Spectral Assignments and Reference Data Tetrapterosides A and B, two new oleanane-type saponins from Tetrapleura tetraptera

Abstract

From the stem bark of Tetrapleura tetraptera, two new oleanane-type saponins, tetrapteroside A 3-O-{6-O-[(2E,6S)-2,6-dimethyl-6-hydroxyocta-2,7-dienoyl]-β-D-glucopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 3)-β-D-glucopyranosyl-(1 → 4)-[β-D-glucopyranosyl-(1 → 2)]-β-D-glucopyranosyl}-3,27-dihydroxyoleanolic acid (1), and tetrapteroside B 3-O-{β-D-glucopyranosyl-(1 → 2)-6-O-[(E)-feruloyl]-β-D-glucopyranosyl-(1 → 3)-β-D-glucopyranosyl-(1 → 4)-[β-D-glucopyranosyl-(1 → 2)]-β-D-glucopyranosyl}-3,27-dihydroxyoleanolic acid (2), were isolated. Further extractions from the roots led to the isolation of four known oleanane-type saponins. Their structures were elucidated by the combination of mass spectrometry (MS), one and two-dimensional NMR experiments.

Full text

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Spectral Assignments and Reference Data
Received: 12 June 2008Revised: 27 October 2008Accepted: 31 October 2008 Published online in Wiley Interscience: 19 January 2009
(www.interscience.com) DOI 10.1002/mrc.2381
Tetrapterosides A and B,two new
oleanane-type saponins from Tetrapleura
tetraptera
Olivier Placide Not´ e,a,bAnne-Claire Mitaine-Offer,aTomofumi Miyamoto,c
Thomas Paululat,dDieudonn´ e EmmanuelPegnyembbandMarie-Aleth
Lacaille-Duboisa∗
From the stem bark of Tetrapleura tetraptera, two new oleanane-type saponins, tetrapteroside A 3-O-{6-O-[(2E,6S)-
2,6-dimethyl-6-hydroxyocta-2,7-dienoyl]-β-D-glucopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 3)-β-D-glucopyranosyl-(1 →
4)-[β-D-glucopyranosyl-(1 → 2)]-β-D-glucopyranosyl}-3,27-dihydroxyoleanolic acid (1), and tetrapteroside B 3-O-{β-D-
glucopyranosyl-(1 → 2)-6-O-[(E)-feruloyl]-β-D-glucopyranosyl-(1 → 3)-β-D-glucopyranosyl-(1 → 4)-[β-D-glucopyranosyl-(1 →
2)]-β-D-glucopyranosyl}-3,27-dihydroxyoleanolic acid (2), were isolated. Further extractions from the roots led to the isolation
of four known oleanane-type saponins. Their structures were elucidated by the combination of mass spectrometry (MS), one
and two-dimensionalNMR experiments. Copyright c ? 2009 John Wiley& Sons,Ltd.
Keywords: NMR;1H;13C; 2D NMR; triterpene saponins; tetrapteroside A; tetrapteroside B; Mimosaceae; Tetrapleuratetraptera
Introduction
In a continuation of the study on bioactive triterpene saponins
from Cameroonian medicinal plants,[1–5]we have examined the
saponin fraction of the stem bark and roots of Tetrapleura
tetraptera Taub. (Mimosaceae), locally named Aridan. This ro-
bust tree from the forest of tropical Africa is well-known as
remedies in the traditional medicine,[6]and for its strong mol-
luscicidal activity. The previous chemical analysis of the fruit
and stem bark led to the isolation of oleanane-type triterpene
saponins like aridanin, a N-acetylglycoside triterpenoid.[7,8]In
this paper, we report the isolation by successive chromato-
graphic steps of two new triterpene saponins, tetrapterosides
A (1) and B (2) (Fig. 1) from the stem bark and four known
saponinsfromtheroots,[7,8]3-O-(2-acetamido-2-deoxy-β-D-gluco-
pyranosyl)oleanolic acid (aridanin), 3-O-(2-acetamido-2-deoxy-
β-D-glucopyranosyl)echinocystic acid, 3-O-[β-D-glucopyranosyl-
(1 → 6)-2-acetamido-2-deoxy-β-D-glucopyranosyl]oleanolic acid,
and 3-O-sulfoechinocystic acid. Their structures were established
mainlybytwo-dimensional(2D)NMR(COSY,TOCSY,NOESY,HSQC,
HMBC) and mass spectrometry.
Results and Discussion
Compound 1, a white amorphous powder, exhibited in high-
resolution electrospray ionization mass spectrometry (HR-ESIMS)
(positive-ion mode) a pseudo-molecular ion peak at m/z =
1471,7090 [M + Na]+(calcd 1471,7085), consistent with a
molecular formula of C70H112O31Na. Its fast-atom bombardment
mass spectrum (FABMS) (negative-ion mode) showed a quasi-
molecular ion peak at m/z = 1447 [M − H]−, indicating a
molecular weight of 1448. Other significant fragment ion peaks
wereobservedatm/z=1281[(M−H)−166]−,1119[(M−H)–166
−162]−,957[(M−H)–166−162−162]−,and795[(M−H)–166−
162 − 162 − 162]−, corresponding to the successive loss of one
monoterpenoyl moiety, and three hexosyl moieties, respectively.
The
of a saponin structure composed by an aglycon part and
an oligosaccharidic part. For the aglycon moiety, the1H-NMR
spectrum displayed signals for six angular methyl groups as
singlets, one olefinic proton at δH 5.77 (br t, J = 3 Hz, H-12),
one oxygen bearing methine protons at δH3.08 (dd, J = 11.4,
3.8 Hz, H-3), and one primary alcoholic function at C-27 position,
δH 3.72 (d, J = 10.5 Hz), 3.98. In the13C-NMR spectrum, the
deshielded signal at δC 89.5 (C-3) in comparison with the free
aglycon, suggested a glycosidic linkage at C-3. The structure
of the aglycon of 1 was thus recognized to be the triterpene
3,27-dihydroxyoleanolic acid by1H-NMR and13C-NMR analyses
1H-NMR spectrum of 1 showed characteristic signals
∗Correspondence to: Marie-Aleth Lacaille-Dubois, Laboratoire de Pharmacog-
nosie,UMIB,UPRESEA3660,Facult´ edePharmacie,Universit´ edeBourgogne,7
bd.JeanneD’Arc,BP87900,21079Dijoncedex,France.
E-mail:m-a.lacaille-dubois@u-bourgogne.fr
a LaboratoiredePharmacognosie,UMIB,UPRESEA3660,Facult´ edePharmacie,
Universit´ e de Bourgogne, 7 bd. Jeanne D’ Arc, BP 87900, 21079 Dijon cedex,
France
b D´ epartement de Chimie Organique, Facult´ e des Sciences, Universit´ e de
Yaound´ e,BP812Yaound´ e,Cameroun
c Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka
812-8582,Japan
d Universit¨ at Siegen FB8, OC- II (AK Ihmels) Adolf-Reichwein-Str. 2 D- 57068
Siegen,Germany
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O. P. Not´ e etal.
COOH
CH2OH
O
O
OHO
O
OH
1
2
3
GlcI
R1
R2
H
H
OH
OH
1
2
3
4
5
6
γ
α
β
OCH3
O
HO
HO
OH
OH
O
HO
O
OH
OH
O
HO
HO
O
OR1
O
HO
HO
OH
OR2
FA
MT
H
O
OH
1
2
3
4
5
6
7
8
9
10
GlcII
GlcIII
GlcIV
GlcV
Figure 1. Structures of 1 and 2.
O
O
OHO
O
OH
3
GlcI
O
HO
HO
OH
OH
O
HO
O
OH
OH
O
HO
HO
O
OH
O
HO
HO
OH
OMT
GlcII
GlcIII
GlcIV
GlcV
1
O
O
OHO
O
OH
3
GlcI
O
HO
HO
OH
OH
O
HO
O
OH
OH
O
HO
HO
O
OFA
O
HO
HO
OH
OH
GlcII
GlcIII
GlcIV
GlcV
2
Figure 2. Important HMBC (
) and NOESY (
) correlations for 1 and 2.
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Spectral Assignments and Reference Data
Table 1.
aglycons of 1 and 2 in pyridine-d5(δ in ppm, J in Hz)
1H-NMR (600 MHz) and
13C-NMR(150 MHz) data of the
Compound 1
Compound 2
No
1H-NMR
13C-NMR
1H-NMR
13C-NMR
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18 3.27 dd, J = 12.5, 2.5
191.25, 1.68 t, J = 12.5
20
21 1.75, 1.89
221.65, nd
231.21 s
241.01 s
25 0.71 s
26 0.89 s
27 3.72 d, J = 10.5, 3.98
28
290.81 s
300.93 s
0.83, 1.28
1.75, 1.95
38.3
26.0
89.5
39.3
55.3
18.4
33.8
40.0
48.2
36.7
23.6
127.6
139.7
47.5
24.1
23.5
46.3
41.4
45.3
30.6
33.2
32.8
27.6
16.4
15.4
18.5
64.1
179.5
32.9
23.6
0.87, 1.31
1.76, 1.96
38.2
26.0
89.6
39.5
55.3
18.2
33.8
40.0
48.2
36.7
23.4
127.1
139.8
47.5
23.9
23.6
46.4
41.5
45.4
30.6
33.1
32.9
27.7
16.4
15.5
18.5
63.9
179.5
32.9
23.6
3.08 dd, J = 11.4, 3.8
–
0.81
1.24, 1.51
1.04, 1.25
–
2.05
–
1.88, 2.04
5.77 br t, J = 3.0
–
–
nd
nd
–
3.12 dd, J = 11.6, 3.8
–
0.83
1.24, 1.50
1.08, nd
–
2.07
–
1.94, 2.03
5.84 br t, J = 3.0
–
–
nd
nd
–
3.35 dd, J = 12.1, 2.4
1.28, 1.70 t, J = 12.1
–
1.76, 1.95
1.69, nd
1.22 s
1.04 s
0.74 s
0.90 s
3.73 d, J = 10.0, 4.03
–
0.83 s
0.96 s
–
–
Overlapped proton NMR signals are reported without designated
multiplicity.
Nd, not determined.
(Table 1) using the correlations observed in COSY, NOESY, HSQC,
andHMBCspectra,andwasinfullagreementwithliteraturedata.[9]
For the oligosaccharidic chain, the1H-NMR spectrum showed five
anomericprotonsatδH4.72(d,J=7.1 Hz),4.91(d,J=7.6 Hz),4.95
(d, J = 7.6 Hz), 5.17 (d, J = 7.8 Hz), and 5.44 (d, J = 7.6 Hz), which
gave correlations, in the HSQC spectrum, with anomeric carbon
signals at δC104.3, 103.2, 104.3, 105.7, and 102.2, respectively.
The ring protons of the monosaccharide residues were assigned
starting from the readily identifiable anomeric protons by means
of COSY, TOCSY, NOESY, HSQC, and HMBC experiments (Table 2).
Unitsoffiveß-D-glucopyranosyl(Glc)wereidentified.Therelatively
large3JH−1,H−2values of the anomeric protons of Glc (7.1–7.8 Hz)
indicatedaß-anomericorientation.TheDconfigurationofGlcwas
determined by gaschromatography (GC) analysis.[10]Correlations
observed in the HMBC spectrum between signals at δH 4.72
(GlcI-1) and δC89.5 (C-3) confirmed the substitution at the C-3
position of the aglycone by a ß-D-glucopyranosyl moiety (GlcI).
Signals at δC78.8 (GlcI-2) and δC80.2 (GlcI-4) in comparison with a
terminal ß-D-glucopyranosyl moiety suggested a 2,4 substitution
of GlcI by GlcII and GlcIII, respectively. This is confirmed by cross
peaks in the NOESY spectrum between δH4.27 (GlcI-2) and an
anomeric signal at δH5.44 (GlcII-1), and between δH4.07 (GlcI-
4) and another anomeric signal at δH 4.95 (GlcIII-1) (Fig. 2). In
the HMBC spectrum, the correlation between δH 3.74 (GlcIII-3)
and δC 103.2 (GlcIV-1), and the reverse correlation between δH
(GlcIV-1) and δC 90.1 (GlcIII-3), and the correlation between δH
3.96 (GlcIV-2) and δC105.7 (GlcV-1), revealed the (1 → 3) linkage
between GlcIV and GlcIII and the (1 → 2) linkage between GlcV
and Glc IV (Fig. 2). Moreover, the deshielded signal of GlcV-6 at
δC63.9 and δH4.71, 5.13 showed an acylation at this position.
After subtraction of the signals of the oligosaccharidic chain
linked at the C-3 position, signals of a monoterpenoyl residue
still remained, which acylated the GlcV-6 position (Table 3). Its
NMR data are in accordance with those described in literature[11]
for a (2E,6S)-2,6-dimethyl-6-hydroxyocta-2,7-dienoyl unit, already
foundinacylatedsaponinsisolatedfromplantsoftheMimosaceae
family.[11,12]
Thestructureof1wasthusestablishedas3-O-{6-O-[(2E,6S)-2,6-
dimethyl-6-hydroxyocta-2,7-dienoyl]-β-D-glucopyranosyl-(1
2)-β-D-glucopyranosyl-(1 → 3)-β-D-glucopyranosyl-(1 → 4)-
[β-D-glucopyranosyl-(1 → 2)]-β-D-glucopyranosyl}-3,27-dihydro-
xyoleanolic acid, a new oleanane-type glycoside named
tetrapteroside A.
Compound 2, a white amorphous powder, exhibited in HR-
ESIMS (positive-ion mode) a pseudo-molecular ion peak at m/z
= 1481,6572 [M + Na]+(calcd 1481,6565), consistent with a
molecular formula of C70H106O32Na. Its FABMS (negative-ion
mode) showed a quasi-molecular ion peak at m/z = 1457 [M
− H]−, indicating a molecular weight of 1458. Other significant
fragmentionpeakswereobservedatm/z=1281[(M−H)−176]−,
1119[(M−H)–176−162]−,and957[(M−H)–176−162−162]−,
corresponding to the successive loss of one feruloyl moiety, and
two hexosyl moieties, respectively.
The1H- and13C-NMR signals of 2 assigned from the 2D NMR
spectra were almost superimposable on those of 1 except for
the acyl moiety and the position of acylation (Tables 1, 2 and 3).
Actually, inside the same oligosaccharidic chain, a substitution is
observed at GlcIV-6 position with signals at δC63.9 and δH4.91,
5.18, instead of GlcV-6 position in 1. Moreover, characteristic NMR
signalsofa(E)feruloylunitwerefoundcorrespondingtoprevious
data from the literature (Table 3).[13]
On the basis of the above results, the structure of com-
pound 2 was elucidated as 3-O-{β-D-glucopyranosyl-(1 → 2)-6-
O-[(E)-feruloyl]-β-D-glucopyranosyl-(1 → 3)-β-D-glucopyranosyl-
(1 → 4)-[β-D-glucopyranosyl-(1 → 2)]-β-D-glucopyranosyl}-3,27-
dihydroxyoleanolic acid (2), a new saponin named tetraptero-
side B.
→
Experimental
General
Optical rotations values were recorded on a AA-OR automatic
polarimeter. HR-ESIMS (positive-ion mode) was carried out on a
Q-TOF 1-micromass spectrometer. FABMS were conducted in the
negative-ionmodeonaJeolSX-102instrument.Medium-pressure
liquid chromatography (MPLC) was performed on a Gilson pump
M 305, with B¨ uchi glass column (460 mm × 25 mm and 460 mm
× 15 mm), a B¨ uchi precolumn (110 mm × 15 mm), using silicagel
60 (Merck, 15 −40 µm). Vacuum liquid chromatography (VLC)
was carried out using reversed-phase RP-18 (25 −40 µm) and
silica gel 60 (63 −200 µm) (Merck). TLC and HPTLC employed
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O. P. Not´ e etal.
Table 2.
(δ in ppm, J in Hz)
1H-NMR (600 MHz) and13C-NMR (150 MHz) data of the sugar moietiesof 1 and 2 in pyridine-d5
Compound1
Compound 2
No
1H-NMR
13C-NMR
1H-NMR
13C-NMR
GlcI-1
2
3
4
5
6
GlcII-1
2
3
4
5
6
GlcIII-1
2
3
4
5
6
GlcIV-1
2
3
4
5
6
4.72 d, J = 7.1
4.27 t, J = 8.3
4.23 t, J = 9.3
4.07 d, J = 9.3
3.80
4.04, 4.33
5.44 d, J = 7.6
3.93
4.15 t, J = 9.0
4.03
3.74
4.32, 4.40 dd, J = 11.3, 2.0
4.95 d, J = 7.6
3.94
3.74
3.79
3.90
4.13, 4.33
4.91 d, J = 7.6
3.96
4.22 d, J = 9.3
3.98
3.90
4.11, 4.44 br d, J = 10.2
104.3
78.8
77.1
80.2
75.6
62.4
102.2
74.5
77.4
71.0
77.2
61.5
104.3
74.5
90.1
70.0
78.0
61.9
103.2
84.5
76.9
70.8
78.0
62.0
4.71 d, J = 7.6
4.31
4.20
4.11
3.68
4.06, 4.38
5.50 d, J = 7.4
4.04
4.20
3.85 t, J = 8.1
3.81
4.33, 4.43
5.00 d, J = 7.8
4.00
3.84 t, J = 7.4
4.08
3.95
4.20, 4.40
5.02 d, J = 7.1
4.03
4.27
3.98
4.10
4.91 dd, J = 11.4, 5.9
5.18 dd, J = 11.2, 6.4
5.26 d, J = 7.8
4.10
4.25
4.04
nd
4.15, 4.43
104.1
79.0
77.2
79.9
75.5
62.3
102.3
74.0
77.2
69.8
77.0
61.2
104.1
74.3
89.9
70.5
78.0
61.8
103.0
84.6
76.8
70.5
75.2
63.9
GlcV-1
2
3
4
5
6
5.17 d, J = 7.8
4.03
4.17
3.79
3.99
4.71, 5.13 br d, J = 11.9
105.7
75.9
77.3
70.2
75.9
63.9
105.8
75.2
77.0
70.5
nd
61.8
Overlapped proton NMR signals are reported without designated multiplicity.
Nd, not determined.
precoated silica gel 60F254 plates (Merck). The following TLC
solvent system CHCl3–MeOH−AcOH −H2O (60:32:0.5:6.5) was
used.ThesprayreagentwasKomarowskyreagent,amixture(5:1)
of p-hydroxybenzaldehyde (2% in MeOH) and ethanolic H2SO4
(50%).
Plantmaterial
The stem bark of T. tetraptera was collected at Eloundem, near
Yaound´ e, Cameroon, in November 2006, and identified by Dr P.
Nana, botanist of the National Herbarium of Cameroon (NHC),
Yaound´ e, where a voucher specimen (No. 5567) was deposited.
NMR spectroscopy
The NMR spectra were recorded on a Varian UNITY Inova
600 spectrometer equipped with 5-mm probes. Samples were
dissolved in 160 ml of C5D5N and 20 ml of D2O and transferred
into 5-mm NMR tubes (Shigemi). The1H and13C-NMR spectra (at
600 and 150 MHz respectively) were measured at 303 K. Chemical
shifts are given on the δ scale and referenced to the residual
solvent signals (δH = 7.19, δC = 123.5). Coupling constants (J)
are in Hz. For 2D experiments, Varian software using pulse field
gradient were applied. The pulse conditions in C5D5N were as
follows: for the1H-NMR spectrum, observation frequency (OF)
= 599.88 MHz, acquisition time (AQ) = 4.202 s, relaxation delay
(RD) = 5.0 s, 90 pulse width = 10.0 µs, spectral width (SW) =
7798.8 Hz, Fourier transform (FT) size = 65 536; for the13C-
NMR spectrum, OF = 150.854 MHz, AQ = 0.453 s, RD = 1.547 s,
90 pulse width = 15.8 µs, SW = 36 182.7 Hz, line broadening
(LB) = 1.0 Hz, FT size = 65 536; for the COSY spectrum, AQ =
0.131, F2 = 2048, F1 = 256, RD = 0.369, SW = 7798.8 Hz; for
the NOESY spectrum, AQ = 0.131, F2= 2048, F1= 256, RD =
0.369, SW = 7798.8 Hz, mixing time = 500 ms; for the TOCSY
spectrum, AQ = 0.131, F2= 2048, F1= 256, RD = 0.369, SW =
7798.8 Hz, mixing time = 60 ms; for the HSQC spectrum, AQ =
0.131, RD =0.369, F1= 36 182.7 Hz, F1= 7798.8 Hz; for the HMBC
spectrum, AQ = 0.131, spectra frequency (SF) = 599.880 MHz,
RD = 0.369, delay time (DE) = 50 ms, F1 = 36 182.7 Hz, F1 =
7798.8 Hz.
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Spectral Assignments and Reference Data
Table 3.
in ppm, J in Hz)
1H-NMR (600 MHz) and13C-NMR (150 MHz) data of the monoterpenoylmoiety (MT) of 1 and the feruloyl moiety (FA) of 2 in pyridine-d5(δ
Compound 1
Compound 2
NO
1H-NMR
13C-NMR
1H-NMR
13C-NMR
MT-1
2
3
4
5
6
7
8
9
10
FA-α
β
γ
1
2
3
4
5
6
3-OMe
–
–
168.0
127.6
143.6
23.8
41.0
72.1
145.7
111.6
12.5
27.8
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
7.12 t, J = 7.3
2.33, 2.44
1.70, 1.79
–
6.08 dd, J = 17.4, 10.7
5.11 d, J = 10.9, 5.43 d, J = 17.8
1.93 s
1.42 s
–
–
–
–
–
–
–
–
–
–
167.5
114.8
145.4
127.0
111.0
148.8
150.0
115.5
123.1
55.8
6.69 d, J = 15.7
8.01 d, J = 15.7
–
7.28 s
–
–
7.23 d, J = 8.1
7.25 d, J = 8.1
3.91 s
Overlappedproton NMR signals are reported without designated multiplicity.
Extractionand isolation
The dried powdered stem barks of T. tetraptera (300 g) were
extracted with MeOH (400 ml × 3, 3 h) in a soxhlet. This MeOH
extract was concentrated to dryness and gave a dark residue
(13 g) which was dissolved in water (250 ml) and partitioned with
n-BuOH saturated with water (250 ml × 3). The n-BuOH extract
(6.1 g) was subjected to VLC on RP-18 (25 −40 µm) with H2O
containingincreasingamountsofMeOH.Thefractionselutedwith
H2O–MeOH (5:5) (240 mg) and pure MeOH were combined and
submitted to VLC on silica gel (CHCl3–MeOH−H2O, 60:32:6.5).
Theresultingresidue(1.6 g)wasthensubjectedtosuccessiveMPLC
on silica gel column eluted with CHCl3–MeOH−H2O (60:32:6.5
and 70:30:5) yielding compounds 1 (7.7 mg) and 2 (9.5 mg).
The dried powdered roots of T. tetraptera (300 g) were ex-
tacted according to the same protocol. Then-BuOH extract (3.1 g)
was submitted to successive MPLC on silica gel column eluted
with CHCl3–MeOH−H2O (40:10:1) to afford aridanin (14.7 mg),
3-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)echinocystic acid
(15.5 mg),3-O-[β-D-glucopyranosyl-(1
deoxy-β-D-glucopyranosyl]oleanolic acid (11.6 mg), and 3-O-
sulfoechinocystic acid (8.7 mg).
Tetrapteroside A (1): Amorphous powder. [α]D25−30◦(c =
0.50, MeOH). For1H-NMR and13C-NMR data, see Tables 1–3. HR-
ESIMS m/z = 1471,7090 [M + Na]+(calcd 1471,7085). FABMS m/z
= 1447 [M − H]−, 1281 [(M − H) −166]−, 1119 [(M − H) −166 −
162]−, 957[(M −H)–166 −162−162]−,795[(M− H)–166 −162
− 162 − 162]−.
Tetrapteroside B (2): Amorphous powder. [α]D25−45◦(c =
0.64, MeOH). For1H-NMR and13C-NMR data, see Tables 1–3. HR-
ESIMS m/z = 1481,6572 [M + Na]+(calcd 1481,6565). FABMS
→
6)-2-acetamido-2-
m/z = 1457 [M − H]−, 1281 [(M − H)–176]−, 1119 [(M − H)–176
− 162]−, 957 [(M − H)–176 − 162 − 162]−.
Acid hydrolysis
Two milligrams of each saponin was refluxed with 2-N aqueous
CF3COOH(5 ml)for2 h.AfterextractionwithCH2Cl2(3×5 ml),the
aqueous layer was repeatedly evaporated to dryness with MeOH
until neutral. One glucose was identified by comparison with an
authentic sample on TLC in CHCl3–MeOH−H2O (8:5:1). The D
configurationofglucosewasdeterminedbyGCanalysisusingthe
method previously described.[10]
Acknowledgement
TheauthorsaregratefultoDrP.NanaoftheNationalHerbariumof
Cameroon (NHC) for the identification and collection of the plant.
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