Saponins and acylated saponins from Dizygotheca kerchoveana

Article abstract of DOI:10.1016/j.phytochem.2004.08.049

Nine saponins and two acylated saponins were isolated from the leaves and the stem of branches of Dizygotheca kerchoveana. Their structures were established by chemical and spectroscopic means. Four new triterpenoid saponins were isolated from the leaves and stem of branches of Dizygotheca kerchoveana along with seven known ones. The new saponins were respectively characterized as 3-O-[β-d-glucopyranosyl-(1 → 3)]-[β-d-glucopyranosyl-(1 → 2)]-α-l-arabinopyranosyl echinocystic acid, 3-O-[β-d-glucopyranosyl- (1 → 3)]-[β-d-glucopyranosyl-(1 → 2)]-α-l-arabinopyranosyl echinocystic acid 28-O-[α-l-rhamnopyranosyl-(1 → 4)-β-d- glucopyranosyl-(1 → 6)-β-d-glucopyranosyl] ester, 3-O-[β-d-3-O- trans-p-coumaroyl-glucopyranosyl-(1 → 3)]-[β-d-glucopyranosyl-(1 → 2)]-α-l-arabinopyranosyl echinocystic acid 28-O-[α-l- rhamnopyranosyl-(1 → 4)-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl] ester and 3-O-[β-d-3-O-cis-p-coumaroyl- glucopyranosyl-(1 → 3)]-[β-d-glucopyranosyl-(1 → 2)]-α-l-arabinopyranosyl echinocystic acid 28-O-[α-l- rhamnopyranosyl-(1 → 4)-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl] ester. Their structures were elucidated by 1D and 2D NMR experiments, FAB-MS as well as chemical means.

Full text of DOI:10.1016/j.phytochem.2004.08.049

PHYTOCHEMISTRY
Phytochemistry 65 (2004) 3089–3095
www.elsevier.com/locate/phytochem
Saponins and acylated saponins from Dizygotheca kerchoveana
a,
b
c
c
c
F.R. Melek *, Toshio Miyase , S.M. Abdel-Khalik , I.I. Mahmoud , S.A. Mina
a
Chemistry of Natural Products Department, National Research Centre, Dokki, 12622 Cairo, Egypt
b
School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422 – 8526, Japan
c
Pharmacognosy Department, Faculty of Pharmacy, Helwan University, Cairo, Egypt
Received 20 April 2004; received in revised form 11 August 2004
Available online 2 October 2004
Abstract
Four new triterpenoid saponins were isolated from the leaves and stem of branches of Dizygotheca kerchoveana along with seven
known ones. The new saponins were respectively characterized as 3-O-[b-
D
-glucopyranosyl-(1 ! 3)]-[b-
-glucopyranosyl-(1 ! 2)]-a-
-glucopyranosyl] ester, 3-O-[b-
-arabinopyranosyl echinocystic acid 28-O-[a-
and 3-O-[b- -3-O-cis-p-coumaroyl-glucopyranosyl-
-rhamnopyranosyl-(1 ! 4)-b- -glucopy-
-glucopyranosyl] ester. Their structures were elucidated by 1D and 2D NMR experiments, FAB-MS as well
D
-glucopyranosyl-(1 ! 2)]-
-arabinopyranosyl
-3-O-trans-
-rhamnopy-
a-
L
-arabinopyranosyl echinocystic acid, 3-O-[b- -glucopyranosyl-(1 ! 3)]-[b-
D
L
echinocystic acid 28-O-[a-
L
-rhamnopyranosyl-(1 ! D4)-b-
D
-glucopyranosyl-(1 ! 6)-b-
D
D
p-coumaroyl-glucopyranosyl-(1 ! 3)]-[b-
D
-glucopyranosyl-(1 ! 2)]-a-
L
L
ranosyl-(1 ! 4)-b-
(1 ! 3)]-[b- -glucopyranosyl-(1 ! 2)]-a-
ranosyl-(1 ! 6)-b-
as chemical means.
ꢀ 2004 Elsevier Ltd. All rights reserved.
D
-glucopyranosyl-(1 ! 6)-b-
D
-glucopyranosyl]
ester
D
D
L
-arabinopyranosyl echinocystic acid 28-O-[a-
L
D
D
Keywords: Dizygotheca kerchoveana; Araliaceae; Triterpenoid saponins
1. Introduction
2. Results and discussion
Dizygotheca species (Araliaceae) are ornamental
trees cultivated in public gardens in Egypt. In our pre-
vious studies on the family members, we have de-
scribed the isolation and structures of several
numbers of saponins (Melek et al., 2002, 2003a,b,
2004). As a part of our continuing search for bioactive
saponins from plants grown in Egypt, we present in
this report the isolation and structure elucidation of
eleven saponins including four new ones, from the
leaves and stem of branches of Dizygotheca kerchove-
ana Hort – Veitch.
The leaves and stem of branches of D. kerchoveana
with the same saponin content (TLC profile) were mixed
and extracted with methanol. The crude saponin mixture
obtained from the methanolic extract was subjected to
column chromatography on a porous polymer polysty-
rene resin (Diaion HP-20), silica gel and Sephadex LH-
20 followed by HPLC to give eleven saponins (1–4,
6–12) including four new ones 1–4. The seven known
saponins 6, 3-O-a-
yarenko et al., 2000); 7, 3-O-a-
nocystic acid (Grishkovets et al., 2001); calenduloside E
8, 3-Ob- -glucuronopyranosyl-oleanolic acid (Hu et al.,
1995); 9, 3-O-a- -arabinopyranosyl-ursolic acid 28-O-
a- -gentiobiosyl ester
-rhamnopyranosyl-(1 ! 4)-b-
(Stolyarenko et al., 2000); ciwujianoside C3 10, 3-O-a-
-arabinopyranosyl-oleanolic acid 28-O-a- -rhamnopy-
L
-arabinopyranosyl-ursolic acid (Stol-
L
-arabinopyranosyl-echi-
D
L
L
D
*
Corresponding author. Tel.: +20 2558 4005; fax: +20 2337 0931.
E-mail address: frmelek@link.net (F.R. Melek).
L
L
0031-9422/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2004.08.049

3090
F.R. Melek et al. / Phytochemistry 65 (2004) 3089–3095
ranosyl-(1 ! 4)-b-
D
-gentiobiosyl ester (Shao et al.,
-arabinopyranosyl-echinocystic acid
-gentiobiosyl
ester (Melek et al., 2002) and hederasaponin F 12, 3-O-b-
sulphate-oleanolic acid 28-O-a-
-rhamnopyranosyl-(1 !
4)-b- -gentiobiosyl ester (Elias et al., 1991), together
preparation of their thiazolidine derivatives (Hara et al.,
1986). The positive FAB-mass spectrum of 1 displayed
the quasi-molecular ion peak [M + Na]+ at m/z 951 cor-
responding to a molecular formula C47H76O18 and com-
patible with a trisaccharide glycoside containing an
aglycone of molecular mass 472 and three sugar units,
one pentose and two hexoses. The resonances due to se-
ven sp3 carbons at d 15.6, 16.8, 17.5, 24.8, 27.3, 28.1,
33.4 and two sp2 carbons at d 122.5 and 145.2 in the
13C NMR spectrum of 1 coupled with the information
1988); 11, 3-O-a-
L
28-O-a-
L
-rhamnopyranosyl-(1 ! 4)-b-
D
L
D
with oleanolic acid 5, were isolated and identified from
their NMR data and comparison with the literature val-
ues. The NMR data of the new saponins 1–4 are pre-
sented in Tables 1 and 2.
COOR
OH
H
1
OH
O
O
OH
O
O
HO
RO
OH
O
OH
O
HO
HO
OH
R
H
H
R1
H
1
2
α-L-Rhap-(1→4)-ß-D-Glcp-(1→6)-ß-D-Glcp-
H
C
O
O H
C
C
3
4
α-L-Rhap-(1→4)-ß-D-Glcp-(1→6)-ß-D-Glcp-
H
O
O H
C
C
C
α
→ →
-L-Rhap-(1 4)-ß-D-Glcp-(1 6)-ß-D-Glcp-
H
H
R5
R4
H
COOR3
R2
R1O
R1
H
R2
R3
H
H
H
H
R4
H
R5
CH3
H
5
6
H
H
α-L-Arap
α-L-Arap
CH3
H
7
OH
H
CH3
CH3
H
8
ß-D-GlcpA
H
9
α-L-Arap
H
α-L-Rhap-(1→4)-ß-D-Glcp-(1 6)-ß-D-Glcp-
CH3
H
→
10
11
12
-L-Arap
-L-Arap
SO3H
H
-L-Rhap-(1 4)-ß-D-Glcp-(1 6)-ß-D-Glcp-
CH3
CH3
CH3
α
α
→
→
α
α
α
OH
H
-L-Rhap-(1→4)-ß-D-Glcp-(1→6)-ß-D-Glcp-
H
-L-Rhap-(1 4)-ß-D-Glcp-(1 6)-ß-D-Glcp-
H
→
→
1
Acid hydrolysis of compounds 1 with 2 M hydrochlo-
ric acid in dioxane (1:1) yielded the sugar components
arabinose, and -glucose, identified by GC analysis after
from the H NMR spectrum (seven methyl proton sin-
glets and a broad triplet vinyl proton at d 5.63) sug-
gested 1 to be a triterpenoid glycoside with an
L
-
D

F.R. Melek et al. / Phytochemistry 65 (2004) 3089–3095
3091
Table 1
1H NMR spectral data for compounds 1–4 in pyridine-d5
Aglycone
1
2
3
4
3
3.27 (dd, 12.0, 4.5)
0.81 (d, 12.0)
5.63 (brt, 3.5)
5.24 (brs)
3.26 (dd, 12.0, 4.5)
0.80 (d, 12.0)
5.59 (brt, 3.0)
5.27 (brs)
3.25 (dd, 12.0, 4.5)
0.80 (d, 11.5)
5.59 (brt, 3.0)
5.27 (brs)
3.25 (dd, 11.5, 4.0)
0.81(d, 11.5)
5.58 (brt, 3.0)
5.27 (brs)
5
12
16
18
23
24
25
26
27
29
30
3.61 (dd, 14.0, 3.5)
1.25 (s)
1.08 (s)
3.50 (dd, 14.0, 3.5)
1.24 (s)
1.09 (s)
3.50 (dd, 14.0, 3.5)
1.23 (s)
1.10 (s)
3.49 (dd, 14.0, 3.5)
1.22 (s)
1.09 (s)
0.87 (s)
1.03 (s)
0.91 (s)
1.11 (s)
0.90 (s)
1.11 (s)
0.90 (s)
1.11 (s)
1.84 (s)
1.07 (s)
1.18 (s)
1.80 (s)
0.99 (s)
1.06 (s)
1.81 (s)
0.99 (s)
1.06 (s)
1.80 (s)
0.99 (s)
1.06 (s)
3-O-sugar
Ara
1
4.79 (d, 6.7)
4.72
4.33
4.78 (d, 6.7)
4.72
4.33
4.77 (d, 6.7)
4.68
4.29
4.76 (d, 6.7)
4.68
4.28
2
3
4
4.48
3.67(brs, 11.5)
4.18
4.48
3.67
4.50
3.68
4.47
3.66
5
5Ô
4.18
4.18
4.15
Glc I at Ara C2
1
2
3
4
5
6
60
5.49 (d, 8.0)
4.03
4.19
5.49 (d, 8.0)
4.03
4.18
5.43 (d, 8.0)
4.02
4.12
5.40 (d, 8.0)
4.00
4.13
4.14
4.13
3.70
4.09
3.69
4.09
3.68
3.70 (m)
4.27
4.35
4.27
4.34
4.25
4.36
4.24
4.35
Glc II at Ara C3
1
2
3
4
5
6
60
5.28 (d, 8.0)
3.99
4.20
5.28 (d, 8.0)
3.99
4.20
5.28 (d, 8.0)
4.10
5.98
5.24 (d, 8.0)
4.05
5.93
4.18
4.18
4.39
4.32
3.93
3.93 (m)
4.31
4.46
3.93 (m)
4.31
4.47
3.97 (m)
4.35
4.45
4.33
4.42
28-O-sugar
Glc III
1
2
3
4
5
6
60
6.22 (d, 8.5)
4.05
4.17
6.22 (d, 8.5)
4.05
4.17
6.22 (d, 8.5)
4.05
4.17
4.22
4.08
4.23
4.08
4.23
4.08
4.30
4.64
4.30
4.64
4.30
4.63
Glc IV
1
2
3
4
5
6
60
4.96 (d, 8.0)
3.91
4.15
4.96 (d, 8.0)
3.91
4.13
4.96 (d, 8.0)
3.91
4.12
4.37
4.37
3.66
4.37
3.66 (m)
4.08
4.20
3.66 (m)
4.06
4.22
4.08
4.20
Rha
1
2
3
4
5
6
5.82 (brs)
5.82 (d, 2.0)
5.82 (d, 2.0)
4.66 (dd, 3.0, 2.0)
4.52 (dd, 9.0, 3.5)
4.30
4.65 (dd, 3.0, 1.5)
4.52 (dd, 9.0, 3.5)
4.30
4.65 (dd, 3.0, 2.0)
4.52 (dd, 9.5, 3.5)
4.30
4.92 (dq, 9.0, 6.5)
1.69 (d, 6.5)
4.92 (dq, 9.0, 6.5)
1.69 (d, 6.5)
4.92 (dq, 9.0, 6.0)
1.69 (d, 6.0)
(continued on next page)

3092
F.R. Melek et al. / Phytochemistry 65 (2004) 3089–3095
Table 1 (continued)
Aglycone
1
2
3
4
p-Coumaroyl moiety
2,6
3,5
a
7.48 (d, 8.8)
7.13 (d, 8.8)
6.53 (d, 16.0)
7.88 (d, 16.0)
8.07 (d, 8.8)
7.12 (d, 8.8)
5.96 (d, 12.5)
6.83 (d, 12.5)
b
1
Values in parantheses are H–1H splittings in cases where these are clearly resolved. Ara, a-
rhamonpyranose.
L
-arabinopyranose; Glc, b-
D
-glucopyranose; Rha, a-L-
aglycone of olean-12-ene skeleton. In comparison of the
13C NMR spectral data with those reported for various
echinocystic acid glycosides (Nagao et al., 1993), 1 was
suggested to be an echinocystic acid derivative with
three sugar moieties attached at C-3 (C-3, 89.1 ppm;
C-28, 180 ppm). The three sugar anomeric proton sig-
nals appeared in the H NMR spectrum at d 4.79 (d,
J = 6.7 Hz), 5.49 (d, J = 8.0 Hz) and 5.28 (d, J = 8.0
Hz) were assigned to one L-arabinose and two D-glucose
ety, it was apparent that the six sugars were present in
two saccharide chains, one attached to C-3 (d 89.0)
and the other to C-28 (d 176.0). The unambiguous iden-
tification of the individual spin systems associated with
the six monosaccharides and the assignments of their
NMR resonances were established by the combined
use of 1D and 2D NMR techniques (HOHAHA,
1H–1H COSY, NOE, HMQC, HMBC). All monosac-
charides were in the pyranose form and the anomeric
1
3
units, respectively. The HOHAHA and NOE difference
experiments of 1 allowed the identification of the spin
systems of the three monosaccharides. The complemen-
configurations, as determined from JH-1,H-2, were a-
for arabinose and b- for glucoses. The anomeric config-
uration of rhamnose was determined as a- from the d
value of C-3 and C-5 (Agrawal, 1992). The structure
of the oligosaccharide chain at C-28 of the echinocystic
1
tary data from the H–1H COSY spectrum defined the
sequential assignments of the proton resonances and
the intra-residue NOEs was used to discriminate be-
tween resonances arising from H-3 and H-4 of glucose
unit. The corresponding 13C resonances of the individ-
ual monosaccaride, deduced from the HMQC spectrum
acid moiety was characterized as 28-O-a-
ranosyl -glucopyranosyl-(1 ! 6)-b-
(1 ! 4)-b-
copyranosyl based on the HMBC correlations between
resonances of Glc IV C-4 (d 78.5) and Rha (a-
L
-rhamnopy-
D
D
-glu-
L
-
of 1, revealed the presence of an inner a-
nose (Ara) substituted at C-2 and C-3 as well as two ter-
minal b- -glucopyranoses (Glc). The HMBC
L
-arabinopyra-
rhamnopyranose) H-1 (d 5.82), Glc III C-6 (d 69.4)
and Glc IV H-1 (d 4.96), C-28 (d 176.0) of the triterpene
moiety and Glc III H-1 (d 6.22). The other HMBC cor-
relations were found the same as those observed for 3-O-
sugar chain of 1, indicating the presence of identical tri-
saccharide unit attached to C-3 of the echinocystic acid
moiety in 1 and 2. Saponins with the same triose moiety
at C-28 were reported by us from other Araliaceae mem-
bers (Melek et al., 2004). Based on the above studies, the
D
experiment of 1 was used to verify the branched nature
of the trisaccharide chain at C-3 of the triterpene moiety
and exhibited cross peaks correlating the resonances due
to C-3 (d 89.1) of the echinocystic acid moiety and Ara
H-1 (d 4.79), Ara C-2 (d 77.5) and Glc I H-1(d 5.49), Ara
C-3 (d 83.4) and Glc II H-1 (d 5.28). The deduced link-
ages were also in agreement with the observed inter-res-
idue NOEs. Therefore, compound 1 was assigned to the
structure of compound 2 was concluded to be 3-O-[b-
glucopyranosyl-(1 ! 3)]-[b-
a- -arabinopyranosyl echinocystic acid 28-O-[a-
rhamnopyranosyl-(1 ! 4)-b-
b- -glucopyranosyl] ester.
Compound 3 (C74H112O34) exhibited [M + Na]+ion
D
-
D
-glucopyranosyl-(1 ! 2)]-
structure of 3-O-[b-
D
-glucopyranosyl-(1 ! 3)]-[b-
D
-glu-
echino-
L
L
-
copyranosyl-(1 ! 2)]-a-
L
-arabinopyranosyl
D
-glucopyranosyl-(1 ! 6)-
cystic acid.
D
Compound 2 exhibited [M + Na]+ ion peak at m/z
1421 in its FAB-mass spectrum which allowed us to pro-
pose a molecular formula C65H106O32. Analysis of the
1H and 13C NMR spectra of 2 in comparison with those
of 1, defined the nature of 2 as another echinocystis acid
saponin. The presence of six monosaccharide units was
indicated from six anomeric proton resonances in the
1H NMR spectrum at d 4.78 (d, J = 6.7 Hz), 5.49 (d,
J = 8.0 Hz), 5.28 (d, J = 8.0 Hz), 6.22 (d, J = 8.5 Hz),
4.96 (d, J = 8.0 Hz) and 5.82 (brs), attributable to one
peak at m/z 1567 in the FAB-mass spectrum and differed
from the formula of compound 2 by supplementary 146
uma. Analysis of 1D and 2D NMR of 3, in comparison
with those of 2, indicated the presence of echinocystic
acid moiety and six monosaccharides present in two sac-
charide units similar in composition and linkages to
those of 2. The UV spectrum of 3 exhibited absorption
maxima at 312, 290 (sh) and 288 nm characteristic for
cinnamoyl chromophore. The 1H–1H correlations in
1
L
-arabinose, four
D
-glucoses and one
L
-rhamnose,
the H–1H COSY spectrum allowed us to identify addi-
respectively, after acid hydrolysis. From the assigned
13C NMR resonances due to the echinocystic acid moi-
tional resonances due to trans-olefinic protons of the
cinammoyl moiety, which appeared as two doublets at

F.R. Melek et al. / Phytochemistry 65 (2004) 3089–3095
3093
Table 2
13C NMR spectral data for compounds 1–4 in pyridine-d5
Table 2 (continued)
Aglycone
1
2
3
4
Aglycone
1
2
3
4
4
161.4
115.8
145.1
167.4
160.5
115.9
143.6
166.8
3
89.1
89.0
89.3
89.3
a
b
CO
12
13
16
23
24
25
26
27
28
29
30
122.5
145.2
74.8
28.1
16.8
15.6
17.5
27.3
180.0
33.4
24.8
122.8
144.5
74.4
28.1
16.8
15.8
17.6
27.3
176.0
33.2
24.7
122.8
144.5
74.4
28.1
16.8
15.8
17.6
27.3
176.0
33.2
24.8
122.8
144.5
74.4
28.0
16.8
15.8
17.6
27.2
176.0
33.2
24.8
d 6.53 and 7.88 (1H each, J = 16.0 Hz) and p-substituted
benzene ring protons at d 7.13 and 7.48 (2H each, d,
J = 8.8 Hz). Their associated 13C resonances assigned
from the HMQC and HMBC experiments, were at d
115.8 and 145.1 for the olefinic carbons and d 126.2
(C-1), 130.6 (C-2, C-6), 116.8 (C-3, C-5), 161.4 (C-4)
for the aromatic ring together with a carbonyl ester res-
onance at d 167.4. Acid hydrolysis of 3 afforded sugar
components identical to those obtained from 2. Mild
alkaline hydrolysis of 3 with 1% KOH yielded p-couma-
ric acid and a deacylated saponin identical to 2. The
above findings indicated that the structural difference
between the sugar part of 3 and 2 was confined to the
presence of a trans-p-coumaroyl group in 3. The loca-
tion of this group at C-3 of Glc II was suggested by
the deshielded value of Glc II H-3 (Dd + 1.78 ppm) com-
pared to saponin 2. Furthermore, the downfield d value
of Glc II C-3 resonance at 79.2 ppm (a-effect) and the
upfield d value of both C-2 at 73.4 ppm and C-4 at
69.5 ppm (b-effect), relative to the corresponding values
in 2, can be explained if the hydroxyl at C-3 of Glc II in
3 was esterified. Further supporting information for the
site of esterification was derived from the HMBC exper-
iment of 3 which exhibited cross peaks correlating the
resonance of the Glc II H-3 (d 5.98) and the resonance
due to Glc II C-2 (d 73.4), Glc II C-4 (d 69.4) and car-
bonyl ester group (d 167.4) of the trans-p-coumaroyl
moiety. Therefore the structure of 3 was elucidated as
3-O-sugar
Ara
1
105.5
77.5
83.4
68.8
66.0
105.4
77.4
83.3
68.8
65.9
105.4
77.4
84.1
68.6
65.9
105.4
77.4
84.3
68.6
65.9
2
3
4
5
Glc I
1
2
3
4
5
6
104.4
76.2
78.7
72.5
77.5
63.3
104.4
76.2
78.5
72.5
77.3
63.3
104.5
76.1
78.4
72.6
77.3
63.4
104.5
76.0
78.4
72.6
77.3
63.4
Glc II
1
2
3
4
5
6
105.0
75.3
78.5
71.6
78.4
62.6
105.0
75.2
78.7
71.6
78.4
62.6
104.9
73.4
79.2
69.5
78.4
62.1
104.9
73.2
79.0
69.5
78.4
62.1
28-O-sugar
Glc III
1
2
3
4
5
6
95.8
74.0
78.8
71.0
78.1
69.4
95.8
74.0
78.7
71.0
78.1
69.5
95.8
74.0
78.7
71.0
78.1
69.5
3-O-[b-
[b-
-glucopyranosyl-(1 ! 2)]-a-
inocystic acid 28-O-[a-
-rhamnopyranosyl-(1 ! 4)-b-
glucopyranosyl-(1 ! 6)-b- -glucopyrsanosyl] ester.
D
-3-O-trans-p-coumaroyl-glucopyranosyl-(1 ! 3)]-
D
L
-arabinopyranosy ech-
L
D-
D
Glc IV
Compound 4 was another echinocystic acid hexasac-
charide from NMR analysis. Upon acid hydrolysis, 4 af-
forded sugar components identical to those obtained
from 2 and 3. The analysis of 1D and 2D NMR of the
sugar part of 4 and 3 revealed similarity with regard
to the structure of the individual monosaccharides and
their linkages. The only structural difference between 4
and 3 was due to the geometry of the double bond be-
tween the olefinic carbons of the p-coumaroyl moiety,
being cis in 4 instead of trans in 3. This was evident from
the position of the two doublets attributable to the cis
olefinic protons at d 5.96 and 6.83 (1H each, J = 12.0
1
2
3
4
5
6
105.0
75.3
76.5
78.5
77.2
61.4
105.0
75.3
76.5
78.4
77.2
61.4
105.0
75.3
76.6
78.5
77.2
61.4
Rha
1
2
3
4
5
6
102.8
72.6
72.8
74.0
70.4
18.5
102.8
72.6
72.8
74.0
70.4
18.5
102.8
72.6
72.8
74.0
70.4
18.5
1
Hz) in the H–1H COSY spectrum of 4. The p-substi-
p-Coumaroyl moiety
1
tuted benzene ring protons appeared as two doublets
at d7.12 and 8.07 (2H each, d, J = 8.8 Hz). Mild alkaline
hydrolysis of 4 using 1% KOH afforded p-coumaric acid
126.2
130.6
116.8
126.6
133.8
116.7
2,6
3,5

3094
F.R. Melek et al. / Phytochemistry 65 (2004) 3089–3095
and deacylated saponin 2. As expected, the [M + Na]+
ion peak was observed at m/z 1567 in the FAB-mass
spectrum of 4. Thus, the structure of 4 was elucidated
3.3. Extraction and isolation
A mixture of dried and powdered leaves and stem of
branches of D. kerchoveana (1.5 kg) was extracted with
CHCl3 twice then extracted with MeOH twice at room
temperature. The combined methanolic extract (95.5 g)
was concentrated and dropped into a 10-fold amount
of (CH3)2CO to precipitate the crude saponin mixture
(16.5 g). The mixture was dissolved in water and the
aqueous solution was passed through a porous polymer
gel column (Misubishi Diaion HP-20). The column was
then eluted with water, 50%, 75% and 100% MeOH. The
75% MeOH eluate. (650 mg) was chromatographed on a
silica gel column eluted with CHCl3–MeOH (65:35) fol-
lowed by Sephadex LH-20 column eluted with methanol
to give 12 (109 mg). The MeOH eluate (5.6 g) was
chromatographed on a silica gel column eluted with
CHCl3–MeOH–H2O (80:18:2–61:33:6) to give 17 frac-
tions (A–Q). Fraction A (224 mg) afforded oleanolic acid
5 (26 mg) after silica gel column chromatography eluted
with CHCl3:MeOH (98:2). Fraction B (420 mg), C (221
mg), E (223 mg), F (589 mg), I (336 mg), L (193 mg) and
P (166 mg) were subjected to silica gel column chroma-
tography eluted with CHCl3–MeOH–H2O mixs. of
increasing polarity. The purification was finally per-
formed over HPLC [ODS, 0.2 · 2.5 cm; solvent, CH3-
CN–H2O (25:75–55:45) + 0.05 TFA; monitored at 205
nm]. Fraction A gave 5 (18 mg), fraction B afforded 6
(10 mg) and 7 (9 mg), fraction C gave 8 (38 mg), fraction
E afforded 1 (18 mg), fraction F gave 9 (22 mg) and 10
(29 mg), fraction I afforded 11(22 mg), fraction L gave 3
(10 mg) and 4 (16 mg) and fraction P afforded 2 (18 mg).
as 3-O-[b-
3)]-[b-
-glucopyranosyl-(1 ! 2)]-a-
echinocystic acid 28-O-[a-
-rhamnopyranosyl-(1 ! 4)-
b- -glucopyranosyl] ester.
-glucopyranosyl-(1 ! 6)-b-
Saponins contained sugar units esterified with varia-
D
-3-O-cis-p-coumaroyl-glucopyranosyl-(1 !
D
L
-arabinopyranosyl
L
D
D
ble organic acids were reported from several plant spe-
cies. Acylated glycosides of echinocystic acid have
been isolated from the roots of Tragopogon porrifolius
(Warashina et al., 1991).
It is worthnoting that saponins with a-
ranosyl as an inner glycosyl unit at C-3 of the triterpene
moiety and a- -glucopy-
-rhamnopyranosyl-(1 ! 4)-b-
ranosyl-(1 ! 6)-b- -glucopyrano syl as the ester glyco-
L-arabinopy-
L
D
D
syl unit at C-28, have been reported not only from
genera of Araliaceae family but also from genera of
other families such as Ranunculaceae (Ansari et al.,
1988; Shao et al., 1995), Boraginaceae (Mohammad
et al., 1995) Berberidaceae (Chen et al., 1997), Lardiza-
balaceae (Chandel and Rastogi, 1980) and Podophylla-
ceae (Chandel and Rastogi, 1980; Agarwal and
Rastogi, 1974). This probably indicates similar biosyn-
thetic pathways in these plants.
The saponin mixture of D. kerchoveana was found to
exhibit a weak molluscicidal activity (LC90 = 27.9 ppm).
Another species, D. elegantissima from Egypt was stud-
ied and showed saponins, which possessed similar activ-
ity. One of them is a saponin with the same glycosidic
chain at C-28 (Abdel-Gawad et al., 1997).
3. Experimental
3.4. Saponin (1)
23
Amorphous powder ½aꢀ þ 19:5 (C = 0.94, MeOH);
3.1. General
D
FAB-MS m/z: 951 [C47H76O18 + Na]+, 905, 817, 717,
1
Optical rotations were measured with Jascodip – 1000
digital polarimeter. MS spectra were measured on Jeol
JMS-SX 102 mass spectrometer. NMR spectra were re-
corded on Jeol GSX-500 FT NMR spectrometer. Chem-
ical shifts are given on the d scale with TMS as internal
standard. UV spectra were recorded on Hitachi U-2010
spectrophotometer. HPLC was performed on a Jasco
system 800 instruments. GC analysis was carried out
on Hitachi G-3000 gas chromatograph.
687, 593, 455. H and 13C NMR: see Tables 1 and 2.
3.5. Saponin (2)
23
Amorphous powder ½aꢀ ꢁ 13:0 (C = 0.94, MeOH);
D
FAB-MS m/z: 1421 [C65H106O32 + Na]+, 1112, 959,
1
836, 717, 613. H and 13C NMR: see Tables 1 and 2.
3.6. Saponin (3)
23
Amorphous powder ½aꢀ þ 4:9 (C = 0.96, MeOH).
3.2. Plant material
D
MeOH
max
UV k
nm : 228; 290 ðshÞ, 312. FAB-MS m/z: 1567
1
The leaves and stem of branches of D. kerchoveana
were collected from a public garden in Cairo, Egypt.
Mrs T. Labib, head specialist for plant identfication in
El-Orman public garden, Cairo, Egypt, confirmed plant
identification. The herbarium voucher specimen (Melek,
F. 25) was deposited in the Herbarium of NRC
(CAIRC).
[C74H112O34 + Na]+, 1403, 910, 836, 717, 613. H and
13C NMR: see Tables 1 and 2.
3.7. Saponin (4)
23
Amorphous powder ½aꢀ ꢁ 14:9 (C = 0.84, MeOH).
D
MeOH
max
UV k
nm : 227 ðshÞ; 290 ðshÞ; and 310. FAB-MS

F.R. Melek et al. / Phytochemistry 65 (2004) 3089–3095
3095
1
m/z: 1567 [C74H112O34 + Na]+, 1403, 836, 717, 613. H
and 13C NMR: see Tables 1 and 2.
Grishkovets, V.I., Sobolev, E.A., Shashkov, A.S., Chirva, V.Ya., 2001.
Triterpenoid glycosides of Fatsia japonica. II. Isolation and
structure of glycosides from the leaves. Chemistry of Natural
Compounds 36 (5), 501–505.
3.8. General method for acid hydrolysis
Hara, S., Okabe, H., Mihashi, K., 1986. Separation of aldose
enantiomers by gas–liquid chromatography. Chemical and Phar-
maceutical Bulletin 34, 1843–1844.
Acid hydrolysis and detection of the aglycones and
sugar constituents of 1–4 were performed as previously
described by Melek et al., 2002, 2003a,b.
Hu, M., Ogawa, K., Sashida, Y., Xiao, P., 1995. Triterpenoid
glucuronide saponins from root bark of Aralia armata. Phyto-
chemistry 39, 179–184.
Melek, F.R., Miyase, T., Abdel-Khalik, S.M., Hetta, M.H., Mah-
moud, I.I., 2002. Triterpenoid saponins from Oreopanax guatem-
alensis. Phytochemistry 60, 185–195.
3.9. Mild alkaline hydrolysis of 3 and 4
Melek, F.R., Miyase, T., Ghaly, N.S., 2003a. Triterpenoid saponins
from Meryta lanceolata. Phytochemistry 62, 557–562.
Melek, F.R., Miyase, T., Abdel-khalik, S.M., El-Gindi, M.R., 2003b.
Triterpenoid saponins from Schefflera arboricola. Phytochemistry
63, 401–407.
Each compound (5 mg) was hydrolysed separately with
1% KOH at room temperature for 1 h. the mix was neu-
tralized with dilute HCl and extracted with Et2O. The
Et2O afforded p-coumaric acid identified by silica gel
TLC (toluene–ether 1:1, satd. With HOAC 10%). The
aq. layer was extracted with n-BuOH to give saponin 2.
Melek, F.R., Miyase, T., Ghaly, N.S., Yousif, M.F., 2004.
Further saponins from Meryta lanceolata. Phytochemistry 65,
909–914.
Mohammad, F.V., Noorwala, M., Ahmad, V.U., Sener, B., 1995. A
bidesmosidic hederagenin hexasaccharide from the roots of Sym-
phytum officinale. Phytochemistry 40, 213–218.
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