Triterpene saponins from the pericarps of Akebia trifoliata

Article abstract of DOI:10.1248/cpb.c12-00448

Eleven new triterpene saponin components (1-11) were isolated from the MeOH extract of pericarp of Akebia trifoliata (THUNB.) KOIDZ. Each of their structures was determined using NMR techniques and mass spectrometry.

Full text of DOI:10.1248/cpb.c12-00448

1264
Regular Article
Chem. Pharm. Bull. 60(10) 1264–1274 (2012)
Vol. 60, No. 10
Triterpene Saponins from the Pericarps of Akebia trifoliata
,b
Syuji Iwanaga,a Tsutomu Warashina,* and Toshio Miyasea
b
a School of Pharmaceutical Sciences, University of Shizuoka; and Institute for Environmental Sciences, University of
Shizuoka; 52–1 Yada, Suruga-ku, Shizuoka 422–8526, Japan. Received May 16, 2012; accepted June 19, 2012
Eleven new triterpene saponin components (1–11) were isolated from the MeOH extract of pericarp of
Akebia trifoliata (THUNB.) KOIDZ. Each of their structures was determined using NMR techniques and mass
spectrometry.
Key words Akebia trifoliata; Lardizabalaceae; triterpene saponin; akemisaponin
Akebia trifoliata (THUNB.) KOIDZ. (Lardizabalaceae) is used 16.8), a carbonyl carbon signal (δ 176.3), two olefinic carbon
as a diuretic and an antiphlogistic in traditional Chinese med- signals (δ 144.0, 122.6), and two hydroxymethine carbon sig-
icine. Use of the stem of A. trifoliata, known by the Japanese nals (δ 83.6, 68.5). These results indicated that the aglycone
name moku-tsu, is described by the Pharmacopoeia of Japan. of 1 was an olean-12-en-28-oic acid derivative. Based on the
Phytochemical analyses were previously carried out on Akebia second dimensional (2D) NMR [1H-detected heteronuclear
1–3)
1
quinata (HOUTT.) DECNE
and stems of A. trifoliata4,5) but multiple-bond connectivity (HMBC), HMQC, and H–1H shift
only a few chemical papers have been published on other parts correlation spectroscopy (1H–1H COSY)] measurements of 1,
of A. trifoliata, therefore we analyzed pericarps of A. trifoli- the methyl proton and carbon signals at δ 1.24, 29.2 and δ
ata. We now report on eleven new triterpene saponin compo- 1.07, 17.4 were assigned to H-23, C-23 and H-24, C-24, and
nents of the pericarps of A. trifoliata.
additionally, two sets of hydroxymethine proton and carbon
signals at δ 4.08, 68.5 and δ 3.37, 83.6 belonged to H-2, C-2
and H-3 and C-3. As the multiplicity and coupling constant
Results and Discussion
The dried pericarps of A. trifoliata were extracted with of the H-3 signal (d, J=9.0Hz) showed that H-2 and H-3 were
hot MeOH. The concentrated MeOH extract was separated both di-axial, the hydroxy groups at C-2 and C-3 were consid-
into ether-soluble, ethyl acetate-soluble and water-soluble ered to retain the α- and β-orientations, respectively. Thus, the
fractions. The water-soluble fraction was passed through a aglycone of 1 was determined as 2α,3β-dihydroxyolean-12-
Diaion HP-20 column, eluted with MeOH–H2O (1:1) and en-28-oic acid.8) On acid hydrolysis, 1 gave a sugar moiety
MeOH. A portion of the MeOH eluate was subjected to re- composed of D-glucose, L-rhamnose and D-xylose (2:1:1).9)
1
versed-phase preparative HPLC, affording compounds 1–15 The H-NMR signals of the sugar moiety were assigned as
including eleven new compounds. By comparing their NMR shown in Table 1 from the results of homonuclear Hartmann–
data with those in the literature, compounds 12–15 were iden- Hahn (HOHAHA) difference experiments. The anomeric
tified as scheffoleoside A (2α,3β,23-trihydroxyolean-12-en-28- configurations of D-glucose and D-xylose were identified as
oic acid O-α-L-rhamnopyranosyl-(1→4)-O-β-D-glucopyra- the both β-form from the coupling constant of anomeric pro-
nosyl-(1→6)-β-D-glucopyranosyl ester) (12),4,6) asiaticoside ton signals (J=8.0Hz), and that of L-rhamnose, as the α-form
(2α,3β,23-trihydroxyurs-12-en-28-oic acid O-α-L-rhamnopyra- from the chemical shift of an anomeric proton signal (δ 5.80)
1
nosyl-(1→4)-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl in the H-NMR spectrum.10) Observation of anomeric proton
ester) (13),4,6,7) 2α,3β,23-trihydroxy-30-norolean-12,20(29)- and carbon signals of the β-D-glucopyranosyl group at δ 6.19
dien-28-oic acid O-α-L-rhamnopyranosyl-(1→4)-O-β-D-glu- and 95.5 indicated that this group was esterified at C-28 of the
copyranosyl-(1→6)-β-D-glucopyranosyl ester (14),4) and aglycone, which was confirmed by the long-range correlation
2α,3β,23-trihydroxyolean-12-en-28-oic acid O-β-D-xylopyra- between H-1 of this β-D-glucopyranosyl group (δ 6.19) and
nosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→4)-O-β-D-glucopyra- C-28 of the aglycone (δ 176.3). Other sugar linkages were de-
nosyl-(1→6)-β-D-glucopyranosyl ester (15).4)
termined based on the consequences of rotating frame nuclear
Akemisaponin A (1) had the molecular formula C53H86O22 Overhauser effect (ROE) difference experiments irradiating
as determined by positive mode high resolution (HR)-FAB-MS each anomeric proton signal. ROEs were observed between δ
1
(m/z 1097.5518 [M+Na]+). The H-NMR spectrum showed sig- 5.20 (H-1 of β-D-xylopyranose) and δ 4.56 (H-3 of α-L-rham-
nals for seven tertiary methyl groups [δ 1.24, 1.21, 1.10, 1.07, nopyranose), δ 5.80 (H-1 of α-L-rhamnopyranose) and δ 4.39
1.03, 0.90×2 (each 3H, s)], an olefinic proton [δ 5.41 (1H, dd, (H-4 of β-D-glucopyranose), and δ 4.93 (H-1 of β-D-glucopyra-
J=3.0, 3.0Hz)], two hydroxymethine protons [δ 4.08 (over- nose) and δ 4.62, 4.29 (H-6 of C-28-linked β-D-glucopyra-
lapped) and 3.37 (1H, d, J=9.0Hz)], and a deshielding me- nose). On the basis of the above evidence, 1 was established
thine proton [δ 3.17 (1H, brd, J=14.0Hz)] with four anomeric to be 2α,3β-dihydroxyolean-12-en-28-oic acid O-β-D-xylopyra-
protons [δ 6.19 (1H, d, J=8.0Hz), 5.80 (1H, brs), 5.20 (1H, d, nosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→4)-O-β-D-glucopyra-
J=8.0Hz), and 4.93 (1H, d, J=8.0Hz)]. Regarding the agly- nosyl-(1→6)-β-D-glucopyranosyl ester.
1
cone moiety of 1, the 13C-NMR and the H-detected heteronu-
The molecular formula of akemisaponin B (2) was deduced
clear multiple-quantum coherency (HMQC) spectra indicated as C52H82O22 from positive mode HR-FAB-MS (m/z 1081.5209
1
seven methyl carbon signals (δ 33.0, 29.2, 25.9, 23.6, 17.4×2, [M+Na]+). The H- and 13C-NMR spectra of the sugar moiety
in 2 were in good agreement with those of 1. However, the
The authors declare no conflict of interest.
NMR signals arising from the ring E part of the aglycone
*To whom correspondence should be addressed. e-mail: warashin@u-shizuoka-ken.ac.jp
© 2012 The Pharmaceutical Society of Japan

October 2012
1265
Table 1. 1H- and 13C-NMR Spectroscopic Data (400, 100MHz) of Compounds 1–11
1
2
Position
δH (J in Hz)
1.27a)
δC
HMBC (H to C)
ROE
δH (J in Hz)
δC
HMBC (H to C)
ROE
1
47.6
1.27a)
47.9
2.24 (dd, 4.0, 12.0)
4.08a)
2, 3, 5, 10
2.24a)
2
3
4
5
6
68.5
83.6
39.6
55.8
18.7
4.26a)
69.8
84.0
39.9
56.1
19.0
3.37 (d, 9.0)
2, 4, 23, 24
3.37 (d, 9.0)
2, 4, 23, 24
24
1.01a)
1.39a)
1.51a)
1.37a)
1.53a)
1.01 (brd, 11.0)
1.55a)
7
33.9
1.34a)
1.51a)
33.0
8
9
39.8
48.0
38.4
23.8
40.3
48.3
38.7
24.1
1.77a)
1.78 (dd, 9.0, 9.0)
1.99a)
8, 10, 26
9, 14, 18
10
11
1.93a)
2.00 (brd, 8.0)
5.41 (dd, 3.0, 3.0)
12, 13
12
13
14
15
122.6
144.0
42.1
9, 14, 18
5.45 (dd, 3.0, 3.0)
123.1
143.7
42.3
1.15a)
2.30a)
2.07a)
28.1
1.10a)
2.32a)
1.88a)
2.02a)
28.7
16
23.2
26.1
17
18
46.9
41.5
45.3
42.4
3.17 (brd, 14.0)
13
3.13 (brdd, 9.0,9.0)
12, 13, 14, 16,
17, 19
19
1.25a)
1.74a)
46.1
1.90a)
2.33a)
36.9
20
21
22
30.6
32.4
33.0
132.7
117.6
36.9
1.12a)
5.22 (brs)
2.22a)
1.76a)
1.91a)
2.52 (brd, 18.0)
1.24 (s)
23
1.24 (s)
1.07 (s)
1.03 (s)
1.10 (s)
1.21 (s)
29.2
17.4
16.8
17.4
25.9
176.3
33.0
23.6
3, 4, 5, 24
3, 4, 5, 23
1, 5, 9, 10
7, 8, 9, 14
8, 13, 14, 15
29.4
17.6
17.0
17.7
27.0
176.2
23.2
3, 4, 5, 24
3, 4, 5, 23
1, 5, 9, 10
7, 8, 9, 14
8, 13, 14, 15
24
1.08 (s)
25
1.04 (s)
26
1.11 (s)
27
1.26 (s)
28
29
0.90 (s)
0.90 (s)
19, 20, 21, 30
19, 20, 21, 29
1.61 (brs)
19, 20, 21
28
30
Glc (at C-28)
1
6.19 (d, 8.0)
4.09 (dd, 8.0, 9.0)
4.17 (dd, 9.0, 9.0)
4.26 (dd, 9.0, 9.0)
4.05a)
95.5
73.7
78.5
70.7
77.8
69.1
28
6.18 (d, 8.0)
95.8
73.9
78.7
71.3
77.9
68.8
2
4.10 (dd, 8.0, 9.0)
4.18 (dd, 9.0, 9.0)
4.26 (dd, 9.0, 9.0)
4.04 (m)
3
4
5
6a
4.29a)
4.28a)
6b
4.62 (brd, 11.0)
4.63 (brd, 11.0)
Glc′ (at C-6 of Glc)
1
4.93 (d, 8.0)
104.7
Glc-6
Glc-6a, 6b
4.91 (d, 8.0)
105.1
Glc-6
Glc-6a, 6b
Glc′-2, 3, 5
Glc′-2, 3, 5
2
3.89 (dd, 8.0, 9.0)
4.12 (dd, 9.0, 9.0)
4.39 (dd, 9.0, 9.0)
3.58 (m)
75.2
76.2
77.4
76.9
61.1
3.91 (dd, 8.0, 9.0)
4.14 (dd, 9.0, 9.0)
4.41 (dd, 9.0, 9.0)
3.59 (m)
75.4
76.6
78.1
77.2
61.6
3
4
5
6a
4.05 (dd, 2.0, 13.0)
4.17 (brd, 13.0)
4.05 (brd, 10.0)
4.16a)
6b
Rha
1
5.80 (brs)
102.2
Glc′-4
Glc′-4, 6a
5.83 (brs)
102.5
Glc′-4
Glc′-4, 6a, 6b
Rha-2, 3, 5
Rha-2
Rha-2, 3, 5
Rha-2
2
4.81 (brs)
71.9
83.0
72.7
69.8
18.2
4.82 (brs)
72.1
83.2
72.9
70.2
18.4
3
4.56 (dd, 3.0, 9.0)
4.45 (dd, 9.0, 9.0)
5.00 (dq, 9.0, 6.0)
1.63 (d, 6.0)
4.58 (dd, 3.0, 9.0)
4.46 (dd, 9.0, 9.0)
5.03 (dq, 9.0, 6.0)
1.64 (d, 6.0)
4
5
6
Xyl
1
5.20 (d, 8.0)
107.0
Rha-3
Rha-3, 4
Xyl-3, 5
5.21 (d, 8.0)
107.1
Rha-3
Rha-3, 4
Xyl-3, 5
2
3
4
5
4.01 (dd, 8.0, 9.0)
4.08 (dd, 9.0, 9.0)
4.12 (m)
75.4
78.1
70.8
67.1
4.02 (dd, 8.0, 9.0)
4.09 (dd, 9.0, 9.0)
4.11 (m)
75.5
78.2
71.1
67.2
3.57 (dd, 10.0, 10.0)
4.23 (dd, 5.0, 10.0)
3.56 (dd, 10.0, 10.0)
4.23 (dd, 5.0, 10.0)

1266
Vol. 60, No. 10
Table 1. 1H- and 13C-NMR Spectroscopic Data (400, 100MHz) of Compounds 1–11
3
4
Position
δH (J in Hz)
δC
HMBC (H to C)
2, 4, 23, 24
ROE
δH (J in Hz)
1.36a)
2.28a)
4.23a)
4.18a)
δC
HMBC (H to C)
ROE
1
1.26a)
47.9
47.8
2.23a)
2
3
4
5
6
4.08a)
68.7
83.9
39.9
56.0
19.0
69.0
78.4
43.7
48.1
18.7
3.37 (d, 9)
0.99a)
1.78 (brd, 12.0)
1.47 (m)
4, 10, 24, 25
7
1.33a)
1.49a)
33.0
1.31a)
1.65a)
32.6
8
9
40.2
48.3
38.7
24.0
40.2
48.3
38.5
24.0
1.77 (dd, 9.0, 9.0)
1.98 (brd, 9.0)
8, 10, 26
12, 13
1.89a)
10
11
1.89a)
2.01a)
12
13
14
15
5.44 (dd, 3.0, 3.0)
123.1
143.6
42.2
9, 14, 18
5.44 (dd, 3.0, 3.0)
123.1
143.7
42.2
9, 11, 14, 18
1.12a)
2.30a)
1.87a)
2.02a)
28.7
1.05a)
2.30a)
1.84a)
2.01a)
28.7
16
26.0
26.0
17
18
19
45.3
42.3
36.9
45.2
42.2
36.9
3.15 (dd, 9.0, 9.0)
1.89a)
2.25a)
12, 13, 14, 16, 19
3.12 (dd, 9.0, 9.0)
1.88a)
2.24a)
14, 16, 17, 19
20
21
22
132.7
117.5
36.8
132.6
117.5
36.7
5.23 (brs)
2.19a)
5.20 (brs)
2.19a)
2.54 (brd, 18.0)
1.24 (s)
2.52 (brd, 18.0)
3.68 (d, 10.0)
4.17a)
23
29.4
3, 4, 5, 24
66.7
3, 4, 5, 24
24
1.08 (s)
1.04 (s)
1.11 (s)
1.25 (s)
17.7
17.0
17.7
27.1
176.3
23.3
3, 4, 5, 23
1, 5, 9, 10
7, 8, 9, 14
8, 13, 14, 15
1.07 (s)
14.3
17.5
17.7
27.0
176.2
23.3
3, 4, 5, 23
1, 5, 9, 10
7, 8, 14
25
1.12 (s)
26
1.14 (s)
27
1.20 (s)
8, 13, 14, 15
28
29
1.62 (brs)
19, 20, 21
28
1.59 (brs)
19, 20, 21
28
30
Glc (at C-28)
1
6.16 (d, 8.0)
95.8
73.9
78.7
71.1
78.0
69.6
6.18 (d, 8.0)
4.09 (dd, 8.0, 9.0)
4.19 (dd, 9.0, 9.0)
4.27 (dd, 9.0, 9.0)
4.16a)
95.8
73.9
78.7
71.1
77.9
69.6
2
4.12 (dd, 8.0, 9.0)
4.18 (dd, 9.0, 9.0)
4.25 (dd, 9.0, 9.0)
4.05 (m)
3
4
5
6a
4.27a)
4.28a)
6b
4.63 (brd, 12.0)
4.63 (brd, 11.0)
Glc′ (at C-6 of Glc)
1
4.94 (d, 8.0)
105.0
Glc-6
Glc-6a, 6b
4.91 (d, 8.0)
105.2
Glc-6
Glc-6a, 6b
Glc′-2, 3, 5
Glc′-2, 3, 5
2
3.91 (dd, 8.0, 9.0)
4.11 (dd, 9.0, 9.0)
4.34 (dd, 9.0, 9.0)
3.65 (m)
75.4
76.6
78.6
77.2
61.5
3.91 (dd, 8.0, 8.0)
4.14 (dd, 8.0, 9.0)
4.42 (dd, 9.0, 9.0)
3.61 (m)
75.5
76.5
77.6
77.2
61.4
3
4
5
6a
4.06 (dd, 3.0, 13.0)
4.19 (brd, 13.0)
4.06 (brd, 10.0)
4.19a)
6b
Rha
1
5.78 (brs)
102.8
Glc′-4
Glc′-4, 6a
5.83 (brs)
102.5
Glc′-4
Glc′-4, 6a, 6b
Rha-2, 3, 5
Rha-2
Rha-2, 3, 5
Rha-2
2
4.62 (brs)
72.6
72.8
74.0
70.4
18.6
4.83 (brs)
72.2
83.4
73.0
70.1
18.5
3
4.49 (dd, 3.0, 9.0)
4.27 (dd, 9.0, 9.0)
4.87 (dq, 9.0, 6.0)
1.66 (d, 6.0)
4.59 (dd, 3.0, 9.0)
4.47 (dd, 9.0, 9.0)
5.03 (dq, 9.0, 6.0)
1.65 (d, 6.0)
4
5
6
Xyl
1
5.21 (d, 8.0)
107.3
Rha-3
Rha-3, 4
Xyl-3, 5
2
3
4
5
4.03 (dd, 8.0, 8.0)
4.10 (dd, 8.0, 9.0)
4.16 (m)
75.7
78.4
71.1
67.3
3.57 (dd, 10.0, 11.0)

October 2012
1267
Table 1. 1H- and 13C-NMR Spectroscopic Data (400, 100MHz) of Compounds 1–11
4.24 (dd, 5.0, 10.0)
5
6
Position
δH (J in Hz)
1.35a)
2.26a)
4.17a)
4.06a)
δC
HMBC (H to C)
ROE
δH (J in Hz)
δC
HMBC (H to C)
ROE
1
47.8
1.38a)
2.29a)
4.17a)
4.03a)
47.7
2
3
4
5
69.0
78.0
43.7
48.1
68.1
77.2
56.6
48.2
1.77 (brd, 12.0)
1.46 (m)
3, 4, 7, 9, 10, 23,
24, 25
1.62a)
6
7
18.7
32.6
1.07a)
1.50a)
1.22a)
1.51a)
20.7
32.2
1.31a)
1.65a)
8
9
40.3
48.3
38.5
24.1
40.2
48.2
38.5
24.0
1.88a)
1.90a)
10
11
1.87a)
1.99a)
1.92a)
2.00a)
12
13
14
15
5.43 (dd, 3.0, 3.0)
123.1
143.7
42.2
11, 14, 18
5.45 (dd, 3.0, 3.0)
122.8
143.7
42.2
9, 11, 14, 18
1.05a)
2.27a)
1.84a)
1.98a)
28.7
1.06a)
2.27a)
1.89a)
2.03a)
28.6
16
26.0
25.9
17
18
45.2
42.2
45.2
42.2
3.12 (dd, 9.0, 9.0)
12, 13, 14, 16, 17,
19, 28
3.14 (dd, 9.0, 9.0)
7, 12, 13, 14, 19
19
1.87a)
2.24a)
36.9
1.91a)
2.26a)
36.8
20
21
22
132.7
117.5
36.8
132.5
117.5
36.7
5.21 (brs)
2.18a)
17, 19, 29
5.23a)
2.21a)
2.53 (brd, 18.0)
3.66 (d, 11.0)
4.15a)
21
2.53 (brd, 18.0)
9.62 (s)
23
66.8
3, 4, 5, 24
206.5
4
24
1.06 (s)
14.4
17.6
17.7
27.1
176.3
23.3
3, 4, 5, 23
1, 5, 9, 10
7, 8, 9, 14
8, 13, 14, 15
1.43 (s)
1.07 (s)
1.09 (s)
1.26 (s)
10.7
17.2
17.5
27.0
176.1
23.3
3, 4, 5, 23
1, 5, 9, 10
7, 8, 9, 14
8, 13, 14, 15
25
1.11 (s)
26
1.13 (s)
27
1.19 (s)
28
29
1.60 (brs)
19, 20, 21
1.62 (brs)
19, 20, 21
28
30
Glc (at C-28)
1
6.16 (d, 8.0)
95.8
28
6.18 (d, 8.0)
95.7
Glc-3
2
4.09 (dd, 8.0, 9.0)
4.18 (dd, 9.0, 9.0)
4.25 (dd, 9.0, 9.0)
4.05a)
73.9
78.4
71.1
78.7
69.6
4.09 (dd, 8.0, 9.0)
4.18 (dd, 9.0, 9.0)
4.28 (dd, 9.0, 9.0)
4.03 (m)
73.9
78.7
71.0
77.9
69.6
3
4
5
6a
4.27a)
4.25a)
6b
4.63 (brd, 11.0)
4.64 (brd, 10.0)
Glc′ (at C-6 of Glc)
1
4.94 (d, 8.0)
105.0
Glc-6
Glc-6a, 6b
4.91 (d, 8.0)
105.2
Glc-6
Glc-6a
Glc′-2, 5
Glc′-2, 3, 5
Glc′-3, 5
2
3.91 (dd, 8.0, 9.0)
4.12 (dd, 9.0, 9.0)
4.34 (dd, 9.0, 9.0)
3.65 (m)
75.4
76.6
78.6
77.2
61.5
3.92 (dd, 8.0, 9.0)
4.14 (dd, 9.0, 9.0)
4.43 (dd, 9.0, 9.0)
3.61 (m)
75.4
76.5
77.5
77.2
61.4
3
4
5
6a
4.06 (dd, 2.0, 13.0)
4.20 (brd, 13.0)
4.07 (dd, 2.0, 13.0)
4.19 (brd, 13.0)
6b
Rha
1
5.77 (brs)
102.8
Glc′-4
Glc′-4, 6a, 6b
5.84 (brs)
102.4
Glc′-4
Glc′-4, 6a
Rha-2, 3, 5
Rha-2
Rha-2, 3, 5
Rha-2
2
4.61 (brs)
72.6
72.8
74.0
70.4
18.6
4.84 (brs)
72.1
83.4
73.0
70.1
18.4
3
4.49 (dd, 2.0, 9.0)
4.27 (dd, 9.0, 9.0)
4.87 (dq, 9.0, 6.0)
1.66 (d, 6.0)
4.59 (dd, 3.0, 9.0)
4.47 (dd, 9.0, 9.0)
5.04 (dq, 9.0, 6.0)
1.65 (d, 6.0)
4
5
6
Xyl
1
5.22 (d, 8.0)
107.3
Rha-3
Rha-3, 4
Xyl-3, 5
2
3
4
5
4.03 (dd, 9.0, 9.0)
4.09 (dd, 9.0, 9.0)
4.17 (m)
75.7
78.4
71.1
67.3
3.57 (dd, 9.0, 11.0)

1268
Vol. 60, No. 10
Table 1. 1H- and 13C-NMR Spectroscopic Data (400, 100MHz) of Compounds 1–11
4.24 (dd, 5.0, 11.0)
7
8
Position
δH (J in Hz)
δC
HMBC (H to C)
25
ROE
δH (J in Hz)
δC
HMBC (H to C)
2, 3, 10, 25
ROE
1
1.37 (dd, 12.0, 12.0)
2.27a)
4.21a)
47.7
1.30 (brdd, 12.0,12.0)
2.29a)
4.05a)
48.2
2
3
4
5
6
68.2
77.2
56.6
48.2
20.8
68.8
84.0
39.8
56.2
18.9
4.01 (d, 9.0)
2, 4, 23
3.37 (d, 9.0)
2, 4, 23, 24
1.60a)
1.02a)
1.39a)
1.04a)
1.48a)
7
1.20 (brd, 10.0)
1.49a)
32.3
9, 26
1.39a)
1.53a)
33.9
8
9
40.2
48.2
38.5
24.0
39.8
48.4
38.7
24.0
1.87a)
1.75a)
10
11
1.89a)
1.99a)
1.92a)
1.97a)
5.55 (dd, 3.0, 3.0)b)
12
13
14
15
5.44 (dd, 3.0, 3.0)
122.8
143.7
42.2
9, 14, 18
11, 14, 18
143.0
43.3
28.1
1.06a)
2.26a)
1.85a)
2.00a)
28.6
1.19a)
2.28a)
1.75a)
2.00a)
16
25.9
26.4
17
18
19
45.2
42.2
36.9
46.1
46.0
3.13 (dd, 9.0, 9.0)
1.90a)
2.25a)
12, 13, 17, 19
3.65 (brs)
5.23a)
19, 28
18
129.5
20
21
22
132.6
116.3
36.7
130.0
23.2
32.7
5.23 (brs)
2.19a)
1.77a)
2.53 (brd, 18.0)
9.61 (s)
2.02a)
23
206.5
10.8
17.2
17.6
27.1
176.2
23.3
4, 24
1.24 (s)
1.07 (s)
1.04 (s)
1.12 (s)
1.16 (s)
29.4
17.6
17.3
17.9
23.6
176.3
23.1
3, 4, 5, 24
3, 4, 5, 23
1, 5, 9, 10
7, 8, 9, 14
8, 13, 14, 15
24
1.41 (s)
3, 4, 5, 23
1, 5, 9, 10
7, 8, 9, 14
8, 13, 14, 15
25
1.06 (s)
26
1.07 (s)
27
1.25 (s)
28
29
1.63 (brs)
19, 20, 21
28
1.60 (brs)
19, 20, 21
28
30
Glc (at C-28)
1
6.16 (d, 8.0)
4.09 (dd, 8.0, 9.0)
4.18 (dd, 9.0, 9.0)
4.26 (dd, 9.0, 9.0)
4.05a)
95.8
73.9
78.7
71.0
78.0
69.5
6.24 (d, 8.0)
95.9
74.1
78.7
71.3
78.0
69.8
2
4.09 (dd, 8.0, 9.0)
4.19 (dd, 9.0, 9.0)
4.29 (dd, 9.0, 9.0)
4.03 (m)
3
4
5
6a
6b
4.30a)
4.26a)
4.63 (brd, 11.0)
4.63 (brd, 11.0)
Glc′ (at C-6 of
Glc)
1
4.94 (d, 8.0)
105.0
Glc-6
Glc-6a, 6b
4.91 (d, 8.0)
105.1
Glc-6
Glc-6a, 6b
Glc′-2, 3, 5
Glc′-5
Glc′-2, 3, 5
2
3.91 (dd, 8.0, 9.0)
4.12 (dd, 9.0, 9.0)
4.35 (dd, 9.0, 9.0)
3.65 (m)
75.3
76.6
78.5
77.2
61.5
3.92 (dd, 8.0, 9.0)
4.15 (dd, 9.0, 9.0)
4.44 (dd, 9.0, 9.0)
3.61 (m)
75.4
76.6
77.9
77.2
61.6
3
4
5
6a
4.07 (dd, 2.0, 12.0)
4.20 (brd, 12.0)
4.04 (brd, 11.0)
4.18a)
6b
Rha
1
5.79 (brs)
102.8
Glc′-4
Glc′-4, 6a
5.82 (brs)
102.5
Glc′-4
Glc′-4, 6a, 6b
Rha-2, 3, 5
Rha-2
Rha-2, 3, 5
Rha-2
2
4.63 (brs)
72.6
72.8
74.0
70.4
18.6
4.85 (brs)
72.1
83.3
72.9
70.1
18.4
3
4.50 (dd, 2.0, 9.0)
4.28 (dd, 9.0, 9.0)
4.88 (dq, 9.0, 6.0)
1.67 (d, 6.0)
4.60 (dd, 3.0, 9.0)
4.48 (dd, 9.0, 9.0)
5.05 (dq, 9.0, 6.0)
1.65 (d, 6.0)
4
5
6
Xyl
1
5.22 (d, 8.0)
107.1
Rha-3
Rha-3, 4
Xyl-3, 5
2
3
4
5
4.03 (dd, 9.0, 9.0)
4.10 (dd, 9.0, 9.0)
4.15 (m)
75.5
78.2
71.1
67.2
3.57 (dd, 10.0, 10.0)

October 2012
1269
Table 1. 1H- and 13C-NMR Spectroscopic Data (400, 100MHz) of Compounds 1–11
4.24 (dd, 5.0, 10.0)
9
10
Position
δH (J in Hz)
δC
HMBC (H to C)
2, 4, 23, 24
ROE
δH (J in Hz)
δC
HMBC (H to C)
2, 4, 23, 24
ROE
1
1.25a)
47.9
1.25a)
47.9
2.22a)
2.22a)
2
3
4
5
6
4.08a)
68.7
83.9
39.9
56.0
19.0
4.08a)
68.7
83.9
39.9
56.0
19.0
3.38 (d, 9.0)
3.38 (d, 9.0)
1.00a)
1.39a)
1.00a)
1.38a)
7
1.36a)
33.2
1.35a)
33.2
1.52 (m)
6, 8
1.50 (m)
6, 8
8
9
40.1
48.2
38.6
24.0
40.1
48.2
38.6
24.0
1.74a)
1.73 (dd, .9.0, 9.0)
1.95 (brd, 9.0)
8, 10, 26
12, 13
10
11
1.73a)
1.96a)
5.43 (dd, 3.0, 3.0)b)
12
13
14
15
9, 14, 18
5.42 (dd, 3.0, 3.0)b)
9, 14, 18
143.6
42.3
28.3
143.6
42.2
28.3
1.17a)
2.30a)
2.04a)
2.18a)
1.17a)
2.29 (brdd, 13.0, 13.0)
16
23.6
2.04a)
2.18a)
23.6
17
18
19
47.4
47.7
41.8
47.5
47.6
41.8
3.10 (dd, 5.0, 13.0)
2.19a)
14, 16, 17
18, 20, 29
3.10 (dd, 4.0, 8.0)
2.19a)
14, 17
2.57 (dd, 13.0, 13.0)
2.57 (dd, 13.0, 13.0)
18, 20, 29
20
21
148.5
30.2
148.4
30.2
2.08a)
2.07a)
2.20a)
2.20a)
22
1.72a)
2.03a)
37.7
1.73a)
2.03a)
37.7
23
24
25
26
27
28
29
1.25 (s)
1.07 (s)
1.02 (s)
1.09 (s)
1.19 (s)
29.4
17.7
3, 4, 5, 24
3, 4, 5, 23
1, 5, 9, 10
7, 8, 9, 14
8, 13, 14, 15
1.25 (s)
1.07 (s)
1.02 (s)
1.08 (s)
1.19 (s)
29.4
17.7
3, 4, 5, 24
3, 4, 5, 23
1, 5, 9, 10
7, 8, 9, 14
8, 13, 14, 15
17.1
17.1
17.7
17.7
26.1
26.1
175.9
107.4
175.9
107.5
4.68 (brs)
4.74 (brs)
19, 21
19, 21
4.68 (brs)
4.74 (brs)
19, 21
19, 21
30
Glc (at C-28)
1
6.16 (d, 8.0)
95.8
73.9
78.7
71.0
78.0
69.5
28
6.15 (d, 8.0)
95.8
73.9
78.7
71.0
78.0
69.5
28
2
4.08 (dd, 8.0, 9.0)
4.17 (dd, 9.0, 9.0)
4.26 (dd, 9.0, 9.0)
4.04 (m)
4.08 (dd, 8.0, 9.0)
4.17 (dd, 9.0, 9.0)
4.25 (dd, 9.0, 9.0)
4.03 (m)
3
4
5
6a
4.27a)
4.28a)
6b
4.61 (brd, 11.0)
4.62 (brd, 11.0)
Glc′ (at C-6 of Glc)
1
4.92 (d, 8.0)
105.0
Glc-6
Glc-6a, 6b
4.94 (d, 8.0)
104.9
Glc-6
Glc-6a, 6b
Glc′-3, 5
Glc′-2, 3, 5
2
3.90 (dd, 8.0, 9.0)
4.13 (dd, 9.0, 9.0)
4.41 (dd, 9.0, 9.0)
3.59 (brd, 9.0)
75.5
76.5
77.5
77.2
61.4
3.91 (dd, 8.0, 9.0)
4.12 (dd, 9.0, 9.0)
4.35 (dd, 9.0, 9.0)
3.65 (m)
75.3
76.6
78.5
77.2
61.5
3
4
5
6a
4.12 (dd, 2.0, 13.0)
4.38 (brd, 13.0)
4.07 (dd, 2.0, 12.0)
4.20 (brd, 12.0)
6b
Rha
1
5.82 (brs)
102.4
Glc′-4
Glc′-4, 6a, 6b
5.79 (brs)
102.8
Glc′-4
Glc′-4, 6a, 6b
Rha-2, 3, 5
Rha-2
Rha-2, 3, 5
Rha-2
2
4.83 (brs)
72.2
83.4
73.0
70.1
18.5
4.63 (brs)
72.6
72.8
74.0
70.4
18.5
3
4.49 (dd, 2.0, 9.0)
4.46 (dd, 9.0, 9.0)
5.01 (dq, 9.0, 6.0)
1.64 (d, 6.0)
4.51 (dd, 2.0, 9.0)
4.29 (dd, 9.0, 9.0)
4.89 (dq, 9.0, 6.0)
1.67 (d, 6.0)
4
5
6
Xyl
1
5.22 (d, 8.0)
107.3
Rha-3
Rha-3, 4
Xyl-3, 5
2
3
4
5
4.03 (dd, 8.0, 9.0)
4.01 (dd, 9.0, 9.0)
4.12 (m)
75.7
78.4
71.1
67.3
3.57 (dd, 10.0, 10.0)

1270
Vol. 60, No. 10
Table 1. 1H- and 13C-NMR Spectroscopic Data (400, 100MHz) of Compounds 1–11
4.24 (dd, 5.0, 10.0)
11
Position
δH (J in Hz)
1.35a)
δC
HMBC (H to C)
2, 5, 10, 25
ROE
1
47.7
2.27a)
4.21a)
4.05a)
2
3
4
5
6
68.1
77.2
56.6
48.2
20.7
1.61 (brd, 10.0)
1.07a)
4, 6, 7, 10, 24, 25
1.49a)
7
1.21a)
32.5
1.50a)
8
9
40.1
48.1
38.5
23.9
1.85 (brd, 8.0)
10
11
1.84a)
1.94 (brd, 8.0)
5.42 (dd, 3.0, 3.0)
9, 12, 13
9, 11, 14
12
13
14
15
123.1
143.6
42.3
1.05a)
2.27a)
2.05a)
2.20a)
28.2
16
23.6
17
18
47.4
47.6
3.10 (dd, 4.0, 13.0)
12, 13, 14, 16,
17, 28
19
2.20a)
41.7
2.56 (dd, 13.0, 13.0)
18, 20, 29
20
21
148.4
30.2
2.08a)
2.23a)
22
1.74 (dd, 4.0, 13.0)
2.03a)
37.7
16, 17, 28
23
24
25
26
27
28
29
9.64 (s)
1.42 (s)
1.05 (s)
1.05 (s)
1.18 (s)
206.5
10.8
4, 24
3, 4, 5, 23
1, 5, 9, 10
7, 9, 14
17.2
17.6
26.1
8, 13, 14, 15
175.8
107.5
4.69 (brs)
4.75 (brs)
19, 21
19, 21
30
Glc (at C-28)
1
6.17 (d, 8.0)
95.8
73.9
78.7
71.0
78.0
69.4
28, Glc-5
2
4.08 (dd, 8.0, 9.0)
4.16 (dd, 9.0, 9.0)
4.25 (dd, 9.0, 9.0)
4.05 (m)
3
4
5
6a
6b
4.29a)
4.62 (brd, 12.0)
Glc′ (at C-6 of
Glc)
1
4.95 (d, 8.0)
104.9
Glc-6
Glc-6a, 6b
Glc′-5
Glc′-2, 3, 5
2
3.92 (dd, 8.0, 8.0)
4.12 (dd, 8.0, 9.0)
4.36 (dd, 9.0, 9.0)
3.66 (m)
75.3
76.6
78.5
77.2
61.5
3
4
5
6a
4.07a)
6b
4.21 (brd, 11.0)
Rha
1
5.80 (brs)
102.8
Glc′-4
Glc′-4, 6a, 6b
Rha-2, 3, 5
Rha-2
2
4.64 (brs)
72.6
72.8
74.0
70.4
18.5
3
4.52 (dd, 3.0, 9.0)
4.29 (dd, 9.0, 9.0)
4.91 (dq, 9.0, 6.0)
1.68 (d, 6.0)
4
5
6
Xyl
1
2
3
4
5
Sugar proton signals were assigned from the HOHAHA difference spectra. a) Overlapped with other signals in each column. b) Overlapped with pyridine-d5 signals at δ 123.

October 2012
1271
Chart 1. Structure of Compounds 1–15
were different from those of 1; the first difference was the related to C-28 of the aglycone. The deduced molecular for-
lack of a tertiary methyl signal, and the second, the pres- mula was less than that of 2 by C5H8O4, corresponding to the
ence of a trisubstituted olefinic group [δ 5.22 (1H, brs) and absence of a xylopyranosyl unit. Therefore, 3 was determined
δ 117.6, 132.7] other than that of 12-en and a methyl group as 2α,3β-dihydroxy-30-norolean-12,20(21)-dien-28-oic acid
on a double bond [δ 1.61 (3H, brs) and δ 23.2]. In the HMBC O-α-L-rhamnopyranosyl-(1→4)-O-β-D-glucopyranosyl-(1→
measurements, this methyl group (δ 1.61) showed long-range 6)-β-D-glucopyranosyl ester. This sugar sequence was sup-
correlations with a methylene carbon (δ 36.9) as well as the ported by the ROE difference experiments irradiating each
2
olefinic carbons (δ 132.7, 117.6). In addition, a JHCC between anomeric proton and HMBC measurements.
the deshielding methine proton (δ 3.13, H-18) and the above
Akemisaponins D (4) and F (6) were found to have the
methylene carbon (δ 36.9) suggested that this methylene car- molecular formula C52H82O23 and C52H80O23, respectively,
bon was assigned to C-19. Thus, the aglycone moiety of 2 was by positive HR-FAB-MS (m/z 1097.5139 [M+Na]+ and m/z
considered to possess the trisubstituted double bond between 1095.4987 [M+Na]+). When the 13C-NMR spectra of 4 and 6
C-20 and C-21, and the methyl group at the C-29 position. were compared with that of 2, the data for the sugar moiety
These findings were quite consistent with those of 2α,3β-dihy- and a majority of the aglycone were consistent. Moreover, the
droxy-30-norolean-12,20(21)-dien-28-oic acid.4) Accordingly, 2 molecular formula of 4 had one more oxygen atom than that
1
was determined as 2α,3β-dihydroxy-30-norolean-12,20(21)-di- of 2. The H- and 13C-NMR spectral data for 4 showed two
en-28-oic acid O-β-D-xylopyranosyl-(1→3)-O-α-L-rhamnopyra- hydroxymethyl proton signals at δ 3.68 (1H, d, J=10.0Hz)
nosyl-(1→4)-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl and δ 4.17 (overlapped), and the corresponding hydroxymethyl
ester.
carbon signal at δ 66.7. The above hydroxymethyl proton (δ
The molecular formula of akemisaponin C (3) was re- 3.68) and the methyl protons (δ 1.07) exhibited long-range
vealed to be C47H74O18 by positive HR-FAB-MS (m/z 949.4765 correlations to C-3 (δ 78.4), C-4 (δ 43.7), and C-5 (δ 48.1) in
1
[M+Na]+). The H- and 13C-NMR spectra showed that 3 had the HMBC measurement, suggesting that these hydroxymethyl
the same aglycone, 2α,3β-dihydroxy-30-norolean-12,20(21)- and methyl groups were assigned to C-23 and C-24. Because
dien-28-oic acid, as 2, but differed in the oligosaccharide part the 13C-NMR spectral data around the ring A part of 4 were

1272
Vol. 60, No. 10
in good agreement with those of 126) and 13,6) but were differ- 6)-β-D-glucopyranosyl ester.
ent from those of scheffoleoside F,6) the hydroxymethyl group
The molecular formula of akemisaponin J (10) was deter-
and methyl group were assigned to C-23 and C-24, respec- mined by positive HR-FAB-MS (m/z 949.4748 [M+Na]+) to
1
tively. Thus, 4 was elucidated to be 2α,3β,23-trihydroxy-30- be C47H74O18, which was the same as that of 3. The H- and
norolean-12,20(21)-dien-28-oic acid O-β-D-xylopyranosyl-(1→ 13C-NMR spectra showed that 10 had the same aglycone,
3)-O-α-L-rhamnopyranosyl-(1→4)-O-β-D-glucopyranosyl-(1→ 2α,3β-dihydroxy-30-norolean-12,20(29)-dien-28-oic acid, as
6)-β-D-glucopyranosyl ester. For 6, the 1H- and 13C-NMR 9, and the sugar moiety was consistent with those of 3. Thus,
spectral data suggested an aldehyde proton signal at δ 9.62 10 was identified as 2α,3β-dihydroxy-30-norolean-12,20(29)-
(1H, s), and the corresponding aldehyde carbon signal at δ dien-28-oic acid O-α-L-rhamnopyranosyl-(1→4)-O-β-D-glu-
206.5. These signals were consistent with those of scheffur- copyranosyl-(1→6)-β-D-glucopyranosyl ester.
soside B6) and scheffoleoside B.6) Accordingly, this aldehyde
Akemisaponin K (11) was shown to have the molecular
group was also assignable to C-23, and 6 was identified formula C47H72O19 by positive HR-FAB-MS (m/z 963.4584
as 2α,3β-dihydroxy-23-oxo-30-norolean-12,20(21)-dien-28-oic [M+Na]+). All the spectral properties of 11 showed a close
acid O-β-D-xylopyranosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→ similarity to those of 10. On comparison of the 1H- and
4)-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester.
13C-NMR spectra of 11 with those of 10, the C-23 terti-
Akemisaponins E (5) and G (7) were shown to have the mo- ary methyl group observed in 10 at δ 1.25 and δ 29.4 were
lecular formula C47H74O19 and C47H72O19, respectively, on the replaced by signals assignable to an aldehyde group at δ
basis of positive HR-FAB-MS (m/z 965.4739 [M+Na]+ and m/z 9.64 and δ 206.5 in 11. Thus, the structure of 11 was char-
1
963.4542 [M+Na]+). The consistency of the H- and 13C-NMR acterized as 2α,3β-dihydroxy-23-oxo-30-norolean-12,20(29)-
spectral data due to the aglycone moieties of 5 and 7 with dien-28-oic acid O-α-L-rhamnopyranosyl-(1→4)-O-β-D-glu-
those of 4 and 6 suggested that 5 and 7 possessed 2α,3β,23-tri- copyranosyl-(1→6)-β-D-glucopyranosyl ester.
hydroxy-30-norolean-12,20(21)-dien-28-oic acid and 2α,3β-
We now report the isolation and structural elucidation of
dihydroxy-23-oxo-30-norolean-12,20(21)-dien-28-oic acid as eleven new triterpene saponins from the pericarps of A. trifo-
each aglycone. Moreover, the sugar sequences in 5 and 7 were liata. The stems of A. trifoliata afforded several bisdesmoside-
found to be the same as in 3 on comparison of NMR spectral triterpene saponins possessing sugar sequences at the C-3
data. Hence, the structures of 5 and 7 were established as and C-28 positions together with monodesmosides.4,5) In spite
shown in Chart 1.
of this detailed investigation, the pericarps of A. trifoliate
Akemisaponin H (8) was proposed to have the molecular yielded only monodesmoside-tritepene saponins having sugars
formula C52H82O22 on the basis of positive mode HR-FAB-MS at C-28. Bisdesmosides were not obtained. However, among
(m/z 1081.5189 [M+Na]+), which was the same as that of 2. the saponins in the stems and pericarps of A. trifoliata, the
The compound 8 also exhibited a trisubstituted double bond [δ sugar sequences at C-28 were consistent. Of the 15 triterpene
5.23 (overlapped) and δ 129.5, 130.0] and a methyl group on a saponins from the pericarps of A. trifoliate, six belonged to
double bond [δ 1.60 (3H, brs) and δ 23.1] the same as 2 in its the 30-norolean-20(21)-en type triterpene saponins. This type
NMR spectra. Moreover, H-18, which was observed at δ 3.13 of triterpene saponins is known to exist the stems of A. trifo-
(1H, brdd, J=9.0, 9.0Hz) in 2, was displayed as a broad sin- liata,4) but few 30-norolean-20(21)-en type triterpene saponins
glet signal at δ 3.65 in 8. H-18 showed a long-range correlation are known from other plants.11) Thus, this type-triterpene
to the above sp2 methine carbon (δ 129.5), together with C-28 saponin may be characteristic of A. trifoliate. 30-Norole-
(δ 176.3) in the HMBC measurements, suggesting that 8 had an-19-en type triterpene saponins had not been obtained from
a part of 19-en. Therefore, the aglycone of 8 was determined any other plants before, and this is the first report. 30-No-
as 2α,3β-dihydroxy-30-norolean-12,19-dien-28-oic acid. The rolean-20(29)-en type triterpene saponins were shown in the
structure of 8 is shown in Chart 1.
Araliaceaus12) and Eupteleaceaus families,13) together with the
The molecular formula of akemisaponin I (9) was deduced Lardizabalaceaus family.4,5,14–16)
as C52H82O22 from positive mode HR-FAB-MS (m/z 1081.5195
1
[M+Na]+). Regarding the aglycone moiety of 9, the H-NMR
Experimental
General Procedures Optical rotations were measured
spectrum revealed signals for five tertiary methyl groups [δ
1
1.25, 1.19, 1.09, 1.07, and 1.02 (each 3H, s)], an olefinic proton on a JASCO P-2200 digital polarimeter. H- (400MHz) and
[δ 5.43 (1H, dd, J=3.0, 3.0Hz)], and two characteristic ex- 13C- (100MHz) NMR spectra were recorded on a JEOL
omethylene protons [δ 4.74 (1H, brs) and 4.68 (1H, brs)]. The α-400 FT-NMR spectrometer, and chemical shifts are given
13C-NMR spectrum also showed signals for the exomethylene as δ values with tetramethylsilane (TMS) as the internal stan-
group at δ 107.4 and 148.5. The long-range correlations from dard at 35°C in pyridine-d5. Inverse-detected heteronuclear
these exomethylene protons to C-19 (δ 41.8) and C-21 (δ 30.2) correlations were measured using HMQC (optimized for
n
in the HMBC measurement indicated that this exomethylene 1JC–H=145Hz) and HMBC (optimized for JC–H=8Hz) pulse
group was assigned to C-29. Moreover, the 13C-NMR spectral sequences with a pulse field gradient. HR-FAB-MS data were
data due to the aglycone moiety of 9 were superimposable on obtained on a JEOL JMS 700 mass spectrometer in the posi-
those of 2α,3β-dihydroxy-30-norolean-12,20(29)-dien-28-oic tive mode using m-nitrobenzyl alcohol as the matrix. Prepara-
acid4) except for the C-28 position. The 1H- and 13C-NMR tive HPLC was performed on a JASCO 800. GC was run on a
spectral data due to the sugar moiety were in good agreement Hitachi G-3000 gas chromatograph.
with those of 1, suggesting that 9 possessed the same sugar
Plant Material The pericarps of A. trifoliata were col-
sequence as 1. Hence, 9 was assigned as 2α,3β-dihydroxy-30- lected in the medicinal plant garden of the University of
norolean-12,20(29)-dien-28-oic acid O-β-D-xylopyranosyl-(1→ Shizuoka, Shizuoka, Japan, in November 2008. The plant was
3)-O-α-L-rhamnopyranosyl-(1→4)-O-β-D-glucopyranosyl-(1→ identified by Dr. Toshio Miyase, a professor at the university.

October 2012
1273
A voucher specimen (No. 20081125) was deposited at the uni- 1095.4987 [M+Na]+ (Calcd for C52H80O23Na: 1095.4989).
versity’s herbarium.
Akemisaponin G (7): Amorphous solid. [α]D22 −16 (c=0.90,
Extraction and Isolation The dried pericarps of A. tri- pyridine). 1H- and 13C-NMR: Table 1. HR-FAB-MS m/z
foliata (dried weight 1.0kg) were extracted with hot MeOH 963.4542 [M+Na]+ (Calcd for C47H72O19Na: 963.4567).
twice. The extract was concentrated under reduced pressure,
Akemisaponin H (8): Amorphous solid. [α]D22 −48 (c=1.56,
and the dark brown concentrate (417g) was suspended in hot pyridine). 1H- and 13C-NMR: Table 1. HR-FAB-MS m/z
water and extracted with ether continuously for 50h. After the 1081.5189 [M+Na]+ (Calcd for C52H82O22Na: 1081.5197).
ether layer was removed and concentrated (concentrate weight
Akemisaponin I (9): Amorphous solid. [α]D22 +1.0 (c=1.03,
33g), the water layer was extracted with ethyl acetate continu- pyridine). 1H- and 13C-NMR: Table 1. HR-FAB-MS m/z
ously for 120h. The ethyl acetate layer was removed and con- 1081.5195 [M+Na]+ (Calcd for C52H82O22Na: 1081.5197).
centrated (concentrate weight 43g), and the water layer was
Akemisaponin J (10): Amorphous solid. [α]D22 +14 (c=1.02,
fractionated on a Mitsubishi Diaion HP-20 column (9×30cm), pyridine). 1H- and 13C-NMR: Table 1. HR-FAB-MS m/z
eluting with water (10L), MeOH–H2O (1:1, v/v) (5L) and 949.4748 [M+Na]+ (Calcd for C47H74O18Na: 949.4774).
MeOH (5L). The fraction eluted with MeOH was concentrated
Akemisaponin K (11): Amorphous solid. [α]D22 +24 (c=1.04,
under reduced pressure to give a brown concentrate (30g). pyridine). 1H- and 13C-NMR: Table 1. HR-FAB-MS m/z
Four grams of the MeOH eluate was subjected to preparative 963.4584 [M+Na]+ (Calcd for C47H72O19Na: 963.4567).
HPLC [column, Tosoh TSKgel ODS-80Ts 5.5×180cm; sol-
Acid Hydrolysis and GC Analysis Compounds 1–11
vent, CH3CN–H2O (75:25–65:35, v/v) linear gradient, UV (ca. 1mg) were dissolved in dioxane (200µL) and 1M HCl
205nm] to give 16 fractions (Frs. A–P). The 74:26 solvent (200µL), respectively. The solution was heated at 100°C for
gave Fr. E [akemisaponin E (5) (670.3mg)], the 73:27 sol- 1h. After hydrolysis, this reaction mixture was diluted with
vent gave Fr. G [2α,3β,23-trihydroxyolean-12-en-28-oic acid H2O and extracted with EtOAc. The H2O layer was neutral-
O-β-D-xylopyranosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→4)-O- ized with an Amberlite IRA-60E column, and the eluate was
β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (15)4) concentrated dry. The residue was stirred with D-cysteine
(727.2mg)], the 72:28 solvent gave Fr. H [scheffoleoside A methyl ester hydrochloride (3mg) in pyridine (25µL) at 60°C
(12)4,6) (202.5mg)] and Fr. I [asiaticoside (13)4,6,7) (116mg)], for 1.5h. After warming, the trimethylsilylation reagent (hex-
the 70:30 solvent gave Fr. K [akemisaponin G (7) (84.3mg)], amethyldisilazane and trimethylsilyl chloride) was added,
the 68:32 solvent gave Fr. L [akemisaponin I (9) (23mg)], the and the warming at 60°C was continued for another 30min.
67:33 solvent gave Fr. N [akemisaponin J (10) (44.5mg)], the The precipitates were centrifuged, and the supernatant was
66:34 solvent gave Fr. O [akemisaponin C (3) (54.8mg)], and subjected to GC analysis. GC conditions: column, GL Sci-
the 65:35 solvent gave Fr. P [akemisaponin A (1) (243.9mg)]. ences capillary column TC-1 0.25mm×30m; carrier gas, N2;
Then, Fr. M (83.7mg) was subjected to semipreparative HPLC column temperature 230°C; tR 18.7min (D-glucose), 18.0min
[column, Develosil C30-UG-5, 2×25cm; solvent, CH3CN– (L-glucose), 10.9min (D-xylose), 10.3min (L-xylose), 12.6min
H2O (70:30, v/v), UV 205nm] to give akemisaponin B (2) (L-rhamnose), 12.3min (D-rhamnose). D-Glucose and L-rham-
(44.2mg) and akemisaponin H (8) (15.1mg). A part (150mg) nose were detected in all compounds. D-Xylose was found in
of Fr. C (408mg) was subjected to semipreparative HPLC 1, 2, 4, 6, 8, and 9. Retention times for L-glucose, L-xylose and
[column, Develosil C30-UG-5, 2×25cm; solvent, CH3CN– D-rhamnose were obtained from their enantiomer made by
H2O (75:25, v/v), UV 205nm] to give akemisaponin D using L-cysteine methyl ester hydrochloride.
(4) (33.6mg) and 2α,3β,23-trihydroxy-30-norolean-12,20(29)-
dien-28-oic acid O-α-L-rhamnopyranosyl-(1→4)-O-β-D-glu-
copyranosyl-(1→6)-β-D-glucopyranosyl ester (14)4) (71.2mg).
Fraction J (70.2mg) was subjected to semipreparative HPLC
[column, Cosmosil 5PE-MS, 2×25cm; solvent, CH3CN–H2O
(75:25, v/v), UV 205nm] to give akemisaponin F (6) (20mg)
and akemisaponin K (11) (38.1mg).
References
1) Ikuta A., Itokawa H., Phytochemistry, 27, 3809–3810 (1988).
2) Higuchi R., Kawasaki T., Chem. Pharm. Bull., 24, 1021–1032
(1976).
3) Jiang D., Gao Q. P., Shi S. P., Tu P. F., Chem. Pharm. Bull., 54,
595–597 (2006).
4) Mimaki Y., Kuroda M., Yokosuka A., Harada H., Fukushima M.,
Sashida Y., Chem. Pharm. Bull., 51, 960–965 (2003).
5) Gao H., Wang Z., Phytochemistry, 67, 2697–2705 (2006).
6) Maeda C., Ohtani K., Kasai R., Yamasaki K., Duc N. M., Nham N.
T., Cu N. K. Q., Phytochemistry, 37, 1131–1137 (1994).
7) Mahato S. B., Sahu N. P., Luger P., Müller E., J. Chem. Soc., Perkin
Trans. 2, 1987, 1509–1515 (1987).
8) Singh B., Singh V. P., Pandey V. B., Rücher G., Planta Med., 61,
576–577 (1995).
9) Hara S., Okabe H., Mihashi K., Chem. Pharm. Bull., 34, 1843–1845
(1986).
10) Kasai R., Okihara M., Asakawa J., Mizutani K., Tanaka O., Tetra-
hedron, 35, 1427–1432 (1979).
11) Gao H., Wang Z., Yao Z.-H., Wu N., Dong H.-J., Li J., Wang N.-L.,
Ye W.-C., Yao X.-S., Helv. Chim. Acta, 91, 451–459 (2008).
12) Jiang W., Li W., Han L., Liu L., Zhang Q., Zhang S., Nikaido T.,
Koike K., J. Nat. Prod., 69, 1577–1581 (2006).
Akemisaponin A (1): Amorphous solid. [α]D22 −17 (c=1.09,
pyridine). 1H- and 13C-NMR: Table 1. HR-FAB-MS m/z
1097.5518 [M+Na]+ (Calcd for C53H86O22Na: 1097.5510).
Akemisaponin B (2): Amorphous solid. [α]D22 −35 (c=0.98,
pyridine). 1H- and 13C-NMR: Table 1. HR-FAB-MS m/z
1081.5209 [M+Na]+ (Calcd for C52H82O22Na: 1081.5197).
Akemisaponin C (3): Amorphous solid. [α]D22 −29 (c=1.07,
pyridine). 1H- and 13C-NMR: Table 1. HR-FAB-MS m/z
949.4765 [M+Na]+ (Calcd for C47H74O18Na: 949.4774).
Akemisaponin D (4): Amorphous solid. [α]D22 −33 (c=0.98,
pyridine). 1H- and 13C-NMR: Table 1. HR-FAB-MS m/z
1097.5139 [M+Na]+ (Calcd for C52H82O23Na: 1097.5146).
Akemisaponin E (5): Amorphous solid. [α]D22 −32 (c=1.01,
pyridine). 1H- and 13C-NMR: Table 1. HR-FAB-MS m/z
965.4739 [M+Na]+ (Calcd for C47H74O19Na: 965.4723).
Akemisaponin F (6): Amorphous solid. [α]D22 −25 (c=1.84, 13) Murakami T., Oominami H., Matsuda H., Yoshikawa M., Chem.
pyridine). 1H- and 13C-NMR: Table 1. HR-FAB-MS m/z
Pharm. Bull., 49, 741–746 (2001).

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Vol. 60, No. 10
14) Wang H.-B., Yu D.-Q., Liang X.-T., Watanabe N., Tamai M., Omura
S., Planta Med., 55, 303–306 (1989).
15) Gao H., Wang Z., Yang L., Yu Y., Yao Z.-H., Wang N.-L., Zhou
G.-X., Ye W.-C., Yao X.-S., Magn. Reson. Chem., 46, 630–637
(2008).
16) Gao H., Zhao F., Chen G.-D., Chen S.-D., Yu Y., Yao Z.-H., Lau
B. W. C., Wang Z., Li J., Yao X.-S., Phytochemistry, 70, 795–806
(2009).