Clerodane and 19-norclerodane diterpenoids from the tubers of Dioscorea antaly

Article abstract of DOI:10.1016/j.phytochem.2010.03.014

Two clerodane diterpenoids, antadiosbulbins A and B and two 19-norclerodane diterpenes, 8-epidiosbulbins E and G along with the known diosbulbin E as well as nine known phenolics including five phenanthrenes and stilbenes and four flavonoids were isolated from the ethyl acetate soluble part of the methanolic extract of the tubers of Dioscorea antaly, a yam endemic to Madagascar. Structures were determined by analysis of the spectral data, mainly 2D-NMR and mass spectrometry.

Full text of DOI:10.1016/j.phytochem.2010.03.014

Phytochemistry 71 (2010) 1007–1013
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Clerodane and 19-norclerodane diterpenoids from the tubers of Dioscorea antaly
a
a
b
Lolona Rakotobe a,b, Lengo Mambu a, , Alexandre Deville , Lionel Dubost , Victor Jeannoda ,
*
Danielle Rakoto b, Bernard Bodo a,
**
a FRE 3206 CNRS-MNHN, Molécules de Communication et Adaptation des Micro-organismes, Muséum National d’Histoire Naturelle, 63 rue Buffon, 75005 Paris, France
b Département de Biochimie Fondamentale et Appliquée, Faculté des Sciences, Université d’Antananarivo, Madagascar
a r t i c l e i n f o
a b s t r a c t
Article history:
Two clerodane diterpenoids, antadiosbulbins A and B and two 19-norclerodane diterpenes, 8-epidiosbul-
bins E and G along with the known diosbulbin E as well as nine known phenolics including five phenan-
threnes and stilbenes and four flavonoids were isolated from the ethyl acetate soluble part of the
methanolic extract of the tubers of Dioscorea antaly, a yam endemic to Madagascar. Structures were
determined by analysis of the spectral data, mainly 2D-NMR and mass spectrometry.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 27 August 2009
Received in revised form 11 March 2010
Available online 10 April 2010
Keywords:
Dioscorea antaly
Dioscoreaceae
19-Norclerodane
Clerodane
Diterpenoid
NMR
15
15
1. Introduction
O
O
14
16
14
16
Dioscorea antaly Jum. and H. Perrier (Dioscoreaceae) is a liana
endemic to Madagascar and found in the West and North-West re-
gions. In times of scarcity, its tubers are used as food after prere-
quisite detoxification to remove bitters and toxic principles. As a
part of our research program devoted to chemical knowledge and
possible commercial use of Madagascar yams, we have chemically
investigated this species. The present article describes the isolation
from the EtOAc soluble part of the methanol extract of the tubers,
and structure determination of the four new diterpenoids, two
clerodanes, antadiosbulbins A (1) and B (2), and two furanoid 19-
norclerodanes, 8-epidiosbulbin E (3) and 8-epidiosbulbin G (4) to-
gether with the complete 1H and 13C NMR assignments for the
known diterpenoid diosbulbin E (5) (Ida et al., 1978a). In addition,
nine known phenolics were isolated, which were five stilbene or
phenanthrene derivatives, i.e. 3,7-dihydroxy-2,4-dimethoxyphe-
nanthrene (Leong et al., 1997), (E)-piceatannol (Brinker and Seigler,
1991), 3,4,30,50-tetrahydroxy-dihydrostilbene or dihydropiceatan-
nol (Mannila et al., 1993; Matsuda et al., 2001), the latter being ob-
tained for the first time from a natural source, cassigarol D (Baba
13
12
13
12
H
H
20
H32C0
H
H3C
11
11
O
O
H
1
17
1
17
9
9
H
H
8
8
2
O
O
10
2
3
O
10
H
H
19
O
O
5
5
3
7
7
4
4
6
6
H
H
OH
HO
18
18
21
H
OCH3
O
O
Antadiosbulbin A (1): 8α-H
Antadiosbulbin B (2): 8β-H
8-Epidiosbulbin E (3): 8β-H
Diosbulbin E (5): 8α-H
15
O
14
11
16
13
12
H
20
H3C
O
H
1
H
17
9
HO
8
O
et al., 1992), scirpusin
flavonoids, catechin (Davies et al., 1996), 3-O-[b-
(6 ? 1)- -rhamnose]-kaempferol (Kartnig and Bucar-Stachel,
1991; Markham et al., 1978), kaempferol and quercetin.
B
(Nakajima et al., 1978), and four
2
10
H
D
-glucose-
5
3
7
a
-L
6
4
H
H
H
O
O
18
* Corresponding author. Tel.: +33 (0)1 40 79 31 29; fax: +33 (0)1 40 79 31 35.
** Corresponding author. Tel.: +33 (0)1 40 79 31 29; fax: +33 (0)1 40 79 31 35.
E-mail addresses: mambu@mnhn.fr (L. Mambu), bodo@mnhn.fr (B. Bodo).
8-Epidiosbulbin G (4): 8β-H
Diosbulbin G (7): 8α-H
0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2010.03.014

1008
L. Rakotobe et al. / Phytochemistry 71 (2010) 1007–1013
Table 1
NMR data for antadiosbulbins A (1) and B (2) (400 MHz for 1H).
Position Antadiosbulbin A (1) in CD3OD
Antadiosbulbin A (1) in CDCl3
Antadiosbulbin B (2) in CD3OD
Antadiosbulbin B (2) in CDCl3
dC
dH
Mult. J (Hz)
dC
dH
Mult. J (Hz)
dC
dH
Mult. J (Hz)
dC
dH
Mult. J (Hz)
1a
28.1
2.32 dddd 13.4; 11.2;
27.1
2.31
m
28.0
2.34 dddd 13.5; 11.2;
27.1
2.27
m
5.2; 2.1
5.2; 2.2
b
2
3a
b
4
5
1.63 dd
4.88 dd
13.4; 8.2
5.2; 5.2
14.8
1.57 dd
4.90 dd
2.70 ddd
13.3; 8.8
5.2; 5.2
14.9; 1.1; 1.1
1.62 dd
4.92 dd
13.5; 8.3
5.2; 5.2
15.0
1.57
4.90 dd
2.50 dd
2.31
–
–
m
74.3
40.2
72.1
38.6
74.1
39.8
71.8
38.2
5.2; 5.2
15.0; 0.7
2.77
d
2.76
d
2.13 ddd
–
–
14.8; 5.2; 2.1
2.26
–
m
2.16 ddd
–
–
15.0; 5.2; 2.2
m
76.5
53.4
24.1
75.9
52.2
23.1
77.0
53.3
21.6
76.1
51.8
20.5
–
6eq
2.62 ddd
15.1; 3.9; 3.0
15.1; 14.5; 5.0
2.34
m
m
2.40 dddd 14.8; 3.5;
2.10
m
m
3.5; 0.8
14.8; 14.2; 4.0
ax
2.06 ddd
2.14
1.90 ddd
2.10
7eq
19.7
1.91 dddd 14.8; 5.0; 3.0;
18.3
2.04 dddd 14.2; 4.8; 3.2; 19.9
2.30 dddd 14.3; 4.0;
3.5; 3.3
1.75 dddd 14.3; 14.2;
4.5; 3.5
19.0
2.39 dddd 14.0; 3.8;
3.0
1.56 dddd 14.8; 14.5;
12.4; 3.9
3.2
1.44 dddd 14.2; 14.2;
12.0; 3.2
3.8; 3.8
ax
1.55
m
m
m
8
9
10
11eq
ax
48.0
37.2
47.8
44.0
2.78 dd
–
12.4; 3.0
46.7
36.0
46.2
43.6
2.48 dd
–
12.0; 3.2
48.1
36.3
38.8
42.2
2.53 dd
–
3.5; 3.3
47.3
35.0
37.1
41.7
2.36
–
2.11
2.20 dd
2.07 dd
1.86 dd
5.50 dd
–
11.2; 8.2
14.5; 6.3
14.5; 12.0
12.0; 6.3
2.09
m
2.08 dd
2.09 dd
1.86 dd
5.48 dd
–
11.2; 8.3
14.8; 3.8
14.8; 12.5
12.5; 3.8
1.94 dd
1.83 dd
5.33 dd
–
14.1; 6.4
14.1; 11.0
11.0; 6.4
2.03 dd
1.75 dd
5.27 dd
–
14.8; 3.5
14.8; 12.3
12.3; 3.5
12
13
71.6
125.9
69.9
124.0
72.3
126.5
70.2
124.6
14
15
16
109.7 6.48 dd
145.1 7.48 dd
141.6 7.56 ddd
1.8; 0.8
1.8; 1.6
1.6; 0.9; 0.8
108.4 6.37 dd
143.8 7.40 dd
139.6 7.42 ddd
1.9; 0.9
1.9; 1.7
1.7; 0.9; 0.9
109.6 6.53 dd
144.9 7.48 dd
141.5 7.59 ddd
1.9; 0.9
1.9; 1.7
1.7; 0.9; 0.8
108.3 6.38 dd
143.7 7.39 dd
139.8 7.43 ddd
1.9; 0.9
1.9; 1.7
1.7; 0.9
17
18
19
20
OMe
4-OH
176.7
174.0
177.2
20.6
53.3
–
–
–
–
0.90
3.80
–
172.9
173.9
172.1
20.2
53.5
–
–
–
–
0.83
3.83
3.45
174.5
174.0
177.0
24.0
53.4
–
–
–
–
0.98
3.82
–
171.0
173.6
172.3
24.1
53.4
–
–
–
s
s
s
s
s
s
s
0.98
3.85
3.44
s
s
s
in Fig. 1, and further assembled from the cross-peaks observed in
the HMBC spectrum. The C@O signal at dC 174.0 (C-18) was as-
signed as a methyl ester at C-18 because of the correlations with
2. Results and discussion
The dried and ground tubers of D. antaly were defatted with
cyclohexane and then extracted with MeOH at room temperature.
After concentration to dryness, the MeOH extract was partitioned
between EtOAc and water. The EtOAc soluble part on fractionation
by a combination of column and MPL chromatographies on silica
gel, C-18 reverse phase and Sephadex LH-20, led to the isolation
of the four new compounds antadiosbulbins A (1, 3 mg) and B (2,
3 mg), 8-epidiosbulbins E (3, 534 mg) and G (4, 26 mg), together
with the known diosbulbin E (5, 16 mg) and the nine phenolic
substances.
4
protons at dH 2.77 (H-3eq) and 4.88 (H-2; JH–C W coupling) and
the methoxyl at 3.80 (CH3-21) pointing C(O)-4 at dC 76.5. Similarly
the C@O at dC 177.2 was assigned as C-19 due to the correlations
with protons at dH 2.62 and 2.06 (CH2-6) and 2.20 (CH-10), and
the correlation with the oxymethine proton at 4.88 (H-2) indicated
the lactone ring closure to C-2. The second lactone C@O at dC 176.7
was assigned to C-17 because it correlated with protons at dH 1.91,
1.56 (CH2-7) and 2.78 (H-8), while its correlation with the proton
at dH 5.50 (H-12) indicated the second lactone ring closure to
C-12. The protons of the methyl at dH 0.90 (C-20) were strongly
correlated with carbons at dC 48.0 (C-8), 37.2 (C-9), 47.8 (C-10)
and 44.0 (C-11) indicating this methyl to be linked to C-9, hence
sub-structures a–c could be assembled as shown in Fig. 1. The fur-
an group was linked to C-12, because the quaternary carbon of this
ring at dC 125.9 (C-13) correlated with the protons at dH 5.50
(H-12) and 1.86 (H-11ax). Other HMBC data confirmed this assem-
blage of sub-structures (a–d) to form the proposed planar structure
for 1 (Fig. 1). This structure was confirmed by analysis of the
1D- and 2D-NMR spectra of 1 in solution in CDCl3 and especially
the linkage of the free hydroxyl group at dH 3.45 to C-4 (Table 1).
The large coupling constant of H-12 with H-11ax (3J = 11.0 Hz),
indicated they were in a trans relationship on the half-chair C-ring.
A trans-diaxial disposition was observed for H-8, which showed a
large coupling (3J = 12.4 Hz) with H-7ax and a gauche coupling
(3J = 3.0 Hz) with H-7eq. The large coupling constant of H-10
(3J = 11.2 Hz) with one of the CH2-1 protons indicated it was axial
on the B-ring. Methine H-2 gave gauche couplings with only one
proton of each of its vicinal methylenes at C-1 (H-1a, at lower
fields) and C-3 (H-3b, at higher fields) and in addition a W coupling
(2.1 Hz) was observed between these two protons (Table 1). These
Compound 1 was an optically active ½a D20
ꢁ45° (c 0.7, CHCl3)
ꢀ
colourless amorphous solid. Its HR-ESI-MS showed the protonated
molecular ion [M+H]+ at m/z 405.1519 (calc. for C21H25O8:
405.1548) a molecular formula indicative of ten degrees of unsat-
uration. The IR spectrum of 1 displayed absorption bands at 1740
(–COO–), 3447 (OH), and 3150 and 1506 cmꢁ1 suggesting the pres-
ence of a furan ring. The 13C J-modulated NMR spectrum (CD3OD)
of 1 exhibited the resonances of 21 carbons consisting of one
methyl, one methoxyl at dC 53.3, five methylenes, seven methines
including three sp2 carbons at dC 109.7, 141.6 and 145.1, seven qua-
ternary carbons including three carbonyls at dC 174.0, 176.7 and
177.2 and one ethylenic carbon at 125.9 (Table 1). Taking into
account the ten degrees of unsaturation, compound 1 should in-
clude five rings. The 1H NMR spectrum (CD3OD) displayed typical
signals of a methyl singlet at dY 0.90, the methoxyl of an ester at
dY 3.80, three ethylenic protons at dY 7.56, 7.48 and 6.48 and two
sp3 oxymethine protons at dY 4.88 and 5.50 (Table 1).
Detailed analysis of the 1H–1H COSY and HSQC spectra estab-
lished the presence of the four sub-structures: a (˜CH–CH2–
CH(O)–CH2–), b (–CH2–CH2–CH—), c (–CH2–CH(O)–) and d (a
monosubstituted furan ring) which are marked with bold bonds

L. Rakotobe et al. / Phytochemistry 71 (2010) 1007–1013
1009
a
-axial as in 1. This equatorial disposition of H-8 on the b-face
O
d
was confirmed from the NOESY spectrum where this proton did
not give cross-peak with H-12, but gave a strong one with the
methyl CH3-20 which was also correlated with H-1b, H-3a, H-7ax
(Fig. 3). The other NOE data indicated for the various chiral centers
the same relative configurations as for antadiosbulbin A. With a
cis-fused A(cyclohexane)/B-ring and a cis-fused B/C-ring the struc-
ture of 2 was established as methyl 15,16-epoxy-4-hydroxyclero-
13(16),14-diene-17,12;19,2-diolide-18-carboxylate and the name
antadiosbulbin B was proposed for this new compound 2, which
is thus the 8-epimer of antadiosbulbin A.
D
H
c
H3C
O
C
H
O
a
O
H
B
O
O
A
b
Compound 3 was a colourless amorphous solid, ½a D20
ꢁ16° (c 0.3,
ꢀ
HO
CHCl3), with the molecular formula C19H22O6 based on the proton-
ated molecular ion [M+H]+ at m/z 347.1485 (calc. 347.1493 for
C19H23O6) indicative of nine degrees of unsaturation. Detailed anal-
yses of the 2D-NMR spectra allowed full assignment of protons and
carbons and indicated that 3 was a furanoid 19-norclerodane
derivative (Table 2). The spin systems observed on the 1H–1H COSY
spectrum (Fig. 4) were connected from the HMBC spectrum to form
the norclerodane nucleus and determine the functionalities loca-
tion. From the NOE data and 1H–1H vicinal coupling constants, pro-
ton H-2 was b-equatorial due to its gauche J values (JH2–H1eq = 4.9
and JH2–H3eq = 5.5 Hz) and H-4 was also b-equatorial because of
its small coupling constants (J = 5.1 and 1.1 Hz) with H-3eq and
H-5ax on the A-ring which is now a chair (Fig. 5). The coupling
of H-5 with H-10 (J = 12.5 Hz) and the lack of NOE interaction be-
tween them indicated they were in a trans-diaxial disposition and
hence the junction between the A- and B-rings was trans. Proton H-
OCH3
Fig. 1. Sub-structures (a–d), key COSY (bold bonds) and HMBC (arrows) correla-
tions for antadiosbulbin A (1).
data, together with the NOEs observed in both solvents (CDCl3 and
CD3OD) between a-face protons (H-8 with H-7eq, H-10 and H-12;
H-10 with H-6ax), or the b-face protons (CH3-20 with H-1b, H-3a,
H-7ax, H-11eq and H-14) whereas H-6eq was correlated with the
hydroxyl group at C-4, and the OCH3-21 with H-3a, H-7ax and
CH3-20, led to the relative configuration proposed in Fig. 2. Hence
the structure (relative configuration) of 1 was established as
methyl 15,16-epoxy-4-hydroxyclero-13(16),14-diene-17,12;19,2-
diolide-18-carboxylate and the name antadiosbulbin A was pro-
posed for this novel furano-clerodane with two d-lactone rings
bridging carbonyl C-19 to C-2 and carbonyl C-17 to C-12.
12 was a-axial because of its large coupling constant (J = 12.3 Hz)
Antadiosbulbin B (2) was an optically active ½a D20
ꢁ28° (c 0.4,
ꢀ
with b-axial H-11. The small coupling constants of H-6 and H-8
with the two protons at CH2-7 indicated that they were both b-
equatorial. Finally NOEs were observed between b-face protons
(H-8 with CH3-20, H-7ax and H-11ax; CH3-20 with H-5 and H-
1ax; H-1ax with H-3ax; H-6 with H-4 and H-5), and between a-
face protons H-10 and H-12 (Fig. 5). The structure was confirmed
CHCl3) colourless amorphous solid. It depicted the same molecular
formula C21H24O8 as antadiobulbin A (1) and had similar IR and
NMR (in CD3OD as well as in CDCl3) spectra, suggesting they were
isomers (Table 1). Analysis of the COSY spectrum of 2 led to the
same sub-structures a–d as 1 (Fig. 1). When compared with those
of antadiosbulbin A, the chemical shifts of carbons C-6, C-9, C-10,
by analysis of the 2D-NMR spectra in C5D5N (Table 2). Hence com-
pound
3 was 15,16-epoxy-6a-hydroxy-19-nor-clero-13(16),14-
C-11 and C-17 of 2 (in CDCl3) were shifted upfield, with
ꢁ1.0, ꢁ9.1, ꢁ1.9 and ꢁ1.9 ppm, whereas those of C-7, C-8 and C-20
were shifted downfield with dC +0.6, +1.6 and +3.9, respectively
(Table 1), with the upfield shift of C-10 being notable. The second
main distinction between the two compounds were the coupling
constants of H-8 (CD3OD), one large and one small for 1 (3J = 12.4
and 3.0 Hz) and two small for 2 (3J = 3.5 and 3.3 Hz) due to its b-
equatorial disposition with respect to the B-ring in 2, instead of
D
dC = ꢁ2.6,
diene-17,12;18,2-diolide, the epimer at C-8 of diosbulbin E (5), a
substance previously isolated from Dioscorea bulbifera L. (forma
spontanea Makino et Nemoto) (Ida et al., 1978a), and thus named
8-epidiosbulbin E. Acetylation of 3 by acetic anhydride in pyridine
afforded in good yield the mono-acetate 6, which had spectral data
D
identical with those of 8-epidiosbulbin
E
acetate previously
21
18
20
15
3
4
9
18
3
D
11
1
A
2
14
10
16
20
4
B
5
7
8
13
C
12
16
1
6
2
13
9
10
A
5
12
19
11
B
D
7
17
8
6
C
19
15
14
Fig. 2. Selected NOESY correlations (blue lines) and proposed 3D-structure for
antadiosbulbin A (1). (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
Fig. 3. Selected NOESY correlations (blue lines) and proposed 3D-structure for
antadiosbulbin B (2). (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)

1010
L. Rakotobe et al. / Phytochemistry 71 (2010) 1007–1013
Table 2
NMR data for 8-epidiosbulbin E (3) and diosbulbin E (5) (400 MHz for 1H).
Position
8-Epidiosbulbin E (3) in CDCl3
8-Epidiosbulbin E (3) in C5D5N
Diosbulbin E (5) in CDCl3
dC
dH
Mult.
J (Hz)
dC
dH
Mult.
J (Hz)
dC
dH
Mult.
J (Hz)
1eq
ax
2
3eq
ax
4
5
6
7eq
ax
8
28.2
2.10
1.45
4.85
2.52
1.77
2.65
1.80
4.14
2.75
1.88
2.25
–
2.71
2.08
1.74
5.49
–
6.40
7.37
7.45
–
–
1.10
2.30
dddd
ddd
dd
dddd
d
brdd
ddd
ddd
ddd
ddd
dd
13.3; 6.0; 4.9; 1.7
13.3; 12.2; 0.9
5.5; 4.9
11.6; 5.5; 5.1; 1.7
11.6
28.4
1.97
1.31
4.73
2.32
1.62
2.63
1.75
4.19
2.99
1.91
2.41
–
3.09
2.04
1.80
5.56
–
6.53
7.56
7.59
–
–
0.98
5.69
dddd
ddd
dd
dddd
d
12.3; 6.0; 5.0; 1.5
12.3, 12.1; 1.2
5.5; 5.0
11.5; 5.5; 5.5; 1.5
11.5
28.5
2.12
1.42
4.92
2.53
1.78
2.68
1.72
4.23
2.14
1.75
3.20
–
2.39
2.13
1.77
5.36
–
6.39
7.40
7.44
–
–
0.98
2.35
brd
13.2
13.2; 12.5; 0.7
5.3; 5.0
11.7; 5.5; 5.3; 1.9
11.7
5.4; 1.5
12.4; 2.0; 1.5
2.6; 2.6; 2.0
14.6; 2.6
14.6; 12.2; 2.6
12.2; 3.6
ddd
brdd
dddd
brd
brdd
ddd
ddd
brdd
ddd
dd
78.0
38.3
77.6
38.6
78.4
38.6
43.3
42.7
69.4
29.7
5.1; 1.1
43.6
43.4
68.8
31.2
dd
5.5; 1.8
43.6
42.9
69.4
28.9
12.5; 2.0; 1.1
2.5; 2.2; 2.0
14.7; 3.2; 2.2
14.7; 6.3; 2.5
6.3; 2.1
ddd
brdddd
ddd
ddd
dd
12.1; 1.8; 1.5
3.4; 2.2; 1.5; 1.5
14.2; 3.4; 2.0
14.2; 6.5; 2.2
6.5; 2.0
45.5
34.6
29.7
39.6
45.9
34.8
30.1
39.5
41.0
35.9
39.0
42.3
9
10
11eq
ax
12
13
14
15
16
17
18
20
OH
ddd
dd
dd
12.5; 12.2; 6.0
15.4; 3.1
15.4; 12.3
12.3; 3.1
ddd
dd
dd
12.1; 12.1; 6.0
14.6; 3.1
14.6; 12.6
12.6; 3.1
ddd
dd
dd
12.5; 12.4; 5.5
15.0; 6.1
15.0; 11.2
11.2; 6.1
69.4
dd
69.5
dd
70.0
dd
125.0
108.5
143.5
139.7
171.8
179.2
22.0
126.4
109.4
143.9
140.4
171.8
178.3
21.9
124.2
108.5
143.7
139.6
174.2
180.0
18.3
dd
dd
ddd
1.8; 0.9
1.8; 1.7
1.7; 0.9; 0.5
dd
brs
brs
1.9; 0.8
dd
dd
dd
1.7; 1.0
1.7; 1.7
1.7; 1.0
s
brs
s
s
s
brs
–
–
–
O
for 5 has so far appeared in the literature and we now report its
complete 1H and 13C NMR assignments (Table 2). Compared with
8-epidiosbulbin E (3), C-7, C-8 and C-20 of 5 (in CDCl3) were shifted
upfield by ꢁ0.8, ꢁ4.5 and ꢁ3.7 ppm, whereas C-9, C-10, C-11 and
C-17 were shifted downfield by +1.3, +9.3, +2.7 and +2.4 ppm
d
H
c
H3C
H
O
(Table 2). Again, a change from H-8b to H-8a results in a shift of
H
a
about dC +10 ppm for C-10. The NMR data for 6-O-coumaroyl deriv-
ative of diosbulbin E recently isolated from D. bulbifera, are in
agreement with the above observation (Wang et al., 2009). Thus
3 is the 8b-epimer of diosbulbin E (5).
O
H
O
b
H
OH
Compound 4, another optically active colourless amorphous so-
H
lid, ½a 2D0
ꢁ20° (c 0.3, CHCl3) with molecular formula C19H22O6
ꢀ
H
exhibited the protonated molecular ion [M+H]+ at m/z: 347.1403
(calc. 347.1493 for C19H23O6). All the 2D-NMR data indicated that
4 had the same planar structure as diosbulbin G (7) (Ida et al.,
1978b). The junction of the A- and B-chair rings was trans due to
O
Fig. 4. Sub-structures (a–d) and key COSY (bold bonds) and HMBC (arrows)
correlations for 8-epidiosbulbin E (3).
the trans-diaxial disposition of H-5 and H-10 (3JH-5/H-10
=
12.2 Hz). NOEs were depicted between equatorial H-2 and its gem-
inal-hydroxyl and its vicinal protons H-1eq, H-1ax, H-3eq and
H-3ax (Fig. 6). NOEs were also observed between b-face protons
(H-6 with H-4eq, H-5ax and H-7ax; H-4eq with H-5ax and
20
11
9
H-3ax; CH3-20 with H-1ax, H-5, H-8) and between
(H-12 with H-10). Thus compound 4 differed from diosbulbin G in
the configuration of C-8, H-8 being b in 4 instead of
. The full 13
a-face protons
1
3
2
16
a
C
B
C
10
8
A
5
NMR assignment of diosbulbin G (7) in Pyr-d5, has been published
by Ternai and co-workers (Lentini et al., 1986). It is interesting that
the chemical shift of C-10 in 4 where H-8 is b, is more than 10 ppm
12
7
D
4
15
13
6
17
14
downfield (
D
dC +13.5) from its shift in diosbulbin G where it is
a.
18
The b-orientation of H-8, which implies the
a-orientation of the
C-17 carbonyl group in 8-epidiosbulbin G (4), induces steric con-
straints which are responsible for this shift, is also true for anta-
diosbulbin B and 8-epidiosbulbin E. Compound 4 may by thought
of as an isomer of 8-epidiosbulbin E (3) from which it differs in
Fig. 5. Selected NOESY correlations (blue lines) and proposed 3D-structure for 8-
epidiosbulbin E (3). (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
the direction of cyclization of the C-18 carboxyl which forms a
lactone involving C-6 instead of C-2.
c-
isolated from D. bulbifera L. var. sativa (Murray et al., 1984; Shriram
et al., 2008). Diosbulbin E (5) has been also isolated in the present
work and its absolute configuration was established earlier by cir-
cular dichroism (Ida et al., 1978a). However, no detailed NMR data
Dioscorea species are known to biosynthesize diterpenoids of
the clerodane or of the 19-norclerodane types which are responsi-
ble of the bitter taste, in addition to alkaloids such as dioscorine
and steroid sapogenins responsible for their toxicity (Sautour

L. Rakotobe et al. / Phytochemistry 71 (2010) 1007–1013
1011
Column chromatography was performed on 200–400 mesh silica
gel 60 (Merck). Preparative medium-pressure liquid chromatogra-
phy (MPLC) was performed with a pump K-120 (Knauer) and
20
Flashsmart cartridges (Si and C-18 gels 20–40 lm, AIT, France).
1
16
9
11
10
2
3.2. Plant material
C
D
15
A
8
B
13
3
5
12
Tubers of D. antaly Jum. and H. Perrier were collected in May
2004 in the Menabe region near Morondava (Beroboka) located
600 km South-West Antananarivo (Madagascar). The plant was
identified by Prof. V.H. Jeannoda and voucher specimens (MT 066
to MT 069) were deposited at the Herbarium of the Department
of Botany, University of Antananarivo.
14
7
4
6
17
18
3.3. Extraction and isolation
Fig. 6. Selected NOESY correlations (blue lines) and proposed 3D-structure for
8-epidiosbulbin G (4). (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
Tubers were washed, cut into small pieces, air-dried and milled.
The powdered plant material (546 g) was extracted successively
with cyclohexane (4 ꢂ 250 ml) and MeOH (4 ꢂ 250 ml) at rt and
concentrated to dryness under reduced pressure. The crude meth-
anolic extract (15.7 g) was partitioned between EtOAc (200 ml)
and water (300 ml) to yield EtOAc (6.2 g) and aqueous (9 g) ex-
tracts after evaporation of solvents. The EtOAc-soluble extract
(6.2 g) was further chromatographed over silica gel column
(300 g) eluted with a cyclohexane/EtOAc gradient of increasing
polarity, followed by EtOAc–MeOH gradient to afford 27 fractions
(500 ml each). Fractions 11, 12 and 13 (290 mg) which showed
similar profiles on TLC were grouped together and purified on
Sephadex LH-20 eluted with MeOH/CH2Cl2:90/10 to yield 3,7-
dihydroxy-2,4-dimethoxyphenanthrene (17 mg).
Purification of fraction 16 (560 mg) by CC on Sephadex LH-20
eluted with MeOH and then by MPLC yielded dihydroxypiceatan-
nol (25 mg), piceatannol (20 mg), compound 1 (7 mg) and a mix-
ture of compound 1 and 2 (161 mg). This mixture was further
separated by isochratic MPLC using CH2Cl2/EtOAc:98/2 at flow rate
of 1 ml/min and furnished further 25 mg of compound 2. Fraction
17 (1.18 g) was subjected to repeated column chromatography
on Sephadex LH-20 eluted with MeOH to furnish 23 sub-fractions
(90 ml each) from which sub-fractions 17-7 and 17-23 contained
pure catechin (150 mg) and cassigarol D (162 mg), respectively.
Flash separation of sub-fraction 17-3 on MPLC with gradient
elution CH2Cl2/MeOH:99/1 afforded diosbulbin E (5, 16 mg), antab-
iosbulbins A (1, 3 mg) and B (2, 3 mg). Purification of sub-fraction
17-16 (15 mg) on a C-18 reverse phase flash column (3 g) on MPLC
using ACN/H2O + 0.1% TFA (20:80) yielded scircupsin B (1 mg).
Recrystallisation of fraction 19 (1473 mg) in MeOH yielded 8-
epidiosbulbin E (3, 534 mg) and that of fraction 20 (559 mg) yielded
8-epidiosbulbin G (4, 26 mg). By successive repurification on Sepha-
et al., 2007). The bitter taste of some Nepalese species has been as-
signed to bitter components identified as furanoid norditerpenes,
especially to diosbulbin B (Bhandari and Kawabata, 2005). Only
diterpenoids were isolated from D. antaly in the course of our work
and no saponins were detected, which suggests that D. antaly and
D. bulbifera are close taxonomically and distinct from other Diosco-
rea species. Only one saponin, a pennogenin glycoside, has been
isolated from D. bulbifera var. sativa (Teponno et al., 2006), all other
phytochemical investigations on D. bulbifera have not produced
any steroid sapogenins (Wang et al., 2009). The water-soluble ex-
tract of the tubers of D. antaly was slightly toxic when medaka
fishes were incubated in a medium containing this extract, with
LD50 around 0.86 mg/ml (Rakotobe et al., 2010).
In summary, two C-8 epimers antadiosbulbins A and B, as well
as two new 19-norclerodanes, 8-epidiosbulbins E and G have thus
been isolated from the tubers of D. antaly in addition to the known
diosbulbin E. A general scheme for the biosynthesis of Dioscorea
diterpenes from geranyl–geranyl diphosphate might lead to a tri-
hydroxylated tri-carboxylic acid intermediate, in which the asym-
metric center at C-8,
a to an acid function might undergo racemi-
zation (Fig. 7). Cyclization could then yield antadiosbulbins A and
B. After decarboxylation, the resulting 19-norclerodane might fur-
ther be cyclized to yield either diosbubin E and 8-epidiosbulbin E
by forming of a lactone ring between the carboxylic function at
C-18 and the alcohol at C-2 or diosbulbin G and its epimer at C-8
by cyclization with the alcohol group at C-6.
3. Experimental
3.1. General experimental procedures
Optical rotations were measured on a Perkin Elmer model 341
dex LH-20 followed by MPLC of fraction 21 afforded (3-O-[b-
D-
glucose-(6 ? 1)- -rhamnose]-kaempferol) (32 mg). Kaempferol
a-L
(12 mg) and quercetin (6 mg) were obtained from fraction 22
(313 mg) by chromatography on Sephadex LH-20 eluted with
MeOH.
polarimeter at 20 °C and the [a] .
values are given in deg cm2 gꢁ1
D
IR spectra were taken on a Shimadzu FTIR-8400S spectrophotome-
ter. Mass spectra data were recorded using on an API Q-STAR Pul-
sar I of Applied Biosystems. 13C NMR spectra were recorded on an
AC 300 BRUKER spectrometer operating at 75.47 MHz (for 13C). 1H
and 2D-NMR spectra were on an Avance-400 BRUKER spectrome-
ter operating at 400.13, equipped with 1H-broad-band reverse gra-
dient probe head. The 1H and 13C NMR chemical shifts are given in
ppm relative to TMS, with coupling constants (J) reported in Hz. For
the HMBC experiments, the delay (1/2J) was 70 ms and for the
NOESY experiments the mixing time was 150 ms. TLC was carried
out on precoated Si gel 60 F254 plates (Merck). Spots were detected
under UV (254 and 366 nm) before spraying with phosphomolyb-
dic acid solution in EtOH or Liebermann–Burchard reagent or van-
illin–sulfuric solution followed by heating the plate at 110 °C.
3.3.1. Antadiosbulbin A (1)
Colourless amorphous solid; ½a D20
ꢁ45° (c 0.7, CHCl3); HR-ESI-
ꢀ
MS positive mode, m/z: 405.1519 [M+H]+, C21H25O8 (calc.:
405.1548); IR (CHCl3) mmax (cmꢁ1): 3447, 3150, 2953, 2928, 1740,
1506, 1448, 1368, 1265, 1157, 1115, 1082; 1H and 13C NMR data,
see Table 1.
3.3.2. Antadiosbulbin B (2)
Colourless amorphous solid; ½a D20
ꢁ28° (c 0.4, CHCl3); HR-ESI-
ꢀ
MS positive mode, m/z: 405.1527 [M+H]+, C21H25O8 (calc.:
405.1548); IR (CHCl3) mmax (cmꢁ1): 3447, 3150, 2953, 1750, 1734,
1505, 1464, 1437, 1204, 1113; 1H and 13C NMR data, see Table 1.

1012
L. Rakotobe et al. / Phytochemistry 71 (2010) 1007–1013
15
OPP
OPP
OPP
O
16
14
13
12
11
20
H3C
H
H3C
H
CH3
H
CH3
9
1
4
17
COOH
8
2
3
10
5
8
7
H
H
6
18
19COOH
CH3
18
19
HOOC
Geranyl-geranyl diphosphate
15
O
O
14
16
13
12
H
H32C0
12
11
-CO2
OH
17COOH
H3C
H
O
1
H
9
H
10
17
2
8
HO
2
8
O
H
H
5
O
3
7
6
4
6
18
HOOC
H
O
O
H
OH
19 COOH
18
H
OH
O
H
Diosbulbin E:
α−H
H
12
-CO2
12
8-Epidiosbulbin E: β−H
H3C
H3C
H
O
O
H
17
17
H
O
HO
8
8
2
O
O
2
H
H
19
O
4
4
6
H
H
H
HO
18
O
21
18
OCH3
O
O
Antadiosbulbin A: α−H
Antadiosbulbin B: β−H
Diosbulbin G:
α−H
8-Epidiosbulbin G: β−H
Fig. 7. Plausible pathway for the biosynthesis of the Dioscorea diterpenoids.
Table 3
3.3.3. 8-Epidiosbulbin E (3)
13C and 1H NMR data for 8-epidiosbulbin G (4) in Pyr-d5 (400.13 MHz for 1H).
Colourless amorphous solid; ½a D20
ꢁ16° (c 0.3, CHCl3); HR-ESI-
ꢀ
MS positive mode, m/z: 347.1485 [M+H]+, C19H23O6 (calc.:
347.1493); IR (CHCl3) mmax (cmꢁ1): 3636, 3150, 2926, 1744, 1725,
1505, 1204, 1109; 1H and 13C NMR data, see Table 2.
Position
8-Epidiosbulbin G (4)
dC
dH
Mult.
J (Hz)
1eq
ax
2
3eq
ax
4
5
6
7eq
ax
8
30.6
1.88
1.16
4.35
2.54
1.79
2.85
2.22
4.48
3.00
2.18
2.51
–
2.65
2.23
1.83
5.82
–
6.51
7.54
7.59
–
–
0.97
5.10
dm
ddd
m
12.7
12.7; 12.2; 2.2
64.5
30.3
3.3.4. 8-Epidiosbulbin G (4)
Colourless amorphous solid; ½a D20
ꢁ20° (c 0.3, CHCl3); HR-ESI-
ꢀ
dm
ddd
ddd
ddd
ddd
ddd
ddd
dd
14.8
14.8; 7.8; 3.7
7.8; 6.2; 1.5
12.2; 6.2; 4.9
5.9; 4.9; 4.0
15.3; 5.9; 4.0
15.3; 5.9; 5.9
5.9; 5.9
MS positive mode, m/z: 347.1497 [M+H]+, C19H23O6 (calc.:
40.5
38.5
76.2
25.8
347.1493); 1H and 13C NMR data see Table 3.
3.3.5. Diosbulbin E (5)
Colourless amorphous solid; HR-ESI-MS positive mode, m/z:
347.1496 [M+H]+, C19H23O6 (calc.: 347.1493); IR (CHCl3) mmax
(cmꢁ1): 3484, 3150, 2930, 1769, 1718, 1503, 1298, 1209, 1147,
1076, 1024; 1H and 13C NMR data, see Table 2.
46.3
33.5
28.7
40.4
9
10
11eq
ax
12
13
14
15
16
17
18
20
2-OH
ddd
dd
dd
12.2; 12.2; 2.2
14.6; 2.8
14.6; 12.5
12.5; 2.8
70.2
dd
126.1
109.3
144.0
140.4
172.1
178.2
21.6
3.3.6. 8-Epidiosbulbin E acetate (6)
dd
dd
brs
2.0; 1.0
2.0; 1.8
Compound 3 (5 mg) was added to a mixture of 1 ml of pyridine
and 1 ml of Ac2O at room temperature and stirred for 24 h. To
accelerate the acetylation, 7 mg of dimethylaminopyridine (DMAP)
were added to the reaction mixture and stirring was maintained
for 24 h; then 10 ml of aqueous HCl (5%) were added to the reac-
tion mixture. After extraction with CH2Cl2 and washing with
s
s
–

L. Rakotobe et al. / Phytochemistry 71 (2010) 1007–1013
1013
Brinker, A.M., Seigler, D.S., 1991. Isolation and identification of piceatannol as a
phytoalexin from sugarcane. Phytochemistry 30, 3229–3232.
10 ml H2O the organic layers were dried over Na2SO4 and concen-
trated under vacuum. Purification on silica gel column chromatog-
raphy (cyclohexane/EtOAc:95/5) afforded 3 mg of 8-epidiosbulbin
E acetate (6).
Davies, A.L., Cai, Y., Davies, A.P., Lewis, J.R., 1996. 1H and 13C NMR assignments of
some green tea polyphenols. Magn. Reson. Chem. 34, 887–890.
Ida, Y., Kubo, S., Fujita, M., Komori, T., Kawasaki, T., 1978a. Furanoid-norditerpene
aus Pflanzen der Familie Dioscoreaceae, V. Struktur der Diosbulbine D, E, F, G
und H. Liebigs Ann. Chem. 1978, 818–833.
Compound 6: colourless amorphous solid; ESI-MS positive
mode, m/z: 389.1607 [M+H]+, C21H25O7 (calc.: 389.1599); 13C
NMR (CDCl3): 28.2 (C-1), 76.4 (C-2), 38.6 (C-3), 42.0 (C-4), 41.2
(C-5), 69.0 (C-6), 26.8 (C-7), 45.6 (C-8), 34.5 (C-9), 31.6 (C-10),
39.7 (C-11), 69.6 (C-12), 124.9 (C-13), 108.6 (C-14), 143.9 (C-15),
139.9 (C-16), 170.8 (C-17), 175.7 (C-18), 21.9 (C-20), 170.7 (C-
21), 20.9 (C-22). 1H NMR (CDCl3): 2.10 (1H, brddd 13.3, 6.0, 5.0,
H-1a), 1.46 (1H, ddd 13.3, 12.0, 1.1, H-1b); 4.85 (1H, dd 5.5, 4.7,
H-2); 2.51 (1H, dddd 11.4, 5.7, 5.5, 1.7, H-3a); 1.77 (1H, d 11.5,
H-3b); 2.57 (1H, dd 5.8, 1.6, H-4); 1.92 (1H, ddd 12.3, 2.0, 2.0, H-
5); 5.15 (1H, brddd 2.8, 2.7, 2.7, H-6); 2.83 (1H, ddd 15.1, 3.1, 2.1,
H-7a); 1.87 (1H, ddd 15.1, 6.2, 2.6, H-7b); 2.31 (1H, dd 6.2, 2.1,
H-8); 2.60 (1H, ddd 12.3, 12.0, 4.0, H-10); 2.11 (1H, dd 14.7, 3.3,
H-11a); 1.78 (1H, dd 14.7, 12.6, H-11b); 5.51 (1H, dd 12.6, 3.3, H-
12); 6.41 (1H, brdd 1.9, 0.8, H-14); 7.40 (1H, brdd 1.9, 1.6, H-15);
7.48 (1H, ddd 1.6, 0.8, 0.8, H-16); 1.16 (1H, s, H-20); 1.99 (1H, s,
H-22).
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Acknowledgements
This work which is part of the ‘‘Fond d’Appui au Développement de
l’Enseignement Supérieur” (FADES – Project), was also supported by
Grants from the French Ministère Français de la Coopération and the
‘‘Service de Coopération et d’Action Culturelle (SCAC)” of the France
Embassy at Antananarivo (Madagascar). It was also supported by
the CNRS International Group of Research (GDRI) ‘‘Biodiversité des
îles de l’Océan Indien” which is gratefully acknowledged.
Rakotobe, L., Berkal, M., Huet, H., Djediat, C., Jeannoda, V., Bodo, B., Mambu, L.,
Crespeau, F., Edery, M., 2010. Effects of Madagascar yam extracts, Dioscorea
antaly, on embryo-larval development of medaka fish, Oryzias latipes. Toxicon
55, 87–91.
Sautour, M., Mitaine-Offer, A.-C., Lacaille-Dubois, M.-A., 2007. The Dioscorea genus:
a review of bioactive steroid saponins. J. Nat. Med. 61, 91–101.
Shriram, V., Jahagirdar, S., Latha, C., Kumar, V., Puranik, V., Rojatkar, S.,
Dhakephalkar, P.K., Shitole, M.G., 2008. A potential plasmid-curing agent, 8-
epidiosbulbin E acetate, from Dioscorea bulbifera L. against multidrug-resistant
bacteria. Int. J. Antimicrob. Agents 32, 405–410.
Teponno, R.B., Tapondjou, A.L., Gatsing, D., Djoukeng, J.D., Abou-Mansour, E.,
Tabacchi, R., Tane, P., Stoeckli-Evans, H., Lontsi, D., 2006. Bafoudiosbulbins A
and B, two anti-salmonellal clerodane diterpenoids from Dioscorea bulbifera L.
var. sativa. Phytochemistry 67, 1957–1963.
Wang, G., Liu, J.-S., Lin, B.-B., Wang, G.-K., Liu, J.-K., 2009. Two new
furanoid norditerpenes from Dioscorea bulbifera. Chem. Pharm. Bull. 57,
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