Three new lignan glycosides from the fruits of Forsythia suspense

Article abstract of DOI:10.1080/10286020.2014.917631

Two new lignan glycosides forsythiayanoside A (1) and forsythiayanoside B (2), one new stereoisomer of lignan glycoside (+)-8-hydroxyepipinoresinol-4-O- β-D-glucopyranoside (3), together with seven known compounds (4-10), were isolated from the fruits of Forsythia suspense (Thunb.) Vahl. Compounds 4 and 10 were isolated from this species for the first time. The structures of 1-3 were elucidated on the basis of chemical and spectral analyses, including 1D and 2D NMR data and HR-ESI-MS. The absolute configurations were determined by the circular dichroism method. All isolates were tested for their cytotoxicities against five human cancer cell lines (A549, Colo- 205, Hep-3B, HL60, and KB). In particular, compound 3 showed significant cytotoxicity with IC₅₀ values of 9.48, 7.75, 0.59, 4.06, and 38.38 μM, respectively.

Full text of DOI:10.1080/10286020.2014.917631

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Three new lignan glycosides from the
fruits of Forsythia suspense
Xin-Jia Yanab, Ying Pengc, Zhi-Xiang Liuab, Jing Wenab, Qing-Bo
Liuab, Ling-Zhi Liab & Shao-Jiang Songab
a School of Traditional Chinese Materia Medica, Shenyang
Pharmaceutical University, Shenyang 110016, China
b Key Laboratory of Structure-Based Drug Design & Discovery
(Ministry of Education), Shenyang Pharmaceutical University,
Shenyang 110016, China
c School of Pharmacy, Shenyang Pharmaceutical University,
Shenyang 110016, China
Published online: 19 May 2014.
To cite this article: Xin-Jia Yan, Ying Peng, Zhi-Xiang Liu, Jing Wen, Qing-Bo Liu, Ling-Zhi Li &
Shao-Jiang Song (2014) Three new lignan glycosides from the fruits of Forsythia suspense, Journal
of Asian Natural Products Research, 16:6, 602-610, DOI: 10.1080/10286020.2014.917631
To link to this article: http://dx.doi.org/10.1080/10286020.2014.917631
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Journal of Asian Natural Products Research, 2014
Vol. 16, No. 6, 602–610, http://dx.doi.org/10.1080/10286020.2014.917631
Three new lignan glycosides from the fruits of Forsythia suspense
Xin-Jia Yanab, Ying Pengc, Zhi-Xiang Liuab, Jing Wenab, Qing-Bo Liuab, Ling-Zhi Liab
and Shao-Jiang Songab*
aSchool of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang
110016, China; bKey Laboratory of Structure-Based Drug Design & Discovery (Ministry of
Education), Shenyang Pharmaceutical University, Shenyang 110016, China; cSchool of Pharmacy,
Shenyang Pharmaceutical University, Shenyang 110016, China
(Received 22 January 2014; final version received 21 April 2014)
Two new lignan glycosides forsythiayanoside A (1) and forsythiayanoside B (2), one
new stereoisomer of lignan glycoside (þ)-8-hydroxyepipinoresinol-4-O-b-D-gluco-
pyranoside (3), together with seven known compounds (4–10), were isolated from the
fruits of Forsythia suspense (Thunb.) Vahl. Compounds 4 and 10 were isolated from
this species for the first time. The structures of 1–3 were elucidated on the basis of
chemical and spectral analyses, including 1D and 2D NMR data and HR-ESI-MS. The
absolute configurations were determined by the circular dichroism method. All isolates
were tested for their cytotoxicities against five human cancer cell lines (A549, Colo-
205, Hep-3B, HL60, and KB). In particular, compound 3 showed significant
cytotoxicity with IC50 values of 9.48, 7.75, 0.59, 4.06, and 38.38 mM, respectively.
Keywords: Forsythia suspense; lignan glycosides; NMR; CD; cytotoxic activity
1. Introduction
analysis and comparison with those of
literature values. Meanwhile, all com-
pounds were evaluated for their cytotoxi-
cities against A549, Colo-205, Hep-3B,
HL60, and KB cancer cell lines. Com-
pound 3 showed significant cytotoxicity
with IC50 values of 9.48, 7.75, 0.59, 4.06,
and 38.38 mM, respectively, as compared
with positive controls (5-fluorouracil:
25.72, 39.30, 4.80, 11.52, and 44.35 mM,
respectively; paclitaxel: 0.43, 7.80, 2.09,
0.01, and 1.00 mM, respectively).
Forsythia suspense (Thunb.) Vahl, a
member of the family Oleaceae, is widely
distributed in China including Henan,
Shanxi, and Shandong Provinces. Its fruits
are one of the most important original
plants of Traditional Chinese Medicine
which have been used for antibacterial,
antiviral, anti-inflammation, diuretic, and
antidotal purposes in oriental medicine [1].
The literature survey revealed that many
kinds of compounds had been isolated
from this plant, such as caffeoyl glyco-
sides, phenylethanoid glycosides, lignans,
terpenoids, alkaloids, and cyclohexy-
lethanes [2,3]. In this paper, we describe
the isolation and the structural elucidation
of those new lignan glycosides, along with
seven known compounds obtained from
the 50% EtOH extract of F. suspense.
Their structures (Figure 1) were estab-
lished by extensive spectroscopic data
2. Results and discussion
Compound 1 was obtained as a colorless
solid (MeOH), with the molecular formula
C27H34O12 as determined by the HR-ESI-
MS at m/z 573.1942 [M þ Na]þ, indicat-
ing 11 degrees of unsaturation. The 1H
NMR spectrum of compound 1 (Table 1)
displayed six aromatic proton signals at dH
*Corresponding author. Email: songsj99@163.com
q 2014 Taylor & Francis

Journal of Asian Natural Products Research
603
OCH3
3′
OCH3
3
H3CO
O
1′
1
HO
HO
OH
OCH3
9
9′
7′
9′
7
1
1′
9
O
7
O
7′
HO
HO
HO
HO
HO
HO
O
O
O
1″
O
1″
OH
3
3′
H3CO
H3CO
OH
1
2
OH
O
7′
9
1′
OCH3
3′
HO
1
H
9′
O
HO
HO
7
O
O
1″
3
HO
H3CO
OH
3
Figure 1. The structures of compounds 1, 2, and 3.
7.47 (1H, d, J ¼ 1.7 Hz, H-2), 7.04 (1H, d,
J ¼ 8.6 Hz, H-5), 7.64 (1H, dd, J ¼ 8.6,
1.7 Hz, H-6), 6.94 (1H, d, J ¼ 1.5 Hz, H-
20), 7.01 (1H, d, J ¼ 8.5 Hz, H-50), and
6.83 (1H, dd, J ¼ 8.5, 1.5 Hz, H-60), which
were assigned to two 1,3,4-trisubstituted
H-8), 4.56 (1H, d, J ¼ 8.0 Hz, H-70), and
2.49–2.52 (1H, m, H-80), three methoxyl
groups at dH 3.72 (3H, s), 3.78 (3H, s), and
3.80 (3H, s), and a glucopyranosyl
anomeric proton at dH 4.85 (1H, d,
J ¼ 7.2 Hz, H-100). The 13C NMR spectrum
of 1 (Table 1) showed 27 carbon signals,
apart from a glucose unit (dC 100.5, 73.7,
77.4, 70.2, 77.3, and 61.1) and three
methoxy carbons at dC 56.0, 56.1, and
56.2, the remaining 18 carbons could be
assigned to a tetrahydrofuranoid lignan
1
benzene rings. In addition, the H NMR
spectrum also revealed two oxymethylene
protons at dH 3.97 (1H, dd, J ¼ 8.4,
5.2 Hz, H-9b), 4.09 (1H, t, J ¼ 8.4 Hz,
H-9a), and 3.44–3.50 (2H, m, H-90), three
methine protons at dH 4.14–4.17 (1H, m,
OCH3
H3CO
OCH3
O
HO
HO
OH
OCH3
O
O
HO
HO
HO
HO
O
O
O
O
HO
HO
H3CO
H3CO
OH
OH
1
2
OH
O
O
OCH3
HO
H
HO
HO
HO
O
O
H3CO
OH
3
Figure 2. Key HMBC ( ! ) correlations of 1, 2, and 3.

604
X.-J. Yan et al.
Table 1. 1H NMR (600 MHz) and 13C NMR (150 MHz) spectral data of compounds 1 and 2 in
DMSO-d6.
1
2
No.
dH
dC
dH
dC
1
2
3
4
5
6
7
8
9a
9b
10
20
30
40
50
60
70
80
900a
900b
1
200
300
400
500
600
129.7
111.1
149.1
153.7
111.4
123.6
198.3
49.3
136.5
111.1
149.25
146.19
115.6
118.7
83.0
7.47 (d, 1.7)
6.92 (d, 1.6)
7.04 (d, 8.6)
7.64 (dd, 8.6, 1.7)
7.04 (d, 8.4)
6.82 (dd, 8.4, 1.6)
4.56 (d, 7.1)
1.62–1.65 (m)
2.93–2.95 (m)
3.06–3.08 (m)
4.14–4.17 (m)
4.09 (t, 8.4)
3.97 (dd, 8.4, 5.2)
52.7
60.9
70.5
135.9
111.3
149.2
146.4
115.5
119.1
83.2
131.2
111.5
148.0
146.6
115.6
120.8
85.7
6.94 (d, 1.5)
6.75 (d, 1.7)
7.01 (d, 8.5)
6.83 (dd, 8.5, 1.5)
4.56 (d, 8.0)
2.49-2.52 (m)
3.46–3.48 (m)
6.72 (d, 8.0)
6.62 (dd, 8.0, 1.7)
3.91 (d, 9.2)
2.39–2.43 (m)
3.82 (dd, 8.8, 7.3)
4.05 (dd, 8.8, 4.4)
4.88 (d, 7.3)
3.25 (m)
53.6
60.3
48.5
70.8
4.85 (d, 7.2)
3.21 (m)
3.25 (m)
3.12 (m)
3.22 (m)
3.62 (m)
3.41 (m)
3.72
100.5
73.7
77.4
70.2
77.3
61.1
100.6
73.7
77.4
70.1
77.5
61.2
3.26 (m)
3.16 (m)
3.29 (m)
3.46 (m)
3.67 (m)
3.77
3.73
3.06
ZOCH3
ZOCH3
ZOCH3
56.1
56.0
56.2
56.2
56.0
56.2
3.78
3.80
Note: Coupling constants (J) in Hz are given in parentheses; chemical shift values are expressed in ppm.
skeleton [4]. The fact that the carbonyl
group is connected to a part of the phenyl
group was indicated by a carbonyl carbon
signal at dC 198.3 and the conjugated
property absorption peak shown in the IR
spectrum at 1665 cm21. All of the above
spectroscopic data were similar to those of
vladinol D [5] with the exception of a
methoxy and a glucose unit. Careful
analysis of the correlations in the HSQC
and HMBC spectra led to the establish-
ment of the planar structure of compound 1
(Figure 1). The hydroxymethyl was linked
to C-80, which was deduced from the long-
rang correlations in the HMBC spectrum
(Figure 2) from the protons at dH 3.46–
3.48 (2H, m) to C-8 (dC 49.3), C-80 (dC
53.6), and C-70 (dC 83.2). The correlations
from H-70 at dH 4.56 (1H, d, J ¼ 8.0 Hz) to
C-20 (dC 111.3), C-60 (dC 119.1), and C-10
(dC 135.9) suggested that C-70 of tetra-
hydrofuran was connected with C-10 of the
benzene ring. The correlations from H-9 at
dH 3.97 (1H, dd, J ¼ 8.4, 5.2 Hz, H-9b),
4.09 (1H, t, J ¼ 8.4 Hz, H-9a) to C-7 (dC
198.3) indicated that the carbonyl carbon
linked to C-8 of the tetrahydrofuran ring.
The relative configuration of 1 was
1
determined by the analysis of H NMR
data and ROSEY correlations (Figure 3).
The chemical shift of H-70 at dH 4.56 (1H,
d, J ¼ 8.0 Hz) suggested that H-70 and

Journal of Asian Natural Products Research
605
9′
7′
8
2′
8′
9a
7
1
Figure 3. Key ROSEY correlations for 1.
H-80 were trans-oriented (for a cis-
orientation of substituents at C-70 and C-
80, the signal of H-70 was at around
5.5 ppm) [6,7], and this was supported by a
ROSEY correlation between H-20 and H-
80. Based on ROSEY correlations of H-8/
H-70, H-8/H-90, and H-70/H-90, H-8 and H-
70 were placed on the same side of the
tetrahydrofuran ring (trans-orientation of
H-8 and H-80). The absolute configuration
of 1 was established by the analysis of its
circular dichroism (CD) spectrum. A
negative Cotton effect at 321 nm and a
positive Cotton effect at 294 nm were
shown in the CD spectrum of 1, indicating
the (8S,70S,80R)-configuration [4,8] in 1.
The anomeric proton at dH 4.85 (1H, d,
J ¼ 7.2 Hz, H-100) indicated the b-con-
figuration of glucose unit. The component
sugar was determined to be D-glucopyr-
anose based on comparison of the acid
hydrolyzate of 1 with authentic D-gluco-
pyranose by thin layer chromatography
(TLC) and gas chromatography (GC)
analyses [9]. In the HMBC spectrum
(Figure 2), the anomeric proton at dH
4.85 showed a long-range correlation with
the aromatic carbon signal at dC 146.4
which was assigned to C-40. Thus, the
structure of was determined as
(8S,70S,80R)-40,90-dihydroxy-3,4,30-tri-
methoxy-70,9-epoxylignan-7-oxo-40-O-
b-D-glucopyranoside, named for-
sythiayanoside A.
1
Compound 2 was obtained as a sticky
oil (MeOH) and possessed a molecular
formula C27H36O12, as revealed from its
HR-ESI-MS analysis (m/z 575.2098
[M þ Na]þ), indicating 10 degrees of
unsaturation. The 1H NMR spectrum
(Table 1) showed proton signals of two
ABX systems of aromatic protons [dH 6.92
(1H, d, J ¼ 1.6 Hz, H-2), 7.04 (1H, d,
J ¼ 8.4 Hz, H-5), 6.82 (1H, dd, J ¼ 8.4,
1.6 Hz, H-6), 6.75 (1H, d, J ¼ 1.7 Hz, H-
20), 6.72 (1H, d, J ¼ 8.0 Hz, H-50), and
6.62 (1H, dd, J ¼ 8.0, 1.7 Hz, H-60)], two
aromatic CH3O groups [dH 3.73 (3H, s)
and 3.77 (3H, s)], indicating the presence
of two trisubstituted benzene rings. Six
oxygenated aliphatic protons [dH 4.56 (1H,
d, J ¼ 7.1 Hz, H-7), 2.93–2.95 (1H, m,
H-9a), 3.06–3.08 (1H, m, H-9b), 3.91
(1H, d, J ¼ 9.2 Hz, H-70), 3.82 (1H, dd,
J ¼ 8.8, 7.3 Hz, H-90a), and 4.05 (1H, dd,
J ¼ 8.8, 4.4 Hz, H-90b)], together with two

606
X.-J. Yan et al.
9
8′
7
8
1
7′
9′
Figure 4. Key NOESY correlations for 2.
aliphatic protons [dH 1.62–1.65 (1H, m,
H-8) and 2.39–2.43 (1H, m, H-80)] were
evidences for the presence of a tetrahy-
drofuranoid lignan skeleton which were
similar with compound 1. In the 13C NMR
spectrum (Table 1), apart from the 12
aromatic, a glucose unit, and three
methoxyl signals, there were six sp3
C-atoms, including two oxymethines [dC
83.0 and 85.7] and two oxymethylenes [dC
60.9 and 70.8]. The remaining CH3O
group was located at C-70, based on the
HMBC correlation (Figure 2) between
CH3O group protons at dH 3.06 (3H, s) and
C-70 (dC 85.7). The correlations from H-7
at dH 4.56 (1H, d, J ¼ 7.1 Hz) to C-2 (dC
111.1), C-6 (dC 118.7), and C-1 (dC 136.5)
suggested that C-7 of tetrahydrofuran was
connected with C-1 of the benzene ring.
The correlations from H-90 at dH 3.82 (1H,
dd, J ¼ 8.8, 7.3 Hz, H-90a), 4.05 (1H, dd,
J ¼ 8.8, 4.4 Hz, H-90b) to C-70 (dC 85.7),
from H-70 at dH 3.91 (1H, d, J ¼ 9.2 Hz) to
C-80 (dC 48.5), C-90 (dC 70.8), C-20 (dC
115.5), and C-60 (dC 120.8) indicated that
C-70 linked to C-20 of the benzene and C-80
of the tetrahydrofuran ring.
the H-7 chemical shift and NOESY
experiment. According to a signal of H-7
at dH 4.56, the orientation of H-7/H-8 of
compound 2 was determined to be trans
[10,11]. All trans-orientation of H-7/H-8/
H-80 was determined by the cross-peak of
H-7/H-9, H-7/H-80, and H-9/H-80 in the
NOESY spectrum (Figure 4). The CD
spectrum of 2 showed two positive peaks
at 240 and 283 nm similar to those of
analogous compound tinosposide B [12],
indicating that C-7, C-8, and C-80 in 2 have
S, R, and S-configurations, respectively.
However, there were no definitive con-
clusions on the relationships between the
absolute configuration of C-70 and CD in
this series of tetrahydrofuranoid lignans
[11,13]. Acid hydrolysis of 2 afforded
D-glucose by TLC and GC analyses [9].
The b-configuration anomeric proton of
glucose at dH 4.88 (1H, d, J ¼ 7.3 Hz)
showed HMBC correlations with the
aromatic C-4 at dC 146.1. Accordingly,
compound
2
was established as
(7S,8R,80S)-4,9,40-trihydroxy-3,30,70-tri-
methoxy-7,90-epoxylignan-4-O-b-D-glu-
copyranoside, named forsythiayanoside B.
Compound 3 was obtained as a yellow
amorphous powder. Its molecular formula
The relative configuration in the
tetrahydrofuran ring was determined by

Journal of Asian Natural Products Research
607
Table 2. 1H NMR (400 MHz) and 13C NMR
(100 MHz) spectral data of compound 3 in
DMSO-d6.
peaks of H-7 with C-9, C-80, C-90, and C-1
and H-70 with C-90, C-8, C-9, and C-10
supported 7.O.90 and 70.O.9 oxygen
bridges in the ditetrahydrofuran ring
moiety. The relative and absolute con-
figurations of the aglycone of compound 3
were determined by comparison of its
NMR data and CD spectrum with those of
known analogous compounds [14,15]. The
13C NMR spectrum was important in
establishing the orientation of the aryl
groups. For an equatorial guaiacyl group,
C-10 appeared in downfield at around dC
131.3 in DMSO-d6, while for an axial
guaiacyl group, C-10 appeared in upfield at
around dC 129.6 [3,14]. Thus, considering
the solvent effect on the aryl group, the
value of C-10 at dC 129.9 in compound 3
accounted for the appearance of axial
guaiacyl group. These spectroscopic data
suggested that 3 and known compound
(þ)-1-hydroxypinoresinol-40-O-b-D-glu-
copyranoside [14] are epimers at C-70.
Thus, the relative configuration could be
proposed as 7,8-cis-8,80-cis-70,80-trans.
The CD spectrum of compound 3 was
identical with that of (þ)-1-hydroxy-6-
epipinoresinol which has the absolute
configuration 7R, 8S, 70R, and 80R [14–
16]. Therefore, the aglycon of compound 3
was (þ)-1-hydroxy-6-epipinoresinol.
Acid hydrolysis of 3 afforded D-glucose
by TLC and GC analyses [9]. The b-
configuration anomeric proton of glucose
at dH 4.87 (1H, d, J ¼ 7.3 Hz) showed
HMBC correlation with the aromatic
C-atom C-4 (dC 146.4). Accordingly,
compound 3 was established as (þ)-8-
hydroxyepipinoresinol-4-O-b-D-glucopyr-
anoside.
3
No.
dH
dC
1
2
3
4
5
6
7
8
9
131.4
112.9
147.8
146.4
115.6
120.3
88.8
7.00 (d, 1.8)
6.75 (d, 8.2)
6.89 (dd, 8.2, 1.8)
4.29 (s)
90.6
75.9
3.50 (d, 9.0)
4.16 (d, 9.0)
10
20
30
40
50
60
70
80
90
129.9
110.2
148.7
145.8
115.0
118.3
81.1
6.86 (d, 1.0)
7.03 (d, 8.6)
6.73 (dd, 8.6, 1.0)
5.08 (d, 5.2)
2.01–3.04 (m)
3.03–3.05 (m)
3.82–3.85 (m)
4.87 (d, 7.3)
3.17 (m)
57.6
67.9
100
200
300
400
500
600
100.7
77.4
73.7
70.2
77.3
61.1
3.26 (m)
3.17 (m)
3.28 (m)
3.47 (m)
3.68 (m)
3.75
3.76
ZOCH3
ZOCH3
56.0
56.0
Note: Coupling constants (J) in Hz are given in
parentheses; chemical shift values are expressed in
ppm.
was established as C26H32O12 by HR-ESI-
MS analysis (m/z 559.1791 [M þ Na]þ).
1
The H and 13C NMR signals (Table 2)
were assigned by the DEPT, HSQC, and
HMBC analyses, indicating the presence
of one glucosyl and two guaiacyl groups.
Two characteristic downfield CH signals
at dH 5.08 (1H, d, J ¼ 5.2 Hz) and 4.29
(1H, s) and six characteristic aliphatic
carbon signals at dC 81.1 (CH), 57.6 (CH),
67.9 (CH2), 88.8 (CH), 90.6 (CH), and
75.9 (CH2) suggested that compound 3
was a 8-hydroxyfuranoid lignan. In the
HMBC spectrum (Figure 2), the cross-
The known compounds were readily
identified as glochidioboside (4) [17], (þ)-
pinoresinol-4-O-b-D-glucopyranoside (5)
[18], (þ)-epipinoresinol-4-O-b-D-gluco-
pyranoside (6) [18], (þ)-epipinoresinol-
40-O-b-D-glucopyranoside (7) [18], (þ)-
phyllrin (8) [18], (–)-matairesinoside (9)
[18], and (þ)-isolariciresino-9-O-b-D-glu-
copyranoside (10) [19] by comparing

608
X.-J. Yan et al.
NMR spectral data with those reported in
the literature.
column
(30 m £ 320 mm £ 0.25 mm).
Sephadex LH-20 (20–100 mm, Pharmacia
Fine Chemical Co. Ltd, Piscataway, NJ,
USA), silica gel (200–300 mesh, Qingdao
Marine Chemistry Ltd, Qingdao, China),
macroporous resin (D101, Cangzhou Bon
Adsorber Technology Co., Ltd, Cangzhou,
China), and Cosmosil octadecyl silane
(ODS; 40–80 mm, Nacalai Tosoh, Inc.,
Uetikon, Switzerland) were used for
column chromatography (CC). TLC was
conducted on silica gel GF254 (Qingdao
Marine Chemistry Ltd).
All isolated compounds were evalu-
ated for their cytotoxic activities against
lung cancer (A549), colon cancer (Colo-
205), hepatoma (Hep-3B), leucocythemia
(HL-60), and cervical cancer (KB) cell
lines by the MTT method [20], using
5-fluorouracil and paclitaxel as positive
controls. Compound 3 showed cytotox-
icity against the five human cancer cell
lines with IC50 values of 9.48, 7.75, 0.59,
4.06, and 38.38 mM, respectively, and was
more cytotoxic than 5-fluorouracil (25.72,
39.30, 4.80, 11.52, and 44.35 mM,
respectively). Meanwhile, compound 3
displayed similar cytotoxicity to paclitaxel
(IC50 ¼ 7.80 mM) against Colo-205 cells
and more cytotoxic than paclitaxel
(IC50 ¼ 2.09 mM) against Hep-3B cells.
Other isolates (1, 2, 4–10) are inactive.
3.2 Plant material
The fruits (8.3 kg) of F. suspense were
collected from Henan Province of China in
June 2009, and authenticated by Prof. Jin-
Cai Lu, Department of Pharmacognosy,
Shenyang Pharmaceutical University. The
voucher specimen (No. 20091011) is kept
in the Nature Products Laboratory of
Shenyang Pharmaceutical University, She-
nyang, China.
3. Experimental
3.1 General experimental procedures
Optical rotations were measured on a
JASCOP-1020 Polarimeter (Jasco Co.,
Tokyo, Japan). UV spectra were carried
out on a Shimadzu UV-1700 spectropho-
tometer (Shimadzu Corporation, Kyoto,
Japan). IR spectra were obtained on a
Shimadzu ftir-8400s spectrophotometer
(Shimadzu Corporation). NMR spectra
were recorded on Bruker ARX-400 and
ARX-600 instruments (Bruker Co., Bill-
erica, MA, USA). HR-ESI-TOF-MS
experiments were carried out on a Micro
TOF spectrometer (Bruker Co., Karlsruhe,
Germany). CD spectra were measured by
Bio-Logic (MOS 450) spectrophotometer.
High-performance liquid chromatography
(HPLC) preparation was carried out on a
Hitachi preparative HPLC system (Hitachi
Ltd, Tokyo, Japan) equipped with Refrac-
tive Index Detector (L-2490) and prep-
ODS (10 mm £ 250 mm). GC was done on
an Agilent 7890A Gas Chromatograph
(Agilent Technologies, Inc., Santa Clara,
CA, USA) equipped with HP-5 capillary
3.3 Extraction and isolation
The fruits of F. suspense (8.3 kg) were
crushed to pieces and refluxed with 50%
EtOH for three times. The extract was
evaporated under reduced pressure to
afford a residue (350 g). The residue was
suspended in H2O and then successively
extracted with CHCl3, EtOAc, and
n-butanol. The n-butanol extract was
evaporated in vacuo to give a residue
(100 g), which was chromatographed over
D101 macroporous resin and a gradient of
aqueous EtOH (water, 30%, 60%, and
95%, v/v) to yield four major fractions
(A–D). Fraction C (55.0 g) was subjected
to silica gel CC (12 £ 60 cm) with a
gradient mixture of CH2Cl2 –MeOH
(100:0–50:50) to afford five fractions
(1–5). Fraction 2 (22.4 g) was further
purified over an ODS column chromatog-
raphy (4 £ 45 cm) using MeOH and H2O
as the mobile phases with a gradient from

Journal of Asian Natural Products Research
609
20% to 60% to afford fractions F2-1–F2-8
based on HPLC analysis. F2-2 (6.1 g) was
subjected to another silica gel CC (2
£ 30 cm) and eluted with CH2Cl2:MeOH:
3.3.2 Forsythiayanoside B (2)
25
Sticky oil (MeOH); ½aꢀD 210.3 (c ¼
0.05, MeOH). IR (KBr) vmax (cm21):
3440, 2918, 2850, 1631, 1466, 1384, 1128,
and 1075; UV (MeOH) lmax: 207.0, 227.4,
and 278.6 nm; CD (MeOH): D1205.1 nm
24.80, D1240.0 nm þ4.69, D1283.4 nm
þ8.73; for 1H and 13C NMR spectral
data (DMSO-d6), see Table 1; HR-ESI-
MS: m/z 575.2098 [M þ Na]þ (calcd for
C27H36O12Na, 575.2099).
water (8:2:0.25) to afford fractions F2-2-1
–
F2-2-5 based on TLC analysis. F2-2-2
(500 mg) was subjected to preparative
HPLC eluted with CH3OH–H2O (15:85)
at 3 ml/min (tR 15 and 17 min) to yield
compounds 4 (15 mg) and 9 (36 mg). F2-2-3
(200 mg) was subjected to semi-prepara-
tive HPLC eluted with CH3CN–H2O
(10:90) at 3.5 ml/min (tR 15 min) to yield
2 (10 mg), 3.5 ml/min (tR 20 min) to
yield 3 (9 mg), and 3.5 ml/min (tR
23 min) to yield 7 (15 mg). F2-3 (2.0 g)
was subjected to semi-preparative HPLC
eluted with CH3CN–H2O (20:80) at
3.5 ml/min (tR 20 min) to yield 1 (20 mg).
Fraction 3 (18.0 g) was further purified
over an ODS column chromatography (4
£ 45 cm) using MeOH and H2O as
the mobile phase with a gradient from
10% to 60% to afford fractions F3-1–F3-4
based on HPLC analysis. F3-2 (10.0 g)
was subjected to semi-preparative HPLC
eluted with CH3CN–H2O (25:75) at
3.3.3 (þ)-8-Hydroxyepipinoresinol-4-
O-b-D-glucopyranoside (3)
25
Yellow amorphous powder (MeOH); ½aꢀD
28.1 (c ¼ 0.05, MeOH). IR (KBr) vmax
(cm21): 3441, 2919, 2850, 1632, 1384,
and 1129; UV (MeOH) lmax: 207.2, 227.0,
and 278.2 nm; CD (MeOH): D1218.4 nm
23.72, D1236.0 nm þ4.23, D1278.2 nm
27.21; for 1H and 13C NMR spectral
data (DMSO-d6), see Table 2; HR-ESI-
MS: m/z 559.1791 [M þ Na]þ (calcd for
C26H32O12Na, 559.1786).
3.4 Acid hydrolysis of compounds 1–3
3.5 ml/min (tR 16 min) to yield
5
(30 mg), 3.5 ml/min (tR 23 min) to yield 6
(20 mg), and 3.5 ml/min (tR 40 min) to
yield 10 (10 mg). Fraction 4 (2.0 g)
was subjected to semi-preparative
HPLC eluted with CH3CN–H2O (25:75)
at 3.5 ml/min (tR 18 min) to yield 8
(100 mg).
Each compound (3.0 mg) was hydrolyzed
with 2 M HCl (5.0 ml), heated for 4 h at
958C and extracted with CHCl3 (3
£ 5.0 ml). Then, the aqueous layer was
concentrated in vacuo to appropriate
volume, and the solution was examined
by TLC (EtOAc–BuOH–H2O–HOAc,
4:4:1:1), and compared with the authentic
sample, glucose was detected. Each
remaining aqueous layer was concentrated
to dryness to give a residue, which was
dissolved in pyridine (1.0 ml), and then
L-cysteine methyl ester hydrochloride
(2.0 mg) was added to the solution. The
mixture was heated at 608C for 2 h, and
0.5 ml of N-tremethylsilyimidazole was
added, followed by heating at 608C for 2 h.
The reaction product was subjected to GC
analysis on Agilent 7890A (HP-5, 30 m
£ 320 mm, 0.25 mm) with flame ionization
detector detection. Column temperature:
3.3.1 Forsythiayanoside A (1)
25
Colorless solid (MeOH); ½aꢀD 212.4
(c ¼ 0.10, MeOH). IR (KBr) vmax
(cm21): 3405, 2919, 2850, 1665, 1594,
1514, 1384, 1263, and 1046; UV (MeOH)
lmax: 206.6, 227.6, 275.6, and 302.2 nm;
CD (MeOH): D1235.2 nm 210.17,
D1276.0 nm 25.83, D1294.0 nm þ3.48,
1
D1321.0 nm 21.25; for H and 13C NMR
spectral data (DMSO-d6), see Table 1;
HR-ESI-MS: m/z 573.1942 [M þ Na]þ
(calcd for C27H34O12Na, 573.1942).

610
X.-J. Yan et al.
120–2808C with the rate of 88C/min, and
the carrier gas was N2 (1.4 ml/min);
injection temperature: 2508C; injection
volume: 1 ml. The absolute configuration
of the monosaccharides was confirmed to
be D-Glu by comparison of the retention
time of its Me3Si ethers with standard
sample [tR (D-glucose) ¼ 25.873 min].
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3.5 Cytotoxicity assay
Human malignant leucocythemia cell lines
HL-60, hepatoma cell lines Hep-3B, colon
cancer cell lines Colo-205, cervical cancer
lines KB, and lung cancer cell lines A549
were obtained from the National Center
for Medical Culture Collection (Shanghai,
China). They were routinely cultured in
Roswell Park Memorial Institute (RPMI)
1640 or Dulbecco’s modified Eagle
medium supplemented with 10% fetal
bovine serum and maintained at 378C in
a humidified incubator with 5% CO2. The
in vitro cell viability effects of compounds
were determined by MTT assay [20]. The
cells (1 £ 105 cells/ml) were seeded into
96-well culture plates. After overnight
incubation, the cells were treated with
various concentrations of agents for 72 h.
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phosphate-buffered saline) was added to
each well, and the plates were incubated
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Acknowledgment
Authors thank Mrs Wen Li and Mr Yi Sha of
Shenyang Pharmaceutical University for the
recording of NMR spectra.