Quinoid glycosides from Forsythia suspensa

Article abstract of DOI:10.1016/j.phytochem.2014.04.010

Phytochemical investigation on Forsythia suspensa (Thunb.) Vahl afforded 10 compounds, including quinoid glycosides, lignan glycosides, phenylethanoid glycoside and allylbenzene glycoside together with 13 known ones. Their structures were established based on extensive spectroscopic data analyses, including IR, UV, HRESIMS, 1D NMR and 2D NMR. Absolute configurations were determined by ECD calculation method and chemical degradation. In addition, all compounds were evaluated for their antiviral activity against influenza A (H1N1) virus and several were further evaluated against respiratory syncytial virus (RSV) in vitro. Among them, two previously known compounds showed significant activities against RSV with EC₅₀ values of 3.43 and 6.72 μM.

Full text of DOI:10.1016/j.phytochem.2014.04.010

Phytochemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Quinoid glycosides from Forsythia suspensa
Chang Li a,1, Yi Dai b,d,1, Shu-Xiang Zhang c, Ying-Hui Duan b,d, Ming-Li Liu b,d, Liu-Yuan Chen b,d
,
Xin-Sheng Yao a,b,d,
⇑
a College of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China
b Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, People’s Republic of China
c Beijing American Oriental Pharmaceutical Institute, Beijing 100176, People’s Republic of China
d Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou 510632, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 May 2013
Received in revised form 26 February 2014
Available online xxxx
Phytochemical investigation on Forsythia suspensa (Thunb.) Vahl afforded 10 compounds, including
quinoid glycosides, lignan glycosides, phenylethanoid glycoside and allylbenzene glycoside together with
13 known ones. Their structures were established based on extensive spectroscopic data analyses,
including IR, UV, HRESIMS, 1D NMR and 2D NMR. Absolute configurations were determined by ECD
calculation method and chemical degradation. In addition, all compounds were evaluated for their
antiviral activity against influenza A (H1N1) virus and several were further evaluated against respiratory
syncytial virus (RSV) in vitro. Among them, two previously known compounds showed significant
Keywords:
Forsythia suspensa
Quinoid glycosides
Lignan glycosides
Antiviral activity
activities against RSV with EC50 values of 3.43 and 6.72 lM.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
Forsythia suspensa (Thunb.) Vahl is widely distributed in China,
Japan, Korea and many European countries. The dried fruit of this
plant, locally named ‘Lianqiao’, is a well known herbal medicine
in China for the treatment of pyrexia and infections (Cui et al.,
2010). A number of phenylethanoid glycosides (Wang et al.,
2009), lignan glycosides (Chang et al., 2008; Piao et al., 2008),
quinoid glycosides (Ming et al., 1998), triterpenes (Rouf et al.,
2001) and alkaloids (Zhang et al., 2002) have been isolated from
the fruit of this plant. Some of them possess various bioactivities,
including antiviral (Li et al., 2011), antibacterial (Qu et al., 2012)
and anti-inflammatory activities (Dai et al., 2009). Our investiga-
tions on the fruit of F. suspensa in searching for antiviral
components led to the isolation of 10 new compounds, along with
13 known ones. Herein, we mainly described the isolation and
structural elucidation of the new compounds, as well as the
antiviral activities against influenza A (H1N1) virus and respiratory
syncytial virus (RSV).
The dried fruit of F. suspensa was extracted with EtOH–H2O
(60:40), and then the extract was subjected to various column
chromatographies to yield 10 new compounds, forsythensides
G–L (1–6) and forsythialansides A–D (7–10) (Fig. 1). Moreover,
13 known compounds were also isolated, and their structures
were identified as cornoside (11) (Lu et al., 2009), forsythenside
A (12) (Ming et al., 1998), icariside E4 (13) (Miyase et al.,
1989), dihydrodehydrodiconiferyl alcohol-4-O-b-D-glucoside (14)
(Ou yang et al., 2011), calceolarioside B (15) (Nicoletti et al.,
1986), forsythoside A (16) (Wang et al., 2009), sasanquin (17)
(Zhao et al., 1999), epipinoresinol-4-O-b-
(Nishibe et al., 1984), phillyrin (19) (Nishibe et al., 1984), epipi-
noresinol-4-O-b- -glucoside (20) (Rahman et al., 1990), pinoresi-
nol-4-O-b- -glucoside (21) (Kim et al., 2005), pinoresinol (22)
D-glucoside (18)
D
D
(Li et al., 2012) and syringaresinol-4-O-b-D-glucoside (23) (Li
et al., 2001), respectively.
Compound 1 was obtained as light yellow gum. Its molecular
formula was deduced to be C24H34O10 by HRESIMS. The 13C NMR
spectrum showed 24 carbon signals. The characteristic chemical
shift of one carbonyl group (d 187.8), along with four olefinic car-
bons (d 154.4 ꢀ 2, 128.0, 127.9), two methylene carbons (d 65.8,
41.0) and a quaternary carbon (d 69.2) indicated that compound
1 had a 1-hydroxy-1-hydroxyethyl-2,5-cyclohexadienone moiety
(Wang et al., 2008). This was further confirmed by HMBC correla-
tions (Fig. 2).
⇑
Corresponding author at: College of Traditional Chinese Materia Medica,
Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China.
Tel.: +86 20 85225849; fax: +86 20 85221559.
E-mail address: tyaoxs@jnu.edu.cn (X.-S. Yao).
These co-authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.phytochem.2014.04.010
0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Li, C., et al. Quinoid glycosides from Forsythia suspensa. Phytochemistry (2014), http://dx.doi.org/10.1016/
j.phytochem.2014.04.010

2
C. Li et al. / Phytochemistry xxx (2014) xxx–xxx
Fig. 1. Structural formulae of compounds 1–10.
{½a 2D0
ꢁ
+45.3 (CHCl3)}, which indicated that the absolute configura-
O
tion of C-400 was R (Nakanishi et al., 2005). Thus, the structure of 1
was deduced as 6-O-[(R)-oleuropeoyl]-1-O-[2-(1-hydroxy-4-oxo-
OH
2,5-cyclohexadien-1-yl)ethyl]-b-D-glucopyranose, trivially named
forsythenside G.
O
OH
HO
HO
Compounds 2–5 were also isolated as light yellow gums. The
molecular formula of 2, C24H38O10, was established by analysis of
HRESIMS data. The 13C NMR and DEPT-135 spectroscopic data of
2 were similar to those of 1 except that the two olefinic groups (d
154.4 ꢀ 2, 128.0, 127.9) in 1, were replaced by four methylenes (d
37.9 ꢀ 2, 37.8 ꢀ 2) in 2, which suggested that the 2,5-cyclohexadi-
enone moiety in 1 was replaced by a cyclohexanone moiety in 2.
Methanolysis of 2 with NaOMe in MeOH also gave (+)-oleuropeic
O
O
OH
O
1
Fig. 2. Key HMBC (?) and 1H, 1H-COSY (
) correlations of 1.
acid (1a) {½a 2D0
ꢁ
+38.0 (CHCl3)}; therefore, the absolute configuration
of C-400 in 2 was assigned to be R. Thus, compound 2 was assigned as
6-O-[(R)-oleuropeoyl]-1-O-[2-(1-hydroxy-4-oxocyclohexyl)ethyl]-
The anomeric carbon resonance at d 104.4 showed the existence
of a monosaccharide moiety. Acid hydrolysis and HPLC analysis
were performed according to the method of Tanaka et al. (2007),
b-D-glucopyranose, it was given the trivial name forsythenside H.
which suggested that the monosaccharide was D-glucose. More-
over, the b configuration was prompted by the large coupling con-
stant of the anomeric proton (d 4.24, d, J = 7.8 Hz).
Compound 3 had the same molecular formula with 1, which
was determined by HRESIMS data. The 13C NMR and DEPT-135
data of 3 also showed the presence of a monosaccharide moiety
and a 1-hydroxy-1-hydroxyethyl-2,5-cyclohexadienone unit, as
same as compound 1. The remaining 10 carbon signals consisted
In addition, typical carbon signals at d 131.2, 141.5 and 168.7
showed the existence of an
a,b-unsaturated carbonyl group. In
1
the 1H, 1H-COSY spectrum, the 1H, H spin system (H200/H300, H300/
H400, H400/H500, and H500/H600) enabled deduction of the fragment –
CH–CH2–CH–CH2–CH2–. In the HMBC spectrum, correlations of
H-200/C-600, C-700 and C-400, and those of H-900/C-800 and C-400 sug-
gested the presence of an oleuropeic acid unit (Tian et al., 2009).
Moreover, the linkages of three moieties were established on the
basis of HMBC correlations of H-10/C-8 and H-60/C-700 (Fig. 2).
The absolute configuration of 1 was determined by chemical
degradation and optical rotation analysis. Methanolysis of 1 was
performed with NaOMe in MeOH. After neutralization with
HCOOH, the EtOAc extract was obtained and evaporated to
dryness, then the residue was dissolved in MeOH and further
purified by preparative HPLC to yield (+) oleuropeic acid (1a)
of one
a,b-unsaturated carbonyl group (d 168.6, 139.3, 130.6),
two methyls (d 17.3, 17.2), three methylenes (d 35.6, 31.4, 22.4),
one methine (d 38.6) and one quaternary carbon signal (d 72.4).
Those resonances demonstrated the presence of a 6-hydroxy-6-
isopropylcyclohex-1-enecarboxylic acid moiety (Dai et al., 2005).
Furthermore, the linkages of the three moieties were based on
the HMBC correlations of H-10/C-8, and H-60/C-700.
Methanolysis of 3 was also performed with NaOMe in MeOH to
yield 6-hydroxy-6-isopropylcyclohex-1-enecarboxylic acid (3a).
The electronic circular dichroism (ECD) spectrum of 3a was calcu-
lated by time dependent density functional theory (TDDFT). The
calculated ECD data matched well with the experimental ECD data
of 3a (Fig. 3), allowing the assignment of the absolute configuration
Please cite this article in press as: Li, C., et al. Quinoid glycosides from Forsythia suspensa. Phytochemistry (2014), http://dx.doi.org/10.1016/
j.phytochem.2014.04.010

C. Li et al. / Phytochemistry xxx (2014) xxx–xxx
3
6
4
the characteristic signals of a feruloyl moiety observed in the
13C NMR spectrum (Table 1). Thus, compound 4 was elucidated
as 6-O-feruloyl-1-O-[2-(1-hydroxy-4-oxo-2,5-cyclohexadien-1-yl)
Experimental of 3a
Calculated of 3a
ethyl]-b-D-glucopyranose, named forsythenside J.
Compound 5 had a molecular formula of C29H36O14 by HRESIMS
data analysis. Its 1H NMR spectrum exhibited characteristic proton
signals of a p-hydroxycinnamoyl moiety at d 6.81 (2H, d, J = 8.6 Hz),
7.47 (2H, d, J = 8.6 Hz), 7.67 (1H, d, J = 15.9 Hz), and 6.37 (1H, d,
J = 15.9 Hz). Additionally, one aromatic ABX coupling system was
observed at d 6.69 (1H, d, J = 1.9 Hz), 6.68 (1H, d, J = 8.1 Hz) and
6.57 (1H, d, J = 1.9, 8.1 Hz). These signals, together with two cou-
pled methylene protons at d 3.98, 3.71 (each 1H, m) and 2.78
(2H, m), indicated the existence of a phenylethanol moiety.
Moreover, the 1H NMR spectrum showed two anomeric proton
signals at d 4.37 (1H, d, J = 7.8 Hz) and 4.64 (1H, d, J = 1.2 Hz). Acid
2
0
-2
-4
-6
hydrolysis of 5 gave D-glucose and L-rhamnose as sugar residues.
200
250
300
350
The 13C NMR spectrum also displayed carbon signals belonged to
one p-hydroxycinnamoyl and one phenylethanol moiety, besides
those of glucose and rhamnose moieties. The linkages were estab-
lished on the basis of the HMBC correlations of H-10/C-8, H-40/C-9000
and H-100/C-60. Therefore, compound 5 was assigned as 3,4-dihy-
Wavelength (nm)
Fig. 3. Experimental and calculated ECD spectra for 3a.
droxy-b-phenethyl-O-
hydroxycinnamoyl-b-
a
D
-
L
-rhamnopyranosyl-(1?6)-40-O-p-
-glucopyranoside, named forsythenside K.
of 600 in 3 as R. Therefore, compound 3 was elucidated as 6-O-[(R)-
6-hydroxy-6-isopropylcyclohex-1-enecarbonyl]-1-O-[2-(1-hydroxy-
4-oxo-2,5-cyclohexadien-1-yl)ethyl]-b-D-glucopyranose, named
Compound 6 was obtained as yellow amorphous powder. Its
molecular formula was determined to be C20H28O11 on the basis
of HRESIMS data. The 1H NMR spectrum indicated the presence
of a 1,3,4-trisubstituted benzene ring [d 6.68 (1H, d, J = 2.1 Hz),
7.13(1H, d, J = 8.2 Hz), 6.63 (1H, dd, J = 2.1, 8.2 Hz)] and a terminal
olefinic bond [d 5.03 (2H, m), 5.93 (1H, ddt, J = 6.7, 10.1, 16.9 Hz)].
In addition, two anomeric protons at d 4.70 (1H, d, J = 7.5 Hz), 4.33
(1H, d, J = 7.3 Hz) suggested that there were two monosaccharide
forsythenside I.
Compound 4 had a molecular formula of C24H28O11 by HRESIMS.
Compared to the NMR spectroscopic data of compound 4 with those
of 1, the same glucose moiety and the same 1-hydroxy-1-hydroxy-
ethyl-2,5-cyclohexadienone unit were deduced. In addition, the 1H
NMR spectrum had a methyl proton signal at 3.90 (3H, s) and
typical proton resonances at d 7.23 (1H, d, J = 1.8 Hz), d 6.82 (1H,
d, J = 8.2 Hz), d 7.09 (1H, dd, J = 1.8, 8.2 Hz) and d 7.64 (1H, d,
J = 15.9 Hz), d 6.40 (1H, d, J = 15.9 Hz), which suggested the
presence of one feruloyl moiety. This was further confirmed by
moieties. Acid hydrolysis of 6 gave D-glucose and D-xylose as sugar
residues. Moreover, The 13C NMR spectroscopic data of the
aglycone moiety in Table 2 were in agreement with those of 3,
4-dihydroxy-1-allylbenzene moiety (Ly et al., 2002). The linkages
Table 1
1H and 13C NMR spectroscopic data for compounds 1–4 (Measured in CD3OD).
Position
Compound 1a
dC, type
Compound 2b
dC, type
Compound 3b
dC, type
Compound 4a
dC, type
dH (J in Hz)
dH (J in Hz)
dH (J in Hz)
dH (J in Hz)
1
2
3
4
5
6
7
8
69.2, C
70.3, C
69.2, C
69.2, C
154.4, CH
128.0, CH
187.8, C
127.9, CH
154.4, CH
41.0, CH2
65.8, CH2
7.01, m
6.10,d (10.1)
37.8, CH2
37.9, CH2
214.8, C
37.9, CH2
37.8, CH2
42.0, CH2
67.1, CH2
2.00, m; 1.84, m
2.66, m; 2.17, m
154.5, CH
128.0, CH
187.8, C
127.9, CH
154.4, CH
41.0, CH2
65.8, CH2
7.01, m
6.10,d (10.2)
154.4, CH
128.0, CH
187.8, C
127.9, CH
154.3, CH
41.0, CH2
65.9, CH2
6.98, dd (10.2)
6.08, dd (10.2)
6.10, d (10.1)
7.01, m
2.66, m; 2.17, m
2.00, m; 1.84, m
1.91, m
4.03, m
3.77, m
4.30,d (7.8)
3.16, m
3.36, m
3.35, m
3.49, m
6.10, d (10.2)
7.01, m
2.04, t (6.5)
3.91, m
3.63, m
4.23, d (7.8)
3.14, m
3.33, m
3.27, m
3.46, m
4.46, dd (2.1, 11.9)
4.21, m
6.08, dd (10.2)
6.98, dd (10.2)
2.05, t (6.5)
3.91, m
3.65, m
4.25, d (7.8)
3.18, m
3.36, m
3.35, m
3.53, m
4.50, dd (2.2, 11.9)
4.33, m
2.04, t (6.5)
3.91,dt (6.6, 10.1)
3.64, dt (6.6, 10.1)
4.24, d (7.8)
3.15, m
3.35, m
3.31, m
3.47, m
4.45, dd (2.0, 11.9)
4.21, dd (6.1, 11.9)
10
20
30
40
50
60
104.4, CH
75.0, CH
77.9, CH
71.7, CH
75.4, CH
64.6, CH2
104.5, CH
75.1, CH
78.0, CH
71.7, CH
75.5, CH
64.6, CH2
104.3, CH
75.0, CH
77.9, CH
71.7, CH
75.4, CH
64.6, CH2
104.4, CH
75.0, CH
77.9, CH
71.7, CH
75.4, CH
64.6, CH2
4.45, dd (2.2, 11.9)
4.25, m
100
131.2, C
141.5, CH
28.6, CH2
45.5, CH
24.5, CH2
26.3, CH2
168.7, C
72.8, C
131.2, C
141.5,CH
28.6, CH2
45.5, CH
24.6, CH2
26.4, CH2
168.7, C
72.8, C
130.6, C
139.3, CH
35.6, CH2
22.4, CH2
31.4, CH2
72.4, C
168.6, C
38.6, CH
17.3, CH3
17.2, CH3
127.7, C
111.7, CH
149.4, C
200
7.03, m
2.36, m; 2.04, m
1.55, m
2.03, m; 1.24, m
2.52, m; 2.18, m
7.04, m
2.34, m; 2.02, m
1.53, m
2.00, m; 1.21, m
2.51, m; 2.14, m
6.93, m
2.33, m; 2.16, m
2.37, m
7.23, d (1.8)
300
400
150.7, C
500
1.74, m; 1.55, m
116.5, CH
124.3, CH
147.1, CH
115.3, CH
169.1, C
6.82, d (8.2)
600
7.09, dd (1.8, 8.2)
7.64, d (15.9)
6.40, d (15.9)
700
800
1.65, m
0.98, d (6.9)
0.95, d (6.9)
900
27.1, CH3
26.4, CH3
1.19, s
1.19, s
27.0, CH3
26.5, CH3
1.18, s
1.18, s
1000
300-OCH3
56.5, CH3
3.90, s
a
300 MHz for 1H NMR, 75 MHz for 13C NMR.
400 MHz for 1H NMR, 100 MHz for 13C NMR.
b
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4
C. Li et al. / Phytochemistry xxx (2014) xxx–xxx
Table 2
1H and 13C NMR spectroscopic data for compounds 5–6 (Measured in CD3OD).
Position
Compound 5a
dC, type
Compound 6a
dC, type
dH (J in Hz)
dH (J in Hz)
1
2
3
4
5
6
7
8
131.4, C
116.3, CH
146.1, C
137.3, C
117.3, CH
148.2, C
6.69, d (1.9)
6.68, d (2.1)
144.7, C
145.0, C
117.1, CH
121.3, CH
36.7, CH2
72.4, CH2
6.68, d (8.1)
6.57, d (1.9, 8.1)
2.78, m
119.2, CH
121.2, CH
40.6, CH2
139.0, CH
7.13, d (8.2)
6.63, dd (2.1, 8.2)
3.24, m
3.98, m
5.93, ddt (6.7, 10.1, 16.9)
3.71, m
9
115.7, CH2
104.5, CH
74.8, CH
77.6, CH
71.4, CH
77.3, CH
69.8, CH2
5.03, m
4.70, d (7.5)
3.45, m
3.27, m
3.40, m
10
20
30
40
50
60
104.5, CH
75.2, CH
75.8, CH
72.2, CH
74.9, CH
67.7, CH2
4.37, d (7.8)
3.31, m
3.64, m
4.94, m
3.60, m
3.57, m
4.12, dd (2.0, 11.5) 3.76, m
3.75, m
3.49, m
4.64, d (1.2)
3.85, m
3.68, m
3.35, m
100
200
300
400
500
102.1, CH
72.1, CH
72.3, CH
73.9, CH
69.8, CH
105.4, CH
75.0, CH
77.4, CH
71.2, CH
66.9, CH2
4.33, d (7.3)
3.20, m
3.43, m
3.47, m
3.84, m
3.62, m
3.16, m
600
18.0, CH3
127.1, C
116.9, CH
131.3, CH
161.4, C
131.3, CH
116.9, CH
147.2, CH
114.8, CH
168.3, C
1.20, d (6.2)
1000
2000
3000
4000
5000
6000
7000
8000
9000
6.81, d (8.6)
7.47, d (8.6)
7.47, d (8.6)
6.81, d (8.6)
7.67, d (15.9)
6.37, d (15.9)
a
300 MHz for 1H NMR, 75 MHz for 13C NMR.
of each moiety were established based on HMBC correlations of H-
O
10/C-4 and H-100/C-60. Thus, the structure of 6 was elucidated as 3,4-
H3CO
H3CO
O
dihydroxy-1-allylbenzene-4-O-b-
glucopyranoside, trivially named forsythenside L.
Compound 7, yellow gum, had molecular formula of
27H34O12 by HRESIMS. Its 1H NMR spectrum gave three methoxy
D-xylopyranosyl-(1?6)-b-D-
HO
a
C
HO
HO
OCH3
groups at d 3.88, 3.89 and 3.91 (each 3H, s), and one anomeric pro-
ton signal at d 4.89 (1H, d, J = 7.5 Hz). Signals belonging to two
pairs of 1,3,4-trisubstituted benzene rings were also observed at
d 7.61 (1H, d, J = 2.0 Hz), 7.07 (1H, d, J = 8.5 Hz), 7.72 (1H, dd,
J = 2.0, 8.5 Hz), and at d 7.14 (1H, d, J = 2.0 Hz), 7.14 (1H, d,
J = 8.3 Hz), 6.95 (1H, dd, J = 2.0, 8.3 Hz). The 13C NMR and DEPT-
135 spectra of 7 showed 27 carbon signals [three primary, three
secondary, fourteen tertiary, and seven quaternary (including one
O
O
HO
OH
7
Fig. 4. Key HMBC (?) and 1H, 1H-COSY (
) correlations of 7.
ketone) carbon signals]. Acid hydrolysis of 7 gave
sugar residue. From the above evidence, compound
D
-glucose as
was
correlation between H-70/H-8 was observed. Thus, the relative con-
figuration of H-70/H-8 was determined as being a trans form. The
calculated ECD spectrum for the aglycone of 7 was not in suffi-
ciently good agreement with the experimental ECD spectrum of
7. In addition, hydrolysis of compound 7 to obtain the aglycone
of 7 was not successful. Thus, the absolute configuration of 7 was
not determined. Compound 7 was deduced as 3,30,4-trimethoxy-
7
proposed as a lignan glycoside with three methoxy groups. The
13C NMR spectroscopic data of the aglycone were similar to those
of forsythialan B (Piao et al., 2008), except that the 3-methoxy-
5-hydroxyphenyl moiety in forsythialan B was replaced by the
3-methoxy-4-hydroxyphenyl moiety in 7. In addition, the glucose
moiety was located at C-40 of the aglycone due to the HMBC
correlation of H-100/C-40 (Fig. 4).
70,9-epoxylignan-90-ol-7-one-40-O-b-
D-glucopyranoside, trivially
named forsythialanside A.
The relative configurations of H-70/H-80 were determined on the
basis of the characteristic chemical shift of H-70. The trans configu-
ration often gave the signal of H-70 at d 4.7, whereas the cis config-
uration gave a relative downfield shift at d 5.5 (Guan et al., 2008).
The chemical shift of H-70 of 7 was observed at d 4.69, indicating
the trans configuration of H-70/H-80. The NOESY spectrum dis-
played a correlation between H-8 and H-80. However, no NOESY
Compound 8 was obtained as yellow gum. Its molecular for-
mula was determined to be C26H32O12 on the basis of HRESIMS
data. The 1D NMR spectroscopic data of 8 resembled those of
compound 7, except for the absence of one methoxy group of the
aglycone. In the HMBC spectrum, the correlation between the
anomeric proton (d 5.06) and O-linked carbon (d 152.6) suggested
the glucose moiety was linked to the C-4 of the aglycone.
Please cite this article in press as: Li, C., et al. Quinoid glycosides from Forsythia suspensa. Phytochemistry (2014), http://dx.doi.org/10.1016/
j.phytochem.2014.04.010

C. Li et al. / Phytochemistry xxx (2014) xxx–xxx
5
The chemical shift of H-70 at d 4.64 suggested a trans configura-
tion for H-70/H-80. As no correlation between H-70 and H-8 in the
NOESY spectrum was observed, the relative configuration of
H-70/H-8 was determined to be trans. Consequently, compound 8
was elucidated as 40-hydroxy-3,30dimethoxy-70,9-epoxylignan-90-
15
10
5
Calculated of 9a
Experimental of 9a
Experimental of 9
ol-7-one-4-O-b-D-glucopyranoside, namely forsythialanside B.
Compound 9 was isolated as yellow gum. Its molecular formula
was deduced to be C32H44O15 based on HRESIMS data. 1H NMR
0
spectrum showed two anomeric protons at
J = 7.6 Hz) and 4.64 (1H, d, J = 1.5 Hz). Acid hydrolysis of 9 yielded
-glucose and
-rhamnose as sugar residues. 13C NMR spectrum
d 4.88 (1H, d,
-5
D
L
showed 32 carbon signals. The resonances of the aglycone were
similar to those of dihydrodehydrodiconiferyl alcohol (Takeda
et al., 1998), which was further confirmed by analysis of the 2D
NMR spectra (Fig. 5). Furthermore, the glucose and rhamnose moi-
ety were located at C-4 and C-90 of the aglycone, respectively, due
to the obvious HMBC correlations of H-100/C-4 and H-1000/C-90.
According to the literature (Sergio et al., 2006), the chemical
shift of H-7 was a competent tool to determine the relative config-
uration of H-7 and H-8 of benzofuran-type neolignans. The chem-
ical shift at d 5.84 suggested a cis configuration, while relative
upfield chemical shift at d 5.57 indicated a trans form. The chemical
shift of H-7 in 9 was observed at d 5.55 (1H, d, J = 5.9 Hz), suggest-
ing a trans configuration of H-7/H-8. Acid hydrolysis of 9 with 2 M
HCl at 90 °C produced dihydrodehydrodiconiferyl alcohol (9a).
Then, the calculation of ECD of 9a was applied in combination with
the experimental ECD data. The calculated ECD spectrum matched
very well with the experimental ECD spectrum of 9a (Fig. 6), indi-
cating the 7S, 8R configuration of 9. Therefore, compound 9 was
-10
-15
200
250
300
350
Wavelength (nm)
Fig. 6. Experimental and calculated ECD spectra for 9a.
activities with EC50 values of 153.9 and 166.4
within 500 M).
lM (no cytotoxicity
l
3. Conclusions
Phenylethanoid glycosides, lignan glycosides, quinoid glyco-
sides were the typical constituents of F. suspensa. Our current study
reported the isolation and elucidation of four new quinoid
glycosides, one new phenylethanoid glycoside, one new allylben-
zene glycoside and four new lignan glycosides, which added the
diversity of these ingredients. Moreover, a number of oleuropeic
acid glucose esters were reported from the family Myrtaceae
(Goodger and Woodrow, 2011), but quinoid glycosides with oleu-
ropeic acid (1–2) were found for the first time from the Oleaceae
family.
Antiviral activity evaluation of the compounds suggested that
five compounds (4, 5, 7, 15, 16) were active in inhibiting RSV. Three
phenylethanoid glycosides showed good activity especially.
Among them, the antiviral activity of forsythoside A (16) has pre-
viously been reported (Ma et al., 2012), but the result here first
assigned as (7S, 8R)-dihydrodehydrodiconiferyl alcohol-4-O-b-D-
glucopyranoside-90-O-
a-L-rhamnopyranoside, named forsythialan-
side C.
Compound 10 was also afforded as yellow gum. Its molecular
formula was determined to be C32H44O14 by HRESIMS. The 1D
NMR spectroscopic data of 10 were similar to those of compound
9, except that the glucose moiety was replaced by a rhamnose
moiety. Acid hydrolysis of 10 gave L-rhamnose as sugar residue.
The chemical shift of H-7 was at d 5.55 (1H, d, J = 6.0 Hz), which
demonstrated the trans configuration of H-7 and H-8. Acid hydro-
lysis of 10 with 2 M HCl at 90 °C also yielded 9a. The calculated
ECD spectrum of 9a matched the experimental result very well,
allowing the assignment of the absolute configuration of 10 as
depicted. The structure of compound 10 was deduced to be
reported its anti-RSV activity with an EC50 value of 6.72 lM. Fur-
thermore, the possible relationships between the chemical struc-
tures of phenylethanoid glycosides and their anti-RSV activities
were not reported before, comparison of compound 5 with 15
and 16 found that the more phenolic hydroxyl groups the com-
pound had, the better activity it showed. In conclusion, compounds
isolated from F. suspensa showed structural diversity. Antiviral
activity suggested that phenylethanoid glycosides were signifi-
cantly active against RSV in vitro, thereby providing a basis for
the activity of this plant.
(7S, 8R)-dihydrodehydrodiconiferyl alcohol-4,90-di-O-
a-L-rhamno-
pyranoside, trivially named forsythialanside D.
All compounds were evaluated for their antiviral activities
against influenza A (H1N1) virus in vitro. Among them, only com-
pounds 8, 10 and 15 showed mild activities, of which the EC50 val-
ues were at 41.1, 60.5 and 91.8
lM with no cytotoxicities within
500 M, respectively. In addition, the antiviral activities against
l
RSV of compounds 1–10, 15–16 were also tested. As a result,
compounds 15 and 16 showed significant activities with EC50 val-
ues of 3.43
lM (CC50 = 193.2 lM) and 6.72 lM (CC50 = 230.0 lM),
while compound 5 showed moderate activity with EC50 values of
4. Experimental section
36.2
lM (CC50 = 259.7
lM), compounds 4 and 7 showed weak
4.1. General experimental procedures
OH
Optical rotations were measured on JASCO P-1020 spectrome-
ter, UV spectra were recorded on
a JASCO V-550 UV/Vis
ORha
spectrometer, CD spectra were acquired on a JASCO J-810 spectro-
polarimeter, whereas IR spectra were obtained using a JASCO FT/
IR-480 plus spectrometer. 1D and 2D NMR spectra were measured
GlcO
O
H3CO
OCH3
on
a Bruker AV-300/400 spectrometer. HRESIMS data were
determined by a Waters Synapt G2 MS mass spectrometer. Open
column chromatography (CC) was performed using silica gel
(200–300 mesh, Qingdao Haiyang Chemical Goup Corp. Qingdao,
9
Fig. 5. Key HMBC (?) and 1H, 1H-COSY (
) correlations of 9.
Please cite this article in press as: Li, C., et al. Quinoid glycosides from Forsythia suspensa. Phytochemistry (2014), http://dx.doi.org/10.1016/
j.phytochem.2014.04.010

6
C. Li et al. / Phytochemistry xxx (2014) xxx–xxx
People’s Republic of China), ODS (50
(Tosoh, Japan). Thin-layer chromatography (TLC) was performed
using precoated silica gel plates (silica gel GF254, 1 mm, Yantai).
l
m, YMC, Japan), and HW-40
4.3.2. Forsythenside H (2)
Light yellow gum; ½a D20
ꢃ58.6 (c 0.5, MeOH); UV (MeOH) kmax
ꢁ
(log
e
) 218 (3.86) nm; CD (MeOH) 230 (
D
e
ꢃ0.31) nm; IR (KBr) mmax
3411, 2926, 1705 cmꢃ1. For 1H and 13C NMR spectroscopic data, see
Table 1; HRESIMS m/z 509.2375 [M+Na]+ (calcd for C24H38O10Na,
509.2363).
4.2. Plant material
Plant material provided by Heilongjiang Provincial Songhuaji-
ang Pharmaceutical Co., Ltd., was identified as the fruit of F. suspen-
sa by Professor Guang-Xiong Zhou (College of Pharmacy, Jinan
University). A voucher specimen (20090919FS) is deposited at
the Institute of Traditional Chinese Medicine and Natural Products,
Jinan University, Guangzhou, China.
4.3.3. Forsythenside I (3)
Light yellow gum; ½a D20
ꢁ
ꢃ8.5 (c 0.2, MeOH); UV (MeOH) kmax
ꢃ0.35) nm; IR (KBr) mmax
(loge) 212 (4.02) nm; CD (MeOH) 227 (De
3426, 2925, 1645 cmꢃ1. For 1H and 13C NMR spectroscopic data, see
Table 1; HRESIMS m/z 481.2054 [MꢃH]ꢃ (calcd for C24H33O10
,
481.2074).
4.3. Extraction and isolation
4.3.4. Forsythenside J (4)
The dried fruit of F. suspensa (2 kg) was extracted with EtOH–
H2O (60:40, v/v) twice, under conditions of reflux, two hours each
time, and then the combined extracts were concentrated under
reduced pressure to yield a brownish extract (540 g). An aliquot
(ꢂ500 g) of the latter was applied to a Diaion HP-20 resin column
eluted with EtOH–H2O in gradient. The 95% EtOH–H2O eluate (D,
100 g) was then subjected to silica-gel CC, eluted with a CHCl3–
MeOH gradient to give 10 fractions (D1–D10). Fraction D1 (CHCl3–
MeOH 10:0 eluate, 2.8 g) was subjected to CC over Sephadex LH-20
and silica gel to give compounds 11 (8.0 mg), and 22 (33.2 mg).
Fraction D5 (CHCl3–MeOH 9:1 eluate, 3.3 g) was subjected to
ODS CC, eluted with a MeOH–H2O gradient, to yield 10 subfrac-
tions (D5A–D5J). Subfraction D5B (MeOH–H2O (30:70, V/V) eluate,
183.7 mg) was subjected to HW-40 CC eluted with a MeOH–H2O
gradient (20:80?60:40, V/V–). The fine fraction D5B3 (MeOH–
H2O (20:80, V/V) eluate, 96.5 mg) was purified by preparative HPLC
Light yellow gum; ½a D20
ꢃ31.6 (c 0.25, MeOH); UV (MeOH) kmax
ꢁ
(loge) 234 (4.06), 296 (3.90), 323 (4.05) nm; IR (KBr) mmax 3284,
2928, 1675 cmꢃ1. For 1H and 13C NMR spectroscopic data, see
Table 1; HRESIMS m/z 491.1523 [MꢃH]ꢃ (calcd for C24H27O11
,
491.1553).
4.3.5. Forsythenside K (5)
Light yellow gum; ½a D20
ꢃ11.2 (c 0.5, MeOH); UV (MeOH) kmax
ꢁ
(loge) 203 (4.34), 226 (4.11), 291 (4.09), 314 (4.16) nm; IR (KBr)
mmax 3410, 2925, 1697, 1601 cmꢃ1. For 1H and 13C NMR spectro-
scopic data, see Table 2; HRESIMS m/z 607.2038 [MꢃH]ꢃ (calcd
for C29H35O14, 607.2027).
4.3.6. Forsythenside L (6)
Yellow amorphous powder; ½a D20
ꢃ54.6 (c 0.5, MeOH); UV
ꢁ
(Ultimate™ XB-C18 5
lm, 21.2 ꢀ 250 mm, Welch) with MeOH–
(MeOH) kmax (loge) 205 (4.52), 254 (4.56) nm; IR (KBr) mmax
H2O (30:70, V/V) to afford compounds 4 (15.2 mg), 12(20.2 mg)
and 21 (47.9 mg). The fine fraction D5B2 (MeOH–H2O (20:80, V/
V) eluate, 21.6 mg) was also purified by preparative HPLC with
MeOH–H2O (30:70, V/V) to yield compounds 7 (11.6 mg) and 8
(6.5 mg). Subfraction D5C (MeOH–H2O (50:50, V/V) eluate,
442.4 mg) was further separated by HW-40 CC eluted with a
MeOH–H2O gradient. The fine fraction D5C1 and D5C3 (MeOH–
H2O (20:80, V/V) eluate, 184.7 and 43.4 mg) were subjected to
ODS CC eluted with MeOH–H2O (30:70, V/V), and further purified
by preparative HPLC with MeOH–H2O (30:70, V/V) to afford com-
pounds 13 (4.0 mg), 1 (8.0 mg), 2 (4.0 mg) and 3 (3.6 mg), respec-
tively. Fraction D8 (CHCl3–MeOH, 80:20 V/V, eluate, 3.3 g) was
applied to ODS CC eluted with a MeOH–H2O gradient. Subfraction
D8C and D8D (MeOH–H2O (50:50, V/V) eluate, 541.2 and
184.4 mg) were subjected to HW-40 CC, and then purifed by
preparative HPLC with MeOH–H2O (40:60, V/V) to yield
3400, 2884, 1074 cmꢃ1; For 1H and 13C NMR spectroscopic data,
see Table 2; HRESIMS m/z 443.1555 [MꢃH]ꢃ (calcd for C20H27O11
,
443.1553).
4.3.7. Forsythialanside A (7)
Yellow gum; ½a 2D0
ꢃ36.8 (c 0.25, MeOH); UV (MeOH) k
ꢁ
max
(loge) 229 (4.18), 276 (3.92), 304 (3.72) nm; CD (MeOH) 240 (De
ꢃ0.22), 272 (
D
e
ꢃ0.30), 313 (
D
e
ꢃ0.25) nm; IR (KBr) mmax 3301,
2869, 1685 cmꢃ1; For 1H and 13C NMR spectroscopic data, see
Table 3; HRESIMS m/z 573.1947 [M+Na]+ (calcd for C27H34O12Na,
573.1948).
4.3.8. Forsythialanside B (8)
Yellow gum; ½a 2D0
ꢃ23.2 (c 0.25, MeOH); UV (MeOH) k
ꢁ
max
(loge) 227 (3.65), 273 (3.42), 305 (3.16) nm; CD (MeOH) 244 (De
ꢃ0.42), 266 (
D
e
ꢃ0.30), 320 (
D
e
ꢃ0.40) nm; IR (KBr) mmax 3328,
compounds 17 (13.7 mg),
5 (20.0 mg), 15 (34.3 mg) and 10
1671 cmꢃ1. For 1H and 13C NMR spectroscopic data, see Table 3;
HRESIMS m/z 559.1785 [M+Na]+ (calcd for C26H32O12Na,
559.1757).
(7.9 mg), respectively. Fraction D9 (CHCl3–MeOH 70:30, V/V
eluate, 9.1 g) was divided into 6 fractions by ODS CC with a
MeOH–H2O gradient. Subfraction D9D (MeOH–H2O (40:60, V/V)
eluate, 1.2 g) was separated using HW-40 CC, and purified by pre-
parative HPLC with MeOH–H2O (40:60, V/V) to afford compounds
16 (105.2 mg), 6 (14.3 mg) and 9 (6.4 mg). By similar procedures,
compounds 14 (3.1 mg), 18 (11.6 mg), 19 (31.8 mg), 20 (11.8 mg)
and 23 (21.9 mg) were isolated from fraction D2 (CHCl3–MeOH,
90:10, V/V, eluate, 3.0 g) and D3 (CHCl3–MeOH, 90:10, V/V eluate,
3.1 g), respectively.
4.3.9. Forsythialanside C (9)
Yellow gum; ½a 2D0
ꢁ
ꢃ53.5 (c 0.6, MeOH); UV (MeOH) kmax (log
e)
206 (4.31), 230 (3.92), 279 (3.50) nm; CD (MeOH) 238 (De
ꢃ5.42),
275 (
D
e
ꢃ1.26) nm; IR (KBr)
m
max 3397, 2926, 2346, 1619 cmꢃ1. For
1H and 13C NMR spectroscopic data, see Table 3; HRESIMS m/z
691.2565 [M+Na]+ (calcd for C32H44O15Na, 691.2578).
4.3.1. Forsythenside G (1)
4.3.10. Forsythialanside D (10)
Light yellow gum; ½a D20
ꢁ
ꢃ40.4 (c 0.7, MeOH); UV (MeOH) kmax
Yellow gum; ½a 2D0
ꢁ
–32.8 (c 0.4, MeOH); UV (MeOH) kmax (loge)
206 (4.57), 233 (4.08), 279 (3.71) nm; CD (MeOH) 241 (De
(log
e
) 223 (4.19) nm; CD (MeOH) 250 (
D
e
ꢃ0.19) nm; IR (KBr) mmax
3428, 2925, 1675 cmꢃ1. For 1H and 13C NMR spectroscopic data, see
Table 1; HRESIMS m/z 505.2042 [M+Na]+ (calcd for C24H34O10Na,
505.2050).
ꢃ0.79), 277 (
D
e
ꢃ0.54) nm; IR (KBr) mmax 3425, 2925, 2359,
1617 cmꢃ1; For 1H and 13C NMR spectroscopic data, see Table 3);
HRESIMS m/z 651.2630 [MꢃH]ꢃ (calcd for C32H43O14, 651.2653).
Please cite this article in press as: Li, C., et al. Quinoid glycosides from Forsythia suspensa. Phytochemistry (2014), http://dx.doi.org/10.1016/
j.phytochem.2014.04.010

C. Li et al. / Phytochemistry xxx (2014) xxx–xxx
7
Table 3
1H and 13C NMR spectroscopic data for compounds 7–10 (Measured in CD3OD).
Position
Compound 7a
dC, type
Compound 8b
dC, type
Compound 9b
dC, type
Compound 10b
dC, type
dH (J in Hz)
dH (J in Hz)
dH (J in Hz)
dH (J in Hz)
1
2
3
4
5
6
7
8
9
130.9, C
112.2, CH
150.6, C
155.3, C
111.8, CH
124.8, CH
200.2, C
50.4, CH
71.8, CH2
132.5, C
113.0, CH
150.8, C
152.6, C
116.3, CH
124.3, CH
200.4, C
50.5, CH
71.6, CH2
138.3, C
111.2, CH
151.0, C
147.6, C
118.1, CH
119.4, CH
88.5, CH
55.7, CH
65.1, CH2
138.8, C
111.2, CH
152.1, C
146.6, C
119.6, CH
119.1, CH
88.5, CH
55.6, CH
65.1, CH2
7.61, d (2.0)
7.66, d (1.9)
7.03, d (1.9)
7.03, d (1.9)
7.07, d (8.5)
7.72, dd (2.0, 8.5)
7.26, d (8.5)
7.69, dd (1.9, 8.5)
7.14, d (8.3)
6.93, dd (1.9, 8.3)
5.55, d (5.9)
3.45, m
7.08, d (8.2)
6.92, dd (1.9, 8.2)
5.55, d (6.0)
3.46, m
4.26, m
4.22, m
4.27, m
4.23, dd (6.3, 13.0) 4.17, m
3.84, m
3.84, m
3.76, m
3.76, m
10
20
30
40
50
60
70
80
90
137.3, C
112.1, CH
150.9, C
147.7, C
117.7, CH
120.7, CH
85.0, CH
54.7, CH
61.4, CH2
133.5, C
111.4, CH
149.1, C
147.5, C
115.9, CH
120.9, CH
85.4, CH
54.6, CH
61.4, CH2
136.7, C
114.1, CH
145.3, C
147.5, C
129.6, C
118.1, CH
33.1, CH2
32.6, CH2
67.6, CH2
136.7, C
114.0, CH
145.3,C
147.6, C
129.6, C
118.1, CH
33.1, CH2
32.6, CH2
67.6, CH2
7.14, d (2.0)
7.05, d (1.8)
6.72, brs
6.72, brs
7.14, d (8.3)
6.95, dd (2.0, 8.3)
4.69, d (8.0)
2.69, m
6.76, d (8.1)
6.84, dd (1.8, 8.1)
4.64, d (8.4)
2.70, m
6.72, brs
2.67, m
1.88, m
3.66, m
3.38, m
4.88, d (7.6)
3.47, m
3.39, m
3.38, m
3.46, m
3.85, m
3.68, m
4.64, d (1.5)
3.81, m
3.67, m
3.35, m
3.56, m
1.21, d (6.2)
3.87, s
6.72, brs
2.64, m
1.88, m
3.66, m
3.38, m
5.34, d (1.6)
4.05, m
3.86, m
3.43, m
3.46, m
3.65, m
3.63, m
100
200
300
400
500
600
102.8, CH
74.9, CH
78.2, CH
71.3, CH
77.8, CH
62.5, CH2
4.89, d (7.5)
3.50, m
3.47, m
3.41, m
3.48, m
3.87, m
3.72, m
101.9, CH
74.7, CH
78.4, CH
71.2, CH
77.9, CH
62.5, CH2
5.06, d (7.4)
3.51, m
3.48, m
3.40, m
3.49, m
3.90, m
3.67, m
102.8, CH
74.9, CH
78.2, CH
71.3, CH
77.9, CH
62.5, CH2
101.4, CH
72.2, CH
72.0, CH
73.8, CH
70.8, CH
18.0, CH3
1.20, d (6.2)
1000
101.8, CH
72.5, CH
72.4, CH
74.0, CH
69.8, CH
18.0, CH3
56.8, CH3
56.7, CH3
101.7, CH
72.5, CH
72.4, CH
74.0, CH
69.8, CH
18.0, CH3
56.8, CH3
56.4, CH3
4.64, d (1.5)
3.81, m
3.65, m
3.36, m
3.56, m
1.20, d (6.2)
3.87, s
3.80, s
2000
3000
4000
5000
6000
30-OCH3
3-OCH3
4-OCH3
56.4, CH3
56.5, CH3
56.7, CH3
3.88, s
3.89, s
3.91, s
56.4, CH3
56.7, CH3
3.88, s
3.93, s
3.83, s
a
300 MHz for 1H NMR, 75 MHz for 13C NMR.
400 MHz for 1H NMR, 100 MHz for 13C NMR.
b
4.4. Acid hydrolysis and HPLC analysis of compounds 1–10
EtOAc, the EtOAc layer was evaporated to dryness and dissolved
in MeOH. Then the mixture was subjected to preparative HPLC
The absolute configuration of the sugar units were determined
by the method of Tanaka et al. (Tu et al., 2011). Compounds 1–5
(cosmosil C18-MS-II column, 250 ꢀ 4.6 mm i.d., 5
lm at 35 °C with
MeOH–H2O, 40:60, V/V) to give (+)-oleuropeic acid (1a). 1a
and 7–8 afforded
-glucose (tR = 19.236 min) and
compound 9 liberated -glucose (tR = 19.236 min) and
D
-glucose (tR = 19.236 min); compound 6 gave
-xylose (tR = 22.351 min);
-rhamnose
-rhamnose (tR =
(0.6 mg): colorless oil, ½a D20
ꢁ
+45.3 (c 0.3, CHCl3). 1H NMR (CD3OD):
6.75 (1H, brs, H-2), 2.28 (1H, m, H-3a) and 1.96 (1H, m, H-3b), 1.53
(1H, m, H-4), 2.00 (1H, m, H-5a) and 1.21 (1H, m, H-5b), 2.49 (1H,
m, H-6a) and 2.12 (1H, m, H-6b), 1.17 (6H, s, H-9 and H-10). Meth-
D
D
D
L
(tR = 32.407 min), compound 10 afforded
L
32.407 min).
anolysis of 2 also gave (1a) {½a D20
ꢁ
+38.0 (c 0.25, CHCl3)}. Similar
methanolysis of 3 (1.6 mg) gave 6-hydroxy-6-isopropylcyclohex-
1-enecarboxylic acid (3a). 3a (0.5 mg): colorless oil, CD (207 nm,
4.5. Acid hydrolysis of 9 and 10
D
e
ꢃ0.06), 1H NMR (CD3OD): 6.68 (1H, brs, H-2), 2.30 (1H, m,
H-3a) and 2.12 (1H, m, H-3b), 2.36 (2H, m, H-4), 1.72 (1H, m,
H-5a) and 1.55 (1H, m, H-5b), 1.65 (1H, m, H-8), 0.87 (3H, d,
J = 7.6 Hz, H-9), 0.89 (3H, d, J = 7.6 Hz, H-10).
A solution of 9 (1.5 mg) was hydrolyzed with 2 M HCl for 2 h at
90 °C. The mixture was then extracted with EtOAc, the EtOAc layer
was evaporated to dryness and dissolved in MeOH, with the mix-
ture subjected to p-HPLC (Cosmosil 18-MS-II column,
C
250 ꢀ 4.6 mm i.d.,
5
l
m) at 35 °C with a 50:50 mixture of
4.7. Computation methods
MeOH–H2O containing 0.1% HCO2H) to give 9a. Its structure was
confirmed by analysis of HRESIMS and 1H NMR spectroscopic data
(Jiang et al., 2005). Acid hydrolysis of 10 (2.0 mg) also gave 9a.
The conformational search of 3a and 9a was performed using
the Random search method, MMFF94 force field and charged with
MMFF94 as implemented in the program package Sybyl 8.0. 27 and
14 minimum geometries within an energy of 10 kcal/mol above
the presumably global minimum, each for 3a and 9a respectively.
These were selected and further optimized by using DFT at the
B3LYP/6-31G(d) level as implemented in the Gaussian 09 software.
The stable conformers obtained were used to CD calculation by
4.6. Methanolysis of compounds 1–3
A solution of 1 (2.0 mg) in 0.02 M NaOMe–MeOH (1 ml) was
placed at room temperature for 12 h respectively (Tian et al.,
2009). After neutralizing with HCOOH (5M) and extracting with
Please cite this article in press as: Li, C., et al. Quinoid glycosides from Forsythia suspensa. Phytochemistry (2014), http://dx.doi.org/10.1016/
j.phytochem.2014.04.010

8
C. Li et al. / Phytochemistry xxx (2014) xxx–xxx
TDDFT [B3LYP/6-31G (d)] method with 50 excited states in gas
phase. The overall predicted CD curves of 3a and 9a were
simulated as sums of Gaussian functions in accordance to the
Boltzmann weighting with the half width at 0.2 eV, which revealed
good agreement to the measured CD curves.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.
2014.04.010.
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