Triterpenoid saponins from Aesculus sylvatica W. Bartram

Article abstract of DOI:10.1016/j.phytol.2015.09.011

16 triterpenoid saponins including two new compounds were isolated from the seeds of A esculus sylvatica W. Bartram. The two new saponins were assigned as 3-O-[β-D-glucopyranosyl-(1 → 2)]-α-L-arabinofuranosyl-(1 → 3)-β-D-glucuronopyranosyl-21,22-O-ditigloyl-3β,16α,21β,22α,24,28 hexahydroxyolean-12-ene (aesculioside S1, 1) and 3-O-[β-D-glucopyranosyl-(1 → 2)]-α-L-arabinofuranosyl-(1 → 3)-β-D-glucuronopyranosyl-21-O-tigloyl-22-O-angeloyl 3β,16α,21β,22α,24,28-hexahydroxyolean-12-ene (aesculioside S2, 2). Aesculioside S1 and S2 displayed moderate cytotoxicity against human non-small cell lung cancer cells (A549) and prostate cancer cells (PC3) (GI₅₀ ranged from 8.7 to 18.2 μM). The structural analysis of the saponins isolated from Aesculus supports the taxonomic placement of A. sylvatica under the section Pavia of Aesculus genus.

Full text of DOI:10.1016/j.phytol.2015.09.011

Phytochemistry Letters 14 (2015) 111–114
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Triterpenoid saponins from Aesculus sylvatica W. Bartram
Wei Yuan , Ping Wang , Zushang Su , Ruixin Gaoa,b, Shiyou Lia,*
a
b
a
a
National Center for Pharmaceutical Crops, Arthur Temple College of Forestry and Agriculture, Stephen F. Austin State University, Nacogdoches, TX 75962-
6
109, USA
b
College of Life Science, Northeast Forestry University, 26 Hexing Road, Harbin, Heilongjiang 150040, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 19 June 2015
Received in revised form 11 September 2015
Accepted 21 September 2015
Available online xxx
16 triterpenoid saponins including two new compounds were isolated from the seeds of A esculus
sylvatica W. Bartram. The two new saponins were assigned as 3-O-[
-L-arabinofuranosyl-(1 !3)-
2)]- -D-glucuronopyranosyl-21,22-O-ditigloyl-3
hexahydroxyolean-12-ene (aesculioside S1, 1) and 3-O-[
-D-glucopyranosyl-(1 !2)]-
-D-glucuronopyranosyl-21-O-tigloyl-22-O-angeloyl ,16 ,21 ,22a
b
b
-D-glucopyranosyl-(1
,16 ,21 ,22 ,24,28
-L-arabinofur-
,24,28-hexahy-
!
a
b
a
b
a
b
a
anosyl-(1 !3)-
b
3b
a
b
droxyolean-12-ene (aesculioside S2, 2). Aesculioside S1 and S2 displayed moderate cytotoxicity
Keywords:
against human non-small cell lung cancer cells (A549) and prostate cancer cells (PC3) (GI50 ranged from
Aesculus sylvatica
Painted buckeye
Triterpenoid saponins
Cytotoxicity
8.7 to 18.2 mM). The structural analysis of the saponins isolated from Aesculus supports the taxonomic
placement of A. sylvatica under the section Pavia of Aesculus genus.
ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Chemotaxonomy
1. Introduction
2. Results and discussion
Aesculus L. is a genus of Sapindaceae with 12 shrub or small
deciduous tree species in the northern hemisphere (Zhang et al.,
010). As a remarkable example of an intercontinental disjunction
From 400 g A. sylvatica seeds, two new oleanane saponins (1–2)
together with 14 known saponins (3–16) were isolated (Fig. 1). By
analysis of their NMR spectral data, the known saponins were
identified as aesculioside IIe (3) (Zhang and Li, 2007), aesculioside
IIf (4) (Zhang and Li, 2007), aesculioside IIg (5) (Zhang and Li,
2007), aesculioside IIh (6) (Zhang and Li, 2007), aesculioside IIk (7)
(Zhang and Li, 2007), aesculioside IIj (8) (Zhang and Li, 2007),
aesculioside IIId (9) (Zhang and Li, 2007), aesculioside IIIe (10)
(Zhang and Li, 2007), aesculioside IIIf (11) (Zhang and Li, 2007),
aesculioside G6 (12) (Yuan et al., 2012), xanifolia-Y8 (13)
(Chan et al., 2008; Voutquenne et al., 2005), aesculioside G9
(14) (Yuan et al., 2012), aesculioside G10 (15) (Yuan et al., 2012),
and xanifolia-Y10 (16) (Voutquenne et al., 2005). Known saponins
3–11 were previously reported in A. pavia (Zhang et al., 2006). 12,
14 and 15 were reported in A. glabra (Yuan et al., 2012). 13 was
reported from A. pavia, A. glabra, Xanthoceras sorbifolia, and
Harpullia austro-caledonica (Chan et al., 2008; Voutquenne et al.,
2005; Yuan et al., 2012; Zhang et al., 2006).16 was reported from A.
pavia, X. sorbifolia, and H. austro-caledonica (Chan et al., 2008;
Voutquenne et al., 2005; Yuan et al., 2012; Zhang et al., 2006).
The structures of the two new saponins (1-2) were elucidated by
extensive analysis of their spectroscopic data as well as chemical
analysis.
2
genus, the taxonomy, biogeography, and evolution of Aesculus have
attracted great attention from botanists (Harris and Xiang, 2009;
Harris et al., 2009; Modliszewski et al., 2006; Xiang et al., 1998).
The phylogenetic and biogeographic analysis of Aesculus was made
using molecular and morphological data with some controversial
results (Harris et al., 2009; Xiang et al., 1998). Since 2004, we have
conducted extensive phytochemical investigations of North
America Aesculus species to identify bioactive saponins against
human cancer (Yuan et al., 2012, 2013; Zhang and Li, 2007; Zhang
et al., 2010, 2006). 61 polyhydroxyoleanene saponins including
5
6 previously unknown compounds were reported from A. pavia L.
and A. glabra Willd. distributed in eastern North America and A.
californica (Spach) Nutt. distributed in western North America. The
phytochemical data of these Aesculus species formed a basis for
medical uses of Aesculus and its triterpenoid saponins and
provided new evidences for understanding systematics and
evolutionary pattern of the genus. In the present study, we report
6 triterpenoid saponins isolated from Aesculus sylvatica W.
Bartram (commonly known as painted buckeye), another species
of Aesculus in the southeastern USA.
1
87 24
The molecular formula of 1 was determined to be C57H O
ꢀ
13
from its HR-ESI-MS data ([M-H] m/z, 1155.5589) and C NMR
spectrum (Table 1). Similar to the saponins previously reported
from the genus Aesculus, the NMR data of 1 displayed signals of a
*
Corresponding author. Fax: +1 936 468 7058.
E-mail address: lis@sfasu.edu (S. Li).
http://dx.doi.org/10.1016/j.phytol.2015.09.011
874-3900/ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1

112
W. Yuan et al. / Phytochemistry Letters 14 (2015) 111–114
3
JH1, H2 coupling constant (1H, d, J = 7.0). The 1H-NMR and 13C-
NMR signal assignments of a-arabinofuranosyl were in accordance
with those of previous reported Aesculus saponins. The HMBC
correlation of glucuronic acid anomeric protons at 4.94 (1H, d, J
7.2 Hz) with the downfield shift signal of C-3 at 91.2 established
d
H
=
d
C
the linkage of glucuronic acid to C-3 of the aglycone. The
0
attachment of D-glucopyranosyl and L-arabinofuranosyl to C-2
0
and C-3 of D-glucuronopyranosyl acid were determined by their
0
HMBC correlations, H-1 of Glc-p and Ara-f (
d
H
5.52 and 6.08) to C-2
0
(d
C
78.0) and C-3 (
d
C
86.3) of GlcA, respectively. Therefore, the
-D-glucopyranosyl-(1 !2)]-
-D-glucuronopyranosyl-21,22-O-
,24,28-hexahydroxyolean-12-ene (aes-
structure of 1 was assigned as 3-O-[
-L-arabinofuranosyl-(1 !3)-
ditigloyl-3 ,16 ,21 ,22
culioside S1).
Compound 2 was assigned a molecular formula of C57
b
a
b
b
a
b
a
H
87
O
24
ꢀ
from its negative HR-ESI-MS ion peak at m/z 1155.5583 [M-H] ,
same as aesculioside S1 (1). By detailed analysis and comparison of
1
13
H NMR, C and HSQC spectra, the signals of 2 were almost
superimposable to those of 1 in terms of the triterpenoid aglycone
and oligosaccharide side chain. The trisaccharide groups of 2 were
also confirmed as D-glucuronic acid, D-glucose and L-arabinose by
acid hydrolysis and HPLC analysis. Compared with 1, one of the
1
tigloyl groups apparently disappeared in 2. The H-NMR spectra of
2
H
showed a set of characteristic angeloyl signals at d 5.87 (1H, q,
J = 7.1 Hz), 2.03 (3H, d, J = 7.1 Hz), and 1.89 (3H, s). The angeloyl
groups were established as C-22 substitution by HMBC correla-
tions of angeloyl carboxyl carbons at
corresponding H-22 at 6.40 (3H, d, J = 10.2 Hz). The structure
of 2 was then elucidated as 3-O-[ -L-
-D-glucopyranosyl-(1!2)]-
arabinofuranosyl-(1 !3)- -D-glucuronopyranosyl-21-O-tigloyl-
2-O-angeloyl-3 ,16 ,21 ,22 ,24,28-hexahydroxyolean-12-ene
aesculioside S2).
Aesculioside S1 (1) and S2 (2) were evaluated for their
cytotoxicity against human lung adenocarcinoma epithelial cell
A549) and human prostate cancer cells (PC3). Aesculioside S1 (1)
exhibited cytotoxic activity against A549 and PC3 with GI50 values
of 18.2 ꢁ 4.3 and 13.6 ꢁ 2.1 M, respectively (doxorubicin as
positive control, GI50 against A549 and PC3 for 0.58ꢁ0.04 and
.81 ꢁ0.06 M, respectively). The cytotoxicity of Aesculioside S2
2) against A549 and PC3 was evaluated at GI50 values of
M, respectively. The cytotoxicity data of
C
d 168.5 with the
d
H
b
a
b
b
2
(
b
a
a
Fig. 1. The structures of triterpenoid saponins 1–16 from Aesculus sylvatica.
(
3
0-carbon olean-12-ene type aglycone, two five carbon side chains
and a trisaccharide chain. Following detailed analyses of the NMR
spectroscopic data (COSY, HSQC, HMBC and NOESY) and compari-
son with literature data, the triterpenoid skeleton was identified as
protoaescigenin or 3b,16a,21b,22a,24,28-hexahydroxyolean-12-
ene as in compounds 6 and 13 (Voutquenne et al., 2005; Zhang and
Li, 2007). The relatively high field shift of the H-23 methyl signal at
m
0
(
m
11.7 ꢁ1.3 and 8.7 ꢁ2.5
m
these polyhydroxyoleanene saponins are consistent with the
similar compounds isolated from A. pavia and A. glabra (Yuan et al.,
d
H
0.66 and downfield shift of C-3 at
4 hydroxylation. The relatively high field shift of H-7 (
.55) and the relatively low field shift observed for methyl in
position 27 ( 1.85 and 27.6, respectively) revealed no hydroxyl
substitution at C-15 but oxygen bearing at C-16. There were two
d
C
91.2 indicated a C-
2
1
d , 1.27,
H
2
012; Zhang and Li, 2007,2006).
Our previous investigations found that the structures of
d
H
d
C
triterpenoid saponins isolated from A. pavia and A. glabra of the
section Pavia in eastern North America can be easily distinguished
from those from other sections of Aesculus in Asia, Europe and
western North America and all saponins in these two species are
characterized by trisaccharide chains with an arabinofuranosyl
unit fixed to C-3 of the glucuronopyranosyl unit (Yuan et al., 2012;
Zhang et al., 2010). A. sylvatica is usually placed in the section Pavia
with A. pavia, A. glabra, and A. flava distributed in the same region.
All 16 saponins isolated from A. sylvatica have an arabinofuranosyl
unit affixed to C-3 of the glucuronopyranosyl unit in the
trisaccharide chain. This result supports the placement of A. pavia,
A. glabra, and A. sylvatica under the same Pavia section.
As reported before, all the saponins in A. glabra have no
hydroxyl group at C-24 (Yuan et al., 2012). However, saponins in
both A. sylvatica and A. pavia have either hydroxyl or no hydroxyl
group at C-24. Based on analysis of the saponin structures, A.
sylvatica may be positioned more closely to A. pavia than to A.
glabra within the Pavia section (Fig. 2). This phylogenetic
relationship among A. pavia, A. glabra, and A. sylvatica drawn from
the saponin structure similarity agrees with the phylogeny derived
from combined analysis of internal transcribed spacer (ITS)
1
sets of tigloyl group signals in H-NMR spectra of 1 (
J = 7.1),1.63 (3H, d, J = 7.1), and 1.93 (3H, s) and 6.95 (1H, q, J = 7.1),
.45 (3H, d, J = 7.1), and 1.84 (3H, s), respectively). By analysis of
H
d 7.07 (1H, q,
d
H
1
COSY, HSQC, and HMBC spectra, the signals of the two tigloyl
groups were assigned unambiguously at C-21 and C-22, respec-
tively. The presence of trisaccharide residues were indicated by
three anomeric proton signals at
d, J = 7.0 Hz), 6.08 (1H, br s) and the corresponding anomeric
carbons at 104.2, 103.3, and 110.7. Acid hydrolysis of 1 afforded
H
d 4.94 (1H, d, J = 7.2 Hz), 5.52 (1H,
d
C
D-glucuronic acid, D-glucose and L-arabinose as analyzed follow-
ing aldose derivatization and HPLC analysis (Tanaka et al., 2007;
Yuan et al., 2013). By HSQC, DQF-COSY, HMBC and 2D-TOCSY, it was
possible to characterize one
pyranosyl and one -arabinofuranosyl moieties. The glucuronic
acid was determined to be in pyranose form from the C NMR
spectroscopic data. The -anomeric configuration of glucuronic
acid was determined according to the 3JH1, H2 coupling constants
1H, d, J = 7.2). The D-glucopyranosyl was determined to be in
-pyranose form according to its 13C-NMR spectroscopic data and
b-glucuronopyranosyl, one b-gluco-
a
13
b
(
b

W. Yuan et al. / Phytochemistry Letters 14 (2015) 111–114
113
Table 1
1
H NMR and 13C NMR spectroscopic data for compounds 1–2 (400 MHz in pyridine-d
5
).
1
2
1
2
d
H
d
C
d
H
d
C
d
H
d
C
d
H
d
C
1
2
3
4
5
6
7
8
9
0.84 m, 1.36 m
1.96 m, 2.31 m
3.45 (dd, 11.5, 3.0)
–
38.8
26.6
91.2
43.7
55.7
18.3
33.2
40.6
46.3
37.2
23.9
123.3
143.0
41.5
35.1
68.7
48.6
40.0
0.80 m, 1.37 m
1.91 m, 2.30 m
3.44 (dd, 11.0, 3.0)
–
38.8
26.9
91.8
43.7
56.3
18.9
33.5
40.4
47.2
36.7
24.3
123.9
143.6
41.8
35.2
68.9
48.4
40.6
C
1
2
3
4
5
C
1
2
3
4
5
C
1
2
3
4
5
21
22
3
Tig
–
Tig
–
168.4
129.9
137.1
14.5
168.2
129.8
137.1
14.1
–
–
7.07 (q, 7.1)
1.63 (d, 7.1)
1.93 s
Tig
–
7.09 (q, 7.0)
1.64 (d, 7.0)
1.94 s
Ang
–
0.89 m
1.55 m
1.27 m, 1.55 m
–
0.88
1.53
1.27, 1.55
–
12.7
12.9
168.8
129.4
137.5
14.0
168.5
129.5
136.6
16.2
1.69 m
-
1.76 m, 1.86 m
5.44
–brs
–
1.67
–
–
–
1
0
6.95 (q, 7.1)
1.45 (d, 7.1)
1.84 s
5.87 (q, 7.1)
2.03 (d, 7.1)
1.89 s
1
1
1.74 m, 1.84 m
5.42 br s
1
2
12.7
21.3
1
3
–
GlcA-p
GlcA-p
1
4
–
4.94 (d, 7.2)
4.27 (dd, 8.9, 7.2)
4.26 (t, 8.9)
4.50 (t, 8.9)
4.57 (d, 8.9)
104.2
78.0
86.3
71.5
77.1
4.92 (d, 7.4)
4.27 (dd, 8.2, 7.4)
4.27 (t, 8.2)
4.51 (t, 8.2)
4.58 (d, 8.2)
105.1
78.5
87.0
72.0
77.9
1
5
1.64 m, 1.88 (1H, d, 10.0)
4.51 m
–
1.61 m, 1.82 (1H, d, 11.0)
4.51 m
1
6
1
7
–
18
3.13 (dd,
3.13 (dd,
13.6, 3.2)
1.14 m, 3.31 (t, 13.6)
–
13.1, 2.9)
1
9
1.46 m, 3.16 (t, 13.1)
–
47.2
37.1
79.0
73.5
22.1
63.1
15.4
16.9
47.8
36.9
79.5
73.8
22.6
63.5
15.9
17.1
6
C
1
2
3
4
5
6
172.0
172.1
2
0
2
0
Glc-p
Glc-p
2
1
6.74 (d, 10.0)
6.35 (d, 10.0)
1.37 s
3.31, 4.31 (d, 11.0)
0.66 s
6.69 (d, 10.2)
6.40 (d, 10.2)
1.37 s
3.32, 4.31 (d, 10.5)
0.65 s
5.52 (d, 7.0)
4.08 (dd, 9.0, 7.0)
4.27 (t, 9.0)
4.59 (dd, 9.0, 8.7)
3.66 m
103.3
75.2
78.0
69.2
78.0
61.2
5.52 (d, 7.0)
4.08 (dd, 9.1, 7.0)
4.27 (t, 9.1)
4.58 (dd, 9.1, 8.6)
3.66 m
104.0
75.6
78.5
69.7
78.6
61.9
2
2
2
2
2
2
3
4
5
6
0.81 s
0.81 s
4.36 m, 4.48 (dd,
4.35 m, 4.47(dd,
11.2, 7.3)
10.9, 7.3)
27
28
29
30
1.85 s
3.42, 3.66 (d, 11.0)
1.14 s
27.6
63.1
29.5
20.1
1.85 s
3.44, 3.67 (d, 11.0)
1.13 s
28.0
64.1
29.9
20.7
C
1
2
3
4
3
0
Ara-f
6.08 (br s)
5.00 m
4.81 br d, 5.8
4.89 (td,
Ara-f
6.08 (br s)
5.00 m
4.82 br d, 5.6
4.89 (td,
110.7
83.5
77.1
111.8
84.0
78.0
85.8
1.37 s
1.35 s
85.4
5
.8, 3.4)
5.6, 3.4)
5
4.21 (dd, 11.1, 3.4), 4.36 m
62.2
4.18 (dd, 10.5, 3.4), 4.34 m
62.9
sequence and morphological dataset (Xiang et al., 1998). In a more
recent reconstructed phylogeny inferred from a combination of
DNA sequence (LFY, trnHK, rps16, matK and ITS), morphology and
fossil information, A. pavia, A. glabra, and A. sylvatica were still
placed in the same eastern North America section Pavia. However,
within the section Pavia, A. pavia was placed as the sister of the
group of A. sylvatica and A. glabra (Harris et al., 2009). More new
emerging data contribution seemed to make the taxonomy of
Aesculus genus more complicated and controversial at this point.
Further investigations are needed to resolve the long time
taxonomy controversy.
Saponins from A. californica have more diversified structures
which share more similar features to eastern Asian species than to
eastern North American species (Yuan et al., 2013). This chemical
evidence agrees to its closer relationship to the Asian clade in
phylogeny derived from DNA and morphology data (Harris et al.,
into two different monotypic sections Macrothyrsus and Parryana,
respectively. The chemical investigation of these two species will
provide important clues to understand the systematics and
evolutionary pattern of the genus Aesculus.
3. Experimental
3.1. General experimental procedures
NMR experiments were performed on a JEOL ECS 400 spec-
trometer, with spectroscopic data referenced to the solvent used.
HR-mass spectra were acquired using a Waters Q-Tof Premier
mass spectrometer. HPLC analysis was performed on an Agilent
1260HPLC system using Agilent ODS columns (Column A:
Poroshell 120 EC-C18, 2.7
mm). Preparative HPLC was performed
with a Lab-Alliance Series III Isocratic HPLC System using an
2
009). According to analysis of the saponins isolated from Aesculus
Alltima C18 column (Column B: 250 ꢂ 22 mm, 10
mm, Grace).
to date, we expect that A. flava in the section Pavia have similar
saponins with the other species in the same section. Due to their
distinctive morphological features, other two American Aesculus
species, A. parviflora Walter and A. parryi A. Gray are usually placed
3.2. Plant material
Seeds of A. sylvatica were collected from Georgia, USA in 2012.
The plant sample was identified by Dr. Shiyou Li and the voucher
specimen (Ga-Fayetteville-NNN-10122012-AS-Fr) was deposited
at the National Center for Pharmaceutical Crops at Stephen F.
Austin State University, USA.
A. sylvatica
Eastern North America
A. pavia
Section Pavia
3
.3. Extraction and isolation
A. glabra
The seeds of A. sylvatica (400 g, dry weight) were ground to a
coarse powder using a Thomas Model 4 Wiley Mill. The powder
was percolated with 4L methanol using a 75 ꢂ 600 mm glass
1
Fig. 2. The phylogenic relationship among three eastern North America Aesculus
species A. pavia,A. glabra, and A. sylvatica derived from their saponin structure data.

114
W. Yuan et al. / Phytochemistry Letters 14 (2015) 111–114
column. After evaporated under reduced pressure, the methanol
extracts were pooled, concentrated and partitioned with n-hexane
to remove compounds of low polarity (n-hexane phase, 7 g). The
lower layer extracts were dried to give 19 g powder as the raw
saponin fraction. The raw saponin was ground and mixed with 30 g
octadecyl-functionalized silica gel (C18) and loaded onto a pre-
equilibrated open ODS column (75 ꢂ 200 mm). The column was
eluted with 45%, 65%, 80% and 100% methanol in water (each for
2007; Yuan et al., 2013). By retention time comparison and co-
HPLC, the absolute configuration of sugars in each hydrolysis
was identified as D-glucose, L-Arabinose and D-Glucuronic acid
(Rt observed at 15.1, 16.3 and 17.9 min, respectively).
3.7. Cytotoxicity assays
The cytotoxicity assays were performed using a Cell Counting
Kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan) as
previously described (Wang et al., 2010). Cytotoxicity was reported
1
L). The eluents were collected with a Spectra/Chrom CF-2 Fraction
Collector (Houston, TX, USA) (50 mL for each fraction) and
analyzed with Agilent 1260HPLC (Column A: 45% acetonitrile in
as GI50 (GI = [1-(T
before the treatment. T
D
-T
0
/T
C
-T
0
)] ꢂ 100. T
0
was the initial reading
represents the readings of the
0
.5% HOAc water, 0.5 mL/min, 210 nm). Sub-fractions were
C
or T
D
subjected for further preparative HPLC separation according to
the analysis results. The sub-fractions 7-9 were combined and
separated by preparative HPLC system (column B, 40% acetonitrile
in 0.5% HOAc water, 3 mL/min, 210 nm) to yield 3 (54.3 min, 3.7 mg)
and 4 (65.0 min, 6.3 mg). The sub-fractions 21–25 furnished 5
untreated control or with the tested compounds. Doxorubicin was
used as the positive cytotoxicity control (GI50 against A549 and PC3
for 0.58 ꢁ 0.04 and 0.81 ꢁ0.06
mM, respectively).
Acknowledgments
(
54.3 min, 2.8 mg) and 6 (65.0 min, 7.1 mg) (column B, 46%
acetonitrile in 0.5% HOAc water, 3 mL/min, 210 nm). 7 (54.3 min,
1.4 mg) and 8 (65.0 min, 7.1 mg) were isolated from the sub-
This study was funded by USDA grants (2008-38928-19308 and
2009-38928-19744). The NMR spectral analysis was conducted
using a JEOL ECS-400 NMR spectrometer purchased through the
Research Development Program at Stephen F. Austin State
University. The authors would like to thank Vic Parcell of University
of Iowa for HRESI-MS analysis; Dr. David Kulhavy of Stephen F.
Austin State University for reviewing the manuscript.
1
fractions 28 and 29 by preparative HPLC system (column B, 46%
acetonitrile in 0.5% HOAc water, 3 mL/min, 210 nm). The sub-
fractions 32–33 were combined and separated by a preparative
HPLC system (column B, 50% acetonitrile in 0.5% HOAc water, 3 mL/
min, 210 nm) to afford 9 (50.8 min, 7.4 mg),10 (53.4 min, 8.6 mg),11
(
(
57.3 min, 9.6 mg) and 12 (60.5 min, 7.5 mg).1 (55.1 min,10.4 mg), 2
57.8 min, 11.7 mg), and 13 (60.7 min, 8.0 mg) were prepared from
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fraction 39 give 14 (56.3 min, 6.3 mg),15 (59.8 min,12.8 mg), and 16
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3
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.5. 3-O-[
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b
,16a
,21
b
,22a
,24,28-hexahydroxyolean-12-ene (aesculioside S2, 2)
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Acid hydrolysis of aesculioside S1 (1) and S2 (2) and sugar
determination followed a method described before (Tanaka et al.,