Cytotoxic triterpenoid saponins from the fruits of Aesculus pavia L.

Article abstract of DOI:10.1016/j.phytochem.2007.05.020

Continued chemical investigation on the fruits of North American Aesculus pavia L. resulted in the isolation and identification of 13 polyhydroxyoleanene pentacyclic triterpenoid saponins, named aesculiosides IIe-IIk (1-7), and IIIa-IIIf (8-13), together with 18 known compounds: aesculiosides Ia-Ie (14-18), IIa-IId (19-22), IVa-IVc (23-25), 3-O-[β-d-galactopyranosyl(1 → 2)]-α-l-arabinofuranosyl(1 → 3)-β-d-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α,16α,21β,22α,28-hexahydroxyolean-12-ene (26), 3-O-[β-d-glucopyranosyl(1 → 2)]-α-l-arabinofuranosyl(1 → 3)-β-d-glucuronopyranosyl-21,22-O-diangeloyl-3β,16α,21β,22α,24β,28-hexahydroxyolean-12-ene (27), 3-O-[β-d-galactopyranosyl(1 → 2)]-α-l-arabinofuranosyl(1 → 3)-β-d-glucuronopyranosyl-21,22-O-diangeloyl-3β,16α,21β,22α,28-pentahydroxyolean-12-ene (28), R₁-barrigenol (29), scopolin (30), and 5-methoxyscopolin (31). The structures of these compounds were elucidated by spectroscopic and chemical analyses. Compounds 14-22 and 26-28 were tested in vitro for their activity against 59 cell lines from nine different human cancers including leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate, and breast. It was found that compounds with two-acyl groups at C-21 and C-22 had cytotoxic activity for all cell lines tested with GI₅₀ 0.175-8.71 μM, while compounds without acyl groups at C-21 and C-22 had weak or no cytotoxic activity. These results suggest that the acyl groups at C-21 and C-22 are essential for their activity.

Full text of DOI:10.1016/j.phytochem.2007.05.020

PHYTOCHEMISTRY
Phytochemistry 68 (2007) 2075–2086
www.elsevier.com/locate/phytochem
Cytotoxic triterpenoid saponins from the fruits of Aesculus pavia L.
*
Zhizhen Zhang, Shiyou Li
National Center for Pharmaceutical Crops, Arthur Temple College of Forestry and Agriculture, Stephen F. Austin State University,
Nacogdoches, Texas 75962-6109, USA
Received 30 November 2006; received in revised form 26 February 2007
Available online 27 June 2007
Abstract
Continued chemical investigation on the fruits of North American Aesculus pavia L. resulted in the isolation and identification of 13
polyhydroxyoleanene pentacyclic triterpenoid saponins, named aesculiosides IIe–IIk (1–7), and IIIa–IIIf (8–13), together with 18 known
compounds: aesculiosides Ia–Ie (14–18), IIa–IId (19–22), IVa–IVc (23–25), 3-O-[b-D-galactopyranosyl(1 ! 2)]-a-L-arabinofurano-
syl(1 ! 3)-b-D-glucuronopyranosyl-21,22-O-diangeloyl-3b,15a,16a,21b,22a,28-hexahydroxyolean-12-ene (26), 3-O-[b-D-glucopyrano-
syl(1 ! 2)]-a-L-arabinofuranosyl(1 ! 3)-b-D-glucuronopyranosyl-21,22-O-diangeloyl-3b,16a,21b,22a,24b,28-hexahydroxyolean-12-ene
(27), 3-O-[b-D-galactopyranosyl(1 ! 2)]-a-L-arabinofuranosyl(1 ! 3)-b-D-glucuronopyranosyl-21,22-O-diangeloyl-3b,16a,21b,22a,28-
pentahydroxyolean-12-ene (28), R1-barrigenol (29), scopolin (30), and 5-methoxyscopolin (31). The structures of these compounds were
elucidated by spectroscopic and chemical analyses. Compounds 14–22 and 26–28 were tested in vitro for their activity against 59 cell lines
from nine different human cancers including leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate, and breast.
It was found that compounds with two-acyl groups at C-21 and C-22 had cytotoxic activity for all cell lines tested with GI50 0.175–
8.71 lM, while compounds without acyl groups at C-21 and C-22 had weak or no cytotoxic activity. These results suggest that the acyl
groups at C-21 and C-22 are essential for their activity.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Aesculus pavia L.; Hippocastanaceae; Cytotoxic activity; Triterpenoid saponins; Aesculiosides
1. Introduction
structure elucidation of these new compounds and the
cytotoxic activity of some saponins.
Aesculus pavia L., known as ‘‘red buckeyes’’ or ‘‘scarlet
buckeye’’, is a shrubby or small tree species naturally dis-
tributed in the southeastern United States (Little, 1980).
Previous work on this plant indicated the presence of
carotenoids (Neamtu and Bodea, 1974) and sapogenins
(Schrutka-Rechtenstamm et al., 1988). We recently isolated
and identified 12 new polyhydroxyoleanene pentacyclic tri-
terpenoid saponins aesculiosides Ia–Ie, IIa–IId, IVa–IVc
from a plant located in Nacogdoches, Texas (Zhang
et al., 2006). From a plant of different provence growing
in the same location, we surprisedly isolated 13 new triter-
penoid saponins named aesculiosides IIe–IIk (1–7) and
IIa–IIIf (8–13). In this paper, we report the isolation and
2. Results and discussion
The column fraction Aes-P from an ethanol extract of
the fruits of A. pavia L. was separated by a low-pressure
column of ODS to give four fractions Aes-I, Aes-II, Aes-
III, and Aes-IV. Further separation of Aes-I, Aes-II, Aes-
III and Aes-IV was achieved by preparative MPLC and
HPLC using a preparative ODS column and an analytical
ODS column to give compounds 1–31, respectively (see
Section 4).
Known compounds 14–18, 30, and 31 were obtained
from fraction Aes-I, and identified by NMR and MS
analysis as aesculiolisides Ia–Ie (14–18) (Zhang et al.,
2006), scopolin (30) (Zhang et al., 2005), and 5-methoxy-
*
Corresponding author. Tel.: +1 936 468 2071; fax: +1 936 468 7058.
E-mail address: lis@sfasu.edu (S. Li).
0031-9422/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2007.05.020

2076
Z. Zhang, S. Li / Phytochemistry 68 (2007) 2075–2086
scopolin (31) (Wagner and Bladt, 1975). While known com-
pounds 23–29 were obtained from fraction Aes-IV and
identified by NMR and MS analysis as aesculiolisides
IVa–IVc (23–25) (Zhang et al., 2006), 3-O-[b-D-galactopyr-
anosyl (1 ! 2)]-a-L-arabinofuranosyl(1 ! 3)-b-D-glucur-
2006), and R1-barrigenol (29) (Zhang et al., 2006),
respectively.
Compounds 1–7 and 19–22 were obtained from fraction
Aes-II as colorless powders. Compounds 19–22 were iden-
tified by NMR and MS analysis and comparison with ref-
erence data as known saponins, aesculiosides IIa–IId
(19–22) (Zhang et al., 2006).
Each of the compounds 1–7 contained either a tigeloyl
or an angeloyl group at C-21 and an acetyl group at C-
22, as indicated by the characteristic NMR signals of these
groups (Tables 1 and 3) (Yoshikawa et al., 1996, 1998;
Zhang et al., 1999, 2006). The downfield chemical shifts
at d 6.59–6.62 (1H, d, 10.0–10.1 Hz) and d 6.22–6.27
(1H, d, 10.0–10.1 Hz) were assigned to H-21 and H-22,
onopyranosyl-21,22-O-diangeloyl-3b,15a,16a,
21b,22a,
28-hexahydroxyolean-12-ene (26) (Chan et al., 2005), 3-O-
[b-D-glucopyranosyl(1 ! 2)]-a-L-arabinofuranosyl(1 ! 3)-
b-D-glucuronopyranosyl-21,22-O-diangeloyl-3b,16a,21b,
22a,24b,28-hexahydroxyolean-12-ene (27) (Voutquenne
et al., 2005; Zhang et al., 2006), 3-O-[b-D-galactopyranosyl-
(1 ! 2)]-a-L-arabinofuranosyl(1 ! 3)-b-D-glucuronopyrano-
syl-21,22-O-diangeloyl-3b,16a,21b,22a,28-pentahydroxy-
olean-12-ene (28) (Voutquenne et al., 2005; Zhang et al.,
Table 1
13C NMR spectroscopic data for the aglycone moieties of 1–13 (150 MHz in pyridine-d5)a
Carbon
1
2
3
4
5
6
7
8
9
10
38.7
11
38.3
12
38.5
13
38.6
1
2
3
4
5
6
7
8
38.4
26.5
91.3
43.5
55.6
18.4
36.4
40.2
46.9
36.6
23.7
124.9
143.5
47.5
67.5
72.7
48.1
40.6
46.6
36.3
78.9
73.6
21.9
63.0
15.4
17.1
20.9
62.8
29.3
19.9
38.8
26.6
89.8
39.6
55.5
18.7
36.6
40.8
47.1
36.9
23.9
125.3
143.5
47.7
67.4
73.4
48.1
41.0
46.8
36.3
79.2
73.4
27.9
16.7
15.7
17.5
21.0
63.4
29.4
20.0
38.8
26.6
89.9
39.6
55.6
18.8
36.6
40.9
47.1
36.9
23.9
125.4
143.6
47.7
67.5
72.8
48.3
41.4
46.8
36.1
78.9
73.7
27.9
16.8
15.7
17.5
20.6
63.2
29.4
20.1
38.4
26.4
91.2
43.6
55.8
18.3
32.9
40.1
46.7
36.5
23.7
123.6
143.2
41.6
34.4
67.8
48.1
40.2
47.2
36.3
79.1
74.0
22.0
63.1
15.5
16.5
27.2
63.5
29.5
19.9
38.4
26.5
91.5
43.6
56.0
18.4
33.1
39.9
46.7
36.6
24.0
123.5
142.8
41.6
34.6
68.0
48.0
40.1
47.1
36.3
78.9
74.3
22.2
63.2
15.5
16.6
27.4
63.8
29.4
20.2
38.4
26.5
89.7
39.8
55.9
18.3
32.9
40.0
46.8
36.7
23.4
123.5
142.9
41.5
34.2
68.0
47.9
40.1
47.0
36.2
79.0
74.1
27.7
16.5
15.5
16.8
27.1
63.4
29.4
19.9
38.6
26.3
89.7
39.7
55.6
18.1
33.0
40.0
46.8
36.7
23.6
123.5
142.8
41.6
34.3
67.9
47.8
40.1
47.0
36.2
78.9
74.0
27.7
16.5
15.5
16.8
27.2
63.6
29.4
20.0
38.3
26.5
91.0
43.5
55.9
18.3
33.0
39.6
46.8
36.5
23.6
123.7
142.8
41.8
34.5
67.9
48.1
40.2
47.3
36.2
80.9
70.9
22.0
63.1
15.5
16.6
27.1
66.3
29.6
19.9
38.5
26.5
91.2
43.6
56.0
18.5
36.5
40.5
47.1
36.6
23.5
125.3
143.5
47.5
67.6
73.2
48.1
40.7
46.9
36.2
79.0
73.3
22.1
63.6
15.8
17.5
20.9
63.3
29.7
20.1
26.5
89.6
39.9
55.6
18.6
36.6
40.0
47.0
36.7
23.6
125.3
143.6
47.6
67.5
73.1
48.0
40.6
46.9
36.2
79.0
73.4
27.9
16.7
15.6
17.4
20.9
63.1
29.5
19.9
26.3
91.0
43.5
55.7
18.4
36.5
39.9
46.9
36.5
23.7
124.9
143.0
47.5
67.4
73.3
48.0
40.6
46.7
36.2
78.9
73.6
22.0
63.1
15.5
17.1
20.7
62.9
29.3
19.9
26.4
91.1
43.6
55.8
18.6
36.5
40.0
46.9
36.7
23.7
125.2
143.1
47.6
67.5
73.3
47.9
40.6
46.7
36.2
78.9
73.4
22.0
63.2
15.5
17.0
20.8
63.0
29.3
19.9
26.5
89.6
39.7
55.8
18.5
36.6
40.6
46.9
36.8
23.6
125.1
143.5
47.6
67.6
73.6
48.1
40.9
46.8
36.3
79.0
73.8
27.8
16.8
15.7
17.4
20.8
63.0
29.3
19.9
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
C21
1
2
3
4
Tig
Tig
Ang
Tig
Ang
Tig
Ang
Ang
Ang
Tig
Tig
Tig
Tig
168.0
129.4
137.0
14.0
168.0
129.3
137.0
14.2
167.9
129.1
137.2
15.8
168.1
129.2
136.8
13.9
167.8
128.9
137.1
15.9
167.9
129.0
136.8
13.9
168.0
129.0
137.0
15.6
168.0
129.6
136.9
15.6
167.9
129.7
137.0
15.6
168.2
129.9
136.9
14.1
168.2
129.8
136.8
13.8
168.0
129.7
136.8
13.9
167.9
129.8
137.0
14.0
5
12.3
12.4
21.0
12.2
21.0
12.3
20.9
20.6
20.6
12.3
12.1
12.2
12.3
C22 or C28
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Mp
Mp
Tig
Ang
Tig
1
2
3
4
5
a
170.8
20.7
170.8
20.7
170.7
20.9
170.8
20.8
170.9
20.8
170.6
20.6
170.6
20.7
170.7
20.7
176.9
34.6
19.2
19.5
177.0
34.7
19.3
19.5
168.5
129.3
137.0
13.6
168.3
129.5
136.9
15.3
168.3
129.5
137.1
13.9
11.9
20.4
12.2
Assignments were based on COSY, HMQC, and HMBC experiments.

Z. Zhang, S. Li / Phytochemistry 68 (2007) 2075–2086
2077
Table 2
13C NMR spectroscopic data for the sugar moieties of 1–13 (150 MHz in pyridine-d5)a
Carbon
1
2
3
4
5
6
7
8
9
10
11
12
13
GlcA-p
1
2
3
4
5
6
104.5
78.0
86.2
69.5
77.1
171.9
105.1
78.1
86.2
69.7
77.1
172.0
105.4
78.0
86.3
69.7
77.2
171.9
104.5
78.0
86.3
71.6
77.1
172.0
104.8
78.2
86.6
71.7
77.3
171.9
104.2
78.2
86.1
69.5
77.3
171.9
104.8
78.2
86.1
69.5
78.5
171.9
104.5
78.2
86.1
69.5
76.7
171.9
105.0
78.4
86.3
69.6
77.3
169.1
104.9
78.4
86.2
69.7
77.3
169.0
104.2
78.1
86.3
69.5
77.2
171.9
104.2
78.2
86.2
69.5
77.1
171.9
104.6
78.5
86.3
69.6
77.2
171.9
Glc-p
103.7
75.0
78.0
69.5
78.2
61.2
Gal-p
104.7
73.9
75.0
69.7
76.6
61.8
Gal-p
104.7
73.4
75.1
69.7
76.6
61.8
Glc-p
103.6
75.2
78.1
69.5
78.2
61.2
Glc-p
103.9
75.5
78.0
69.5
78.3
61.3
Gal-p
104.3
73.2
75.0
69.7
76.4
61.9
Gal-p
104.5
73.3
75.0
69.5
76.4
61.9
Glc-p
103.5
75.3
78.2
69.5
78.1
61.5
Glc-p
103.7
75.5
78.4
69.6
78.6
61.6
Gal-p
104.6
73.2
75.0
69.7
76.5
61.9
Glc-p
103.5
75.3
78.2
69.5
78.2
61.7
Glc-p
104.0
75.3
78.2
69.5
78.3
61.7
Gal-p
104.4
73.5
75.0
69.5
76.6
62.0
1
2
3
4
5
6
Ara-f
1
2
3
4
5
a
111.2
83.2
77.5
85.0
62.3
111.0
83.5
77.5
85.4
62.3
111.0
83.5
77.7
85.4
62.4
110.9
83.4
77.5
85.2
62.3
111.2
83.6
77.5
85.4
62.3
110.6
83.2
77.5
85.2
62.5
110.8
83.3
77.6
85.2
62.3
110.9
83.1
77.9
85.1
62.7
110.3
83.6
77.8
85.5
63.0
110.1
83.6
77.9
85.4
62.6
110.0
83.5
77.6
85.2
62.4
110.8
83.6
77.6
85.2
62.5
110.7
83.5
77.9
85.6
62.3
Assignments were based on COSY, HMQC, and HMBC experiments.
1
respectively, based on the HMBC correlations. The ange-
loyl or tigenoyl group at C-21 and the acetyl group at C-
22 were established by HMBC correlations of H-21 of
the aglycone with C-1 of the angeloyl or tigenoyl group,
and H-22 of the aglycone with C-1 of the acetyl group. Full
assignments of the 13C and 1H NMR signals were achieved
2006). The H and 13C NMR spectra of 1 displayed char-
acteristic signals (Tables 1 and 3) of a tigeloyl group (Yos-
hikawa et al., 1996, 1998; Zhang et al., 1999) and an acetyl
group (dC 170.8, 20.7 and dH 2.00) (Tables 1 and 3). As
observed in the HMBC spectrum, the long-range correla-
tions of H-21 (d 6.59, 1H, d, 10.0 Hz) of the aglycone with
C-1 (d 168.0) of the tigeloyl unit, and H-22 (d 6.27, 1H, d,
10.0 Hz) of the aglycone with C-1 of the acetyl unit estab-
lished that the tigeloyl and acetyl groups were attached to
C-21 and C-22, respectively. The downfield chemical shifts
of H-21 and H-22 also indicated that C-21 and C-22 were
the acyl positions. The identity of the monosaccharides
and the sequence of the trisaccharide chain were deter-
mined by a combination of 1H–1H COSY, HMQC,
HMBC, and ROESY experiments, and by comparison
with reference data (Zhang et al., 2006). After the assign-
ments of the protons and protonated carbons were estab-
lished (Tables 2 and 4), the three sugar units were
identified as glucuronic acid, glucose and arabinose (Yos-
hikawa et al., 1996, 1998; Zhang et al., 1999, 2006). From
1
by a combination of H, 13C, COSY, HMQC, and HMBC
spectroscopic analyses.
Aesculioside IIe (1) was assigned a molecular formula of
C54H84O24, as deduced from the main [M + Na]+ ion m/z
at 1139.5248 in the positive HRESIMS as well as from
its NMR spectroscopic data. The NMR data (Tables 1–4)
of 1 were characteristic of a polyhydroxyoleanene triter-
penoid glycoside. After detailed NMR spectra interpreta-
tion, the aglycone was identified as 3b,15a,16a,21b,
22a,24b,28-heptahydroxyolean-12-ene (1a) (Zhang et al.,
2006). The stereochemistry of the aglycone was confirmed
by a NOESY experiment (Fig. 1) (Tang et al., 2004). Com-
pound 1 exhibited three sugar anomeric protons at d 4.80
(1H, d, J = 7.8 Hz), 5.47 (1H, d, J = 7.6 Hz), 6.06 (1H, br
s) (Table 4) and three protonated carbons at d 104.5,
103.7, 111.2 (Table 2) in HMQC spectrum. The 13C
NMR spectrum of 1 displayed 54 carbon signals, from
which 30 were assigned to the aglycone part, 17 to the
trisaccharide moiety, five to a tigeloyl group, and the
remaining two to an acetyl group. Alkaline hydrolysis of
1 liberated prosapogenin that was identified as aesculioli-
side Ib (15) (Zhang et al., 2006) by co-HPLC analysis with
the authentic sample. Acid hydrolysis of 1 produced three
monosaccharide sugars (D-glucuronic acid (GlcA), D-glu-
cose (Glu), and L-arabinose (Ara)), which were identified
by co-TLC analysis and by measurement of optical rota-
tion after separation by preparative TLC (Zhang et al.,
their 13C NMR spectroscopic data and JH1,H2 coupling
3
constants (Tables 2 and 4), the glucuronic acid and glucose
units were determined to be in the pyranose form with a
b-anomeric configuration, and arabinose to be in the
a-furanose form (Zhang et al., 2006). The whole sugar
sequence of 1 was deduced from the HMBC and ROESY
information as described in Fig. 1. The structure of
aesculioside IIe (1) was established as 3-O-[b-D-glucopyr-
anosyl(1 ! 2)]-a-L-arabinofuranosyl(1 ! 3)-b-D-glucuron-
opyranosyl-21-O-tigeloyl-22-O-acetyl-3b,15a,16a,21b,22a,
24b,28-heptahydroxyolean-12-ene.
Aesculioside IIf (2) gave a [M + Na]+ ion m/z at
1123.5299 in the positive HRESIMS, 16 mass units lower

2078
Z. Zhang, S. Li / Phytochemistry 68 (2007) 2075–2086
Table 3
1H NMR spectroscopic data for the aglycone moieties of 1–7 (600 MHz in pyridine-d5)a
Proton
1
2
3
4
5
6
7
1
0.85, 1.43
0.81, 1.40
0.81, 1.42
0.82, 1.32
0.78, 1.40
0.82, 1.42
0.84, 1.41
2
2.16 (2H, m)
2.12 (2H, m)
2.18 (2H, m)
2.28 (2H, m)
2.29 (2H, m)
2.12 (2H, m)
2.14 (2H, m)
3
3.45 (1H, dd-like) 3.27 (1H, dd-like) 3.29 (1H, dd-like) 3.44 (1H, dd-like) 3.44 (1H, dd-like) 3.30 (1H, dd-like) 3.28 (1H, dd-like)
5
0.90
0.82
0.82
0.84
0.86
0.80
0.80
6
7
9
1.60, 1.62
2.01, 2.08
1.67
1.59, 1.62
2.05, 2.12
1.70
1.57, 1.59
2.00, 2.07
1.66
1.51, 1.56
1.26, 1.53
1.72
1.53, 1.55
1.28, 1.53
1.67
1.50, 1.57
1.30, 1.58
1.72
1.51, 1.53
1.30, 1.57
1.72
11
12
15
16
18
19
21
1.90, 1.95
5.52 (1H, br s)
4.20
4.45
3.08
1.80, 1.90
5.51 (1H, br s)
4.19
4.45
3.05
1.46, 3.06
6.57 (1H, d,
10.0 Hz)
6.25 (1H, d,
10.0 Hz)
1.29 (3H, s)
1.19 (3H, s)
1.73, 1.87
5.51 (1H, br s)
4.21
4.52
3.03
1.43, 3.07
6.61 (1H, d,
10.1 Hz)
6.22 (1H, d,
10.1 Hz)
1.30 (3H, s)
1.19 (3H, s)
1.72, 1.90
5.40 (1H, br s)
1.61, 1.87
4.46
1.64, 1.87
5.39 (1H, br s)
1.61, 1.90
4.47
1.72, 1.90
5.44 (1H, br s)
1.62, 1.88
4.47
1.77, 1.88
5.41 (1H, br s)
1.64, 1.85
4.47
3.07
3.04
3.09
3.07
1.43, 2.09
6.59 (1H, d,
10.0 Hz)
6.27 (1H, d,
10.0 Hz)
1.31 (3H, s)
3.32 (1H, d,
10.0 Hz), 4.26
0.87 (3H, s)
1.03 (3H, s)
1.86 (3H, s)
3.47, 3.72 (each,
1H, d, 10.0 Hz)
1.14 (3H, s)
1.36 (3H, s)
1.40, 3.08
6.59 (1H, d,
10.0 Hz)
6.26 (1H, d,
10.0 Hz)
1.35 (3H, s)
3.31 (1H, d,
10.5 Hz), 4.25
0.65 (3H, s)
0.80 (3H, s)
1.83 (3H, s)
3.39, 3.65 (each,
1H, d, 10.8 Hz)
1.11 (3H, s)
1.34 (3H, s)
1.42, 3.10
6.62 (1H, d,
10.1 Hz)
6.22 (1H, d,
10.1 Hz)
1.36 (3H, s)
3.32 (1H, d,
10.6 Hz), 4.31
0.65 (3H, s)
0.81 (3H, s)
1.83 (3H, s)
3.39, 3.66 (each,
1H, d, 10.6 Hz)
1.10 (3H, s)
1.32 (3H, s)
1.50, 3.10
6.60 (1H, d,
10.0 Hz)
6.27 (1H, d,
10.0 Hz)
1.29 (3H, s)
1.18 (3H, s)
1.42, 3.10
6.62 (1H, d,
10.0 Hz)
6.22 (1H, d,
10.0 Hz)
1.31 (3H, s)
1.19 (3H, s)
22
23
24
25
26
27
28
0.85 (3H, s)
1.01 (3H, s)
1.85 (3H, s)
3.45, 3.65 (each,
1H, d, 10.0 Hz)
1.12 (3H, s)
1.34 (3H, s)
0.86 (3H, s)
1.03 (3H, s)
1.86 (3H, s)
3.46, 3.71 (each,
1H, d, 10.9 Hz)
1.12 (3H, s)
1.33 (3H, s)
0.84 (3H, s)
0.87 (3H, s)
1.85 (3H, s)
3.40, 3.64 (each,
1H, d, 10.6 Hz)
1.12 (3H, s)
1.36 (3H, s)
0.83 (3H, s)
0.86 (3H, s)
1.84 (3H, s)
3.39, 3.65 (each,
1H, d, 10.3 Hz)
1.11 (3H, s)
1.33 (3H, s)
29
30
C21
3
Tig
7.13 (1H, q,
7.0 Hz)
Tig
7.11 (1H, q,
7.0 Hz)
Ang
5.98 (1H, q,
7.2 Hz)
Tig
7.11 (1H, q,
7.0 Hz)
Ang
5.98 (1H, q,
7.2 Hz)
Tig
7.0 (1H, q,
7.0 Hz)
Ang
5.99 (1H, q,
7.1 Hz)
4
5
1.67 (3H, d,
7.0 Hz)
1.99 (3H, s)
1.66 (3H, d,
7.0 Hz)
1.97 (3H, s)
2.13 (3H, d,
7.2Hz)
2.04 (3H, s)
1.66 (3H, q,
7.0 Hz)
1.97 (3H, s)
2.12 (3H, d,
7.2Hz)
2.04 (3H, s)
1.66 (3H, d,
7.0 Hz)
1.99 (3H, s)
2.11 (3H, d,
7.1 Hz)
2.04 (3H, s)
C22
2
Ac
Ac
Ac
Ac
Ac
Ac
Ac
2.00 (3H, s)
2.03 (3H, s)
1.99 (3H, s)
1.92 (3H, s)
1.94 (3H, s)
1.92 (3H, s)
1.94 (3H, s)
a
Assignments were based on COSY, HMQC, and HMBC experiments. Due to severe overlapping in the 1H spectrum only detectable relative J (Hz) are
reported.
than that of 1, implying the absence of an oxygen-bearing
it was concluded that the glucose in 1 was replaced in 2
by a galactose, based on the differences of their 13C
NMR chemical shifts (Table 2) (Yoshikawa et al., 1996,
1998; Zhang et al., 1999, 2006). The linkage of the trisac-
charide moiety 2 were also deduced from the HMBC and
NOESY information as depicted for 1. The structure of
aesculioside IIf (2) was established as 3-O-[b-D-galactopyr-
anosyl(1 ! 2)]-a-L-arabinofuranosyl(1 ! 3)-b-D-glucuron-
opyranosyl-21-O-tigeloyl-22-O-acetyl-3b,15a,16a,21b,22a,
28-hexahydroxyolean-12-ene.
Aesculioside IIg (3) had the same molecular formula
of C54H84O23 as 2 deduced from the [M + Na]+ ion m/z
at 1123.5295 in the positive HRESIMS. An extensive
NMR spectroscopic study suggested that 3 and 2 differ
structurally only in the acyl substitute at C-21. The char-
acteristic NMR signals at dC 14.2 (C-4), 12.4 (C-5) and
dH 7.11 (1H, q, J = 7.0 Hz, H-3), 1.66 (3H, q, J =
7.0 Hz, H-4) of the tigeloyl group in 2 were replaced
function in 2. The 13C NMR spectrum of 2 also showed
54 signals as 1, of which 30 were assigned to the aglycone,
17 to the oligosaccharide moiety, and the remaining seven
to two other groups identified from the NMR spectro-
scopic data as tigeloyl (Yoshikawa et al., 1996, 1998;
Zhang et al., 1999) and acetyl groups (Tables 1 and 3).
Detailed NMR spectroscopic analysis indicated that the
aglycone of 2 was somewhat different from that of 1
based on the different chemical shifts of C-4, C-23, and
C-24 (Table 1), due to the substituent at C-24. The
–CH2OH group at C-24 in 1 was replaced by a methyl
group in 2, thus the aglycone of 2 was assigned as R1-
barrigenol (2a). The presence of three sugar units in 2
was indicated by the anomeric protons at d 4.84 (1H, d,
J = 7.2 Hz), 5.34 (1H, d, J = 7.2 Hz), and 6.06 (1H, br
s), and the protonated carbons at d 105.1, 104.7, and
111.0 observed in the HMQC spectrum. By NMR analysis,

Z. Zhang, S. Li / Phytochemistry 68 (2007) 2075–2086
2079
by the characteristic NMR resonances at dC 15.8 (C-4),
21.0 (C-5) and dH 5.98 (1H, q, J = 7.2 Hz, H-3), 2.13
(3H, q, J = 7.2 Hz, H-4) of an angeloyl group in 3, indi-
cating 3 had an angeloyl substitute at C-21. Accordingly,
the structure of aesculioside IIg (3) was established as
3-O-[b-D-galactopyranosyl(1 ! 2)]-a-L-arabinofuranosyl (1 ! 3)-
b-D-glucuronopyranosyl-21-O-angeloyl-22-O-acetyl-3b,
15a,16a,21b,22a,28-hexahydroxyolean-12-ene.
glucopyranosyl(1 ! 2)]-a-L-arabinofuranosyl(1 ! 3)-b-D-
glucuronopyranosyl-21-O-angeloyl-22-O-acetyl-3b,16a,21b,
22a,24b,28-hexahydroxyolean-12-ene, respectively.
The same molecular formula of C54H84O22 for aesculiosides
IIj (6) and IIk (7) were determined by their HRESIMS and 13
C
NMR data. The NMR data (Tables 1–4) of 6 and 7 were also
characteristic of the polyhydroxyoleanene triterpenoid glyco-
sides with the same aglycone 6a and a same trisaccharide unit
as well as two-acyl groups. Alkaline hydrolysis of 6 and 7
produced the prosapogenin 16, identified by co-HPLC
analysis with the authentic sample as 3-O-[b-D-galactopyrano-
syl(1 ! 2)]- a-L-arabinofuranosyl(1 ! 3)-b-D-glucurono-
pyranosyl-3b,16a,21b,22a,28-pentahydroxyolean-12-ene
(aesculioliside Ic). A detailed NMR produced spectral anal-
ysis indicated that compounds 6 and 7 differ structurally
only in the acyl substitute at C-21 in the same way as the
differences between 4 and 5, that is, 6 has a tigeloyl group at
C-21, and 7 has an angeloyl group at C-21. Thus, the struc-
tures of aesculiosides IIj (6) and IIk (7) were established as
3-O-[b-D-galactopyranosyl-(1 ! 2)]-a-L-arabinofuranosyl-
(1 ! 3)-b-D-glucuronopyr-anosyl-21-O-tigeloyl-22-O-acetyl-
3b, 16a, 21b, 22a, 28-pentahydroxyolean-12-ene, and
3-O-[b-D-galactopyranosyl-(1 ! 2)]-a-L-arabinofuranosyl-
(1 ! 3)-b-D-glucuronopyranosyl-21-O-angeloyl-22-O-acetyl-
3b,16a,21b,22a,28-pentahydroxyolean-12-ene, respectively.
Compounds 8–13 were obtained from the fraction
Aes-III as colorless powders. Aesculioside IIIa (8) had the
same molecular formula of C54H84O23 as 5. Alkaline
hydrolysis of 8 also produced compound 17 as 5, indicating
that the two saponins share a same prosapogenin, that is
3-O-[b-D-glucopyranosyl(1 ! 2)]-a-L-arabinofuranosyl-
Aesculiosides IIh (4) and IIi (5) had the same molecular
formula of C54H84O23 as deduced from their HRESIMS
and NMR spectroscopic data. The NMR spectral reso-
nances due to the aglycone and the oligosaccharide moiety
of 4 were very similar to those of 5, suggesting that 4 and 5
share the same aglycone, which was assigned as protoaesc-
igenin 4a (Yoshikawa et al., 1996, 1998; Zhang et al., 1999,
2006) by NMR analysis, and the same trisaccharide chain.
Alkaline hydrolysis of 4 and 5 both produced the prosapo-
genin 17, identified by co-HPLC analysis with the authentic
sample as 3-O-[b-D-glucopyranosyl(1 ! 2)]-a-L-arabino-
furanosyl(1 ! 3)-b-D-glucuronopyranosyl-3b,16a,21b,22a,
24b,28-hexahydroxyolean-12-ene (aesculioliside Id). Com-
pounds 4 and 5 are structurally different only in the acyl
substitute at C-21, similar to the difference between 2 and
3. The acyl substituents at C-21 in 4 and 5 were identified
as tigeloyl and angeloyl, respectively, based on their char-
acteristic NMR data (Tables 1 and 3) (Yoshikawa et al.,
1996, 1998; Zhang et al., 1999). Therefore, the structure
of aesculiosides IIh (4) and IIi (5) were established as 3-
O-[b-D-glucopyranosyl(1 ! 2)]-a-L-arabinofuranosyl(1 ! 3)-
b-D-glucuronopyranosyl-21-O-tigeloyl-22-O-acetyl-3b,16a,
21b,22a,24b,28-hexahydroxyolean-12-ene, and 3-O-[b-D-
Table 4
1H NMR spectroscopic data for the sugar moieties of 1–7 (600 MHz in pyridine-d5)a
Proton
1
2
3
4
5
6
7
GlcA-P
1
4.80 (1H, d,
7.8 Hz)
4.25
4.26
4.50
4.84 (1H, d,
7.2 Hz)
4.24
4.27
4.57
4.87 (1H, d,
7.2 Hz)
4.23
4.25
4.59
4.90 (1H, d,
7.6 Hz)
4.26
4.27
4.48
4.92 (1H, d,
7.4 Hz)
4.25
4.27
4.52
4.85 (1H, d,
7.6 Hz)
4.23
4.25
4.59
4.87 (1H, d,
7.6 Hz)
4.24
4.25
4.61
2
3
4
5
4.57
4.53
4.43
4.57
4.58
4.58
4.46
Glc-p
Gal-p
5.34 (1H, d,
7.2 Hz)
4.41
Gal-p
5.35 (1H, d,
7.8 Hz)
4.48
Glc-p
Glc-p
Gal-p
5.31 (1H, d,
7.2 Hz)
4.40
Gal-p
5.34 (1H, d,
7.6 Hz)
4.53
1
5.47 (1H, d,
7.6 Hz)
4.06
5.50 (1H, d,
7.6 Hz)
4.06
5.52 (1H, d,
7.6 Hz)
4.07
2
3
4
5
6
4.25
4.57
3.64
4.30, 4.40
4.11
4.57
3.96
4.40, 4.55
4.07
4.59
3.97
4.46, 4.52
4.26
4.58
3.65
4.29, 4.36
4.29
4.59
3.63
4.35, 4.49
4.09
4.59
3.95
4.40, 4.44
4.12
4.62
3.62
4.48, 4.57
Ara-f
1
2
3
4
5
a
6.06 (1H, br s)
4.84
4.60
4.77
4.19, 4.32
6.06 (1H, br s)
4.99
4.80
4.86
4.17, 4.31
6.07 (1H, br s)
4.91
4.79
4.82
4.12, 4.27
6.08 (1H, br s)
4.99
4.79
4.87
4.17, 4.30
6.09 (1H, br s)
5.01
4.82
4.87
4.17, 4.33
6.01 (1H, br s)
4.92
4.64
4.90
4.17, 4.40
6.05 (1H, br s)
4.96
4.77
4.87
4.18, 4.36
Assignments were based on COSY, HMQC, and HMBC experiments. Due to severe overlapping in the 1H spectrum only detectable relative J (Hz) are
reported.

2080
Z. Zhang, S. Li / Phytochemistry 68 (2007) 2075–2086
O
O
O
O
CH2OH
OH
OH
COOH
O
O
O
O
OH
OH
HO
OH
OH
HO
O
O
C
H
H
H
OH
OH
OH
Fig. 1. Key HMBC and NOE correlations of aesculioside IIe (1).
(1 ! 3)-b-D-glucuronopyranosyl-3b,16a,21b, 22a,24b,28-
hexahydroxyolean-12-ene (aesculioliside Id). Detailed
NMR spectroscopic analyses indicated that compounds 8
and 5 are structurally different in the position of their acetyl
groups. Based on long-range correlations in the HMBC
spectrum of 8, the carbon signals at d 80.9 and 66.3 were
assigned to C-21 and C-28, and the proton resonances at
d 6.49 (1H, d, 9.8 Hz) and 4.28, 4.30 (each 1H, d,
10.3 Hz) to H-21 and H-28. The HMBC correlations of
H-21 with C-1 (d 168.0) of the angeloyl group and H-28
with C-1 (d 170.7) of the acetyl group established C-21
and C-28 were the positions of the acylation. Therefore,
the structure of aesculioside IIIa (8) was determined as
3-O-[b-D-glucopyranosyl(1 ! 2)]-a-L-arabinofuranosyl(1 ! 3)-
b-D-glucuronopyranosyl-21-O-angeloyl-28-O-acetyl-3b,16a,
21b,22a,24b,28-hexahydroxy-olean-12-ene.
that 10 is structurally different from 9 in three substitu-
ents at C-21, C-24 of the aglycone, and C-2 of the Glc
A. The angeloyl group at C-21, -CH2OH at C-24 and
the glucose residue at C-2 of the Glc A in 9 were
replaced in 10 by a tigeloyl group, -CH3 and galactose,
respectively, as indicated by their NMR data (Tables 1,
2, 5, 6) as previously discussed. The full structure
assignment was confirmed by COSY, HMQC and HMBC
correlations. Therefore, the structure of aesculioside IIIc
(10) was determined as 3-O-[b-D-galactopyranosyl(1 ! 2)]-
a-L-arabinofuranosyl(1 ! 3)-b-D-glucuronopyranosyl-
21-O-tigeloyl-22-O-2-methypropanoyl-3b,15a,16a,21b,
22a,28-hexahydroxyolean-12-ene.
Aesculiosides IIId (11) and IIIe (12) had the same molec-
ular formula of C57H88O24 as deduced from their HRE-
SIMS and 13C NMR data. NMR data analysis of 11 and
12 indicated that 11 and 12 had a common aglycone (1a),
a common trisaccharide chain as well as a common tigeloyl
at C-21 as those of 1. Alkaline hydrolysis of 11 and 12 gave
the same prosapogenin (15) as 1. Further NMR spectro-
scopic comparison of 1, 11, and 12 indicated that these
three compounds are structurally different only in the acyl
group at C-22. In the NMR spectra of 11, the signals at d
170.8 and 20.6 for the acetyl group in 1 were absent,
instead of the characteristic NMR signals of a tigeloyl
group signals at dC 13.6 (C-4), 11.9 (C-5) and dH
6.81(1H, q, J = 7.0 Hz, H-3), 1.34 (3H, q, J = 7.0 Hz, H-
4) (Tables 1 and 5) (Yoshikawa et al., 1996, 1998; Zhang
et al., 1999). Similarly, the NMR spectra of 12 displayed
characteristic NMR signals of an angeloyl group signals
at dC 15.3 (C-4), 20.4 (C-5) and dH 5.76 (1H, q,
J = 7.0 Hz, H-3), 1.95 (3H, q, J = 7.0 Hz, H-4) (Tables 1
and 5) (Yoshikawa et al., 1996, 1998; Zhang et al., 1999).
Consequently, the structure of aesculiosides IIId (11) and
IIIe (12) were established as 3-O-[b-D-glucopyranosyl(1 ! 2)]-
a-L-arabinofuranosyl(1 ! 3)-b-D-glucuronopyranosyl-
21-O-tigeloyl-22-O-tigeloyl-3b, 15a,16a, 21b, 22a,24b,
28-heptahydroxyolean-12-ene, and 3-O-[b-D-glucopyrano-
The HRESIMS of 9 displayed an extensive [M + Na]+
ion m/z at 1167.5538, corresponding to the molecular for-
mula C56H88O24. Comparison of NMR spectroscopic data
of 9 and 1 indicated that both compounds had the same
aglycone 1a and the same trisaccharide unit. However,
compound 9 had two different acyl groups, which were
characterized as angeloyl (Zhang et al., 1999) and 2-methy-
propanoyl (Matsushita et al., 2004), respectively, on the
basis of their NMR data (Tables 1 and 5) and by compar-
ison with the literature data (Matsushita et al., 2004; Zhang
et al., 1999). As described in Fig. 2, the linkage of the wo-
acyl groups of angeloyl at C-21 and 2-methypropanoyl at
22 were established by long-range correlations. Thus, the
structure of aesculioside IIIb (9) was determined as
3-O-[b-D-glucopyranosyl(1 ! 2)]-a-L-arabinofuranosyl(1 !
3)-b-D-glucuronopyranosyl-21-O-angeloyl-22-O-2-methy-
propanoyl-3b,15a,16a,21b,22a,24b,28-heptahydroxyolean-
12-ene.
Aesculioside IIIc (10) had a [M + Na]+ ion m/z at
1151.4027, corresponding to the molecular formula
C56H88O23, one oxygen atom less than 9. Detailed com-
parison of NMR data of 10 with those of 9 indicated

Z. Zhang, S. Li / Phytochemistry 68 (2007) 2075–2086
2081
Table 5
1H NMR spectroscopic data for the aglycone moieties of 8–13 (600 MHz in pyridine-d5)a
Proton
8
9
10
11
12
13
1
2
3
5
0.80, 1.37
0.83, 1.42
2.13 (2H, m)
3.42 (1H, dd-like)
0.85
0.84, 1.45
2.13 (2H, m)
3.23 (1H, dd-like)
0.85
0.81, 1.43
2.43 (2H, m)
3.41 (1H, dd-like)
0.90
0.82, 1.30
2.41 (2H, m)
3.42 (1H, dd-like)
0.86
0.83, 1.43
2.43 (2H, m)
3.30 (1H, dd-like)
0.82
2.15 (2H, m)
3.42 (1H, dd-like)
0.85
6
7
9
1.50, 1.53
1.26, 1.53
1.67
1.51, 1.56
2.05, 2.17
1.69
1.53, 1.61
2.08, 2.18
1.72
1.56, 1.59
2.00, 2.12
1.67
1.60, 1.62
1.93, 2.11
1.65
1.57, 1.60
2.02, 2.12
1.70
11
12
15
16
18
19
21
22
23
24
1.74, 1.88
5.44 (1H, br s)
1.64, 1.88
4.75
3.12
1.73, 1.88
5.53 (1H, br s)
4.23
4.46
3.07
1.75, 1.89
5.55 (1H, br s)
4.24
4.47
3.09
1.75, 1.89
5.55 (1H, br s)
4.23
4.47
3.12
1.75, 1.86
5.54 (1H, br s)
4.23
4.46
3.10
1.81, 1.95
5.57 (1H, br s)
4.25
4.47
3.13
1.39, 3.14
1.43, 3.10
1.47, 3.10
1.46, 3.14
1.47, 3.12
1.48, 3.15
6.49 (1H, d, 9.8 Hz)
4.48 (1H, d, 9.8 Hz)
1.32 (3H, s)
3.29 (1H, d,
10.0 Hz), 4.30
0.66 (3H, s)
0.93 (3H, s)
1.82 (3H, s)
6.67 (1H, d, 10.0 Hz) 6.65 (1H, d, 10.2 Hz) 6.73 (1H, d, 10.2 Hz) 6.69 (1H, d, 10.1 Hz) 6.76 (1H, d, 10.0 Hz)
6.25 (1H, d, 10.0 Hz) 6.29 (1H, d, 10.2 Hz) 6.34 (1H, d, 10.2 Hz) 6.39 (1H, d, 10.1 Hz) 6.36 (1H, d, 10.2 Hz)
1.34 (3H, s)
3.33 (1H, d,
10.0 Hz), 4.34
0.70 (3H, s)
0.99 (3H, s)
1.87 (3H, s)
1.30 (3H, s)
1.21 (3H, s)
1.29 (3H, s)
3.31 (1H, d,
10.0 Hz), 4.34
0.68 (3H, s)
0.97 (3H, s)
1.87 (3H, s)
1.30 (3H, s)
3.31 (1H, d,
10.0 Hz), 4.34
0.68 (3H, s)
0.97 (3H, s)
1.87 (3H, s)
1.27 (3H, s)
1.18 (3H, s)
25
26
27
28
0.88 (3H, s)
1.05 (3H, s)
1.88 (3H, s)
0.87 (3H, s)
1.04 (3H, s)
1.88 (3H, s)
4.28, 4.30 (each, 1H, 3.50, 4.75 (each, 1H, 3.49, 4.76 (each, 1H, 3.48, 3.75 (each, 1H, 3.54, 3.77 (each, 1H, 3.50, 3.77 (each, 1H,
d, 10.3 Hz)
1.13 (3H, s)
1.32 (3H, s)
d, 10.0 Hz)
1.14 (3H, s)
1.36 (3H, s)
d, 10.2 Hz)
1.15 (3H, s)
1.36 (3H, s)
d, 10.8 Hz)
1.15 (3H, s)
1.37 (3H, s)
d, 10.3 Hz)
1.15 (3H, s)
1.38 (3H, s)
d, 10.6 Hz)
1.17 (3H, s)
1.39 (3H, s)
29
30
C21
3
4
Ang
Ang
Tig
Tig
Tig
Tig
5.92 (1H, q, 7.0 Hz)
2.06 (3H, d, 7.0 Hz)
2.01 (3H, s)
6.08 (1H, q, 7.0 Hz)
2.17 (3H, d, 7.0 Hz)
2.07 (3H, s)
7.12 (1H, q, 7.0 Hz)
1.69 (3H, d, 7.0 Hz)
1.99 (3H, s)
7.06 (1H, q, 7.0 Hz)
1.62 (3H, d, 7.0 Hz)
1.93 (3H, s)
7.10 (1H, q, 7.0 Hz)
1.65 (3H, d, 7.0 Hz)
1.96 (3H, s)
7.07 (1H, q, 7.0 Hz)
1.63 (3H, d, 7.0 Hz)
1.94 (3H, s)
5
C22 or
Ac
Mp
Mp
Tig
Ang
Tig
C28
2
3
4
5
a
1.99 (3H, s)
2.35 (1H, m)
1.05 (3H, t, 7.0 Hz)
1.01 (3H, t, 7.0 Hz)
2.36 (1H, m)
1.03 (3H, t, 7.0 Hz)
1.01 (3H, t, 7.0 Hz)
6.81 (1H, q, 7.0 Hz)
1.34 (3H, d, 7.0 Hz)
1.76 (3H, s)
5.76 (1H, q, 7.0 Hz)
1.95 (3H, d, 7.0 Hz)
1.76 (3H, s)
6.81 (1H, q, 7.0 Hz)
1.34 (3H, d, 7.0 Hz)
1.94 (3H, s)
Assignments were based on COSY, HMQC, and HMBC experiments. Due to severe overlapping in the 1H spectrum only detectable relative J (Hz) are
reported.
syl(1 ! 2)]-a-L-arabinofuranosyl(1 ! 3)-b-D-glucuron-
Aesculioside IIIf (13) was assigned a molecular formula
of C57H88O23 deduced from HRESIMS data. A detailed
NMR spectroscopic study suggested that compounds 13
opyranosyl-21-O-tigeloyl-22-O-angeloyl-3b, 15a,16a,21b,22a,
24b,28-heptahydroxyolean-12-ene, respectively.
O
O
O
CH2OH
O
OH
OH
COOH
O
O
O
O
OH
OH
HO
OH
OH
HO
O
C
H
H
H
O
OH
OH
OH
Fig. 2. Key HMBC and NOE correlations of aesculioside IIIb (9).

2082
Z. Zhang, S. Li / Phytochemistry 68 (2007) 2075–2086
Table 6
1H NMR spectroscopic data for the sugar moieties of 8–13 (600 MHz in pyridine-d5)a
Proton
8
9
10
11
12
13
GlcA-p
1
2
3
4
5
4.80 (1H, d, 7.6 Hz)
4.78 (1H, d, 7.5Hz)
4.85 (1H, d, 7.5Hz)
4.82 (1H, d, 7.6 Hz)
4.86 (1H, d, 7.5 Hz)
4.86 (1H, d, 7.5 Hz)
4.25
4.27
4.58
4.40
4.25
4.27
4.58
4.40
4.28
4.29
4.57
4.40
4.25
4.27
4.53
4.40
4.25
4.27
4.54
4.40
4.26
4.27
4.6
4.40
Glc-p
Glc-p
Gal-p
Glc-p
Glc-p
Gal-p
1
2
3
4
5
6
5.47 (1H, d, 7.6 Hz)
4.06
4.28
4.50
3.65
4.33, 4.48
5.47 (1H, d, 7.6 Hz)
4.06
4.26
4.58
3.60
4359, 4.48
5.35 (1H, d, 7.6 Hz)
4.48
4.11
4.57
3.96
4.49, 4.53
5.43 (1H, d, 7.5 Hz)
4.04
4.27
4.53
3.62
4.34, 4.46
5.42 (1H, d, 7.5 Hz)
4.03
4.25
4.54
3.63
4.35, 4.46
5.30 (1H, d, 7.5 Hz)
4.50
4.09
4.56
3.94
4.47, 4.52
Ara-f
1
2
3
4
5
a
6.11 (1H, br s)
4.98
4.71
4.92
4.17, 4.35
6.10 (1H, br s)
4.97
4.79
4.93
4.17, 4.37
6.09 (1H, br s)
4.98
4.71
4.93
4.17, 4.40
6.04 (1H, br s)
4.95
4.70
4.90
4.19, 4.34
6.04 (1H, br s)
4.95
4.73
4.89
4.18, 4.31
6.04 (1H, br s)
5.01
4.65
4.97
4.17, 4.35
Assignments were based on COSY, HMQC, and HMBC experiments.
and 2 shared a same aglycone (2a) and a same trisaccharide
moiety, but differed only in the acyl substituent at C-22. The
acyl substituent at C-22 in 13 was determined as tigeloyl on
the basis of its characteristic NMR data (Tables 1 and 5)
(Yoshikawa et al., 1996, 1998; Zhang et al., 1999). Accord-
ingly, the structure of aesculioside IIIf (13) was established
as 3-O-[b-D-galactopyranosyl(1 ! 2)]-a-L-arabinofurano-
syl(1 ! 3)-b-D-glucuronopyranosyl-21-O-tigeloyl-22-O-tige-
loyl-3b,15a,16a,21b,22a,28-hexahydroxyolean-12-ene.
The compounds 14–22 and 26–28 from the fruits of
A. pavia were in vitro screened for cytotoxicity against
non-small cell lung cancer (NCI-H460), CNS cancer
(SF-268), and breast cancer (MCF7). Five positive com-
pounds (21, 22, 26, 27, and 28) were further evaluated
against 59 cell lines from nine different human cancers
including leukemia, non-small cell lung, colon, CNS,
melanoma, ovarian, renal, prostate, and breast. Com-
pounds 26–28 with two-acyl groups at C-21 and C-22
showed activity with GI50 0.175–8.71 lM against almost
all tumor cell lines tested (Table 7). Compounds 21
and 22 with only one acyl group at C-21 also showed
activity with GI50 3.48 lM to >25 lM against the cell
lines tested (Table 7).
chemical investigation on this plant collected in different
location further supported this conclusion. It is worthwhile
to mention that compounds 23–28 are the main com-
pounds in the saponin fraction based on our previous
and current investigations. Interestingly, compounds
26–28 with two-acyl groups at C-21 and C-22 showed
activity against almost all tumor cell lines tested. But, com-
pounds 14–18 without acyl group at C-21 and C-22 showed
no or weak activity. These results suggest that the acyl
groups at C-21 and C-22 are important for the activity of
these triterpenoid saponins.
4. Experimental
4.1. General experimental procedures
NMR experiments were performed on a Bruker
600 MHz NMR instrument. NMR data were reported as
d (ppm) values and referenced to the solvent used. HRE-
SIMS were measured on a PE SCIEX QSTAR LC/MS/
MS spectrometer. Octadecyl-functionalized silica gel
(Aldrich) was used for low-pressure chromatography.
HPLC analysis was performed on a Hewlett Packard Series
1100 with a HP 1100 diode array detector using a Hypersil
ODS column (column A, 250 · 4.6 mm, 5 lm, Supelco;
detector: 208 nm; flow rate: 0.5 mL/min; solvent A:
CH3CN–H2O, 30:70 + 0.1% AcOH; solvent B: CH3CN–
H2O, 40:60 + 0.1% AcOH; solvent C: CH3CN–H2O,
42:58 + 0.1% acetic acid). Preparative HPLC was per-
formed with an Acuflow Series III pump connected with
an Acutect 500 UV/VIS detector using an Econosil ODS
column (column B, 250 · 22 mm, 10 lm, Alltech; detector:
208 nm; flow rate: 3.0 mL/min; solvents A, B, and C).
3. Conclusions
Our previous chemical investigation on this plant
showed that the structures of saponins isolated from North
American A. pavia are different from those isolated from
Eurasian A. hippocastanum and A. chinensis in their oligo-
saccharide moieties, suggesting a different chemotaxo-
nomic feature between North American A. pavia and
Eurasian Aesculus species (Zhang et al., 2006). Current

Z. Zhang, S. Li / Phytochemistry 68 (2007) 2075–2086
2083
Table 7
Cytotoxicity data of compounds 21, 22 and 26–28 (GI50, lM)a
Compounds (NSC#)
21 (D736862)
22 (D736863)
26 (D736859)
27 (D736857)
28 (D736858)
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
–
20
3.44
5.71
6.65
–
4.73
0.866
0.175
0.961
3.64
–
2.43
0.333
2.53
1.50
5.99
–
2.51
0.649
5.06
4.96
–
–
3.60
7.40
14.1
18.4
>25
3.71
Non-small cell lung cancer
A549/ATCC
EKVX
Hop-62
Hop-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
4.69
4.99
5.09
–
5.59
5.12
4.47
4.52
4.52
4.65
>25
10.8
>25
>25
10.1
20.1
>25
3.55
4.27
4.24
3.98
3.32
4.86
4.59
4.03
4.54
4.53
4.22
3.78
2.76
1.52
5.72
4.68
4.89
3.99
4.55
6.59
5.71
4.32
2.84
6.63
4.37
7.98
4.37
5.40
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
3.96
5.24
6.84
9.05
4.97
4.26
4.45
5.50
6.64
>25
>25
>25
22.6
>25
3.28
–
2.42
2.56
2.75
5.51
1.94
3.56
4.62
3.56
8.71
3.63
7.49
3.46
5.61
6.61
4.04
2.43
3.80
3.74
2.77
KM12
SW-620
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
4.72
4.83
6.35
3.71
5.25
4.39
>25
>25
7.72
>25
>25
>25
4.22
3.44
4.05
2.68
4.62
4.74
2.76
4.63
2.64
2.19
3.75
6.42
3.48
4.83
3.18
2.97
3.27
8.54
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
4.44
5.15
4.67
4.19
4.68
5.43
4.41
4.86
>25
>25
>25
>25
>25
>25
–
3.44
4.25
4.14
4.35
5.34
3.48
4.62
4.10
4.63
5.70
3.28
4.47
4.40
4.58
5.22
5.03
6.16
8.18
3.31
4.56
4.15
4.78
5.77
5.75
>25
Ovarian cancer
IGROV1
4.27
5.26
4.96
4.17
4.57
3.76
19.4
>25
7.90
7.41
>25
>25
3.36
4.51
5.46
5.07
3.35
4.01
4.57
4.89
3.67
4.04
2.62
4.70
5.29
6.43
3.32
4.94
4.55
4.70
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Renal cancer
786-0
A498
ACHN
CAK1-1
RXF-393
SN12C
TK-10
4.42
4.35
3.86
4.43
4.72
4.35
4.68
3.75
>25
5.38
>25
14.9
–
10.9
>25
4.09
5.30
2.20
3.50
5.05
2.68
3.60
4.36
2.92
3.02
6.14
4.96
5.64
–
3.68
5.14
3.37
3.98
5.11
4.66
6.90
3.57
4.00
6.71
3.84
UO-31
Prostate cancer
PC-3
DU-145
–
5.75
6.73
>25
4.27
4.07
1.72
4.51
3.43
4.44
(continued on next page)

2084
Z. Zhang, S. Li / Phytochemistry 68 (2007) 2075–2086
Table 7 (continued)
Compounds (NSC#)
21 (D736862)
22 (D736863)
26 (D736859)
27 (D736857)
28 (D736858)
Breast cancer
MCF7
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
MDA-MB-435
BT-549
T-47D
4.80
–
>25
>25
11.5
23.3
>25
7.05
>25
4.75
–
2.57
6.14
3.84
–
4.21
–
1.78
2.82
1.94
–
4.70
–
3.34
1.68
1.41
–
3.64
3.48
6.02
7.83
4.29
4.62
3.56
4.31
a
GI50 (concentration at 50% growth inhibition) values are identical to the IC50 (the half maximal inhibitory concentration) values.
D-Glucose, D-galactose, L-arabinose and D-glucuronic acid
were purchased from Aldrich.
19–22, of which
1
(20.2 mg, tR 16.8 min) and 19
(9.8 mg, tR 16.8 min) were from Sf-IIa; 2 (13.0 mg, tR
18.6 min) and 20 (10.0 mg, tR 20.3 min) from Sf-IIb; 3
(9.7 mg, tR 20.9 min) and 21 (10.5 mg, tR 22.9 min) from
Sf-IIc; 4 (10.1 mg, tR 24.7 min) and 22 (2.6 mg, tR
4.2. Plant material
Fruits of A. pavia L. were collected from a single plant,
in Nacogdoches, Texas, on October 6, 2004, and were iden-
tified by Shiyou Li. A voucher specimen (CMPR-
AP2004106) was deposited at the National Center for
Pharmaceutical Crops of Stephen F. Austin State Univer-
sity, USA. This plant was different in location from the
one previously investigated (CMPR-2003101) (Zhang
et al., 2006).
26.6 min) from Sf-IId;
5 (10.6 mg, tR 28.9 min), 6
(10.1 mg, tR 29.1 min) and 7 (6.5 mg, tR 31.4 min) from
Sf-IIe. Similarly, the fraction Aes-III was separated by
preparative MPLC (column B and solvent C) to afford
Aes-IIIa (4.3 mg, tR 70.0 min), 11 (3.0 mg, tR 78.5 min),
12 (2.0 mg, tR 84.0 min), and 13 (2.5 mg, tR 98.0 min).
Ase-IIIa was further isolated by HPLC (column A and
solvent C) to yield 8 (1.3 mg, tR 26.0 min), 9 (1.2 mg,
tR 27.9 min), and 10 (1.0 mg, tR 29.2 min). Compounds
23–29 were obtained from fraction Aes-IV using the
same purified methods as our previous publication
(Zhang et al., 2006).
4.3. Extraction and isolation
Air-dried fruits (450 g) were ground to a coarse pow-
der and percolated five times with EtOH–H2O (95:5, %).
EtOH–H2O solubles were then concentrated under vac-
uum to give an extract (50.0 g). Part of this extract
(30.0 g) was suspended in H2O and partitioned succes-
sively with CHCl3 and n-BuOH. The n-butanol-soluble
fraction (16.0 g) was applied to a column of Diaion
HP-20 eluting with MeOH–H2O (3:7) and MeOH–H2O
(80–20). The latter was concentrated to afford a fraction
(coded Aes-P), and then subjected to ODS cc eluting
with a MeOH–H2O gradient (4:6; 1:1, 6:4, and 8:2) to
give four fractions: Aes-I (MeOH–H2O (4:6) elution),
Aes-II (MeOH–H2O (1:1) elution), Aes-III (MeOH–
H2O (6:4) elution), and Aes-IV (MeOH–H2O (8:2) elu-
tion). Fraction Aes-I was subjected to additional ODS
cc eluting with MeOH–H2O (35:65) to furnish Aes-Ia
and Aes-Ib. Aes-Ib was separated by preparative MPLC
(column B and solvent A) to give 14 (15.6 mg, tR
48.1 min), 15 (5.8 mg, tR 42.6 min), 16 (17.5 mg, tR
68.4 min), 17 (11.6 mg, tR 57.6 min), and 18 (2.5 mg, tR
80.3 min). Compounds 30 and 31 were isolated from
Aes-Ia by Sephadex LH-20 cc eluting with MeOH. Frac-
tion Aes-II was separated by preparative MPLC (column
B and solvent B) to give five subfractions: Sf-IIa (tR
49.3 min), Sf-IIb (tR 55.6 min), Sf-IIc (tR 60.9 min), Sf-
IId (tR 74.5. min), and Sf-IIe (tR 79.6 min). These five
subfractions (Sf IIa–IIe) were further separated by HPLC
(column A and solvent B) to give compounds 1–7 and
4.4. Aesculioside IIe (1)
Colorless powder from CH3CN–H2O, tR 16.8 min (col-
umn A, solvent B). HRESIMS: m/z 1139.5248 [M + Na]+
(calcd. for C54H84NaO24, 1139.5250). For 13C and 1H
NMR spectroscopic data, see Tables 1–4.
4.5. Aesculioside IIf (2)
Colorless powder from CH3CN–H2O, tR 18.6 min (col-
umn A, solvent B). HRESIMS: m/z 1123.5299 [M + Na]+
(calcd. for C54H84NaO23, 1123.5301). For 13C and 1H
NMR spectroscopic data, see Tables 1–4.
4.6. Aesculioside IIg (3)
Colorless powder from CH3CN–H2O, tR 20.3 min (col-
umn A, solvent B). HRESIMS: m/z 1123.5295 [M + Na]+
(calcd. for C54H84NaO23, 1123.5301). For 13C and 1H
NMR spectroscopic data, see Tables 1–4.
4.7. Aesculioside IIh (4)
Colorless powder from CH3CN–H2O, tR 24.7 min (col-
umn A, solvent B). HRESIMS: m/z 1123.5295 [M + Na]+

Z. Zhang, S. Li / Phytochemistry 68 (2007) 2075–2086
2085
1
(calcd. for C54H84NaO23, 1123.5301). For 13C and 1H
NMR spectroscopic data, see Tables 1–4.
For 13C and H NMR spectroscopic data, see Tables 1,
2, 5, and 6.
4.8. Aesculioside IIi (5)
4.15. Aesculioside IIIe (12)
Colorless powder from CH3CN–H2O, tR 28.9 min
(column A, solvent B). HRESIMS: m/z 1123.5299 [M +
Na]+ (calcd. for C54H84NaO23, 1123.5301), 1139.5164
[M + K]+ (calcd. for C54H84KO23, 1139.5041). For 13C
Colorless powder from CH3CN–H2O, tR 33.7 min (col-
umn A, solvent C). HRESIMS: m/z 1179.5578
[M + Na]+ (calcd. for C57H88NaO24, 1179.5563),
1195.5400 [M + K]+ (calcd. for C57H88KO24, 1195.5303).
1
1
and H NMR spectroscopic data, see Tables 1–4.
For 13C and H NMR spectroscopic data, see Tables 1,
2, 5, 6.
4.9. Aesculioside IIj (6)
4.16. Aesculioside IIIf (13)
Colorless powder from CH3CN–H2O, tR 28.9 min (col-
umn A, solvent B). HRESIMS: m/ 1107.5347 [M + Na]+
(calcd. for C54H84NaO22, 1107.5352), 1123.5190
[M + K]+ (calcd. for C54H84KO22, 1123.5091). For 13C
Colorless powder from CH3CN–H2O, tR 35.5 min (col-
umn A, solvent C). HRESIMS: m/z 1163.5603
[M + Na]+ (calcd. for C57H88NaO23, 1163.5614),
1179.5311 [M + K]+ (calcd. for C57H88KO23, 1179.5354).
1
and H NMR spectroscopic data, see Tables 1–4.
1
For 13C and H NMR spectroscopic data, see Tables 1,
4.10. Aesculioside IIk (7)
2, 5, and 6.
Colorless powder from CH3CN–H2O, tR 31.4 min (col-
umn A, solvent B). HRESIMS: m/ 1107.5348 [M + Na]+
(calcd. for C54H84NaO22, 1107.5352), 1123.5226
[M + K]+ (calcd. for C54H84KO22, 1123.5091). For 13C
4.17. Acid hydrolysis of compound 1
Compound 1 (15 mg) was refluxed with 1 mL 1 M HCl
(dioxane–H2O, 1:1) at 80 ꢁC for 2 h. After the dioxane
was removed, the solution was extracted with CHCl3–
MeOH (7:3, 1 mL · 3). The monosaccharide portion was
neutralized by passing through an ion-exchange resin
(Amberlite MB-3) column, and then freeze-dried. Three
monosaccharides were identified using authentic samples
by TLC in MeCOEt–iso-PrOH–Me2CO–H2O (20:10:7:6)
as glucose, arabinose, and glucuronic acid. After prepara-
tive TLC of the sugar mixture in this solvent, the optical
rotation of each purified sugar was measured (Zhang
et al., 2006).
1
and H NMR spectroscopic data, see Tables 1–4.
4.11. Aesculioside IIIa (8)
Colorless powder from CH3CN–H2O, tR 26.0 min (col-
umn A, solvent C). HRESIMS: m/z 1123.5306
[M + Na]+ (calcd. for C54H84NaO23, 1123.5301),
1139.5218 [M + K]+ (calcd. for C54H84KO23, 1139.5041).
1
For 13C and H NMR spectroscopic data, see Tables 1,
2, 5, and 6.
4.12. Aesculioside IIIb (9)
4.18. Alkaline hydrolysis of compounds 1, 4–7, and 11–13
Colorless powder from CH3CN–H2O, tR 27.9 min
(column A, solvent C). HRESIMS: m/z 1167.5538
[M + Na]+ (calcd. for C56H88NaO24, 1167.5563). For
Compound 1 (3.0 mg) was refluxed with 1 mL 0.8 M
NaOH at 80 ꢁC for 4 h. After cooling, the reaction
mixture was neutralized with 1 M HCl and then extracted
with n-BuOH (2 mL · 3). The organic layers were com-
bined and then evaporated to dryness under a vacuum.
The residue was subjected to HPLC purification affording
a prosapogenin (15, 2.3 mg), which was identified by co-
HPLC analysis with an authentic sample. By the same
method, 4 and 5 afforded 17, 6 and 7 afforded 16, 11 and
12 afforded 15, and 13 afforded 14.
1
13C and H NMR spectroscopic data, see Tables 1, 2, 5,
and 6.
4.13. Aesculioside IIIc (10)
Colorless powder from CH3CN–H2O, tR 29.2 min (col-
umn A, solvent C). HRESIMS: m/z 1151.4027
[M + Na]+ (calcd. for C56H88NaO23, 1151.5614). For 13C
and 1H NMR spectroscopic data, see Tables 1, 2, 5, and 6.
4.19. Cytotoxicity assay
4.14. Aesculioside IIId (11)
Cytotoxicity of compounds 14–22 and 26–28 was
assayed by National Cancer Institute (NCI) using the
Methodology of The In Vitro Cancer Screen (National
Cancer Institute, 2007).
Colorless powder from CH3CN–H2O, tR 32.2 min (col-
umn A, solvent C). HRESIMS: m/z 1179.5568
[M + Na]+ (calcd. for C57H88NaO24, 1179.5563),
1195.5281 [M + K]+ (calcd. for C57H88KO24, 1195.5303).

2086
Z. Zhang, S. Li / Phytochemistry 68 (2007) 2075–2086
29
30
20
Acknowledgements
OR3
OR4
19
18
21
22
12
26
This study is partially funded by the US CDC Grant
(R01 CI000315-01). The authors thank: Camptotech Inc.’s
cytotoxicity testing at the National Cancer Institute (NCI);
The Keck/IMD NMR Center, which is funded by the
W.M. Keck Foundation and the University of Houston;
Dr. Youlin Xia for NMR analysis assistance; Dr. Shane Ti-
chy for HRESI-MS analysis, and Stacy Ownby, Dr. Wei
Yuan, Dr. Ping Wang, and Dr. Wanli Zhang for manu-
script preparation.
13
14
11
25
17
28
H
9
CH2OR5
OH
16
15
1
2
3
10
8
H
GlcA-p
COOH
O
5
7
27
R2
4
6
Ara-f
O
OH
O
O
H
24 R1
23
HO
OH
OR6
OH
O
HO
GlcA-p:
β
-D-glucuronopyranosyl acid
-D-galactopyranosyl
-D-glucopyranosyl
HO
OH
OH
Gal-p:
Glc-p:
Ara-f:
β
O
OH
OH
Glc-p
β
α
-L-arabinofuranosyl
OH
OH
Gal-p
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