which
were in accordance with the fragment ion obtained after
MS/MS analysis of the peak at m/z 718 (Scheme 2). The
analyses of the NMR and mass spectroscopy data led to the
unambiguous assignment of structure 2 as (2R)-2-hydroxy-
N-[(2S,3S,4R,26E)-1,3,4-trihydroxy-26-triaconten-2-yl]te-
tradecanamide trivially named triumfettamide A.
Fig. 1 COSY and HMBC correlations of compound 1
H
H
H
COOH
O
H
HO
O
H
H
H
Fig. 2 NOESY interactions of compound 1
Since, the protons at dH 1.13 (H-25) and 1.65 (H-9) have a
spatial configuration b and a, respectively [10], it was
noted that the one at dH 1.13 interacted with the protons at
dH 2.00 (H-11), 2.45 (H-4) and 4.20 (H-2). This meant that
the four protons have the same spatial orientation. The lack
of methyl group C-24 and the equatorial position of C-23
were confirmed by the correlations observed between the
signal at dH 1.65 and the protons at dH 0.89, 0.97 and 1.27.
The combination of the foregoing data led us to identify 1
as 2a-19a-dihydroxy-3-oxo-23-nor-urs-12-en-28-oic acid.
Compound 2 was obtained as a dark solid from a mix-
ture of methylene chloride:methanol (19:1, v/v). Its posi-
tive HR-ESI-MS showed a pseudomolecular ion at m/z
732.6447 [M?Na]? (calc. 732.6476), corresponding to the
formula C44H87NO5 accounting for two double bond
equivalents. The FT-IR spectrum of 2 showed character-
istic absorption bands for a free hydroxyl (3,334 cm-1) and
an amide carbonyl function (1,624 cm-1) [11]. The H-
1
and 13C-NMR spectral data of 2 exhibited a set of signals
which were in accordance with those of phytoceramides
[11]. In fact, the presence of a NH group at dH 8.51 (d,
J = 8.0 Hz) which was exchangeable with deuterated
water was observed as well as two multiplets of two ole-
finic protons at dH 5.40 (m) and 5.50 (m), a broad singlet at
dH 1.20 (methylene protons), a triplet of 6H at dH 0.80
(J = 8.0 Hz, two terminal methyl groups) and two carbinol
protons at dH [4.38 (dd, J = 4.8, 10.8 Hz); 4.45 (dd,
J = 8.0, 10.8 Hz)]. The NMR spectrum of 2 (Table 2) also
showed carbon signals at dC 175.8, 52.8 and the range
23.9–33.9, further supporting the fatty-acid amine nature of
2. In addition, we observed the resonances for three oxy-
methine protons at dH/dC 4.22 (m)/73.1, 4.30 (dd, J = 4.8,
7.2 Hz)/78.1 and 4.58 (dd, J = 3.6, 7.8 Hz)/72.8
Compound 3 was isolated as a brown solid from a
mixture of methylene chloride:methanol (9:1, v/v). Its
positive mode HR-ESI-MS exhibited a pseudomolecular
ion at m/z 894.6938 [M?H]?, corresponding to a molecular
formula C52H95O10N (C52H96O10N, calc. 894.7029) indi-
cating six double bond equivalents. The FTIR spectrum of
3 showed broad absorption bands for hydroxyl function
(3,336 cm-1), strong absorptions for secondary amides
123

1174
J Am Oil Chem Soc (2010) 87:1167–1177
b
H
HO
O
HO
O
a
NH OH
b
NH OH
H
H
a
OH OH
OH OH
Na
m/z 732
Na
m/z 664
b
- CH2
O
NH
O
NH
OH OH
OH OH
Na
Na
m/z 682
- CH2
m/z 696
O
NH
- CH2
O
NH
OH OH
OH OH
Na
Na
m/z 668
m/z 654
Scheme 1 Fragment ions giving the position of the double bond and other information on 2
Na
Na
H
H
O
HO
O
HO
O
H
NH
NH
O
- NaH
O
NH
OH OH
OH OH
OH OH
m/z 316
m/z 718.6320
m/z 340
-C2H4
H
O
O
NH
OH OH
m/z 288
Scheme 2 Fragment ions from MS/MS justifying the identity of the fatty acid side chain and those of the long chain base in the compound 2
(1,622, 1,546 and 3,205 cm-1) and aliphatic chains (1,469,
2,849 and 2,918 cm-1). The presence of a nitrogen-
attached carbon was confirmed by the signal at dC 51.6 and
a carbonyl signal at dC 177.2 in the APT spectrum. 3 gave a
positive test result with Molish reagent suggesting the
presence of a sugar moiety. Its NMR spectra (Table 3)
showed the anomeric proton at dH 4.27 (1H, d, J = 8.7 Hz;
104.7) and a set of carbon signals at dC 75.0, 77.9, 71.6,
78.0, 62.6 consistent with b-D-glucopyranoside. This cor-
roborated with the peak observed on HR-ESI-MS/MS at
m/z 574.4852 (calc. 574.4830) resulting from [C52H95O10N-
H2O-C10H20-C6H10O5 ? H]? (Scheme 3). We observed
also, the characteristic signals close to those of phyto-
sphingosine type possessing a 2R-hydroxyfatty acid side
chain [12]. So, an intense signal of CH2 sequence charac-
teristic of long aliphatic chains was observed in the NMR
spectra between dH 1.21 and 1.38 (br s) and a triplet of
terminal CH3 groups at dH/dC (6H, 0.90, t, J = 6.9 Hz)/
14.5. Furthermore, an oxymethylene at dH/dC 3.82 (dd,
J = 9.6, 11.2 Hz) and 4.08 (dd, J = 4.8, 11.2 Hz)/69.7 and
123

J Am Oil Chem Soc (2010) 87:1167–1177
1175
C9H19
C15H31
C15H31
C15H31
HO
-H2O
OH
HO
-C10H20
OH
HO
-C6H10O5
O
OH
O
O
N
HO
HO
OH
OH
O
O
O
N
H
O
N
HO
HO
OH
OH
HO
O
H
H
H
H
m/z 894
m/z 736
m/z 574
C7H15
C15H31
C6H13
C15H31
C5H11
C15H31
HO
OH
-C2H4
HO
-CH2
HO
-CH2
HO
OH
OH
O
O
O
N
OH
OH
O
O
O
N
OH
HO
HO
HO
O
O
O
N
OH
H
HO
HO
OH
OH
OH
H
H
OH
m/z 866
C5H11
OH
m/z 852
C9H19
m/z 838
C5H11
HO
-C10H20
OH
O
HO
- C16H28
OH
OH
O
O
H
N
HO
HO
OH
N
O
O
O
H
HO
HO
OH
OH
OH
OH
m/z 754
m/z 534
Scheme 3 Fragment ions giving indications (sugar and the position of the double bond) of the structure of 3
three oxymethines at dH/dC 3.61(dd, J = 5.8, 9.7 Hz)/75.5,
4.03 (m)/73.0 and 4.12 (m)/73.1 were observed. Beside this
information, two ethylenic protons in trans-geometry [12]
at dH/dC 5.49 (dd, J = 7.4, 14.6 Hz)/132.0 and 5.75 (br d,
J = 14.6 Hz)/134.4 were observed together with four
olefinic ones in cis-configuration [11] between 5.37 and
5.42 ppm (m, J = 4.8, 7.4 Hz)/130.7, 130.8, 130.9, 131.2.
From the foregoing data, the structure of 3 was suggested
to be a polyunsaturated b-D-glucopyranosyl sphingolipid.
Pertinent interactions observed in the HMBC spectrum
(Table 3) from the proton of oxymethine at dH 4.03 (m,
73.0) to C=O and a set of methylene group justifying that
the fatty acyl part is a-hydroxylated. Two carbinol protons
Ha-10 (3.82), Hb-10 (4.08) correlated with the anomeric
carbon C-10000 led us to attach the sugar moiety at C-10.
Moreover, the long chain base was considered to be of
phytosphingosine type because of correlations between the
proton H-3 (3.61 (m); dC 75.5) and the carbons C-10 (69.7),
C-2 (51.6), C-4 (32.9) and C-6 (73.1). An interesting cor-
relation observed from the proton H-6 (4.12) to the carbon
at dC 54.8 suggested that this ceramide was an azacyclane
derivative. The same proton H-6 showed correlation with
the olefinic carbons in trans-configuration (132.0; 134.4)
confirming their position in long chain base side. The
correlation between H-6 (4.02)/dC 54.8 and the carbons at
dC 177.2 and 51.6 added to the comparison of spectro-
scopic data with those presented by Vanderlan et al. [14]
and Yamashita et al. [15] ascertained that 3 was a pipe-
ridinic derivative.The COSY spectrum (Fig. 4) revealed
additional interactions between the proton at dH 4.12 and
those at dH 5.49, 5.75, 4.00; the proton H-5 (4.02) corre-
lated with the ones at dH 4.25, 3.61 and 1.55.
Assuming the absolute configurations of phytoceramide
to be 20R, and 2S [16], the NOESY spectrum showed
important interactions between H-6 (4.02) and those at dH
3.82, 4.08 and 3.61; H-2 (4.25) interacted with the protons
H-5 (4.12) and H-4 (1.70) (Fig. 4). These observations
indicated that the correlating protons were close in the
space and led us to define the absolute configuration of the
other carbons to be 3R, 5R and 6S.
The fragment ion at m/z 534 obtained from ESI-MS/MS
was used to locate the double bonds in the piperidine side
chain (Scheme 3). This position was evident after acetyla-
tion and oxidative cleavage of double bonds (Scheme 4).
The organic phase obtained, was analyzed by MALDI-TOF-
MS giving five interesting peaks at 193 [C11H22O ? Na]?,
123

1176
J Am Oil Chem Soc (2010) 87:1167–1177
Fig. 4 COSY and NOESY
interactions of compound 3
OH
H
H
H
O
O
HO
HO
H
O
HO
R=
N
R
H
H
H
OH
H
H
H
OH
H
OH
COSY
NOESY
C9H19
C9H19
C15H31
C15H31
HO
AcO
(AcO)2O/C5H5N (1:1)
Room T. /1H
OH
OAc
OH
OAc
O
O
O
N
O
O
O
N
HO
HO
AcO
AcO
OH
OAc
OH
OAc
O
AcO
O
OsO4 (cat)-NaIO4 (4eq)
pyridine (2eq); dioxane/H2O (3:1)
OAc
OAc
+
+
OHCC15H31
OHCC10H21
O
O
O
N
AcO
AcO
OAc
OAc
m/z 787
Scheme 4 Oxidative cleavage of double bonds in compound 3
209 [C11H22O ? K]?, 241 [C16H32O ? Na],?, 263
[C16H32O ? K]? and 810 [C35H49O19N ? Na]?. The last
value justified the belief that 3 had seven free alcohol
functions.
Methanolysis solution (aqueous 0.9 N hydrochloric
acid:methanol) of 3 was neutralized with sodium carbonate
aqueous solution and extracted with chloroform to yield
methyl-2-hydroxy-8Z-tetracos-8-enoate which was identi-
fied after LC-ESI-MS analysis with the peak at m/z 396
(retention time 10.66 min). From the foregoing data,
compound 3 was identified as being (2R,8Z)-2-hydroxy-
{(2S,3R,5R,6S)-3,5-dihydroxy-6-[(1E,5Z)-hexadeca-1,5-
dienyl]-2-(b-D-glucopyranosyloxy)methylpiperidine-1-yl}
tetracos-8-enamide.
Fig. 5 Result of antioxidant test of flash chromatographic fractions
methanolic one (G). Reduction of FeIII-TPTZ to FeII-TPTZ
was attributable to other secondary metabolites present in
trace amounts in the organic extract because not all isolated
compounds displayed reducing activity. Although the iso-
lated compounds were not active, some ursolic acid deriv-
atives have been reported as being radical scavenging
natural products [17]. In addition, lupeol induces apoptotic
death of human pancreatic adenocarcinoma cells [18] and
tormentic acid has been reported as being an anti-inflam-
matory compound [19]. In addition to this wide activity
spectrum, ceramide and its derivatives play a significant role
in the apoptosis induction of some cancer cells lines [20].
1
The H- and 13C-NMR data of the known compounds
were similar with those reported in literature.
In the aim to valorise this edible plant, the antioxidant
activity of fractions obtained from flash chromatography,
evaluated by the FRAP method, was used to carry out this
biological activity. Two of them showed a good activity in
comparison to those where trolox was used as the standard
(Fig. 5). It was noted that, among the four extracts from
leaves, one (B0) did not show any activity and the active
fraction from stems (the part used as food) was the
123

J Am Oil Chem Soc (2010) 87:1167–1177
1177
Acknowledgments L. P. Sandjo would like to thank EGIDE and
SCAC (Service de Cooperation et d’Actions Culturelles: French
Grant Government No. 607832K) for their financial support. I would
10. Shoko T, Yoko I, Eri K, Yoshie T, Hideyuki I, Tsutomu H,
Hiroshi S, Harukuni T, Hoyoku N, Daigo S, Susumu S, Takashi Y
(2002) Production of bioactive triterpenes by Eriobotrya japonica
calli. Phytochemistry 59:315–323
´
also like to thank the LSMCL (Laboratoire de Spectrometrie de
Masse et Chimie Laser) for the MALDI-TOF-MS analyses and Serge
Schneider for the LC-MS analyses.
11. Gao JM, Dong ZJ, Liu JK (2001) Cyanoxanthamide, a new cer-
amide from the Basidiomycetes Russula cyanoxantha. Lipids
36:175–180
12. Jung JH, Lee C-O, Kim YC, Kang SS (1996) New bioactive
cerebrosides from Arisaema amurense. J Nat Prod 59:319–322
13. Hsu FF, Turk J (2001) Structural determination of glyco-
sphingolipids as lithiated adducts by electrospray ionization mass
spectrometry using low-energy collisional-activated dissociation
on a triple stage quadrupole instrument. J Am Soc Mass Spect
12:61–79
14. da Vanderlan SB, Leslie GAA, David GIK (1995) Bioactive and
other piperidine alkaloids from Cassia leptophylla. Tetrahedron
51:5929–5934
15. Toru Y, Kayo Y, Haruhisa K, Yukihiko K, Alison AW, Robert
JN, George WJF, Naoki A (2002) New polyhydroxylated pyr-
rolidine, piperidine, and pyrrolizidine alkaloids from Scilla sibi-
rica. J Nat Prod 65:1875–1881
16. Wu ZP, Chen Y, Xia B, Wang M, Dong YF, Feng X (2009) Two
novel ceramides with a phytosphingolipid and a tertiary amide
structure from Zephyranthes candida. Lipids 44:63–70
17. Brigida D, Antonio F, Pietro M, Palma O, Severina P (2006)
Annurcoic acid: A new antioxidant ursane triterpene from fruits
of cv. Annurca apple. Food Chem 98:285–290
18. Mohammad S, Satwinderjeet K, Mee-Hyang K, Vaqar MA,
Farrukh A, Hasan M (2005) Lupeol, a fruit and vegetable based
triterpene, induces apoptotic death of human pancreatic adeno-
carcinoma cells via inhibition of Ras signaling pathway. Carci-
nogenesis 26:1956–1964
19. Chikako M, Kiyomi I, Mitsuru H, Kengo S, Hiromi Y, Yoshiyuki
M (2002) Novel anti-inflammatory compounds from Rubus sie-
boldii, triterpenoids, are inhibitors of mammalian DNA poly-
merases. Biochim et Biophys Acta 1596:193–200
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