Two new lignans from the fruits of Forsythia suspensa

Article abstract of DOI:10.1080/10286020.2019.1666828

Two new lignans, wikstronoside B (1) and forsysesquinorlignan (2), were isolated from the fruits of Forsythia suspensa, along with two known sesquineolignans, hedyotol A and hedyotol C (3 and 4). The structures of new compounds were established via extensive spectroscopy techniques, including UV, IR, HRESIMS, NMR, and ECD. Compounds 3 and 4 were isolated from this plant for the first time. Their anti-inflammatory effects were evaluated via a detection model with LPS-induced murine macrophage RAW264.7 cells, and compound 3 showed a moderate activity.

Full text of DOI:10.1080/10286020.2019.1666828

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: https://www.tandfonline.com/loi/ganp20
Two new lignans from the fruits of Forsythia
suspensa
Jun Cao, Si-Yuan Shao, Xu Zhang, Xiang Yuan, Zi-Ming Feng, Jian-Shuang
Jiang, Ya-Nan Yang & Pei-Cheng Zhang
To cite this article: Jun Cao, Si-Yuan Shao, Xu Zhang, Xiang Yuan, Zi-Ming Feng, Jian-Shuang
Jiang, Ya-Nan Yang & Pei-Cheng Zhang (2019): Two new lignans from the fruits of Forsythia
suspensa, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2019.1666828
To link to this article: https://doi.org/10.1080/10286020.2019.1666828
Published online: 20 Sep 2019.
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2019.1666828
Two new lignans from the fruits of Forsythia suspensa
Jun Cao, Si-Yuan Shao, Xu Zhang, Xiang Yuan, Zi-Ming Feng, Jian-Shuang Jiang,
Ya-Nan Yang 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, China
ABSTRACT
ARTICLE HISTORY
Received 12 July 2019
Accepted 8 September 2019
Two new lignans, wikstronoside B (1) and forsysesquinorlignan
(2), were isolated from the fruits of Forsythia suspensa, along with
two known sesquineolignans, hedyotol A and hedyotol C (3 and
4). The structures of new compounds were established via exten-
sive spectroscopy techniques, including UV, IR, HRESIMS, NMR,
and ECD. Compounds 3 and 4 were isolated from this plant for
the first time. Their anti-inflammatory effects were evaluated via a
detection model with LPS-induced murine macrophage RAW264.7
cells, and compound 3 showed a moderate activity.
KEYWORDS
Oleaceae; Forsythia
suspensa; sesquinorlignan
1. Introduction
As a traditional Chinese medicine, the fruits of Forsythia suspensa (Tunb.) Vahl are
mainly used to treat inflammation, pyrexia and ulcers [1]. According to the previous
studies, the compounds from F. suspensa are mainly phenylethanoid glycosides [2,3],
triterpenoids [4], lignans [5], and flavonoids [6], in which some compounds show
good pharmacological activities, such as anti-inflammatory [7–9], antioxidant [9],
antibacterial [10], and neuroprotective effects [11]. In our continuing search for bio-
active constituents from F. suspensa, four lignans (1–4) were isolated and identified.
Compounds 1 and 2 were new lignans and compounds 3 and 4 were obtained from
CONTACT Ya-Nan Yang
yyn@imm.ac.cn; Pei-Cheng Zhang
pczhang@imm.ac.cn
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
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
J. CAO ET AL.
Figure 1. Chemical structures of compounds 1, 1a, and 2.
the plant for first time (Figure 1). Their anti-inflammatory effects against LPS-
induced NO production in murine macrophage RAW264.7 cells were estimated.
2. Results and discussion
Compound 1 was obtained as a yellow amorphous powder, and its molecular formula
was determined as C26H32O11 by HRESIMS at m/z 521.2030 [M þ Na]þ. The IR spec-
trum showed absorption bands attributed to hydroxy groups (3420 cmꢀ1), carbonyl
1
groups (1669 cmꢀ1), and aromatic rings (1593, 1514, 1419 cmꢀ1). The H-NMR spec-
trum of 1 revealed the presence of two sets of ABX system aromatic protons at dH 7.51
(1H, brs), 7.21 (1H, d, J¼ 8.5 Hz), 7.62 (1H, brd, J ¼ 8.5 Hz), 6.87 (1H, brs), and 6.73
(2H, brs), two methoxy signals at dH 3.83 (3H, s) and 3.75 (3H, s), one methyl signal at
dH 0.93 (3H, d, J ¼ 6.5 Hz), three methine signals at dH 4.28 (1H, d, J ¼ 9.5 Hz),
4.04–4.06 (1H, m), and 2.30–2.32 (1H, m), one methylene group at dH 4.19 (1H, t,
J ¼ 8.5 Hz) and 3.92 (1H, dd, J¼ 15.0, 8.5 Hz). In addition, one anomeric proton at dH
1
5.08 (1H, d, J ¼ 7.0 Hz) was also observed from the H-NMR spectrum, and the remain-
ing resonances were assigned to a glucosyl unit. After acid hydrolysis, the sugar unit of
1 was confirmed to be b-D-glucose by GC analysis after chiral derivatization (retention
time of 29.6 min). The 13C-NMR spectrum of 1 showed 12 aromatic carbons from dC
151.0 to 110.4, one carbonyl at dC 197.8 in the downfield region. With the exception of
the glucosyl signals, two methoxy signals (dC 55.6 and 55.6), three methine signals (dC
87.9, 53.2, and 45.4), one oxymethylene signal (dC 69.6) and one methyl signal (dC 14.7)
were observed in the upfield region. HMBC correlations of H-8/C-1, H-9/C-7, H-80/C-
10, and H-90/C-70 revealed the presence of two C6-C3 units. Furthermore, the correla-
tions of H-70 with C-8 and C-9 indicated these two units formed a furan ring, and the
correlation of H-100/C-4 indicated the glucosyl unit was linked to C-4 (Figure 2). The
relative configurations of H-70/H-80 and H-8/H-80 were determined as trans-configura-
tions via ROSEY correlations of H-90 with H-8 and H-70 (Figure 3). Considering the
numerous conformations stemming from the single glucosyl bonds, aglycone (1a) was
used for ECD calculation, and the calculated ECD data of (8S, 70S, 80S)-1a matched with
the experimental data for 1a (Figure 4). Thus, the absolute configuration of 1 was deter-
mined as 8S, 70S, 80S. Accordingly, the structure of 1 was assigned as shown and named
wikstronoside B (Figure 1).
Compound 2 was obtained as a brown amorphous powder, and its molecular for-
mula was determined as C24H28O10 by HRESIMS at m/z 475.1603 [M - H]-. The IR
spectrum showed absorption bands attributed to hydroxy groups (3454 cmꢀ1), car-
1
bonyl groups (1767 cmꢀ1), and aromatic rings (1593, 1514, 1461 cmꢀ1). The H-NMR

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Figure 2. Key HMBC correlations of compounds 1 and 2.
Figure 3. Key ROSEY correlations of compound 1.
Figure 4. Experimental and calculated ECD spectrum of 1a.
spectrum of 2 revealed the presence of one set of ABX system aromatic protons at dH
6.97 (1H, d, J ¼ 2.0 Hz), 6.84 (1H, d, J ¼ 8.0 Hz), and 6.72 (1H, dd, J ¼ 8.0, 2.0 Hz)
and two meta-substituted aromatic protons at dH 6.69 (2H, s). In addition, three
methoxy signals at dH 3.87 (6H, s) and 3.82 (3H, s), three oxymethine signals at dH
4.97 (1H, d, J ¼ 6.5 Hz), 4.72 (1H, d, J ¼ 6.0 Hz), and 4.15 (1H, overlap), two methine
signals at dH 3.54–3.56 (1H, m) and 3.16–3.18 (1H, m), three methylene groups at dH
4.57 (1H, dd, J ¼ 9.5, 7.0 Hz), 4.43 (1H, dd, J ¼ 9.5, 2.0 Hz), 4.31 (1H, dd, J ¼ 9.0,

4
J. CAO ET AL.
2.0 Hz), 4.12 (1H, dd, J ¼ 9.0, 3.5 Hz), 3.78 (1H, dd, J ¼ 12.0, 4.0 Hz), and 3.37 (1H,
1
dd, J ¼ 12.0, 3.5 Hz) were also observed in the H-NMR spectrum. The 13C-NMR
spectrum of 2 showed twelve aromatic carbons from dC 154.4 to 104.1, one carbonyl
at dC 181.0 in the downfiled region, three methoxy signals (dC 56.7, 56.7, and 56.3),
three oxymethine signals (dC 88.5, 87.5, and 74.2), two methine signals (dC 49.8 and
47.5), three oxymethylene signals (dC 72.0, 71.3, and 61.0) in the upfield region. The
similar planar structure was established via comparing the NMR data with the corre-
sponding those of hedyotol C [12], it’s worth to note that compound 2 had one more
carbonyl group and lacked one phenyl and methoxy group, that is the carbonyl in 2
replaced the aromatic ring in hedyotol C. The HMBC correlations of 3-OMe (dH
3.87) with C-3 (dC 154.3), 5-OMe (dH 3.87) with C-5 (dC 154.3), and 300-OMe (dH
3.82) with C-300 (dC 148.6) confirmed the three methoxy groups were located at C-3,
C-5, and C-300, respectively (Figure 2).
Comparison of the NMR data between 2 and hedyotol C demonstrated that they
had same relative configuration [13], and the relative configuration of H-700 and H-800
00
was determined as threo-configuration via the chemical shift difference (DH9 >0.25)
[12], and the C-8/C-80 of furofurans lignans in F. suspensa were all in R/R
configurations. Thus, the configuration of compound 2 was determined as
0
00
00
ꢁ
ꢁ
7S,8R,8 R,7 S ,8 R , and named forsysesquinorlignan (Figure 1).
In addition, compounds 3 and 4 were identified as hedyotol A and hedyotol C by
comparison of spectroscopic data with those of the corresponding literature [13,14].
The detection model with LPS-induced murine macrophage RAW264.7 cells
showed that their NO inhibition rates were 0.4%, 3.1%, 50.7%, and 9.9% at a concen-
tration of 10 lM, respectively. Comparison with the positive control, curcumin
(10 lM, 73.0%), showed that compound 3 had a moderate anti-inflammatory activity.
3. Experimental
3.1. General experimental procedures
The optical rotations, UV and ECD were measured on JASCO P-2000, JASCO V-650,
and JASCO J-815 spectrometers (JASCO, Easton, MD, USA), respectively. IR spectra
were recorded on a Nicolet 5700 spectrometer using an FT-IR microscope transmis-
sion method (Thermo Scientific, Waltham, MA, USA). NMR spectra were obtained
on a Bruker AVIII-500 spectrometer (Bruker-Biospin, Billerica, MA, USA). HRESIMS
were performed on an Agilent 6520 HPLC-Q-TOF (Agilent Technologies,
Waldbronn, Germany). GC experiments were performed using an Agilent 7890A ser-
ies system with a capillary column, HP-5 (60 m ꢂ 0.25 mm, with a 0.25 lm film;
Dikma Technologies Inc., Beijing, China). The reversed-phase preparative HPLC was
performed utilizing a Shimadzu LC-10AT instrument equipped with a YMC-Pack
ODS-A column (250 mm ꢂ 20 mm, 5 lm; YMC Corp., Kyoto, Japan). Silica gel
(100–200 mesh, Qingdao Marine Chemical Inc. Qingdao, China), RP-C18 (50 lm,
YMC, Kyoto, Japan) and Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala,
Sweden) were employed for column chromatography. The HPLC experiments were
performed using an Agilent 1260 system with an Apollo C18 column (250 ꢂ 4.6 mm,
5 lm, Grace Davison).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Table 1. 1H and 13C NMR Spectral data for compounds 1 and 2 (500/125 MHz, d in ppm, J in Hz).
1(DMSO-d6)
2(CD3OD)
Position
dH
dC
dH
dC
1
2
3
4
5
6
7
8
130.5
112.2
148.8
151.0
114.2
122.8
197.8
53.2
137.6
104.1
154.3
137.0
154.3
104.1
87.5
7.51 brs
6.69 s
7.21 d (8.5)
7.62 brd (8.5)
6.69 s
4.72 d (6.0)
3.16–3.18 m
4.04–4.06 m
49.8
9
4.19 t (8.5) 3.92 dd (15.0, 8.5)
69.6
4.57 dd (9.5, 7.0) 4.43 dd (9.5, 2.0)
72.0
1’
2’
3’
4’
5’
6’
7’
8’
9’
1’’
2’’
3’’
4’’
5’’
6’’
131.3
110.4
147.5
146.2
115.1
119.2
87.9
45.4
14.7
99.4
73.0
6.87 brs
6.73 brs
6.73 brs
4.28 d (9.5)
2.30–2.32 m
0.93 d (6.5)
5.08 d (7.0)
3.26–3.28 m
3.27–3.29 m
3.15–3.17 m
3.36–3.38 m
3.65 brd (10.5)
3.42–3.44 m
181.0
47.5
71.3
133.5
111.4
148.6
147.0
115.7
120.6
3.54–3.56 m
4.31 dd (9.0, 2.0) 4.12 dd (9.0, 3.5)
6.97 d (2.0)
76.8
69.5
77.2
60.5
6.84 d (8.0)
6.72 d (8.0, 2.0)
7’’
8’’
9’’
3-OMe
5-OMe
3’-OMe
3’’-OMe
4.97 d (6.5)
4.15 overlap
3.78 dd (12.0, 4.0) 3.37 dd (12.0, 3.5)
3.87 s
74.2
88.5
61.0
56.7
56.7
3.83 s
3.75 s
55.6
55.6
3.87 s
3.82 s
56.3
3.2. Plant material
The fruits of Forsythia suspensa were collected in December 2011 from Yuncheng
City of Shanxi Province, China. The plant material was identified by associate profes-
sor Lin Ma (Institute of Materia Medica, Peking Union Medical College and Chinese
Academy of Medical Sciences). A voucher specimen numbered ID-S-2597 was depos-
ited at the Herbarium of the Department of Natural Medicinal Chemistry, Institute of
Materia Medica, Chinese Academy of Medical Sciences.
3.3. Extraction and isolation
The fruits of F. suspensa (90.0 kg) were extracted with 75% EtOH (200 L) under reflux
three times and filtered. The filtrate was evaporated in vacuo to give a crude extract
(12.6 kg), which was suspended in H2O and extracted successively with petroleum
ether, EtOAc, and n-BuOH.
The n-BuOH portion (4 kg) was suspended in water (40 L) to obtain an water sol-
uble layer, which was concentrated to yield an aqueous portion (1.5 kg). The portion
was separated on a HP-20 resin column eluting with a mixture of EtOH-H2O to
obtain five fractions (0%, 15%, 30%, 50%, 95%). The 50% ethanol solution (392.0 g)

6
J. CAO ET AL.
was separated on a HP-20 resin column eluting with a mixture gradient of EtOH-
H2O (0%, 10%, 20%, 30%, 40%, 50%, 95%) to obtain seven fractions (A–G).
Separation of fraction G (65.0 g) was performed on a Sephadex LH-20 column in a
mixture gradient of CH3OH-H2O (from 30% to 50%) to give 14 fractions (G1-G14).
Fraction G13 (1.7 g) was further performed on a Sephadex LH-20 column in a mix-
ture gradient of CH3OH-H2O (from 30% to 50%) to give nine fractions (G13.1-
G13.9). Fraction G13.9 (54.0 mg) was purified using reversed-phase preparative HPLC
with MeOH-H2O (45:55) as the mobile phase to yield 1 (tR 14.7 min, 4.0 mg).
The EtOAc fraction (3.0 kg) was chromatographically separated on a silica gel column
eluted with EtOAc/acetone (from 100:0 to 0:100) and methanol to give 12 fractions (frac-
tions 1–12). Fraction 10 (318.0 g) was further subjected to a silica gel column eluted with
a gradient of CH2Cl2-MeOH (100:0–0:100), yielding fractions 10.1–10.15. Fractions 10.3
and 10.4 were further merged into three fractions (fractions A–C). Fraction B (1.1 g) was
further chromatographed over ODS eluting with mixtures of MeOH-H2O (15%, 25%,
30%, 35%, 40%, 45%, 70%, 100%) to yield fractions B1–B16. Fraction B4 (34.0 mg) was
purified using reversed-phase preparative HPLC with MeOH-H2O (35:65) as the mobile
phase to yield 2 (tR 19.3 min, 4.5 mg). Fraction B10 (47.0 mg) was purified using reversed-
phase preparative HPLC with CH3CN-H2O (35:65) as the mobile phase to yield 3 (tR
13.7 min, 10.0 mg). Fraction B14 (79.0 mg) was purified using reversed-phase preparative
HPLC with CH3CN-H2O (35:65) as the mobile phase to yield 4 (tR 15.3 min, 6.0 mg).
3.3.1. Wikstronoside B (1)
20
Yellow amorphous powder; ½aꢃD –30.0 (c 0.10, MeOH); UV (MeOH) kmax (log e) 275
(4.16), 223 (4.47) nm; ECD (CH3OH) De208 nm þ0.52, De228 nm –1.01, De299 nm þ0.79;
IR ꢀmax 3420, 2960, 1669, 1593, 1514, 1419, 1267, 1075 cmꢀ1; H and 13C NMR spec-
1
tral data, see Table 1; HRESIMS: m/z 521.2030 [M þ Na]þ (calcd for
C26H32NaO11, 521.2017).
3.3.2. Sesquinorlignan (2)
20
Brown amorphous powder; ½aꢃD þ25.0 (c 0.10, MeOH); UV (MeOH) kmax (log e) 279.2
(3.70), 231.6 (4.23), 205.2(4.81) nm; IR ꢀmax 3454, 1767, 1593, 1514, 1461, 1234, 1034 cmꢀ1;
1H and 13C NMR spectral data, see Table 1; HRESIMS: m/z 475.1603 [M - H]- (calcd for
C24H27O10, 475.1604).
3.4. Anti-inflammatory assay
The experimental method was carried out according to the literature [15].
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This project was financially supported by the National Natural Science Foundation of China
(81673313) and the Innovation Fund for Medical Sciences (2016-I2M-1-010).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
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