Chevalierinoside a: A new isoflavonoid glycoside from the stem bark of Antidesma chevalieri Beille (euphorbiaceae)

Article abstract of DOI:10.4314/bcse.v28i2.16

Chevalierinoside A (1), a new isoflavonoid glycoside determined as biochanin A 7-O- [α-L-rhamnopyranosyl-1→6-β-D-apiofuranosyl- 1→2-β-D-glucopyranoside], together with the known friedelin (2), friedelan-3β-ol (3) and betulinic acid (4), were isolated from

Full text of DOI:10.4314/bcse.v28i2.16

Bull. Chem. Soc. Ethiop. 2014, 28(2), 309-314.
Printed in Ethiopia
DOI: http://dx.doi.org/10.4314/bcse.v28i2.16
ISSN 1011-3924
 2014 Chemical Society of Ethiopia
SHORT COMMUNICATION
CHEVALIERINOSIDE A: A NEW ISOFLAVONOID GLYCOSIDE FROM THE STEM
BARK OF ANTIDESMA CHEVALIERI BEILLE (EUPHORBIACEAE)
Marie Geneviève Djouossi1, Perrin Lanversin Foning Tebou1, Florence Déclaire Mabou1, David
Ngnokam1*, Leon A. Tapondjou1, Dominique Harakat2 and Laurence Voutquenne-
Nazabadioko3
1Faculty of Science, Department of Chemistry, University of Dschang, P.O. Box 67, Dschang
Cameroon
2Service Commun d’Analyses, Institut de Chimie Moléculaire de Reims (ICMR), CNRS UMR
7312, Bat. 18 B. P. 1039, 51687 Reims Cedex 2, France
3Groupe Isolement et Structure, Institut de Chimie Moléculaire de Reims (ICMR), CNRS UMR
7312, Bat. 18 B. P. 1039, 51687 Reims Cedex 2, France
(Received April 30, 2013; revised December 5, 2013)
ABSTRACT. Chevalierinoside A (1), a new isoflavonoid glycoside determined as biochanin A 7-ꢀ-
[
-L-rhamnopyranosyl-1→6-ꢁ-D-apiofuranosyl-1→2-ꢁ-D-glucopyranoside], together with the
known friedelin (2), friedelan-3 -ol (3) and betulinic acid (4), were isolated from the stem bark of
ꢂꢃꢄꢅꢆꢇꢈꢉꢊꢋ ꢌꢍꢇꢎꢊꢏꢅꢇꢐꢅꢋ Beille. Their structures were established by direct interpretation of their
spectral data, mainly TOF-HRESIMS, 1D-NMR (1H, 13C and DEPT) and 2D-NMR (COSY, ROESY,
TOCSY, HSQC and HMBC), and by comparison with the literature.
KEY WORDS: ꢂꢃꢄꢅꢆꢇꢈꢉꢊꢋꢌꢍꢇꢎꢊꢏꢅꢇꢐꢅ, Euphorbiaceae, Isoflavonoid glycoside, Chevalierinoside A
INTRODUCTION
The genus Antidesma (Euphorbiaceae) contains about 170 species, forest shrubs and trees,
distributed in the world [1, 2]. Antidesma chevalieri Beille (syn. Antidesma laciniatum Muell.
Arg. var. laciniatum) is a tree of about 9 m high encountered in Africa, particularly in
Cameroon, Democratic Republic of Congo and Equatorial Guinea [3], and occupies a prominent
position in traditional African medicine [4]. Previous studies on the genus afforded terpenoids,
steroids, flavonoids [2] and alkaloids [5], some of which possessed various biological activities
[5-7]. Particularly, squalene, (E)-phyt-2-en-1-ol, sitosterol and amentoflavone are the only
compounds previously isolated from the leaves of Antidesma laciniatum Muell. Arg. [2].
Continuing with the phytochemical investigation of plants of the genus Antidesma, we herein
report the isolation and structure elucidation of a novel isoflavonoid glycoside, chevalierinoside
A (1) from the methanol extract of the stem bark of A. chevalieri Beille.
EXPERIMENTAL
Melting points were recorded with a Reichert microscope and are uncorrected. Optical rotations
1
were measured on a Belligham and Stanley ADP 220 polarimeter. H and 13C NMR spectra
were recorded on a Bruker Avance III 600 spectrometer equipped with a cryoplatform (1H at
600 MHz and 13C at 150 MHz). 2D-NMR experiments were performed using standard Bruker
microprograms (Xwin-NMR version 2.1 software). Chemical shifts (
__________
δ) are reported in parts per
*Corresponding author. E-mail: dngnokam@yahoo.fr

310
Marie Geneviève Djouossiꢋꢇꢄꢋꢊꢏꢑ
million (ppm) with the solvent signals as reference relative to TMS (δ = 0) as internal standard,
while the coupling constants (J values) are given in Hertz (Hz). The IR spectra were recorded
with a Shimadzu FT-IR-8400S spectrophotometer. UV spectra were determined as methanol
solution with
a Cary 50 UV/VIS Spectrophotometer. TOF-ESIMS and HR-TOF-ESI
experiments were performed using a Micromass Q-TOF micro instrument (Manchester, UK)
with an electrospray source. The samples were introduced by direct infusion in a solution of
MeOH at a rate of 5 µL min-1. Column chromatography was run on Merck silica gel 60 (70-230
mesh) and gel permeation on Sephadex LH-20 while TLC was carried out on silica gel GF254
pre-coated plates with detection accomplished by spraying with 50% H2SO4 followed by heating
at 100 oC, or by visualizing with an UV lamp at 254 and 365 nm.
Plant material. The stem bark of Antidesma chevalieri Beille was collected at Bansoa, Menoua
Division, West Region of Cameroon, in July 2011. Authentication was done by Mr Victor Nana,
a botanist of the Cameroon National Herbarium, Yaoundé, where a voucher specimen (No.
9667/SRF/Cam) has been deposited.
Extraction and isolation. The dried and powdered plant material (3 kg) was extracted by
percolation with methanol (3 x 10 L, 72 h) at room temperature affording 50 g of crude extract
after evaporation of the solvent under vacuum. A portion (47 g) of this extract was subjected to
silica gel column chromatography eluting with gradient mixtures of n-hexane-EtOAc (10:0, 9:1,
4:1, 7:3, 1:1, 3:7 and 0:10) followed by EtOAc-MeOH (19:1, 9:1, 17:3, 4:1, 1:1 and 0:10). 40
fractions of 300 mL each were collected and combined on the basis of their TLC profiles to give
5 major fractions: F1 (1-8, 12.9 g), F2 (9-16; 7.7 g), F3 (17-27; 2.3 g), F4 (28-35; 3.1 g) and F5
(36-40; 2.2 g). F1 and F2 were respectively purified on silica gel column chromatography eluted
with n-hexane-EtOAc (19:1 and 9:1 for F1, 4:1, 3:2, and 1:1 for F2). 18 sub-fractions of 25 mL
each were collected from F1. Re-crystallization of sub-fractions 7-8 and 10-11 gave friedelin
(20 mg) and friedelan-3β-ol (18 mg) respectively. From F2, 23 sub-fractions of 25 mL each
were collected affording betulinic acid (33 mg) after re-crystallization of sub-fractions 6-8. F4
was passed through sephadex LH-20 column eluted with methanol. 20 sub-fractions of 10 mL
each were collected. Sub-fractions 12-18 (D) were combined and passed through silica gel
column chromatography eluted with EtOAc-MeOH (19:1 and 9:1). Purification of D3
(combined fractions 10-14 from D) through sephadex LH-20 eluted with methanol yielded
compound 1 (31 mg). Attempts of purifications of fractions F3 and F5 failed.
23
Chevalierinoside A (1). Yellowish gum, [α]D - 4o (c 0.53, MeOH). IR (NaCl) νmax (cm-1):
3500-3200 (OH); 1654 (C = O), 1608, 1583 (aromatic). 1H- and 13C-NMR data, see Table 1.
TOF-ESIMS m/z: 1471.5 [2M+Na]+, 747.2 [M+Na]+. TOF-ESIMS-MS 747 m/z: 747.2
[M+Na]+, 615.2 [(M+Na) - 132]+, 469.1 [(M+Na) – 132 - 146]+, 307.1 [(M+Na) - 132 - 146 -
162]+, TOF-HRESIMS m/z: 747.2105 [M+Na]+, calcd. for C33H40O18Na: 747.2112.
Acid hydrolysis and GC-MS analysis. Compound 1 (10 mg) was dissolved in MeOH-2 N HCl
(1:4) (10 mL) and refluxed at 80 °C for 3 h. After removal of MeOH under reduced pressure,
the aqueous layer was extracted with CH2Cl2 (3 x 5 mL). The combined CH2Cl2 extracts were
washed with H2O and evaporated to dryness to afford the aglycon (2 mg) identified as 5,7-
dihydroxy-4’-methoxyisoflavone by comparison of its 1D-NMR data with those of literature [8].
The aqueous layer was neutralized by dilute NaOH. The sugar components were analyzed by co-
TLC with the mixture CHCl3/MeOH/H2O (7:3:0.2). After spraying, apiose gave a weak yellow
spot at Rf 0.78, rhamnose gave a green spot at Rf 0.75 and glucose gave a blue spot at Rf 0.70.
The previous aqueous layer was concentrated to dryness. The residue obtained was
dissolved in pyridine (1 mL), then (CH3)3SiNHSi(CH3)3 (1 mL) was added. After 10 min at
Bull. Chem. Soc. Ethiop. 2014, 28(2)

Short Communication
311
room temperature, the solution was blown to dryness with a stream of nitrogen. The residue was
dissolved in diethyl ether then subjected to GC-MS analysis.
GC-MS experiments were carried out on an MD 800 instrument. Trimethylsilyl ether
derivatives were separated using an HP Ac-5 capillary column (0.25 x 30 m). Nitrogen was used
as the carrier gas. The initial column oven temperature was 180 °C, then increased at 5 °C min-1
to a final value of 240 °C. The sugars were determined by comparison of retention times (tR)
with standard sugars: tR (min) Glc 6.87, Rha 4.32, Api 2.80.
RESULTS AND DISCUSSION
The high resolution time of flight electrospray ionisation mass spectrometry (HR-TOF-ESI-MS)
of compound 1 (Figure 1) exhibited a pseudo-molecular ion peak at m/z 747.2105 [M+Na]+
(calculated. 747.2112) consistent with a molecular formula C33H40O18 and confirmed by 13C
NMR and DEPT analysis. This was in accord with an isoflavone having one hydroxyl, one
methoxyl, and one pentosyl-deoxyhexosyl-hexosyl substitutions. The TOF-ESIMSMS of the
[M+Na]+ ion peak at m/z 747 gave significant fragment ion peaks observed at m/z 615 [(M+Na)-
132]+, 469.1 [(M+Na)-132-146]+, and 307 [(M+Na)-132-146-162]+ corresponding, respectively,
to the successive loss of one pentosyl, one deoxyhexosyl and one hexosyl moieties from the
pseudomolecular ion. The UV spectrum showed two maxima absorption at 300 and 260 nm,
characteristic of isoflavone. The IR spectrum showed absorption bands for hydroxyl (3500-3200
cm-1), carbonyl (1654 cm-1) and aromatic (1608 and 1583 cm-1) functionalities. The NMR
spectral data for the aglycon moiety were in agreement with those of biochanin A [8]. In the 1H
NMR spectrum of 1, signals at
δ 7.93 (H-2, s), 6.56 (H-8, d, J = 2.2 Hz), 6.41 (H-6, d, J = 2.2
Hz), 7.39 (H-2’/H-6’, d, J = 8.7 Hz) and 6.92 (H-3’/H-5’, d, J = 8.7 Hz) indicated substitutions
on carbons 5, 7 and 4’ of the isoflavone. The signal at δΗ 3.78 (s) correlated to the 13C NMR
signal at δC 159.7 in the HMBC spectrum (see Table 1 and Figure 2) suggesting that the
methoxyl group was located at C-4’. After acid hydrolysis of 1, glucose, apiose and rhamnose
1
were detected by TLC and the aglycon was identified (ESIMS, H- and 13C- NMR) as 5,7-
dihydroxy-4’-methoxyisoflavone (biochanin A) [8]. The 1H NMR spectrum of 1 displayed three
sugar anomeric protons at
δ
correlations with three anomeric carbon atoms at
4.88 (d, J = 7.7 Hz), 4.69 (brs) and 5.33 (d, J = 1.6 Hz) giving
99.2, 100.4 and 109.5, respectively in the
δ
HSQC spectrum (Table 1) confirming that this compound contains three sugar units. Complete
assignments of each sugar proton system were achieved by analysis of H-1H COSY, ROESY
and TOCSY spectra (see the main correlations on Figure 3) while carbons were assigned from
1
HSQC and HMBC spectra. Starting from the anomeric proton at
rhamnopyranosyl (Rha) with its methyl signal observed at H 1.18 (d, J = 6.0 Hz) was identified
after evaluation of the spin-spin couplings and carbon chemical shifts. Starting from the
anomeric proton at 4.88 (d, J = 7.7 Hz), a seven spin system was identified corresponding to
an inner -glucopyranosyl (Glc) unit. The deshielding signals of carbons C-2’’ ( C 77.6) and C-
6’’ ( C 66.5) suggest a disubstitution of this unit. From the last anomeric signal at 5.33 (d, J =
1.6 Hz), the structure of one terminal -apiofuranosyl (Api) was elucidated. The analysis of
COSY spectra showed correlations between proton H-1’’’’ and proton H-2’’’’ ( 3.91, d, J =
δH 4.69 (brs), a terminal α-
δ
δ
β
δ
δ
δ
β
δ
1.6 Hz) and between protons of two isolated AB system, one at 3.80 and 3.90 (d, J = 9.8 Hz, H-
4’’’’) and the second at 3.53 (s, 2H, H-5’’’’). In the HMBC spectra the proton H-1’’’’ was
correlated with the methylene C-4’’’’ (δC 74.2) and a quaternary carbon C-3’’’’ (δC 79.1), the
proton H-2’’’’ was also correlated with carbons C-3’’’’ and C-4’’’’ and carbons C-2’’’’, C-3’’’’
and C-4’’’’ were correlated with the methylene H-5’’’’ (δH 3.53). The rOe effect observed
between H-2’’’’ and H-5’’’’ confirmed the structure of the apiofuronosyl moiety [8]. The sugar
units were confirmed by thin layer chromatography after hydrolysis. The D-configuration for
Bull. Chem. Soc. Ethiop. 2014, 28(2)

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Marie Geneviève Djouossiꢋꢇꢄꢋꢊꢏꢑ
glucose and apiose, and L-configuration for rhamnose were suggested by comparison of the
NMR data of 1 with those of related compounds (including biochanin A 7-O- -L-
rhamnopyranosyl-1→6- -D-glucopyranoside and biochanin A 7-O- -D-apiofuranosyl-1→5-
-D-glucopyranoside) [9-12], and were confirmed by GC-MS after
α
β
β
β-
D-apiofuranosyl-1→6-
β
derivatization.
Table 1. 1H (600 MHz) and 13C (150 MHz) NMR data, and HMBC correlations of compound 1, in CD3OD.
δ 13
C
δ
1H (mult , J)
7.93 (s)
No
2
HMBC (H → C)
3, 4, 9, 1’
153.5
123.6
181.0
162.0
100.1
162.9
95.1
3
4
5
6
6.41 (d, 2.2)
6.56 (d, 2.2)
5, 7, 8, 10
6, 7, 9, 10
7
8
9
157.7
107.1
122.8
130.1
114.0
159.7
114.0
130.1
55.3
10
1’
2’
7.39 (d, 8.7)
6.92 (d, 8.7)
3, 4’
3’
1’, 4’, 5’
4’
5’
6.92 (d, 8.7)
7.39 (d, 8.7)
3.78 (s)
1’, 4’, 3’
3, 4’
6’
4’-OMe
Glc
1’’
2’’
3’’
4’’
5’’
6’’
4’
99.2
77.6
77.0
70.0
75.6
66.5
4.88 (d, 7.7)
3.62 (m)
7, Glc-3’’, Glc-5’’
Glc-1’’, Api-1’’’’
Glc-1’’, Glc-5’’
3.54 (m)
3.35 (t, 9.1)
3.51 (m)
Glc-3’’, Glc-5’’, Glc-6’’
4.00 (d, 9.2)
3.53 (d, 9.2)
Glc-4’’, Rha-1’’’
Glc-4’’, Rha-1’’’
Rha
1’’’
2’’’
3’’’
4’’’
5’’’
6’’’
Api
1’’’’
2’’’’
3’’’’
4’’’’
100.4
70.4
71.3
72.9
68.3
17.2
4.69 (brs)
Glc-6’’, Rha-2’’’, Rha-3’’’, Rha-5’’’
Rha-3’’’, Rha-4’’’
3.87 (dm, 3.7)
3.73 (dd, 9.5, 3.7)
3.30 (t, 3.5)
3.60 (m)
Rha-4’’’, Rha-5’’’
Rha-3’’’, Rha-5’’’, Rha-6’’’
1.18 (d, 6.0)
Rha-4’’’, Rha-5’’’
109.5
76.6
79.1
74.2
5.33 (d, 1.6)
3.91 (d, 1.6)
Glc-2’’, Api-3’’’’, Api-4’’’’
Api-4’’’’
3.80 (d, 9.9)
3.90 (d, 9.9)
3.53 (s)
Api-1’’’’, Api-2’’’’, Api-3’’’’, Api-5’’’’
Api-1’’’’, Api-2’’’’, Api-3’’’’, Api-5’’’’
5’’’’
65.0
In the HMBC spectrum correlation observed between H-1’’ (
162.9) of the aglycon indicated that Glc was linked at C-7 of the aglycon. Furthermore the
HMBC correlations observed between H-1’’’’ ( 5.33) of Api and C-2’’ ( 77.6) of Glc and
between H-1’’’ ( 4.69) of Rha and C-6’’ ( 66.5) of Glc established the connectivities between
δ 4.88) of Glc and C-7 (δ
δ
δ
δ
δ
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313
Api and Glc and between Glc and Rha (see Table 1 and Figure 2). These were confirmed by
ROESY correlations (see Figure 3) observed between H-2’’ ( 3.63) of Glc and H-1’’’’ ( 5.33)
of Api, and between H-1’’’ ( 4.69) of Rha and 2H-6’’ ( 4.00/3.53) of Glc. Hence, the
oligosaccharide at C-7 of the aglycon was established as O-[ -L-rhamnopyranosyl-1→6- -D-
apiofuranosyl-1→2- -D-glucopyranoside]. Thus, the structure of compound 1 was established
as biochanin 7-O-[ -L-rhamnopyranosyl-1→6- -D-apiofuranosyl-1→2- -D-
glucopyranoside] a new isoflavonoid glycoside named chevalierinoside A.
δ
δ
δ
δ
α
β
β
A
α
β
β
Structures of compounds 2-4 (Figure 1) were determined by means of Co TLC,
spectroscopic data, and by comparative analysis of their physical and spectral data with those
reported in the literature for friedelin [13], friedelan-3
respectively.
6''
β
-ol [13] and betulinic acid [14],
5''
2''
4''
5'''
3'''
6'''
4'''
1'''
3''
1''
2'''
4''''
3''''
1''''
2''''
5''''
29
30
30
20
29
20
21
27
12
8
18
17
28
25
26
1
COOH
28
14
26
16
10
2
25
3
5
27
3
6
R
HO
24
2 R = O
3 R = αH, βOH
4
23
24
23
Figure 1. Structures of the isolated compounds.
H3C
OH
H
H
O
HO
HO
O
H
H
H
O
HO
HO
H
H
O
O
H
H
H
O
O
H
H
H
H
H
H
OH
O
OMe
H
HO
OH
OH
H
Figure 2. Important HMBC correlations of compound 1.
Bull. Chem. Soc. Ethiop. 2014, 28(2)

314
Marie Geneviève Djouossiꢋꢇꢄꢋꢊꢏꢑ
H
OH
H
H
O
HO
HO
O
H
H
O
HO
H
H
O
O
H
HO
H
O
O
H
H
H
H
OH
O
OMe
H
HO
OH
OH
H
Figure 3. Key ROESY correlations of compound 1.
ACKNOWLEDGMENTS
The authors are grateful to the University of Dschang for financing some consumables used in
this work.
REFERENCES
1. Pax, F.; Hoffmann, P. Euphorbiaceae-Phyllanthoideae-Phyllanthae in: Das
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(Ed.), Crown Agents: London; 1958; pp 374-375.
4. Schmelzer, G.H. Antidesma laciniatum Mull. Arg. in: Ressources végétales de l’Afrique
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13.
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Bull. Chem. Soc. Ethiop. 2014, 28(2)

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