Triterpene glycosides from the aerial parts of Larrea tridentata

Article abstract of DOI:10.1016/j.phytochem.2010.09.012

Chemical study of the aerial parts of Larrea tridentata (Zygophyllaceae) resulted in the isolation of 25 triterpene glycosides, 13 of which were previously unknown. Their structures were determined on the basis of comprehensive spectroscopic analyses, including 2D NMR spectroscopy, and hydrolytic cleavage followed by chromatographic and spectroscopic analyses. This is the first systematic phytochemical study of L. tridentata with attention paid to its triterpene glycoside constituents. The isolated compounds were evaluated for their cytotoxic activity against HL-60 human promyelocytic leukemia cells.

Full text of DOI:10.1016/j.phytochem.2010.09.012

Phytochemistry 71 (2010) 2157–2167
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Triterpene glycosides from the aerial parts of Larrea tridentata
⇑
Maki Jitsuno, Yoshihiro Mimaki
Tokyo University of Pharmacy and Life Sciences, School of Pharmacy, 1432-1, Horinouchi, Hachioji, Tokyo 192-0392, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 July 2010
Received in revised form 9 August 2010
Available online 25 October 2010
Chemical study of the aerial parts of Larrea tridentata (Zygophyllaceae) resulted in the isolation of 25 tri-
terpene glycosides, 13 of which were previously unknown. Their structures were determined on the basis
of comprehensive spectroscopic analyses, including 2D NMR spectroscopy, and hydrolytic cleavage fol-
lowed by chromatographic and spectroscopic analyses. This is the first systematic phytochemical study
of L. tridentata with attention paid to its triterpene glycoside constituents. The isolated compounds were
evaluated for their cytotoxic activity against HL-60 human promyelocytic leukemia cells.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Larrea tridentata
Zygophyllaceae
Triterpene glycosides
HL-60 cells
Cytotoxic activity
1. Introduction
(940 g) was subjected to porous-polymer polystyrene resin (Diaion
HP-20) chromatography and successively eluted with MeOH in
Larrea tridentata (Sesse. & Moc. Ex DC.) Coville. (Zygophyllaceae)
is an evergreen shrub that grows in the desert areas of the Americas.
The aerial parts (leaves and stems) of this plant are called Chaparral
and are an alternative herbal medicine used for the treatment of
various cancers, tuberculosis, menstrual pains, and diabetes in the
United States (Lambert et al., 2005). Fragmentary phytochemical
studies have been carried out on L. tridentata, and lignans such as
nordihydroguaiaretic acid, flavonoids, and triterpenes (Abou-Gazar
et al., 2004) have been isolated and identified. The present investi-
gation of the aerial parts of L. tridentata, with particular attention
paid to its triterpene glycoside constituents, resulted in the isola-
tion of 25 triterpene glycosides, 13 of which were found to be
new compounds. This paper is a report on the structural determina-
tion of the new compounds on the basis of comprehensive spectro-
scopic analyses, including 2D NMR spectroscopy, and hydrolytic
cleavage followed by chromatographic and spectroscopic analyses.
The isolated compounds were evaluated for their cytotoxic activity
against HL-60 human promyelocytic leukemia cells.
H2O (3:7) MeOH–H2O (1:1), MeOH, EtOH, and EtOAc. The MeOH
eluate fraction (477 g), in which triterpene glycosides were en-
riched, was subjected to silica gel column chromatography and
octadecylsilanized (ODS) silica gel, as well as preparative HPLC,
yielding compounds 1–25. By comparison of the physical and spec-
troscopic data with literature values, 3–8, 11, and 21–25 were
identified as 3-[(O-b-
syl)oxy]olean-12-en-28-oic acid (3; guaianin N) (Stolyarenko
et al., 2000), 3-[(O-b- -glucopyranosyl-(1?3)- -arabinopyrano-
syl)oxy]olean-12-en-28-oic acid b- -glucopyranosyl ester (4; guaiacin
B) (Ahmad et al., 1989), 3-[(O-b- -xylopyranosyl-(1?3)-b- -glu-
curonopyranosyl)oxy]olean-12-en-28-oic acid b- -glucopyranosyl
ester (5; quinoside D) (Mizui et al., 1990), 3-[(O-b- -glucopyrano-
syl-(1?3)-O-[ -rhamnopyranosyl-(1?2)]- -arabinopyrano-
syl)oxy]olean-12-en-28-oic acid (6; patriniaglycoside B-II) (Shao
et al., 1989), 3-[(O-b- -glucopyranosyl-(1?3)-O-[ -rhamnopyr-
anosyl-(1?2)]- -arabinopyranosyl)oxy]olean-12-en-28-oic acid
b- -glucopyranosyl ester (7; nudicaucin C) (Konishi et al., 1998),
3-[(O-b- -glucopyranosyl-(1?3)-O-[ -rhamnopyranosyl-(1?2)]-
-arabinopyranosyl)oxy]olean-12-en-28-oic acid O-b- -gluco-
pyranosyl-(1?6)-b- -glucopyranosyl ester (8; mateglycoside A)
(Sugimoto et al., 2009), 3-[(b- -xylopyranosyl)oxy]olean-12-ene-
28,29-dioic acid (11) (Yu et al., 1995), 3b-hydroxy-30-noroleana-
12,20(29)-dien-28-oic acid b- -glucopyranosyl ester (21) (Li et al.,
2009), 3-[(O-b- -glucopyranosyl-(1?3)- -arabinopyranosyl)oxy]-
30-noroleana-12,20(29)-dien-28-oic acid b- -glucopyranosyl ester
(22; guaiacin A) (Ahmad et al., 1989), 3-[(O- -rhamnopyranosyl-
(1?2)- -arabinopyranosyl)oxy]-30-noroleana-12,20(29)-dien-
28-oic acid b- -glucopyranosyl ester (23; guaianin A2) (Ahmad
D-glucopyranosyl-(1?3)-a-L-arabinopyrano-
D
a-L
D
D
D
D
D
a
-
L
a-L
D
a-L
a-L
D
D
a-L
a-
L
D
D
2. Results and discussion
D
2.1. Structure elucidation
D
D
a-L
The aerial parts of L. tridentata (3.0 kg) were extracted with
MeOH under conditions of reflux. The concentrated MeOH extract
D
a
-L
a
-L
⇑
Corresponding author. Tel.: +81 42 676 4573; fax: +81 42 676 4579.
E-mail address: mimakiy@toyaku.ac.jp (Y. Mimaki).
D
0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2010.09.012

Table 1
1H and 13C NMR chemical shift assignments for the sugar moieties of compounds 1, 2, 9, 10, and 12–20 in C5D5N.
1
2
9
10
12
13
14
1H
13C
1H
13C
1H
13C
1H
13C
1H
13C
1H
13C
1H
13C
Ara
1
2
4.74 d (7.4) 107.4 Ara
4.54 dd (9.1, 71.8
7.4)
4.18 dd (9.1, 84.1
3.2)
1
2
5.24 d (3.8) 103.7 Xyl
5.54 dd (3.8, 75.8
3.8)
1
2
4.83 d (7.5)
4.02 dd (8.7,
7.5)
4.17 dd (8.7,
8.7)
107.6 Xyl
75.5
1
2
4.78 d (7.5)
4.04 dd (8.6,
7.5)
4.14 dd (8.6,
8.6)
107.1 Ara
74.2
1
2
4.77 d (7.5) 107.4 Ara
4.61 dd (9.0, 72.0
7.5)
1
2
4.79 d (7.4) 107.5 Ara
1
2
4.73 d (7.5) 107.5
4.56 dd (9.2, 71.8
7.5)
4.22 dd (9.2, 83.9
3.3)
4.40 dd
(8.4, 7.4)
4.18 m
72.9
74.7
3
4
3
4
4.71 br s
79.9
3
4
78.6
3
4
88.5
69.6
3
4
4.24 m
84.2
3
4
3
4
4.39 br s
69.3
67.0
4.62 m
67.0
63.3
4.24 m
71.3
67.1
4.11 m
4.45 br s
69.4
67.0
4.33 br s
69.6
66.8
4.44 br s
69.4
67.1
5a 4.20 br d
(11.5)
5a 4.30 br d
(8.9)
5a 4.39 br d
(11.5)
5a 4.33 dd (11.1, 66.4
5a 4.23 br d
(12.0)
5a 4.32 br d
(11.1)
5a 4.20 br d
(11.4)
4.9)
5b 3.77 br d
(11.5)
5b 3.78 br d
(8.9)
5b 3.78 dd (11.5,
10.9)
5b 3.69 dd (11.1,
10.5)
5b 3.76 br d
(12.0)
5b 3.84 br d
(11.1)
5b 3.75 br d
(11.4)
Glc
1
2
5.29 d (7.8) 105.9 Glc
1
2
5.21 d (7.7) 104.4 Glc
1
2
6.36 d (8.1)
4.23 dd (8.9,
8.1)
4.31 dd (8.9,
8.9)
4.39 dd (9.3,
8.9)
4.06 m
95.8 Glc
74.1
1
2
5.32 d (7.8)
4.09 dd (8.8,
7.8)
4.26 dd (8.8,
8.8)
4.22 dd (9.1,
8.8)
4.04 m
105.8 Glc
75.5
1
2
5.41 d (8.0) 106.4 Glc
4.05 dd (8.3, 75.8
8.0)
4.27 dd (8.5, 78.4
8.3)
1
2
6.30 d (8.1)
4.20 dd
(8.8, 8.1)
4.28 dd
(8.8, 8.8)
4.34 dd
(8.8, 9.2)
4.02 m
95.8 Glc
74.1
1
2
5.43 d (7.8) 106.1
4.03 dd (8.7,
75.6
4.00 dd (8.4,
74.7
4.06 dd (9.5,
73.5
7.8)
7.7)
7.8)
3
4
5
4.42 dd (9.4, 76.8
8.7)
5.23 dd (9.4, 75.8
9.4)
3
4
5
4.18 dd (9.5, 77.3
8.4)
3
4
5
78.9
71.1
79.4
3
4
5
78.3
71.7
78.7
3
4
5
3
4
5
78.9
71.1
79.3
62.3
3
4
5
5.87 dd (9.5, 79.3
9.5)
4.32 dd (9.5, 69.3
9.6)
4.16 dd (9.5,
8.9)
3.85 m
70.9
78.1
61.7
4.24 dd (8.5, 71.6
8.0)
3.84 br d
(9.4)
76.4
61.6
4.01 m
78.7
3.96 br d
(9.6)
78.4
62.0
6a 4.50 dd
(11.7, 3.3)
6b 4.30 dd
(11.7, 2.7)
6a 4.37 dd
(11.2, 3.4)
6b 4.30 dd
(11.2, 5.9)
6a 4.50 dd (11.9, 62.2
6a 4.54 dd (11.6, 62.5
6a 4.56 dd
(12.0, 2.0)
6b 4.40 dd
(12.0, 5.0)
62.7
6a 4.46 br d
(11.7)
6b 4.39 br d
(11.7)
6a 4.49 dd
(12.0, 2.1)
6b 4.40 br d
2.3)
2.2)
6b 4.43 dd (11.9,
4.4)
6b 4.32 dd (11.6,
5.5)
(12.0)
Glc0
1
2
6.34 d (8.0)
4.21 dd (9.0, 74.1
8.0)
95.8 Glc0
1
2
6.31 d (8.1)
4.20 dd (9.0,
8.1)
95.7
74.1
Ac
1.95 s
21.2
170.8
3
4
5
4.29 dd (9.0, 78.9
9.0)
4.37 dd (9.0, 71.1
9.0)
3
4
5
4.28 dd (9.0, 78.9
9.0)
4.35 dd (9.0, 71.1
9.0)
Glc0
1
2
3
4
5
6.29 d (8.2)
95.8
4.04 m
79.3
62.2
4.03 br d
(9.2)
79.3
62.2
4.19 dd (8.8, 74.1
8.2)
4.28 dd (8.8, 78.8
8.8)
4.33 dd (9.1, 71.2
8.8)
4.02 br d
(9.1)
6a 4.48 dd
(11.7, 2.1)
6b 4.42 dd
(11.7, 2.9)
6a 4.46 dd
(12.2, 2.2)
6b 4.40 dd
(12.2, 4.1)
79.3
62.3
6a 4.46 dd
(12.2, 2.7)
6b 4.38 br d
(12.2)
15
16
17
18
19
20
1H
13C
1H
13C
1H
13C
1H
13C
1H
13C
1H
13C
Ara
1
2
4.77 d (7.5)
4.60 dd (8.3,
7.5)
107.5 Ara
71.8
1
2
4.74 d (7.5)
4.53 dd (8.5,
7.5)
107.3 Ara
71.8
1
2
4.81 d (6.0)
4.61 dd (6.0,
8.0)
105.1 Ara
74.8
1
2
4.88 d (5.4)
4.65 dd (6.3,
5.4)
104.7 Ara
74.8
1
2
4.87 d (5.4)
4.65 dd (6.3,
5.4)
104.6 Ara
74.7
1
2
4.88 d (5.5)
4.66 dd (6.4,
5.5)
104.6
74.8
3
4
4.22 m
4.49 br s
84.1
69.4
67.1
3
4
4.19 m
4.37 br s
84.0
69.2
67.0
3
4
4.21 m
4.45 br s
82.6
68.6
65.5
3
4
4.34 m
4.53 br s
81.9
68.1
64.7
3
4
4.34 m
4.53 br s
81.9
68.1
64.7
3
4
4.34 m
4.54 br s
82.0
68.2
64.8
5a 4.28 br d
(11.0)
5a 3.77 br d
(11.6)
5a 4.19 br d
(10.5)
5a 3.75 br d (9.7)
5a 3.74 dd (11.5,
1.9)
5a 3.75 dd (9.7,
1.4)
5b 3.83 br d
(11.0)
5b 4.19 br d
(11.6)
5b 3.73 br d
(10.5)
5b 4.22 dd (9.7,
5b 4.23 br d
(11.5)
5b 4.24 br d (9.7)
5.0)

Glc
1
2
5.35 d (8.0)
4.00 dd (9.0,
8.0)
106.2 Glc
75.5
1
2
5.29 d (7.8)
4.07 dd (8.6,
7.8)
105.8 Rha
75.4
1
2
6.15 s
4.74 br d (2.0)
101.7 Rha
72.3
1
2
6.14 s
4.73 br s
101.9 Rha
72.4
1
2
6.14 s
4.73 br s
101.9 Rha
72.4
1
2
6.15 s
4.73 br s
102.0
72.4
3
4
5
4.22 dd (9.5,
9.0)
4.03 dd (9.5,
9.5)
78.1
75.3
75.5
64.7
3
4
5
4.40 dd (9.1,
8.6)
5.24 dd (9.4,
9.1)
76.6
75.7
76.3
61.4
3
4
5
6
4.60 dd (9.5,
2.0)
4.24 dd (10.0,
9.5)
4.60 dq (10.0,
6.0)
1.60 d (6.0)
72.3
73.9
69.9
18.5
3
4
5
6
4.60 dd (9.4,
3.7)
4.28 dd (9.8,
9.4)
4.60 dd (9.8,
6.1)
1.64 d (6.1)
72.5
73.9
70.1
18.6
3
4
5
6
4.60 dd (9.3,
3.3)
4.28 dd (9.4,
9.3)
4.59 dq (9.4,
6.1)
1.64 d (6.1)
72.5
73.9
70.1
18.6
3
4
5
6
4.60 dd (9.2,
2.9)
4.28 dd (9.9,
9.2)
4.59 dq (9.9,
6.2)
1.64 d (6.2)
72.6
73.9
70.1
18.6
4.04 m
3.81 br d (9.4)
6a 4.92 br d
(11.5)
6a 4.49 dd (12.6,
2.6)
6b 4.75 dd (11.5,
5.5)
6b 4.27 br d
(12.6)
Glc
1
2
5.02 d (7.5)
104.5 Glc
74.8
1
2
5.11 d (7.7)
104.6 Glc
74.9
1
2
5.11 d (7.7)
104.6 Glc
75.0
1
2
5.12 d (7.8)
104.6
75.0
Ac
1.97 s
20.7 Glc0
170.8
1
2
3
6.28 d (8.1)
95.8
3.89 dd (9.0,
7.5)
3.96 dd (8.3,
7.7)
3.96 dd (8.3,
7.7)
3.96 dd (8.2,
7.8)
4.19 dd (8.7,
8.1)
4.27 dd (8.7,
8.7)
74.1
78.8
3
4
4.32 dd (9.0,
9.5)
5.15 dd (9.5,
9.5)
76.5
75.7
3
4
4.16 dd (8.8,
8.3)
4.19 dd (8.8,
6.5)
78.2
71.4
3
4
4.19 dd (8.8,
8.3)
4.16 dd (8.8,
8.7)
78.2
71.4
3
4
4.18 dd (9.3,
8.2)
4.17 dd (9.3,
8.7)
78.2
71.5
Glc0
1
2
3
4
5
6.30 d (8.0)
95.8
74.1
78.8
71.2
4
5
4.33 dd (9.1,
8.7)
4.01 br d (9.1)
71.2
79.3
62.3
5
3.79 br d (9.5)
76.3
61.5
5
3.91 m
78.6
62.5
5
3.93 m
78.6
62.5
5
3.93 m
78.6
62.5
4.19 dd (8.8,
8.0)
4.28 dd (9.0,
8.8)
4.34 dd (9.0,
9.0)
4.04 m
6a 4.47 br d
(13.0)
6b 4.28 br d
(13.0)
6a 4.47 dd (11.4,
1.9)
6b 4.33 br d
(11.4)
6a 4.50 dd (11.8,
2.2)
6b 4.33 br d
(11.8)
6a 4.50 dd (11.6,
2.2)
6b 4.34 br d
(11.6)
6a 4.45 dd (11.1,
2.5)
6b 4.38 dd (11.1,
4.0)
79.3
62.3
Glc0
1
2
6.30 d (8.0)
4.19 dd (9.0,
8.0)
95.9 Glc0
74.1
1
2
6.22 d (8.1)
4.12 dd (8.7,
8.1)
95.8 Glc0
73.8
1
2
6.34 d (8.1)
4.23 dd (8.5,
8.1)
95.8 Glc0
74.1
1
2
6.35 d (8.1)
4.22 dd (8.7,
8.1)
95.8
74.1
6a 4.46 br d
(11.0)
6b 4.39 dd (11.0,
3.0)
3
4
5
4.28 dd (9.0,
9.0)
4.34 dd (9.0,
9.5)
78.9
71.2
3
4
5
4.21 dd (8.7,
8.5)
4.32 dd (9.3,
8.5)
78.7
70.9
3
4
5
4.30 dd (9.2,
8.5)
4.37 dd (9.2,
9.2)
78.9
71.1
3
4
5
4.30 dd (9.1,
8.7)
4.37 dd (9.1,
9.0)
78.9
71.1
4.02 br d (9.5)
79.3
62.3
4.09 m
77.9
69.5
4.03 m
79.4
62.1
4.06 m
79.3
62.3
6a 4.46 dd (12.0,
6a 4.70 br d
(10.5)
6a 4.45 dd (11.9,
6a 4.49 dd (11.9,
2.5)
2.3)
2.5)
6b 4.39 dd (12.0,
4.5)
6b 4.31 br d
(10.5)
6b 4.39 dd (11.9,
4.1)
6b 4.42 dd (11.9,
4.4)
Glc00
1
2
5.01 d (7.8)
4.01 dd (8.2,
7.8)
105.3
75.1
3
4
5
4.19 dd (8.3,
8.2)
4.22 dd (8.3,
8.2)
78.5
71.5
3.90 m
78.5
62.7
6a 4.49 dd (11.8,
2.4)
6b 4.36 dd (11.8,
5.2)

2160
M. Jitsuno, Y. Mimaki / Phytochemistry 71 (2010) 2157–2167
et al., 1988), 3-[(O-b-
D
-glucopyranosyl-(1?3)-O-[
a
-
L
-rhamnopyr-
shown to be involved in a linkage at C-4 of Glc because the signals
assignable to H-4 and C-4 were markedly displaced downfield at dH
5.23 (dd, J = 9.4, 9.4 Hz; +0.86 ppm) and dC 75.8 (+4.2 ppm), respec-
tively, when the 1H and 13C NMR spectra of 1 were compared with
those of 4. Accordingly, the structure of 1 was determined to be
anosyl-(1?2)]-
dien-28-oic acid (24; guaiacin C) (Ahmad et al., 1990), and 3-[(O-b-
-glucopyranosyl-(1?3)-O-[ -rhamnopyranosyl-(1?2)]-
arabinopyranosyl)oxy]-30-noroleana-12,20(29)-dien-28-oic acid
b- -glucopyranosyl ester (25; guaiacin D) (Ahmad et al., 1990),
a-L-arabinopyranosyl)oxy]-30-noroleana-12,20(29)-
D
a
-
L
a-L-
D
3-[(O-(4-O-sulfo-b-
D
-glucopyranosyl)-(1?3)-
a
-
L
-arabinopyrano-
respectively.
syl)oxy]olean-12-en-28-oic acid b-D-glucopyranosyl ester sodium
Compound 1 was obtained as an amorphous solid. Its molecular
formula was determined to be C47H75NaO20S by HRESI-TOFMS (m/z
1037.4329 [M + Na]+). The 1H NMR spectrum of 1 was typical of a
triterpene triglycoside based upon 3b-hydroxyolean-12-en-28-oic
acid, exhibiting seven three-proton singlet signals at d 1.29, 1.27,
1.11, 0.96, 0.92, 0.90, and 0.87, an olefinic proton signal at d 5.44
(br s), and three anomeric proton (H-1) signals at d 6.34 (d,
J = 8.0 Hz), 5.29 (d, J = 7.8 Hz), and 4.74 (d, J = 7.4 Hz). Acid hydroly-
sis of 1 with 1.0 M HCl in dioxane–H2O (1:1) gave 3b-hydroxyole-
salt.
Compound 2 was shown to have a molecular formula of
C47H75NaO20S on the basis of HRESI-TOFMS, which was the same
as that of 1. The spectroscopic properties of 2 were essentially
analogous to those of 1, and acid hydrolysis of 2 furnished 1a,
L-arabinose, and D-glucose, together with sulfuric acid. These data
implied that 2 was a positional isomer of 1 in regard to the sulfate
group. When the 1H and 13C NMR spectra of 2 were compared with
those of 4, the signals attributable to H-2 and C-2 were moved
downfield at dH 5.54 (dd, J = 3.8, 3.8 Hz; +0.94 ppm) and dC 75.8
(+3.9 ppm), respectively. Accordingly, the structure of 2 was deter-
an-12-en-28-oic acid (1a) as the aglycone, and
L-arabinose and
D-glucose as the carbohydrate moieties. The monosaccharides,
including their absolute configurations, were identified by direct
HPLC analysis of the hydrolysate using an optical rotation (OR)
detector. Furthermore, when 0.5 M BaCl2 aqueous solution was
added to the hydrolysate, it gave white precipitate, indicating the
presence of sulfate ion. From the above spectroscopic and chemical
data, together with the information from the IR spectrum of 1,
exhibiting the strong absorption at 1259 cmꢀ1 (S = O), 1 was de-
duced to be an olean-12-en-28-oic acid triglycoside with a sodium
sulfate group. Analyses of the 1H and 13C NMR spectra of 1 (Tables
1 and 2) suggested that the sugar moieties and their linkage posi-
tions to the aglycone of 1 were the same as those of the concomi-
tantly isolated compound (4) in this study. This was ascertained by
mined to be 3-[(O-b-
nopyranosyl)oxy]olean-12-en-28-oic acid b-
D
-glucopyranosyl-(1?3)-2-O-sulfo-
a-L-arabi-
D
-glucopyranosyl
3
ester sodium salt. The small JH-1,H-2 coupling constant (3.8 Hz),
as well as HMBC correlations between H-1 at dH 5.24 and C-3 at
dC 79.9/C-5 at dC 63.3 suggested that the arabinopyranosyl moiety
of 2 had a 1C4 conformation to reduce the steric hindrance caused
by the C-2 sulfate group. However, in the NOESY spectrum of 2,
H-1 showed NOE correlations with H-2, H-3 at d 4.71 (br s), and
H-5ax (H-5a) at d 3.78 (br d, J = 8.9 Hz) (Fig. 1). These data indi-
cated that the arabinopyranosyl group is present as the 1C4 and
4C1 forms in equilibrium with rapid conformational exchange
(Kuroda et al., 2002).
HMBC correlations between H-1 of one b-
(Glc) at dH 5.29 and C-3 of -arabinopyranosyl unit (Ara) at dC
84.1, H-1 of the other b-
-glucopyranosyl unit (Glc0) at dH 6.34
and C-28 of the aglycone at dC 176.5, and between H-1 of Ara at
dH 4.74 and C-3 of the aglycone at dC 88.7. The sulfate group was
D
-glucopyranosyl unit
Compounds 9 and 10 had the same molecular formula of
a
-L
C
41H64O14 as determined by their HRESI-TOFMS data, and gave
the common hydrolysates 3b-hydroxyolean-12-ene-28,29-dioic
acid (9a; seratagenic acid) (Yu et al., 1995), -glucose, and -xylose
on acid hydrolysis. These results, as well as 1H and 13C NMR
D
D
D
Table 2
13C NMR chemical shift assignments for the aglycone moiety of compounds 1, 2, 9, 10, and 12–20 in C5D5N.
Position
1
2
9
10
38.7
12
38.8
13
38.8
14
38.8
15
38.8
16
38.8
17
39.1
18
39.0
19
39.0
20
39.0
1
2
3
4
5
6
7
8
38.8
26.7
88.7
39.6
55.8
18.5
32.5
39.9
48.0
37.0
23.8
122.9
144.1
42.1
28.3
23.4
47.0
41.7
46.2
30.8
34.0
33.1
28.1
16.9
15.6
17.5
26.1
176.5
33.1
23.7
38.9
26.4
88.4
39.6
55.7
18.4
32.5
39.9
48.0
37.0
23.8
38.8
26.7
88.6
39.6
55.8
18.5
33.1
39.9
48.0
37.0
23.8
123.1
143.6
42.1
28.2
23.5
46.9
40.8
40.8
42.3
29.1
31.7
28.2
17.0
15.6
17.5
26.0
176.2
180.9
19.9
26.7
88.7
39.6
55.8
18.5
33.1
39.8
48.0
37.0
23.8
123.1
144.3
42.2
28.3
23.8
46.6
41.1
41.1
42.6
29.3
32.4
28.0
17.0
15.5
17.4
26.1
179.9
181.0
20.0
26.7
88.7
39.6
55.9
18.5
33.2
39.8
48.0
37.0
23.8
123.0
144.1
42.1
28.3
23.8
47.0
47.9
42.0
149.1
30.4
38.4
28.2
17.0
15.5
17.3
26.1
179.4
107.1
26.6
88.7
39.6
55.8
18.5
33.1
39.9
48.0
37.0
23.8
123.1
143.4
42.1
28.2
23.5
47.3
47.6
41.7
148.5
30.1
37.6
28.2
16.9
15.6
17.5
26.0
175.7
107.3
26.7
88.8
39.6
55.8
18.5
33.1
39.9
48.0
37.0
23.8
123.1
143.4
42.1
28.1
23.5
47.3
47.6
41.7
148.5
30.1
37.6
28.1
16.9
15.5
17.4
26.0
175.7
107.3
26.7
88.7
39.6
55.9
18.5
33.1
39.9
48.0
37.0
23.8
123.1
143.4
42.1
28.2
23.5
47.3
47.6
41.7
148.5
30.1
37.6
28.1
17.0
15.6
17.5
26.0
175.7
107.3
26.7
88.7
39.6
55.8
18.5
33.1
39.9
48.0
37.0
23.8
123.1
143.4
42.1
28.2
23.5
47.3
47.6
41.7
148.4
30.1
37.6
28.1
16.9
15.6
17.4
26.0
175.8
107.3
26.7
88.3
39.6
56.0
18.5
33.1
39.9
48.0
37.0
23.8
123.1
143.4
42.1
28.2
23.6
47.3
47.6
41.7
148.5
30.1
37.6
28.1
17.0
15.7
17.5
26.0
175.7
107.3
26.6
88.2
39.6
56.0
18.5
33.1
39.9
48.0
37.0
23.7
123.1
143.4
42.1
28.2
23.5
47.3
47.5
41.7
148.4
30.1
37.6
28.1
17.0
15.7
17.5
26.0
175.8
107.3
26.6
88.1
39.6
56.0
18.5
33.1
39.9
48.1
37.0
23.8
122.5
144.3
42.2
28.4
23.3
46.8
41.1
46.2
67.9
34.2
31.9
28.1
17.0
15.6
17.5
26.1
176.5
32.0
26.6
88.2
39.6
56.0
18.5
33.1
39.9
48.0
37.0
23.7
123.1
143.6
42.1
28.2
23.6
47.1
44.1
47.8
69.6
35.9
34.5
28.1
17.0
15.7
17.5
26.0
176.3
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
122.8
144.1
42.1
28.2
23.4
47.0
41.7
46.2
30.7
34.0
33.1
28.3
17.0
15.6
17.4
26.1
176.5
33.1
23.6
25.6

M. Jitsuno, Y. Mimaki / Phytochemistry 71 (2010) 2157–2167
2161
HO
O
Glc
O
H
H
O
HMBC
O
C5
O
H
O
H1
HO
C3
H5ax
H2
O
Glc
H1
NOE
SO3Na
H3
H
O
NOE
SO3Na
Fig. 1. HMBC and NOE correlations of the arabinopyranosyl moiety of 2.
30
29
spectroscopic data, indicated that 9 and 10 were closely related triter-
pene diglycoside and had a b- -glucopyranosyl unit (Glc) [dH-1 6.36
(d, J = 8.1 Hz) in 9; dH-1 5.32 (d, J = 7.8 Hz) in 10] and a b- -xylopyr-
D
D
12
26
18
anosyl unit (Xyl) [dH-1 4.83 (d, J = 7.5 Hz) in 9; dH-1 4.78 (d,
J = 7.5 Hz) in 10] in each molecule. The C-3 oxymethine carbon
and C-28 carbonyl carbon were observed at d 88.6 and 176.2,
respectively, in the 13C NMR spectrum of 9, which suggested that
9 was a bisdesmosidic triterpene. In the HMBC spectrum of 9,
long-range correlations were observed between H-1 of Glc at dH
6.36 and C-28 of the aglycone at dC 176.2, and between H-1 of
Xyl at dH 4.83 and C-3 of the aglycone at dC 88.6. On the other hand,
the HMBC spectrum of 10 showed long-range correlations between
H-1 of Glc at dH 5.32 and C-3 of Xyl at dC 88.5, and between H-1 of
Xyl at dH 4.78 and C-3 of the aglycone at dC 88.7. The structures of 9
25
H
COOR1
28
HO
HO
HO
H
27
O
O
R3O
3
O
O
H
OH
24 23
OR2
R1
Glc
Glc
H
R2
H
R3
1
2
3
4
6
7
8
SO3Na
SO3Na
H
H
H
H
H
H
and 10 were assigned as 3-[(b-
28,29-dioic acid 28-b- -glucopyranosyl ester and 3-[(O-b-
-xylopyranosyl)oxy]olean-12-ene-28,29-dioic
D-xylopyranosyl)oxy]olean-12-ene-
H
D
D
-gluco-
Glc
H
H
pyranosyl-(1?3)-b-
D
Rha
Rha
Rha
acid, respectively.
Glc
Compounds 12 and 13 had the same molecular formula of
40H62O12 on the basis of HRESI-TOFMS, and gave -glucose and
-arabinose on acid hydrolysis. The 1H NMR spectrum of 12 showed
Glc-(1 ¨6)-Glc
C
L
D
signals for an exomethylene group at d 4.82 and 4.76 (each s), as
well as signals for five tertiary methyl groups at d 1.33, 1.29,
0.99 ꢁ 2, and 0.84 (each s), an olefinic proton at d 5.50 (br s), and
two anomeric protons at d 5.41 (d, J = 8.0 Hz) and 4.77 (d,
J = 7.5 Hz), which were characteristic of a triterpene glycoside with
the 3b-hydroxy-30-norolean-12-en-28-oic acid framework. The
molecular formula of 12 was less than that of the known 30-nortri-
terpene bisdesmoside (22) by C6H10O5, corresponding to the lack of
one hexose. Alkaline treatment of 22 with 4% KOH in EtOH yielded
O
H
HOOC
H
O
HO
O
O
H
O
HO
HO
OH
HO
OH
O
HO
O
HO
12. Thus, the structure of 12 was elucidated as 3-[(O-b-
D-glucopyr-
OH
anosyl-(1?3)- -arabinopyranosyl)oxy]-30-noroleana-12,20(29)-
a-L
5
dien-28-oic acid. Compound 13 was an isomer of 12 in regard to
the monosaccharide linkage positions. In the HMBC spectrum of
13, H-1 of an
showed a long-range correlation with C-3 of the 30-nortriterpene
aglycone at dC 88.7, whereas H-1 of a b- -glucopyranosyl unit at
dH 6.30 (d, J = 8.1 Hz) had a correlation with C-28 of the aglycone
at dC 175.7. The structure of 13 was identified as 3-[( -arabino-
a-L-arabinopyranosyl unit at dH 4.79 (d, J = 7.4 Hz)
29
30
COOH
D
12
a-L
H
COOR1
pyranosyl)oxy]-30-noroleana-12,20(29)-dien-28-oic acid b-D-glu-
copyranosyl ester.
H
O
Compounds 14 and 15, which had the same molecular formula
of C48H74O18, were established to contain an acetyl group in each
molecule by examination of the 1H and 13C NMR spectra [dH 1.95
(3H, s)/dC 170.8 (C = O) and 21.2 (Me) in 14; dH 1.97 (3H, s)/dC
170.8 (C = O) and 20.7 (Me) in 15]. Alkaline hydrolysis of 14 and
15 gave the same hydrolysate (22). When the 1H NMR spectra of
14 and 15 were compared with that of 22, H-3 of the b-
anosyl unit (Glc) attached to C-3 of the -arabinopyranosyl unit
was shifted to a lower field by 1.6 ppm in 14, while H-6a and
H-6b of Glc were moved downfield by 0.46 and 0.36 ppm, respec-
tively, in 15. Furthermore, the carbonyl carbon of the acetyl group
of 14 at dC 170.8 showed a long-range correlation with H-3 of Glc at
HO
O
R2O
H
OH
R1
Glc
H
R2
H
9
10
11
D-glucopyr-
Glc
H
a-L
H
dH 5.87 (dd, J = 9.5, 9.5 Hz), and that of 15 at dC 170.8 had correla-
tions with H2-6 at dH 4.92 (br d, J = 11.5 Hz) and 4.75 (dd, J = 11.5,

2162
M. Jitsuno, Y. Mimaki / Phytochemistry 71 (2010) 2157–2167
5.5 Hz) in the HMBC spectra of 14 and 15. Thus, the structures of 14
and 15 were formulated as 3-[(O-(3-O-acetyl-b- -glucopyranosyl)-
(1?3)- -arabinopyranosyl)oxy]-30-noroleana-12,20(29)-dien-
28-oic acid b- -glucopyranosyl ester and 3-[(O-(6-O-acetyl-b-
glucopyranosyl)-(1?3)- -arabinopyranosyl)oxy]-30-noroleana-
12,20(29)-dien-28-oic acid b- -glucopyranosyl ester, respectively.
Compound 16 exhibited a molecular formula of C46H71NaO20
the aglycone at dC 175.8. HMBC correlations were also observed
between H-1 of Rha at dH 6.14 and C-2 of Ara at dC 74.8, H-1 of
Glc at dH 5.11 and C-3 of Ara at dC 81.9, and between H-1 of Ara at
dH 4.88 and C-3 of the aglycone at dC 88.2. The structure of 18 was
D
a
-L
D
D-
a
-
L
determined to be 3-[(O-b-D-glucopyranosyl-(1?3)-O-[a-L-rhamno-
D
pyranosyl-(1?2)]- -arabinopyranosyl)oxy]-30-noroleana-12,20(29)-
a-L
S
dien-28-oic acid O-b-
D
-glucopyranosyl-(1?6)-b-
D-glucopyranosyl
on the basis of HRESI-TOFMS. The 1H NMR spectroscopic features
of 16 were essentially analogous to those of 22, showing signals
for an exomethylene group at d 4.77 and 4.70 (each s), as well as
resonances for five tertiary methyl groups at d 1.29, 1.25, 1.09,
0.95, and 0.86 (each s), an olefinic proton at d 5.45 (br s), and three
anomeric protons at d 6.28 (d, J = 8.1 Hz), 5.29 (d, J = 7.8 Hz), and
4.74 (d, J = 7.5 Hz). The presence of a sodium sulfate group in 16
was shown by not only the deduced molecular formula, but also
the prominent absorption at 1259 cmꢀ1 in the IR spectrum and
the results of acid hydrolysis, giving sulfuric acid, as well as
ester.
29
20
H
COOR1
HO
R5O
R3O
H
O
O
R4O
O
O
H
D
-glucose and
one b- -glucopyranosyl unit (Glc) at dH 5.29 and C-3 of
nopyranosyl unit (Ara) at dC 84.0, H-1 of the other b- -glucopyran-
L
-arabinose. HMBC correlations between H-1 of
OH
OR2
D
a-L
-arabi-
D
R1
R2
H
R3
H
R4
R5
osyl unit (Glc0) at dH 6.28 and C-28 of the aglycone at at dC 175.8,
and between H-1 of Ara at dH 4.74 and C-3 of the aglycone at dC
88.7 gave evidence that the sugar moieties of 16 were the same
as those of 22. When the 1H and 13C NMR spectra of 16 were com-
pared with those of 22, the signals assignable to H-4 and C-4 of Glc
were markedly displaced downfield at dH 5.24 (dd, J = 9.4, 9.1 Hz;
+0.91 ppm) and dC 75.7 (+4.1 ppm), respectively, allowing a sodium
sulfate group to be located at the C-4 hydroxy group of Glc in 16.
12
H
H
H
H
H
H
14
15
16
17
18
22
24
25
Glc
Glc
Glc
Glc
H
Ac
H
H
Ac
H
H
H
SO3Na
Rha
Rha
H
H
SO3Na
H
Glc-(1 ¨6)-Glc
H
H
H
H
H
H
Glc
H
H
H
The structure of 16 was determined to be 3-[(O-(4-O-sulfo-b-
copyranosyl)-(1?3)- -arabinopyranosyl)oxy]-30-noroleana-
12,20(29)-dien-28-oic acid b- -glucopyranosyl ester sodium salt.
D-glu-
a
-L
Rha
Rha
H
H
D
Glc
H
H
Compound 17 was found to have a molecular formula of
C52H81NaO24S as determined by HRESI-TOFMS, which was higher
than that of 16, with the difference corresponding to a C6H10O4.
The 1H NMR spectrum showed signals for four anomeric protons
at d 6.30 (d, J = 8.0 Hz), 6.15 (s), 5.02 (d, J = 7.5 Hz), and 4.81 (d,
J = 6.0 Hz), together with signals for the 30-nortriterpene aglycone.
On the basis of the spectral properties of 17 and the results of acid
O
H
hydrolysis experiments, in which 17 yielded
D-glucose, L-arabinose,
H
L-rhamnose, and sulfuric acid, 17 was shown to be a triterpene gly-
RO
coside with a sodium sulfate group closely related to 16; however,
the sugar chain attached to C-3 of the aglycone was made up of
three monosaccharides and differed from that of 16 by the pres-
H
HO
O
HO
O
HO
ence of an additional
a-L-rhamnopyranosyl unit (Rha). In the
OH
HMBC spectrum of 17, H-1 of Rha at dH 6.15 exhibited a long-range
correlation with C-2 of Ara at dC 74.8. HMBC correlations were also
observed between H-1 of Glc at dH 5.02 and C-3 of Ara at dC 82.6, H-
1 of Glc0 at dH 6.30 and C-28 at dC 175.7, and between H-1 of Ara dH
4.81 and C-3 of the aglycone at dC 88.3. The H-4 and C-4 signals of
Glc appeared at dH 5.15 (dd, J = 9.5, 9.5 Hz) and dC 75.7, respec-
tively. The structure of 17 was characterized as 3-[(O-(4-O-sulfo-
R
13
21
Ara
H
Compound 19 had a molecular formula of C52H84O22 on the ba-
sis of HRESI-TOFMS. It was shown to be a 30-nortriterpene deriva-
tive related to 25, having the same sugar moieties at C-3 and C-28
of the aglycone as 25 by the 1H and 13C NMR spectra. However, dif-
ferences were recognized in the NMR signals arising from the
E-ring moiety of the hexacyclic skeleton. Instead of the signals for
the C-20(29) exomethylene group, signals assignable to a methyl
group at dH 1.43 (3H, s) and dC 32.0, and a quaternary carbon bear-
ing an oxygen atom at d 67.9 were observed. Long-range correla-
tions between dH 1.43 (Me-29) and dC 46.2 (C-19)/dC 67.9 (C-20)/
dC 34.2 (C-21) in the HMBC spectrum indicated that 19 was a
20(29)-saturated derivative with the introduction of a hydroxy
group to C-20. The C-20b configuration of the hydroxy group was
determined by an NOE correlation between Me-29 and H-19ax at
d 2.04 (dd, J = 13.3, 13.3 Hz) in the NOESY spectrum of 19. The
significant downfield shifts of the 1H NMR signals for H-18 and
b-
arabinopyranosyl)oxy]-30-noroleana-12,20(29)-dien-28-oic acid
b- -glucopyranosyl ester sodium salt.
Compound 18 was deduced to be C58H92O26 on the basis of
HRESI-TOFMS, which was higher than that of 25 by C6H10O5, corre-
sponding to a hexose unit. The 1H NMR spectrum contained signals
for five anomeric protons at d 6.22 (d, J = 8.1 Hz), 6.14 (s), 5.11 (d,
J = 7.7 Hz), 5.01 (d, J = 7.8 Hz), and 4.88 (d, J = 5.4 Hz), together with
signals for the 30-nortriterpene aglycone. Acid hydrolysis of 18
D-glucopyranosyl)-(1?3)-O-[a-L-rhamnopyranosyl-(1?2)]-a-L-
D
gave
D-glucose, L-arabinose, and L-rhamnose. These data and com-
parison of the 13C NMR spectrum of 18 with that of 25 suggested
that 18 was related to 25 with an additional b-D-glucopyranosyl
unit (Glc00). In the HMBC spectrum of 18, H-1 of Glc00 at dH 5.01
showed a long-range correlation with C-6 of the inner Glc0 at dC
69.5, of which H-1 at dH 6.22 exhibited a correlation with C-28 of

M. Jitsuno, Y. Mimaki / Phytochemistry 71 (2010) 2157–2167
2163
Table 3
H-22ax, which were observed at d 3.80 (dd, J = 13.4, 4.2 Hz) and
Cytotoxic activity of compounds 1–25 against HL-60 leukemia cells.
2.57 (m), respectively, were ascribed to the 1,3-diaxial interactions
with the C-20b hydroxy group. The structure of 19 was assigned as
Compound
IC50
(l
M)a
Compound
IC50 (lM)
3-[(O-b-
-arabinopyranosyl)oxy]-20b-hydroxy-30-norolean-12-en-28-oic
acid b- -glucopyranosyl ester.
D-glucopyranosyl-(1?3)-O-[a-L-rhamnopyranosyl-(1?2)]-
b
1
2
3
4
5
6
7
8
–
15
16
17
18
19
20
21
22
23
24
25
–
–
–
–
–
–
a-L
–
18.6 0.12
–
–
6.9 0.11
–
–
–
–
–
–
D
Compound 20 exhibited a molecular formula of C52H84O22 on
the basis of HRESI-TOFMS, which was the same as that of 19. The
spectroscopic data of 20 was very similar to those of 19 except
for the signals arising from C-20 and its neighboring protons and
carbons, suggesting that 20 was a stereoisomer of 19 in regard to
the C-20 hydroxy group. In the NOESY spectrum of 20, NOE corre-
lations were detected between Me-30 at d 1.46 (s) and H-18 at d
3.25 (dd, J = 13.7, 4.1 Hz)/H-22ax at d 1.85 (m). The structure of
10.1 0.20
–
4.1 0.37
–
–
9
10
11
12
13
14
6.0 1.47
–
Etoposide
Cisplatin
0.33 0.01
1.1 0.03
20 was elucidated as 3-[(O-b-D-glucopyranosyl-(1?3)-O-[a-L-
rhamnopyranosyl-(1?2)]- -arabinopyranosyl)oxy]-20
29-norolean-12-en-28-oic acid b-D-glucopyranosyl ester.
a
-
L
a
-hydroxy-
a
Data represent the mean value SEM of three experiments performed in
triplicate.
b
Means IC50 >20 lM.
OH
20
of 18.6, 6.9, 6.0, 10.1, and 4.1
(positive controls) had IC50 values of 0.33 and 1.1
l
M, when etoposide and cisplatin
O
lM, respectively.
H
O
The other compounds (1, 2, 4, 5, 7–12, 14–20, 22, 24, and 25) were
not cytotoxic to HL-60 cells at sample concentrations of 20–
HO
HO
H
O
O
O
HO
100
lM. In the glycosides of 3b-hydroxyolean-12-en-28-oic acid
O
O
HO
H
(oleanolic acid), only the monodesmosides with
a
glucosyl-
OH
HO
(1?3)-arabinosyl and a glucosyl-(1?3)-[rhamnosyl-(1?2)]-arabi-
nosyl unit at C-3 of the aglycone (3 and 6) exhibited cytotoxic
activity against HL-60 cells; however, the corresponding 30-nor-
12,20(29)-diene derivatives (akebonoic acid derivatives) (12 and
24) were not cytotoxic. On the other hand, akebonoic acid 28-O-
glucoside (monodesmoside) (21), and its 3-O-arabinoside (13)
and 3-O-[rhamnosyl-(1?2)-arabinoside] (bisdesmosides) (23)
showed cytotoxicity against HL-60 cells. The above results sug-
gested that the structures of both the aglycone and sugar moieties
contributed to the appearance of cytotoxicity in these glycosides.
HO
O
Me
O
HO
HO
OH
HO
OH
19
20
20β-OH
20α-OH
2.3. Conclusions
O
H
Fragmentary phytochemical studies of L. tridentata (Zygophyll-
aceae) have been carried out, and lignans, flavonoids, and triter-
penes have been isolated and identified (Abou-Gazar et al.,
2004). The present investigation of the aerial part of L. tridentata,
with attention paid to its triterpene glycoside constituents, re-
sulted in the isolation of a total of 25 triterpene glycosides includ-
ing 13 new compounds. The new compounds (1, 2, 16, and 17) are
triterpene glycosides with a sulfate group at their sugar moieties.
Triterpene glycosides with a sulfate group have been also isolated
from other plants belonging to the Zygophyllaceae family such as
Fagonia indica (Shaker et al., 2000), F. arabica (Perrone et al.,
2007), and Zygophyllum fabago (Feng et al., 2009). Among the 25
compounds, five showed cytotoxic activity, which may partially ac-
count for the alternative use of L. tridentata to treat cancers.
HO
HO
H
O
O
O
H
HO
O
HO
O
Me
O
HO
HO
OH
HO
OH
23
HO
HO
HO
O
O
HO
Ara:
Glc:
HO
OH
OH
3. Experimental
3.1. General
Me
Rha:
O
HO
Optical rotations were measured using a JASCO P-1030 (Tokyo,
Japan) automatic digital polarimeter. IR and CD spectra were re-
corded on a JASCO FT-IR 620 and a JASCO V-520 spectrophotome-
ter, respectively. NMR spectra were recorded on either a Bruker
DPX-400 spectrometer (400 MHz for 1H NMR, Karlsruhe, Germany)
or a Bruker DRX-500 spectrometer (500 MHz for 1H NMR) using
standard Bruker pulse programs. Chemical shifts are given as d
HO
2.2. Cytotoxic activity
OH
The isolated compounds were evaluated for their cytotoxic
activity against HL-60 cells (Table 3). Compounds 3, 6, 13, 21,
and 23 showed moderate cytotoxicity with respective IC50 values

2164
M. Jitsuno, Y. Mimaki / Phytochemistry 71 (2010) 2157–2167
values with reference to tetramethylsilane (TMS) as an internal
standard. ESIMS data were obtained on a Waters-Micromass LCT
mass spectrometer (Manchester, UK). Diaion HP-20 (Mitsubishi-
Chemical, Tokyo, Japan), silica gel (Fuji-Silysia Chemical, Aichi,
Japan), and ODS silica gel (Nacalai Tesque, Kyoto, Japan) were used
for column chromatography. TLC was carried out on Silica gel 60
F254 (0.25 or 0.5 mm thick, Merck, Darmstadt, Germany) and RP-
18 F254S (0.25 mm thick, Merck) plates, and spots were visualized
by spraying with 10% H2SO4 in H2O followed by heating. HPLC
was performed with a system composed of a CCPM pump (Tosoh,
Tokyo, Japan), a CCP PX-8010 controller (Tosoh), a RI-8010 (Tosoh)
or a Shodex OR-2 (Showa-Denko, Tokyo, Japan) detector, and a
Rheodyne injection port. A Capcell Pak C18 AQ column (10 mm
preparative HPLC using MeOH–H2O (17:3). Fraction VI-7 was ap-
plied to ODS silica gel CC eluted with MeOH–H2O (11:9; 2:1;
4:1) and MeCN–H2O (2:5; 1:2; 2:3), and then silica gel CC with
CHCl3–MeOH–H2O (40:10:1; 30:10:1) to give a mixture of 1 and
5, 7 (23.6 mg), 19 (9.3 mg), 20 (5.0 mg), 21 (7.4 mg), and 25
(8.5 mg). Compounds 1 and 5 were separated by preparative TLC
using CHCl3–MeOH–H2O (7:4:1) to furnish 1 (9.7 mg) and 5
(16.5 mg). Fraction VI-9 was applied to an ODS silica gel column
eluted with MeOH–H2O (1:1; 2:1) and silica gel with CHCl3–
MeOH–H2O (14:6:1; 75:40:7) to yield 16 (36.4 mg) and 17 with
few impurities. Compound 17 (8.3 mg) was purified by preparative
HPLC using MeCN–MeOH–H2O (1:1:3). Fraction VI-10 was sub-
jected to CC on ODS silica gel eluted with MeOH–H2O (1:1; 2:1),
and then silica gel with CHCl3–MeOH–H2O (14:6:1; 7:4:1) to gave
8 (8.7 mg) and 18 (92.3 mg) as pure compounds, and 2 with few
impurities. Compound 2 (19.5 mg) was refined by preparative
TLC using CHCl3–MeOH–H2O (20:10:1).
i.d. ꢁ 250 mm, 5
lm, Shiseido, Tokyo, Japan) was employed for
preparative HPLC. The following materials and reagents were used
for cell culture assay; microplate reader, Spectra Classic, Tecan
(Salzburg, Austria); 96-well flat-bottom plate, Iwaki Glass (Chiba,
Japan); HL-60 cells, Human Science Research Resources Bank (JCRB
0085, Osaka, Japan); fetal bovine serum (FBS), Bio-Whittaker
(Walkersville, MD, USA); RPMI 1640 medium, etoposide, cisplatin,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bro-
mide (MTT), and 0.25% Tripsin–EDTA solution, Sigma (St. Louis,
MO, USA); Dulbecco’s modified Eagle medium (DMEM) medium,
penicillin G sodium salt, and streptomycin sulfate, Gibco (Grand Is-
land, NY, USA). All other chemicals used were of biochemical re-
agent grade.
3.4. Compound 1
3-[(O-(4-O-Sulfo-b-
D-glucopyranosyl)-(1?3)-a-L-arabinopyr-
anosyl)oxy]olean-12-en-28-oic acid b-
D
25
-glucopyranosyl ester so-
20.1 (c 0.10; MeOH); IR
dium salt (1); amorphous solid; [
a]
D
mmax (film) cmꢀ1: 3389 (OH), 2927 and 2878 (CH), 1746 (C = O),
1651 (C = C), 1259 (S = O), 1078 (C–O); 1H NMR (500 MHz,
C5D5N): d 5.44 (1H, br s, H-12), 1.29 (3H, s, Me-23), 1.27 (3H, s,
Me-27), 1.11 (3H, s, Me-26), 0.96 (3H, s, Me-24), 0.92 (3H, s, Me-
29), 0.90 (3H, s, Me-30), 0.87 (3H, s, Me-25): For 1H NMR spectro-
scopic data of the sugar moieties, see Table 1; for 13C NMR
(125 MHz, C5D5N) spectroscopic data, see Tables 1 and 2; HRESI-
TOFMS m/z: 1037.4329 [M + Na]+ (Calcd for C47H75Na2O20S:
1037.4368).
3.2. Plant material
The aerial parts of L. tridentata were obtained from Richters, On-
tario, Canada. A small amount of the sample is preserved in our
laboratory (07-003-LR).
3.3. Extraction and isolation
3.5. Acid hydrolysis of 1
The aerial parts of L. tridentata (3.0 kg of dry weight) were ex-
tracted with hot MeOH (77 l). After removing the solvent, the
MeOH extract (940 g), which showed cytotoxic activity against
A solution of 1 (7.1 mg) in 1 M HCl (dioxane–H2O, 1:1; 2 ml)
was heated at 95 °C for 1 h under Ar atmosphere. After the reaction
mixture was diluted with H2O, it was extracted with EtOAc satu-
rated with H2O (10 ml ꢁ 3). The EtOAc extract was subjected to sil-
ica gel CC eluted with hexane–Me2CO (4:1) to yield oleanolic acid
(1.9 mg). The H2O residue was neutralized by passage through an
Amberlite IRA-96SB (Organo, Tokyo, Japan) column and passed
through a Sep-Pak C18 cartridge (Waters, Milford, MA, USA), which
was then analyzed by HPLC under the following conditions: col-
HL-60 cells (IC50 6.8 lg/ml), was passed through a Diaion HP-20
column and successively eluted with MeOH–H2O (3:7), MeOH–
H2O (1:1), MeOH, EtOH, and EtOAc. The MeOH, EtOH, and EtOAc
eluate fractions exhibited cytotoxic activity against HL-60 cells
(IC50 2.7, 2.4, and 11.0
phy (CC) of the MeOH eluate portion (477 g) on silica gel and elu-
tion with stepwise gradient mixture of CHCl3–MeOH–H2O
lg/ml, respectively). Column chromatogra-
a
umn, Capcell Pak NH2 UG 80 (4.6 mm i.d. ꢁ 250 mm, 5
ido); solvent, MeCN–H2O (17:3); flow rate, 1.0 ml/min; detection,
RI and OR. The identification of -arabinose and -glucose present
lm, Shise-
(19:1:0; 9:1:0; 40:10:1), and finally with MeOH alone, gave six
fractions (I–VI). Fraction V was subjected to silica gel CC eluted
with CHCl3–MeOH–H2O (9:1:0; 40:10:1) to collect 11 subfractions
(V-1–11). Fraction V-2 was applied to ODS silica gel CC eluted with
MeCN–H2O (2:5; 1:2; 1:1) and MeOH–H2O (2:1), and then silica gel
CC with CHCl3–MeOH (9:1) to afford 11 (15.3 mg). Fraction V-9
was subjected to CC on ODS silica gel eluted with MeCN–H2O
(2:5; 1:1) and MeOH–H2O (3:2), and then silica gel with CHCl3–
MeOH–H2O (40:10:1) to afford 9 (14.0 mg), 10 (13.4 mg), 12
(6.2 mg), 13 (12.6 mg), and 15 (9.4 mg) as pure compounds, and
3 with few impurities. Compound 3 (12.0 mg) was purified by pre-
parative HPLC using MeOH–H2O (9:1). Fraction VI was chromato-
graphed on silica gel eluted with CHCl3–MeOH–H2O (40:10:1;
20:10:1; 7:4:1) to collect 10 subfractions (VI-1–10). Fraction VI-2
was applied to ODS silica gel CC eluted with MeOH–H2O (2:1;
4:1) and silica gel CC with CHCl3–MeOH–H2O (50:10:1; 40:10:1)
to afford 14 (28.2 mg) and 23 (4.5 mg). Fraction VI-4 was subjected
to CC on ODS silica gel eluted with MeOH–H2O (7:3; 4:1), and then
silica gel with CHCl3–MeOH–H2O (30:10:1) to afford 4 (71.1 mg),
15 (5.2 mg), 22 (75.4 mg), and 24 (226 mg) as pure compounds,
and 6 with few impurities. Compound 6 (2.0 mg) was purified by
L
D
in the sugar fraction was carried out by comparison of their reten-
tion times and optical rotations with those of authentic samples. tR
(min): 8.53 (L-arabinose, positive optical rotation), 14.04 (D-glu-
cose, positive optical rotation).
3.6. Compound 2
3-[(O-b-
syl)oxy]olean-12-en-28-oic acid b-
salt (2); amorphous solid; [ m
25 2.6 (c 0.10; MeOH); IR
D
-Glucopyranosyl-(1?3)-2-O-sulfo-
-glucopyranosyl ester sodium
max (film)
a-L-arabinopyrano-
D
a
]
D
cmꢀ1: 3418 (OH), 2944 and 2880 (CH), 1733 (C = O), 1645 (C = C),
1259 (S = O), 1075 (C–O); 1H NMR (500 MHz, C5D5N): d 5.42 (1H,
br s, H-12), 1.33 (3H, s, Me-23), 1.25 (3H, s, Me-27), 1.14 (3H, s,
Me-26), 1.07 (3H, s, Me-24), 0.91 (3H, s, Me-29), 0.88 (3H, s,
Me-30), 0.83 (3H, s, Me-25): For 1H NMR spectroscopic data of
the sugar moieties, see Table 1; for 13C NMR (125 MHz, C5D5N)
spectroscopic data, see Tables
1 and 2; HRESI-TOFMS m/z:
1037.4353 [M + Na]+ (Calcd for C47H75Na2O20S: 1037.4368).

M. Jitsuno, Y. Mimaki / Phytochemistry 71 (2010) 2157–2167
2165
3.7. Acid hydrolysis of 2
1H, s, H-29), 1.33 (3H, s, Me-23), 1.29 (3H, s, Me-27), 0.99 (3H, s,
Me-26), 0.99 (3H, s, Me-24), 0.84 (3H, s, Me-25): For 1H NMR spec-
troscopic data of the sugar moiety, see Table 1; for 13C NMR
(125 MHz, C5D5N) spectroscopic data, see Tables 1 and 2; HRESI-
TOFMS m/z: 757.4138 [M + Na]+ (Calcd for C40H62NaO12: 757.4139).
A solution of 2 (6.3 mg) was subjected to acid hydrolysis as de-
scribed for 1 to give oleanolic acid (2.3 mg) and a sugar fraction.
HPLC analysis of the sugar fraction under the same conditions as
in the case of 1 showed the presence of
L-arabinose and
D-glucose.
tR (min): 9.45 ( -arabinose, positive optical rotation), 16.27 (
L
D
-glu-
3.13. Acid hydrolysis of 12
cose, positive optical rotation).
A solution of 12 (3.2 mg) was subjected to acid hydrolysis as de-
scribed for 1 to give a sugar fraction. HPLC analysis of the sugar
fraction under the same conditions as in the case of 1 showed
3.8. Compound 9
3-[(b-
D
-Xylopyranosyl)oxy]olean-12-ene-28,29-dioic acid 28-b-
the presence of
nose, positive optical rotation), 16.22 (
rotation).
L
-arabinose and
D
-glucose. tR (min): 9.43 (
L
-arabi-
25
D
-glucopyranosyl ester (9); amorphous solid; [
a]
ꢀ2.1 (c 0.10;
D-glucose, positive optical
D
MeOH); IR mmax (film) cmꢀ1: 3390 (OH), 2939 and 2877 (CH),
1730 (C = O), 1651 (C = C), 1074 (C–O); 1H NMR (500 MHz,
C5D5N): d 5.50 (1H, br s, H-12), 1.46 (3H, s, Me-30), 1.29 (3H, s,
Me-23), 1.27 (3H, s, Me-27), 1.12 (3H, s, Me-26), 0.99 (3H, s, Me-
24), 0.88 (3H, s, Me-25): For 1H NMR spectroscopic data of the
sugar moieties, see Table 1; for 13C NMR (125 MHz, C5D5N) spec-
troscopic data, see Tables 1 and 2; HRESI-TOFMS m/z: 803.4208
[M + Na]+ (Calcd for C41H64NaO14: 803.4194).
3.14. Preparation of 12 from 22
Compound 22 (11.2 mg) was treated with 4% KOH in EtOH
(10.0 ml) at 80 °C for 2 h. The reaction mixture was neutralized
by passage through an Amberlite IR-120B (Oregano, Tokyo, Japan)
and purified by silica gel CC eluted with CHCl3–MeOH–H2O
(50:10:1) to give 12 (7.3 mg).
3.9. Acid hydrolysis of 9
3.15. Compound 13
A solution of 9 (4.9 mg) in 0.2 M HCl (dioxane–H2O, 1:1; 2 ml)
was heated at 95 °C for 5 h under Ar atmosphere. After the reaction
mixture was diluted with H2O, it was extracted with EtOAc satu-
rated with H2O (10 ml ꢁ 3). The EtOAc extract was applied to silica
gel CC eluted with CHCl3–MeOH (19:1) to yield serratagenic acid
(2.2 mg) (Yu et al., 1995). The H2O residue was neutralized by
passage through an Amberlite IRA-96SB column and passed
through a Sep-Pak C18 cartridge to give a sugar fraction. HPLC
analysis of the sugar fraction under the same conditions as in the
3-[(
28-oic acid b-
a-L-Arabinopyranosyl)oxy]-30-noroleana-12,20(29)-dien-
25
D-glucopyranosyl ester (13); amorphous solid; [a]
D
73.3 (c 0.10; MeOH); IR mmax (film) cmꢀ1: 3390 (OH), 2941 and
2879 (CH), 1733 (C = O), 1651 (C = C), 1074 (C–O); 1H NMR
(500 MHz, C5D5N): d 5.45 (1H, br s, H-12), 4.77 and 4.70 (each
1H, s, H2-29), 1.29 (3H, s, Me-23), 1.24 (3H, s, Me-27), 1.09 (3H, s,
Me-26), 0.96 (3H, s, Me-24), 0.87 (3H, s, Me-25): For 1H NMR spec-
troscopic data of the sugar moieties, see Table 1; for 13C NMR
(125 MHz, C5D5N) spectroscopic data, see Tables 1 and 2; HRESI-
TOFMS m/z: 757.4103 [M + Na]+ (Calcd for C40H62NaO12: 757.4139).
case of 1 showed the presence of
D
-xylose and
D-glucose. tR
(min): 10.08 ( -xylose, positive optical rotation), 16.39 (
D
D
-glucose,
positive optical rotation).
3.16. Acid hydrolysis of 13
3.10. Compound 10
A solution of 13 (3.2 mg) was subjected to acid hydrolysis as de-
scribed for 1 to give a sugar fraction. HPLC analysis of the sugar
fraction under the same conditions as in the case of 1 showed
3-[(O-b-
D-Glucopyranosyl-(1?3)-b-D-xylopyranosyl)oxy]olean-
25
12-ene-28,29-dioic acid (10); amorphous solid; [
a
]
D
ꢀ13.2 (c
0.043; MeOH); IR mmax (film) cmꢀ1: 3390 (OH), 2928 and 2877
(CH), 1691 (C = O), 1647 (C = C), 1075 (C–O); 1H NMR (500 MHz,
C5D5N): d 5.55 (1H, br s, H-12), 1.60 (3H, s, Me-30), 1.29 (3H, s,
Me-23), 1.31 (3H, s, Me-27), 1.01 (3H, s, Me-26), 0.99 (3H, s, Me-
24), 0.85 (3H, s, Me-25): For 1H NMR spectroscopic data of the
sugar moiety, see Table 1; for 13C NMR (125 MHz, C5D5N) spectro-
scopic data, see Tables 1 and 2; HRESI-TOFMS m/z: 803.4159
[M + Na]+ (Calcd for C41H64NaO14: 803.4194).
the presence of
L-arabinose and
D
-glucose. tR (min): 9.41 (
L
-arabi-
nose, positive optical rotation), 16.19 (
rotation).
D-glucose, positive optical
3.17. Compound 14
3-[(O-(3-O-Acetyl-b-
anosyl)oxy]-30-noroleana-12,20(29)-dien-28-oic acid b-
pyranosyl ester (14); amorphous solid;
D
-glucopyranosyl)-(1?3)-
a
-
L
-arabinopyr-
-gluco-
16.3 (c 0.10;
D
25
[a]
D
3.11. Acid hydrolysis of 10
MeOH); IR mmax (film) cmꢀ1: 3389 (OH), 2932 and 2878 (CH),
1732 (C = O), 1651 (C = C), 1076 (C–O); 1H NMR (500 MHz,
C5D5N): d 5.44 (1H, br s, H-12), 4.77 and 4.70 (each 1H, s, H2-29),
1.31 (3H, s, Me-23), 1.24 (3H, s, Me-27), 1.09 (3H, s, Me-26), 0.96
(3H, s, Me-24), 0.85 (3H, s, Me-25): For 1H NMR spectroscopic data
of the sugar moieties, see Table 1; for 13C NMR (125 MHz, C5D5N)
A solution of 10 (3.1 mg) was subjected to acid hydrolysis as de-
scribed for 9 to give serratagenic acid (0.7 mg) and a sugar fraction.
HPLC analysis of the sugar fraction under the same conditions as in
the case of 1 showed the presence of
D-xylose and
D-glucose. tR
(min): 10.02 ( -xylose, positive optical rotation), 16.19 (
positive optical rotation).
D
D
-glucose,
spectroscopic data, see Tables 1 and 2; HRESI-TOFMS m/z:
961.4713 [M + Na]+ (Calcd for C48H74NaO18: 961.4773).
3.12. Compound 12
3.18. Alkaline hydrolysis of 14
3-[(O-b-
D
-Glucopyranosyl-(1?3)-
a
-
L
-arabinopyranosyl)oxy]-
A solution of 14 (4.3 mg) was treated with 10% KOH (dioxane–
H2O, 1:1; 2 ml) at room temperature for 4 h. The reaction mixture
was neutralized by passage through an Amberlite IR-120B column
and purified by silica gel CC eluted with CHCl3–MeOH–H2O
(30:10:1) to yield 22 (1.1 mg).
30-noroleana-12,20(29)-dien-28-oic acid (12); amorphous solid;
25
[
a]
15.9 (c 0.033; MeOH); IR mmax (film) cmꢀ1: 3365 (OH),
D
2927 and 2876 (CH), 1683 (C = O), 1647 (C = C), 1077 (C–O); 1H
NMR (500 MHz, C5D5N): d 5.50 (1H, br s, H-12), 4.82 and 4.76 (each

2166
M. Jitsuno, Y. Mimaki / Phytochemistry 71 (2010) 2157–2167
3.19. Compound 15
3-[(O-(6-O-Acetyl-b-
anosyl)oxy]-30-noroleana-12,20(29)-dien-28-oic acid b-
pyranosyl ester (15); amorphous solid;
3.25. Compound 18
D
-glucopyranosyl)-(1?3)-
a
-
L
-arabinopyr-
-gluco-
36.5 (c 0.10;
3-[(O-b-
(1?2)]- -arabinopyranosyl)oxy]-30-noroleana-12,20(29)-dien-
28-oic acid O-b- -glucopyranosyl-(1?6)-b- -glucopyranosyl ester
(18); amorphous solid; [ 6.9 (c 0.10; MeOH); IR mmax (film)
D-Glucopyranosyl-(1?3)-O-[a-L-rhamnopyranosyl-
D
a-L
25
[a
]
D
D
D
MeOH); IR mmax (film) cmꢀ1: 3389 (OH), 2927 and 2879 (CH),
1739 (C = O), 1651 (C = C), 1076 (C–O); 1H NMR (500 MHz,
C5D5N): d 5.45 (1H, br s, H-12), 4.77 and 4.70 (each 1H, s, H2-29),
1.31 (3H, s, Me-23), 1.25 (3H, s, Me-27), 1.09 (3H, s, Me-26), 0.98
(3H, s, Me-24), 0.86 (3H, s, Me-25): For 1H NMR spectroscopic data
of the sugar moieties, see Table 1; for 13C NMR (125 MHz, C5D5N)
a
]
D
25
cmꢀ1: 3390 (OH), 2935 and 2877 (CH), 1733 (C = O), 1651 (C = C),
1074 (C–O); 1H NMR (500 MHz, C5D5N): d 5.42 (1H, br s, H-12),
4.73 and 4.70 (each 1H, s, H2-29), 1.23 (3H, s, Me-27), 1.21 (3H, s,
Me-23), 1.12 (3H, s, Me-24), 1.08 (3H, s, Me-26), 0.87 (3H, s, Me-
25): For 1H NMR spectroscopic data of the sugar moieties, see
Table 1; for 13C NMR (125 MHz, C5D5N) spectroscopic data, see
Tables 1 and 2; HRESI-TOFMS m/z: 1227.5760 [M + Na]+ (Calcd
for C58H92NaO26: 1227.5775).
spectroscopic data, see Tables
1 and 2; HRESI-TOFMS m/z:
961.4722 [M + Na]+ (Calcd for C48H74NaO18: 961.4773).
3.20. Alkaline hydrolysis of 15
3.26. Acid hydrolysis of 18
Compound 15 (3.8 mg) was subjected to alkaline hydrolysis as
described for 14 to give 22 (1.1 mg).
A solution of 18 (8.1 mg) was subjected to acid hydrolysis as de-
scribed for 1 to give a sugar fraction. HPLC analysis of the sugar
fraction under the same conditions as in the case of 1 showed
3.21. Compound 16
the presence of
L
-rhamnose,
L
-arabinose, and
D
-glucose. tR (min):
-arabinose, po-
sitive optical rotation), 16.27 (D-glucose, positive optical rotation).
3-[(O-(4-O-Sulfo-b-
D
-glucopyranosyl)-(1?3)-
a
-
L
-arabinopyr-
8.10 ( -rhamnose, negative optical rotation), 9.49 (L
L
anosyl)oxy]-30-noroleana-12,20(29)-dien-28-oic acid b-
D
25
-gluco-
49.5 (c
pyranosyl ester sodium salt (16); amorphous solid; [
a]
D
0.095; MeOH); IR mmax (film) cmꢀ1: 3409 (OH), 2937 and 2877
(CH), 1732 (C = O), 1651 (C = C), 1259 (S = O), 1076 (C–O); 1H
NMR (500 MHz, C5D5N): d 5.45 (1H, br s, H-12), 4.77 and 4.70 (each
1H, s, H2-29), 1.29 (3H, s, Me-23), 1.25 (3H, s, Me-27), 1.09 (3H, s,
Me-26), 0.95 (3H, s, Me-24), 0.86 (3H, s, Me-25): For 1H NMR spec-
troscopic data of the sugar moieties, see Table 1; for 13C NMR
(125 MHz, C5D5N) spectroscopic data, see Tables 1 and 2; HRESI-
TOFMS m/z: 1021.4078 [M + Na]+ (Calcd for C46H71Na2O20S:
1021.4055).
3.27. Compound 19
3-[(O-b-
(1?2)]- -arabinopyranosyl)oxy]-20b-hydroxy-30-norolean-12-
en-28-oic acid b-
D-Glucopyranosyl-(1?3)-O-[a-L-rhamnopyranosyl-
a
-L
D
-glucopyranosyl ester (19); amorphous solid;
25
[a]
D
ꢀ10.2 (c 0.10; MeOH); IR mmax (film) cmꢀ1: 3364 (OH),
2925 and 2854 (CH), 1731 (C = O), 1652 (C = C), 1076 (C–O); 1H
NMR (500 MHz, C5D5N): d 5.48 (1H, br s, H-12), 3.80 (1H, dd,
J = 13.4, 4.2 Hz, H-18), 2.57 (1H, m, H-22ax), 2.04 (1H, dd, J = 13.3,
13.3 Hz, H-19ax), 1.89 (1H, br d, J = 16.0 Hz, H-22eq), 1.84 (1H,
dd, J = 13.3, 4.2 Hz, H-19eq), 1.43 (3H, s, Me-29), 1.30 (3H, s, Me-
27), 1.20 (3H, s, Me-23), 1.12 (3H, s, Me-24), 1.12 (3H, s, Me-26),
0.85 (3H, s, Me-25): For 1H NMR spectroscopic data of the sugar
moieties, see Table 1; for 13C NMR (125 MHz, C5D5N) spectroscopic
data, see Tables 1 and 2; HRESI-TOFMS m/z: 1083.5345 [M + Na]+
(Calcd for C52H84NaO22: 1083.5352).
3.22. Acid hydrolysis of 16
A solution of 16 (6.4 mg) was subjected to acid hydrolysis as de-
scribed for 1 to give a sugar fraction. HPLC analysis of the sugar
fraction under the same conditions as in the case of 1 showed
the presence of
L-arabinose and
D
-glucose. tR (min): 9.61 (
L
-arabi-
nose, positive optical rotation), 16.55 (
rotation).
D-glucose, positive optical
3.28. Acid hydrolysis of 19
3.23. Compound 17
A solution of 19 (7.0 mg) in 0.2 M HCl (dioxane–H2O, 1:1; 2 ml)
was heated at 95 °C for 1 h under Ar atmosphere. After the reaction
mixture was diluted with H2O, it was extracted with EtOAc satu-
rated with H2O (10 ml ꢁ 3). The H2O residue was neutralized by
passage through an Amberlite IRA-96SB column and passed
through a Sep-Pak C18 cartridge to give a sugar fraction. HPLC
analysis of the sugar fraction under the same conditions as in the
3-[(O-(4-O-Sulfo-b-
pyranosyl-(1?2)]- -arabinopyranosyl)oxy]-30-noroleana-12,20
(29)-dien-28-dioic acid b- -glucopyranosyl ester sodium salt (17);
26.7 (c 0.10; MeOH); IR mmax (film) cmꢀ1
D-glucopyranosyl)-(1?3)-O-[a-L-rhamno-
a
-L
D
25
amorphous solid; [
a
]
D
:
3417 (OH), 2937 and 2882 (CH), 1748 (C = O), 1651 (C = C), 1261
(S = O), 1074 (C–O); 1H NMR (500 MHz, C5D5N): d 5.44 (1H, br s,
H-12), 4.77 and 4.70 (each 1H, s, H2-29), 1.24 (3H, s, Me-27), 1.21
(3H, s, Me-23), 1.11 (3H, s, Me-24), 1.08 (3H, s, Me-26), 0.86 (3H,
s, Me-25): For 1H NMR spectroscopic data of the sugar moieties,
see Table 1; for 13C NMR (125 MHz, C5D5N) spectroscopic data,
see Tables 1 and 2; HRESI-TOFMS m/z: 1167.4736 [M + Na]+ (Calcd
for C52H81Na2O24S: 1167.4634).
case of 1 showed the presence of
-glucose. tR (min): 7.25 ( -rhamnose, negative optical rotation),
8.59 ( -arabinose, positive optical rotation), 14.06 ( -glucose, posi-
L-rhamnose, L-arabinose, and
D
L
L
D
tive optical rotation).
3.29. Compound 20
3-[(O-b-
(1?2)]- -arabinopyranosyl)oxy]-20
en-28-oic acid b-
D
-Glucopyranosyl-(1?3)-O-[
a-L-rhamnopyranosyl-
3.24. Acid hydrolysis of 17
a
-
L
a
-hydroxy-29-norolean-12-
D
-glucopyranosyl ester. (20); amorphous solid;
ꢀ2.7 (c 0.10; MeOH); IR mmax (film) cmꢀ1: 3364 (OH), 2925
25
A solution of 17 (6.1 mg) was subjected to acid hydrolysis as de-
scribed for 1 to give a sugar fraction. HPLC analysis of the sugar
fraction under the same conditions as in the case of 1 showed
[a]
D
and 2878 (CH), 1731 (C = O), 1651 (C = C), 1075 (C–O); 1H NMR
(500 MHz, C5D5N): d 5.49 (1H, br s, H-12), 3.25 (1H, dd, J = 13.7,
4.1 Hz, H-18), 1.96 (1H, br d, J = 13.5 Hz, H-22eq), 1.85 (1H, m,
H-22ax), 1.46 (3H, s, Me-30), 1.23 (3H, s, Me-27), 1.20 (3H, s, Me-
23), 1.12 (3H, s, Me-24), 1.09 (3H, s, Me-26), 0.85 (3H, s, Me-25):
the presence of
8.09 ( -rhamnose, negative optical rotation), 9.67 (
sitive optical rotation), 16.63 ( -glucose, positive optical rotation).
L
-rhamnose,
L
-arabinose, and
D
-glucose. tR (min):
L
L-arabinose, po-
D

M. Jitsuno, Y. Mimaki / Phytochemistry 71 (2010) 2157–2167
2167
For 1H NMR spectroscopic data of the sugar moieties, see Table 1;
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Acknowledgements
This work was partially supported by Grant-in-Aid for Scientific
Research (C) (No. 21590023) from The Ministry of Education, Cul-
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