Chemical structures of constituents from the seeds of Cassia auriculata

Article abstract of DOI:10.1016/j.tet.2015.07.045

Five new diterpene glycosides, auricuosides I (1), II (2), III (3), IV (4), and V (5), a new flavonol glycoside, auricuoflavonoside I (6), and a new megastigmane glycoside, auricuomegastigmane I (7), were isolated from the seeds of Cassia auriculata, together with 11 known constituents. The chemical structures of seven new constituents were characterized on the basis of chemical and physicochemical evidence. In the present study, auricuosides I (1)-V (5) were obtained from the seeds of C. auriculata. To the best of our knowledge, there is no report about the isolation of kaurenolides with hydroxy group at 14-position from plants. This finding is interesting from the perspective of chemotaxonomy and biosynthesis of kaurenolide.

Full text of DOI:10.1016/j.tet.2015.07.045

Accepted Manuscript
Chemical structures of constituents from the seeds of Cassia auriculata
Yi Zhang, Seikou Nakamura, Souichi Nakashima, Tao Wang, Masayuki Yoshikawa,
Hisashi Matsuda
PII:
S0040-4020(15)01090-X
10.1016/j.tet.2015.07.045
TET 26990
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 27 May 2015
Revised Date: 11 July 2015
Accepted Date: 15 July 2015
Please cite this article as: Zhang Y, Nakamura S, Nakashima S, Wang T, Yoshikawa M, Matsuda
H, Chemical structures of constituents from the seeds of Cassia auriculata, Tetrahedron (2015), doi:
10.1016/j.tet.2015.07.045.
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ACCEPTED MANUSCRIPT
Tetrahedron (Full Paper)
(Total 18, Legends 1, Figure 4, Table 3, Graphical abstract 1)
Chemical structures of constituents
from the seeds of Cassia auriculata
Yi Zhanga,b, Seikou Nakamuraa, Souichi Nakashimaa,c, Tao Wanga,b,
Masayuki Yoshikawaa, Hisashi Matsudaa,*
aKyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
bTianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of
Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, China
cN.T.H Co., Ltd., 4F Sky-ebisu Bldg, 1-8-11 Ebisu, Shibuya-ku, Tokyo 150-0013, Japan
Abstract− Five new diterpene glycosides, auricuosides I (1), II (2), III (3), IV (4),
and V (5), a new flavonol glycoside, auricuoflavonoside I (6), and a new
megastigmane glycoside, auricuomegastigmane I (7), were isolated from the seeds of
Cassia auriculata, together with 11 known constituents. The chemical structures of
seven new constituents were characterized on the basis of chemical and
physicochemical evidence. In the present study, auricuosides I (1)–V (5) were
obtained from the seeds of C. auriculata. To the best of our knowledge, there is no
report about the isolation of kaurenolides with hydroxy group at 14-position from
plants. This finding is interesting from the perspective of chemotaxonomy and
biosynthesis of kaurenolide.
Key words: Cassia auriculata; auricuoside; Ayurvedic traditional medicine; diterpene
glycoside, Leguminosae
*To whom correspondence should be addressed.
Tel: +81-75-595-4633, Fax: +81-75-595-4768, E-mail: matsuda@mb.kyoto-phu.ac.jp
1

ACCEPTED MANUSCRIPT
1. Introduction
Cassia auriculata belongs to the family Leguminosae and is mainly distributed in
India, Sri Lanka, etc. This plant is used for the treatment of diabetes, rheumatism,
asthma, and skin diseases in the Ayurvedic system of traditional Indian medicine.1–5
Previously, we have isolated constituents such as flavonols and flavanol dimers from
the leaves of C. auriculata.6 On the other hand, several constituents such as fatty
acids from the seeds of this plant have been characterized on the basis of GC-MS
data.7 However, the detailed chemical constituents of the seeds of C. auriculata have
not been characterized. In the course of our studies on the constituents of traditional
Asian medicine,8−14 we examined the constituents of the seeds of C. auriculata. In
this paper, we describe the isolation and structure elucidation of five new diterpene
glycosides named auricuosides I (1), II (2), III (3), IV (4), and V (5), a new flavonol
glycoside named auricuoflavonoside I (6), and a new megastigmane glycoside named
auricuomegastigmane I (7).
2. Results and Discussion
The MeOH extract (11.0% from the seeds of C. auriculata) was partitioned between
n-hexane-H2O (1:1, v/v) to furnish an n-hexane-soluble fraction (1.3%) and an
aqueous layer (9.7%). The aqueous layer was subjected to Diaion HP-20 column
chromatography to give H2O- and MeOH-eluted fractions. The MeOH-eluted
fraction was subjected to normal-phase and reversed-phase silica gel column
chromatography (CC) and repeated HPLC to give auricuosides I (1), II (2), III (3), IV
(4), V (5), a new flavonol glycoside, auricuoflavonoside I (6), and a new
megastigmane glycoside, auricuomegastigmane I (7), together with isorhamnetin-3-
O-rutinoside (8),15 rutin (9),15 quercetin 3-O-
O- -glucopyranosyl(1→2)[ -rhamnopyranosyl(1→6))
(11),16 5-methoxyluteolin 7-O- -glucopyranoside (12),17 7,4'-dihydroxy-5,3'-
β
-D
-glucopyranoside (10),15 quercetin 3-
β
-D
α
-L
β-D
-glucopyranoside
β-D
2

ACCEPTED MANUSCRIPT
dimethoxyflavone 7-O-
diglucoside),19 lucenin II (15),20 stellarin 2 (16),21 (3S,5R,9S)-megastigma-6,7-dien-
3,5,9-triol 9-O-
-glucopyranoside (17),22 5-[(1R,3R,5R,8S)-3,8-dihydroxy-1,5-
β-D
-glucopyranoside (13),18 vicenin II (14, apigenin 6,8-C-
β-D
dimethyl-6-oxabicyclo[3,2,1]oct-8yl]-3-methyl-(2Z,4E)-2,4-pentadienoic acid (18),23
and adenosine (Fig. 1).
Fig. 1 Structures of constituents from the seeds of C. auriculata
3

ACCEPTED MANUSCRIPT
Auricuosides I (1) and II (2), which were obtained as a white powder with negative
optical rotations (1: [α] α]
26 –10.7, 2: [ 26 –7.2 in MeOH), showed absorption bands
D D
suggestive of the glycosidic structures (1 and 2: 3450 and 1078 cm–1) together with
absorption bands indicative of ester, ketone, and olefin functions (1: 1763, 1709, and
1655 cm–1, 2: 1773, 1707, and 1655 cm–1) in their IR spectra. Positive-ion FABMS
of 1 and 2 revealed a quasimolecular ion peak [M + Na]+ at m/z 547, from which the
common molecular formula C26H36O11 was determined via each HRMS and 13C
NMR data. Acid hydrolysis of 1 and 2 with 1.0
M aqueous HCl liberated D-glucose,
which was identified by HPLC analysis using an optical rotation detector. The 1H and
13C NMR spectra (Table 1 and 2, pyridine-d5) of 1 and 2, the assignments of which
were made with the aid of DEPT, DQF COSY, HMQC, and HMBC experiments,
showed signals assignable to two methyls [δH 1: 1.30, 1.60 (3H each, all s, H3-20, 18),
2: 1.36, 1.41 (3H each, all s, H3-20, 18)], two olefinic protons [δH 1: 5.26, 5.28 (1H
each, br s, H2-17), 2: 5.25, 5.27 (1H each, br s, H2-17)], four methines bearing an
oxygen function [δH 1: 4.23 (1H, s like, H-14), 4.49 (1H, m, H-2), 5.00 (1H, dd, J =
6.2, 6.2 Hz, H-6), 5.11 (1H, d, J = 6.2 Hz, H-7), 2: 4.29 (1H, s like, H-14), 4.31 (1H,
m, H-2), 5.17 (1H, d, J = 4.9 Hz, H-7), 5.40 (1H, dd, J = 4.9, 4.9 Hz, H-6)], β-D-
glucopyranosyl moiety [δH 1: 4.93 (1H, d, J = 7.4 Hz, H-1'), 2: 5.21 (1H, d, J = 7.4
Hz, H-1')], and two carbonyl carbons [δC 1: 182.1, 207.9 (C-19, 11), 2: 181.3, 207.9
(C-19, 11)]. As shown in Fig. 2, the DQF COSY experiments of 1 indicated the
presence of partial structures (written in bold) and the HMBC experiments of 1
yielded long-range correlations between the following protons and carbons: H2-1 and
C-20; H2-3 and C-18; H-5 and C-18, 19, 20; H-6 and C-7; H-7 and C-6, 15; H-9 and
C-8, 10, 11, 20; H2-12 and C-14, 16; H-14 and C-8, 12, 16; H2-15 and C-7, 14, 16;
H2-17 and C-13; H3-18 and C-3, 4, 5, 19; H3-20 and C-1, 5, 9; H-1' and C-2. Thus,
the planar structure of 1 was shown to be kaurenolide derivative with a glucose at 2-
position and a hydroxy group at 14-position. On the other hand, on the basis of the
detail examination of DQF COSY and HMBC experiments (Fig. 2), the planner
structure on diterpene moiety of 2 found to be the same as that of 1. The connectivity
4

ACCEPTED MANUSCRIPT
of a glucose in 2 was characterized by HMBC experiment, which showed long-range
correlations between the following protons and carbons: H-7 and C-1'; H-1' and C-7
(Fig. 2). Next, the stereostructures on diterpene moieties of 1 and 2 were
characterized by the NOESY experiment, which showed NOE correlations between
the following proton pairs: H-1α and H-2, H3-20; H-2 and H3-20; H-5 and H-6, H-9,
H3-18; H-6 and H3-18; H-7 and H-14, H3-20; H-14 and H3-20. Consequently, the
chemical structures of auricuosides I (1) and II (2) were determined as shown,
respectively.
Fig. 2. Selected DQF, HMBC and NOESY correlations of 1 and 2
Auricuosides III (3), IV (4), and V (5), were obtained as a white powder with
negative optical rotations (3: [α] α] α]
24 –12.7, 4: [ 27 –5.6, 5: [ 27 –11.2 in MeOH).
D D D
Absorption bands due to hydroxy, ester, olefin, and ketone (only for 4) components
were shown in their IR spectra. FABMS in the positive-ion mode of 3–5 revealed
quasimolecular ion peaks (m/z 549 [M+Na]+ for 3, 531 [M+Na]+ for 4, 533 [M+Na]+
for 5) from which those molecular formulas (C26H38O11 of 3, C26H36O10 of 4,
C26H38O10 of 5) were determined via each HRMS and 13C NMR data. Acid
hydrolysis of 3−5 with 1.0
M aqueous HCl liberated D-glucose, which was identified
by HPLC analysis using an optical rotation detector.
5

ACCEPTED MANUSCRIPT
Table 1
1H NMR Data of auricuosides I–V (1–5) in pyridine-d5
1
3.07 (dd, 6.8, 13.7)
1.59 (m)
2
3.03 (dd, 4.9, 12.2)
1.38 (m)
3
2.38 (dd, 4.9, 12.2)
1.32 (dd, 8.6, 12.2)
4.34 (m)
4
5
position
2.39 (m)
0.99 (m)
1.53 (m)
1.33 (m)
2.16 (m)
1.33 (m)
1.71 (m)
0.94 (m)
1.56 (m)
1.37 (m)
2.26 (m)
1.30 (m)
1
1
2
2
3
3
5
6
7
9
α
β
α
β
α
β
4.49 (m)
4.31 (m)
2.52 (dd, 4.8, 15.1)
2.04 (dd, 6.2, 15.1)
2.25 (d, 6.2)
2.78 (dd, 4.9, 14.7)
1.73 (dd, 8.6, 14.7)
2.17 (d, 4.9)
2.91 (dd, 4.9, 13.5)
1.71 (dd, 8.6, 13.5)
2.18 (d, 4.9)
1.85 (d, 6.1)
5.32 (dd, 6.1, 6.1)
5.13 (d, 6.1)
2.29 (s)
1.91 (d, 4.9)
5.00 (dd, 6.2, 6.2)
5.11 (d, 6.2)
5.40 (dd, 4.9, 4.9)
5.17 (d, 4.9)
5.39 (dd like, ca. 5, 5)
5.40 (d like, ca. 5)
1.54 (br s)
5.31 (dd, 4.9, 4.9)
5.35 (d, 4.9)
2.42 (s)
2.45 (s)
1.37 (m)
4.47 (m)
4.39 (m)
11
12
12
13
14
15
15
17
2.73 (dd, 6.8, 17.2)
2.45 (d like, 17.2)
2.92 (d like, ca. 7)
4.23 (s like)
2.75 (dd, 7.3, 17.1)
2.43 (m)
2.51 (dd like, 9.8, 14.6)
1.93 (dd like, 6.1, 14.6)
2.94 (d like, ca. 10)
5.84 (s like)
2.74 (dd, 7.3, 17.1)
2.44 (d like, 17.1)
2.95 (d like, 7.3)
4.28 (s like)
2.54 (dd like, 8.6, 14.7)
1.94 (dd like, 6.1, 14.7)
2.94 (d like, ca. 10)
5.83 (s like)
α
β
2.95 (d like, 7.3)
4.29 (s like)
3.83 (br d, 13.5)
2.41 (br d, 13.5)
5.25 (br s)
3.77 (br d, 15.8)
2.26 (br d, 15.8)
5.26 (br s)
3.75 (br d, 15.0)
2.14 (br d, 15.0)
5.16 (br s)
3.83 (br d, 14.7)
2.38 (br d, 14.7)
5.26 (br s)
3.73 (d like, ca. 16)
2.09 (d like, 15.9)
5.18 (br s)
α
β
5.28 (br s)
5.27 (br s)
5.22 (br s)
5.28 (br s)
5.23 (br s)
18
20
1'
1.60 (s)
1.41 (s)
1.37 (s)
1.19 (s)
1.19 (s)
1.30 (s)
1.36 (s)
1.56 (s)
1.29 (s)
1.47 (s)
4.93 (d, 7.4)
3.97 (dd, 7.4, 8.2)
4.19 (m)
5.21 (d, 7.4)
3.99 (dd, 7.4, 8.6)
4.16 (m)
5.23 (d, 7.3)
5.21 (d, 7.3)
5.20 (d, 8.6)
4.02 (dd, 7.3, 8.0)
4.18 (m)
4.00 (dd, ca. 7, 9)
4.16 (m)
4.01 (dd, 8.6, 8.6)
4.17 (m)
2'
3'
4.20 (m)
4.20 (m)
4.19 (m)
4.20 (dd, 8.6, 8.6)
3.96 (m)
4.18 (m)
4'
3.87 (m)
3.93 (m)
3.96 (m)
3.95 (m)
5'
4.33 (dd, 5.5, 11.7)
4.47 (dd, 2.5, 11.7)
4.26 (dd, 4.9, 11.0)
4.43 (dd, 2.5, 11.0)
4.26 (dd, ca. 5, 11)
4.46 (m)
4.27 (dd, 4.9, 11.0)
4.45 (dd, 2.5, 11.0)
4.26 (dd, 4.9, 12.2)
4.46 (dd, 2.5, 12.2)
6''
The numbers shown in parentheses are J values.
Table 2
13C NMR Data of auricuosides I–V (1–5) in pyridine-d5
position
position
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
45.4
71.8
33.3
41.9
50.2
82.9
72.2
49.5
69.8
34.9
207.9
49.8
46.9
46.6
63.3
38.8
42.4
50.7
82.5
82.6
50.0
68.9
35.4
207.9
49.8
46.5
47.9
63.4
39.7
42.6
51.7
83.4
84.3
49.6
61.8
37.2
63.7
45.1
47.3
36.6
18.0
28.7
41.4
50.9
82.5
82.7
49.8
69.4
34.1
207.8
49.9
46.6
37.6
18.0
29.0
41.2
51.9
83.4
84.4
49.3
62.0
35.4
63.6
45.2
47.3
84.5
39.9
155.5
111.1
26.7
182.1
21.3
102.6
75.4
78.8
71.8
78.4
62.8
84.0
41.0
155.4
111.0
26.6
181.3
20.5
105.3
75.1
78.8
71.7
78.6
62.8
78.6
40.5
159.6
108.8
26.6
181.6
23.6
105.7
75.2
79.0
71.9
78.5
63.0
84.0
41.0
155.6 159.7
110.9 108.7
25.3
181.5 181.8
20.6 23.1
105.3 105.6
75.1
78.9
71.8
78.7
62.9
78.6
40.5
25.8
1'
2'
3'
4'
5'
6''
75.2
79.0
71.9
78.5
63.0
6

ACCEPTED MANUSCRIPT
1
The H and 13C NMR spectra (Table 1 and 2, pyridine-d5) of 3 showed signals
assignable to two methyls [δH 1.37, 1.56 (3H each, all s, H3-18, 20)], two olefinic
protons [δH 5.16, 5.22 (1H each, br s, H2-17)], five methines bearing an oxygen
function,
β-D-glucopyranosyl moiety [δH 5.23 (1H, d, J = 7.3 Hz, H-1')], and a
carbonyl carbons [δC 181.6 (C-19)]. The proton and carbon signals in the 1H and 13C
NMR spectra of 3 were similar to those of 2, except for the signals around 11-
position. The positions of three hydroxy groups and a glucosyloxy moiety were
1
confirmed based on DQF COSY and HMBC spectroscopy. (Fig. 3). The H NMR
13
and C NMR spectra (Table 1 and 2, pyridine-d5) of 4 showed signals due to two
methyls, two olefinic protons, three methines bearing an oxygen function, a β-D-
glucopyranosyl moiety, and two carbonyl carbons [δC 181.5, 207.8 (C-19, 11)]. The
proton and carbon signals in the 1H and 13C NMR spectra of 4 were superimposable
on those of 2, except for the signals around 2-position.
HO
O
HO
HO
HO
HO
HO
O
H
O
H
O
H
O
O
O
O
O
O
HO
HO
HO
O
O
O
OH
OH
OH
HO
HO
HO
OH
OH
OH
3
5
4
11
11
9
1
1
2
1
2
15
9
9
5
20
5
5
14
14
18
14
7
7
7
20
18
18
20
6
6
4
5
3
NOESY
HMBC
DQF COSY
Fig. 3. Selected DQF, HMBC and NOESY correlations of 3, 4, and 5
On the other hand, the proton and carbon signals in the 1H and 13C NMR spectra of 5
were superimposable on those of 3, except for the signals around 2-position. On the
7

ACCEPTED MANUSCRIPT
basis of this evidence and the detail examination of DQF COSY and HMBC
experiments (Fig 3). Next, the stereostructures on diterpene moieties of 3–5 were
characterized by the detail NOESY experiment. Consequently, the chemical
structures of auricuosides III (3), IV (4), and V (5) were determined as shown,
respectively.
Fig. 4. Selected DQF, HMBC and NOESY correlations of 6 and 7
Auricuoflavonoside I (6), was obtained as a pale yellow powder with negative optical
rotation ([α]
26 –118.2 in MeOH). FABMS in the positive- and negative-ion mode of
D
6 revealed quasimolecular ion peaks (m/z 631 [M+Na]+ and m/z 607 [M–H]–) from
13
which the molecular formula C28H32O15 was determined via HRMS and C NMR
data. Acid hydrolysis of 6 with 1.0
M
aqueous HCl liberated
D
-glucose and
D
-apiose,
1
which were identified by HPLC analysis using an optical rotation detector. The H
(DMSO-d6) and 13C NMR (Table 3) spectra of 6 showed signals ascribable to a
lutelin part together with two methoxy groups [δH 3.84, 3.88 (3H each, both s, 5, 3'-
OMe)], a β-D-glucopyranosyl moiety [δH 5.17 (1H, d, J = 8.3 Hz, H-1'')], and a β-D-
apiofuranosyl moiety [δH 5.38 (1H, s like, H-1''')]. The proton and carbon signals of
the glycosides of 6 in the 1H and 13C NMR spectra were superimposable on those of
known compound, 3,7,3'-tri-O-methylquercetin 4'-O-
glucopyranoside.24 The postions of two methoxy groups, a
moiety, and a -apiofuranosyl moiety were confirmed based on DQF COSY,
β
-D
-apiofuranosyl(1→2)-
β-D-
β-D
-glucopyranosyl
β-D
8

ACCEPTED MANUSCRIPT
HMBC, and NOESY spectroscopy (Fig. 4). Consequently, the chemical structures of
Auricuoflavonoside I (6) was determined as 5,3'-di-O-methyllutelin 7-O-β-D-
apiofuranosyl(1→2)-
β
-D
-glucopyranoside.
Table 3
a
13C NMR Data of 6 and 7 and in DMSO-d6 or CD3OD b
6a
6 b
6a
6 b
7 b
7 b
position
position
position
position
1
2
3
4
5
6
7
8
9
10
11
12
13
1'
2'
3'
4'
5'
2
3
4
5
6
7
8
9
10
1'
2'
3'
4'
5'
6'
160.1
106.3
175.6
160.1
96.9
164.0
107.0
180.1
162.1
98.5
CH3O-5
CH3O-3'
1''
55.9
55.8
98.4
75.5
76.9
70.0
77.2
60.6
108.5
76.0
79.2
73.9
64.3
56.7
56.7
39.2
42.7
65.9
132.0
138.7
77.1
130.7
136.3
68.8
23.9
24.5
24.7
70.6
102.6
75.1
78.2
71.7
62.8
100.6
78.7c
78.6c
71.5
2''
3''
4''
5''
6''
1'''
2'''
3'''
161.2
96.2
163.8
97.7
78.5c
62.6
158.5
108.9
121.5
109.7
147.9
150.1
115.6
119.5
160.8
110.2
123.5
110.5
149.5
151.9
116.8
121.5
110.9
78.1
80.7
75.4
65.9
4'''
5'''
cinterchangable
Auricuomegastigmane I (7), was obtained as an amorphous powder with negative
27
D
optical rotation ([
α
]
–2.5 in MeOH). FABMS in the positive-ion mode of 7
revealed quasimolecular ion peak (m/z 427 [M+Na]+) from which the molecular
formula C19H32O9 was determined via HRMS and 13C NMR data. Acid hydrolysis of
7 liberated D
-glucose. The 1H (CD3OD) and 13C NMR (Table 3) spectra of 7 showed
signals ascribable to a megastigmane part {three methyls [δH 0.91, 0.95 (3H each, all
s, H3-11, 12), 1.24 (3H, d, J = 6.4 Hz, H3-10)], three olefinic protons [δH 5.67 (1H,
dd, J = 1.3, 15.6 Hz, H-7), 5.83 (1H, dd, J = 5.5, 15.6 Hz, H-8), 5.93 (1H, br s, H-4)],
two methines bearing an oxygen function [δH 4.22 (1H, m, H-3), 4.33 (1H, m, H-9)]}
together with a
β
-
D
-glucopyranosyl moiety [δH 4.29 (d, J = 7.6 Hz, H-1'')]. The
-glucopyranosyl
planner structure of the megastigmane part and the position of a
β-D
moiety were confirmed based on DQF COSY and HMBC spectroscopy (Fig. 4). The
stereostructure on the ring part of 7 was characterized by the NOESY experiment,
which showed NOE correlations between the following proton pairs: H-2β and H-3,
9

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H3-11; H-3 and H3-11; H-7 and H3-11. Consequently, the chemical structure of
auricuomegastigmane I (7) was determined as shown.25
In conclusion, five new diterpene glycosides, auricuosides I (1), II (2), III (3), IV (4),
V (5), a new flavonol glycoside, auricuoflavonoside I (6), and a new megastigmane
glycoside, auricuomegastigmane I (7), were isolated from the seeds of Cassia
auriculata. In the present study, auricuosides I–V (1–5), which were kaurenolides
with hydroxy group at 14-position, were obtained from the seeds of C. auriculata. To
the best of our knowledge, there is no report about the isolation of kaurenolides with
hydroxy group at 14-position from plants. This finding is interesting from the
perspective of chemotaxonomy and biosynthesis of kaurenolides.
3. Experimental
The following instruments were used to obtain physical data: specific rotations,
Horiba SEPA-300 digital polarimeter (l = 5 cm); IR spectra, Shimadzu FTIR-8100
spectrometer; CD spectra, JASCO J-720WI spectrometer; UV spectra, Shimadzu
UV-1600 spectrometer; EIMS and HREIMS, JEOL JMS-GCMATE mass
spectrometer; FABMS and HRFABMS, a JEOL JMS-SX 102A mass spectrometer;
1H NMR spectra, JNM-LA500 (500 MHz) and JEOL JNM-ECA600 (600 MHz)
spectrometers; 13C NMR spectra, JNM-LA500 (125 MHz) and JEOL JNM-ECA600
(150 MHz) spectrometers; HPLC detector, Shimadzu RID-6A refractive index
detector and SPD-10A UV-VIS detectors; and HPLC column, Cosmosil 5C18-MS-II
(Nacalai Tesque Inc., 250 × 4.6 mm i.d.) and (250 × 20 mm i.d.) columns were used
for analytical and preparative purposes, respectively. The following experimental
materials were used for chromatography: normal-phase silica gel column
chromatography (CC), Silica gel BW-200 (Fuji Silysia Chemical, Ltd., 150–350
mesh); reversed-phase silica gel column chromatography, Chromatorex ODS
DM1020T (Fuji Silysia Chemical, Ltd., 100–200 mesh); TLC, precoated TLC plates
10

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with Silica gel 60F254 (Merck, 0.25 mm) (ordinary phase) and Silica gel RP-18 F254S
(Merck, 0.25 mm) (reversed phase); reversed-phase HPTLC, precoated TLC plates
with Silica gel RP-18 WF254S (Merck, 0.25 mm); and detection was achieved by
spraying with 1% Ce(SO4)2–10% aqueous H2SO4 followed by heating.
3.1. Plant Material
The seeds of C. auriculata were purchased from N.T.H Co., Ltd. A voucher of the
plant is on file in our laboratory [CAS-1].
3.2. Extraction and Isolation
The seeds of C. auriculata (3.93 kg) were crushed and extracted three times with
MeOH under reflux for 3 h. Evaporation of the solvent under reduced pressure
provided the MeOH extract (430.6 g, 10.96%). A part of the extract (399.0 g) was
partitioned into an n-hexane-H2O (1:1, v/v) mixture to furnish a n-hexane-soluble
fraction (46.4 g, 1.27%) and an aqueous phase (350.8 g, 9.63%) soluble fractions. A
part of the aqueous phase (322.7 g) was subjected to Diaion HP-20 CC (3.0 kg, H2O
→ MeOH) to give H2O- (248.2 g) and MeOH- (63.2 g) eluted fractions. The MeOH-
eluted fraction (60.0 g) was was subjected to ordinary-phase silica gel column
chromatography (CC) [1.5 kg, CHCl → CHCl3–MeOH (20:1, v/v]] → CHCl3–
3
MeOH–H2O (10:3:1 → 7:3:1 → 6:4:1, v/v/v, low layer) → MeOH] to give 9
fractions {Fr. 1–Fr. 4, Fr. 5 (10.4 g), Fr. 6 (5.7 g), Fr. 7 (16.7 g), Fr. 8 (16.3 g), Fr. 9}.
Fraction 5 (10.4 g) was subjected to reversed-phase silica gel CC [300 g, MeOH–
H2O (20:80 → 30:70 → 40:60 → 50:50 → 60:40 → 70:30 → 80:20, v/v) → MeOH]
to give 13 fractions [Fr. 5-1, Fr. 5-2 (522 mg), Fr. 5-3 (282 mg), Fr. 5-4 (783 mg), Fr.
5-5 (1991 mg), Fr. 5-6–5-13]. Fraction 5-2 (522 mg) was purified by HPLC [MeOH–
H2O (20:80, v/v), CH3CN–MeOH–H2O (8:8:84, v/v/v)] to give 1 (3.6 mg,
0.00011%), 2 (25 mg, 0.00079%), and adenosine (16 mg, 0.00049%). Fraction 5-3
(282 mg) was purified by HPLC [MeOH–H2O (25:75, v/v), CH3CN–MeOH–H2O
(9:9:82, v/v/v)] to give 2 (23 mg, 0.00072%), 3 (4.1 mg, 0.00013%), and 18 (6.6 mg,
11

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0.00021%). Fraction 5-4 (783 mg) was purified by HPLC [MeOH–H2O (35:65 and
30:70, v/v)] to give 2 (7.3 mg, 0.00023%). Fraction 5-5 (1991 mg) was purified by
HPLC [MeOH–H2O (45:55 and 30:70, v/v) and CH3CN–MeOH–H2O (19:8:73,
v/v/v)] to give 4 (8.2 mg, 0.00026%) and 5 (14 mg, 0.00043%). Fraction 6 (5.7 g)
was subjected to reversed-phase silica gel CC [200 g, MeOH–H2O (10:90 → 20:80
→ 30:70 → 40:60 → 50:50 → 60:40 → 70:30 → 80:20, v/v) → MeOH]] to give 8
fractions [Fr. 6-1, Fr. 6-2 (300 mg), Fr. 6-3, Fr. 6-4 (436 mg), Fr. 6-5 (231 mg), Fr.6-
6–6-8]. Fraction 6-2 (300 mg) was purified by HPLC [MeOH–H2O (25:75, v/v) and
CH3CN–MeOH–H2O (8:8:84, v/v/v)] to give 3 (9.4 mg, 0.00030%), 7 (3.6 mg,
0.00011%), 17 (6.4 mg, 0.00020%). Fraction 6-4 (436 mg) was purified by HPLC
[MeOH–H2O (35:65, v/v) and CH3CN–MeOH–H2O (13:8:79, v/v/v)] to give 6 (5.0
mg, 0.00016%), 10 (47 mg, 0.0015%), and 13 (76 mg, 0.0024%). Fraction 6-5 (231
mg) was purified by HPLC [MeOH–H2O (40:60, v/v)] to give 8 (63 mg, 0.0020%).
Fraction 7 (16.7 g) was subjected to reversed-phase silica gel CC [480 g, MeOH–
H2O (10:90 → 20:80 → 30:70 → 40:60 → 50:50 → 60:40 → 70:30, v/v) → MeOH]
to give 12 fractions [Fr. 7-1–7-1, Fr. 7-5 (428 mg), Fr. 7-6, Fr. 7-7 (1004 mg), Fr.7-
8–7-12]. Fraction 7-5 (428 mg) was purified by HPLC [MeOH–H2O (30:70, v/v) and
CH3CN–MeOH–H2O (11:9:80)] to give 12 (2.6 mg, 0.00008%). Fraction 7-7 (360
mg) was purified by HPLC [MeOH–H2O (35:65, v/v)] to give 9 (73 mg, 0.0064%).
Fraction 8 (16.3 g) was subjected to reversed-phase silica gel CC [480 g, MeOH–
H2O (20:80 → 30:70 → 40:60 → 50:50, v/v) → MeOH] to give 12 fractions [Fr. 8-
1–8-4, Fr. 8-5 (3355 mg), Fr. 8-6, Fr. 8-7, Fr. 8-8 (1175 mg), Fr.8-9–8-12]. Fraction
8-5 (244 mg) was purified by HPLC [MeOH–H2O (35:65, v/v)] to give 14 (59 mg,
0.025%), 15 (35 mg, 0.015%), and 16 (108 mg, 0.046%). Fraction 8-8 (1175 mg)
was purified by HPLC [MeOH–H2O (35:65, v/v) and CH3CN–MeOH–H2O
(11:9:80)] to give 11 (7.2 mg, 0.00023%). The known compounds were identified by
1
comparison of their physical data ([
values.
α
]D, H NMR, 13C NMR, MS) with reported
12

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3.2.1. Auricuoside I (1). White powder; [α]
26 –10.7 (c 0.27, MeOH); IR (KBr) vmax
D
3450, 2965, 1763, 1709, 1655, 1078 cm–1; 1H NMR (pyridine-d5, 600 MHz)
δ given
13
in Table 1; C NMR (pyridine-d5, 150 MHz)
δ
given in Table 2; NOE correlations
and H-2, H3-20; H-2 and H3-20; H-3 and
and H3-18; H-5 and H-6, H-9, H3-18; H-6 and H3-18; H-7 and H-14,
H3-20; H-14 and H-12
, 13, H3-20; positive-ion FABMS m/z 547 [M + Na]+;
HRFABMS m/z 547.2148 (calcd for C26H36O11Na [M + Na]+, 547.2155).
between the following proton pairs: H-1
α
α
H3-20; H-3
β
α
3.2.2. Auricuoside II (2). White powder; [α]
26 –7.2 (c 0.99, MeOH); IR (KBr) vmax
D
3450, 2940, 1773, 1707, 1655, 1078 cm–1; 1H NMR (pyridine-d5, 600 MHz)
δ given
13
in Table 1; C NMR (pyridine-d5, 150 MHz)
δ
given in Table 2; NOE correlations
and H-2, H3-20; H-2 and H3-20; H-3 and
H3-18; H-5 and H-6, H-9, H3-18; H-6 and H3-18; H-7 and H-14, H3-20; H-14 and H-
12
, 13, H3-20; positive-ion FABMS m/z 547 [M + Na]+; HRFABMS m/z 547.2160
(calcd for C26H36O11Na [M + Na]+, 547.2155).
between the following proton pairs: H-1
α
β
α
3.2.3. Auricuoside III (3). White powder; [α]
24 –12.7 (c 0.64, MeOH); IR (KBr)
D
vmax 3450, 2930, 1773, 1655, 1076 cm–1; 1H NMR (pyridine-d5, 600 MHz)
δ given in
13
Table 1; C NMR (pyridine-d5, 150 MHz)
δ
given in Table 2; NOE correlations
and H-2, 11; H-1 and H-3 , 9, 11; H-2
and H3-18; H-5 and H-6, H-9, H3-18; H-6 and H3-18; H-7 and H-14,
H3-20; H-9 and H-11, 15 ; H-11 and H-12 , 12 ; H-12 and H-13, 14; H-13 and H-
between the following proton pairs: H-1
α
β
β
and H3-20; H-3
β
β
α
β
α
14; H-14 and H3-20; positive-ion FABMS m/z 549 [M + Na]+; HRFABMS m/z
549.2318 (calcd for C26H38O11Na [M + Na]+, 549.2312).
3.2.4. Auricuoside IV (4). White powder; [α]
27 –5.6 (c 0.44, MeOH); IR (KBr) vmax
D
3450, 2932, 1767, 1701, 1655, 1078 cm–1; 1H NMR (pyridine-d5, 600 MHz)
δ given
13
in Table 1; C NMR (pyridine-d5, 150 MHz)
δ
given in Table 2; NOE correlations
between the following proton pairs: H-1
α
and H-2 ; H-1 and H-3 , 5, 9; H-2 and
α
β
β
α
13

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H-3
H-12
[M + Na]+; HRFABMS m/z 531.2206 (calcd for C26H36O10Na [M + Na]+, 531.2199).
α
; H-5 and H-6, 9, H3-18; H-6 and H3-18; H-7 and H-14, H3-20; H-9 and H-15β;
α
and H-13, 14; H-13 and H-14; H-14 and H3-20; positive-ion FABMS m/z 531
3.2.5. Auricuoside V (5). White powder; [α]
27 –11.2 (c 0.84, MeOH); IR (KBr) vmax
D
1
3450, 2930, 1775, 1655, 1078 cm–1; H NMR (pyridine-d5, 600 MHz)
δ given in
13
Table 1; C NMR (pyridine-d5, 150 MHz)
δ
given in Table 2; NOE correlations
and H-11; H-1 and H-2 , 5; H-2 and H-
, H3-18; H-6 and H3-18; H-7 and H3-20; H-9 and H-11,
; H-12 and H-13, 14; H-13 and H-14; H-14 and H3-20;
between the following proton pairs: H-1
; H-5 and H-1 , 6, 15
; H-11 and H-12 , 12
α
β
β
α
3
α
β
β
15
β
α
β
α
positive-ion FABMS m/z 533 [M + Na]+; HRFABMS m/z 533.2372 (calcd for
C26H38O10Na [M + Na]+, 533.2363).
3.2.6. Auricuoflavonoside I (6). Pale yellow powder; [α]
26 –118.2 (c 0.17, MeOH);
D
IR (KBr) vmax 3425, 2924, 1655, 1561, 1540, 1458, 1074 cm–1; UV λmax (MeOH) nm
1
(log
ε
): 210 (4.66), 243 (4.40), 265 (4.32), 337 (4.45); H NMR (DMSO-d6, 600
MHz)
δ
3.44, 3.74 (1H each, both m, H-6''), 3.68, 3.99 (1H each, both d, J = 9.7 Hz,
H2-4'''), 3.84, 3.88 (3H each, both s, 5, 3'-OMe), 5.17 (1H, d, J = 8.3 Hz, H-1''), 5.38
(1H, s like, H-1'''), 6.57 (1H, d, J = 2.1 Hz, H-6), 6.70 (1H, s, H-3), 6.91 (2H, m, H-8,
5'), 7.50 (2H, m, H-1', 6'); 1H NMR (CD3OD, 600 MHz)
δ 3.73, 4.01 (1H each, both
d, J = 9.6 Hz, H2-4'''), 3.85 (2H, m, H2-6''), 3.82, 3.86 (3H each, both s, 5, 3'-OMe),
5.07 (1H, d, J = 7.6 Hz, H-1''), 5.38 (1H, d, J = 2.1 Hz, H-1'''), 6.50 (1H, s, H-3), 6.57
(1H, d, J = 2.0 Hz, H-6), 6.82 (1H, d, J = 8.3 Hz, H-5'), 6.84 (1H, d, J = 2.1 Hz, H-8),
7.36 (1H, d, J = 2.1 Hz, H-2'), 7.40 (1H, d, J = 2.1, 8.3 Hz, H-6'); 13C NMR (DMSO-
d6 and CD3OD, 150 MHz)
δ given in Table 3; positive-ion FABMS m/z 631 [M +
Na]+; negative-ion FABMS m/z 607 [M – H]–; HRFABMS m/z 631.1633 (calcd for
C28H32O15Na [M + Na]+, 631.1639).
14

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3.2.7. Auricuomegastigmane I (7). Amorphous powder; [α]
27 –2.5 (c 0.17, MeOH);
D
1
IR (KBr) vmax 3420, 2924, 1655, 1084 cm–1; H NMR (CD3OD, 500 MHz)
δ 0.91,
0.95 (3H each, all s, H3-11, 12), 1.24 (3H, d, J = 6.4 Hz, H3-10), 1.64 (1H, dd, J = 6.1,
12.9 Hz, H-2 ), 1.79 (1H, dd, J = 8.3, 12.9 Hz, H-2 ), 4.04, 4.36 (1H each, both br d,
β
α
J = 12.8 Hz, H2-13), 4.22 (1H, m, H-3), 4.29 (d, J = 7.6 Hz, H-1''), 4.33 (1H, m, H-9),
5.67 (1H, dd, J = 1.3, 15.6, H-7), 5.83 (1H, dd, J = 5.5, 15.6, H-8), 5.93 (1H, br s, H-
13
4); C NMR (CD3OD, 125 MHz)
δ given in Table 3; positive-ion FABMS m/z 427
[M + Na]+; HRFABMS m/z 427.1950 (calcd for C19H32O9Na [M + Na]+, 427.1944).
3.2.8. Acid Hydrolyses
A solution of 1–7 (1.0 mg) in 1 M HCl–1,4-dioxane (1:1, v/v, 1.0 mL) was heated
under reflux for 3 h. After cooling, the reaction mixture was extracted with EtOAc.
The aqueous layer was subjected to HPLC analysis under the following conditions,
respectively: HPLC column, Kaseisorb LC NH2-60-5, 4.6 mm i.d. × 250 mm (Tokyo
Kasei Co., Ltd., Tokyo, Japan); detection, optical rotation [Shodex OR-2 (Showa
Denko Co., Ltd., Tokyo, Japan); mobile phase, CH3CN-H2O (85:15, v/v); flow rate
0.8 mL/min]. Identifications of
aqueous layer were carried out by comparison of their retention times and optical
rotations with those of authentic samples [tR: -apiose, 6.3 min (positive optical
rotation); -glucose, 11.5 min (positive optical rotation)].
D-apiose for 6 and D-glucose for 1–7 present in the
D
D
Acknowledgments
This work was partly supported in part by a Ministry of Education, Culture, Sports,
Science and Technology (MEXT)-Supported Program for the Strategic Research
Foundation at Private Universities.
References and Notes
1
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15

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14 Matsumoto, T.; Nakamura, S.; Nakashima, S.; Yoshikawa, M.; Fujimoto, K.;
16

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25 The stereostructure at 9-position of 7 was not determined.
17

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Legends
Fig. 1. Structures of constituents from the seeds of C. auriculata
Fig. 2. Selected DQF, HMBC and NOESY correlations of 1 and 2
Fig. 3. Selected DQF, HMBC and NOESY correlations of 3, 4, and 5
Fig. 4. Selected DQF, HMBC and NOESY correlations of 6 and 7
18

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Graphical Abstract
19

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Supporting Information
Chemical structures of constituents
from the seeds of Cassia auriculata
Yi Zhang, Seikou Nakamura, Souichi Nakashima, Tao Wang,
Masayuki Yoshikawa, Hisashi Matsuda*
NMR spectrum of new compounds
compound 1
compound 3
compound 5
compound 7
P2–5
compound 2
compound 4
compound 6
P5−8
P9−12
P16−19
P25−27
P12−15
P19−24
1

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auricuoside I (1) 1H NMR (pyridine-d5)
auricuoside I (1) 13 C NMR (pyridine-d5)
2

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auricuoside I (1) DEPT 135 (pyridine-d5)
auricuoside I (1) DQF COSY (pyridine-d5)
3

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auricuoside I (1) HMQC (pyridine-d5)
auricuoside I (1) HMBC (pyridine-d5)
4

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auricuoside I (1) NOESY (pyridine-d5)
auricuoside II (2) 1H NMR (pyridine-d5)
5

ACCEPTED MANUSCRIPT
auricuoside II (2) 13 C NMR (pyridine-d5)
auricuoside II (2) DEPT 135 (pyridine-d5)
6

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auricuoside II (2) DQF COSY (pyridine-d5)
auricuoside II (2) HMQC (pyridine-d5)
7

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auricuoside II (2) HMBC (pyridine-d5)
auricuoside II (2) NOESY (pyridine-d5)
8

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auricuoside III (3) 1H NMR (pyridine-d5)
auricuoside III (3) 13 C NMR (pyridine-d5)
9

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auricuoside III (3) DEPT 135 (pyridine-d5)
auricuoside III (3) DQF COSY (pyridine-d5)
10