Neolignan and megastigmane glucosides from the aerial parts of Isodon japonicus with cell protective effects on BaP-induced cytotoxicity

Article abstract of DOI:10.1016/j.phytochem.2017.02.007

Six neolignan glucosides, named isodonosides I–VI, and a megastigmane glucoside named isodonmegastigmane I, were isolated together with 15 known compounds from the methanolic extract of aerial parts of Isodon japonicus cultivated in Tokushima, Japan. The chemical structures of the compounds were elucidated based on their MS and NMR spectroscopic analysis. The absolute configurations of the neolignan and megastigmane glucosides were determined by derivatizations, by ECD (electronic circular dicroism) Cotton effect approximation, and by the modified Mosher's method. In addition, a significant cell protective effects of neolignan glucosides on benzo[a]pyrene-induced cytotoxicity was found.

Full text of DOI:10.1016/j.phytochem.2017.02.007

Phytochemistry xxx (2017) 1e8
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Neolignan and megastigmane glucosides from the aerial parts of
Isodon japonicus with cell protective effects on BaP-induced
cytotoxicity
Takahiro Matsumoto, Seikou Nakamura, Souichi Nakashima, Tomoe Ohta, Keiko Ogawa,
*
Masashi Fukaya, Junko Tsukioka, Tomohiro Hasei, Tetsushi Watanabe, Hisashi Matsuda
Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto, 607-8412, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 August 2016
Received in revised form
Six neolignan glucosides, named isodonosides IeVI, and a megastigmane glucoside named iso-
donmegastigmane I, were isolated together with 15 known compounds from the methanolic extract of
aerial parts of Isodon japonicus cultivated in Tokushima, Japan. The chemical structures of the compounds
were elucidated based on their MS and NMR spectroscopic analysis. The absolute configurations of the
neolignan and megastigmane glucosides were determined by derivatizations, by ECD (electronic circular
dicroism) Cotton effect approximation, and by the modified Mosher's method. In addition, a significant
cell protective effects of neolignan glucosides on benzo[a]pyrene-induced cytotoxicity was found.
25 January 2017
Accepted 6 February 2017
Available online xxx
Keywords:
Isodon japonicus
Lamiaceae
©
2017 Elsevier Ltd. All rights reserved.
Isodonoside
Isodonmegastigmane
Glucoside
Neolignan
Megastigmane
1
. Introduction
et al., 2016), the glycoside constituents of the aerial parts of I.
Japonicus were examined. Herein, the isolation of glucosides 1e22
and the structure elucidation of six new neolignan glucosides,
named isodonosides I (1), II (2), III (3), IV (4), V (5), and VI (6), and a
new megastigmane glucoside named isodonmegastigmane I (7), as
well as the protective effects of neolignan glucosides on benzo[a]
pyrene-induced cytotoxicity in HT1080 cells are described.
Isodon japonicus (Burm. f.) H. Hara (Lamiacaeae) is a perennial
plant with a wide distribution in China and Japan (Lim et al., 2010).
The aerial parts of I. japonicus have been used as a traditional
medicine for treatment of gastrointestinal disorders, tumors, and
inflammatory diseases (Tanaka et al., 2014). Previous reports
described the structures of diterpenoids (Takeda et al., 1990), fla-
vonoids (Hong et al., 2009), and lignans (Agata et al., 1989) as
constituents of I. japonicus. More than 40 ent-kaurane diterpenoids
were identified in phytochemical studies, and some of these com-
pounds have cytotoxic and antibacterial activities (Hou et al., 2012).
In contrast, there have been few reports about glycosides as con-
stituents of I. japonicus except for only a few flavonoid glycosides
and a diterpene glycoside (Xiang et al., 2015). Based on this infor-
mation and as a part of our ongoing research program for discovery
of bioactive constituents of Asian traditional medicines (Nakamura
et al., 2012, 2013; Matsumoto et al., 2013, 2014a, 2014b; Matsuda
2. Results and discussion
2.1. Isolation of constituents from the aerial parts of I. japonicus
A methanolic extract of the dried aerial parts (8.36%) of I. japo-
nicus (cultivated in Tokushima, Japan) was partitioned between
EtOAc and H O (1:1, v/v) to furnish an EtOAc-soluble fraction
3.45%) and an aqueous layer (4.91%). The latter was further
extracted with n-BuOH to give n-BuOH- (0.24%) and H O- (4.67%)
2
(
2
soluble fractions. The n-BuOH soluble fraction was subjected to
normal- and reversed-phase silica-gel column chromatography and
repeated HPLC. From the n-BuOH-soluble fraction, six new neo-
lignan glucosides, isodonosides I (1), II (2), III (3), IV (4), V (5), and VI
*
Corresponding author.
E-mail address: matsuda@mb.kyoto-phu.ac.jp (H. Matsuda).
http://dx.doi.org/10.1016/j.phytochem.2017.02.007
031-9422/© 2017 Elsevier Ltd. All rights reserved.
0
Please cite this article in press as: Matsumoto, T., et al., Neolignan and megastigmane glucosides from the aerial parts of Isodon japonicus with
cell protective effects on BaP-induced cytotoxicity, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.007

2
T. Matsumoto et al. / Phytochemistry xxx (2017) 1e8
(6), and a megastigmane glucoside, isodonmegastigmane I (7),
double bond on the sidechain (Lundgren et al., 1985) (see Fig. 1).
were isolated together with fifteen known compounds, (7R,8S)-
Isodonosides III (3), IV (4), and V (5) were determined as
0
erythro-guaiacylglycerol-
b
-O-4 -sinapyl ether 9-O-
b
-D-glucopyr-
C
26
32
H O
11 on the basis of their HRMS results. The presence of a D-
0
anoside (8) (Machida et al., 2014), (7S,8S)-threo-4,9,9 -trihydroxy-
glucose moiety was indicated from acid hydrolysis of 3, 4, and 5 as
0
1
13
8
-O-4 -neolignan-7-O-
b
-D-glucopyranoside (9) (Matsuda and
same as 1 and 2. By comparison of the H NMR and C NMR
(Table 2) spectra of 3, 4, and 5 with previous reports, the overall
structure of the neolignan moieties of 3, 4, and 5 were determined
to be the same as the known compound 4-O-methylcedrusin
(Pieters et al., 1993). The relative configurations of 3, 4, and 5 at the
0
0
Kikuchi, 1996), (7S,8S)-threo-4,9,9 -trihydroxy-8-O-4 -neolignan-
0
9
-O-b-D-glucopyranoside (10) (Matsuda and Kikuchi, 1996),
dehydrodiconiferyl-glucoside D (11) (Binns et al., 1987), dihy-
drodehydrodiconiferyl alcohol-9-O- -D-glucopyranoside (12)
Otsuka et al., 2000), (7S,8R)-dihydrodehydrodiconiferyl alcohol-3-
b
0
0
(
C-7 and C-8 positions were also determined as S , R by the NOESY
experiment (Fig. 2). The absolute configurations of the neolignan
moiety of 3, 4, and 5 at C-7 and C-8 were also confirmed on the
basis of the ECD Cotton effects (Schramm et al., 1979). The ECD
O-b-D-glucopyranoside (13) (Yang et al., 2012), (7S,8S)-dihy-
drodehydrodiconiferyl alcohol-9-O-
b
-D-glucopyranoside (14) (Lee
0
et al., 2014), (ꢀ)-secoisolariciresinol-9 -O-
b
-D-glucopyranoside
-ionol-9-O- -D-gluco-
pyranoside (16) (Pabst et al., 1992), (6R,9R)-3-oxo- -ionol-9-O- -D-
glucopyranoside (17) (Pabst et al., 1992), (6S,9S)-9-O- -D-gluco-
pyranosyloxy-6-hydroxy-3-oxo- -ionol (18) (Calis et al., 2002), 3-
hydroxy-5,6-epoxy- -ionol-9-O- -D-glucopyranosyloxy-6-
hydroxy-3-oxo- -ionol (19) (Harput et al., 2002), (4R)-(Z)-3-
methyl-2-(pent-2-en-1-yl)cyclopent-2-en-1-one-4-O- -D-gluco-
pyranoside (20) (Yamamura et al., 1998), phenylethyl-2-O- -D-
glucopyranoside (21) (Miyase et al., 1982), and methylsalicylate-2-
O- -D-glucopyranoside (22) (Karrer and Weidmann, 1920),
(15) (Inoshiri et al., 1987), (6R,9S)-3-oxo-
a
b
spectrum (MeOH) of 3 [203 nm (
D
ε þ13.9), 210 nm (
ε ꢀ1.9)], 4 [203 nm ( ε þ14.4), 212 nm
ε þ0.4), 239 nm ( ε ꢀ4.8)], and 5 [202 nm
ε ꢀ16.5), 227 nm ( ε þ3.5), 242 nm ( ε ꢀ1.8)]
D
ε ꢀ15.3),
a
b
228 nm (
D
ε þ3.3), 239 nm (
ε ꢀ7.1), 225 nm (
ε þ20.6), 211 nm (
D
D
b
(
(
D
D
D
D
D
D
a
D
b
b
were identical to that of the known compound (7R, 8S)-dihy-
drodehydrodiconiferyl alcohol 4-O- -D-glucopyranoside [203 nm
a
b
b
(D
ε þ6.9), 211 nm (
D
ε ꢀ5.3), 226 nm (
D
ε þ2.3), 243 nm ( ε ꢀ1.1)]
D
b
(Machida et al., 2009). In addition, the ECD maxima of 3, 4, and 5
appear to be opposite to those of the 7S, 8R known compounds 12
b
[204 nm (
D
ε ꢀ1.6), 212 nm (
ε þ0.6)] and 13 [203 nm (
ε ꢀ1.5), 242 nm (
D
D
ε þ1.8), 223 nm (
ε ꢀ12.5), 212 nm (
ε þ1.8)]. Thus, the absolute configurations of
D
D
ε ꢀ0.4), 240 nm
1
13
respectively. The H NMR and C NMR spectra and optical rotation
of the known compounds were identical with reported data.
(
(
D
D
ε þ0.6), 226 nm
D
the neolignan moiety at the C-7 and C-8 positions were determined
to be 7R,8S. Based on this evidence, the chemical structures of
isodonosides III (3), IV (4), and V (5) were characterized as shown.
Among the isolated dihydrobenzofuran neolignans, 4-methoxy-
substituted neolignans, such as 3, 4, and 5, are less common than 4-
hydroxy-substituted neolignans.
2.2. Structures of isodonosides IeVI (1e6) and
isodonmegastigmane I (7)
Isodonosides I (1) and II (2) were isolated as white powders with
2
5
25
negative optical rotations (1: [
a
]
D
ꢀ46.1, 2: [
a]
D
ꢀ35.3 in
MeOH). Their IR spectra showed absorption bands due to hydroxy
Isodonoside VI (6) was isolated as a white powder with a
2
5
groups, aromatic rings, and ether functionalities (1: 3400, 1583 and
negative optical rotation ([
a
]
D
ꢀ18.2 in MeOH). Its molecular
ꢀ1
ꢀ1
1
034 cm , 2: 3395, 1583 and 1034 cm ). Their molecular
formula (C25 11) was determined from the positive-ion FABMS
32
H O
1
13
formulae (C27
H
38
O
13 of 1, C26
H
36
O
12 of 2) were determined from
and by HRMS measurement. From the H NMR and C NMR
(Table 2) spectra of 6, the overall structure was determined to be
the same as 3, except for the methoxy group at the C-4 position.
NOESY correlations were observed between H-7 and H-9 (Fig. 2).
Therefore, the relative configuration of 6 at the 7 and 8-positions
þ
the quasimolecular ion peaks (m/z 593 [MþNa] for 1, 563
þ
[
MþNa] for 2) in the positive-ion FABMS and by HRMS mea-
2 4
surement. Acid hydrolysis of 1 and 2 with 5% aqueous H SO in 1,4-
dioxane yielded D-glucose. D-Glucose was identified by HPLC of the
0
0
tolylthiocarbamoyl thiazolidine derivatives (Tanaka et al., 2007).
were determined as S ,R . Next, the ECD (MeOH) maxima of 6
[202 nm ( ε þ10.0), 223 nm ( ε ꢀ1.4), 240 nm
ε ꢀ5.2), 210 nm (
ε þ1.8)] were identical to that of the known compounds 12 and
1
The H NMR (methanol-d
4
) spectra of 1 and 2, which were assigned
D
D
D
by various NMR experiments, showed signals assignable to 1: a
,3,4-trisubstituted benzenring, 1,3,4,5-tetrasubstituted benzene
(D
1
13. In addition, the ECD maxima of 6 appears to be opposite to those
of compounds 3, 4, and 5. Thus, the absolute configurations of the
neolignan moiety at C-7 and C-8 positions were determined to be
7S,8R. From this evidence, the chemical structures of isodonoside VI
(6) were characterized as shown. The structural differences of 6
from known compound 13 were in the position of the glucose
moiety.
ring, three methoxy groups, two oxymethines, two oxymethylenes,
two methylenes, and a glucose moiety. 2: two 1,3,4-trisubstituted
benzene rings, two methoxy groups, two oxymethines, two oxy-
methylenes, two methylenes, and a glucose moiety. According to
the DQF COSY and HMBC spectroscopy, the overall structure of 1
was elucidated as
a
7-glucose-substituted bursenolignan
(
Jutiviboonsuk et al., 2005). Enzymatic hydrolysis of 1 and 2 affor-
Isodonmegastigmane I (7), ([
a
]25
D
ꢀ39.3 in MeOH) was isolated
1
ded the aglycones 1a and 2a. H NMR signals, HR-EI MS, and optical
rotation of 1a were in agreement with that of bursenolignan
as a white powder. Its IR spectrum gave absorption bands at 3350,
ꢀ
1
1259 and 1038 cm , suggesting the presence of hydroxy,
carbonyl, and ether functionalities, respectively. Its molecular
(
Jutiviboonsuk et al., 2005). The erythro configuration of 1 and 2
was also confirmed by the coupling constants (1a: J7,8 ¼ 5.0 in
methanol-d
formula was established to be C19
40 7
H O by the positive-ion FABMS
4
, 2a: J7,8 ¼ 4.5 in chloroform-d) (Miyase et al., 1987;
and by HRMS measurement. Acid hydrolysis of 7 with 5% aqueous
1
Machida et al., 2014). In addition, the absolute configurations of
the neolignan moiety of 1 and 2 at C-7 and C-8 were also confirmed
to be 7S, 8R on the basis of the negative electronic circular di-
2 4 4
H SO -1,4-dioxane yielded D-glucose. The H NMR (methanol-d )
spectra of 7 suggested its megastigmane moiety (an olefin, an
oxymethine, two methylenes, four methyl groups), and a glucose
moiety. From the DQF COSY and HMBC spectroscopy of 7, the
overall structure was determined to be the same as the known
compound 9-hydroxy-5,7-megastigmadien-4-one, except for the
glucose moiety at the C-9 position (Prelog and Meier, 1950). The
absolute configuration of the 9-position in 7 was characterized by
the application of the modified Mosher's method. Namely, enzy-
matic hydrolysis of 7 afforded the aglycone 7a, treatment of which
chroism (ECD) Cotton effects of 1 and 2 [1: 241 nm (
40 nm (
ε ꢀ1.1)] (Huo et al., 2008; Arnoldi and Merlini, 1985).
Consequently, the chemical structures of isodonosides I (1) and II
2) were characterized as shown. There are many reports about 7-
Dε ꢀ4.3) 2:
2
D
(
0
0
or 9 -glucose-substituted 8-O-4 neolignans without a double bond
on the sidechains such as 9 and 10. On the other hand, there is only
one report on 9-glucose-substituted 8-O-4 neolignans without a
0
Please cite this article in press as: Matsumoto, T., et al., Neolignan and megastigmane glucosides from the aerial parts of Isodon japonicus with
cell protective effects on BaP-induced cytotoxicity, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.007

T. Matsumoto et al. / Phytochemistry xxx (2017) 1e8
3
Table 1
1
H NMR Spectroscopic data for compounds 1e6.
Position
1
2
3
4
5
6
d
H
(J in Hz)
d
H
(J in Hz)
d
H
(J in Hz)
d
H
(J in Hz)
d
H
(J in Hz)
d
H
(J in Hz)
2
5
6
7
8
9
6.96 (d, 2.0)
6.70 (d, 8.0)
6.74 (dd, 8.0, 2.0)
4.84 (d, 4.5)
4.42 (m)
7.00 (d, 2.0)
6.70 (d, 8.0)
7.05 (d, 2.0)
6.90 (d, 8.5)
7.00 (dd, 8.5, 2.0)
5.63 (d, 6.0)
3.60 (m)
6.97 (d, 1.7)
6.90 (d, 6.9)
6.95 (dd, 6.9, 1.7)
5.53 (d, 5.2)
3.50 (m)
7.00 (d, 2.0)
6.78 (d, 6.5)
6.90 (dd, 6.5, 2.0)
5.51 (d, 6.0)
3.43 (m)
7.01 (d, 2.0)
6.74 (d, 8.0)
6.86 (dd, 8.0, 2.0)
5.56 (d, 6.5)
3.62 (m)
4.10 (t-like, 9.5)
3.85 (m)
6.55 (s)
6.82 (dd, 8.0, 2.0)
a
4.83
4.49 (m)
4.10 (dd, 11.0, 3.0)
3.31 (dd, 10.0, 5.5)
3.74 (t-like, 9.5)
3.76 (m)
3.74 (t-like, 7.5)
3.82 (m)
0
0
0
0
0
0
2
5
6
7
8
9
6.53 (s)
6.77 (d, 2.0)
6.90 (d, 8.0)
6.65 (dd, 8.0, 2.0)
2.59 (t, 8.0)
1.78 (m)
6.63 (s)
6.91 (s)
6.59 (s)
6.53 (s)
6.56 (s)
6.79 (s)
6.62 (s)
6.66 (s)
2.63 (t, 7.5)
1.82 (m)
3.57 (t, 6.5)
3.82 (s)
2.55 (t, 9.5)
1.76 (m)
3.54 (t, 6.5)
3.80 (s)
2.60 (t, 6.9)
1.79 (t-like, 6.9)
3.55 (t, 5.2)
3.79 (s)b
2.60 (t, 8.0)
1.86 (t-like, 8.0)
3.59 (t, 6.5)
3.74 (s)
2.55 (t, 8.0)
1.78 (m)
3.54 (t, 6.5)
3.82 (s)
3.54 (t, 8.0)
3.79 (s)
3
4
3
5
1
2
3
4
5
6
-OMe
-OMe
3.81 (s)
3.80 (s)b
3.79 (s)
0
0
-OMe
-OMe
3.81 (s)
3.81 (s)
4.29 (d, 7.5)
3.17 (dd, 8.0, 7.5)
3.30 (m)
3.29 (m)
3.30 (m)
3.63 (m)
3.76 (s)
0
0
0
0
0
0
0
0
0
0
0
0
4.32 (d, 8.0)
3.20 (t, 8.0)
3.34 (m)
3.27 (m)
3.34 (m)
4.35 (d, 7.5)
3.23 (t, 7.5)
3.27 (m)
3.28 (m)
3.27 (m)
5.00 (d, 6.3)
3.42 (m)
3.42 (m)
3.38 (m)
3.78 (m)
3.68 (m)
3.83 (m)
4.23 (d, 7.5)
3.18 (dd, 9.0, 7.5)
3.34 (m)
3.26 (m)
3.35 (m)
4.34 (d, 7.5)
3.22 (t, 7.5)
3.28 (m)
3.29 (m)
3.34 (m)
3.82 (m)
3.84 (m)
3.85 (m)
3.87 (m)
3.65 (m)
3.78 (m)
3.68 (m)
3.87 (m)
3.79 (m)
Measured in methanol-d
4
.
a
Overlapped signal.
b
Interchangeable.
Table 2
1
3C NMR Spectroscopic data for compounds 1e6.
Position
1
2
3
4
5
6
Position
1
2
3
4
5
6
0
0
0
0
0
0
0
0
0
1
2
3
4
5
6
7
8
9
133.2
111.4
148.7
146.8
115.7
120.7
73.8
133.9
111.9
148.7
147.0
115.6
121.1
73.8
136.5
110.8
150.5
150.1
112.8
119.4
88.4
135.8
110.9
150.7
150.5
112.9
119.8
89.2
136.5
110.7
150.6
150.2
112.9
119.4
88.4
134.9
110.8
149.0
147.3
116.0
119.8
88.9
1
2
3
4
5
6
7
8
9
139.9
106.9
154.3
134.6
154.3
106.9
33.4
138.0
114.1
151.8
147.0
119.6
121.8
32.7
136.8
116.9
142.0
146.4
129.3
117.1
32.7
137.2
118.2
142.4
147.5
130.4
119.6
32.7
136.7
116.9
141.9
146.5
129.6
117.2
32.7
136.8
116.9
141.9
146.4
129.6
117.1
32.7
86.1
69.6
85.0
69.8
53.7
72.6
55.2
64.9
55.8
65.2
53.2
72.4
35.4
62.2
35.5
62.2
35.8
62.3
35.6
62.2
32.9
69.9
35.8
62.3
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
6
105.0
75.2
77.8
71.5
78.0
105.1
75.2
77.9
71.6
78.0
104.6
75.2
78.1
71.7
78.3
102.8
74.9
77.8
71.4
78.2
104.4
75.2
77.9
71.7
78.1
104.2
75.2
78.1
71.7
78.2
3-OMe
4-OMe
56.4
56.4
56.5
56.5
56.5
56.5
56.5
56.4
56.4
0
3 -OMe
56.7
56.7
56.5
0
5 -OMe
62.6
62.7
62.8
62.5
62.8
62.8
4
Measured in methanol-d .
with (ꢀ)-
a
-methoxy-
a
-(trifluoromethyl) phenylacetyl chloride
2.3. The protective effects of constituents on benzo[a]pyrene-
induced cytotoxicity
[
(ꢀ)-MTPA-Cl] in pyridine yielded (S)-MTPA ester 7b. The (R)-
MTPA ester 7c was derived from 7 by treatment with (þ)-MTPA-Cl
in pyridine. As shown in Fig. 3, the signals due to protons attached
to the 10-positions in 7b were observed at lower fields compared
with those of 7c [Dd: positive], while the signals due to protons
attached to the 4, 5, 7, 8, 11, 12, and 13-positions in the (S)-MTPA
ester (7b) were observed at higher field compared with those of
the (R)-MTPA ester (7c) [Dd: negative]. Thus, the absolute config-
uration at the 9-position in 7 was determined to be R. The absolute
configulation of 7a was different from known compound with
same overall structure (S)-9-hydroxy-5,7-megastigmadien-4-one
Benzo[a]pyrene (BaP) is a well-known environmental carcin-
ogen that promotes oxidative stress and DNA damage and plays a
critical role in lung carcinogenesis (Stephen and Steven, 1993).
Chemopreventive agents against BaP can decrease the level of
DNA damage, and should therefore decrease the risk of cancer
(Zhu et al., 2014). In the present study, to find chemopreventive
agents, cell protective effects of glucoside constituents from I.
japonicus were evaluatedon human fibroblasts fibrosarcoma cell
line (HT1080). Among the isolates, neolignan glucosides 4 [re-
(
D'Abrosca et al., 2004). Based on this evidence, the chemical
covery: 23.2% (P < 0.01) at 10
10 M], 10 [recovery: 14.1% (P < 0.05) at 5.0
51.9% (P < 0.01) at 10 M], and 19 [recovery: 75.3% (P < 0.01) at
M] showed significant protective against benzo[a]pyrene-
m
M], 9 [recovery: 37.0% (P < 0.01) at
structure of isodonmegastigmane I (7) was characterized as
shown.
m
mM], 16 [recovery:
m
1
0 m
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4
T. Matsumoto et al. / Phytochemistry xxx (2017) 1e8
Fig. 1. Structures of compounds isolated from I. japonicus.
induced cytotoxicity (Supporting information S5). In this study, no
structure-activity relationship was clarified among the isolated
compounds.
4. Experimental section
4.1. General experimental procedures
The following instruments were used to obtain physical data:
specific rotations, a Horiba SEPA-300 digital polarimeter (l ¼ 5 cm);
IR spectra, a Thermo Electron Nexus 470; FABMS and HRFABMS, a
JEOL JMS-SX 102A mass spectrometer; EIMS and HREIMS, a JEOL
3
. Conclusion
Six new neolignan glucosides isodonosides I (1), II (2), III (3), IV
1
JMS-GCMATE mass spectrometer; H-NMR spectra, JEOL JNM-LA
(4), V (5), and VI (6) and a new megastigmane glucoside, iso-
5
00 (500 MHz) and JNM-ECA 600K (600 MHz) spectrometers;
C-NMR spectra, JEOL JNM-LA 500 (125 MHz) and JNM-ECA 600K
donmegastigmane I (7), were isolated from aerial parts of I. japo-
nicus cultivated in Tokushima, Japan. The absolute configurations of
the new compounds were elucidated by derivatization, by ECD
Cotton effect approximations, and by modified Mosher's method. In
this study, the erythro type (1 and 2) and threo type (8e10), the
trans configuration at the benzofuran ring (3e6 and 11e13), as well
as the cis configuration of the benzofuran ring (14) neolignans were
isolated. Aglycones of new compounds 1 and 2 were elucidated as
enantiomers of aglycones of known compound 8. From these re-
sults, it is concluded that the absolute stereo structure of these
neolignans resulted from non-specified phenoxy radical couplings
in this plant. On the other hand, because of steric effects, neolignans
with a cis-dihydrobenzofuran ring were comparatively difficult to
be biosynthesized in plants. In addition, significant protective ef-
fects of neolignan glucosides on benzo[a]pyrene-induced cytotox-
icity were found.
13
(
(
150 MHz) spectrometers; NOESY spectra, JEOL JNM-ECA 600K
600 MHz) spectrometer; ECD spectra, a Jasco J-1500 circular di-
chroism spectrometer; HPLC, a Shimadzu SPD-10AVP UV-VIS de-
tector. YMC Triart PFP (250 ꢁ 4.6 mm i.d. and 250 ꢁ 10 mm i.d.),
YMC Pack Ph (250 ꢁ 4.6 mm i.d. and 250 ꢁ 20 mm i.d.), YMC Triart
PFP (250 ꢁ 4.6 mm i.d. and 250 ꢁ 10 mm i.d.), COSMOSIL 5PFP
(
(
250 ꢁ 4.6 mm i.d. and 250 ꢁ 20 mm i.d.), COSMOSIL 5C18-MS-II
250 ꢁ 4.6 mm i.d., 250 ꢁ 10 mm i.d. and 250 ꢁ 20 mm i.d.) and
COSMOSIL 5C18-PAQ (250 ꢁ 4.6 mm i.d. and 250 ꢁ 20 mm i.d.)
columns were used for analytical and preparative purposes.
The following experimental materials were used for chroma-
tography: normal-phase silica gel column chromatography (CC),
silica gel BW-200 (Fuji Silysia Chemical, Ltd., 150e350 mesh);
reversed-phase silica gel CC, Chromatorex ODS DM1020T (Fuji
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T. Matsumoto et al. / Phytochemistry xxx (2017) 1e8
5
Fig. 2. Important 2D NMR correlations of compounds 1e7.
Tenkaido Co. Ltd (Osaka, Japan) in September 2015. A voucher
specimen is on file in our laboratory (KPU IJ-2016-1).
4.4. Extraction and isolation
Dried aerial parts of I. japonicus (11.4 kg) were extracted with
MeOH (90 L ꢁ 3) under conditions of reflux for 3 h. Evaporation of
the combined extracts under reduced pressure provided a MeOH
extract (953.4 g, 8.36%). The latter was partitioned into an EtOAc-
Fig. 3. Determination of the absolute configurations of 9-position in 7.
H
2
O (1:1, v/v) mixture to furnish an EtOAc- soluble fraction
393.3 g, 3.45%) and an aqueous phase. The latter was further
extracted with n-BuOH to give a n-BuOH-soluble fraction (27.4 g,
0.24%) and an H O-soluble fraction (532.3 g, 4.67%). The n-BuOH-
soluble fraction was subjected to normal phase silica gel CC [1.2 kg,
CHCl / CHCl eMeOH (10:1 / 5:1 / 4:1 / 3:1 / 2:1 / 1:1
(
Silysia Chemical, Ltd., 100e200 mesh); Sephadex LH-20 (GE
Healthcare); TLC, precoated TLC plates with silica gel 60F254 (Merck,
2
0
0
.25 mm) (ordinary phase) and silica gel RP-18 F254S (Merck,
.25 mm) (reversed phase); reversed-phase HPTLC, precoated TLC
3
3
plates with silica gel RP-18 WF254S (Merck, 0.25 mm). Detection of
compounds was achieved by UV irradiation and by spraying with
/ 1:2 / 1:4, v/v) /MeOH] to give seven fractions [Fr.B1eFr.B8].
Fraction B4 (1.6 g) was further separated by reversed phase silica
1
% Ce(SO
4
)
2
e10% aqueous H
2
SO
4
followed by heating.
2
gel CC [40.0 g, MeOH:H O (2:8 / 3:7 / 4:6 / 5:5 / 6:4 /
7:3 / 8:2 / 9:1 v/v) / MeOH] to give twelve fractions [Fr.B4-
1
{
eFr.B4-12]. Fraction B4-4 (131.9 mg) was separated by HPLC,
O:MeCN (85:15, v/v) [YMC Pack Ph (250 ꢁ 20 mm
i.d.)]} followed by {mobile phase: H O:MeCN (85:15, v/v) [COS-
MOSIL 5C18-MS-II (250 ꢁ 10 mm i.d.)]} to give 21 (3.7 mg,
.000032%) and 22 (0.9 mg, 0.0000079%). Fraction B4-6 (227.2 mg)
was separated by HPLC, {mobile phase: H O:MeCN (80:20, v/v)
4.2. Reagents for bioassay
mobile phase: H
2
2
Minimum essential medium Eagle's (MEM) and fetal bovine
serum (FBS) were purchased from Sigma-Aldrich (MO, U.S.A.).
Other reagents were from Wako Pure Chemical Industries, Ltd.
Osaka, Japan). 96-Well microplates were purchased from Greiner
Japan Co., Ltd. (Tokyo, Japan).
0
2
(
[
H
7
COSMOSIL 5C18-MS-II (250 ꢁ 20 mm i.d.)]} and {mobile phase:
2
O:MeCN (77:23, v/v) [YMC triart PFP (250 ꢁ 10 mm i.d.)]}to give
(4.5 mg, 0.000039%), 16 (15.6 mg, 0.00014%), 17 (7.1 mg,
4.3. Plant material
0.000062%), and 20 (1.2 mg, 0.000011%). Fraction B5 (4.5 g) was
further separated by reversed phase silica gel CC [150.0 g,
Dried aerial parts of I. japonicus cultivated in Tokushima, Japan,
2
MeOH:H O (1:9 /2:8 / 3:7 / 4:6 / 5:5 / 6:4 / 7:3
were obtained as commercial products purchased from Tochimoto
/ 8:2 / 9:1 v/v) / MeOH] to give fifteen fractions [Fr.B5-
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6
T. Matsumoto et al. / Phytochemistry xxx (2017) 1e8
þ
1
eFr.B5-15]. Fraction B5-3 (407.3 mg) was further separated by size
FABMS: m/z 545 [MþNa] ; HRFABMS: m/z 545.2005 (Calcd for
þ
exclusion CC (10.0 g, MeOH) to give six fractions [Fr.B5-3-1eFr.B5-
-6]. Fraction Fr.B5-3-1 (44.4 mg) and Fr.B5-3-2 (281.8 mg) were
individually purified by HPLC [COSMOSIL 5C18-MS-II
O:MeCN (87:13, v/v), 3.0 mg,
O:MeCN (87:13, v/v), 3.0 mg, 0.000026%], 19
O:MeCN (87:13, v/v), 15.5 mg, 0.00014%], and eleven fractions
Fr.B5-3-2-1eFr.B5-3-2-11]. Fraction Fr.B5-3-2-11 (9.2 mg) was
O:MeCN (8:92, v/v)
C
26
34
H O
11Na [MþNa] : m/z 545.1999).
3
4.9. Isodonoside V (5)
(
0
250 ꢁ 20 mm i.d.)] to give 2 [H
2
.000026%], 18 [H
2
25
White powder; [
a
]
D
-43.4 (c 0.25, MeOH); IR(KBr):
n
max 3390,
[H
2
ꢀ1
1
3
305, 1604, and 1080 cm ; For H NMR (methanol-d
4
, 500 MHz)
[
13
and C NMR (125 MHz) spectroscopic data, see Tables 1 and 2; UV
further purified by HPLC {mobile phase: H
2
(
(
[
[
MeOH)
l
max 281.6 nm (log ε 3.2); ECD:
D
ε (nm) þ20.6 (202), ꢀ16.5
[
COSMOSIL HILIC (250 ꢁ 10 mm i.d.)]} to give 13 (2.1 mg,
211), þ3.5 (227), ꢀ1.8 (242) (MeOH); positive-ion FABMS: m/z 545
0
.000018%). Fraction Fr.B5-4 (239.8 mg) was applied to HPLC,
O:MeCN (85:15, v/v) [COSMOSIL 5PFP
250 ꢁ 20 mm i.d.)]} and {mobile phase: H O:MeCN (85:15, v/v)
YMC Triart 5C18 (250 ꢁ 10 mm i.d.)]} to give 10 (1.2 mg,
.000011%), 6 (2.7 mg, 0.000024%), and 9 (1.2 mg, 0.000011%).
Fraction Fr.B5-5 (332.1 mg) was subjected to HPLC {mobile phase:
þ
þ
MþNa] ; HRFABMS: m/z 545.2005 (Calcd for
C
26 34
H O11Na
{
(
[
mobile phase:
H
2
MþNa] : m/z 545.1999).
2
4.10. Isodonoside VI (6)
0
25
White powder; [
a
]
D
-18.2 (c 0.27, MeOH); IR(KBr):
n
max 3400,
13
H
2
O:MeCN (80:20, v/v) [COSMOSIL 5C18-MS-II (250 ꢁ 20 mm
ꢀ1
1
1
607, and 1031 cm ; For H NMR (methanol-d
4
, 500 MHz) and
C
i.d.)]} to give 12 (13.0 mg, 0.00011%), 14 (11.6 mg, 0.0001%), and six
fractions [Fr.B5-5-1eFr.B5-5-6]. Fraction Fr.B5-5-1 (21.9 mg) and
Fr.B5-5-2 (34.1 mg) were individually subjected to HPLC [YMC
NMR (125 MHz) spectroscopic data, see Tables 1 and 2; UV (MeOH)
max 282.8 nm (log ε 3.0); ECD:
l
D
ε (nm) ꢀ5.2 (202), þ10.0
(
[
[
210), ꢀ1.4 (223), þ1.8 (240) (MeOH); positive-ion FABMS: m/z 531
Triart PFP (250 ꢁ 10 mm i.d.)] to give 1 [H
.1 mg, 0.000045%], 8 [H O:MeCN (83:17, v/v), 2.9 mg, 0.000025%],
and 4 [H O:MeCN (81:19, v/v), 9.6 mg, 0.000084%]. Fraction Fr.B5-6
340.7 mg) was purified by HPLC {mobile phase: H O:MeCN (80:20,
v/v) [COSMOSIL 5C18-MS-II (250 ꢁ 20 mm i.d.)]}, {mobile phase:
O:MeCN (80:20, v/v) [YMC Triart PFP (250 ꢁ 10 mm i.d.)]}, and
mobile phase: H O:MeCN (80:20, v/v) [COSMOSIL 5C18-MS-II
2
O:MeCN (83:17, v/v),
þ
þ
MþNa] ; HRFABMS: m/z 531.1846 (Calcd for
C
25 32
H O11Na
5
2
MþNa] : m/z 531.1842).
2
(
2
4
.11. Isodonmegastigmane I (7)
H
{
2
2
5
White powder; [
a
]
D
-39.3 (c 0.4, MeOH); IR(KBr):
n
max 3350,
13
2
ꢀ
1
1
1
259, and 1038 cm ; For H NMR (methanol-d
4
, 500 MHz) and
C
(
0
250 ꢁ 10 mm i.d.)]}to give 3 (1.0 mg, 0.0000088%), 5 (2.5 mg,
NMR (125 MHz) spectroscopic data, see Table 3; positive-ion
.000022%), 11 (1.1 mg, 0.0000096%), and 15 (13.5 mg, 0.00012%).
þ
FABMS: m/z 393 [MþNa] ; HRFABMS: m/z 393.1892 (Calcd for
þ
C
19
H
40
O
7
Na [MþNa] : m/z 393.1889).
4.5. Isodonoside I (1)
2
5
White powder; [
a
]
D
-46.1 (c 0.4, MeOH); IR(KBr):
n
max 3400,
13
4.12. Enzymatic hydrolysis of 1 and 2
ꢀ
1
1
1
583, and 1034 cm ; For H NMR (methanol-d
4
, 500 MHz) and
C
NMR (125 MHz) spectroscopic data, see Tables 1 and 2; UV (MeOH)
To the individual solution of 1 (2.0 mg) or 2 (2.0 mg) in 20 mM
l
max 230.0 nm (log ε 3.3), 280.2 nm (log ε 2.9); ECD:
D
ε (nm) ꢀ4.3
acetate buffer (2.5 mL, pH ¼ 5.0) was added
b-glucosidase (1 mg,
from Sweet almond), and cellulase (1 mg). The mixtures were then
þ
(
241) (MeOH); positive-ion FABMS: m/z 593 [MþNa] ; HRFABMS:
þ
ꢂ
m/z 593.2213 (Calcd for C27
38
H O
13Na [MþNa] : m/z 593.2210).
individually stirred for 12 h at 50 C. Each supernatant solution was
concentrated under vacuum to give the correspond residues, which
4
.6. Isodonoside II (2)
3
were subjected to normal phase silica gel CC [0.5 g, CHCl /
MeOH] to give 1a (0.8 mg) or 2a (0.7 mg).
2
5
White powder; [
a
]
D
-35.3 (c 0.3, MeOH); IR(KBr):
n
max 3395,
13
ꢀ1
1
1583, and 1034 cm ; For H NMR (methanol-d
4
, 500 MHz) and
C
Table 3
NMR (125 MHz) spectroscopic data, see Tables 1 and 2; UV (MeOH)
1H and 13C NMR spectroscopic data for compounds 7 and 7a.
lmax 230.4 nm (log ε 3.6), 280.4 nm (log ε 3.2); ECD:
D
ε (nm) ꢀ1.1
þ
Position
7
7a
(
240) (MeOH); positive-ion FABMS: m/z 563 [MþNa] ; HRFABMS:
þ
m/z 563.2108 (Calcd for C26
36
H O
12Na [MþNa] : m/z 563.2104).
d
C
d
H (J in Hz)
d
C
d H (J in Hz)
1
2
3
4
5
6
7
8
9
10
163.7
130.7
201.8
35.1
38.2
36.6
127.5
140.4
77.2
21.0
13.7
27.7
27.7
102.7
75.3
78.0
71.5
78.1
62.6
160.5
130.0
199.4
34.3
37.2
35.4
125.3
140.4
68.7
23.6
13.4
4
.7. Isodonoside III (3)
2
5
2.48 (t, 6.5)
1.85 (t, 6.5)
2.50 (t, 6.5)
1.85 (t, 6.5)
White powder; [
a
]
D
-135.6 (c 0.1, MeOH); IR(KBr):
n
max 3350,
13
ꢀ
1
1
1606, and 1033 cm ; For H NMR (methanol-d
4
, 500 MHz) and
C
NMR (125 MHz) spectroscopic data, see Tables 1 and 2; UV (MeOH)
max 281.4 nm (log ε 2.3); ECD:
6.29 (d, 16.0)
5.75 (dd, 16.0, 6.0)
4.50 (m)
1.35 (d, 6.0)
1.78 (s)
1.17 (s)
1.18 (s)
4.39 (d, 8.0)
3.19 (m)
3.24 (m)
6.20 (d, 16.0)
5.70 (dd, 16.0, 6.0)
4.45 (m)
1.35 (d, 6.0)
1.78 (s)
1.17 (s)
l
Dε (nm) þ13.9 (203), ꢀ15.3
(
[
[
210), þ3.3 (228), ꢀ1.9 (239) (MeOH); positive-ion FABMS: m/z 545
þ
þ
MþNa] ; HRFABMS: m/z 545.1993 (Calcd for
26 34
C H O11Na
1
1
1
1
2
3
4
5
6
1
2
MþNa] : m/z 545.1999).
27.3
27.3
3
1.18 (s)
0
4.8. Isodonoside IV (4)
0
0
0
0
0
2
5
White powder; [
305, 1068, 1033, 1604and 1517 cm ; For H NMR (methanol-d
00 MHz) and 13C NMR (125 MHz) spectroscopic data, see Tables 1
max 281.0 nm (log ε 3.1); ECD:
ε (nm) þ14.4
203), ꢀ7.1 (212), þ0.4 (225), ꢀ4.8 (239) (MeOH); positive-ion
D
a] -24.1 (c 0.48, MeOH); IR(KBr): nmax 3390,
3.34 (m)
3.35 (m)
3.65 (dd, 11.0, 5.5)
ꢀ
1
1
3
5
4
,
3.89 (d-like, 11.0)
and 2; UV (MeOH)
(
l
D
4
Measured in methanol-d .
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cell protective effects on BaP-induced cytotoxicity, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.007

T. Matsumoto et al. / Phytochemistry xxx (2017) 1e8
7
2
5
1
a: Colorless oil; [
a
]
D
þ40.7 (c 0.2, MeOH); positive-ion EIMS m/
Bank (Osaka, Japan). Cells were maintained in MEM supplemented
with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin.
þ
þ
z 408 [M] ; HREIMS m/z 593.2213 (calcd for C27
38
H O
13 [M] ,
5
93.2210).
a: Colorless oil; [
2
5
2
a]
D
þ15.8 (c 0.07, MeOH); positive-ion EIMS
4.17. MTT assay
þ
þ
m/z 378 [M] ; HREIMS m/z 378.1671 (calcd for C26
36
H O
12 [M] ,
3
78.1679).
.13. Enzymatic hydrolysis of 7
To a solution of 7 (3.8 mg) in 20 mM acetate buffer (2 mL,
Human fibrosarcoma HT1080 cells were seeded into 96-well
4
plates (5.0 ꢁ 10 cells/100
mL/well). Cells were treated with
50 M BaP alone or with test samples for 20 h. After that, MTT
4
m
solution (10
m
L, 5 mg/mL) was added to the well and incubated for
ꢂ
4 h at 37 C. After removal of the culture medium, the cells were
dissolved by i-PrOH including 0.04 M HCl and absorbance was
measured at 570 nm with a reference wavelength at 655 nm. Re-
sults expressed the percent of recovery rate compared to the con-
trol- and normal- group.
pH ¼ 5.0) was added hesperidinase (2 mg, from Penicillium sp.
ꢂ
Sigma-Aldrich) and the mixture was stirred for 12 h at 50 C. The
supernatant solution was concentrated under vacuum to give a
residue, which was subjected to normal phase silica gel CC [0.5 g,
3
CHCl / MeOH] to give 7a (1.6 mg).
2
5
1
7
a: Colorless oil; [
a
]
D
-46.1 (c 0.4, MeOH); For H NMR (meth-
4.18. Statistical analyses
1
3
anol-d
4
, 500 MHz) and C NMR (125 MHz) spectroscopic data, see
þ
Table 3; positive-ion EIMS: m/z 208 [M] ; HREIMS m/z 208.1458
Values are expressed as mean ± S.E.M. (n ¼ 4) One-way analysis
of variance followed by Dunnett's test was used for statistical an-
alyses. Probability (p) values less than 0.05 were considered
significant.
þ
(
calcd for C13
20
H O
2
[M] , 208.1463).
4.14. Preparation of the (S)- and (R)-MTPA esters (7b and 7c) of 7a
A solution of 7a (0.8 mg) in pyridine (0.5 ml) was treated with
Acknowledgments
(
ꢀ)-MTPA-Cl (0.02 mL), and the mixture was stirred at r.t. for 12 h.
Removal of the solvent from the reaction mixture under reduced
pressure furnished a residue, which was subjected to normal phase
This research was supported by MEXT (Ministry of Education,
Culture, Sports, Scientific and Technology) - Supported Program for
the Strategic Research Foundation at Private Universities,
2015e2017.
silica gel CC [0.5 g, CHCl
.8 mg). Employing a similar procedure, (R)-MTPA ester (7c, 0.9 mg)
was obtained from 7a (0.8 mg, 0.0059 mmol) using (þ)-MTPA-Cl.
3
/ MeOH] to give (S)-MTPA ester (7b,
0
1
(
S)-MTPA ester (7b). H-NMR (chloroform-d, 500 MHz)
d
2.49 (t,
Appendix A. Supplementary data
J ¼ 6.4, H-4), 1.83 (t, J ¼ 6.4, H-5), 6.22 (d, J ¼ 16.8, H-7), 5.51 (m, H-
8
), 3.76 (m, H-9), 1.49 (d, J ¼ 6.0, H-10), 1.72 (s, H-11), 1.07 (s, H-12),
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.phytochem.2017.02.007.
þ
1
C
.08 (s, H-13); EIMS: m/z 424 [M] ; HREIMS m/z 424.1864 (calcd for
þ
H O
23 27 4
F
3
[M] , m/z 424.1862).
1
(R)-MTPA ester (7c). H-NMR (chloroform-d, 500 MHz)
d
2.50 (t,
References
J ¼ 6.4, H-4), 1.84 (t, J ¼ 6.4, H-5), 6.33 (d, J ¼ 15.2, H-7), 5.62 (m, H-
Agata, I., Hatano, T., Nishibne, S., Okuda, T., 1989. A tetrameric derivative of caffeic
acid from Rabdosia japonica. Phytochemistry 28, 2447e2450.
Arnoldi, A., Merlini, L., 1985. Asymmetric synthesis of 3-methyl-2-phenyl-1,4-
benzodioxanes. Absolute configuration of the neolignans eusiderin and eusi-
derin C and D. Chem. Soc. Perkin Trans. 1, 2555e2557.
8
), 3.76 (m, H-9), 1.44 (d, J ¼ 6.0, H-10), 1.76 (s, H-11), 1.10 (s, H-12),
þ
1
.10 (s, H-13); EIMS: m/z 424 [M] ; HREIMS m/z 424.1864 (calcd for
þ
23 27 4 3
C H O F [M] , m/z 424.1862).
Binns, N.A., Chen, H.R., Wood, N.H., Lynn, G.D., 1987. Cell division promoting activity
of naturally occurring dehydrodiconiferyl glucosides: do cell wall components
control cell division? Proc. Natl. Acad. Sci. U. S. A 84, 980e984.
4.15. Acid hydrolysis of 1e7
To determine the absolute configurations of the constituent
monosaccharids of 1 and 2, the reported method by Tanaka et al.
was used with slight modifications as follows. Compounds 1e7
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a-
(
0.4 mg) were dissolved in 5% aqueous H
v, 2.0 mL), then each solution heated at 90 C for 3 h. After
2
SO
4
in 1,4-dioxane (1:1, v/
ꢂ
extraction with EtOAc ( ꢁ 3), the solutions were individually
ꢀ
neutralized with Amberlite IRA-400 (OH form). After drying in
vacuo, each residue was dissolved in pyridine (0.1 mL) containing L-
ꢂ
cysteine methyl ester hydrochloride (0.5 mg) and heated at 60 C
for 1 h. Each solution of o-tolylisothiocyanate (0.5 mg) in pyridine
ꢂ
(
0.1 mL) was added to each mixture and heated at 60 C for 1 h. The
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COSMOSIL 5C18-MS-II (Nacalai Tesque), 250 ꢁ 4.6 mm i.d. (5 m);
mobile phase: MeCN:H O (18:82, v/v); detection: UV (254 nm);
m
2
ꢂ
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Please cite this article in press as: Matsumoto, T., et al., Neolignan and megastigmane glucosides from the aerial parts of Isodon japonicus with
cell protective effects on BaP-induced cytotoxicity, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.007