Triterpenoid saponins from Echinopsis macrogona (Cactaceae)

Article abstract of DOI:10.1016/j.phytochem.2010.10.004

Triterpene saponins, pachanosides C1, E1, F1 and G1 (1-4), and bridgesides A1, C1, C2, D1, D2, E1 and E2 (5-11) were isolated from Echinopsis macrogona. Compounds 1-4 were saponins with pachanane type triterpene saponins, while the others (5-11) were oleanane type triterpene saponins. While the aglycones of 2-4 and 8-11 were hitherto unknown, the structure of pachanol C was revised in this paper. Their structures were elucidated on the basis of chemical and physicochemical evidence.

Full text of DOI:10.1016/j.phytochem.2010.10.004

Phytochemistry 72 (2011) 136–146
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Triterpenoid saponins from Echinopsis macrogona (Cactaceae)
a
a
a
a
b
a,
⇑
Sachie Okazaki , Kaoru Kinoshita , Satoru Ito , Kiyotaka Koyama , Hiroshi Yuasa , Kunio Takahashi
a
Department of Pharmacognosy and Phytochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose-shi, Tokyo 204-8588, Japan
Research Institute of Evolutionary Biology, 2-4-28 Kamiyoga, Setagaya-ku, Tokyo 158-0098, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 May 2010
Received in revised form 3 August 2010
Available online 1 November 2010
Triterpene saponins, pachanosides C1, E1, F1 and G1 (1–4), and bridgesides A1, C1, C2, D1, D2, E1 and E2
5–11) were isolated from Echinopsis macrogona. Compounds 1–4 were saponins with pachanane type tri-
terpene saponins, while the others (5–11) were oleanane type triterpene saponins. While the aglycones
of 2–4 and 8–11 were hitherto unknown, the structure of pachanol C was revised in this paper. Their
structures were elucidated on the basis of chemical and physicochemical evidence.
Ó 2010 Elsevier Ltd. All rights reserved.
(
Keywords:
Echinopsis macrogona
Cactaceae
Saponins
Pachanane type
Pahanoside
Bridgeside
1
. Introduction
pachanane skeleton triterpene. This paper deals with the isolation
and structure elucidation of these new saponins (1–11).
Triterpene sapogenins from cacti were reported by C. Djerassi in
953–1958 (Djerassi et al., 1953a,b, 1954a–c, 1955, 1956a,b, 1957;
1
2
. Results and discussion
Djerassi and Lippman, 1954; Djerassi and Mills, 1958; Sandoval
et al., 1957). Although 18 triterpene sapogenins were isolated from
cacti, saponins had never been isolated by the Djerassi group. A
new saponin from the cactus, Pereskia grandifolia had, however
been reported, and is the only report about saponins from cacti
Sahu et al., 1974). In previous contributions, 17 new and 20
known triterpenee sapogenins were reported from several cacti
Kinoshita et al., 1992, 1995; Takizawa et al., 1993; Koyama
et al., 1993; Yang et al., 1998), and also a saponin, dumortierino-
side A, from Isolatocereus dumortieri (Kinoshita et al., 2000). More-
over another new one, stellatoside A, was isolated together with
two known saponins from cultured and wild type cactus, Stenoce-
reus stellatus (Imai et al., 2006) and stellatoside B and erucasaponin
A were isolated from wild type cactus, S. eruca (Okazaki et al.,
007). Some triterpene sapogenins from cacti showed antinocicep-
tive (Kinoshita et al., 1998) and antitumor-promotion activities
Kinoshita et al., 1999). In the study herein of saponins from cacti,
1 new triterpene saponins named pachanosides C1, E1, F1 and G1
1–4), and bridgesides A1, C1, C2, D1, D2, E1 and E2 (5–11) were
isolated from Echinopsis macrogona. Among them, compounds 1–
Dry E. macrogona plant tissue was extracted repeatedly with
chloroform and then with methanol. The methanol extract was
passed through a Diaion HP-20 column to adsorb saponins and
remove sugar by eluting with H O. The methanol eluate was
2
(
separated by silica gel and octadecyl silyl silica gel (ODS) column
chromatography and HPLC, yielding pachanosides C1, E1, F1 and
G1 (1–4), and bridgesides A1, C1, C2, D1, D2, E1 and E2 (5–11).
Compounds 1–4 were the first isolated saponins with aglycones
possessing a pachanane skeleton triterpene. Pachanane skeleton
triterpenes, a new skeleton established in our previous studies of
cacti, are characterized by translocation of the C-27 methyl from
C-14 to C-15 (Takizawa et al., 1993; Kinoshita et al., 1995). The
structures of compounds 1–11 were determined on the basis of
spectroscopic data using DEPT, HMQC, HMBC, DQF-COSY( H– H
COSY), HSQC-TOCSY, phase sensitive TOCSY, phase sensitive
NOESY and NOE difference experiments. The C and H NMR spec-
troscopic data of compounds 1–11 are presented in Tables 1 and 2,
respectively.
(
2
1
1
(
1
(
1
3
1
Pachanoside C1 (1), a colorless powder, consists of a pachanane
skeleton triterpene, pachanol C, and three sugars, glucuronic
4
were the first isolated saponins with aglycones possessing a
74
acid, glucose and xylose. Its molecular formula C49H O21 was
determined from the molecular ions observed in the ESI (Negative)
ꢀ
⇑
Corresponding author. Tel.: +81 42 495 8913; fax: +81 42 495 8912.
E-mail address: diamonds@my-pharm.ac.jp (K. Takahashi).
Tof MS (m/z 997.4676 [MꢀH] ) and ESI (positive) Tof MS (m/z
+
13
1021.4620 [M+Na] ) and was confirmed by C NMR and DEPT
0
031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2010.10.004

S. Okazaki et al. / Phytochemistry 72 (2011) 136–146
137
CH2OR3
OR2
CH2OR3
OR2
CH2OAc
OAc
O
COOH
O
O
O
R1O
R1O
RO
R1 R2 R3
R
X
R1 R2 R3
1
2
3
X
X
X
Ac
H
Ac Ac
H
Ac
4
5
6
7
8
9
Y
X
H
Ac
H
H
Y1 Ac
H
X
Y
X
Y
H
H
Ac
Ac
Na+
10
11
Ac Ac
Ac Ac
2
2
2
2
2
2
X:
glcA glc xyl
Y:
glcA glc rha
Y :
glcA- glc rha
1
spectroscopic analysis. The IR spectrum of 1 showed absorptions at
In a previous paper, we reported the structure of pachanol C iso-
lated from Echinopsis pachanoi (Trichocereus pachanoi, Cactaceae)
(Kinoshita et al., 1995), almost all the H and C NMR data of 1
ꢀ1
ꢀ1
3
400 cm (hydroxy) and 1720 and 1738 cm (ester carbonyl).
13
1
1
13
The compound displayed 49 signals in its C NMR spectrum. Its H
NMR spectrum showed the presence of six methyl groups character-
ized by singlets at d 0.81, 1.04, 1.10, 1.28, 1.31, and a doublet at d 1.03
were in good agreement with pachanol C. Although we previously
reported the 14-OH of pachanol C to be in an
a-orientation, its
(
5
J = 7.6 Hz), an acetoxy methyl at d 2.04 and an olefinic proton at d
.56 (br s). The C NMR spectrum showed resonances of a quaternary
structure was the same as aglycone of 1. Thus, we decided to re-
investigate the structure of pachanol C. This time, when examining
the NOESY correlations of pachanol C in great detail, the H-5
methine proton showed an NOE correlation with the H-3 (d 3.36)
and H-9 (d 1.60) methine protons. The 3H-27 at d 1.03 (d,
J = 7.6 Hz) also showed correlations with the H-9 methine proton
by phase sensitive NOESY. From the above, the 3H-27 connected
1
3
carbon at d 90.8, a pair of olefinic carbons at d 124.4 and 133.1, and
three carbonyl carbons at d 170.5, 172.9 and 176.7. The methyl proton
at d 1.03 (d, J = 7.6 Hz) and quaternary carbon at d 90.8 indicated that
the aglycone possesses a pachanane skeleton (Takizawa et al., 1993;
Kinoshita et al., 1995), and its structure was determined by HMQC,
HMBC and DQF-COSY ( H– H COSY) spectra correlations and phase
sensitive NOESY spectroscopic correlations (Fig. 1).
HMBC correlations identified most of the structural elements of
rings A and B, and these were gradually extended to cover the
other rings. Regarding rings C–E, they were ascertained from the
following extensive NMR experiments. H– H COSY correlations
between 2H-16 at d 1.70 (2H, m) and H-15 at d 2.27 (1H, m) and
between H-15 and 3H-27 at d 1.03 (J = 7.6 Hz), and by HMBC cor-
relations between H-16 and C-27 and between 3H-27 and C-15.
Moreover, on the basis of HMQC and HMBC results, the methyl
proton with its doublet signal at d 1.03 (d, J = 7.6 Hz) was located
on C-27 which was attached to C-15. In the HMBC spectrum, the
H-12 olefinic proton at d 5.56 (1H, br s), H-18 at d 2.86 (1H, br d,
J = 11.0 Hz) and H-15 at d 2.27 (1H, m) had cross-peaks with the
quaternary carbon at d 90.8. This information suggested that the
quaternary carbon at d 90.8 was assigned at C-14. From the chem-
ical shift value, this quaternary carbon was assumed to be attached
to an oxygen atom; the fact that 1 has a 6-membered ring lactone
formed by the 14-C oxygen atom and 28-COO like pachanol D
1
1
to C-15 was established to be in an a-orientation. These NOESY
correlations could only be observed if the 14-O functionally was
in a b-orientation. Thus, pachanol C was determined to be the same
as aglycone 1 as confirmed by its NMR, IR and MS data. Moreover,
pachanol D, isolated from I. dumortieri, was determined by spectro-
scopic methods and X-ray diffraction analysis (Kinoshita et al.,
1998), and has a six-membered ring lactone formed by the 14-O
1
1
1
3
and 28-COO as shown in Fig. 3. The C NMR chemical shift of
the A–D rings of pachanol C are in good agreement with the data
for pachanol D. Therefore, pachanol C is 21b-acetyloxy-3b,14b,
30-trihydroxypachan-12-en-28-oic acid 14b,28-lactone and has
been corrected to that shown in Fig. 2.
Thirty-two out of the 49 carbons signals total observed in the
1
3
C NMR spectrum were assigned to the aglycone structure, with
1
the remaining 17 carbons to the oligosaccharide moiety. The
H
1
3
and C NMR spectra of 1 showed three anomeric carbons at d
102.9, 104.9 and 106.4, three anomeric protons at d 5.02 (d,
J = 7.6 Hz), 5.39 (d, J = 7.0 Hz), and 5.58 (d, J = 7.9 Hz), together with
two methylene carbons at d 62.9 and 67.3, and a carbonyl carbon at
d 172.9. The unknown sugar residue possessing a carboxyl carbon
was concluded to be a glucuronic acid on the basis of analysis of
the 2D NMR spectroscopic data (Fig. 3). From the analysis of the
HMQC, HMBC and DQF COSY spectra, the carbonyl carbon at d
(Kinoshita et al., 1995) shown in Fig. 1 was confirmed by IR and
its molecular formula.
The stereochemistry of the aglycone moiety of 1 was proven
from analysis of the phase sensitive NOESY cross-peaks (Fig. 2).
The olefinic proton at d 5.56 showed a cross peak with H-18 at d
0
172.9 was assigned to C-6 , and the methine protons at d 4.33 (d,
2.86, which in turn was correlated with H-22 (ax.) at d 2.51 and
2H-30 at d 3.88 and 4.22, while 2H-30 gave a cross peak with H-
22 (ax.) at d 2.51. These results led to assignment of the hydro-
J = 10.1 Hz), 4.48 (t, J = 10.1 Hz), 4.43 (t, J = 10.1 Hz), 4.23 (m), and
0
0
0
0
5.02 (d, J = 7.6 Hz) were assigned to H-5 , H-4 , H-3 , H-2 and H-
0
1 , respectively. For the second sugar moiety, the methylene carbon
xy-methyl at C-30. The H-5 methine proton at d 0.79 (m) showed
an NOESY correlation with the H-3 and H-9 methine protons and
the H-7 methylene axial proton. The 3H-27 showed important cor-
relations with the H-7 axial proton at d 1.77 (1H, m) and H-9
methine proton at d 1.60 (dd, J = 11.1, 5.3 Hz) by phase sensitive
NOESY. These results suggested the 3H-27 at d 1.03 (3H, d,
at d 62.9, which demonstrated HMQC correlations with protons at
d 4.36 (dd, J = 10.1, 5.2 Hz) and 4.49 (d, J = 10.1 Hz), was assigned to
0
0
the C-6 of glucose. From the DQF COSY analysis, methylene pro-
tons at d 4.36 (dd, J = 10.1, 5.2 Hz) and 4.49 (d, J = 10.1 Hz) and
methine protons at d 3.87 (m), 4.19 (t, J = 9.4), 4.29 (t, J = 9.4),
00
00
4.19 (m) and 5.58 (d, J = 7.9 Hz) were assigned as H-6 to H-1 ,
respectively. For the third sugar moiety, the methylene carbon at
d 67.3, which exhibited HMQC correlations with protons at d
3.69 (t, J = 12.6 Hz) and 4.33 (dd, J = 12.6, 2.7 Hz), was assigned to
J = 7.6 Hz), which connects with C-15, has an
a-orientation and
only the 14-O is in a b-orientation. Thus, the aglycone 1 was deter-
mined as 21-acetyloxy-3b,30-dihydroxy-pachanan-12-en 28,14b-
olide as shown in Fig. 2.
0
00
C-5 of xylose. From the HMQC, HMBC and DQF COSY analyses,

1
38
S. Okazaki et al. / Phytochemistry 72 (2011) 136–146
Table 1
1
3C NMR spectral data for the aglycone moiety of 1–11 in pyrideine-d
6*
or DMSO-d or methanol-d
**
.
5
4
(temp. 50 °C)
*
**
Position
1
2
3
4
5a
6
7
7
8
9
10
11
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
38.9
26.4
89.2
39.6
55.9
18.0
33.5
40.1
47.7
37.1
22.8
124.4
133.1
90.8
37.2
34.3
42.5
37.1
33.4
39.4
75.8
31.7
28.2
16.7
16.3
17.5
20.1
176.7
24.0
61.7
170.5
20.9
38.9
26.5
89.2
39.6
55.9
18.0
33.5
40.1
47.7
37.2
22.8
124.4
133.3
90.6
37.2
34.5
42.4
37.2
33.9
39.3
72.2
35.6
28.1
16.7
16.2
17.5
20.2
177.7
24.6
65.1
38.9
26.5
89.2
39.6
55.9
18.0
33.5
40.1
47.7
37.2
22.8
124.8
132.7
90.8
37.1
34.1
42.3
39.6
33.9
38.1
75.0
31.6
28.2
16.8
16.3
17.5
20.1
176.3
23.6
64.6
170.3
20.7
170.8
20.7
38.8
26.5
89.1
39.2
54.6
19.0
40.9
40.8
56.8
37.2
22.0
124.7
136.9
139.0
126.2
38.7
46.8
40.9
39.2
38.4
75.4
34.8
27.7
16.3
16.6
19.5
24.3
177.9
23.6
64.8
170.3
20.6
170.8
20.6
39.0
26.3
89.8
39.7
56.0
18.2
33.7
40.5
48.5
36.7
23.5
127.9
138.1
45.8
80.1
34.6
46.5
41.6
42.5
41.0
74.5
36.8
28.3
16.7
16.1
19.7
25.2
179.5
25.0
63.2
39.0
26.4
89.0
39.5
55.8
18.1
33.7
40.3
48.4
36.7
23.4
128.1
137.8
45.8
80.1
34.3
46.2
41.3
41.0
40.3
76.3
32.3
28.0
16.7
16.1
19.6
25.2
178.9
24.0
61.2
170.4
20.8
38.3
25.2
88.6
38.7
54.6
17.4
32.9
39.8
45.2
36.1
22.7
127.0
137.4
45.0
79.5
33.5
45.2
40.1
39.6
39.5
75.1
31.3
27.6
16.1
15.8
19.1
24.6
178.3
23.2
60.0
169.9
20.7
40.4
26.9
91.7
40.7
57.2
19.1
34.7
41.6
49.7
38.0
24.5
129.7
138.6
46.9
82.1
35.2
47.5
42.3
41.8
41.0
77.6
32.8
28.8
17.0
16.7
20.2
25.7
181.4
24.1
62.3
172.4
20.9
39.1
26.5
89.0
39.6
55.8
18.1
33.7
40.5
48.4
36.7
23.5
128.0
137.6
45.8
80.1
34.5
46.2
41.3
41.4
40.4
73.2
36.4
28.0
16.8
16.1
19.7
25.2
179.2
24.7
64.6
39.1
26.4
89.7
39.6
55.9
18.1
33.7
40.5
48.5
36.8
23.5
128.0
137.8
45.7
80.1
34.5
46.2
41.3
41.4
40.4
73.2
36.4
28.3
16.7
16.1
19.7
25.2
179.0
24.7
64.6
39.1
26.5
89.0
39.6
55.8
18.1
33.7
40.4
48.4
36.8
23.5
128.4
137.3
45.7
80.1
34.1
46.0
41.0
41.1
39.1
75.6
32.3
28.0
16.8
16.1
19.6
25.3
178.5
23.7
64.0
170.3
20.7
170.8
20.7
39.1
26.4
89.7
39.6
55.9
18.1
33.7
40.4
48.4
36.8
23.5
128.4
137.3
45.7
80.1
34.1
46.0
41.0
41.1
39.1
75.6
32.3
28.3
16.7
16.1
19.6
25.2
178.5
23.6
64.0
170.2
20.7
170.8
20.7
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
C-27
C-28
C-29
C-30
21-CH
21-CH
30-CH
30-CH
3
CO
CO
CO
CO
3
3
171.1
20.8
171.1
20.8
171.1
20.8
3
b-D-glcA
0
1
104.9
81.9
78.0
72.7
77.4
172.9
104.9
81.9
78.0
72.6
77.5
172.6
105.0
82.0
78.0
72.6
77.6
172.5
104.8
81.8
77.7
72.3
77.3
172.3
105.3
78.5
78.6
73.2
76.8
104.9
82.1
78.0
72.6
77.6
172.5
103.7
76.3
72.3
77.7
73.5
105.7
78.8
79.0
74.1
76.6
n.d.
105.9
82.0
78.1
72.7
77.3
173.1
105.1
78.5
78.9
73.9
76.6
174.6
105.0
82.1
78.0
72.6
77.6
172.5
105.3
78.5
78.7
73.5
77.3
176.3
0
2
0
3
0
4
0
5
COOH
COOCH
3
170.6
51.9
COOCH
3
ꢀ
+
COO Na
b-D-glc
172.5
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
6
102.9
84.6
77.8
71.8
77.8
62.9
102.9
84.5
77.8
71.8
77.8
62.9
103.0
84.6
77.9
71.8
77.8
62.9
102.7
84.3
77.9
71.5
77.7
62.6
102.0
78.4
79.4
72.7
77.8
63.3
103.0
84.5
77.9
71.8
77.7
62.9
99.6
77.0
77.6
71.2
76.4
61.9
102.3
79.7
79.5
73.0
78.0
63.8
102.9
84.5
77.9
71.8
77.7
62.8
101.9
78.5
79.4
72.8
77.6
63.4
103.0
84.5
77.9
71.8
77.7
62.9
102.0
78.5
79.4
72.8
77.6
63.4
b-D-xyl
0
0
0
0
0
00
00
00
00
00
1
2
3
4
5
a
1
106.4
75.8
77.6
70.7
67.3
106.4
75.8
77.6
70.7
67.3
106.5
75.8
77.6
70.7
67.3
106.1
75.5
77.3
70.4
67.0
106.4
75.8
77.6
70.7
67.3
106.4
75.8
77.6
70.7
67.3
106.4
75.8
77.8
70.8
67.3
-L-rha
0
00
00
00
00
00
102.0
72.4
73.2
74.3
69.4
19.0
99.9
70.5
70.5
72.3
67.9
18.0
102.0
72.5
72.5
74.4
69.8
18.4
101.9
72.4
72.6
74.3
69.4
18.9
102.0
72.3
72.6
74.3
69.3
18.9
0
2
3
4
5
6
0
0
0
00
’
n.d: signal was not detected.
the methylene protons at d 3.69 (t, J = 12.6 Hz) and d 4.33 (dd,
J = 12.6, 2.7 Hz), and methine protons at 4.14 (m), 4.13 (m), 4.09
The configurations of glucuronic acid, glucose and xylose were
determined using the HPLC method (Tanaka et al., 2007). 1 was
hydrolyzed and converted into the thiazolizine derivatives to aryl-
000
000
(
m) and 5.39 (d, J = 7.0 Hz) were assigned to H-5 to H-1 , respec-
tively. The coupling constants attributed to the trans-diaxial and
phase sensitive NOESY spectrum suggested these three sugars
had b-configurations (b-glcA, b-glc and b-xyl).
thiocarbamate using
anate. Then the reaction mixture was analyzed by C18 HPLC and
the retention time (T ) was compared with derivatives of each D-
L-cysteine methyl ester and O-tolylisothiocy-
R

S. Okazaki et al. / Phytochemistry 72 (2011) 136–146
139
Table 2.1
H NMR spectroscopic data [d
1
6*
**
H
, mult. (J in Hz)] for the aglycone moieties of 1–11. (500 MHz, in pyridine-d
5
or DMSO-d or methanol-d
4
on temp. 50 °C ).
Position
1
2
3
4
5a
6
1
2
3
5
6
7
0.73, 1.38 (each m)
1.84, 2.23 (each m)
3.36 (dd, 11.4, 3.8)
0.79 (m)
1.33, 1.57 (each m)
1.52, 1.77 (each m)
0.74, 1.39 (each m)
1.87, 2.20 (each m)
3.35 (dd, 11.6, 4.6)
0.83 (m)
1.36, 1.57 (each m)
1.81 (m)
0.76, 1.37 (each m)
1.88, 2.20 (each m)
3.36 (dd, 11.7, 4.4)
0.79 (m)
1.31, 1.56 (each m)
1.52 1.79 (each m)
0.67, 1.35 (each m)
1.81, 2.15 (each m)
3.29 (dd, 11.7, 4.1)
0.67 (m)
1.32 1.51 (each m)
1.50 (m)
0.88, 1.39 (each m)
1.82, 2.05 (each m)
3.30 (dd, 11.5, 4.2)
0.80 (m)
NA
1.54 (m)
0.81, 1.36 (each m)
1.84, 2.16 (each m)
3.33 (dd, 11.6, 4.3)
0.76 (brd, 11.6)
1.33, 1.52 (each m)
1.50 (m)
2.56 (brd, 11.9)
9
1.60 (dd, 11.1, 5.3)
1.77, 1.84 (each m)
5.56 (brs)
2.27 (m)
1.70 (m)
1.63 (m)
1.71, 1.87 (each m)
5.71 (brs)
2.31 (m)
1.68 (m)
1.60 (dd, 11.3, 5.5)
1.73, 1.88 (each m)
5.66 (t-like, 2.1)
2.28 (m)
1.08 (m)
1.82 (m)
5.78 (brs)
1.52 (m)
1.76 (m)
5.50 (t-like, 3.5)
4.58 (d, 6.1)
1.93 (d, 12.6, 6.1)
1.48 (m)
1
1
1
1
1
2
5
6
1.71 (dd, 8.1, 2.9)
5.49 (t-like, 3.2)
4.57 (d, 6.1)
1.94 (dd, 12.3, 3.2)
2.75, d (12.3)
2.93 (dd, 13.5, 3.2)
1.81 (m)
2.38 (dd, 13.5, 3.2)
5.13 (dd, 12.8, 4.9)
1.78 (dd, 12.8, 4.9)
2.53 (t, 12.8)
1.28 (s)
1.69 (m)
2.72, 2.82 (each m)
2.72 (d, 12.6)
1
1
8
9
2.86 (brd, 12.6)
1.38 (m)
2.81 (brd, 10.7)
1.42, 2.23 (each m)
2.76 (brd, 12.0)
1.47 (brd, 12.0)
2.18 (m)
5.13 (dd, 13.3, 4.6)
2.00 (dd, 13.3, 4.6)
2.37 (t, 13.3)
1.31 (s)
1.11 (s)
0.81 (s)
1.05 (s)
1.02 (d, 6.4)
1.13 (s)
3.33 (dd, 13.7, 3.4)
1.61 (t, 13.7)
1.96 (m)
5.28 (dd, 12.2, 5.2)
2.06 (m)
2.20 (t, 12.2)
1.26 (s)
1.05 (s)
0.70 (s)
1.03 (s)
2.02 (s)
1.10 (s)
2.88 (dd, 6.8, 3.5)
1.85 (m)
2.56 (dd, 12.6, 4.4)
2.11 (dd, 13.9, 3.5)
3.95 (dd, 12.9.4.7)
1.97 (dd, 12.9, 4.7)
2.79 (t, 12.9)
1.34 (s)
1.06 (s)
0.76 (s)
1.16 (s)
1.22 (s)
2
2
1
2
5.20 (dd, 12.8, 4.7)
2.01 (dd, 12.8, 4.7)
3.95 (dd, 13.2, 4.4)
2.14 (dd, 13.2, 4.4)
2.60 (t, 13.2)
1.30 (s)
1.11 (s)
0.81 (s)
1.07 (s)
1.09 (d, 6.4)
1.39 (s)
2.51 (t, 12.8)
2
2
2
2
2
2
3
3
4
5
6
7
9
0
1.31 (s)
1.10 (s)
0.81 (s)
1.04 (s)
1.03 (d, 7.6)
1.28 (s)
3.88 (d, 11.0)
1.08 (s)
0.78 (s)
1.12 (s)
1.22 (s)
1.53 (s)
4.22 (d, 11.0)
4.45 (d, 11.0)
1.23 (s)
4.65 (s)
4.37 (d, 11.5)
4.47 (d, 11.5)
2.02 (s)
4.42 (d, 11.8)
4,81 (d, 11.8)
2.04 (s)
3.91 (d, 10.7)
4.39 (d, 10.7)
2.03 (s)
4.22 (d, 11.0)
2
3
1-CH
0-CH
3
CO
CO
2.04 (s)
3
2.09 (s)
2.12 (s)
2.12 (s)
b-D-glcA
0
1
5.02 (d, 7.6)
4.23 (m)
4.43 (t, 10.1)
4.48 (t, 10.1)
4.33 (d, 10.1)
5.02 (d, 8.8)
4.24 (t, 8.8)
4.43 (t, 8.8)
4.51 (t, 8.8)
4.60 (d, 8.8)
5.04 (d, 8.8)
4.24 (t, 8.8)
4.44 (t, 8.8)
4.52 (t, 8.8)
4.62 (d, 8.8)
5.00 (d, 8.7)
4.21 (t, 8.7)
4.40 (t, 8.7)
4.48 (t, 8.7)
4.56 (d, 8.7)
4.98 (d, 7.1)
4.50 (m)
4.59 (t, 9.9)
4.29 (m)
4.48 (d, 9.9)
3.72 (s)
5.00 (d, 7.6)
4.22 (m)
4.43 (t, 9.3)
4.52 (t, 9.3)
4.60 (d, 9.3)
0
2
0
3
0
0
4
5
0
6
-COOCH
3
b-D-glc
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
6
5.58 (d, 7.9)
4.19 (m)
4.29 (t, 9.4)
4.19 (t, 9.4)
3.87 (m)
5.58 (d, 8.7)
4.19 (t, 8.7)
4.29 (t, 8.7)
4.20 (t, 8.7)
3.87 (m)
5.58 (d, 7.9)
4.19 (t, 9.2, 7.9)
4.29 (t, 9.2)
4.20 (t, 9.2)
3.87 (m)
5.51 (d, 8.3)
4.14 (t, 8.3)
4.24 (t, 8.3)
4.12 (t, 8.3)
3.83 (m)
5.85 (d, 7.5)
4.30 (dd, 9.0, 7.5)
4.24 (t, 9.0)
4.07 (t, 9.0)
3.85 (m)
5.57 (d, 7.9)
4.19 (m)
4.29 (t, 9.0)
4.20 (m)
3.86 (m)
4.36 (dd, 11.6, 4.0)
4.48 (dd, 11.6, 4.0)
4.36 (dd, 10.1, 5.2)
4.37 (dd, 12.2, 5.2)
4.50 (d, 12.2)
4.36 (dd, 11.7, 4.8)
4.50 (dd, 11.7, 4.8)
4.30 (dd, 11.5, 5.2)
4.45 (brd, 11.5)
4.31 (m)
4.50 (m)
4.49 (d, 10.1)
b-D-xyl
0
0
0
0
0
0
00
00
00
00
00
00
1
2
3
4
5
6
a
1
5.39 (d, 7.0)
4.09 (m)
4.13 (m)
4.14 (m)
3.69 (t, 12.6)
4.33 (dd, 12.6, 2.7)
5.39 (d, 6.7)
4.09 (m)
4.15 (m)
4.12 (m)
3.70 (dd, 11.0, 4.6)
4.33 (dd, 11.0, 4.6)
5.39 (d, 7.0)
4.10 (dd, 9.3, 7.0)
4.13 (m)
4.14 (m)
3.69 (m)
5.34 (d, 7.0)
4.06 (m)
4.12 (t, 8.4)
4.09 (m)
3.67 (t, 10.4)
4.32 (brs, 10.4)
5.40 (d, 6.7)
4.11 (m)
4.13 (m)
4.14 (m)
3.69 (m)
4.33 (m)
4.33 (dd, 11.9, 4.8)
-L-rha
0
00
00
00
00
00
00
6.40 (brs)
0
2
3
4
5
6
4.75 (dd, 3.5, 1.5)
4.66 (dd, 9.3, 3.5)
4.32 (t, 9.3)
5.00 (m)
1.78 (d, 6.1)
0
0
0
0
N.A.: could not assignment.
0
or L-sugar. The peaks at 18.5, 19.2 and 21.4 minutes coincided with
derivatives of -glucose, -glucuronic acid, and -xylose (T of
-glucuronic acid: 19.4, T of -glucose: 18.7, T of -xylose:
1.1), respectively. The linkage of the sugar units at C-3 of pacha-
The linkage of the glc at C-2 of glcA was indicated by cross-peaks
0
0
0
D
D
D
R
between H-1 of glc at d 5.58 and C-2 of glcA at d 81.9 and between
H-2 of glcA at d 4.23 and C-1 of glc at d 102.9. Similarly, the link-
age of the xyl at C-2 of glc was indicated by cross-peaks between
H-1 of xyl at d 5.39 and C-2 of glc at d 84.6, and H-2 of glc at d
4.19 and C-1 of xyl at d 106.4. For the NOESY spectrum, NOE cor-
relations between the methine proton at H-3 (d 3.36) of the agly-
cone and the anomeric proton (d 5.02) of glcA, and between H-2
0
0
D
R
D
R
D
0
0
2
0
00
00
00
nol C and the sequence of the sugar chains were established by the
following HMBC and NOESY spectra. Analysis of the HMBC spec-
0
00
0
trum of 1 established that these were cross-peaks between H-1
0
of glcA at d 5.02 and C-3 of pachanol C at d 89.2 and also between
0
H-3 of the aglycone at d 3.36 and C-1 at d 104.9 of glcA. These re-
(d 4.23) of glcA and the anomeric proton of glc (d 5.58) were ob-
sults indicated that the glcA was connected to C-3 of the aglycone.
served. On the basis of the above, the structure of pachanoside

1
40
S. Okazaki et al. / Phytochemistry 72 (2011) 136–146
Table 2.2
H NMR spectroscopic data [d
1
6*
**
H
, mult. (J inHz)] for the aglycone moieties of 1–11. (500 MHz, in pyridine-d
5
or DMSO-d or methanol-d
4
on temp. 50 °C ).
*
**
Position
7
7
8
9
10
11
1
2
3
5
6
7
9
0.89, 1.56 (each m)
1.54, 1.98 (each m)
2.97 (dd, 11.6, 4.3)
0.75 (m)
1.35, 1.53 (each m)
1.51 (m)
1.03, 1.67 (each m)
1.73, 2.05(each m)
3.22 (m)
0.83 (m)
1.48, 1.61 (each m)
1.61 (m)
0.88, 1.42 (each m)
1.85, 2.23 (each m)
3.33 (dd, 11.6, 4.3)
0.76 (m)
1.33, 1.53 (each m)
1.52 (m)
0.90, 1.40 (each m)
1.84, 2.26 (each m)
3.32 (dd, 11.0, 3.4)
0.78 (m)
1.33, 1.53 (each m)
1.53 (m)
0.89, 1.43 (each m)
1.86, 2.15 (each m)
3.34 (dd, 11.6, 4.3)
0.77 (brs, 11.6)
1.34, 1.53 (each m)
1.53 (m)
0.92, 1.45 (each m)
1.80, 2.16 (each m)
3.30 (dd, 11.4, 3.2)
0.81 (m)
1.34, 1.54 (each m)
1.51 (m)
1.43 (m)
1.55 (t, 8.7)
1.50 (m)
1.52 (m)
1.49 (t, 8.1)
1.54 (m)
1
1
1
1
1
2
5
6
1.83 (m)
5.56 (brs)
4.57 (d, 5.8)
1.85 (m)
1.93 (m)
5.64 (t-like, 3.5)
4.64 (m)
1.99 (dd, 14.3, 5.3)
2.78 (d, 5.3)
2.50 (dd, 13.1, 1.1)
1.70, 1.90 (each m)
1.75 (m)
5.52 (t-like, 3.2)
4.58 (d, 5.8)
1.56 (m)
5.51 (brs)
1.75 (m)
5.46 (t-like, 3.5)
4.56 (d, 6.1)
1.92 (d, 12.5, 6.0)
2.71 (d, 12.5)
2.75 (dd, 10.4, 7.3)
1.81 (m)
1.51, 1.75 (each m)
5.46 (brs)
4.56 (d, 5.9)
1.93 (dd, 12.5, 5.9)
2.70 (d, 12.5)
2.74 (t, 10.1)
1.78 (m)
4.57 (d, 6.7)
1.91 (m)
2.69 (d, 12.5)
2.83 (dd, 13.0, 2.9)
1.77 (m)
1.92 (m)
2.73 (d, 12.5)
2.70 (d, 12.5)
2.84 (dd, 12.9, 3.1)
1.76 (m)
1
1
8
9
2.39 (brd, 13.1)
1.67 (t, 13.1)
1.85 (m)
2
2
1
2
4.72 (dd, 13.0, 5.1)
1.40 (dd, 13.0, 5.1)
4.77 (dd, 13.4, 4.8)
1.51 (dd, 13.4, 4.8)
2.06 (t, 13.4)
1.12 (s)
0.88 (s)
0.99 (s)
1.04 (s)
1.22 (s)
0.97 (s)
3.89 (m)
1.90 (m)
2.65 (t, 13.3)
1.28 (s)
1.09 (s)
0.81 (s)
1.15 (s)
1.23 (s)
1.32 (s)
4.55 (d, 12.1)
4.95 (d, 12.1)
3.88 (dd, 13.3, 4.4)
1.92 (dd, 13.3, 4.4)
2.65 (t, 13.3)
1.33 (s)
1.07 (s)
0.78 (s)
1.15 (s)
1.23 (s)
1.33 (s)
4.45 (d, 11.8)
4.95 (d, 11.8)
5.04 (dd, 12.7, 4.7)
1.76 (m)
2.38 (t, 13.1)
1.29 (s)
1.10 (s)
0.81 (s)
1.13 (s)
1.20 (s)
1.05 (s)
5.03 (dd, 13.0, 4.7)
1.76 (m)
2.37 (t, 13.0)
1.34 (s)
1.08 (s)
0.79 (s)
1.12 (s)
1.20 (s)
1.05 (s)
1.90 (t, 13.0)
2
2
2
2
2
2
3
3
4
5
6
7
9
0
1.03 (s)
0.76 (s)
0.89 (s)
0.91 (s)
1.13 (s)
0.85 (s)
3.28 (d, 10.6)
3.55 (d, 11.0)
3.94 (d, 11.0)
2.03 (s)
4.26 (d, 12.1)
4.74 (d, 12.1)
2.03 (s)
4.26 (d, 11.5)
4.73 (d, 11.5)
2.02 (s)
3.76 (d, 10.6)
2
3
1-CH
0-CH
3
CO
CO
2.00 (s)
3
2.11 (s)
2.12 (s)
2.13 (s)
2.13 (s)
b-D-glcA
0
1
4.13 (d, 8.0)
3.49 (t, 8.0)
3.09 (t, 8.0)
3.40 (t, 8.0)
3.15 (d, 8.0)
4.43 (d, 8.3)
3.70 (t, 8.3)
3.62 (t, 8.3)
4.45 (m)
4.97 (d, 8.3)
4.22 (t, 8.3)
4.42 (t, 8.3)
4.47 (t, 8.3)
4.53 (d, 8.3)
4.95 (d, 7.6)
4.48 (t, 7.6)
4.58 (t, 7.6)
4.29 (m)
5.00 (d, 8.7)
4.22 (t, 8.7)
4.43 (t, 8.7)
4.52 (t, 8.7)
4.59 (d, 8.7)
4.99 (d, 8.8)
4.51 (t, 8.8)
4.60 (t, 8.8)
4.42 (t, 8.8)
4.57 (d, 8.8)
0
2
0
3
0
4
0
5
3.57 (d, 7.6)
4.41 (d, 9.8)
b-D-glc
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
6
4.82 (d, 8.9)
3.16 (t, 8.9)
3.27 (m)
2.90 (t, 9.2)
3.03 (m)
4.90 (d, 7.6)
3.39 (m)
3.47 (t, 9.2)
3.10 (t, 9.2)
3.24 (m)
5.57 (d, 7.6)
4.12 (t, 9.1, 7.6)
4.28 (t, 9.1)
4.03 (t, 9.1)
3.85 (m)
5.82 (d, 7.6)
4.28 (m)
4.22 (t, 9.1)
4.03 (t, 9.1)
3.84 (m)
5.58 (d, 8.4)
4.19 (t, 8.4)
4.29 (t, 8.4)
4.21 (t, 8.4)
3.86 (m)
4.37 (dd, 11.6, 3.8)
4.49 (dd, 11.6, 3.8)
5.83 (d, 7.6)
4.29 (m)
4.23 (t, 9.2)
4.06 (t, 9.2)
3.84 (m)
3.35 (dd,)
3.55 (dd, 11.9, 3.3)
3.83 (dd, 11.9, 3.3)
4.35 (m)
4.47 (m)
4.27 (m)
4.49 (d, 8.5)
4.31 (m)
4.49 (dd, 11.7, 3.2)
3.70 (brd, 11.3)
b-D-xyl
0
0
0
0
0
00
00
00
00
00
1
2
3
4
5
5.39 (d, 6.7)
4.09 (m)
4.12 (m)
4.14 (m)
3.68 (m)
5.40 (d, 6.7)
4.10 (m)
4.12 (m)
4.13 (m)
3.69 (m)
4
.33 (dd, 9.5, 4.6)
4.33 (dd, 11.9, 4.3)
a
-L-rha
0
00
00
00
00
00
00
1
5.02 (s)
3.66 (brs)
3.52 (dd, 10.6, 4.0)
3.16 (t, 10.6)
3.96 (m)
5.22 (d, 1.9)
3.92 (dd, 3.3, 1.9)
3.76 (dd, 9.5, 3.3)
3.39 (t, 9.5)
6.37 (brs)
4.74 (m)
4.69 (dd, 9.4, 3.1)
4.32 (t, 9.4)
5.03 (m)
6.38 (brs)
4.74 (m)
4.69 (dd, 9.1, 3.1)
4.33 (t, 9.1)
5.04 (m)
0
2
3
4
5
6
0
0
0
0
4.12 (m)
1.09 (d, 6,1)
1.26 (d, 6.4)
1.77 (d, 6.1)
1.77 (d, 6.1)
N.A.: could not assignment.
C1 (1) was elucidated as 21b-acetyloxy-3b,14b,30-trihydroxypa-
chan-12-en-28-oic acid 14b,28-lactone (pachanol C) 3-O-b- -xylo-
-glucuronopyra-
on the C-21 (-3.6 ppm) and acetoxy methyl carbon (ꢀ0.1 ppm) and
showed downfield shifts on the C-22 (+3.9 ppm), C-30 (+3.4 ppm)
and carboxyl carbon at d 171.1 (+0.6 ppm). In the HMBC spectrum,
the acetoxy methyl proton at d 2.09 and the methylene proton on
2H-30 at d 4.65 (2H, s) showed correlations with the carboxyl car-
bon at d 171.1. These results indicated that an acetoxyl group was
located at C-30 instead of at C-21 as in 1. This aglycone is thus a
new triterpene, 30-acetyloxy-3b,14b,21b-trihydroxypachan-12-
en-28-oic acid 14b,28-lactone, named pachanol E. After an exten-
sive NMR study, the oligosaccharide moiety of 2 was determined
to be the same as that of 1. The configurations of glucuronic acid,
glucose and xylose were determined using the same method
(Tanaka et al., 2007). Peaks at 18.4, 19.2 and 21.4 minutes
D
pyranosyl (1 ? 2)-b-D-glucopyranosyl (1 ? 2)-b-D
noside.
Pachanoside E1 (2) was isolated as a colorless powder. Its
molecular formula C49 21 was determined from its negative
ion HRFABMS (m/z 997.4641 [MꢀH] ) and confirmed by C-
74
H O
ꢀ
13
13
NMR and DEPT analysis. Its C NMR spectrum contained 49 sig-
1
13
nals. For the H and C NMR spectra of 2, a doublet methyl proton
at d 1.09 (J = 6.4 Hz) and a quaternary carbon at d 90.6 were ob-
served, and it was assumed that the aglycone of 2 possesses a
pachanane skeleton. In comparison with the 13C NMR spectro-
scopic data of the aglycone moiety for 1, 2 exhibited upfield shifts

S. Okazaki et al. / Phytochemistry 72 (2011) 136–146
141
H
δ 3.88, 4.22
δ 5.56
30
COOH
3
H
H
O
δ 2.86
CH OH
2
12
O
H
HO
HO
H
δ 2.51
O
18
H
H
29
OH
H
1
3
δ 1.28
H
H
O
2
8
CH3
H
H
H
δ 0.81
H
O
2
5
9
O
H C
22
OCOCH3
21
HO
HO
3
14
2
6
H
H C
δ 1.38
3
H
H
δ 1.70
O
δ 1.60
H
H δ 5.20
3
H
H
15
H
H
H
5
H
RO
16
O
2
4
δ 3.36
2
7
HO
HO
H C
3
H
δ 0.79
H
CH
3
H
H
δ 1.03
7
H
OH
HMBC
23
δ 1.77
H
CH3
H
δ 1.52
NOESY
DQF-COSY
NOESY
Fig. 1. Phase-sensitive NOESY correlations for the aglycone moiety of pachanoside
C1 (1).
Fig. 3. HMBC, DQF-COSY and phase-sensitive NOESY correlations for the sugar
moiety of pachanoside C1 (1).
coincided with derivatives of
xylose (T of -glucuronic acid: 19.4, T
-xylose: 21.1). Thus, pachanoside E1 (2) was elucidated as 30-
acetyloxy-3b,14b,21b-trihydroxypachan-12-en-28-oic acid
4b,28-lactone (pachanol E) 3-O-b- -xylopyranosyl (1 ? 2)-b-
glucopyranosyl (1 ? 2)-b- -glucuronopyranoside.
Pachanoside F1 (3) was isolated as a colorless powder. Its
molecular formula C51 22 was determined from its negative
ion HRFABMS (m/z 1039.4742) and confirmed by C NMR and
D
-glucose,
D
-glucuronic acid, and
D
-
methyl proton at d 2.02, had a cross-peak with H-21 at d 5.13
(1H, dd, J = 13.3, 4.6 Hz). The additional carbonyl carbon at d
170.8, which has a correlation with another acetoxy methyl at d
2.12, had cross-peaks with 2H-30 at d 4.37 and 4.47 (each 1H, d,
J = 11.5 Hz). These results established that the two acetoxyl groups
were attached to C-21 and C-30. Its aglycone moiety, 21b,30-diace-
tyloxy-3b,14b-dihydroxypachan-12-en-28-oic acid 14b,28-lactone,
is a new triterpene named pachanol F. After an extensive NMR
study, the oligosaccharide moiety of 3 was determined to be the
same as that of 1. The configurations of glucuronic acid, glucose
R
D
R
of -glucose: 18.7, T
D
R
of
D
1
D
D-
D
76
H O
13
1
3
DEPT analysis. The compound displayed 51 signals in its
C
NMR spectrum. For the 1H and C NMR spectra of 3, a doublet
13
and xylose were determined as
D-glucuronic acid, D-glucose and
methyl proton at d 1.02 (J = 6.4 Hz) and a quaternary carbon at d
D-xylose using the same method (Tanaka et al., 2007). Thus, pac-
9
0.8 were observed, and it was similarly assumed that the agly-
hanoside F1 (3) was elucidated as 21b,30-diacetyloxy-3b,14b-
dihydroxypachan-12-en-28-oic acid 14b,28-lactone (pachanol F)
cone of 3 possessed a pachanane skeleton. Unambiguous assign-
ment for the
1
13
H
and
C
NMR signals were made by
a
3-O-b-D-xylopyranosyl (1 ? 2)-b-D-glucopyranosyl (1 ? 2)-b-D-
1
combination of 2D NMR experiments. In comparison with H and
glucuronopyranoside.
1
3
C NMR data of the aglycone moiety for pachanol C, an additional
methyl proton signal at d 2.12 and two carbon signals at d 20.7 and
Pachanoside G1 (4) was isolated as a colorless powder. Its molec-
ular formula C51H74O21 was determined from its negative ion
1
3
13
1
70.8 for an acetoxy group were observed on 3. For the C NMR
HRFABMS (m/z 1039.4747), and its C NMR spectrum had 51 sig-
spectrum, upfield shifts of C-21 (ꢀ0.8 ppm) and downfield shifts
of C-30 (+2.9 ppm) were seen. From the HMBC spectrum, the car-
bonyl carbon at d 170.3, which has a correlation with an acetoxy
nals. The UV spectrum indicated the presence of a conjugated hetero
system in the molecule (kmax 242 and 248 nm). The H NMR spec-
trum showed the presence of six methyl groups characterized by
1
O
OH
O
HO
pachanol D
CH2OH
OCOCH3
CH2OH
OCOCH3
O
O
COOH
OH
HO
HO
reported structure of pachanol C
corrected structure of pachanol C
Fig. 2. Structures of pachanols C and D.

1
42
S. Okazaki et al. / Phytochemistry 72 (2011) 136–146
O
O
of olefinic carbons at d 127.9, 137.9 and a carbonyl carbon at d
1
3
3
0
179.4. The sugar part contained, in the C NMR spectrum, three
CH2O
CH3
CH3
13
anomeric signals at d 101.8, 101.9 and 105.9. Since the C NMR
21
O
spectrum of 5 showed the presence of a carbonyl group consistent
12
1
13
with an uronic acid, 5. Since the H and C NMR spectroscopic data
1
3
COOH
of compound 5 were broad, 5 was converted to its methyl ester
14
13
(
2 2
5a) by treatment with CH N . The C NMR spectrum of 5a dis-
1
5
3
played 50 signals, and two additional carbons at d 51.9 (CH ),
27
RO
and 170.6 (C) were observed. The six methyl protons characterized
by singlets at d 0.76, 1.06, 1.16, 1.22, 1.34 and 1.53 and an olefinic
proton at d 5.50 (t-like, J = 3.5 Hz), coupled with the information
HMBC
DQF-COSY
1
3
from C NMR spectrum (six methyl carbon signals and a pair of
olefinic carbons at d 127.9, 138.1), indicated that the aglycone of
5a possesses an olean-12-ene skeleton. The aglycone portion was
identified as bridgesigenin A, which has a five-membered ring lac-
tone by the HMQC and HMBC correlations of 5a, a common agly-
cone that occurs in Echinopsis bridgesii (Trichocereus bridgesii,
Cactaceae) (Kinoshita et al., 1992). The overall structural assign-
ment was achieved following the same methodology as in 1. The
downfield chemical shift of C-3 (d 89.8) indicated that 5a was a
monodesmoside glycoside. Out of 50 carbon signals observed in
Fig. 4. Key HMBC and DQF-COSY correlations for the aglycone moiety of pachan-
oside G1 (4).
singlets at d 0.70, 1.03, 1.05, 1.10, 1.26 and 2.02, two acetoxy methyl
groups at d 2.04 and 2.12, and an olefinic proton at d 5.78 (br s). The
1
3
C NMR spectrumshowed signals for two pairs ofolefinic carbons at
d 124.7(CH), 126.2(C), 136.9 (C), 139.0 (C) and four carbonyl carbons
at d 170.3, 170.8, 172.3 and 177.9. Its structure was determined by
1
3
HMQC,
HMBC
and
DQF
COSY
the C NMR spectrum of 5a, 30 were assigned to the aglycone part,
spectra correlations (Fig. 4) and phase sensitive NOESY spectral
correlations.
and the remaining 20 carbons to the oligosaccharide moiety. The
H and C NMR spectra of 5a shows three anomeric carbon signals
1
13
A-, B, and E rings of its structure were elucidated following the
same method as for 1. These results led to assignment of five
methyl signals, d 0.70, 1.03, 1.05, 1.10 and 1.26, at 3H-25, -26, -
at d 102.0, 102.0 and 105.3, and three anomeric proton resonances
at d 4.98 (d, J = 7.1 Hz), 5.85 (d, J = 7.5 Hz), 6.40 (br s), together with
a methyl doublet proton signal at d 1.78 (J = 6.1 Hz), suggesting the
occurrence of three sugar units including one rhamnose unit. Two
additional carbons at d 51.9 and 170.6 suggested the presence of an
uronic acid on the basis of the following data. From the analyses of
the HMQC, HMBC and DQF COSY spectra (Fig. 5), the carbonyl car-
bon at d 170.6 had a cross-peak with d 3.72 (3H, s), which in turn
has an HMQC correlation with a methoxyl carbon at d 51.9. An
extensive NMR study permitted assignment of the proton and
the carbon resonances in the respective spectra and indicated pres-
2
4, -29 and -23, respectively, while a carbonyl carbon at d 177.9
could be assigned to C-28. The resonances of two methyl groups
d 2.04 and 2.12) and carbonyl carbons suggested the presence of
(
two acetoxyl groups. From the HMBC correlations, one is located
at C-21 and the other at C-30. Thus, the methyl proton signals at
d 2.02 were deemed to be located on C-27. Regarding rings C and
D, their structures were elucidated from the following extensive
NMR experiments. On the HMBC, H-9 at d 1.08 (1H, m) showed
2
cross-peaks with C-11 (CH
(
1
2
) at d 22.0 and a sp carbon at d 124.7
ence of a terminal
a-L-rhamnose (rha), a 2-substituted glucuronic
2
CH), and H-11 at d 1.82 (1H, m) correlated with sp carbons at d
acid methyl ester and 2-substituted glucose. The anomeric config-
24.7 (CH) and d 136.9 (C); thus, the sp2 carbon at d 124.7 (C)
uration for rhamnose was determined as the
the large JC, H values (J = 173 Hz).
a-configuration from
1
was assigned as C-12 and the quaternary carbon at d 136.9 as C-
3. Furthermore, the methyl proton at d 2.02 (s), which has an
1
The linkage of the sugar units at C-3 of bridgesigenin A and the
sequence of the sugar chains were established by HMQC, HMBC
and NOESY spectra. The HMBC spectrum of 5a indicated cross-
peaks between H-1 of glcA at d 4.98 and C-3 of bridgesigenin A
at d 89.8. This result indicated that the glcA was connected to C-
3 of the aglycone. The linkage of the glc at C-2 of glcA was indicated
by a HMBC cross-peak between H-2 of glcA at d 4.50 and C-1 of glc
HMQC correlations with carbon at d 24.3, was assumed to be at-
2
tached to a sp carbon based on its chemical shift value. In the
HMBC spectrum, the methyl proton at d 2.02 has cross-peaks with
2
two sp carbond at d 126.2 and 139.0 (Fig. 4). The 3H-26 at d 1.03
2
showed a similar correlation with d 139.0. Thus, the sp carbons at
d 139.0 and 126.2 were assigned as C-14 and C-15, respectively.
The above information confirmed that the methyl proton C-27 at
d 2.02 is connected to C-15. This aglycone is a new triterpene,
2
1b,30-diacetyloxy-3b-hydroxypachan-12,14(15)-dien-28oic acid
which has a pachanane skeleton, named pachanol G. After an
extensive NMR spectroscopic study, the oligosaccharide moiety
of 4 was determined to be the same as that of 1. The configurations
of glucuronic acid, glucose and xylose were determined to D-conf-
igrations using the same method (Tanaka et al., 2007). Thus, pac-
hanoside G1 (4) was elucidated as 21b,30-diacetyloxy-3b-
H
COOMe
3
H
O
HO
HO
O
H
OH
H
O
H
H
H
H
H
H
O
HO
HO
hydroxypachan-12,14(15)-dien-28oic acid (pachanol G) 3-O-b-D-
H
xylopyranosyl (1 ? 2)-b-
D
-glucopyranosyl (1 ? 2)-b-
D
-glucuron-
O
H
H
H
opyranoside.
Bridgeside A1 (5) was isolated as a colorless powder. Its molec-
ular formula C48 20 was determined from its negative ion
HRFABMS. The IR spectrum of 5 shows absorption at 3400 cm
H
74
H O
ꢀ
1
H
HMBC
O
H3C
HO
ꢀ
1
(
hydroxy) and 1760 cm (five-membered ring lactone). The com-
H
DQF-COSY
13
1
OH
pound displayed 48 signals in its C NMR spectrum. The H NMR
spectrum showed the presence of six methyl groups characterized
by singlets at d 0.76, 1.07, 1.13, 1.22, 1.33, 1.51 and an olefinic pro-
ton at d 5.49 (br s). The 1 C NMR spectrum showed signals for a pair
NOESY
OH
H
Fig. 5. HMBC, DQF-COSY and phase sensitive NOESY correlations for the sugar
moiety of bridgeside A1 methyl ester (5a).
3

S. Okazaki et al. / Phytochemistry 72 (2011) 136–146
143
at d 102.0. Similarly, the linkage of the rha at C-2 of glc was indi-
cated by cross-peaks between the anomeric proton of rha at d
derivatives of
D
-xylose,
D
-glucose and
D
-glucuronic acid. Thus,
bridgeside C2 (7) was elucidated as 21b-acetyloxy-3b,15b,30-tri-
hydroxyolean-12-en-28-oic acid 15b,28-lactone (bridgesigenin C)
6
.40 and C-2 of glc at d 78.4 and between H-2 of glc at d 4.30
and C-1 of rha at d 102.0. The configurations of glucuronic acid,
glucose and rhamnose were determined using same method (Ta-
3-O-b-
glucuronopyranoside, mono sodium salt.
Bridgeside D1 (8) was isolated as a colorless powder. Its molec-
ular formula C49 21 was determined from its negative ion
HRFABMS. The IR spectrum of
D-xylopyranosyl (1 ? 2)-b-D-glucopyranosyl (1 ? 2)-b-D-
naka et al., 2007). In the case of rhamnose,
verted into the thiazolizine derivatives to arylthiocarbamate
using -cysteine methyl ester hydrochloride instead of -cysteine
methyl ester hydrochloride, the HPLC retention times (T ) coin-
cided with derivatives of -glucuronic acid, -glucose and -rham-
L-rhamnose was con-
74
H O
L
D
8
showed absorptions at
(five-membered ring lac-
ꢀ1
ꢀ1
R
3420 cm
(hydroxy) and 1760 cm
1
3
D
D
L
tone). The compound displayed 49 signals in its C NMR spectrum.
On comparing with the C NMR data of 6, they shared the same
1
3
nose. On the basis of the above evidence, the structure of
bridgeside A1 methyl ester (5a) could be elucidated as bridgesige-
1
3
sugar moiety. Its C NMR spectrum showed upfield shifts of C-
21 at d 73.2 (ꢀ3.1 ppm) and downfield shifts of C-30 at d 64.4
(+3.4 ppm) and carboxyl carbon at d 171.1 (+0.7 ppm). The car-
boxyl carbon at d 171.1, which has a correlation with an acetoxy
methyl signal at d 2.11, had cross-peaks with 2H-30 at d 4.55
and 4.95 (each 1H, d, J = 12.1 Hz) by HMBC. These results indicated
that an acetoxyl group is located at C-30 instead of at C-21 as in 6.
The aglycone is a new tritepene, 30-acetyloxy-3b,15b,21b-trihydr-
oxyolean-12-en-28-oic acid 15b,28-lactone, named bridgesigenin
D, but whose sugar moiety and configuration is the same as 1.
Thus, bridgeside D1 (8) was elucidated as 30-acetyloxy-
3b,15b,21b-trihydroxyolean-12-en-28-oic acid 15b,28-lactone
nin A 3-O-
)-6’-O-methyl-b-
bridgeside A1 (5) was determined as bridgesigenin A 3-O-
a
-
L
-rhamnopyranosyl (1 ? 2)-b-
D-glucopyranosyl(1 ?
2
D
-glucuronopyranoside. Thus, the structure of
a
-L
-
-
rhamnopyranosyl
(1 ? 2)-b-
D-glucopyranosyl
(1 ? 2)-b-D
glucuronopyranoside.
Bridgeside C1 (6) was isolated as a colorless powder. Its molec-
ular formula C49 21 was determined from its negative ion
HRFABMS. The IR spectrum of 6 shows absorptions at 3420 cm
74
H O
ꢀ1
ꢀ
1
(
hydroxy) and 1760 cm (five-membered ring lactone). The com-
13
pound displayed 49 signals in its C NMR spectrum. When com-
pared with the H NMR spectrum of bridgesigenin A (Kinoshita
1
et al., 1992), an additional acetoxy methyl signal at d 2.03 was ob-
(bridgesigenin D) 3-O-b-
syl (1 ? 2)-b- -glucuronopyranoside.
Bridgeside D2 (9) was isolated as a colorless powder. Its molec-
ular formula C50 21 was determined from its negative ion
HRFABMS. Its IR spectrum showed absorptions at 3410 cm (hy-
D-xylopyranosyl (1 ? 2)-b-D-glucopyrano-
1
3
served on 6. The C NMR spectrum showed downfield shifts of C-
1 (+1.8 ppm) and two additional signals at d 20.8 and 170.4. From
D
2
the HMBC spectrum, the carboxyl carbon at d 170.4, which has a
correlation with an acetoxy methyl at d 2.03, showed a cross-peak
with H-21 at d 5.13 (1H, dd, J = 12.8, 4.9 Hz). These results indi-
cated that an acetoxyl group is attached to C-21 as bridgesigenin
C. Thus, the aglycone of 6 was identified as in bridgesigenin C, a
common aglycone that occurs in Echinopsis pachanoi (Trichocereus
pachanoi, Cactaceae) (Kinoshita et al., 1995). After an extensive
NMR spectroscopic analysis, the oligosaccharide moiety of 6 was
determined to be the same as that of 1. The configuration of each
sugars were also the same as 1. Thus, the structure of bridgeside
76
H O
ꢀ
1
ꢀ
1
droxy) and 1760 cm
extensive NMR study, comparison of the signals of 8 and 9 indi-
cated many similarities, except that signals due to the b- -xylopyr-
anosyl unit in 8 were replaced in 9 by a set of resonances assigned
to a terminal -rhamnopyranosyl unit. Its sugar moiety and con-
(five-membered ring lactone). After an
D
a-L
figuration were the same as for bridgeside A1 (5). Thus, bridgeside
D2 (9) was elucidated as 30-acetyloxy-3b,15b,21b-trihydroxyole-
an-12-en-28-oic acid 15b,28-lactone (bridgesigenin D) 3-O-
a-
L-
C1 (6) was elucidated as bridgesigenin C 3-O-b-
1 ? 2)-b- -glucopyranosyl (1 ? 2)-b- -glucuronopyranoside.
Bridgeside C2 (7) was isolated as a colorless powder. Its IR spec-
D
-xylopyranosyl
rhamnopyranosyl
glucuronopyranoside.
(1 ? 2)-b-
D
-glucopyranosyl
(1 ? 2)-b-D
-
(
D
D
Bridgeside E1 (10) was isolated as a colorless powder. Its molec-
ular formula C51 21 was determined from its negative ion
HRFABMS. The IR spectrum of 10 showed absorptions at
ꢀ1
ꢀ1
trum showed absorptions at 3420 cm (hydroxy) and 1760 cm
five-membered ring lactone). Interestingly, 7 was insoluble in pyr-
76
H O
(
1
3
ꢀ1
ꢀ1
idine. While the compound displayed 49 signals in its C NMR
3400 cm
(hydroxy) and 1720 cm
(five-membered ring lac-
1
13
1
spectrum in methanol-d
of 7 shows three anomeric carbon signals at d 102.0, 102.3 and
05.7 and three anomeric proton resonances at d 4.43 (d,
4
(temp.50 °C), the H and C NMR spectra
tone). When compared with the H NMR spectrum of 6, an addi-
tional acetoxy methyl at d 2.13 was observed. The 13C NMR
spectrum showed upfield shifts of C-21 at d 75.6 (ꢀ1.3 ppm),
downfield shifts of C-30 at d 64.0 (+2.8 ppm) and two additional
signals at d 20.7 and 170.8. The carboxyl carbon at d 170.3, which
is correlated with an acetoxy methyl at d 2.03, showed a cross-peak
with H-21 at d 5.04 (1H, dd, J = 12.7, 4.7 Hz), while another carboxy
carbon at d 170.8 which is correlated with an acetoxy methyl at d
2.13 had cross-peaks with 2H-30 at d 4.26 and 4.74 (each 1H, d,
J = 12.1 Hz) by HMBC. These results indicated that acetoxyl groups
were attached to C-21 and C-30. The aglycone part corresponds to
a new genin, 21b,30-diacetyloxy-3b,15b-dihydroxyolean-12-en-
28-oic acid 15b,28-lactone, named bridgesigenin E. After an exten-
sive NMR study, the oligosaccharide moiety of 10 was determined
to be the same as that of 6. The sugar moiety and their configura-
tion were same as 1. Thus, bridgeside E1 (10) was elucidated as in
1
J = 8.3 Hz), 4.90 (d, J = 7.6 Hz), 5.22 (d, J = 1.9 Hz), respectively.
After an extensive NMR study, the stereochemistry of the aglycone
moiety was identified as bridgesigenin C and two sugar moieties
were confirmed as glucose and rhamnose. The remaining sugar
unit seemed to be glucuronic acid from its chemical shifts. But
the carbonyl carbon signal of glucuronic acid in 7 was not observed
in methanol-d
4
(temp. 50 °C). Further analysis of the NMR data in
DMSO-d , indicated the unknown sugar unit was glucuronic acid,
6
and the sequence of the oligosaccharide chain was determined to
be the same as those of 5. The results of FABMS indicated the pres-
ence of a sodium ion in a sample of 7. FABMS (positive ion mode) of
+
7
1
(
[
(magic bullet + NaCl as matrix) gave an ion [MꢁNa+Na] at m/z
75 2
057, in accordance with molecular formula C50H O21Na . FABMS
ꢀ
negative ion mode) showed ions [MꢁNaꢀH] at m/z 1033,
21b,30-diacetyloxy-3b,15b-dihydroxyolean-12-en-28-oic
15b,28-lactone (bridgesigenin E) 3-O-b- -xylopyranosyl (1 ? 2)-
b- -glucopyranosyl (1 ? 2)-b- -glucuronopyranoside.
Bridgeside E2 (11) was isolated as a colorless powder. Its molec-
ular formula C52 22 was determined from its negative ion
HRFABMS. The IR spectrum of 11 shows absorption at 3415 cm
acid
ꢀ
MꢁNaꢀNa] at m/z 1011. Furthermore, HRFABMS (positive ion
D
+
mode) gave ions [MꢁNa+Na] at m/z 1057.4602 (calcd for
D
D
+
1
057.4596, C50
H
75
O
21Na
2
) and [MꢁNa+H] at m/z 1035.4784 (calcd
for 1035.4777, C50
H
76
O
21Na). The above information suggests that
78
H O
ꢀ
1
7
had been isolated as mono sodium salt. The configurations of xy-
lose, glucose and glucuronic acid were determined using the same
method. The retention times (T ) of HPLC were considered with
ꢀ1
(hydroxy) and 1740 cm (five-membered ring lactone). After an
extensive NMR study, comparison between the assigned signals
R

1
44
S. Okazaki et al. / Phytochemistry 72 (2011) 136–146
of 11 and 10 indicated that the only difference was the resonance
due to the sugar unit attached to C-2 of the ester-linked a-rhamn-
E3-2 (98.5 mg), Fr. E3-3 (0.24 g), Fr. E3-4 (0.10 g), Fr. E3-5
(0.19 g) and Fr. E3-6 (43.6 mg)]. Fraction E3-2 (98.5 mg) was puri-
00
opyransly unit. The sugar moiety and their configuration were
3 2
fied by HPLC [CHCl –MeOH–H O (15:6:1), 3.0 mL/min, detector,
same as 5. Thus, bridgeside E2 (11) was elucidated as bridgesigenin
222 nm] to yield pachanoside C1 (1, 16.3 mg, 0.007%). Further-
E 3-O-
b- -glucuronopyranoside.
Concluding remarks: Eleven new saponins from E. macrogona
a
-
L
-rhamnopyranosyl (1 ? 2)-b-
D
-glucopyranosyl (1 ? 2)-
more, fraction E3-3 (0.23 g) was separated by ODS CC using a step-
wise gradient [20 g, MeOH–H O (20:80, v/v) ? MeOH] to give
2
three fractions [Fr. E3-3-1 (16.7 mg), Fr. E3-3-2 (0.21 g) and Fr.
E3-3-3 (20.1 mg)]. Fraction E3-3-2 (0.21 g) was purified by HPLC
2
[MeOH–H O (60:40, v/v), 2.0 mL/min, detector, 222 nm] to yield
pachanoside C1 (1, 32.1 mg, 0.014%).
Fraction D (2.22 g) was applied to a silica gel column using a
stepwise gradient [70 g, CHCl –MeOH–H O (60:10:1) ? MeOH]
3 2
D
were isolated and their structures determined. The Pachanoside
C1, E1, F1 and G1 are the first isolated saponins of the pachan-
ane-type triterpene saponins. We had previously reported pacha-
nol A as a triterpene sapogenin from Trichocereus (Echinopsis)
pachanoi (Kinoshita et al., 1995), and considered that it must be
an artifact by acid hydroloysis of saponins. The aglycone of sapo-
nin, pachanoside G1 (4), is a pachanol A derivative. Thus, pachanol
A and pachanol G were considered as true aglycones of saponins.
to afford six fractions [Fr. D1 (19.5 mg), Fr. D2 (10.7 mg), Fr. D3
(0.52 g), Fr. D4 (0.49 g), Fr. D5 (0.88 g) and Fr. D6 (0.17 g)]. Fraction
D5 (0.88 g) was further separated by ODS column chromatography
3 2 3
using a stepwise gradient [20 g, CH CN–H O (30:70, v/v) ? CH CN]
to give six fractions [Fr. D5-1 (0.33 g), Fr. D5-2 (0.28 g), Fr. D5-3
3
. Experimental
(
(
26.0 mg), Fr. D5-4 (12.6 mg), Fr. D5-5 (13.6 mg) and Fr. D5-6
154.2 mg)]. Fraction D5-1 (0.33 g) underwent ODS CC using a
3.1. General experimental procedures
2
stepwise gradient [20 g, MeOH–H O (40:60, v/v) ? MeOH] to yield
pachanoside B (2, 20.0 mg, 0.009%). Fraction D5-2 (0.28 g) was fur-
ther purified by ODS CC using a stepwise gradient [20 g, MeOH–
Melting points were determined on a Yanagimoto MP micro-
melting point apparatus, and [a] values were determined with a
D
2
H O (40:60, v/v) ? MeOH] to afford 10 fractions [Fr. D5-2-1
JASCO DIP-140 digital polarimeter. IR spectra were measured with
a JASCO A-102 IR spectrophotometer, whereas 1H and 13C NMR
(5.1 mg), Fr. D5-2-2 (7.6 mg), Fr. D5-2-3 (1.4 mg), Fr. D5-2-4
(
(
15.4 mg), Fr. D5-2-5 (29.7 mg), Fr. D5-2-6 (53.2 mg), Fr. D5-2-7
0.12 g), Fr. D5-2-8 (11.6 mg), Fr. D5-2-9 (43.0 mg) and Fr. D5-2-
spectra were recorded using a JEOL AL-400 or JNM LA-500 spec-
trometer in C
an internal standard. Kieselgel-60F254 (MERCK) or RP-18F254S
MERCK)-precoated plates were employed for thin-layer chroma-
tography (TLC). Column chromatography (CC) was carried out on
0–230 mesh silica gel (MERCK) and prep ODS-7515-12A. HPLC
was performed using a PU-2080 Plus pump with a JASCO UV-
075 Plus UV detector. HREIMS and HRFABMS were obtained using
a JOEL JMS-700.
5 5 3 6
D N, CD OD or DMSO-d with tetramethylsilane as
1
0 (3.2 mg)]. Fraction D5-2-5 (29.7 mg) was identified as pachano-
side C1 (1, 38.6 mg, 0.02%). Furthermore, fraction D5-2-6 (53.2 mg)
was separated by ODS CC using a stepwise gradient [20 g, CH CN–
O (30:70, v/v) ? CH CN] to give pachanoside C1 (1, 36.8 mg,
.02%). Fraction D5-2-7 (0.12 g) was separated by ODS CC using a
stepwise gradient [20 g, CH CN–H O (25:75, v/v) ? CH CN] to af-
ford bridgeside C1 (6, 77.1 mg, 0.03%).
Fraction E4 (1.64 g) was subjected to ODS CC using a stepwise
gradient [35 g, CH CN–H O (20:80, v/v) ? CH CN] to afford seven
(
3
H
0
2
3
7
3
2
3
2
3
2
3
3
.2. Plant material
fractions [Fr. E4-1 (0.20 g), Fr. E4-2 (0.51 g), Fr. E4-3 (0.26 g), Fr.
E4-4 (0.15 g), Fr. E4-5 (0.34 g), Fr. E4-6 (70.3 mg) and Fr. E4-7
E. macrogona H. Friedrich and G.D. Rowley, was cultivated orig-
(
59.2 mg)]. Fraction E4-5 (0.34 g) was further separated by silica
gel CC using stepwise gradient [76 g, CHCl -MeOH-H
20:7:1) ? MeOH] to afford five fractions [Fr. E4-5-1 (19.9 mg),
inally at the Japan Cactus Planning Co. (Fukushima City, Fukushi-
ma, Japan). The cactus was identified by Dr. H. Yuasa. A voucher
specimen (MPU-C-02-1) is deposited at our laboratory.
a
3
2
O
(
Fr. E4-5-2 (64.1 mg), Fr. E4-5-3 (58.6 mg), Fr. E4-5-4 (40.5 mg)
and Fr. E4-5-5 (0.15 g)]. Fraction E4-5-3 (58.6 mg) was purified
3
.3. Extraction and isolation
by HPLC [CHCl
22 nm] to give bridgeside C1 (6, 42.5 mg, 0.018%). Furthermore,
fraction E4-5-5 (0.15 g) was purified by HPLC [MeOH–H
3 2
–MeOH–H O (15:6:1), 3.0 mL/min, detector,
2
The dried and powdered whole plants of E. macrogona (229 g)
2
O
were extracted with CHCl
.5 L). The dried MeOH extract (47.0 g, 20.5%) was then applied
to a Diaion HP-20 column which was eluted with H O, MeOH
O (30:70, v/v) and 100% MeOH, successively. The MeOH eluted
fraction (13.3 g, 5.8%) was separated by silica gel CC using a step-
wise gradient [350 g, CHCl –MeOH (50:1) ? CHCl –MeOH–
O ? MeOH] to give eight fractions [Fr. A (0.34 g), Fr. B (0.81 g),
Fr. C (0.34 g), Fr. D (4.18 g), Fr. E (3.80 g), Fr. F (0.96 g), Fr. G
3
(3 ꢂ 3.5 L) and then with MeOH (6 ꢂ
(35:65, v/v), 2.0 mL/min, detector, 222 nm] to yield bridgeside C2
(7, 0.12 g, 0.054%). Fraction E4-3 (0.26 g) was purified by HPLC
[CHCl –MeOH–H O (15:6:1), 3.0 mL/min, detector, 222 nm] to give
3 2
bridgeside D1 (8, 33.2 mg, 0.014%) and bridgeside D2 (9, 0.11 g,
0.047%).
3
2
H
2
3
3
Fraction D3 (0.52 g) underwent ODS CC using a stepwise gradi-
H
2
ent [35 g, MeOH–H
(10, 82.7 mg, 0.036%). Fraction D5-6 (0.15 g) was separated by ODS
CC using stepwise gradient [20 g, MeOH–H (60:40, v/
2
O (40:60, v/v) ? MeOH] to yield bridgeside E1
(
0.22 g) and Fr. H (0.78 g)], respectively.
a
2
O
Fraction F (0.96 g) was further subjected to silica gel CC using a
v) ? MeOH] to afford three fractions [Fr. D5-6-1 (20.9 mg), Fr.
stepwise gradient [32 g, CHCl
give bridgeside A1 (5, 0.27 g, 0.12%), and a portion of 5 (30.5 mg)
was methylated with diazomethane (CH ) and purified by on sil-
ica gel CC [7 g, CHCl –MeOH–H O (60:20:1) ? MeOH] to yield
3
–MeOH–H
2
O (60:10:1) ? MeOH] to
D5-6-2 (0.13 g) and Fr. D5-6-3 (11.4 mg)]. Fraction D5-6-2
(0.13 g) was purified by HPLC [CHCl –MeOH–H O (15:6:1),
3 2
3.0 mL/min, detector, 222 nm] to yield bridgeside E2 (11,
80.7 mg, 0.035%).
2
N
2
3
2
bridgeside A methyl ester (5a, 15.1 mg).
Fraction C (4.18 mg) was separated by ODS CC using a stepwise
Fraction E (3.80 g) was subjected to silica gel CC using a step-
3 2 3
gradient [125 g, CH CN–H O (10:90, v/v) ? CH CN] to afford eight
wise gradient [120 g, CHCl
ford five fractions [Fr. E1 (55.9 mg), Fr. E2 (0.45 g), Fr. E3 (0.85 g),
Fr. E4 (1.64 g) and Fr. E5 (0.55 g)]. Fraction E3 (0.85 g) was further
3
–MeOH–H
2
O (60:20:1) ? MeOH] to af-
fractions [Fr. C1 (0.68 g), Fr. C2 (0.32 g), Fr. C3 (42.6 mg), Fr. C4
(0.12 g), Fr. C5 (46.4 mg), Fr. C6 (0.16 g), Fr. C7 (85.7 mg) and Fr.
C8 (32.9 mg)]. Fraction C6 (0.16 g) was subjected to ODS CC using
separated by ODS CC using a stepwise gradient [20 g, MeOH–H
2
O
2
a stepwise gradient [70 g, MeOH–H O (10:90, v/v) ? MeOH] to
(
40:60, v/v) ? MeOH] to give six fractions [Fr. E3-1 (66.8 mg), Fr.
yield pachanoside F1 (3, 0.26 g, 0.111%) and bridgeside E1 (10,

S. Okazaki et al. / Phytochemistry 72 (2011) 136–146
145
7
1.8 mg, 0.031%). Furthermore, fraction C4 (0.12 g) was purified by
ODS CC using a stepwise gradient [45 g, MeOH–H O (40:60, v/
v) ? MeOH] to afford pachanoside G1 (4, 51.1 mg, 0.022%).
1057.4596,
[M+H] , 1035.4777).
C
50
H
75
O
21Na
2
), 1035.4784 (calcd for
C
50
H
76
O21Na
ꢀ
2
3
.3.9. Bridgeside D1 (8)
Colorless powder (33.2 mg); ½
3
.3.1. Pachanoside C1 (1)
Colorless powder (124 mg); ½
25
a
ꢃ
-17.9 (c 1.00, MeOH); IR m
max
D
2
D
5
a
ꢃ
ꢀ14.7 (c 1.00, MeOH); IR
m
max
(KBr) 3420, 1760, 1730, 1240, 1120, 1080, 1040 cm ; for 1H and
ꢀ1
KBr) 3400, 1738, 1720, 1600, 1240, 1030 cm ; for 1H and 13C
ꢀ1
(
13
C NMR spectroscopic data, see Tables 1 and 2; negative FABMS
NMR spectroscopic data, see Tables 1 and 2; ESI (Negative) Tof
ꢀ
ꢀ
ꢀ
m/z: 997 [MꢀH] , 865 [M-xyl-H] , 703 [M-xyl-glc-H] ; negative
ꢀ
MS m/z: 997.4676 [MꢀH] (calcd for
C
49
H
73
O
21
,
997.4644
ꢀ
HRFABMS m/z: 997.4646 (calcd for C49
H
73
O
21 [MꢀH] , 997.4644).
ꢀ
[
M–H] ), ESI (Positive) Tof MS m/z: 1021.4620 (calcd for
+
C
49 74
H O21Na [M+Na] , 1021.4620).
3
.3.10. Bridgeside D2 (9)
2
5
Colorless powder (107 mg); mp 198–201 °C; ½
aꢃ
-34.5 (c 1.00,
D
3.3.2. Pachanoside E1 (2)
2
D
5
MeOH); IR
m
max (KBr) 3410, 1760, 1740, 1240, 1130, 1080,
Colorless powder (20.0 mg); ½
a
ꢃ
ꢀ14.6 (c 1.00, MeOH); IR
m
max
ꢀ1
1
13
KBr) 3430, 1740, 1720, 1250, 1080, 1040 cm ; for 1H and 13C
ꢀ1
1040 cm ; for H and C NMR spectroscopic data, see Tables 1
(
ꢀ
ꢀ
and 2; negative FABMS m/z: 1011 [MꢀH] , 865 [M-rha-H] , 703
NMR spectroscopic data, see Tables 1 and 2; negative FABMS m/
ꢀ
ꢀ
ꢀ
[M-rha-glc-H] ; negative HRFABMS m/z: 1011.4797 (calcd for
z: 997 [MꢀH] , 865 [M-xyl-H] ; negative HRFABMS m/z:
ꢀ
ꢀ
C
H
50 75
O
21 [MꢀH] , 1011.4801).
9
97.4641 (calcd for C49
H
73
O
21 [MꢀH] , 997.4644).
3
.3.11. Bridgeside E1 (10)
Colorless powder (155 mg); mp 205–208 °C; ½
3
.3.3. Pachanoside F1 (3)
Colorless powder (255 mg); ½
2
D
5
2
5
a
ꢃ
ꢀ28.1 (c 1.00,
ꢀ1
a
ꢃ
ꢀ10.2 (c 1.00, MeOH); IR
m
max
D
(
KBr) 3400, 1740, 1240, 1040 cm ; for 1H and C NMR spectro-
ꢀ1
13
MeOH); IR mmax (KBr) 3400, 1720, 1370, 1230, 1030 cm ; negative
ꢀ
ꢀ
ꢀ
FABMS m/z: 1039 [MꢀH] , 907 [M-xyl-H] , 745 [M-xyl-glc-H] ;
scopic data, see Tables 1 and 2; negative FABMS m/z: 1039
1
13
ꢀ
ꢀ
ꢀ
ꢀ
for H and C NMR spectroscopic data, see Tables 1 and 2; nega-
[
MꢀH] , 907 [M-xyl-H] , 745 [M-xyl-glc-H] ; negative HRFABMS
ꢀ
tive HRFABMS m/z: 1039.4762 (calcd for C51
039.4750).
H
75
O
22 [MꢀH] ,
m/z: 1039.4742 (calcd for C51
H
75
O
22 [MꢀH] , 1039.4750).
1
3
.3.4. Pachanoside G1 (4)
Colorless powder (51.1 mg); ½
2
5
a
ꢃ
ꢀ15.8 (c 1.00, MeOH); IR
m
max
3.3.12. Bridgeside G (11)
D
KBr) 3410, 1730, 1240, 1040 cm ; for 1H and C NMR spectro-
ꢀ1
13
25
D
(
Colorless powder (80.7 mg); ½
aꢃ
ꢀ36.5 (c 1.00, MeOH); IR mmax
ꢀ1 1 13
scopic data, see Tables 1 and 2; negative FABMS m/z: 1039
(KBr) 3415, 1740, 1380, 1240, 1040 cm ; for H and C NMR
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
[
MꢀH] , 907 [M-xyl-H] , 745 [M-xyl-glc-H] , 569 [aglycone moi-
spectroscopic data, see Tables 1 and 2; negative FABMS m/z:
ꢀ
ꢀ
ꢀ
ety-H] ; negative HRFABMS m/z: 1039.4747 (calcd for C51
H
75
O
22
1053 [MꢀH] , 907 [M-rha-H] , 745 [M-rha-glc-H] ; negative
ꢀ
[
MꢀH] , 1039.4750).
HRFABMS m/z: 1053.4902 (calcd for
C
2 77 22
H O
[MꢀH] ,
5
1
053.4907).
3
.3.5. Bridgeside A1 (5)
Colorless powder (272 mg); ½
2
D
5
aꢃ
ꢀ33.5 (c 0.98, MeOH); IR
m
max
3.4. Determination of sugar configuration (Tanaka et al., 2007)
ꢀ1
ꢀ
(
KBr) 3400, 1760, 1600, 1240, 1040 cm ; 969 [MꢀH] , 823 [M-
ꢀ ꢀ ꢀ 1
rha-H] , 661 [M-rha-glc-H] , 485 [aglycone moiety-H] ; H NMR
Saponins (1–11) (each 2 mg) were hydrolyzed by 3.5% HCl
(
pyridine-d
5
) d: 0.76, 1.07, 1.13, 1.22, 1.33, 1.51 (each 3H, s), 1.77
(
0.4 mL) at 110 °C for 2.5 h. The reaction mixture was neutralized
with 0.5 M NaOH. After drying under vacuum, the residue was dis-
solved in pyridine (0.4 mL) containing -cysteine methyl ester
hydrochloride (2 mg) and heated at 60 °C for 1 h. O-Toylisothiocy-
anate (2 L) was then added and the mixture was heated at 60 °C
(
3H, d, J = 5.6 Hz), 3.31 (1H, m), 4.97 (1H, d, J = 6.6 Hz), 5.49 (1H,
13
br s), 5.82 (1H, d, J = 7.1 Hz), 6.37 (1H, br s); C-NMR (pyridine-
) d: 16.1, 16.8, 19.0, 19.7, 25.0, 25.3, 28.3 (each CH ), 63.1
CH ), 63.4 (CH ), 89.7 (CH), 101.8 (CH), 101.9 (CH), 105.1 (CH),
L
d
(
1
9
5
3
2
2
l
27.9 (CH), 137.9 (C), 179.4 (C); negative HRFABMS m/z:
69,4686 (calcd for C48
for 1 h. Each reaction mixture was directly analyzed by reversed-
phase HPLC using a SSC-3461 HPLC Pump with a SSC-5410 UV/
VIS detector (Senshu Scientific Co. Ltd.). An Senshu Pak PEGASIL
ꢀ
H
73
O
20 [MꢀH] , 997.4644).
3
.3.6. Bridgeside A1 methyl ester (5a)
Colorless powder (15.1 mg); for H and C NMR spectroscopic
4
0
H
.6 / ꢂ 250 mm HPLC column was used, (temp, 35 °C; flow,
.8 ml/min; eluate, CH CN–H (25:75) containing 50 mM
PO ). The HPLC column was washed with MeOH after each injec-
tion. The reaction conditions for -glucose, -glucuronic acid,
-xylose and -rhamnose were same as described above. For
rhamnose, -rhamnose dissolved in pyridine (0.4 mL) containing
-cysteine methyl ester hydrochloride (2 mg) was used for the
reaction. The derivative was used for determining retention time
instead of -rhamnose. Retention times for authentic sugar deriva-
tives, -glucose (18.69), -glucose (17.01), -xylose (21.08), -xylose
20.03), -glucuronic acid (19.35), -glucuronic acid (18.60),
rhamnose (33.12) and as -rhamnose (17.74) were used for com-
1
13
3
2
O
ꢀ
data, see Tables 1 and 2; negative FABMS m/z: 983 [MꢀH] .
3
4
D
,
L
D
,
L
D,
3
.3.7. Bridgeside C1 (6)
Colorless powder (105 mg); ½
L
L
D-
2
D
5
aꢃ
ꢀ33.7 (c 1.00, MeOH); IR
m
max
L
KBr) 3420, 1760, 1740, 1260, 1040 cm ; for 1H and C NMR
ꢀ1
13
(
D
spectroscopic data, see Tables 1 and 2; negative FABMS m/z: 997
ꢀ
ꢀ
ꢀ
[
MꢀH] , 865 [M-xyl-H] , 703 [M-xyl-glc-H] ; negative HRFABMS
D
ꢀ
m/z: 997.4645 (calcd for C49
H
73
O
21 [MꢀH] , 969.4644).
D
L
D
L
(
D
L
L-
3
.3.8. Bridgeside C2 (7)
Colorless powder (124 mg); ½
D
2
D
5
aꢃ
ꢀ44.6 (c 1.00, DMSO); IR
m
max
1
parison with retention times from reaction mixtures for each sapo-
nin. Peaks at 18.5, 19.2 and 21.4 minutes of the sugar derivatives
ꢀ1
(
KBr) 3410, 1770, 1730, 1620, 1415, 1395, 1250, 1040 cm ; for H
1
3
and C NMR spectroscopic data, see Tables 1 and 2; positive FAB-
MS m/z: 1057 [(RCOONa)+Na] , 1035 [(RCOONa)+H] ; negative
FABMS m/z: 1033 [(RCOONa)-H] , 1011 [(RCOONa)-Na] , 887
from 1 coincided with derivatives of
and -xylose. Peaks at 18.4, 19.2 and 21.4 of the sugar derivatives
coincided with derivatives of -glucose, -glucuronic acid, and
D-glucose, D-glucuronic acid,
+
+
D
ꢀ
ꢀ
D
D
D-
ꢀ
ꢀ
[
(RCOONa)-rha-H] , 865 [(RCOONa)-rha-Na] , 703 [(RCOONa)-
xylose. For the other saponins (3–11), the same results as com-
pounds 1 and 2 were obtained.
ꢀ
rha-glc-Na] ; positive HRFABMS m/z: 1057.4602 (calcd for

1
46
S. Okazaki et al. / Phytochemistry 72 (2011) 136–146
Djerassi, C., Farkas, E., Liu, L.H., Thomas, G.H., 1955. Terpenoids. XVII. The cactus
Acknowledgments
triterpenes thurberogenin and stellatogenin. J. Am. Chem. Soc. 77, 5330–5336.
Djerassi, C., Henry, J.A., Lemin, A.J., Rios, T., Thomas, G.H., 1956a. Terpenoids. XXIV.
The structure of the cactus triterupene queretaroic acid. J. Am. Chem. Soc. 78,
3783–3787.
Djerassi, C., Robinson, C.H., Thomas, D.B., 1956b. Terpenoids. XXV. The structure of
the cactus triterpene dumortierigenin. J. Am. Chem. Soc. 78, 5685–5691.
Djerassi, C., Burstein, S., Estrada, H., Lemin, A.J., Manjarrez, A., Monsimer, H.G., 1957.
Terpenoids. XXVIII. The triterpene composition of the genus Myrtillocactus. J.
Am. Chem. Soc. 79, 3525–3528.
This work partly was supported by the Sasakawa Scientific Re-
search Grant from The Japan Science Sciety. The authors would like
to thank Mrs. S. Kubota and T. Koseki for the spectroscopic (NMR or
MS) measurements. This work was partially supported by a grant
from the High-Tech Research Center Project, the Ministry of Educa-
tion, Culture, Sports, Science and Technology (MEXT), Japan
Imai, T., Okazaki, S., Kinoshita, K., Koyama, K., Takahashi, K., Yuasa, H., 2006.
Triterpenoid saponins from cultural plants of Stenocereus stellatus (Cactaceae). J.
Nat. Med. 60, 49–53.
(
S0801043).
Kinoshita, K., Koyama, K., Takahashi, K., Kondo, N., Yuasa, H., 1992. New triterpenes
from Trichocereus bridgesii. J. Nat. Prod. 55, 953–955.
Appendix A. Supplementary data
Kinoshita, K., Takizawa, T., Koyama, K., Takahashi, K., Kondo, N., Yuasa, H., 1995.
New triterpenes from Trichocereus pachanoi. J. Nat. Prod. 58, 1739–
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytochem.2010.10.004.
1744.
Kinoshita, K., Akiba, M., Koyama, K., Takahashi, K., Kondo, N., Yuasa, H., 1998.
Antinociceptive effect of triterpenes from cacti. Pharmaceutical. Biol. 36,
50–57.
Kinoshita, K., Koyama, K., Takahashi, K., Nishino, H., 1999. Inhibitory effect of some
triterpenes from cacti on 32Pi-incorporation into phospholipids of HeLa cells
promoted by 12-O-tetradecanoylphorbol-13-acetate. Phytomedicine 6, 73–77.
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