Forsythoneosides A-D, Neuroprotective Phenethanoid and Flavone Glycoside Heterodimers from the Fruits of Forsythia suspensa

Article abstract of DOI:10.1021/acs.jnatprod.5b00372

Forsythoneosides A-D (1-4), four unusual adducts of a flavonoid unit fused to a phenylethanoid glycoside through a pyran ring or carbon-carbon bond, and four new phenylethanoid glycosides (5-8) were isolated from the fruits of Forsythia suspensa, together with nine known compounds. The structures of 1-8, including their absolute configurations, were elucidated by spectroscopic data as well as experimental and calculated electronic circular dichroism analysis. Compounds 2 and 4 inhibited PC12 cell damage induced by rotenone, and increased cell viability from 53.9 ± 7.1% to 70.1 ± 4.0% and 67.9 ± 5.2% at 0.1 μM, respectively.

Full text of DOI:10.1021/acs.jnatprod.5b00372

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Forsythoneosides A−D, Neuroprotective Phenethanoid and Flavone
Glycoside Heterodimers from the Fruits of Forsythia suspensa
Fan Zhang,† Ya-Nan Yang,† Xiu-Yun Song, Si-Yuan Shao, Zi-Ming Feng, Jian-Shuang Jiang, Li Li,
Nai-Hong Chen, and Pei-Cheng Zhang*
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, People’s Republic of China
S
* Supporting Information
ABSTRACT: Forsythoneosides A−D (1−4), four unusual adducts of a flavonoid unit fused to a phenylethanoid glycoside
through a pyran ring or carbon−carbon bond, and four new phenylethanoid glycosides (5−8) were isolated from the fruits of
Forsythia suspensa, together with nine known compounds. The structures of 1−8, including their absolute configurations, were
elucidated by spectroscopic data as well as experimental and calculated electronic circular dichroism analysis. Compounds 2 and
4 inhibited PC12 cell damage induced by rotenone, and increased cell viability from 53.9 7.1% to 70.1 4.0% and 67.9 5.2%
at 0.1 μM, respectively.
Forsythia suspensa (Thunb.) Vahl grows in northern mainland
China, and its fruits, known as Fructus Forsythiae, are used
widely as a traditional Chinese medicine to treat inflammation,
pyrexia, ulcers, gonorrhea, and erysipela.1,2 Over the past few
decades, a series of phenylethanoid glycosides, lignans,
flavonoids, alkaloids, and triterpenoids have been reported
from species of this genus, and a few have been found to exhibit
antioxidant, neuroprotective, cytotoxic, anti-inflammatory,
antibacterial, and antiviral effects.3−10
Of particular interest for this study was the n-BuOH-soluble
portion of the fruits of F. suspensa obtained from a 75% EtOH
extract, which exhibited neuroprotective effects and could
inhibit rotenone-induced damage of pheochromocytoma
(PC12) cells, increasing the cell viability from 54.2 2.8% to
RESULTS AND DISCUSSION
■
Compound 1 was obtained as a yellow, amorphous powder.
The molecular formula, C56H62O31, was established by the
protonated molecular ion peak at m/z 1231.3354 [M + H]+
(calcd for C56H63O31, 1231.3353) in the HRESIMS, indicating
26 degrees of unsaturation. The IR spectrum showed
absorption bands attributed to hydroxy groups (3371 cm−1),
carbonyl groups (1693 cm−1), and aromatic rings (1601 and
1519 cm−1). Analysis of the 1H NMR spectrum (Table 1) of 1
revealed the presence of two sets of ABX system aromatic
protons at δH 7.92 (1H, d, J = 2.5 Hz), 7.10 (1H, d, J = 8.5 Hz),
and 7.95 (1H, dd, J = 8.5, 2.5 Hz) and δH 7.15 (1H, d, J = 2.5
Hz), 6.85 (1H, d, J = 8.5 Hz), and 7.02 (1H, dd, J = 8.5, 2.5
Hz), three singlet aromatic protons at δH 7.12 (1H, s), 6.70
(1H, s), and 6.52 (1H, s), and two olefinic protons at δH 7.64
(1H, d, J = 16.0 Hz) and 6.33 (1H, d, J = 16.0 Hz). In addition,
there were many aliphatic proton signals between δH 5.10 and
1.20, including a methylene signal at δH 3.98 (1H, dd, J = 10.5,
7.5 Hz) and 3.87 (1H, m), a methine signal at δH 4.64 (1H, dd,
J = 7.5, 4.0 Hz), and four anomeric protons at δH 5.10 (1H, d, J
= 7.5 Hz), 4.48 (1H, d, J = 8.0 Hz), 4.74 (1H, br s), and 4.70
78.5
5.0% at a concentration of 10 μg/mL (Table S1,
Supporting Information). Through bioactivity-guided isolation,
four heterodimers of phenylethanoid glycoside and flavonoid
units, i.e., forsythoneosides A−D (1−4), and four new
phenylethanoid glycosides (5−8) were isolated from the fruits
of F. suspensa, together with nine known compounds. Reported
herein are the isolation and structure elucidation of compounds
1−8 and the evaluation of compounds 1−4 as potential
neuroprotective agents in inhibiting cell damage in PC12 cells.
Received: April 29, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b00372
J. Nat. Prod. XXXX, XXX, XXX−XXX

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Chart 1
(1H, br s). After acid hydrolysis, the sugar units of 1 were
confirmed to be D-glucose and L-rhamnose by GC analysis of
the trimethylsilyl L-cysteine derivatives of these compounds
(Figure S1, Supporting Information). The 13C NMR and
HSQC spectra of 1 revealed 56 carbon resonances, including 19
quaternary carbons, 32 methines, three methylenes, and two
methyls. In addition to a caffeoyl unit (nine carbons) and four
hexoses (24 carbons), the remaining 23 carbons could be
assigned to a flavonol aglycone and a phenylethanoid moiety. A
careful comparison of the 1D NMR signals of 1 with the
corresponding data of rutin11 and forsythoside A,12 two main
components isolated from the fruits of F. suspensa, suggested
the presence of a rutin moiety and phenylethanoid glycoside
moiety in 1. The connectivity of these two moieties was
established by analyzing the HMBC spectrum (Figure 1).
Correlations from H-7′ at δH 4.64 (dd, J = 7.5, 4.0 Hz)
(monomer B) to C-7, C-8, and C-9 (monomer A) were
observed, suggesting that the C-7′ of the phenylethanoid unit is
connected to the C-8 of the rutin moiety by a C−C bond. Two
singlet aromatic protons of the phenylethanoid moiety at δH
(Δε) values of 367 (+4.45), 274 (−1.02), and 217 (−2.44).
The 7′R configuration was determined by comparing the
experimental and calculated ECD data.13 Accordingly, the
structure of 1 was assigned as shown, and this compound was
accorded the trivial name forsythoneoside A.
Compound 2 was obtained as a yellow, amorphous powder
and exhibited the same IR, UV, and HRMS characteristics as 1.
The NMR spectroscopic data of 2 were similar to those of 1,
1
except for subtle shifts in the glycosyl chain protons in the H
NMR spectrum. A careful analysis of the 1D and 2D NMR
spectra of 2 suggested this compound to have the same planar
structure as 1 (Tables 1 and 2). The ECD spectrum of 2
exhibited an opposite Cotton effect of that observed for 1
(Figure S26, Supporting Information), and 2 was accorded the
trivial name forsythoneoside B.
According to this spectroscopic data analysis, 1 and 2 are a
pair of diastereoisomers with different configurations at C-7′.
The absolute configurations of C-7′ in 1 and 2 were established
by comparing the experimental and calculated ECD data.
Considering that the flexible glycosyl chains and caffeoyl moiety
far from the chiral center C-7′ have no significant influence on
the Cotton effect at approximately 275 and 370 nm, which are
peaks attributed to the electronic transition from the
benzopyran and flavonol groups, a simplified structure of 1
was used for conformational analysis (Figure 2). After scanning
for stable conformations of 1a and 1b using the MMFF94
molecular mechanics force field, the preferred conformers were
optimized using the time-dependent density functional theory
(TD-DFT) method at the B3LYP/6-31G(d) level (Table S2,
1
7.12 (1H, s) and 6.70 (1H, s) in the H NMR spectrum
suggested that a tetrasubstituted aromatic ring is present in the
phenylethanoid moiety in 1. Furthermore, the presence of a
downfield carbon signal at δC 147.3 and the remaining one
degree of unsaturation implied that an ether bond is formed
between C-6′ and C-7. Therefore, 1 possesses a pyran ring,
which consists of [C-7−C-8−C-7′−C-1′−C-6′−O], formed by
the fusion of the phenylethanoid glycoside moiety to the
flavonol moiety. The ECD spectrum of 1 showed λmax (nm)
B
DOI: 10.1021/acs.jnatprod.5b00372
J. Nat. Prod. XXXX, XXX, XXX−XXX

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1
Supporting Information). The calculated electronic circular
dichroism (ECD) spectra of 1a and 1b were generated by a
Boltzmann weighting of their lowest energy conformers with
different populations. The overall pattern of the calculated
ECD spectra of 1a and 1b was in agreement with the
experimental data obtained for 2 and 1 (Figure 3), respectively.
Therefore, the absolute configurations of C-7′ in forsythoneo-
side A and B were determined to be R and S, respectively.
Compound 3 was obtained as a yellow, amorphous powder.
The molecular formula, C56H64O31, was established by the
protonated molecular ion peak (m/z 1233.3502 [M + H]+) in
the HRESIMS, indicating 25 degrees of unsaturation. The IR
spectrum showed absorption bands attributed to hydroxy
groups (3394 cm−1), carbonyl groups (1694 cm−1), and
aromatic rings (1603 and 1514 cm−1). On comparing the key
NMR data of 3 with those of 1 (Tables 1 and 2), their close
resemblance indicated that 3 was a heterodimer of rutin and
phenylethanoid glycoside, making it structurally similar to 1. In
Table 1. H NMR Spectroscopic Data for 1−4 in Acetic
Acid-d4 (δ in ppm, J in Hz)
a
a
b
b
position
1
2
3
4
6
6.52, s
6.53, s
6.51, s
6.51, s
12
15
16
7.92, d (2.5)
7.10, d (8.5)
7.93, d (2.5)
7.10, d (8.5)
7.74, d (1.8)
6.81, d (8.4)
7.67, d (1.8)
6.83, d (8.4)
7.95, dd (8.5,
2.5)
7.81, dd (8.5,
2.5)
7.29, dd (8.4, 7.33, dd (8.4,
1.8)
1.8)
17
18
19
20
21
22a
22b
23
24
25
26
27
28
2′
5.10, d (7.5)
3.76, m
3.75, m
3.60, m
3.53, m
3.86, m
3.54, m
4.70, br s
3.85, m
3.80, m
3.50, m
3.61, m
1.20, d (6.0)
7.12, s
5.20, d (7.5)
3.75, m
3.76, m
3.64, m
3.58, m
3.83, m
3.56, m
4.76, br s
3.88, m
3.78, m
3.48, m
3.56, m
1.17, d (6.0)
7.15, s
5.04, d (7.8)
3.76, m
3.73, m
3.60, m
3.48, m
3.79, m
3.49, m
4.65, d (1.8)
3.80, m
3.78, m
3.51, m
3.53, m
1.19, d (6.0)
7.00, s
5.05, d (7.2)
3.72, m
3.70, m
3.59, m
3.50, m
3.79, m
3.49, m
4.65, br s
3.81, m
3.78, m
3.52, m
3.58, m
1.20, d (6.0)
7.05, s
1
the H NMR spectrum of 3, a typical β-methylene signal of a
phenylethanol unit at δH 2.64 (m) indicated that the C-7′
position is not substituted. There were two singlet aromatic
protons at δH 7.00 (s) and δH 6.83 (s), illustrating the presence
of a tetrasubstituted aromatic ring in the phenylethanol unit of
3. Focusing on the 13C NMR resonance of C-6′, the chemical
shift at δC 125.1 was distinctly different from that of 1, revealing
that C-6′ of the phenylethanol unit is linked to C-8 of ring A of
rutin via a C−C bond. This result was confirmed by the HMBC
spectrum (Figure 1), in which a key correlation between H-5′
and C-8 was observed. The ECD spectrum of 3 showed the
following values at λmax (nm) (Δε): 342 (+0.83), 264 (+5.52),
and 225 (−13.02). The M configuration was confirmed based
on the experimental and calculated ECD data. Thus, 3 was
accorded the trivial name forsythoneoside C.
Compound 4 was obtained as a yellow, amorphous powder.
Similar NMR data (Tables 1 and 2) and Cotton effects
opposite those of 3 at approximately 220, 270, and 350 nm in
the ECD spectrum (Figure S48, Supporting Information)
suggested that 4 is a diastereoisomer of 3, with the opposite
axial chirality at C-8−C-6′. Compound 4 was accorded the
trivial name forsythoneoside D.
The absolute configurations of 3 and 4 were established by
the same method applied to 1 and 2. Similarly, simplified
structures of 3 were used for the conformational analysis
(Figure 2). A systematic conformational analysis and
optimization were performed for 3a and 3b using the same
method applied to 1a and 1b (Table S3, Supporting
Information). A comparison of the theoretically calculated
and experimental ECD curves (Figure 4) permitted the
assignment of the absolute configurations of M and P to 3
and 4, respectively.
5′
6.70, s
6.66, s
6.83, s
6.81, s
7′
4.64, dd (7.5,
4.0)
4.67, dd (7.5,
4.0)
2.64, m
2.69, m
8′a
3.98, dd (10.5, 4.10, dd (10.5, 3.84, m
3.87, m
7.5)
4.0)
8′b
9′
3.87, m
3.78, m
3.60, m
3.63, m
4.48, d (8.0)
3.56, m
4.50, d (8.0)
3.55, m
4.20, d (7.8)
3.41, m
4.14, d (7.8)
3.44, m
10′
11′
12′
13′
14′a
14′b
15′
16′
17′
18′
19′
20′
2″
3.84, m
3.77, m
3.72, m
3.70, m
4.98, t (10.0)
3.39, m
5.04, t (10.0)
3.69, m
4.93, t (9.6)
3.50, m
4.93, t (9.6)
3.53, m
3.65, d (8.5)
3.51, m
3.70, d (8.5)
3.47, m
3.63, d (8.4)
3.50, m
3.72, d (8.4)
3.55, m
4.74, br s
3.91, m
4.70, br s
3.85, m
4.79, d (1.8)
3.98, m
4.79, br s
3.98, m
3.82, m
3.67, m
3.85, m
3.86, m
3.48, m
3.43, m
3.48, m
3.49, m
3.65, m
3.63, m
3.67, m
3.71, m
1.12, d (6.0)
7.15, d (2.5)
6.85, d (8.5)
1.10, d (6.0)
7.15, d (2.5)
6.85, d (8.5)
1.16, d (6.0)
7.14, d (1.8)
6.85, d (8.4)
1.19, d (6.0)
7.15, d (1.8)
6.86, d (8.4)
5″
6″
7.02, dd (8.5,
2.5)
7.02, dd (8.5,
2.5)
7.02, dd (8.4, 7.01, dd (8.4,
1.8) 1.8)
7″
8″
7.64, d (16.0) 7.64, d (16.0) 7.63, d (16.2) 7.64, d (16.2)
6.33, d (16.0) 6.33, d (16.0) 6.31, d (16.2) 6.32, d (16.2)
a
b
Recorded at 500 MHz. Recorded at 600 MHz.
Figure 1. Key HMBC correlations of 1, 3, 5, and 7.
C
DOI: 10.1021/acs.jnatprod.5b00372
J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. 13C NMR Spectroscopic Assignments for 1−5 in Acetic Acid-d4 and 6−8 in DMSO-d6 (δ in ppm)
a
a
b
b
a
a
a
a
position
1
2
3
4
position
5
6
7
8
2
161.1
138.7
181.5
162.7
102.6
161.5
103.6
156.8
110.0
125.0
119.5
147.0
151.5
118.6
126.9
107.6
77.4
161.4
138.6
181.5
162.7
102.6
161.6
103.9
157.0
110.1
125.2
120.2
147.0
151.2
118.6
126.3
107.0
77.3
160.7
138.0
181.5
163.4
102.3
164.5
110.6
157.1
107.9
125.1
120.4
146.6
151.1
118.2
125.7
107.7
77.5
161.1
138.2
181.9
163.6
102.4
164.5
110.7
157.3
108.2
125.3
120.4
146.7
151.2
118.4
126.1
107.9
77.7
1
130.0
118.0
147.5
153.9
118.2
125.5
199.1
73.8
129.0
115.5
144.9
143.6
116.4
119.4
35.1
124.8
147.6
115.4
113.3
149.6
117.0
30.7
124.8
147.6
115.4
113.3
149.6
117.0
30.7
3
2
4
3
5
4
6
5
7
6
8
7
9
8
70.3
68.6
68.6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
1′
1′
2′
3′
4′
5′
6′
7′
8′
130.0
117.5
147.7
150.6
118.6
125.5
149.7
116.9
170.3
105.7
76.7
128.5
115.0
146.9
147.6
115.9
120.7
145.0
115.7
165.6
102.8
72.8
120.3
131.4
115.3
162.0
115.3
131.4
165.4
120.5
112.3
147.3
151.5
115.2
123.6
165.4
9′
79.7
79.8
79.8
79.8
1″
2″
3″
4″
5″
6″
1‴
2‴
3‴
4‴
5‴
6‴
1⁗
2⁗
3⁗
4⁗
5⁗
6⁗
OCH3-3′
103.0
73.4
76.5
70.0
73.7
63.6
103.0
73.4
76.5
70.2
73.8
64.0
72.4
72.4
72.2
72.7
78.3
78.5
78.1
78.4
77.3
73.9
70.6
70.0
70.5
70.8
73.7
71.2
103.6
73.8
103.6
73.8
103.7
73.7
103.8
73.9
76.8
73.4
69.7
66.8
74.3
74.3
74.3
74.5
103.3
73.7
100.5
70.2
75.9
75.8
75.9
76.1
71.3
71.3
71.3
71.6
74.3
70.5
19.8
19.7
19.7
19.9
75.9
71.8
115.8
119.0
144.0
147.7
106.7
147.3
36.3
115.6
119.3
143.7
147.7
106.7
147.7
36.6
134.2
120.1
147.9
146.0
121.8
125.1
36.2
134.5
120.4
147.9
146.2
122.1
125.2
36.3
71.4
68.4
2′
3′
4′
5′
6′
7′
8′
9′
19.7
17.8
101.5
73.2
75.8
69.7
77.2
77.4
77.7
73.5
73.5
60.7
105.6
76.8
106.3
76.9
105.6
76.4
105.9
76.7
55.3
10′
11′
12′
13′
14′
15′
16′
17′
18′
19′
20′
1″
2″
3″
4″
5″
6″
7″
8″
9″
77.5
77.5
77.3
77.5
73.9
74.1
73.8
73.9
76.3
76.2
76.5
76.8
69.6
69.8
69.3
69.8
103.2
73.7
103.4
73.6
103.1
73.7
103.5
73.5
74.2
74.3
74.3
74.3
75.8
75.8
75.8
76.1
71.3
71.4
71.2
71.5
19.6
19.6
19.6
19.8
130.0
117.6
147.6
150.6
118.6
125.6
149.9
116.9
170.5
130.0
117.6
147.6
150.6
118.5
125.6
149.9
117.0
170.5
130.0
117.5
147.7
150.6
118.6
125.6
149.8
116.9
170.4
130.3
117.8
147.8
150.7
118.8
125.7
149.9
117.3
170.5
a
b
Recorded at 125 MHz. Recorded at 150 MHz.
Compound 5 was obtained as a yellow powder, and its
molecular formula was determined to be C29H34O16 based on
the sodiated molecular ion peak observed at m/z 661.1739 [M
1
+ Na]+ in the HRESIMS. The H NMR spectrum of 5 (Table
D
DOI: 10.1021/acs.jnatprod.5b00372
J. Nat. Prod. XXXX, XXX, XXX−XXX