Fitoterapia 125 (2018) 147–154

Contents lists available at ScienceDirect

Fitoterapia
journal homepage: www.elsevier.com/locate/fitote

Ferulic acid derivatives from Ligusticum chuanxiong T

⁎
Xu Zhang, Bing Han, Zi-Ming Feng, Ya-Nan Yang, Jian-Shuang Jiang, Pei-Cheng Zhang
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences, Peking Union Medical
College, Beijing 100050, People's Republic of China

A R T I C L E I N F O A B S T R A C T

Keywords: Four new ferulic acid derivatives ligusticumacid A-C (1–3) and ligusticumaldehyde A (4), one new dimer li-
Ligusticum chuanxiong gusticumacid D (5), and two novel 8–8′ lignans ligusticumacid E-F (6–7) were isolated from the rhizome of
Ferulic acid derivatives Ligusticum chuanxiong Hort. In particular, compounds 1–2, 5 were rarely phenylpropanoid phenolic acid dimers
Structure elucidation through different polymerization action in natural products. Their structures were established using UV, IR,
Neuroprotective effects
HRESIMS, NMR data. The absolute configurations of 3 was determined by quantum ECD calculation and 4, 6–7
were determined by the ECD exciton chirality method. In addition, all compounds were evaluated for their
neuroprotective effects on human neuroblastoma SH-SY5Y cell injury induced by H2O2. Compound 2 had a
moderate neuroprotective activity and 7 had a weak neuroprotective activity on human neuroblastoma SH-SY5Y
cell injury induced by H2O2 respectively.

1. Introduction 2. Experimental

Ferulic acid (FA), a hydroxyl cinnamic acid, is a natural antioxidant 2.1. General experimental procedures
from plant cell walls. It consisted widely in fruits and vegetables and
was applied to industry, food and medicine [1]. In recent years, the The optical rotations, UV spectra and ECD spectra were recorded
pharmacological activities of FA and FA derivatives were deeply stu- with JASCO P-2000, V650 and J-815 spectrometer (JASCO, Easton,
died on cancer, cardiovascular diseases, diabetes mellitus and skin MD, USA), respectively. The Infrared spectra were measured on Nicolet
disease [2–5]. Especially on Alzheimer's disease, FA and FA derivatives 5700 spectrometer (Thermo Scientific, FL, USA). The NMR spectra were
have been proposed as a potential treatment for their antioxidant and recorded with Bruker 500 MHz (Bruker-Biospin, Billerica, MA, USA).
anti-inflammatory pharmacological properties [6–9]. HRESIMS reports were obtained from Agilent 6520 HPLC-Q-TOF
Ligusticum chuanxiong Hort., named Chuanxiong in traditional (Agilent Technologies, Waldbronn, Germany) and LCMS-IT-TOF system
Chinese medicine, has been used for treating disease for more than a (Shimadzu Scientific Instruments Inc., Kyoto, Japan). Preparative HPLC
thousand years in China [10]. In particular, pharmacological studies was performed using a Shimadzu LC-10AT with a ODS-A column
showed that it had a satisfactory activity to treat neurological disease (250 mm × 20 mm, 5 μm; YMC Corp., Kyoto, Japan). The Agilent 1200
[11]. Phytochemical investigation revealed that phenolic acids, alka- series system was used to carry on the HPLC-DAD analysis with an
loids, and phthalides have been isolated from it [12–27]. Especially, the Apollo C18 column (250 mm × 4.6 mm, 5 μm; Alltech Corp., KY, USA).
phthalides, such as senkyunolides and ligustilides, have been confirmed The Agilent 7890A was used to carry on the GC analysis with a capillary
as the important bioactivity components [12–15]. Additionally, FA is a column, HP-5 (60 m × 0.32 mm, with a 1 μm film; Agilent
high-content constituent that is generally regarded as an index for Technologies Inc., CA, USA). Macroporous resin Diaion HP-20
quality control of the rhizome of Ligusticum chuanxiong and plays an (Mitsubishi Chemical Corp., Tokyo, Japan), RP-C18 (50 μm, YMC Corp.,
important role on its efficacy [28]. Thus it is significative to find more Kyoto, Japan), and Sephadex LH-20 (Pharmacia Fine Chemicals,
FA derivatives for screening cardiovascular activities from Chuanxiong. Uppsala, Sweden) were used to column chromatograph.
In this study, four new FA derivatives (1–4), one new dimer (5) and two
novel 8–8′ lignans (6–7) were isolated from the rhizome of L. chuan- 2.2. Plant material
xiong (Fig. 1). Their structures were elucidated by various spectroscopy
methods. Among them, compound 2 shown a moderate neuroprotective The roots of Ligusticum chuanxiong Hort. were collected from
activity on human neuroblastoma SH-SY5Y cell injury induced by H2O2. Pengzhou Town, Sichuan Province in PRC, in June 2013 and identified

⁎
Corresponding author.
E-mail address: pczhang@imm.ac.cn (P.-C. Zhang).

https://doi.org/10.1016/j.fitote.2018.01.005
Received 2 January 2018; Received in revised form 8 January 2018; Accepted 13 January 2018
0367-326X/ © 2018 Published by Elsevier B.V.
X. Zhang et al. Fitoterapia 125 (2018) 147–154

Fig. 1. Chemical structures of 1–7.

by Professor L. Ma. A voucher specimen (ID-S-2594) was deposited at Table 1

1 13
the Institute of Materia Medica, Peking Union Medical College and H NMR (500 MHz) and C NMR (125 MHz) data of compounds 1–3 in DMSO-d6.
Chinese Academy of Medical Sciences, Beijing, China.
Position 1 2 3

2.3. Extraction and isolation δH (J in Hz) δC δH (J in Hz) δC δH (J in Hz) δC

1 126.0 128.5 128.0

The powdered rhizome of L. chuanxiong Hort. (100.0 kg) was ex- 2 7.40, d (1.5) 113.4 7.42, d (8.5) 128.1 7.30, overlap 112.1
haustively extracted with 80% EtOH under reflux condition. The sol- 3 148.6 6.77, d (8.5) 115.6 144.2
vent was evaporated by reduced pressure and then the residue (23.1 kg) 4 147.7 157.4 149.2
was partitioned successively with EtOAc and n-BuOH. The n-BuOH- 5 7.06, d (8.5) 114.9 6.77, d (8.5) 115.6 127.4
6 7.23, dd (1.5, 123.8 7.42, d (8.5) 128.1 7.24, brs 118.2
soluble portion (1300 g) was applied on a HP-20 column to give five
8.5)
fractions A− E through gradient elution with H2O, 15% ethanol, 30% 7 7.39, s 126.3 7.21, d (16.0) 129.8 7.53, d (16.0) 144.2
ethanol, 50% ethanol, and 95% ethanol, respectively. Fraction C 8 138.5 7.49, d (16.0) 119.7 6.40, d (16.0) 116.6
(103.0 g) was chromatographed over an RP-C18 column, eluting with 9 164.1 167.9
H2O/MeOH (from 100:0 to 0:100) to give 16 fractions (C1 − C16) on 1′ 128.9 130.5 133.5
2′ 7.45, d (1.5) 111.6 7.28, d (1.5) 110.9 7.29, d (8.5) 127.4
the basis of HPLC analyses. Fraction C13 was separated by column 3′ 148.6 152.8 7.03, d (8.5) 116.3
chromatography over a Sephadex LH-20 using H2O as the eluent and 4′ 147.2 144.3 157.4
was further purified by semi-preparative HPLC (MeOH/H2O, 45:55, V/ 5′ 6.69, d (8.5) 113.1 132.1 7.03, d (8.5) 116.3
V, HOAc, 0.02%) to give 4 (4 mg) and 6 (50 mg). Fraction D (48.0 g) 6′ 7.12, dd (1.5, 122.1 7.64, d (1.5) 118.2 7.29, d (8.5) 127.4
8.5)
was chromatographed over an RP-C18 column, eluting with H2O/MeOH
7′ 7.51, d (16.0) 143.8 7.57, d (16.0) 144.1 5.99, d (7.0) 87.0
(from 95:5 to 0:100) to give 24 fractions (D1 − D24) on the basis of 8′ 6.47, d (16.0) 117.6 6.61, d (16.0) 118.6 4.21, d (7.0) 55.5
HPLC and TLC analyses. Fraction D9 was purified by a Sephadex LH-20 9′ 167.8 167.8 171.9
with a gradient of increasing MeOH (0%–100%) in H2O to give 20 1′′ 4.95, d (7.0) 99.4 4.82, d (8.0) 103.7 4.86, d (7.0) 100.3
fractions (D9–1− D9–20). Fraction D9–17 was separated by pre- 2′′ 3.23, m 73.1 3.33, t (8.0) 74.3 3.22, m 73.2
3′′ 3.23, m 76.8 3.22, t (8.0) 76.5 3.25, m 76.6
parative HPLC (MeCN/H2O, 25:75, V/V, HOAc, 0.02%) to give 2
4′′ 3.13, m 69.5 3.16, t (8.0) 69.9 3.15, m 69.6
(137 mg) and 7 (5 mg). Fraction D5 was chromatographed over a 5′′ 3.30, m 77.0 3.05, m 77.2 3.30, m 77.0
Sephadex LH-20 with a gradient of increasing MeOH (30%–100%) in 6′′ 3.41, dd (5.0, 60.6 3.41, dd (5.5, 60.9 3.45, dd (5.5, 60.6
H2O to give 15 fractions (D5–1− D5–15) on the basis of HPLC and TLC 12.0); 3.62, 11.5); 3.60, 11.5); 3.67, d
overlap dd (1.5, 11.5) (11.5)
analyses. Fraction D5–12 was separated by semi-preparative HPLC
3-OCH3 3.91, s 55.9 3.84, s 3.83, s 55.9
(MeCN/H2O, 20:80, V/V, HOAc, 0.02%) to give 1 (23 mg) and 3 3′-OCH3 3.64, s 55.2 7.42, d (8.5) 56.4
(15 mg). Fraction D8 was chromatographed over a Sephadex LH-20
with a gradient of increasing MeOH (0%–100%) in H2O to give 19
fractions (D8–1 −D8–19) on the basis of HPLC and TLC analyses. J = 8.5, 1.5 Hz, H-6′), 6.69 (1H, d, J = 8.5 Hz, H-5′). Two protons of
Fraction D8–14 was separated by semi-preparative HPLC (MeCN/H2O, trans conjugated double bond were presented at δH 7.51 (1H, d,
18:82, V/V, HOAc, 0.02%) to give 5 (8 mg). J = 16.0 Hz, H-7′) and 6.47 (1H, d, J = 16.0 Hz, H-8′). Additionally, a
single peak at δH 7.39 (1H, s, H-7) suggested the presence of a mono-
3. Results and discussion substituted double bond. The presence of multiple protons between δH
3.13 and 3.62 and the presence of a doublet at δH 4.95 (1H, d,
Compound 1 was obtained as a yellow amorphous powder. The J = 7.0 Hz, H-1′′) suggested the occurrence of a glucose moiety. The
13
negative HRESIMS gave the [M - H]− ion peak at m/z 547.1444, in C NMR spectrum of 1 (Table 1) showed 26 carbons, including two
accordance with the molecular formula of C26H28O13. The 1H NMR carbonyl carbons at δC 164.1 (C-9) and 167.8 (C-9′), four olefinic car-
spectrum of 1 (Table 1) presented two ABX systems at δH 7.40 (1H, d, bons, twelve aromatic carbons, and two methoxy groups. In addition,
J = 1.5 Hz, H-2), 7.23 (1H, dd, J = 8.5, 1.5 Hz, H-6), 7.06 (1H, d, six oxygenated carbons that contributed to a glucose moiety were also
J = 8.5 Hz, H-5), and 7.45 (1H, d, J = 1.5 Hz, H-2′), 7.12 (1H, dd, observed at δC 99.4 (C-1′′), 73.1 (C-2′′), 76.8 (C-3′′), 69.5 (C-4′′), 77.0

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Fig. 2. Key HMBC, ROESY and NOE correlations of 1–5 and 7.

8-O-4′-dehydrodiferulic acid and named ligusticumacid A.

Compound 2 was obtained as a yellow amorphous powder. Its
molecular formula was determined to be C24H26O10 by the negative ion
at m/z 473.1438 [M-H]− of HRESIMS. The 1H NMR spectrum (Table 1)
of 2 showed six aromatic protons which contained an AA′BB′ system at
δH 7.42 (2H, d, J = 8.5 Hz, H-2, 6), 6.77 (2H, d, J = 8.5 Hz, H-3, 5) and
two meta-protons at δH 7.64 (1H, d, J = 1.5 Hz, H-6′) and 7.28 (1H, d,
J = 1.5 Hz, H-2′). Two pairs of trans-olefinic protons at δH 7.57 (1H, d,
J = 16.0 Hz, H-7′) and 6.61 (1H, d, J = 16.0 Hz, H-8′); 7.49 (1H, d,
J = 16.0 Hz, H-8) and 7.21 (1H, d, J = 16.0 Hz, H-7). The presence of
multiple protons between δH 3.05 and 3.60 and the presence of an
anomeric proton at δH 4.82 (1H, d, J = 8.0 Hz, H-1′′) suggested the
occurrence of a glucose moiety. The 13C NMR spectrum (Table 1)
showed 24 carbon resonances including one carbonyl carbon, twelve
aromatic carbons, four olefinic carbons, one methoxy carbon, and a set
of glucose carbons.
The HMBC correlations (Fig. 2) of H-7′/C-9′, H-7′/C-6′, H-2′/C-4′
and 3′-OCH3/C-3′ confirmed the presence of a ferulic acid group. The
Fig. 3. Experimental ECD and calculated ECD spectrum of 3 in MeOH. correlations of H-8/C-6′, H-7/C-2, H-2/C-4 and H-5/C-1 revealed that
compound 2 was an 8–5′ neolignan compound with a glucose moiety
which was attached at C-4′ through the correlation H-1′′/C-4′. The
(C-5′′), and 60.6 (C-6′′).
glucose was confirmed as having a D-configuration by the same method
In the HMBC spectrum of 1 (Fig. 2), the correlations of H-7′/C-9′, H-
as used above. The β-configuration was deduced based on the coupling
7′/C-2′, H-6′/C-4′ and 3′-OCH3/C-3′ confirmed the presence of a ferulic
constant (J = 8.0 Hz) of H-1′′. Thus, the structure of 2 was defined as
acid group. The correlations of H-7/C-9, H-7/C-2, H-6/C-4 and 3-
4′-O-β-D-glucopyranosyl-5′-p-hydroxystyryl-ferulic acid and named li-
OCH3/C-3 confirmed the presence of another ferulic acid group. A
gusticumacid B.
singlet proton at δH 7.39 appeared to the presence of an 8-O-4′-linked
Compound 3 was obtained as a white amorphous powder. It had the
structure in combination with the bigger chemical shift of C-8 (δC
molecular formula C25H26O12 by the analysis of its HRESIMS, which
138.5) on ferulic acid chain side. Additionally, H-7 generated a gain
gave a negative ion at m/z 517.1335 [M-H]− (calcd 517.1352). The 1H
when irradiating 3′-OCH3 in the NOE experiment (Fig. 2). So these two
NMR spectrum (Table 1) of 3 showed an AA′BB′ system at δH 7.29 (2H,
ferulic acid groups were conjuncted through the dehydrogenative
d, J = 8.5 Hz, H-2′, 6′) and 7.03 (2H, d, J = 8.5 Hz, H-3′, 5′). Two
coupling of the hydroxyl of C-4′ with C-8. The glucose moiety was at-
trans-olefinic protons at δH 7.53 (1H, d, J = 16.0 Hz, H-7), 6.40 (1H, d,
tached at C-4 by the correlation of H-1′′/C-4 in the HMBC spectrum.
J = 16.0 Hz, H-8) and two aromatic protons at δH 7.30 (1H, overlap, H-
The D-configuration of the glucose moiety was determined by GC ana-
2), 7.24 (1H, brs, H-6) and one methoxy group at δH 3.83 (3H, s) were
lysis after acidic hydrolysis and chiral derivatization (see supporting
assigned to a ferulic acid group. In addition, a set of signals of a glucose
information). The relatively large coupling constant (J = 7.0 Hz) of the
moiety was also presented. The 13C NMR spectrum (Table 1) of 3 dis-
anomeric proton suggested that the glucose moiety was β-configured.
played a carbonyl carbon at δC 167.9 (C-9), two olefinic carbons at δC
Therefore, the structure of 1 was established as 4-O-β-D-glucopyranosyl-

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Table 2 observed. The correlations of H-6/C-8′, H-7′/C-5, H-7′/C-6′ and H-7′/C-

1 13
H NMR (500 MHz) and C NMR (125 MHz) data of compounds 4–5 in DMSO-d6. 9′ indicated that 3 was a benzofuranoid neolignan. In the ROESY ex-
periment of 3 (Fig. 2), the correlations of H-8′/H-2′, H-8′/H-6′ and the
Position 4 5
coupling constant of 7.0 Hz between H-7′ and H-8′ suggested a trans
δH (J in Hz) δC δH (J in Hz) δC configuration of H-7′ and H-8′ [29]. Thus, the absolute configuration of
C-7′ and C-8′ was 7′S,8′S (3a) or 7′R,8′R (3b). The absolute config-
1 130.9 129.2
uration was determined by comparing the experimental and calculated
2 7.42, d (1.0) 112.6 7.43, d (1.5) 121.3
3 144.3 144.0 ECD data. The systematic conformational analysis for 3a and 3b was
4 153.2 150.6 carried out using a molecular mechanics force field (MMFF94) calcu-
5 130.1 7.29, d (8.5) 116.9 lation. The optimized conformations were obtained using the time-de-
6 7.51, d (1.0) 120.6 7.51, dd (1.5, 8.5) 126.0 pendent density functional theory (TD-DFT) method at the B3LYP/6-
7 9.81, s 191.1 7.49, d (15.5) 141.8
31G(d) level. The overall calculated ECD spectra (Fig. 3) were gener-
8 6.41, d (15.5) 118.9
9 168.2 ated by Boltzmann weighting of their lowest energy conformers.
1′ 134.2 129.0 Throughout the entire range of wavelengths, the calculated spectrum of
2′ 6.99, d (2.0) 110.5 7.63, d (9.0) 129.8 (7′S,8′S)-3a matched with the experimental data for 3. The configura-
3′ 149.0 6.93, d (9.0) 117.2
tion of the glucose was confirmed by the aforementioned method.
4′ 146.4 159.1
5′ 7.07, d (8.5) 115.2 6.93, d (9.0) 117.2 Therefore, the structure of 3 was established as (2S,3S)-5-((E)-2-car-
6′ 6.85, dd (8.5, 2.0) 118.2 7.63, d (9.0) 129.8 boxyvinyl)-2-(4-O-β-D-glucopyranosyl- phenyl)-7-methoxy-2,3-dihy-
7′ 5.66, d (6.5) 88.3 7.45, d (15.5) 142.4 drobenzofuran-3-carboxylic acid and named ligusticumacid C.
8′ 3.58, q (6.0) 52.3 6.41, d (15.5) 119.6 Compound 4 was obtained as a white amorphous powder. The [M
9′ 3.72, overlap 62.5 168.2
+ Na]+ ion peak at m/z 515.1527 (calcd 515.1524) gave the molecular
1′′ 4.89, d (7.5) 99.9 5.00, d (8.0) 100.1
2′′ 3.22, m 73.2 3.09, m 73.1 formula C24H28O11. A careful comparison of the IR, UV and NMR data
3′′ 3.27, m 76.9 3.23, t (8.0) 76.8 of 4 and 3 suggested that they had the similar planar structure. In the
4′′ 3.13, m 69.6 3.09, m 69.5 1
H NMR and 13C NMR spectrum (Table 2) of 4, δH 3.72 (2H, overlap, H-
5′′ 3.24, m 77.0 3.33, m 77.2
9′) and δC 62.5 (C-9′) suggested the presence of a methylol instead of
6′′ 3.42, m; 3.63, m 60.6 3.44, m; 3.67, d (10.5) 60.6
3-OCH3 3.86, s 55.8
the carboxyl in 3. Further, the fragment of α,β-unsaturated ketone in 3
3′-OCH3 3.74, s 55.7 was replaced by the aldehyde [δH 9.81 (1H, s); δC 191.1] on the C-1
position of compound 4. In the ROESY experiment of 4 (Fig. 2), the
correlations of H-8′/H-2′, H-8′/H-6′ and the coupling constant of 6.0 Hz
144.2 (C-7) and 116.6 (C-8), twelve aromatic carbons, a methoxy group between H-7′ and H-8′ suggested a trans configuration of H-7′ and H-8′
at δC 55.9 and a set of glucose carbon resonances. Further, a ferulic acid [29]. According to the Harada − Nakanishi nonempirical rule, the ab-
group was deduced by the HMBC experiment (Fig. 2), in which the solute configuration of 4 was determined by the ECD exciton chirality
correlations of H-7/C-9, H-7/C-6, H-2/C-4 and 3-OCH3/C-3 were method [30]. The ECD spectrum of 4 showed the positive first Cotton

Fig. 4. Absolute configurations of compounds 4 as de-

termined by the ECD exciton chirality method.

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Table 3 suggested that the glucose moiety was β-configured. Therefore, the
1 13
H NMR (500 MHz) and C NMR (125 MHz) data of compounds 6–7 in DMSO-d6. structure of 5 was established as 3-O-p-hydroxycinnamoyl-4-O-β-D-
glucopyranosyl-caffeic acid and named ligusticumacid D.
Position 6 7
Compound 6 was obtained as a yellow amorphous powder. The [M
δH (J in Hz) δC δH (J in Hz) δC + Na]+ ion peak at m/z 541.1319 suggested that its molecular formula
was C25H26O12. In the low field regions of 1H NMR spectrum of 6
1 133.2 133.3
(Table 3), two benzene ring moieties were observed including an ABX
2 6.98, d (2.0) 110.3 6.98, d (2.0) 110.0
3 149.0 149.0 system at δH 7.07 (1H, d, J = 8.5 Hz, H-5), 6.76 (1H, dd, J = 8.5,
4 146.7 146.5 2.0 Hz, H-6), 6.98 (1H, d, J = 2.0 Hz, H-2) and an AA′BB′ system at
5 7.07, d (8.0) 115.4 7.12, d (8.5) 114.9 7.56 (2H, d, J = 8.5 Hz, H-2′, 6′), 6.81 (2H, d, J = 8.5 Hz, H-3′, 5′)
6 6.76, dd (2.0, 8.0) 117.5 6.68, dd (2.0, 8.5) 117.2 (Table 3). In addition, a olefinic proton was presented at δH 7.55 (1H, d,
7 5.77, d (2.5) 79.6 5.76, d (2.5) 79.6
J = 2.5 Hz, H-7′). The high field regions of 1H NMR spectrum of 6
8 4.20, t (2.5) 52.5 4.17, t (2.5) 52.6
9 171.3 171.4 contained two methine protons at δH 5.77 (1H, d, J = 2.5 Hz, H-7),
1′ 124.3 124.2 4.20 (1H, t, J = 2.5 Hz, H-8) and a methoxy at δH 3.75 (3H, s, -OCH3).
2′ 7.56, d (8.5) 132.8 7.52, d (8.5) 132.8 The remain protons suggested the presence of a glucose moiety at δH
3′ 6.81, d (8.5) 115.8 6.80, d (8.5) 115.8
4.89 (1H, d, J = 7.5 Hz, H-1′′) and multiple protons between δH 3.13
4′ 160.6 160.0
5′ 6.81, d (8.5) 115.8 6.80, d (8.5) 115.8 and δH 3.62. Corresponding to the 13C NMR spectrum of 6 (Table 3),
6′ 7.56, d (8.5) 132.8 7.52, d (8.5) 132.8 twelve benzene ring carbons, two olefinic carbons, six glucose carbons
7′ 7.55, d (2.5) 139.2 7.57, d (2.5) 139.5 and a methoxy were presented. Additionally, two carbonyl carbons at
8′ 118.6 118.4 δC 171.0 and 171.3 were observed. The assignment of protons and
9′ 171.0 171.0
carbons of 6 were achieved by HSQC, HMBC, and ROESY experiments.
1′′ 4.89, d (7.5) 99.8 5.00, d (7.0) 99.5
2′′ 3.22, overlap 73.1 3.30, overlap 73.0 In the HMBC spectrum, the observation of the correlation between H-7
3′′ 3.24, overlap 76.8 3.31, overlap 76.6 (δH 5.77) and C-9′ (171.0) indicated the occurrence of a five-membered
4′′ 3.13, t (8.5) 69.6 3.25, t (8.5) 69.9 lactone ring in 6. According to the correlations of H-7/C-2, H-7/C-6, H-
5′′ 3.27, overlap 77.0 3.72, overlap 73.8
7/C-9, H-7′/C-2′, H-7′/C-6′, H-7′/C-9′ and H-7′/C-8, the type of 8–8′
6′′ 3.41, dd (7.0, 11.5); 3.62, 60.6 4.13, dd (7.0, 11.5); 4.52, 63.9
dd (2.0, 11.5) dd (2.0, 11.5)
lignans was confirmed. The glucose moiety was determined to attach at
1′′′ 120.4 the C-4 through the correlation of H-1′′/C-4. The methoxy was attached
2′′′ 7.40, d (2.0) 112.6 at the C-3 through the correlation of H-OCH3/C-3. The relatively large
3′′′ 147.4 coupling constant (J = 7.5 Hz) of the anomeric proton suggested that
4′′′ 151.6
the glucose moiety was β-configured. The D-configuration of glucose
5′′′ 6.87, d (8.0) 115.2
6′′′ 7.43, dd (2.0, 8.0) 123.4 was confirmed through the same method as used above.
7′′′ 165.4 The correlation of H-8/H-2, 6 suggested that the relative config-
3-OCH3 3.75, s 55.7 3.74, s 55.6 uration of H-7 and H-8 was trans in the ROESY experiment (Fig. 2). The
3′′′-OCH3 3.79, s 55.7
ECD Cotton effects observed for 6 were attributed to exciton coupling
between the transition dipoles of the cinnamic acid and phenylpro-
pionic acid moieties and could be used to predict the absolute config-
effect at 297 nm and negative second Cotton effect at 232 nm (Fig. 4),
uration. The positive long-wave Cotton effect around λ max = 316 nm
which is consistent with 7′R,8′S-configuration. The correlation from H-
and negative short-wave Cotton effect around λ max = 234 nm in-
1′′ to C-4′ in the HMBC spectrum (Fig. 2) suggested that the glucose
dicated that the front chromophore (cinnamic acid) existed in a
moiety was located at C-4′. The β-configuration was deduced based on
clockwise arrangement relative to the rear chromophore (phenylpro-
the coupling constant (J = 8.0 Hz) of H-1′′. The D-configuration of
pionic acid) (Fig. 5). So, the absolute configuration of compound 6 was
glucose was confirmed through the same method as used above. Thus,
confirmed as 7R,8R. Thus, the structure of 6 was confirmed and named
the structure of 4 was established as (2R,3S)-2-(4-O-β-D-glucopyranosyl-
ligusticumacid E.
3-methoxyphenyl)-3-(hydroxymethyl)-7-methoxy-2,3-dihy-
Compound 7 was also obtained as a yellow amorphous powder. It
drobenzofuran-5-carbaldehyde and named ligusticumaldehyde A.
was formulated as C33H32O15 from its HRESIMS data at m/z 667.1650
Compound 5 was obtained as a yellow amorphous powder. Its
[M - H]−, calcd for C33H31O15 667.1668. Compound 7 and 6 possessed
molecular formula was determined to be C24H24O11 according to the
similar skeleton by comparison the NMR data. However, differences
molecular ion peak [M + Na]+ at m/z 511.1227. The 1H NMR spec-
were apparent for the signals at δC 73.8 (C-5′′) and 63.9 (C-6′′) in the13C
trum of 5 (Table 2) presented an ABX system at δH 7.51 (1H, dd,
NMR spectrum, which suggested the glucose moiety was substituted.
J = 1.5, 8.5 Hz, H-6), 7.43 (1H, d, J = 1.5 Hz, H-2), 7.29 (1H, d,
Comparing the 1H NMR spectrum of 7 and 6, an extra ABX system at δH
J = 8.5 Hz, H-5); an AA′BB′ system at δH 7.63 (2H, d, J = 9.0 Hz, H-2′,
7.43 (1H, dd, J = 8.0, 2.0 Hz, H-6′′′), 7.40 (1H, d, J = 2.0 Hz, H-2′′′),
6′), 6.93 (2H, d, J = 9.0 Hz, H-3′, 5′) and four trans conjugated double
6.87 (1H, d, J = 8.0 Hz, H-5′′′) and a methoxy at δH 3.79 (3H, s, -OCH3)
bond protons at δH 7.49 (1H, d, J = 15.5 Hz, H-7), 6.41 (1H, d,
were observed in compound 7. Corresponding to the 13C NMR spec-
J = 15.5 Hz, H-8), 7.45 (1H, d, J = 15.5 Hz, H-7′), and 6.41 (1H, d,
trum, six extra benzene ring carbons, a carbonyl carbon (δC 165.4), and
J = 15.5 Hz, H-8′). In the 13C NMR spectrum of 5 (Table 2), two car-
a methoxy (δC 55.7) were presented. According to above information, a
bonyl carbons were presented at δC 168.2 (C-9) and 168.2 (C-9′). The
benzoic acid moiety was confirmed. In the HMBC spectrum, the cor-
above information showed that 5 contained a caffeic acid group and a
relation from H-6′′ to C-7′′′ supported the benzoic acid moiety which
p-hydroxycinnamic acid group. When comparing to the corresponding
was attached at the C-6′′ of the glucose moiety. The other correlations
13
C NMR data with β-D-glucopyranosyl 4-O-β-D-glucopyranosylcaffeate
that occurred in the HMBC spectrum of 7 confirmed the same planar
[31], the C-2 (δC 121.3) and C-4 (δC 150.6) were shifted significantly
structure with compound 6. The relatively large coupling constant
downfield and C-3 (δC 144.0) was shifted significantly highfield in
(J = 7.0 Hz) of the anomeric proton suggested that the glucose moiety
compound 5. It illustrated that the C-3 was substituted. In the HMBC
was β-configured too. The D-configuration of glucose was also con-
spectrum of 5 (Fig. 2), the correlation H-1′′/C-4 confirmed the sugar
firmed through the same method as used above.
moiety which was attached at C-4. The D-configuration of the glucose
The same method with 6 was carried out to determine the absolute
moiety was determined by the same method as used above. The rela-
configuration of compound 7. In the ROESY experiment (Fig. 2), the
tively large coupling constant (J = 8.0 Hz) of the anomeric proton
correlation of H-8/H-2, 6 suggested that the relative configuration of C-

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X. Zhang et al. Fitoterapia 125 (2018) 147–154

Fig. 5. Absolute configurations of compounds 6 as de-

termined by the ECD exciton chirality method.

Fig. 6. Absolute configurations of compounds 7 as de-

termined by the ECD exciton chirality method.

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X. Zhang et al. Fitoterapia 125 (2018) 147–154

7 and C-8 was trans too. In the ECD spectra, the positive long-wave 221 (4.56), 266 (4.18), 321 (4.38) nm; [α]20 D + 37 (c 0.1 MeOH);
Cotton effect around λ max = 316 nm and negative short-wave Cotton HRESIMS m/z 667.1650 [M - H]− (calcd 667.1668); IR νmax: 3431,
effect around λ max = 234 nm indicated that the front chromophore 2944, 1715, 1601, 1515, 1453, 1429, 1285, 1225, 1171, 1072,
(cinnamic acid) existed in a clockwise arrangement relative to the rear 764 cm− 1; 1H NMR and 13C NMR see Table 3.
chromophore (phenylpropionic acid) (Fig. 6). So, the absolute config-
uration of compound 7 was also confirmed as 7R,8R. Finally, it was 5. Determination of the absolute configurations of the sugars
named ligusticumacid F.
Compounds 1–7 were tested for their neuroprotective effects on SH- Compounds 1 (2 mg) was dissolved in 1 mol/l CF3COOH (14 ml)
SY5Y cell injury induced by H2O2 with edaravone as a positive control. and then the mixture was heated in 70 °C for 1 h. The mixture was then
The results showed that compound 2 exhibited a moderate neuropro- extracted three times with EtOAc, and the aqueous layer was freeze-
tective effect with the increase cell viability rate of 32.87%, and 7 ex- dried to obtain residue. Using the same method with the literature [32],
hibited a weak neuroprotective effect with the increase cell viability the residue was dissolved in anhydrous pyridine (2 ml), L-Cysteine
rate of 9.86%, compared with the positive control edaravone with in- methyl ester hydrochloride (4 mg) was added, and then the mixture
crease cell viability rate of 24.16%. was heated in a water bath (60 °C) for 1 h. After the reaction solution
was dried under vacuum, N-trimethylsilylimidazole (1 ml) was added,
4. Structure characterization and the solution was heated in a water bath (60 °C) for 1 h and ex-
tracted three times with H2O/n-hexane. Then, the n-hexane layer was
4.1. Ligusticumacid A, 1 analyzed using GC under conditions as follows: injection temperature,
300 °C; detector temperature (FID), 300 °C; capillary column, HP-5
Yellow amorphous powder; UV λmax (MeOH) (log ε): 216 (4.30), (60 m × 0.32 mm, Dikma); start temperature, 200 °C, raised to 260 °C
230 (4.25), 290 (4.34), 323 (4.39) nm; [α]20 D − 41 (c 0.1 MeOH); at a rate of 10 °C/min, and the final temperature maintained for 30 min;
HRESIMS m/z 547.1444 [M - H]− (calcd 547.1457); IR νmax: 3396, and N2 used as the carrier gas. Compounds 2–7 were also measured by
2935, 1694, 1509, 1258, 1075, 816 cm− 1; 1H NMR and 13C NMR see the same procedure and all of the sugars were determined to be D-
Table 1. configuration.

4.2. Ligusticumacid B, 2 6. Neuroprotective activity assay

Yellow amorphous powder; UV λmax (MeOH) (log ε): 209 (4.46), The assay method of neuroprotective effects about compounds refer
232 (4.31), 306 (4.63) nm; [α]20 D + 70 (c 0.1 MeOH); HRESIMS m/z to the procedures of literature [33].
473.1438 [M - H]− (calcd 473.1453); IR νmax: 3333, 2935, 1759, 1692,
1636, 1608, 1579, 1514, 1462, 1261, 1075, 849 cm− 1; 1H NMR and Acknowledgements
13
C NMR see Table 1.
This work was supported by the National Natural Science
4.3. Ligusticumacid C, 3 Foundation of China (No. 81773588).

White amorphous powder; UV λmax (MeOH) (log ε): 203 (4.42), 224 Conflict of interest
(4.46), 322 (4.30) nm; [α]20 D − 62 (c 0.1 MeOH); HRESIMS m/z
517.1335 [M - H]− (calcd 517.1352); IR νmax: 3329, 2921, 1728, 1666, The authors declare no conflicts of interest.
1511, 1236, 1078, 852 cm− 1; 1H NMR and 13C NMR see Table 1.
Appendix A. Supplementary data
4.4. Ligusticumaldehyde A, 4
UV, IR, 1D and 2D NMR, CD, and HRESIMS spectra of the new
White amorphous powder; UV λmax (MeOH) (log ε): 205 (4.69), 231 compounds. Supplementary data associated with this article can be
(4.47), 307 (4.41) nm; [α]20 D − 11 (c 0.1 MeOH); HRESIMS m/z found in the online version, at https://doi.org/10.1016/j.fitote.2018.
515.1527 [M + Na]+ (calcd 515.1524); IR νmax: 3397, 2928, 1677, 01.005.
1592, 1514, 1324, 1137, 1075, 1044 cm− 1; 1H NMR and 13C NMR see
Table 2. References

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