Echinocystic acid 3,16-O-bisglycosides from Albizia procera

Article abstract of DOI:10.1016/j.phytochem.2010.05.004

Three triterpene glycosides and two known ones were isolated from the bark of Albizia procera by using chromatographic techniques. The structures of the compounds were determined to be 3-O-β-d-xylopyranosyl-(1 → 2)-β-d-galactopyranosyl-(1 → 6)-2-acetamido-2-deoxy-β-d- glucopyranosyl echinocystic acid 16-O-β-d-glucopyranoside, 3-O-β-d-xylopyranosyl-(1 → 2)-α-l-arabinopyranosyl-(1 → 6)-2-acetamido-2-deoxy-β-d-glucopyranosyl echinocystic acid 16-O-β-d-glucopyranoside and 3-O-α-l-arabinopyranosyl-(1 → 2)-α-l-arabinopyranosyl-(1 → 6)-2-acetamido-2-deoxy-β-d- glucopyranosyl echinocystic acid 16-O-β-d-glucopyranoside. Their structures were determined by NMR techniques including HOHAHA, ¹H-¹H COSY, ROE, HMQC and HMBC experiments together with FABMS as well as acid hydrolysis. To the best of our knowledge, the new compounds are considered the first examples of echinocystic acid 3,16-O-bisglycosides. In contrast to other cytotoxic echinocystic acid glycosides with N-acetyl glucosamine unit, the new glycosides were found inactive when assayed by MTT method for their cytotoxicities against the human tumor cell lines HEPG2, A549, HT29 and MCF7. The results showed the importance of the free hydroxyl group at the aglycone C-16 for exhibiting cytotoxic properties.

Full text of DOI:10.1016/j.phytochem.2010.05.004

Phytochemistry 71 (2010) 1375–1380
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Echinocystic acid 3,16-O-bisglycosides from Albizia procera
b
c
d
b
Toshio Miyase a, F.R. Melek b, , N.S. Ghaly , Tsutomu Warashina , M. El-Kady , Marian Nabil
*
a School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan
b Chemistry of Natural Compounds Department, National Research Center, Dokki 12622, Giza, Egypt
c Institute for Environmental Sciences, University of Shizuoka, 52-1, Yada, Suruga-ku, Shizuoka 422-8526, Japan
d Chemistry Department, Faculty of Science, Ain-Shams University, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Three triterpene glycosides and two known ones were isolated from the bark of Albizia procera by using
chromatographic techniques. The structures of the compounds were determined to be 3-O-b- -xylopyrano-
syl-(1 ? 2)-b- -galactopyranosyl-(1 ? 6)-2-acetamido-2-deoxy-b- -glucopyranosyl echinocystic acid
16-O-b- -glucopyranoside, 3-O-b- -xylopyranosyl-(1 ? 2)- -arabinopyranosyl-(1 ? 6)-2-acetamido-
2-deoxy-b- -glucopyranosyl echinocystic acid 16-O-b- -glucopyranoside and 3-O- -arabinopyranosyl-
(1 ? 2)- -arabinopyranosyl-(1 ? 6)-2-acetamido-2-deoxy-b- -glucopyranosyl echinocystic acid 16-O-
b-
-glucopyranoside. Their structures were determined by NMR techniques including HOHAHA, 1H-1H
Received 28 February 2010
Received in revised form 15 April 2010
Available online 4 June 2010
D
D
D
D
D
a-L
D
D
a-L
Keywords:
Albizia procera
Leguminosae
a
-
L
D
D
COSY, ROE, HMQC and HMBC experiments together with FABMS as well as acid hydrolysis. To the best of
our knowledge, the new compounds are considered the first examples of echinocystic acid 3,16-O-bisglyco-
sides. In contrast to other cytotoxic echinocystic acid glycosides with N-acetyl glucosamine unit, the new
glycosides were found inactive when assayed by MTT method for their cytotoxicities against the human
tumor cell lines HEPG2, A549, HT29 and MCF7. The results showedthe importance of the free hydroxyl group
at the aglycone C-16 for exhibiting cytotoxic properties.
Triterpene glycosides
Echinocystic acid
Oleanolic acid
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
side (Yoshikawa et al., 1998). Similar antitumor acacic acid glyco-
sides julibrosides with variable monoterpene quinovosyl units at
The genus Albizia (Leguminosae) comprises about 150 species
distributed in Africa, Asia and Central and South America. In tradi-
tional Chinese medicine Albizia julibrissin is used for the treatment
of insomnia, irritability and wounds (Bown, 1995). A. lebbeck was
reported to possess antidysenteric, antiseptic and antitubercular
activities (Chadha, 1985). The Albizia members in Africa are used
in folk medicine for the treatment of rheumatism, cough, diarrhea
and injuries (Watt and Breyer-Brandwijk, 1962). Phytochemical
studies carried out on Albizia species led to the isolation of several
triterpene glycosides. The root bark of A. anthelmintica (Carpani
et al., 1989), the seeds of A. lucida (Orsini et al., 1991), the stems
of A. subdimidiata (Abdel-Kader et al., 2001) and the bark of A. pro-
C-21 and oligosaccharide moities at C-3 (trisaccharide substituted
by N-acetamido unit or tetrasaccharide), were reported from A.
julibrissin (Ikeda et al., 1995; Ma et al., 1996; Liang et al., 2005;
Zheng et al., 2006). Three antitumor acacic acid glycosides grandi-
bracteosides A–C acylated by an o-aminobenzoyl unit were iso-
lated from leaves of A. grandibracteata (Krief et al., 2005).
Cytotoxicity guided fractionation of the extract obtained from the
roots of Madagascan plant A. gummifera, led to the isolation of
three cytotoxic acylated acacic acid glycosides gummiferaosides
A–C (Cao et al., 2007). Two recent reports described the isolation
of further cytotoxic acylated acacic acid glycosides albizosides A–
C from A. chinensis (Liu et al., 2009) and coriariosides A and B from
A. coriaria (Note et al., 2009).
cera (Melek et al., 2007) afforded a number of N-acetyl-D-glucosa-
mine derivatives of oleanolic and echinocystic acids. Three acacic
acid lactone glycosides albiziasaponins A–C were obtained from
the bark of A. lebbeck (Pal et al., 1995). Proceraosides A–D obtained
from the seeds of A. procera were acacic acid glycosides bearing a
common oligosaccharide moiety at C-28 and acylated at C-21 with
a monoterpenic carboxylic acid linked to a monoterpene quinovo-
Albizia procera is a tree cultivated in streets and public gardens
in Egypt. In folk medicine, the bark is considered useful in preg-
nancy and stomachache. As a part of our continuous interest in bio-
active triterpene glycosides from plants grown in Egypt (Miyase
et al., 1996; Melek et al., 2002, 2003a,b, 2004a,b), we reported in
our previous communication (Melek et al., 2007) the isolation
and structure elucidation of four triterpene glycosides from the
bark of A. procera. Further investigation on the glycoside-contain-
ing fraction led to the isolation of further three new glycosides
1–3 and two known ones 6, 7. In this contribution, we describe
* Corresponding author.
E-mail address: frmelek@link.net (F.R. Melek).
0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2010.05.004

1376
T. Miyase et al. / Phytochemistry 71 (2010) 1375–1380
Table 1
the structure determination of 1–3 by NMR analysis using HOHA-
HA, 1H-1H COSY, ROE, HMQC and HMBC techniques, FAB-mass
spectra and acid hydrolysis.
1H NMR spectral data for compounds 1–3 in pyridin-d5.
1
2
3
Aglycone
3
5
3.48 (dd, 12.0, 4.0)
0.81
3.36 (dd, 13.0, 3.0)
0.81
3.36 (dd, 13.0, 3.0)
0.81
2. Results and discussion
12
16
18
19
190
23
24
25
26
27
29
30
5.50 (brt, 3.0)
5.33 (brs)
3.51
1.22
2.69 (t, 13.0)
1.25 (s)
1.00 (s)
0.84 (s)
1.10 (s)
1.89 (s)
5.54
5.34 (brs)
3.54
5.54
5.34 (brs)
3.54
The methanolic extract of the dried bark of A. procera was sub-
jected to a combination of HP-20 and silica gel column chromatog-
raphy followed by HPLC to afford five compounds including three
new ones 1–3.
1.22
1.22
2.68 (t, 13.0)
1.24 (s)
0.99 (s)
0.85 (s)
0.98 (s)
1.89 (s)
0.84 (s)
1.11 (s)
2.68 (t, 13.0)
1.24 (s)
0.99 (s)
0.85 (s)
0.98 (s)
1.89 (s)
0.84 (s)
1.11 (s)
Compound 1 showed in its positive ion FAB-mass spectrum a
quasi-molecular ion peak [M+Na]+ at m/z 1154 corresponding to
a molecular formula C55H89NO23. The 1H NMR spectrum of 1
showed seven angular methyl signals at d 0.80, 0.84, 0.98, 1.00,
1.10, 1.25 and 1.89 and a typical signal for axial proton attached
to a hydroxylate carbon at d 3.48 (dd, J = 12.0, 4.0 Hz). Further fea-
tures were vinylic proton signal at d 5.50 (brt, J = 3.0 Hz) and sugar
proton signals between d 3.59 and d 5.13. In the 13C NMR spectrum
of 1, the signals due to seven sp3 carbons and two sp2 carbons at d
122.5 and 144.9 coupled with the information from 1H NMR spec-
trum, indicated that 1 was a triterpene glycoside with a triterpene
moiety of oleanene skeleton. The deshielded position of the axial
methyl group at C-14 (Me-27, d 1.89) and the presence of a broad
singlet proton signal at d 5.33 (H-16) in the 1H NMR spectrum of 1,
indicated oxygenation at C-16. The assignments of the proton and
the carbon signals of the triterpene moiety were established by the
combined use of 1H–1H COSY, HMQC and HMBC experiments
which allowed identification of the triterpene moiety as echino-
cystic acid, a common triterpene of triterpene glycosides. The sig-
nal at d 179.5 and the carbon signals of D and E rings suggested the
occurrence of a free carboxylic group at C-28. The 3,16 bisdesmos-
idic nature of 1 was deduced from the presence of two deshielded
signals at d 88.6 and d 79.9 assigned to echinocystic acid C-3 and C-
16, respectively. The assignment of C-16 (d 79.9) was based on di-
rect correlation of its signal in the HMQC spectrum to that of H-16
(d 5.33, brs) which in turn showed HMBC correlations to the signals
of C-17 (d 48.5) and C-18 (d 41.7) in the HMBC experiment of 1.
Acid hydrolysis of 1 afforded echinocystic acid detected by TLC
0.80 (s)
0.98 (s)
3-O-sugar
GlcNHAc
1
2
5.07 (d, 8.5)
4.52 (ddd, 8.5, 8.5,
8.2)
4.33 (t, 8.5)
4.02
3.99 (m)
4.33 (dd, 11.0, 3.0)
4.72 (d, 11.0)
5.04 (d, 8.0)
4.50 (ddd, 8.5, 8.2,
8.0)
4.36 (t, 8.5)
4.11
5.04 (d, 8.0)
4.50 (ddd, 8.5, 8.2,
8.0)
4.36 (t, 8.5)
4.11
3
4
5
6
60
4.04 (m)
4.04 (m)
4.19 (dd, 11.0, 4.0)
4.61 (d, 11.0)
2.16 (s)
4.19 (dd, 11.0, 4.0)
4.61 (d, 11.0)
2.16 (s)
Me(CONH) 2.16 (s)
NH
8.87 (d, 8.2)
8.89 (d, 8.2)
8.89 (d, 8.2)
Gal
1
2
3
4
5
6
60
5.10 (d, 7.3)
4.52 (dd, 7.3, 8.0)
4.19 (dd, 8.0, 3.0)
4.55 (d, 2.5)
4.00
4.26
4.37
Ara
1
2
3
4
5.13 (d, 5.0)
4.50 (dd, 5.0, 6.0)
4.36
4.38 (brs)
3.75 (d, 11.0)
4.27 (dd, 11.0, 3.0)
5
50
and HPLC and the sugar components 2-amino-2-deoxy-
D
-glucose,
-xylose. The sugar 2-amino-2-deoxy-
-glucose was identified by paper chromatography and optical
Ara0
1
2
3
4
D-glucose, D-galactose and D
5.08 (d, 5.0)
4.52 (dd, 5.0, 5.5)
4.32
4.33 (brs)
3.72 (d, 10.0)
4.26
D
rotation value while the rest sugars were recognized by GC analy-
sis of their thiazolidine derivatives. The sugar portion of 1 exhib-
ited in the 13C NMR spectrum four anomeric carbon signals at d
101.7, 103.8, 104.7 and 106.8, a methyl carbon signal at d 23.8, a
carbonyl amide carbon signal at d 170.1 and a typical amide carbon
at d 58.1. Four corresponding anomeric proton signals were ob-
served in the HMQC spectrum. The structures of the four monosac-
charide units were deduced from the use of HOHAHA spectrum of
1 which recognized the spin system of each unit. The 1H–1H COSY
and ROE spectra allowed the sequential assignments of the proton
resonances to the individual monosaccharides as reported in Ta-
ble 1. The sugar unit with anomeric signal at d 5.07 (d, J = 8.5 Hz)
was assigned to a 2-acetamido-2-deoxy-glucose unit (Abdel-Kader
et al., 2001; Melek et al., 2007). The sugar with anomeric proton
signal at d 5.10 (d, J = 7.3 Hz) was due to a galactose unit while
5
50
Xyl
1
2
3
4
5.13 (d, 6.0)
4.07 (t, 6.0)
4.01
4.99 (d, 7.0)
4.00 (dd, 7.0, 8.0)
4.04 (t, 8.0)
4.02
4.12
5
50
3.59 (dd, 12.0, 9.0)
4.45 (dd, 12.0, 4.5)
3.60 (dd, 11.0)
4.38 (dd, 11.0, 5.0)
Ara00
1
2
3
4
5.05 (d, 5.5)
4.47 (dd, 5.5, 6.0)
4.10
4.23 (brs)
3.67 (d, 11.0)
4.42
5
50
the remaining sugars with anomeric signals at
d 5.00 (d,
J = 7.0 Hz) and 5.13 (d, J = 6.0 Hz) were attributable to glucose
and xylose units, respectively. The anomeric centers of the sugar
units were all determined to have a b-configuration based on large
3JH1,H2 values. The HMQC experiment allowed assignments of the
sugar carbons and showed that all the sugars were in the pyranose
form. It also allowed the assignments of the interglycosidic link-
ages by comparison of the observed carbon chemical shifts with
those of the corresponding methyl pyranoside (Seo et al., 1978).
16-O-Glc
1
2
3
4
5
6
60
5.00 (d, 7.0)
4.04 (dd, 7.0, 8.0)
4.28 (t, 8.0)
4.23 (t, 9.0)
4.00 (m)
5.01 (d, 7.0)
4.06 (dd, 7.0, 8.0)
4.29 (t, 8.0)
4.23
3.99 (m)
5.01 (d, 7.0)
4.06 (dd, 7.0, 8.0)
4.29 (t, 8.0)
4.23
3.99 (m)
4.35 (dd, 10.0, 3.5)
4.53 (dd, 10.0, 2.0)
4.36 (dd, 11.0, 2.5)
4.54 (dd, 11.0, 2.5)
4.36 (dd, 11.0, 2.5)
4.54 (dd, 11.0, 2.5)

T. Miyase et al. / Phytochemistry 71 (2010) 1375–1380
1377
of 2 and 3 exhibited a [M+Na]+ at m/z 1124 corresponding to a
molecular formula C54H87NO22 and compatible with a tetrasaccha-
ride glycoside containing an aglycone of molecular mass 472 and
four sugar units, one 2-acetamido-deoxy hexose, one hexose and
two pentoses. The 13C NMR spectrum showed one set of signals
due to echinocystic acid very similar to those of 1 but different sig-
nals due to the sugar part. Six anomeric carbon signals at d 101.6,
102.3, 102.5, 104.9, 105.5 and 106.2 correlated in the HMQC spec-
trum with anomeric proton signals at d 5.01 (d, J = 7.0 Hz), 5.08
(d, J = 5.0 Hz), 5.13 (d, J = 5.0 Hz), 5.04 (d, J = 8.0 Hz), 5.05 (d,
J = 5.5 Hz) and 4.99 (d, J = 7.0 Hz), were observed. The HOHAHA,
experiment of 2 and 3 allowed the sub-spectrum of each monosac-
charide unit to be extracted from the crowded overlapped sugar re-
gion. Starting from the anomeric proton signals, the sequential
assignments of the proton resonances to individual monosaccha-
rides were achieved using 1H–1H COSY spectrum with the aid of
ROE experiment. The sugar with anomeric proton signal at d 5.04
(J = 8.0 Hz) was assigned to a 2-acetamido-2-deoxy-b-glucose and
the sugar unit with anomeric proton signal at d 5.01 was attribut-
able to a b-glucose. The anomeric proton signals at d 5.05, 5.08
The absence of any 13C NMR glycosylation shifts for the b-
pyranosyl (Xyl) and b- -glucopyranosyl (Glc) units, suggested their
terminal positions. Glycosylation shifts were observed for C-6 (d
70.1) of the 2-acetamido-2-deoxy-b- -glucopyranosyl unit (Glc-
NHAc) and C-2 (d 82.1) of the b- galactopyranosyl unit (Gal).
D-xylo-
D
D
D
The sequence of the sugar units was unambiguously determined
from HMBC experiment. The HMBC correlation between the sig-
nals of the echinocystic acid C-3 (d 88.6) and H-1 (d 5.07) of Glc-
NHAc indicated direct attachment of this unit to the triterpene
moiety. The correlation between the signal of GlcNHAc C-6 (d
70.1) with that of Gal H-1 (d 5.10) established the connectivity be-
tween the two sugar units as Gal-(1 ? 6)-GlcNHAc and the corre-
lation between the signals of Gal C-2 (d 82.1) and Xyl H-1 (d 5.13)
confirmed the terminal position of the xylose unit. The correlation
between the signals of echinocystic acid C-16 (d 79.9) and Glc H-1
(d 5.00) supported by the reverse correlation between the signals
of H-16 (d 5.33) and Glc C-1 (d 101.7), unambiguously established
the glycosylation of the hydroxyl group at C-16 of the echinocystic
acid moiety. Accordingly, 1 was assigned as the structure of 3-O-b-
D
-xylopyranosyl-(1 ? 2)-b-
ido-2-deoxy-b- -glucopyranosyl echinocystic acid 16-O-b-
glucopyranoside.
D-galactopyranosyl-(1 ? 6)-2-acetam-
and 5.13 were assigned to three L-arabinoses while the remaining
D
D-
anomeric signal at d 4.99 was due to b-xylose. The anomeric centers
of the sugars 2-acetamido-2-deoxy-glucose, glucose and xylose
were each determined to have a b-configuration based on large
3JH-1,H-2. The
a-anomeric configuration of the arabinose was de-
3
duced from its JH-1,H-2 values (5.0–5.5 Hz) expected for an a-arab-
inose in rapid 4C1 M 1C4 conformational exchange. The HMQC
spectrum allowed assignments of the sugar carbons by direct corre-
lation with the assigned proton and showed that all the sugars were
in pyranose form. It also showed the presence of 6-substituted
2-acetamido-2-deoxy-b-
uted
-arabinopyranose (Ara, Ara0), one terminal
anose (Ara00) and one terminal b-
-xylopyranose (Xyl). The sugar
D
-glucopyranose (GlcNHAc), two 2-substi-
a-
L
a-L-arabinopyr-
D
pattern of the 3-O-sugar chains in 2 and 3 were determined by
the HMBC correlations. The cross peak correlated the signals of
echinocystic acid H-3 (d 3.36) and GlcNHAc C-1 (d 104.9), H-6 and
H-60 of GlcNHAc (d 4.19, 4.61) and Ara C-1 (d 102.5) as well as Ara0
C-1 (d 102.3) indicated that both 2 and 3 contained GlcNHAc unit
(one set of NMR signals for 2 and 3) attached to C-3 of echinocystic
acid and substituted at C-2 by Ara unit in 2 and Ara0 unit in 3. Also,
cross peaks correlated the signals of Ara H-2 (d 4.50) and Xyl C-1 (d
106.2) as well as Ara0 H-2 (d 4.52) and Ara00 C-1 (d 105.5) revealed
that the terminal Xyl unit was bound to C-2 of Ara unit in 2 while
the terminal Ara00 unit was connected to C-2 of Ara0 in 3. Accordingly
the structure of 3-O-sugar chain of 2 and 3 were deduced as Xyl-
(1 ? 2)-Ara-(1 ? 6)-GlcNHAc and Ara”-(1 ? 2)-Ara0-(1 ? 6)-Glc-
NHAc, respectively. The attachment of a remaining Glc unit (one
set of NMR signals in 2 and 3) to echinocystic acid C-16 in both 2
and 3, was established from the cross peak correlated the signals
of echinocystic acid H-16 (d 5.34) and Glc C-1 (d 101.6) and after
considering the tetrasaccharide nature of 2 and 3 from the FABMS
data. The close resemblance of sugar data of the 3-O-sugar chain
in 2 and 3 (Tables 1 and 2) and the corresponding ones in 4 and 5
(Melek et al., 2007), lent further support to the sugar pattern in 2
and 3. Therefore saponins 2 and 3 were assigned the structures
of 3-O-b-
2-acetamido-2-deoxy-b-
b- -glucopyranoside and 3-O-
arabinopyranosyl-(1 ? 6)-2-acetamido-2-deoxy-b-
echinocystic acid 16-O-b- -glucopyranoside, respectively.
D
-
xylopyranosyl-(1 ? 2)-
-glucopyranosyl echinocystic acid 16-O-
-arabinopyranosyl-(1 ? 2)-
-glucopyranosyl
a-L-arabinopyranosyl-(1 ? 6)-
D
D
a
-L
a-L-
D
D
Oligosaccharide chains of triterpene glycosides with oleanane
skeleton are usually attached at aglycone C-3 and or C-17 (via C-
28). There are few cases in which short saccharide chains of one
or two sugar residues only are attached at C-4, C-16, C-20, C-21
and C-22 (Vincken et al., 2007). Inspection of the literature re-
vealed that glycosylation at aglycone C-16 was only observed in
Compound 2 and 3 were obtained as inseparable mixture and gave
only one spot on HPTLC but two peaks by HPLC (1:1). Attempts to
separate 2 and 3 by preparative HPLC were unsuccessful. Acid
hydrolysis of 2 and 3 yielded echinocystic acid and the sugar com-
ponents 2-amino-2-deoxy-D-glucose, D-glucose, D-xylose and L-
arabinose identified as mentioned for 1. The FAB-mass spectrum

1378
T. Miyase et al. / Phytochemistry 71 (2010) 1375–1380
Table 2
Table 2 (continued)
13C NMR spectral data for compounds 1–3 in pyridine-d5.
1
2
3
1
2
3
16-O-Glc
1
2
3
4
5
6
Aglycone
1
2
3
4
5
6
7
8
101.7
75.8
79.4
72.4
78.1
63.3
101.6
75.7
79.4
72.4
78.1
63.3
101.6
75.7
79.4
72.4
78.1
63.3
38.7
26.7
88.6
39.4
55.9
18.6
33.5
40.0
47.3
37.1
23.8
122.5
144.9
41.3
30.5
79.9
48.5
41.7
46.5
30.9
36.3
32.6
28.3
17.0
15.5
17.4
27.4
179.5
33.4
24.9
38.7
26.6
89.1
39.3
55.9
18.6
33.5
39.9
47.2
37.1
23.9
122.5
144.9
41.4
30.4
79.8
48.5
41.7
46.5
30.9
36.3
32.6
28.3
17.0
15.5
17.4
27.4
179.5
33.4
24.9
38.7
26.6
89.1
39.3
55.9
18.6
33.5
39.9
47.2
37.1
23.9
122.5
144.9
41.4
30.4
79.8
48.5
41.7
46.5
30.9
36.3
32.6
28.3
17.0
15.5
17.4
27.4
179.5
33.4
24.9
9
a few saiko-saponins isolated from the Labiatae species Clinopodi-
um micranthum (Yamamoto et al., 1993) and the Scrophulariaceae
plant Verbascum sinaiticum (Miyase et al., 1997). Compounds 1–3
represent the first occurrence of triterpene 3,16-O-bisglycosides
in the family Leguminosae. The co-existed metabolites 4 and 5
might represent interesting intermediates in the biosynthesis of
glycosides 2 and 3.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
It is worth noting that triterpene glycosides with 2-acetamido-
2-deoxy-b-D-glucopyranose as an inner glycosyl unit at C-3 of the
triterpene moiety, have been reported before from the other Legu-
minosae members Pithecellobium cubense and P. arboretum (Rip-
perger et al., 1981), Tetrapleura tetraptera (Adesina and Reisch,
1985; Maillard et al., 1989) and Entada phaseoloides (Okada et al.,
1987, 1988a,b).
The known compounds 6 and 7 were identified to be Pithedulo-
side G and Pitheduloside F from NMR data and comparison with
the literature values (Nigam et al., 1997). Detailed 1H NMR data
of 6 and 7 are reported here for the first time (Table 3) based on
1D and 2D NMR analyses.
C-3-O-sugar
Glycosides 1, 2 and 3, 6, 7 were assayed for their cytotoxic activ-
ities against the human tumor cell lines HEPG2, A549, HT29 and
MCF7 using MTT method (Mosmann, 1983). In contrast to the cyto-
toxic glycosides with N-acetyl glucosamine unit 4 and 5 (16-O-
deglucosylated derivatives of 2 and 3), the glycosides 1, 2 and 3
showed no cytotoxic effects against the used cell lines. The results
showed the importance of the free hydroxyl group at C-16 of echi-
nocystic acid for the cytotoxic activity. On the other hand, the ole-
anolic acid glycoside 7 showed no cytotoxic effects while the
glycoside 6 exhibited only weak activity on HEPG2 cell line
GlcNHAc
1
2
3
4
5
6
C@O
104.7
58.1
75.7
72.6
75.8
70.1
170.1
23.8
104.9
58.0
75.7
72.8
76.1
69.6
170.1
23.7
104.9
58.0
75.7
72.8
76.1
69.6
170.1
23.7
Me (CONH)
Gal
1
2
3
4
103.8
82.1
75.3
69.8
76.8
62.1
(57 lg/ml).
5
6
3. Experimental
Ara
1
2
3
4
3.1. General
102.5
80.2
72.9
67.4
64.2
Optical rotations were measured with Jascodip-1000 digital
polarimeter. MS spectra were measured on Jeol JMS-SX 102 mass
spectrometer. NMR spectra were recorded on Jeol GSX-500 FT
NMR spectrometer. Chemical shifts are given on the d-scale with
TMS as internal standard. HPLC was performed on a Jasco system
800 instrument. GC analysis was carried out on Hitachi G-
3000 gas chromatograph.
5
Ara0
1
2
3
4
102.3
79.4
72.9
67.7
64.6
5
3.2. Plant material
Xyl
1
2
3
4
106.8
76.9
77.3
70.9
67.0
106.2
75.2
77.8
70.9
67.2
The bark of A. procera (Benth.) was collected from the zoological
garden in Giza, Egypt in June 2004. Plant identification was con-
firmed by Mrs. T. Labib, head specialist for plant identification in
El-Orman public garden, Giza, Egypt. The Herbarium voucher spec-
imen was deposited in the Herbarium of NRC (CAIRC).
5
Ara00
1
2
3
4
105.5
72.5
74.3
68.9
66.6
3.3. Extraction and isolation
5
Air-dried and powdered bark (750 g) of A. procera was extracted
twice with MeOH followed by once with 50% aqueous methanol.

T. Miyase et al. / Phytochemistry 71 (2010) 1375–1380
1379
The methanolic and aqueous methanolic extracts were combined
and the solvent was evaporated to dryness under reduced pres-
sure. The residue (27 g) was dissolved in water and the aqueous
solution was passed through a porous polymer gel column (Mitsu-
bishi Diaion HP-20). The column was then eluted with water, 50%,
75%, 85%, and 100% MeOH. The 85% MeOH and MeOH eluates were
combined and the material from the combined fraction (5.8 g) was
chromatographed on silica gel column eluted with CHCl3–MeOH–
H2O (70:27:3 – 58:35:7) and finally with MeOH to give 11 fractions
(A–K). Part (183 mg) of fraction A eluted with CHCl3–MeOH–H2O
(70:27:3) was subjected to repeated HPLC [column ODS,
2 mm ꢀ 25 cm; solvent, CH3CN–H2O (20:80 – 25:75); flow rate,
6.5 ml/min; detection, UV, 205 nm] to give 1 (14 mg) and mixture
of 2 and 3 (26 mg). Part (50 mg) of fraction F was subjected to HPLC
[column, capcell pak Ph, 2 mm ꢀ 25 cm; solvent, CH3CN–H2O
(32.5:67.5) + 0.05 TFA; flow rate, 9.5 ml/min; detection, UV,
205 nm] to afford 6 (14 mg) and 7 (10 mg).
3.4. Saponin 1
Amorphous powder ½a D23
ꢂ 15.1 (c = 1.79, MeOH), FABMS m/z:
ꢁ
1154 [C55H89NO23 + Na]+, 1H and 13C NMR (see Tables 1 and 2).
3.5. Saponins 2 and 3
Amorphous powder, FABMS m/z: 1124 [C54H87NO22 + Na]+, 1H
and 13C NMR (see Tables 1 and 2).
3.6. Saponin 6
Amorphous powder ½a D23
ꢁ
+ 7.0 (c = 1.50, MeOH), 1H NMR (see
+ 11.9 (c = 1.01, MeOH), 1H NMR (see
Table 3
1H NMR spectral data for compounds 6 and 7 in pyridin-d5.
Table 3).
6
7
Aglycone
3
5
3.7. Saponin 7
3.46 (dd, 12.0, 4.5)
0.85 (d, 4.0)
5.44 (brt, 3.5)
1.95 (dt, 12.0, 3.0)
2.18
3.27 (dd, 14.0, 4.0)
1.28
1.81
1.31 (s)
1.11 (s)
0.88 (s)
1.00 (s)
3.41 (dd, 12.0, 4.5)
0.86
5.45 (brt, 3.5)
1.95
Amorphous powder ½a D23
ꢁ
12
Table 3).
16
160
18
19
190
23
24
25
26
27
2.18
3.28 (dd, 14.0, 4.0)
1.27
1.81
1.33 (s)
1.11 (s)
0.87 (s)
1.00 (s)
1.31 (s)
0.94 (s)
3.8. General method for acid hydrolysis
A solution of 10 mg of saponin 1 (2 and 3) in 9 ml 2 N HCl and
9 ml CH3OH was heated at 100 °C for 6 h. The mixture was left to
cool, diluted with H2O and extracted with CHCl3. From the CHCl3
layer, the aglycone was detected by TLC using CHCl3–MeOH
(10:1) and HPLC [column, YMC R&D ODS, 4.6 mm ꢀ 25 cm; solvent
MeOH–H2O (9:1) + 0.05% TFA; flow rate, 1 ml/min; detection, UV
205 nm]. The H2O layer was repeatedly diluted with CH3OH and
1.32 (s)
0.94 (s)
1.01 (s)
29
30
1.02 (s)
3-O-sugar
Inner Glc
evaporated to dryness. 2-Amino-2-deoxy-D-glucose hydrochloride
was obtained from the residue by PPC using BuOH–AcOH–H2O
(4:1:5, upper layer) and identified by comparing its PC Rf and
1
2
3
4
5
6
60
4.88 (d, 7.0)
4.24
4.24
4.10
3.99 (m)
4.20
4.88 (d, 7.0)
4.24
4.23
4.13
3.96 (m)
4.21
[a] values with those of authentic sample. The other sugar com-
D
ponents were detected from the H2O Layer by GC analysis after
being converted to their thiazolidine derivatives as described by
Hara et al. (1986); conditions: [column, Supelco SPB™ ꢂ1,
4.60 (dd, 11.5, 2.5)
4.58 (dd, 11.5, 2.5)
0.25 mm ꢀ 27 m; column temperature 215 °C; carrier gas, N2].
D
-
-
Inner Ara
1
2
3
4
5
50
5.12 (d, 5.0)
4.49 (dd, 5.0, 6.0)
4.36
4.31 (brs)
3.75 (dd, 11.0, 2.5)
4.30
5.08 (d, 5.0)
4.52(dd, 5.0, 6.0)
4.33
4.34 (brs)
3.73 (dd, 11.0, 2.5)
4.30
glucose,
glucose,
D
-galactose and
D-xylose were detected from 1 while
D
D
-xylose and -arabinose were detected from 2 and 3.
L
3.9. Cytotoxic assay
The procedure for the cytotoxic assay was performed by MTT
assay as described by Mosmann (1983). In this study, the cell lines
HEPG2, A549, HT29 and MCF7 were used.
Xyl
1
2
4.98 (d, 7.0)
4.01 (dd, 7.0, 8.0)
4.05 (t, 8.0)
3
4
5
50
4.12
References
3.61 (dd, 11.0, 10.0)
4.39 (dd, 11.0, 5.0)
Abdel-Kader, M., Hoch, J., Berger, J.M., Evans, R., Miller, J.S., Wisse, J.H., Mamber,
S.W., Dalton, J.M., Kingston, D.G.I., 2001. Two bioactive saponins from Albizia
subdimidiata from the Suriname rainforest. J. Nat. Prod. 64, 536–539.
Adesina, S.K., Reisch, J., 1985. A triterpenoid glycoside from Tetrapleura tetraptera
fruit. Phytochemistry 24, 3003–3006.
Bown, D., 1995. Encyclopedia of Herbs and their Uses. Dorling Kindersley, London.
p. 232.
Cao, S., Norris, A., Miller, J.S., Ratovoson, F., Razfitsalama, J., Andriantsiferana, R.,
Rasamison, V.E., Tendyke, K., Suh, T., Kingston, D.G.I., 2007. Cytotoxic
triterpenoid saponins of Albizia gummifera from Madagscar rain forest. J. Nat.
Prod. 70, 361–366.
Carpani, G., Orsini, F., Sisti, M., Verotta, L., 1989. Saponins from Albizia anthelmintica.
Phytochemistry 28, 863–866.
Chadha, Y.R., 1985. The Wealth of India. Raw Materials; Publication and Information
Directorate, vol. 1A. Council of Scientific and Industrial Research, New Delhi. p.
126.
Terminal Ara
1
2
3
4
5
50
5.04 (d, 6.5)
4.47
4.12
4.28 (brs)
3.71 (dd, 12.0, 2.0)
4.41 (dd, 12.0, 3.5)
Terminal Glc
1
2
3
4
5
6
60
5.35 (d, 7.5)
4.09 (dd, 7.5, 8.0)
4.22 (t, 8.0)
4.31
3.92 (m)
4.30
5.35 (d, 8.0)
4.10 (t, 8.0)
4.22 (t, 8.0)
4.33
3.91 (m)
4.31
Hara, S., Okabe, H., Mihashi, K., 1986. Separation of aldose enantiomers by gas–
liquid chromatography. Chem. Pharm. Bull. 34, 1843–1845.
4.46
4.45

1380
T. Miyase et al. / Phytochemistry 71 (2010) 1375–1380
Ikeda, T., Fujiwara, S., Araki, K., Kinjo, J., Nohara, T., Ida, Y., Shoji, J., Shingu, T., Isobe,
R., Kajimoto, T., 1995. A cytotoxic saponin with two monoterpenoids from
Albizia julibrissin. Bull. Chem. Soc. Jpn. 68, 3483–3490.
Nigam, S.K., Gopal, M., Uddin, R., Yoshikawa, K., Kawamoto, M., Arihara, S., 1997.
Pithedulosides A–G, oleanane glycosides from Pithecellobium dulce.
Phytochemistry 44, 1329–1334.
Krief, S., Thoison, O., Sevenet, T., Wrangham, R.W., Lavaud, C., 2005. Triterpenoid
saponins anthranilates from Albizia grandibracteata leaves ingested by primates
in Uganda. J. Nat. Prod. 68, 897–903.
Liang, H., Tong, W., Zhao, Y., Cui, J., Tu, G., 2005. An antitumor compound julibroside
J28 from Albizia julibrissin. Bioorg. Med. Chem. Lett. 15, 4493–4495.
Liu, R., Ma, S., Yu, S., Pei, Y., Zhang, S., Chen, X., Zhang, J., 2009. Cytotoxic oleanane
triterpene saponins from Albizia chinensis. J. Nat. Prod. 72, 632–639.
Ma, L., Tu, G., Chen, S., Zhang, R., Lai, L., Xu, X., Tang, Y., 1996. NMR determination of
the structure of julibroside J1. Carbohydr. Res. 281, 35–46.
Note, O.P., Mitaine-offer, A.C., Miyamoto, T., Paululat, T., Mirjolet, J.F., Duchamp, O.,
Pegnyemb, D.E., Lacaille-Dubois, M.A., 2009. Cytotoxic acacic acid glycosides
from the roots of Albizia coriaria. J. Nat. Prod. 72, 1725–1730.
Okada, Y., Shibata, S., Ikekawa, T., Javellana, A.M.J., Kamo, O., 1987. Entada saponin-
III, a saponin isolated from the bark of Entada phaseoloides. Phytochemistry 26,
2789–2796.
Okada, Y., Shibata, S., Javellana, A.M.J., Kamo, O., 1988a. Entada saponins (ES) II
and IV from the bark of Entada phaseoloides. Chem. Pharm. Bull. 36, 1264–
1269.
Maillard, M., Adewunmi, C.O., Hostettman, K., 1989. New triterpenoid N-acetyl
glycosides with molluscicidal activity from Tetrapleura tetraptera TAUB. Helv.
Chim. Acta 72, 668–674.
Okada, Y., Shibata, S., Kamo, O., Okuyama, T., 1988b. 13C NMR spectral studies
of entagenic acid to stablish its structure. Chem. Pharm. Bull. 36, 5028–
5030.
Melek, F.R., Miyase, T., Abdel-Khalik, S.M., Hetta, M.H., Mahmoud, I.I., 2002.
Triterpenoid saponins from Oreopanax guatemalensis. Phytochemistry 60,
185–195.
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.
Melek, F.R., Miyase, T., Ghaly, N.S., Yousif, M.F., 2004a. Further saponins from Meryta
lanceolata. Phytochemistry 65, 909–914.
Orsini, F., Pelizzoni, F., Verotta, L., 1991. Saponins from Albizia lucida.
Phytochemistry 30, 4111–4115.
Pal, B.C., Achari, B., Yoshikawa, K., Arihara, S., 1995. Saponins from Albizia lebbeck.
Phytochemistry 38, 1287–1291.
Ripperger, H., Preiss, A., Schmidt, J., 1981. O(3)-(2-acetylamino-2-deoxy-b-
glucopyranosyl)-oleanolic acid, novel triterpenoid glycoside from two
Pithecellobium species. Phytochemistry 20, 2434–2435.
D-
A
Seo, S., Tomita, Y., Tori, K., Yoshimura, Y., 1978. Determination of the configuration
of secondary hydroxyl group in a chiral secondary alcohol using glycosidation
shifts in carbon-13 nuclear magnetic resonance spectroscopy. J. Am. Chem. Soc.
100, 3331–3339.
Melek, F.R., Miyase, T., Abdel-Khalik, S.M., Mahmoud, I.I., Mina, S.A., 2004b.
Saponins
and
acylated
saponins
from
Dizygotheca
kerchoveana.
Phytochemistry 65, 3089–3095.
Vincken, J.P., Heng, L., Groot, A., Gruppen, H., 2007. Saponins, classification and
occurrence in plant kingdom. Phytochemistry 68, 275–297.
Watt, J.M., Breyer-Brandwijk, M.G., 1962. Medicinal and Poisonous Plants of South
and East Africa, second ed. Livingstone, Edinburgh. p. 553.
Yamamoto, A., Suzuki, H., Miyase, T., Ueno, A., Maeda, T., 1993. Clinoposaponins VI
and VIII, two oleanane-triterpene saponins from Clinopodium micranthum.
Phytochemistry 34, 485–488.
Yoshikawa, K., Satou, Y., Tokunaga, Y., Tanaka, M., Arihara, S., Nigam, S.K., 1998. Four
acylated triterpenoid saponins from Albizia procera. J. Nat. Prod. 61, 440–445.
Zheng, L., Zheng, J., Zhao, Y., Wang, B., Wu, L., Liang, H., 2006. Three anti-tumor
saponins from Albizia julibrissin. Bioorg. Med. Chem. Lett. 16, 2765–2768.
Melek, F.R., Miyase, T., Ghaly, N.S., Nabil, M., 2007. Triterpenoid saponins with N-
acetyl sugar from the bark of Albizia procera. Phytochemistry 68, 1261–1266.
Miyase, T., Melek, F.R., El-Gindi, O.D., Abdel-Khalik, S.M., El-Gindi, M.R., Haggag, M.Y.,
Hilal, S.H., 1996. Saponins from Fagonia arabica. Phytochemistry 41, 1175–1179.
Miyase, T., Horikoshi, C., Yabe, S., Miyasaka, S., Melek, F.R., Kusano, G., 1997.
Saikosaponins homologues from Verbascum spp. The structures of
mulleinsaponins I–VII. Chem. Pharm. Bull. 45, 2029–2033.
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J. Immunol. Methods 65,
55–63.