Glycosides from the bark of Machilus robusta

Article abstract of DOI:10.1080/10286020.2013.785530

Six new glycosidic constituents (1-6), together with 10 known analogs, have been isolated from the bark of Machilus robusta. Structures of the new compounds, including the absolute configurations, were determined by spectroscopic and chemical methods as (

Full text of DOI:10.1080/10286020.2013.785530

This article was downloaded by: [Tulane University]
On: 25 January 2015, At: 03:29
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Asian Natural Products
Research
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/ganp20
Glycosides from the bark of Machilus
robusta
Peng-Bin Bu a , Yan-Ru Li a , Ming Jiang a , Fang Wang a , Xiao-Liang
Wang a , Sheng Lin a , Cheng-Gen Zhu a & Jian-Gong Shi a
a State Key Laboratory of Bioactive Substance and Function of
Natural Medicines, Institute of Materia Medica, Chinese Academy
of Medical Sciences and Peking Union Medical College , Beijing ,
100050 , China
Published online: 24 Apr 2013.
To cite this article: Peng-Bin Bu , Yan-Ru Li , Ming Jiang , Fang Wang , Xiao-Liang Wang , Sheng Lin ,
Cheng-Gen Zhu & Jian-Gong Shi (2013) Glycosides from the bark of Machilus robusta , Journal of
Asian Natural Products Research, 15:5, 482-491, DOI: 10.1080/10286020.2013.785530
To link to this article: http://dx.doi.org/10.1080/10286020.2013.785530
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising
out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

Journal of Asian Natural Products Research, 2013
Vol. 15, No. 5, 482–491, http://dx.doi.org/10.1080/10286020.2013.785530
Glycosides from the bark of Machilus robusta
Peng-Bin Bu, Yan-Ru Li, Ming Jiang, Fang Wang, Xiao-Liang Wang, Sheng Lin*,
Cheng-Gen Zhu and Jian-Gong Shi*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College,
Beijing 100050, China
(Received 12 January 2013; final version received 10 March 2013)
Six new glycosidic constituents (1–6), together with 10 known analogs, have been
isolated from the bark of Machilus robusta. Structures of the new compounds,
including the absolute configurations, were determined by spectroscopic and chemical
methods as (2)-nectandrin B-b-D-glucopyranoside (1), (2)-(7R,70R,8S,80R)-4,
40-dihydroxy-3,30-dimethoxy-7,70-epoxylignan-4-O-b-D-glucopyranoside (2), (2)-
(7R,70R,8S,80R)-4,40-dihydroxy-3,30-dimethoxy-7,70-epoxylignan-40-O-b-D-glucopyra-
noside (3), (2)-(8S,80R)-4,40-dihydroxy-3,30,50-trimethoxylignan-40-O-b-D-glucopyr-
anoside (4), (2)-(7R,8R)-syringylglycerol-8-O-b-D-glucopyranoside (5), and (2)-3-
hydroxy-2-methyl-4-pyrone-3-O-b-D-xylopyranosyl-(1 ! 6)-O-b-D-glucopyranoside
(6), respectively.
Keywords: Machilus robusta; Lauraceae; 7,70-epoxylignan glucosides; ligningluco-
side; syringylglycerol glucoside; 2-methyl-4-pyrone diglycoside
1. Introduction
bark of M. robusta, and some of these
compounds showed activity against HIV-1
replication and PC12 cell damage [12].
Continuing examination of the water
soluble potion of the extract has resulted
in the characterization of six new (1–6)
(Figure 1) and 10 known glycosidic
metabolites. Based on IUPAC recommen-
dations for the nomenclature of lignans
and neolignans [13], compounds 1–3 are
categorized as 7,70-epoxylignan b-D-glu-
cosides, and 4 is a lignan b-D-glucoside.
Compound 5 is a syringylglycerol 8-O-
b-D-glucopyranoside with the 7R,8R-con-
figuration (a pair of the structures with the
same relative configuration was reported
from Alangium premnifolium [14]), and 6
is an uncommon 2-methyl-4-pyrone digly-
coside. In this paper, we describe the
isolation and structure elucidation of these
compounds.
Species of the genus Machilus (Lauraceae)
are sources of secondary metabolites with
interesting chemical structures (lignans,
butanolides, sesquiterpenes, alkaloids, and
flavonoids) and significant bioactivities
[1–3]. Several plants of this genus have
long been used for the treatment of various
diseases including edema, abdominal dis-
tension, pain, and inflammation in China
[4]. As part of a program to assess the
chemical and biological diversity of
several traditional Chinese medicines, we
have investigated three species of the
genus, including M. yaoshansis [5–9], M.
wangchiana [10,11], and M. robusta [12]
widely distributed in the south of China. In
our previous study, 28 metabolites were
characterized from the EtOAc soluble
portion of the ethanolic extract of the
*Corresponding authors. Email: lsznn@126.com; shijg@imm.ac.cn
q 2013 Taylor & Francis

Journal of Asian Natural Products Research
483
9'
8'
7'
9
8
7
OMe
OR2
OMe
OH
1'
3'
MeO
R1O
1
MeO
RO
3
O
O
5'
5
2
R1 = Glu, R2 = H
R1 = H, R2 = Glu
R1 = R2 = H
1
R = Glu
3
1a R = H
2a
7
9
MeO
HO
1
8
3
OH
9
8'
HO
HO
HO
7'
5'
OH
7
9'
O
5
O
1'
1'
3'
1
OH
3'
5'
5
3
MeO
OMe
MeO
OMe
O
OR
OH
5
4
R = Glu
R = H
O
HO
HO
4a
O
1''
O
HO
OH
O
7
3
1'
5
HO
HO
5"
O
1"
Glu =
OH
HO
HO
O
1
3"
6
OH
Figure 1. The structures of compounds 1–6.
2. Results and discussion
H-70), two aliphatic methines at d 2.27
(2H, m, H-8 and H-80), and two aliphatic
methyl groups at d 0.96 (H3-9) and 0.99
(H3-90), as well as characteristic signals
due to a b-glucopyranosyl unit (Table 1)
with an anomeric proton at d 4.84 (d,
J ¼ 7.5 Hz). The 13C NMR and DEPT
spectra of 1 showed carbon signals
corresponding to the above units (Table 1).
The presence of four oxygen-bearing aro-
matic carbons (dC . 145 ppm) in the 13C
NMR spectrum, in combination with the
molecular composition, suggested that 1
was a 7,70-epoxylignan b-glucopyranoside
with two phenolic OH and two aromatic
methoxy groups. This was confirmed by
2D NMR data analysis of 1. Extensive
analysis of gHSQC spectroscopic data of 1
provided unambiguous assignments of
proton and corresponding carbon signals
in the NMR spectra. 1H–1H gCOSY
correlations of H-5/H-6, H-7/H-8/H3-9,
Compound 1 was obtained as a white solid
20
with ½aꢀD 222.5 (c ¼ 0.10, MeOH). The
IR spectrum of 1 showed absorption band
)
for aromatic ring (1601 and 1515 cm21
functional groups. The (þ)-ESI mass
spectrum of 1 exhibited a quasi-molecular
ion peak at m/z 529 [M þ Na]þ and (þ)-
HR-ESI-MS at m/z 529.2049 [M þ Na]þ
indicated the molecular formula
1
C26H34O10. The H NMR spectrum of 1
in MeOH-d4 showed resonances attribu-
table to two 1,3,4-trisubstitued phenyl
rings at d 7.03 (br s, H-2), 7.11 (d,
J ¼ 8.5 Hz, H-5), and 6.93 (br d,
J ¼ 8.5 Hz, H-6), and 6.95 (br s, H-20),
6.74 (d, J ¼ 8.0 Hz, H-50), and 6.83 (br d,
J ¼ 8.0 Hz, H-60) and two aromatic meth-
oxy groups at d 3.79 and 3.80. In addition,
it displayed resonances assignable to two
oxygen-bearing methines at d 4.45 (d,
J ¼ 5.5 Hz, H-7) and 4.43 (d, J ¼ 6.0 Hz,

484
P.-B. Bu et al.
d
d
d
d
d
d
d
d
d
d

Journal of Asian Natural Products Research
485
H-50/H-60, and H-70/H-80/H3-90 verified the
structure units with vicinal coupling
protons in 1. In the HMBC spectrum of
1, long-range correlations from H-7 to C-1,
C-2, C-6, C-8, C-9, C-70, and C-80 and
from H-70 to C-10, C-20, C-60, C-80, C-90, C-
7, and C-8, in combination with their
chemical shifts, confirmed the 7,70-epox-
ylignan skeleton. Meanwhile, HMBC
correlations of C-3 with H-2, H-5 and 3-
OCH3 and correlations of C-30 with H-20
and H-50 and 30-OCH3 demonstrated
that the two OCH3 groups were located
at C-3 and C-30 in 1, respectively.
In addition, an HMBC correlation from
H-100 to C-4 revealed that the b-glucopyr-
anosyl unit was attached to C-4. Thus, the
planar structure of 1 was elucidated as
4,40-dihydroxy-3,30-dimethoxy-7,70-epox-
ylignan-4-O-b-glucopyranoside.
trans configuration [17,19–23]. The elu-
cidation was further confirmed by NOE
enhancements of H3-9 and H3-90 upon
irritation of H-70 in the NOE difference
experiment of 2. Enzymatic hydrolysis
20
of 2 produced glucose {½aꢀD þ 42.1
20
(c ¼ 0.08, H2O)} and 2a {½aꢀD 235.6
(c ¼ 0.16, MeOH)}. The NMR data of 2a
were consistent with those of machilin-I
[17] and 30,300-dimethoxylarreatricin [23],
which have the same relative configur-
ation, but both the absolute configurations
have not been determined. The CD spectra
displayed negative Cotton effects corre-
1
1
sponding to the La and Lb transitions of
the aromatic units at 231 (D1 29.74) and
278 (D1 22.29) nm for 2 and at 231 (D1
20.32) and 272 (D1 20.11) nm for 2a,
which are opposite to those of chicanine
[20]. Since the absolute configuration of
chicanine was determined by chemical
transformation to (2)-galcitin [20] and
the chirality at C-7 and C-70 was reported
to be expected to have a major influence on
the electronic transition of the aromatic
chromaphore [24], the configuration of
2 was assigned as 7R,70R,8S,80R. There-
fore, compound 2 was determined to be
(2)-(7R,70R,8S,80R)-4,40-dihydroxy-3,30-
dimethoxy-7,70-epoxylignan-4-O-b-D-glu-
copyranoside.
In the NOESY spectrum of 1, corre-
lationsofH-7/H3-9andH-70/H3-90 indicated
the 7,8-trans-70,80-trans configuration.
Enzymatic hydrolysis of 1 produced a
sugar and 1a. The sugar was identified as
D-glucose by TLC comparison with an
authentic sample and the positive specific
20
rotation, ½aꢀD þ 45.1 (c ¼ 0.13, H2O) [7].
20
The 1H NMR and specific rotation
{½aꢀD < 0 (c ¼ 0.12, MeOH)} data of 1a
were identical to those of nectandrin B [15–
18]. Therefore, the structure of compound 1
was determined as (2)-nectandrin B-b-D-
glucopyranoside.
Compound 3 showed spectroscopic
data similar to those of 2, except that the
chemical shifts of the phenyl moieties
were changed in the NMR spectra of 3
(Table 1). This suggested that the b-
glucopyranosyl moiety was located at C-40
in 3 instead of at C-4 in 2. The suggestion
was confirmed by HMBC correlations
from H-70 to C-20, C-60, C-80, and C-90;
from H-20 and H-60 to C-70 and C-40; and
from H-100 to C-40, as well as by NOE
enhancements of H3-9 and H3-90 upon
Compound 2 was obtained as a white
solid. The spectroscopic data of 2 indi-
cated that it was an isomer of 1. This was
confirmed by 2D NMR data analysis,
particularly, HMBC correlations from H-
2, H-5, H-6, and H-100 to C-4 confirmed
that the b-glucopyranosyl moiety was
located at C-4 in 2. Comparison of the
NMR data of 2 with those of 1 (Table 1)
indicated that the chemical shifts of H-7
and H3-9 and H-70 and H3-90 were
significantly different from those of 1.
This, combined with the coupling constant
irradiation of H-70 (J7 ,8 ¼ 9.0 Hz). The
CD spectrum of 3 displayed Cotton
effects at 230 (D1 213.9) and 281 (D1
22.6) nm, which is similar to those of 2.
This indicated that 3 had the absolute
configuration identical to that of 2,
0
0
0
0
values of J7,8 (3.5 Hz) and J7 ,8 (9.5 Hz),
demonstrated that 2 had the 7,8-cis-70,80-

486
P.-B. Bu et al.
which was proved by enzymatic hydroly-
20
structure as the reported compounds. The
7,8-threo-configuration of 5 was indicated
by the J7,8 and DdC8–C7 values [25].
However, comparison of the NMR data of
5 and the co-occurring (7S,8S)-syringyl-
glycerol 8-O-b-D-glucopyranoside indi-
cated that H-8 and C-8 in 5 were
shielded by DdH 20.03 ppm and DdC
20.7 ppm, respectively, whereas H-10 was
deshielded by DdH þ 0.15 ppm. This
suggested that 5 had the (7R,8R)-configur-
ation. Enzymatic hydrolysis of 5 yielded
sis of 3 produced D-glucose {½aꢀD þ 42.8
(c ¼ 0.14, H2O)} and 2a. Therefore, the
structure of compound 3 was determined
as (2)-(7R,70R,8S,80R)-4,40-dihydroxy-
3,30-dimethoxy-7,70-epoxylignan-40-O-b-
D-glucopyranoside.
Compound 4, an amorphous powder
20
with ½aꢀD 218.8 (c ¼ 0.12, MeOH),
exhibited a quasi-molecular ion peak at
m/z 545 [M þ Na]þ in the positive mode
ESI-MS. The molecular formula of 4
(C27H38O10) was determined by HR-ESI-
MS at m/z 545.2363 [M þ Na]þ, which
was supported by the NMR data (Table 1).
The NMR spectra of 4 in MeOH-d4
showed resonances attributed to a 1,3,4-
trisubstituted phenyl, a symmetrically
10,30,40,50-tetrasubstituted phenyl, three
aromatic methoxy, two methines, two
methylenes, and two methyls, together
with characteristic signals due to a b-
glucopyranosyl unit. These data suggested
that 4 was a lignan glucoside with the
typical nucleus, which was confirmed by
2D NMR data analysis. In particular,
HMBC correlations from H-20/H-60 and H-
100 to C-40 and from OCH3-30/OCH3-50 to
C-30/C-50 demonstrated unequivocally that
the b-glycopyranosyl moiety and two
methoxy groups were symmetrically sub-
stituted on the same phenyl unit. Enzy-
matic hydrolysis of 4 produced D-glucose
20
b-D-glucose {½aꢀD þ 41.7 (c ¼ 0.16,
1
H2O)} and 5a. The H NMR data of 5a
were identical to those of (7S,8S)-syrin-
gylglycerol, however, the specific rotation
of 5a, ½aꢀ2D0 225.4 (c ¼ 0.25, MeOH), was
opposite to that of (7S,8S)-syringylgly-
cerol [25]. This confirmed that 5 had the
7R,8R-configuration. Therefore, the struc-
ture of compound 5 was assigned as (–)-
(7R,8R)-syringylglycerol 8-O-b-D-gluco-
pyranoside. Although a pair of 7,8-threo-
syringylglycerol 8-O-b-D-glucopyrano-
sides was reported from A. premnifolium
[14], the absolute configuration was not
determined.
Compound 6 was obtained as a
yellowish solid. Its molecular formula
C17H24O12 was determined by positive
mode HR-ESI-MS at m/z 443.1168
[M þ Na]þ combined with the NMR data
(Table 2). The NMR data of 6 were
similar to those of the co-occurring
spatholosineside A, 3-hydroxy-2-methyl-
20
{½aꢀD þ 40.3 (c ¼ 0.08, H2O)} and 4a.
20
1
The H NMR data and specific rotation
{½aꢀD þ 2.5 (c ¼ 0.56, CHCl3)} of 4a
4-pyrone
3-O-a-L-rhamnopyranosyl-
were in good agreement with those of (þ)-
(8S,80R)-4,40-dihydroxy-3,30,50-trimethox-
ylignan isolated from the same plant [12].
Thus, the structure of compound 4 was
characterized as (2)-(8S,80R)-4,40-dihy-
droxy-3,30,50-trimethoxylignan-40-O-b-D-
glucopyranoside.
(1 ! 6)-O-b-D-glucopyranoside [26],
except that the L-rhamnopyranosyl unit in
spatholosineside A was replaced by a b-
glycosyl unit. Acid hydrolysis of 6
produced b-D-xylopyranose and b-D-glu-
copyranose, as identified by GC analysis
of trimethylsiyl L-cysteine of the deriva-
tive of hydrolysate and the authentic
sugars [27]. This suggested that 6 was a
derivative of spatholosineside A with the
b-D-xylopyranosyl unit substituting the L-
rhamnopyranosyl unit. The elucidation
was confirmed by 2D NMR data analysis
Compound 5 was isolated as an
amorphous powder. The spectroscopic
features of 5 were similar to those of 7,8-
threo-syringylglycerol 8-O-b-D-glucopyr-
anosides [14,25]. 2D NMR data analysis
confirmed that 5 had the same planar

Journal of Asian Natural Products Research
487
Table 2. 1H and 13C NMR spectral data (d) for compounds 5 and 6 in MeOH-d4.
5
6a
No.
dH
dC
dH
dC
1
2
3
4
5
6
7
8
9a
132.6
105.9
149.4
136.6
149.4
105.9
75.3
6.65 s
6.65 s
4.60 d (7.5)
3.79 m
3.45 dd (12.0, 3.0)
3.25 dd (12.0, 6.0)
4.46 d (7.0)
3.26 dd (7.0, 9.0)
3.34 dd (9.0, 9.0)
3.26 dd (9.0, 8.5)
3.28 m
3.83 dd (12.0, 1.0)
3.63 dd (12.0, 5.0)
3.79
165.0
143.8
177.5
117.7
157.5
16.2
6.39 d (5.5)
7.94 d (5.5)
2.40 s
86.9
62.6
9b
10
104.5
75.6
78.1
71.8
78.5
62.9
4.73 d (7.0)
105.5
75.7
78.2
71.5
77.8
70.2
20
3.31 dd (7.0, 9.0)
3.33 dd (9.0, 8.5)
3.34 dd (8.5, 9.0)
3.34 m
3.99 brd (11.5)
3.64 dd (11.5, 6.0)
30
40
50
60a
60b
OMe-3/-5
57.1
Notes: 1H NMR data (d) were measured in MeOH-d4 for 5 and 6 at 500 MHz for 1H and 125 MHz for 13C. Proton
coupling constants (J) in Hz are given in parentheses. The assignments were based on 1H–1H COSY, HSQC, and
HMBC experiments.
a Data for the b-D-xylopyranosyl moiety of 6: dH 4.18 (1H, d, J ¼ 7.5 Hz, H-100), 3.10 (1H, dd, J ¼ 7.5 and 9.0 Hz,
H-200), 3.22 (1H, dd, J ¼ 9.0 and 8.5 Hz, H-300), 3.41 (1H, m, H-400), 3.77 (1H, dd, J ¼ 11.5 and 5.0 Hz, H-500a),
and 3.11 (1H, dd, J ¼ 11.5 and 7.0 Hz, H-500b); dC 105.8 (C-100), 75.1 (C-200), 78.0 (C-300), 71.5 (C-400), and 67.2
(C-500).
of 6. In particular, in the HMBC spectrum
of 6, correlations of H-5/C-3 and C-6; H-
6/C-2, C-4, and C-5; and H3-7/C-2 and C-3
verified the 2-methyl-4-pyrone moiety. In
addition, an HMBC correlation of H-10/C-
3 confirmed that the b-D-glucopyranosy-
loxy unit was located at C-3 of the 2-
methyl-4-pyrone moiety, while a corre-
lation of H-100/C-60 proved the location of
the b-D-xylopyranosyloxy unit at C-60 of
the b-D-glucopyranosyloxy unit in 6.
Thus, the structure of compound 6 was
determined as 3-hydroxy-2-methyl-4-pyr-
one 3-O-b-D-xylopyranosyl-(1 ! 6)-O-b-
D-glucopyranoside.
pyranoside [32], (þ)-(8S,80S)-4,40- dihy-
droxy-3,30,5,50-tetramethoxylignan-9,
90-diol 9-O-b-D-xylopyranoside [33], 4-
hydroxy-3-methoxyphenol-b-D-[6-O-(4-
hydroxy-3,5-dimethoxylbenzoate)]-gluco-
pyranoside [34], (2)-2,6-dimethoxy-p-
hydroquinone 1-O-b-D-glucopyranoside
[35], (2)-3,5-dimethoxy-4-hydroxyphe-
nol 1-O-b-D-glucopyranoside [36], (2)-
4-hydroxy-3-methoxyphenyl 1-O-b-D-
xylopyranosyl-(1 ! 6)-b-D-glucopyrano-
side [37], (7S,8S)-syringylglycerol 8-O-b-
D-glucopyranoside [14,25], and spatholo-
sineside A [26].
The known compounds were identified
by comparing the spectroscopic data with
those reported in the corresponding litera-
ture as (þ)-isolariciresinol-90-O-b-xylo-
pyranoside [28,29], (þ)-50-methoxy-
isolariciresinol-90-O-b-xylopyranoside
[30,31], (þ)-lyoniresinol-90-O-b-D-xylo-
3. Experimental
3.1 General experimental procedures
Optical rotations were measured on P-
2000 polarimeter (JASCO, Tokyo, Japan).
UV spectra were measured on a JASCOP-
650 spectrometer (JASCO). CD spectra

488
P.-B. Bu et al.
were recorded on a JASCO J-815 CD
spectrometer (JASCO). IR spectra were
recorded on a Nicolet 5700 FT-IR
microscope instrument (FT-IR microscope
transmission; Thermo Electron Corpor-
ation, Madison, WI, USA). NMR spectra
were obtained at 500 MHz for 1H, and
125 MHz for 13C, respectively, on an
Inova 500 spectrometer (Varian Associ-
ates, Inc., Palo Alto, CA, USA) in MeOH-
d4 with solvent peaks used as references.
ESI-MS and HR-ESI-MS data were
measured using an AccuToFCS JMS-
T100CS spectrometer (Agilent Technol-
ogies, Ltd, Santa Clara, CA, USA).
Column chromatography (CC) was per-
formed with silica gel (200–300 mesh,
Qingdao Marine Chemical, Inc., Qingdao,
China) and Sephadex LH-20 (Pharmacia
Biotech AB, Uppsala, Sweden). HPLC
separation was performed on an instru-
ment consisting of a Waters 600 controller,
a Waters 600 pump, and a Waters 2487
dual l absorbance detector (Waters
Corporation, Milford, MA, USA), with
an Prevail (250 £ 10 mm i.d.) column
packed with C18 (5 mm) (Alltech Associ-
ates, Inc., Deerfield, IL, USA). TLC was
carried out with glass precoated silica gel
GF254 plates (Qingdao Marine Chemical,
Inc.). Spots were visualized under UV
light or by spraying with 7% H2SO4 in
95% EtOH followed by heating. Unless
otherwise noted, all chemicals were
obtained from commercially available
sources and were used without further
purification.
3.3 Extraction and isolation
The air-dried bark of M. robusta (5.3 kg)
was powdered and extracted with 45 liters
of aq. 95% EtOH at room temperature for
3 £ 48 h. The EtOH extract was evapor-
ated under reduced pressure to yield a dark
brown residue (535 g). The residue was
suspended in H2O (2000 ml) and parti-
tioned with EtOAc (8 £ 2000 ml). The aq.
phase (196.3 g) was applied to a HDP100
macroporous adsorbent resin (1000 g,
dried weight) column. A successive
elution of the column with H2O, 30%,
50%, and 95% EtOH (5000 ml each)
yielded four corresponding fractions after
removing of solvents. The fraction (22.3 g)
eluted by 50% EtOH was separated by
medium pressure liquid chromatography
over reversed-phase silica gel eluting with
a gradient of increasing MeOH (30–
100%) in H2O to give fractions A–D on
the basis of TLC analysis. Subsequent
separation of fraction B (5.52 g) on normal
silica gel CC, eluting with a gradient of
increasing MeOH (0–100%) in CHCl3,
afforded fractions B1–B5. Fraction B3
(1.98 g) was chromatographed over Sepha-
dex LH-20, using MeOH:H2O (70:30) as
the eluting solvent, to give three subfrac-
tions B3-1–B3-3. B3-2 (0.30 g) and B3-3
(0.22 g) were separately isolated by
reversed-phase semipreparative HPLC,
using mobile phase of MeOH:H2O
(20:80) (flow rate: 2 ml/min; detection
wavelength: 230 nm) to afford 1 (15.0 mg,
Rt ¼ 34.2), 2 (10.6 mg, Rt ¼ 31.4), and 3
(11.2 mg, Rt ¼ 32.7) from B3-2; and 5
(16.9 mg, Rt ¼ 22.7) and 6 (5.9 mg,
Rt ¼ 26.3) from B3-3. Fraction C (2.82 g)
was separated on normal silica gel CC,
eluting with a gradient of increasing
MeOH (10–50%) in CHCl3, to afford
subfractions C1 –C3. C2 (0.85 g) was
further chromatographed over Sephadex
LH-20, using MeOH as the elution solvent,
and then isolated by reversed-phase
semipreparative HPLC by using
MeOH:H2O (30:70) as mobile phase
3.2 Plant material
The bark of M. robusta was collected at
Dayao Mountain, Guangxi Province,
China in August 2006. The plant was
identified by Mr Guang-Ri Long (Guangxi
Forest Administration, Guangxi, China). A
voucher specimen (No. 041) was deposited
at the Herbarium of the Department of
Natural Product Chemistry, Institute of
Materia Medica.

Journal of Asian Natural Products Research
489
(flow rate: 2 ml/min; detection wave-
length: 254 nm) to yield 4 (20.8 mg,
Rt ¼ 23.9).
(film) nmax 3398, 2964, 1600, 1514, 1460,
1424, 1384, 1268, 1226, 1158, 1123, 1073,
1
and 1039 cm21; for H NMR (MeOH-d4,
500 MHz) and 13C NMR (MeOH-d4,
125 MHz) spectroscopic data, see Table
1; positive-mode ESI-MS: m/z 529
[M þ Na]þ; negative-mode ESI-MS: m/z
505 [M–H]2; HR-ESI-MS: m/z 529.2056
[M þ Na]þ (calcd for C26H34O10Na,
529.2044).
3.3.1 (2)-Nectandrin B-b-D-
glucopyranoside (1)
20
Yellowish solid; ½aꢀD 222.5 (c ¼ 0.10,
MeOH); UV (MeOH) lmax (log 1) 229
(0.18), 279 (0.07) nm; CD (MeOH)
D1210 nm þ4.20, D1233 nm 23.14; IR
(film) nmax 3448, 3389, 2958, 1601, 1515,
1461, 1388, 1270, 1226, 1157, 1126, 1076,
3.3.4 (2)-(8S,80R)-4,40-Dihydroxy-
3,30,50-trimethoxylignan-40-O-b-D-
glucopyranoside (4)
1
and 1049 cm21; for H NMR (MeOH-d4,
500 MHz) and 13C NMR (MeOH-d4,
125 MHz) spectroscopic data, see Table 1;
positive-mode ESI-MS: m/z 529
[M þ Na]þ; negative-mode ESI-MS: m/z
505 [M–H]2; HR-ESI-MS: m/z 529.2049
[M þ Na]þ (calcd for C26H34O10Na,
529.2044).
20
Amorphous powder; ½aꢀD
218.8
(c ¼ 0.12, MeOH); UV (MeOH) lmax
(log 1) 230 (0.32), 278 (0.10) nm; CD
(MeOH) D1217 nm 22.19, D1234 nm þ0.65,
D1278 nm þ0.53; IR (film) nmax 3411, 2958,
1594, 1515, 1462, 1423, 1272, 1243, 1131,
1071, 894, and 816 cm21; for H NMR
1
3.3.2 (2)-(7R,70R,8S,80R)-4,40-
Dihydroxy-3,30-dimethoxy-7,70-
epoxylignan-4-O-b-D-glucopyranoside (2)
(MeOH-d4, 500 MHz) and 13C NMR
(MeOH-d4, 125 MHz) spectroscopic data,
see Table 1; positive-mode ESI-MS: m/z 545
[M þ Na]þ; negative-mode ESI-MS: m/z
521 [M–H]2; HR-ESI-MS: m/z 545.2363
[M þ Na]þ (calcd for C27H38O10Na,
545.2357).
20
Yellowish solid; ½aꢀD 225.3 (c ¼ 0.29,
MeOH); UV (MeOH) lmax (log1) 230
(0.38), 278 (0.16) nm; CD (MeOH)
D1231 nm 29.74, D1278 nm 22.29; IR
(film) nmax 3386, 2965, 1597, 1514, 1456,
1417, 1384, 1272, 1223, 1073, and
1036 cm21; for 1H NMR (MeOH-d4,
500 MHz) and 13C NMR (MeOH-d4,
125 MHz) spectroscopic data, see Table 1;
positive-mode ESI-MS: m/z 529
[M þ Na]þ; negative-mode ESI-MS: m/z
505 [M 2 H]2; HR-ESI-MS: m/z 529.2055
[M þ Na]þ (calcd for C26H34O10Na,
529.2044).
3.3.5 (2)-(7R,8R)-Syringylglycerol
8-O-b-D-glucopyranoside (5)
20
Amorphous powder; ½aꢀD
248.0
(c ¼ 0.53, MeOH); UV (MeOH) lmax
(log 1) 238 (0.04), 281 (0.01) nm; CD
(MeOH) D123 nm þ0.05, D1280 nm þ0.04;
IR (film) nmax 3392, 2940, 1617, 1520,
1466, 1424, 1322, 1222, 1125, 1082, 894,
1
and 837 cm21; for H NMR (MeOH-d4,
3.3.3 (2)-(7R,70R,8S,80R)-4,40-
Dihydroxy-3,30-dimethoxy-7,70-
500 MHz) and 13C NMR (MeOH-d4,
125 MHz) spectroscopic data, see Table
2; positive-mode ESI-MS: m/z 429
[M þ Na]þ; negative-mode ESI-MS: m/z
405 [M–H]2; HR-ESI-MS: m/z 429.1371
[M þ Na]þ (calcd for C17H26O11Na,
429.1367).
epoxylignan-40-O-b-D-glucopyranoside(3)
20
Yellowish solid; ½aꢀD 254.5 (c ¼ 0.46,
MeOH); UV (MeOH) lmax (log 1) 231
(0.33), 281 (0.12) nm; CD (MeOH)
D1231 nm 213.92, D1281 nm 22.61; IR

490
P.-B. Bu et al.
3.3.6 (2)-3-Hydoxy-2-methyl-4-pyrone
3-O-b-D-xylopyranosyl-(1 ! 6)-O-b-D-
glucopyranoside (6)
3.3.8 Acid hydrolysis of 6
Compound 6 (3.5 mg) was refluxed in 2 N
HCl (5.0 ml) at 808C for 6 h. The reaction
mixture was extracted with EtOAc
(3 £ 5 ml). The H2O layer was evaporated
under reduced pressure. After addition
of H2O (5 ml), the acidic solution was
evaporated again, and this procedure
was repeated until a neutral solution was
obtained. The neutral solution was evap-
orated and dried in vacuo to furnish a
monosaccharide residue. The residue was
dissolved in pyridine (0.5 ml) and 2 mg of
L-cysteine methyl ester hydrochloride was
added. The mixture was maintained at
608C for 2 h, evaporated under a stream
of N2, and dried in vacuo. Next, 0.2 ml of
N-trimethylsilylimidazole was added, and
the resultant reaction mixture was main-
tained at 608C for 1 h. The mixture was
partitioned between n-hexane and H2O
(2 ml each), and the n-hexane extract was
analyzed by GC-MS under the following
conditions: capillary column, DB-5
(30 m £ 0.25 mm £ 0.25 mm); detector,
FID; detector temperature, 2808C; injec-
tion temperature, 2508C; initial tempera-
ture, 1008C for 2 min and subsequent
increase to 2808C at the rate of 108C/min;
final temperature, 2808C for 5 min; carrier,
N2 gas. The absolute configurations of the
sugars were determined by comparing the
retention times of their trimethylsilyl-L-
cysteine derivatives with those of authentic
sugars prepared by a similar procedure.
The retention times of the trimethylsilyl-L-
cysteine derivatives of the sugars were as
follows: D-glucose, 19.57 min, and D-
xylopyranose, 17.66 min.
20
Yellowish solid; ½aꢀD 274.0 (c ¼ 0.15,
MeOH); UV (MeOH) lmax (log 1) 257
(0.44), 205 (0.70) nm; IR (film) nmax 3397,
2920, 1651, 1441, 1375, 1287, 1253, 1191,
1108, 1072, 992, 972, and 834 cm21; for
1H NMR (MeOH-d4, 500 MHz) and 13C
NMR (MeOH-d4, 125 MHz) spectroscopic
data, see Table 2; positive-mode ESI-MS:
m/z 443 [M þ Na]þ; negative-mode ESI-
MS: m/z 419 [M–H]2; HR-ESI-MS:
m/z 443.1168 [M þ Na]þ (calcd for
C17H24O12Na, 443.1160).
3.3.7 Enzymatic hydrolysis of 1–5
A solution of each compound (1–5, 5–
8 mg) in H2O (3 ml) was hydrolyzed with
snailase (30 mg, LJ0427B2011Z, Shang-
hai Sangon Biotech Co. Ltd, Shanghai,
China) at 378C for 12 h. The reaction
mixture was extracted with EtOAc
(3 £ 3 ml). The H2O phases of the hydro-
lysates of 1–5 were separately concen-
trated to dryness and then eluted with
CH3CN:H2O (8:1) on a silica gel column
20
to yield glucose with ½aꢀD values that
ranged from þ40.3 to þ45.1 (c ¼ 0.08–
0.16, H2O). The solvent system CHCl3:
MeOH:H2O (8:5:1) was used for TLC
identification of glucose (Rf, 0.30). EtOAc
extracts of the hydrolysates were separ-
ately concentrated to dryness, and then
eluted on a silica gel column with 40–70%
EtOAc in petroleum ether to yield
20
nectandrin B {1a, 1.2 mg, ½aꢀD < 0
(c ¼ 0.12, MeOH)} [14,16,17] from 1,
(2)-(7R,70R,8S,80R)-4,40-dihydroxy-3,30-
20
dimethoxy-7,70-epoxylignan {2a, ½aꢀD
235.6 (c ¼ 0.16, MeOH)} [16,22]
2.0 mg from 2 and 1.6mg from 3, (þ)-
(8S,80R)-4,40-dihydroxy-3,30,50-trimethox-
Acknowledgements
Financial support from the National Natural
Sciences Foundation of China (NNSFC; Grant
Nos 30825044 and 20932007), the Program for
Changjiang Scholars and Innovative Research
Team in University (PCSIRT, Grant No.
IRT1007), and the National Science and
Technology Project of China (No.
2012ZX09301002-002) is acknowledged.
20
ylignan {4a, 5.6 mg, ½aꢀD þ 2.5 (c ¼ 0.56,
20
CHCl3)} [12] from 4, and (2)-(7R,8R)-
syringylglycerol {5a, 2.5 mg, ½aꢀD 225.4
(c ¼ 0.25, MeOH)} [24] from 5,
respectively.

Journal of Asian Natural Products Research
491
[19] A.M. Rimando, J.M. Pezzuto, and N.R.
Farnsworth, J. Nat. Prod. 57, 896 (1994).
[20] J.S. Liu, M.F. Huang, Y.L. Gao, and J.A.
Findlay, Can. J. Chem. 59, 1680 (1981).
[21] H. Achenbach, J. Grob, X.A. Dominguez,
G. Cano, J. Vende Star, L.D.C. Brussolo,
References
[1] P.M. Giang, P.T. Son, K. Matsunami, and H.
Otsuka, Chem. Pharm. Bull. 54, 308 (2006).
[2] M.J. Cheng, I.L. Tsai, S.J. Lee, B.
Jayaprakasam, and I.S. Chen, Phytochem-
istry 66, 1180 (2005).
[3] E.Y. Park, S.M. Shin, C.J. Ma, Y.C. Kim,
and S.G. Kim, Planta Med. 71, 393 (2005).
[4] Jiangsu New Medical College, Dictionary
of Traditional Chinese Medicine (Shang-
hai Science and Technology Publishing
House, Shanghai, 1977), Vol. 1, pp. 114,
1009, and 1423.
´
G. Munoz, F. Salgado, and L. Lopez,
Phytochemistry 26, 1159 (1987).
[22] C. Konno, Z.Z. Lu, H.Z. Xue, C.A.J.
Erdelmeier, D. Meksuriyen, C.T. Che,
G.A. Cordell, D. Doel Soejarto, D.P.
Waller, and H.H.S. Fong, J. Nat. Prod. 53,
396 (1990).
[23] A.K. Parsad, O.D. Tyagi, J. Wengel, P.M.
Boll, C.E. Olsen, K.S. Bisht, A. Singh, A.
Sarangi, R. Kumar, S.C. Jain, and V.S.
Parmar, Phytochemistry 39, 655 (1995).
[24] H. Abou-Gazar, E. Bedir, S. Takamatsu,
D. Ferreira, and I.A. Khan, Phytochem-
istry 65, 2499 (2004).
[25] M.L. Gan, Y.L. Zhang, S. Lin, M.T. Liu,
W.X. Song, J.C. Zi, Y.C. Yang, X.N. Fan,
J.G. Shi, J.F. Hu, J.D. Sun, and N.H.
Chen, J. Nat. Prod. 71, 647 (2008).
[26] T. Yin, H. Liu, F. Wang, G.Z. Tu, H.
Liang, and Y.Y. Zhao, Acta Pharm. Sin.
43, 67 (2008).
[27] M.L. Gan, M.T. Liu, L.S. Gan, S. Lin, B.
Liu, Y.L. Zhang, J.C. Zi, W.X. Song, and
J.G. Shi, J. Nat. Prod. 77, 1373 (2012).
[28] M. Takani, K. Ohya, and K. Takahashi,
Chem. Pharm. Bull. 27, 1422 (1979).
[29] L.N. Lundgren, T. Popoff, and O. Theader,
Phytochemistry 20, 1967 (1981).
[30] S. Inoshiri, M. Sasaki, H. Kohda, H.
Otsuka, and K. Yamasaki, Phytochemistry
26, 2811 (1987).
[31] V. Vecchietti, G. Ferrari, F. Orsini, and F.
Pelizzoni, Phytochemistry 18, 1847(1979).
[32] G. Dada, A. Corbani, P. Mantto, G.
Speranza, and L. Lunazzi, J. Nat. Prod.
52, 1327 (1989).
[33] K. Yoshinari, Y. Sashida, and H. Shimo-
mura, Chem. Pharm. Bull. 37, 3301(1989).
[34] Y.L. Zhang, M.L. Gan, S. Lin, M.T. Liu,
W.X. Song, J.C. Zi, S.J. Wang, S. Li, Y.C.
Yang, and J.G. Shi, J. Nat. Prod. 71, 905
(2008).
[5] M.T. Liu, S. Lin, Y.H. Wang, W.Y. He, S.
Li, S.J. Wang, Y.C. Yang, and J.G. Shi,
Org. Lett. 9, 129 (2007).
[6] M.T. Liu, M. Gan, S. Lin, Y.L. Zhang, J.C.
Zi, W.X. Song, X.N. Fan, Y. Liu, Y.C. Yang,
and J.G. Shi, Org. Lett. 13, 2856 (2011).
[7] M.L. Gan, M.T. Liu, B. Liu, S. Lin, Y.L.
Zhang, J.C. Zi, W.X. Song, Y. Fei, X.G.
Chen, and J.G. Shi, J. Nat. Prod. 74, 2431
(2011).
[8] M.T. Liu, S. Lin, M.L. Gan, M.H. Chen,
L. Li, S.J. Wang, J.C. Zi, X.N. Fan, Y.
Liu, Y.K. Si, Y.C. Yang, X.G. Chen, and
J.G. Shi, Org. Lett. 14, 1004 (2012).
[9] M.L. Gan, M.T. Liu, L.S. Gan, S. Lin, B.
Liu, Y.L. Zhang, J.C. Zi, W.X. Song, and
J.G. Shi, J. Nat. Prod. 75, 1373 (2012).
[10] W. Cheng, C.G. Zhu, W.D. Xu, X.N. Fan,
Y.C. Yang, Y. Li, X.G. Chen, W.J. Wang,
and J.G. Shi, J. Nat. Prod. 72, 2145 (2009).
[11] W. Cheng, C.G. Zhu, W.D. Xu, X.N. Fan,
Y.C. Yang, Y. Li, and J.G. Shi,
J. Asian Nat. Prod. Res. (2012) DOI:
10.1080/10286020.2012.702762.
[12] Y.R. Li, W. Cheng, C.G. Zhu, C.S. Yao,
L. Xiong, Y. Tian, S.J. Wang, S. Lin, J.F.
Hu, Y.C. Yang, Y. Guo, Y. Yang, Y. Li,
Y.H. Yuan, N.H. Chen, and J.G. Shi,
J. Nat. Prod. 74, 1444 (2011).
[13] G.P. Moss, Pure Appl. Chem. 72, 1493
(2000).
[14] H. Kijima, T. Ide, H. Otsuka, C. Ogimi,
E. Hirata, A. Takushi, and Y. Takeda,
Phytochemistry 44, 1551 (1997).
[15] P.W. Le Quesne, J.E. Larrahando, and
R.F. Raffauf, J. Nat. Prod. 43, 353 (1980).
[16] M. Hattori, S. Hada, Y. Kawata, Y.
Tezuka, T. Kikuchi, and T. Namba, Chem.
Pharm. Bull. 35, 3315 (1987).
[17] H. Shimomura, Y. Sashida, and M.
Oohara, Phytochemistry 27, 634 (1988).
[18] B.G. Wang, X. Hong, L. Li, J. Zhou, and
X.J. Hao, Planta Med. 66, 511 (2000).
[35] H. Otsuka, M. Takeuchi, S. Inoshiri, T.
Sato, and K. Yamasaki, Phytochemistry
28, 883 (1989).
[36] M.I. Chung, M.H. Lai, M.H. Yen, R.R.
Wu, and C.N. Lin, Phytochemistry 44,
943 (1997).
[37] J. Kitajima, A. Kamoshita, T. Ishikawa,
A. Takano, T. Fukuda, S. Isoda, and Y.
Ida, Chem. Pharm. Bull. 51, 152 (2003).