Sesquiterpenoids and γ-pyranone derivatives from the whole plant of Erigeron breviscapus and their neuroprotective effects

Article abstract of DOI:10.1016/j.fitote.2019.104288

Four new sesquiterpenes (1–2, 6–7), a new pyranone glycoside (10) along with six known compounds, were isolated from the whole plant of Erigeron breviscapus. Their planar structures were elucidated using extensive spectroscopic analyses. Brevisterpene A (1) and brevisterpene B (2) were proved to be a pair of diastereomer followed by mixtures resolution using chiral HPLC. Their absolute configurations were determined by ECD calculation. The relative configuration of brevisnoside B (7) was elucidated by a combined analysis of NOESY spectrum and computation of ¹³C NMR chemical shifts, and determination of the absolute configurations of 6 and 7 assisted by optical rotation calculations. Compounds 1 and 2 displayed moderate neuroprotective effects against H₂O₂-induced damage in SH-SY5Y cells.

Full text of DOI:10.1016/j.fitote.2019.104288

Fitoterapia138(2019)104288
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Sesquiterpenoids and γ-pyranone derivatives from the whole plant of
Erigeron breviscapus and their neuroprotective effects
Shuang Hana, Ying Liua, Zilin Houa, Xiujia Sunc, Guodong Yaoa, Pinyi Gaoa,b,*, Lingzhi Lia,*
Shaojiang Songa,**
,
a School of Traditional Chinese Materia Medica, Key Laboratory of Computational Chemistry-Based Natural Antitumor Drug Research & Development, Liaoning Province,
Shenyang Pharmaceutical University, Shenyang 110016, People′s Republic of China
b College of Pharmaceutical and Biotechnology Engineering, Institute of Functional Molecules, Shenyang University of Chemical Technology, 11 Street, Shenyang economic
and Technological Development Zone, Shenyang 110142, People′s Republic of China
c Shanghai Key Laboratory of Psychotic Disorders, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai, 200030, 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 sesquiterpenes (1–2, 6–7), a new pyranone glycoside (10) along with six known compounds, were
isolated from the whole plant of Erigeron breviscapus. Their planar structures were elucidated using extensive
spectroscopic analyses. Brevisterpene A (1) and brevisterpene B (2) were proved to be a pair of diastereomer
followed by mixtures resolution using chiral HPLC. Their absolute configurations were determined by ECD
calculation. The relative configuration of brevisnoside B (7) was elucidated by a combined analysis of NOESY
spectrum and computation of 13C NMR chemical shifts, and determination of the absolute configurations of 6
and 7 assisted by optical rotation calculations. Compounds 1 and 2 displayed moderate neuroprotective effects
against H2O2-induced damage in SH-SY5Y cells.
Erigeron breviscapus
Sesquiterpene
ECD calculation
Optical rotation calculation
Neuroprotective effect
1. Introduction
were tested for their neuroprotective activities against H2O2-induced
damage in SH-SY5Y cells. Detailed information regarding the extrac-
Erigeron breviscapus (Vaniot) Hand.-Mazz., a traditional Chinese
herb medicine, is a perennial, clump-forming shrub belonging to the
Compositae family, widely distributed throughout southwest China
[1,2]. The dried whole plant of E. breviscapus is used for the treatment
of cerebrovascular disease, coronary heart disease, cerebral embolism,
paralysis caused by cerebral vascular accidents, and apoplexy [3–5].
Previous studies revealed that E. breviscapus possessed multiple bioac-
tivities including antioxidant, anti-inflammatory, anticoagulant, and
cardiovascular effects [6,7]. Most of the phytochemical studies of this
plant have focused on the main bioactive compounds such as flavonoids
and caffeic acid esters [8].
tion, isolation, structure elucidation, and neuroprotective effects eva-
luation of 1–11 are reported.
2. Experimental section
2.1. General experimental procedures
The ultraviolet (UV) spectra were collected on a Shimadzu UV-1700
spectrophotometer (Kyoto, Japan). Optical rotations were measured on
a JASCO DIP-370 digital polarimeter (Easton, MD, USA). CD spectra
were recorded on
a MOS-450 spectrometer (Bio-Logic Science
To further study the chemical constituents and bioactivities of E.
breviscapus, the investigation toward its ethanol extract was conducted.
Careful chromatographic purification and chemical examination led to
the isolation and characterization of five new compounds, including
four sesquiterpenes and one γ-pyranone glycoside. Compounds 1 and 2
were proved to be a pair of diastereomer. All the isolated compounds
Instruments, Seyssinet-Pariset, France). HRESIMS spectra were carried
out on an Agilent G6520 Q-TOF spectrometer (Santa Clara, CA, USA).
NMR spectra (1D and 2D NMR) were determined on Bruker ARX-300,
ARX-400, and AV-600 spectrometers with trimethylsilane (TMS) as an
internal standard. The chemical shifts were referenced to the residual
DMSO-d6 signal (δC/H 39.50 and 2.50) and CD3OD-d4 (δC/H 49.00 and
* Corresponding Author.
** Corresponding Author at: School of Traditional Chinese Materia Medica, Key Laboratory of Computational Chemistry-Based Natural Antitumor Drug Research &
Development, Liaoning Province, Shenyang Pharmaceutical University, Shenyang 110016, People′s Republic of China.
E-mail addresses: gpy1982@126.com (P. Gao), lilingzhijessie@163.com (L. Li), songsj99@163.com (S. Song).
https://doi.org/10.1016/j.fitote.2019.104288
Received 9 July 2019; Received in revised form 2 August 2019; Accepted 2 August 2019
Availableonline06August2019
0367-326X/©2019ElsevierB.V.Allrightsreserved.

S. Han, et al.
Fitoterapia138(2019)104288
3.31), and expressed as δ values (ppm), with coupling constants (J)
reported in Hz. Column chromatography was performed on silica gel
(100–200 and 200–300 mesh, Qingdao Marine Chemical Factory,
Qingdao, People's Republic of China), Sephadex LH-20 gel (GE
Healthcare, Sweden), polyamide-gel (80–100 mesh, Taizhou Luqiao
Sijia Biochemical Plastic Factory, Zhejiang, China), MCI-gel CHP 20P
(75–150 μm, Tokyo, Japan) and ODS gel (60–80 μm, Merck, Germany).
TLC was carried out on precoated silica gel GF254 plates (Qingdao
Marine Chemical, Inc. China). Semipreparative reversed-phase HPLC
was performed on a Waters 1525 series pumping system equipped with
Brevisnoside B (7): yellow oil; [α]D20 − 39.0 (c 0.10, MeOH); 1H,
13C NMR data (CD3OD), see Tables 1 and 2; HRESIMS m/z 277.1778
[M + Na]+ (calcd for C15H26O3, 277.1774).
Erigeroside 6′-palmitate (10): white, amorphous powder; 1H, 13C
NMR data (DMSO‑d6), see Tables 1 and 2; HRESIMS m/z 557.3005
[M + COOH]− (calcd for C27H44O9, 557.2965).
2.4. Sugar analysis
2.4.1. Acid hydrolysis
a
Waters 2489 UV detector using
a
YMC Pack ODS-A column
Acid hydrolysis was carried out to obtain the monosaccharide of
compound 10 [9]. Compound 10 (2.0 mg) was hydrolyzed with 1 M
aqueous HCl (4 mL) in a thermostatically controlled oil bath at 80 °C for
4 h. After cooling, the reaction mixture was extracted with EtOAc
(3 × 4 mL), and the H2O phase was then concentrated under vacuum
and dried to give a sugar residue. The residue was dissolved in H2O
(1.5 mL) and analyzed by a LCNetII/ADC HPLC with OR-4090 as the
detector and equipped with a Shodex Asahipak NH2P-50 4E column
using an isocratic solvent system of MeCN-H2O (3:1, v/v) at a flow rate
of 0.8 mL/min. The absolute configuration was determined by com-
paring the retention time and peak shape with standard sugar: D-glucose
(tR = 10.20 min) (Fig. S30). The sugar moiety of compound 10 was
identified to be D-glucose.
(250 × 10 mm, 5 μm). Chiral HPLC isolation was run on a Shimadzu
LC-10 CE liquid chromatography instrument equipped with a Shimadzu
SPD-M10A UV–vis detector by using Chiralpak AD-H column
(4.6 mm × 250 mm, 5 μm, Daicel Polymer Ltd., Tokyo, Japan).
2.2. Plant material
The whole plants of E. breviscapus were collected from Yunnan
Province, People's Republic of China, in May 2016. The plant materials
were identified by Professor Jincai Lu (Department of Pharmaceutical
Botany, School of Traditional Chinese Materia Medica, Shenyang
Pharmaceutical University, China). A voucher specimen (accession
number: DZXX-2016-1Y) was maintained at the specimen room of
Shenyang Pharmaceutical University.
2.4.2. Enzymatic hydrolysis
Compound 1 (1.0 mg) and snailase (2.0 mg) were dissolved in 3 mL
of potassium dihydrogen phosphate (KDP) buffer at pH 5.6 and 37 °C
for 5 h [10]. The reaction mixture was extracted with the same volume
of EtOAc three times. The EtOAc extracts and water-soluble layer were
evaporated to dryness in vacuum, respectively. The EtOAc extracts
were purified by semi-preparative HPLC (eluted with MeCN-H2O, 1:1,
v/v) to afford the aglycone of 1 (0.5 mg). Compound 2 (1.1 mg) was
hydrolyzed with the same procedure to afford monosaccharide.
LCNetII/ADC HPLC-OR analysis showed the presence of D-glucose of 1
and 2 (Fig. S30).
2.3. Extraction and isolation
The air-dried aerial parts of E. breviscapus (20 kg) were extracted
thrice with 80% EtOH at room temperature under reflux and evapo-
rated to dryness to obtain a crude extract (4 kg). This crude extract was
suspended in H2O and successively partitioned with CH2Cl2 to give
CH2Cl2-(600 g) and aqueous-soluble (3.2 kg) extracts upon evaporation
in vacuo. The H2O-soluble fraction was dissolved in MeOH and parti-
tioned by macroporous resin column chromatography (CC) eluted with
EtOH/H2O gradient (1:1, 7:3, and 8:2, v/v) to give three fractions Fr.1-
Fr.3. Fr.3 was fractionated by silica gel CC using CH2Cl2/MeOH/H2O
(9:1:0 to 1:1:0.8, v/v) to afford Fr.3.1-Fr.3.2 by TLC analysis. Fr.3.1 was
separated by polyamide column (MeOH/H2O, 6:4 to 8:2, v/v) followed
by Sephadex LH-20 with 60% MeOH/H2O to yield Fr.3.1.1-Fr.3.1.3.
Fr.3.1.2 was further divided into three portions (Fr.3.1.2.1-Fr.3.1.2.3)
by silica gel with CH2Cl2/MeOH, 50:1 to 1:1, v/v. Fr.3.1.2.1 and
Fr.3.1.2.2 were purified by ODS column (MeOH/H2O, 2:8 to 8:2, v/v)
and semipreparative HPLC to obtain 10 (6.0 mg) and 11 (20.0 mg),
respectively. Fr.3.1.2.3 was separated in a similar manner to Fr.3.1.2.1
to give mixed 1 and 2 (8 mg), 3 (3.5 mg), 4 (4.4 mg) and 5 (3.6 mg).
Subsequently, the separation of mixed 1 and 2 by chiral HPLC using a
Daicel Chiralpak AD-H chiral column eluted with n-hexane-isopropanol
(11:1, v/v) detection at 210 nm afforded 1 (2.0 mg) and 2 (2.1 mg).
The dried CH2Cl2 extract was subjected to polyamide CC eluted with
ethanol/H2O (1:9 to 9:1, v/v) followed by MCI gel CC using aqueous
ethanol to yield four fractions (Fr.1-Fr.4). Fr.1-Fr.4 were separated by
using silica gel CC eluted with (CH2Cl2/MeOH) and purified by semi-
preparative HPLC to give 6 (2.5 mg), 7 (2.2 mg), 8 (5.0 mg) and 9
(6.0 mg).
2.5. ECD calculations
The absolute configurations of the aglycone of 1 and 2 were de-
termined by using time-dependent density functional theory (TDDFT)
calculations. Conformational distribution of the optimized structures
was investigated at B3LYP/6-31G(d) and suggested the major con-
formers (> 98%). Energies of the geometric conformations in MeOH
were calculated at the B3LYP/6–311 + G(d) level. The hybrid B3LYP
functions were chose to run the TDDFT calculations, solving for 25
states for per molecule. The ECD of the conformers were performed by
the TDDFT method at the B3LYP/6-31G(d) level with the CPCM model
in methanol solution, and the overall ECD curves were produced by
SpecDis 1.51 [11–13].
2.6. Optical rotation calculations
A conformational search using the Molecular Merck force field
(MMFF) led to the identification of the major conformers (> 95%),
which was performed using the Gaussian 09 program at B3LYP/6-
31G(d) level [14]. Geometry optimization followed by OR calculations
at D-sodium line radiation (wavelength of 589 nm) in MeOH (CPCM)
using the B3LYP functional and the 6–311++G(2d,p) basis set for DFT.
Final calculated ORs were obtained as the result of the Boltzmann-
weighted average.
Brevisterpene A (1): colorless oil; ECD (MeOH) λmax (Δε) 204
(−3.30), 214 (−0.44), 245 (−0.46) nm; 1H, 13C NMR data (DMSO‑d6),
see Tables 1 and 2; HRESIMS m/z 409.2181 [M + Na]+ (calcd for
C20H34O7, 409.2191).
Brevisterpene B (2): colorless oil; ECD (MeOH) λmax (Δε) 202
(−2.99), 222 (+0.50), 243 (−0.61) nm; 1H, 13C NMR data (DMSO‑d6),
see Tables 1 and 2; HRESIMS m/z 409.2181 [M + Na]+ (calcd for
2.7. 13C NMR calculations
C
20H34O7, 409.2191).
Brevisnoside A (6): yellow oil; [α]D20 − 25.0 (c 0.10, MeOH); 1H,
After optimization of the major conformers (> 95%) was performed
using the Gaussian 09 program at B3LYP/6-31G(d) level. Computed
chemical shifts reported in this study were determined using the GIAO
13C NMR data (CD3OD), see Tables 1 and 2; HRESIMS m/z 261.1844
[M + Na]+ (calcd for C15H26O2, 261.1825).
2

S. Han, et al.
Fitoterapia138(2019)104288
Table 1
1H (600 MHz) NMR spectroscopic data for compounds 1, 2, 6, 7 and 10.
No.
1a
2a
6b
7b
10a
1
2
4.13 (2H, m)
4.02 (1H, overlapped)
5.61 (1H, s)
1.40 (1H, t, 12.2)
1.80 (1H, m)
1.40 (1H, t, 12.2)
1.79 (1H, m)
5.54 (1H, t, 6.4)
8.12 (1H, s)
3
4
3.97 (1H, m)
3.96 (1H, m)
1.95 (1H, dd, 17.0, 9.6)
2.39 (1H, m)
1.96 (1H, dd, 16.8, 9.6)
2.39 (1H, dd, 16.8, 5.8)
3.95 (1H, t, 6.8)
3.93 (1H, overlapped)
5
2.25 (2H, t, 6.9)
1.70 (2H, m)
1.42 (1H, m)
2.01 (1H, m)
1.34 (2H, m)
2.09 (2H, m)
5.14 (1H, t, 7.3)
6.40 (1H, d, 5.6)
8.13 (1H, d, 5.9)
6
5.12 (1H, overlapped)
7
5.99 (1H, d, 15.9)
5.22 (1H, dd, 15.9, 7.9)
3.76 (1H, m)
5.99 (1H, d, 15.9)
5.23 (1H, d, 15.9, 7.8)
3.76 (1H, p, 6.6)
1.18 (3H, d, 6.3)
3.18 (3H, s)
8
1.99 (2H, t, 7.5)
9
2.07 (2H, t, 7.5)
10
11
12
13
14
1.17 (3H, d, 6.3)
3.18 (3H, s)
5.09 (1H, overlapped)
1.02 (3H, s)
1.00 (3H, s)
1.67 (3H, s)
1.60 (3H, s)
1.62 (3H, s)
1.67 (3H, s)
0.99 (3H, s)
0.99 (3H, s)
1.61 (3H, s)
1.66 (3H, s)
1.67 (3H, s)
3.93 (1H, overlapped)
4.00 (1H, overlapped)
0.82 (3H, d, 6.9)
15
1′
2′
3′
4′
5′
6′
1.64 (3H, s)
4.28 (1H, d, 7.8)
3.13 (1H, m)
4.28 (1H, d, 7.8)
3.11 (1H, m)
4.85 (1H, d, 7.0)
3.20 (1H, m)
2.91 (1H, m)
2.90 (1H, m)
3.21 (1H, m)
3.04 (1H, m)
3.04 (1H, m)
3.11 (1H,m)
3.09 (1H, m)
3.08 (1H, m)
3.54 (1H, m)
3.44 (1H, dt, 11.6, 5.8)
3.65 (1H, m)
3.43 (1H, dt, 11.7, 5.9)
3.65 (1H, m)
4.04 (1H, dd, 11.9, 6.8)
4.28 (1H, d, 11.8)
5.47 (1H, d, 4.9)
5.22 (1H, d, 4.8)
5.29 (1H, d, 5.5)
Glc-OH
Glc-OH
Glc-OH
1″
4.44 (1H, t, 5.8)
4.95 (1H, d, 4.7)
4.90 (2H, d, 4.9)
4.46 (1H, t, 5.8)
4.94 (1H, d, 4.7)
4.91 (2H, dd, 8.3, 4.9)
2″
2.26 (2H, m)
1.49 (2H, m)
1.22 (24H, m)
3″
4″-15″
16″
a
0.85 (3H, t, 6.9)
Recorded in DMSO-d6.
b
Recorded in methanol-d4.
method in Gaussian 09 at the B3LYP/6–311++G(2d,p) level of theory
[15]. The scaled calculated 13C NMR chemical shifts were obtained
from the following: δscal.calc. = (δcalc. –intercept)/slope. A result was
evaluated in terms of R2, MaxDev and AveDev (Table S5). Among them,
R2 is its coefficient of determination. MaxDev is the maximum absolute
deviation with respect to the experimental chemical shifts δexp. AveDev
spectrum, and four oxygenated methines and an oxygenated methylene.
Correspondingly, analysis of the 13C NMR data and HSQC spectra in-
dicated that 1 contained four olefinic carbons (δC 125.5, C-5; 136.4, C-
6; 128.4, C-7; 136.1, C-8), one O-methyl groups (δC 55.2, C-11), four
methyl groups (δC 21.6, C-10; 21.1, C-14; 28.1, C-12; 29.9, C-13), two
oxymethine carbons (δC 70.0, C-3; 77.6; C-9), two methine groups (δC
45.6, C-2; 38.5, C-4), a quaternary carbon (δC 36.1, C-1), and one
glucose moiety carbon signal (δC 100.7, C-1′; 76.8, C-2′; 73.5, C-3′;
70.1, C-4′; 76.7, C-5′; 61.1, C-6′) (Table 2). Based on detailed analyses
of 2D NMR data, the planar structure of 1 was constructed in Fig. 1. The
HMBC correlations from H-2 to C-1 (δC 36.1), C-3 (δC 70.0), and C-4 (δC
38.5) and from H-4 to C-2 (δC 45.6), C-3 (δC 70.0), C-5 (δC 125.5), and
C-6 (δC 136.4) confirmed the presence of an unsaturated six-membered
ring. Correlations from CH3-12 and CH3-13 to C-1 (δC 36.1), C-2 (δC
45.6), and C-6 (δC 136.4) indicated the attachment of two methyl
groups at C-1. HMBC correlations from CH3-10 to C-8 (δC 136.1), and C-
9 (δC 77.6) and CH3-14 to C-4 (δC 38.4), C-5 (δC 125.5), and C-6 (δC
136.4) justified the assignment of two methyl groups to C-5 and C-9,
respectively. The double bond at C-7/C-8 was also joined at C-6 and C-9
on the basis of correlations from H-7 to C-5 (δC 125.5) and C-9 (δC 77.6)
and H-8 to C-6 (δC 136.4) and C-9 (δC 77.6). The HMBC correlation of
the signal at δH 3.18 with C-9 (δC 77.6) placed methoxy group on C-9.
Due to two separated resonances at some of the carbons (Fig. S28),
compounds 1 and 2 were proved to be a pair of mixed sesquiterpenes,
and it was presented as a single peak in the HPLC using both the C18 or
silica gel columns. Mixed compounds 1 and 2 were separated using a
chiral AD-H column eluted with n-hexane-isopropanol to successfully
obtain pure compounds 1 and 2 (Fig. S29). The molecular formula of
is the average absolute deviation, computed as (1/n) Σni=1 |δscale.calc.
−
δ
exp.|.
2.8. Neuroprotective activity assay
The neuroprotective activities of all isolated compounds (1−11)
toward H2O2-induced SH-SY5Y cells damaged by the MTT assay were
screened following the reported procedures [16].
3. Results and discussion
Brevisterpene A (1) was isolated as yellow oil and found to have a
molecular formula of C20H34O7, based on the HRESIMS ion peak at m/z
409.2181 [M + Na]+ and its 13C NMR data, accounting for four indices
of hydrogen deficiency. The 1H NMR data (DMSO‑d6, Table 1) exhibited
resonances assignable to two olefinic protons (δH 5.99, d, J = 15.9 Hz,
H-7; δH 5.22, dd, J = 15.9, 7.9 Hz, H-8), one methoxyl (δH 3.18, s, CH3-
11), an oxymethine (δH 3.97, m, H-3), four methyl groups (δH 1.17, d,
J = 6.3 Hz, CH3-10; 1.02, s, CH3-12; 0.99, s, CH3-13; 1.66, s, CH3-14).
The presence of a sugar moiety was shown by the appearance of an
anomeric proton doublet at δH 4.28 (1H, d, J = 7.8 Hz, H-1′), which
correlated to its anomeric carbon signal at δC 100.7 (C-1′) in the HMQC
3

S. Han, et al.
Fitoterapia138(2019)104288
Table 2
brevisterpene B (2) was deduced as C20H34O7 from the HRESIMS and
13C NMR spectroscopy, the same as 1. Careful comparison of all the
NMR spectra involving the 1H, 13C, and 2D-NMR values of 1 and 2
revealed that these compounds have an identical planar structure. The
E configuration of the 7,8-diene was deduced from the coupling con-
stant (J7,8 = 15.9 Hz). According to the large coupling constant of the
anomeric proton at δH 4.28 (J = 7.8 Hz, H-1′), the orientation of glu-
cose moiety was deduced to be β-configuration. The glycosidation at C-
3 was determined by a long-range correlation between δH 4.28 (H-1′)
and δC 70.0 (C-3) in the HMBC spectra of 1 and 2. The D configuration
of sugar portion of 1 and 2 were confirmed by the analysis of LCNetII/
ADC HPLC-OR [9,10] (Fig. S30).
13C (100 MHz) NMR spectroscopic data for compounds 1, 2, 6, 7 and 10.
No.
1a
2a
6b
7b
10a
1
36.1
45.6
70.0
38.5
125.5
136.4
128.4
136.1
77.6
21.6
55.2
28.1
29.9
21.1
36.1
45.6
70.0
38.5
125.5
136.4
128.3
136.2
77.6
21.6
55.2
28.0
30.0
21.1
59.2
69.4
127.4
141.0
66.5
21.9
47.4
32.0
36.7
27.0
125.9
131.9
25.9
17.7
66.5
14.5
2
126.2
140.6
78.1
144.0
145.7
172.3
116.2
155.7
3
4
5
34.7
6
121.7
137.8
40.9
7
8
9
27.8
10
11
12
13
14
15
1′
125.4
132.1
25.9
However, the NOESY correlations could not provide sufficient in-
formation to elucidate the unambiguous relative configuration of C-3
and C-9. The absolute configuration of C-3 and C-9 asymmetric center
in the aglycone of 1 and 2 were established by time-dependent Density
Function Theory (TDDFT) calculations of the electronic circular di-
chroism (ECD) spectrum. Quantum chemical calculations were con-
ducted for the (3S, 9S) and (3R, 9R), (3R, 9S) and (3S, 9R) isomers of
the aglycone of 1 and 2 using TDDFT at the B3LYP/6-31G (d) level with
CPCM in methanol, and the experimental ECD spectrum of the aglycone
of 1 matched well with the calculated ECD spectrum of 1a (Fig. 3). And,
the experimental ECD spectrum of the aglycone of 2 matched well with
the calculated ECD spectrum of 2a (Fig. 3). Thus, the absolute config-
urations of the aglycone of 1 and 2 were determined as 3S, 9S and 3S,
9R, respectively. Compounds 1 and 2 were proved to be a pair of dia-
stereomer.
17.7
11.8
16.4
100.7
76.8
73.5
70.1
76.7
61.1
100.7
76.8
73.5
70.1
76.8
61.1
99.9
73.0
76.3
69.7
73.8
63.1
172.7
33.5
24.4
28.4
29.0
22.1
31.3
13.9
2′
3′
4′
5′
6′
1″
2″
3″
4″
5″-13″
14″
15″
16″
The molecular formula of brevisnoside A (6) was established as
C
15H26O2 according to the [M + Na]+ ion peak at m/z 261.1844 in the
a
b
Recorded in DMSO-d6.
(+) HRESIMS spectrum with an index of three hydrogen deficiency.
Recorded in methanol-d4.
Fig. 1. The structures of compounds 1–11.
4

S. Han, et al.
Fitoterapia138(2019)104288
Fig. 2. Key HMBC correlations of compounds 1, 2, 6, 7 and 10.
25
20
15
10
5
25
Calculated ECD 3S9S of 1a
Calculated ECD 3R9R of 1b
Experimental ECD of 1
Calculated ECD 3S9R of 2a
Calculated ECD 3R9S of 2b
Experimental ECD of 2
20
15
10
5
0
0
-5
-5
-10
-15
-20
-25
-10
-15
-20
-25
200
250
300
350
400
200
250
300
350
400
Wavelength / nm
Wavelength / nm
Fig. 3. Electronic circular dichroism (ECD) spectra of compounds 1 and 2 recorded in methanol in the 400–200 nm range. Calculated ECD spectra were shown in red
and blue compared to the experimental values shown in black. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
The 1H, 13C NMR data (Tables 1 and 2) and HSQC analysis of 6 in-
dicated the presence of six olefinic carbons (three are proton-bearing),
four methyls, an oxygenated methenyl, one oxygenated methylene, and
three methylenes. In the HMBC spectrum, the correlations between
CH3-12/CH3-13 to C-10 (δC 125.4)/C-11 (δC 132.1), CH3-15 to C-6 (δC
121.7)/C-7 (δC 137.8)/C-8 (δC 40.9), and CH3-14 to C-2 (δC 126.2)/C-3
(δC 140.6)/C-4 (δC 78.1) were observed. The HMBC correlations of H-1
to C-2 (δC 126.2)/C-3 (δC 140.6), H-2 to C-4 (δC 78.1), H-6 to C-5 (δC
34.7)/C-7 (δC 137.8)/C-8 (δC 40.9), H-9 to C-7 (δC 137.8), and H-10 to
C-9 (δC 27.8)/C-11 (δC 132.1) suggested the planer structure of 6
(Fig. 2). In addition, the NOESY correlations of H-14 with H-1 and H-5,
H-15 with H-5 and H-9 suggested that the double bonds at C-6/7 and C-
2/3 are both E configuration, respectively (Fig. 4).
geometric optimization of each one. The optimized conformers were
subjected to OR calculations in MeOH (CPCM) using the B3LYP func-
tional and the 6–311 + G(d) basis set for DFT. Final calculated ORs
were obtained as the result of the Boltzmann-weighted average (Table
S4). From these results, the negative calculated ORs ([α]D20 − 26.6) of
the (4S)-stereoisomer was a better fit to the experimental OR value
([α]D20 − 25.0, c 0.1, MeOH) than the positive one ([α]D20 + 26.6).
Therefore, the structure of 6 was identified as shown.
Brevisnoside B (7), having a molecular formula of C15H26O3 (m/z
277.1778 [M + Na]+), was also obtained as a yellow oil. Its 1H, 13C
NMR and HSQC spectra showed characteristic resonances of two double
bonds, three methyls, four methylenes (including one oxygenated), one
methine and two oxygenated methines groups (Tables 1 and 2). The key
HMBC correlations from CH3-13 to C-10 (δC 125.9), C-11 (δC 131.9),
and C-12 (δC 25.9); from CH3-12 to C-10 (δC 125.9), C-11 (δC 131.9),
and C-13 (δC 17.7); from CH3-15 to C-6 (δC 47.4), C-7 (δC 32.0), and C-8
(δC 36.7); from H-9 to C-7 (δC 32.0), C-8 (δC 36.7), C-10 (δC 125.9), and
The absolute configuration of 6 was established by comparison of
the experimental and calculated OR value [16]. A conformational
search using the MMFFs force field for the (4S)-stereoisomer and its
enantiomer led to the identification of 32 conformers, followed by
5

S. Han, et al.
Fitoterapia138(2019)104288
Fig. 4. Selected NOESY correlations of 6 and 7.
160.0
140.0
120.0
100.0
80.0
possible stereoisomers (1R*, 4S*, 6R*, 7R* or 1R*, 4S*, 6R*, 7S*) in-
dicated that the potential one is 1R*, 4S*, 6R*, 7S* (R2 = 0.9960,
AveDev = 2.2) better than 1R*, 4S*, 6R*, 7R* (R2 = 0.9953,
AveDev = 2.4) (Fig. 5, Table S5).
y = 0.9284x - 0.2654
R² = 0.996
The absolute configuration of 7 was established by comparison of
the experimental and calculated OR value []. Due to the above NMR
data, the configuration of 7 was identified as one of the two isomers
(1R, 4S, 6R, 7S or 1S, 4R, 6S, 7R). A conformational search using the
MMFFs force field for the (1R, 4S, 6R, 7S)-stereoisomer and its en-
antiomer led to the identification of 17 conformers, followed by geo-
metric optimization of each one. The optimized conformers were sub-
jected to OR calculations in MeOH (CPCM) using the B3LYP functional
and the 6–311++g(2d,p) basis set for DFT. Final calculated ORs were
obtained as the result of the Boltzmann-weighted average (Table S4).
From these results, the negative calculated ORs ([α]D20 − 13.0) of the
(1R, 4S, 6R, 7S)-stereoisomer was a better fit to the experimental OR
60.0
40.0
20.0
0.0
0.0
20.0
40.0
60.0
80.0
100.0 120.0 140.0 160.0
value ([α]D20 − 39.0,
c
0.1, MeOH) than the positive one
([α]D20 + 13.0). Therefore, the structure of 7 was identified as shown.
Erigeroside 6′-palmitate (10) was obtained as an amorphous, white
solid. It has the molecular formula C27H44O9 as deduced from HRESIMS
data with a negative ion peak at m/z 557.3005 [M + COOH]−, corre-
sponding to six indices of hydrogen deficiency. The 1H NMR data of 10
(Table 1) revealed three olefinic signals at δH 8.13 (1H, d, J = 5.9 Hz,
H-6), 8.12 (1H, s, H-2), and 6.40 (1H, d, J = 5.6 Hz, H-5). A char-
acteristic β-glucopyranosyl unit was evidenced at δH 4.85 (1H, d,
J = 7.0 Hz, H-1′), 3.20 (1H, m, H-2′), 3.21 (1H, m, H-3′), 3.11 (1H, m,
H-4′), 3.54 (1H, m, H-5′), 4.29 (1H, d, J = 11.8 Hz, H-6′a), and 4.04
(1H, dd, J = 11.9 Hz, 6.8 Hz, H-6′b), and the exchangeable protons of
δH 5.47, 5.22, and 5.29 were assigned in this hexose ring based on the
HSQC and HMBC correlations (Fig. 2). In addition, the characteristic
signals at δH 0.85 (3H, t, J = 6.9 Hz, H-16″) and 1.22 (24H, m, from H-
5″ to H-13″) indicated the presence of a long alkyl chain. The HRESIMS,
13C NMR data, and HSQC spectrum of 7 revealed the presence of 27
carbon signals, comprising four olefinic carbons (δC 144.0, C-2; 145.7,
C-3; 116.2, C-5; 155.7, C-6), two carbonyl carbons (δC 172.3 C-4; 172.7,
C-1″), a β-glucopyranosyl unit (δC 99.9, C-1′; 73.0, C-2′; 76.3, C-3′; 69.7,
C-4′; 73.8, C-5′; 63.1, C-6′), 14 nonoxygenated methylenes (from δC
22.1 to 33.5, C-2″-C-15″), and one terminal methyl (δC 13.9, C-16″)
(Table 2).
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15
Fig. 5. Experimental and calculated 13C chemical shifts of 7.
C-11
(δC
131.9)
confirmed
the
presence
of
a
(CH3)2C=CHCH2CH2(CH3)- group. The six-membered ring moiety was
further elaborated via the HMBC correlations from H-2 to C-4 (δC 66.5)
and C-6 (δC 47.4), and from H-6 to C-5 (δC 21.9) and C-8 (δC 36.7).
Additionally, a hydroxymethyl group was attached at C-3 according to
the HMBC correlations from H-12 to C-2 (δC 127.4) and C-3 (δC 141.0).
Thus, the planar structure of 7 was elucidated as a bisabolene-type
sesquiterpene.
The relative configuration of 7 was determined by the analysis of
NOESY spectrum in Fig. 4. The NOESY correlations of H-1/H-7, H-4/H-
7 and H-4/CH3-15 indicated their co-facial orientation, which were
arbitrarily assigned as β. However, the orientation of H-6 were assigned
as α. In addition, the relative configuration at C-7 was established by
calculated 13C NMR [17,18]. High-accuracy 13C NMR calculation of two
The structure of 10 was determined mainly by using 2D data
(Fig. 2). The HMBC spectrum of 10 showed correlations of H-2, H-5/C-3
(δC 145.7), C-6 (δC 155.7), H-6/C-2 (δC 144.0), C-4 (δC 172.3), C-5 (δC
116.2), indicating that the existence of a γ-pyrone ring. Correlations of
H-2″/C-1″ (δC 172.7), C-3″ (δC 24.4), C-4″ (δC 28.4), H-3″/C-1″ (δC
172.7), C-2″ (δC 33.5), C-4″ (δC 28.4), and CH3-16″/C-14″ (δC 22.1), C-
6

S. Han, et al.
Fitoterapia138(2019)104288
Fig. 6. The neuroprotective effects of compounds 1–11 against H2O2-induced injury of SH-SY5Y cells. MTT assay was used to determine the cell viability in the
presence or absence of the tested compounds at different concentrations (12.5, 25.0, 50.0 μM).
15″ (δC 31.3) revealed the partial structure of an alkyl hydrophobic
chain starting from the carbonyl (C-1″) group. A key correlation from
H-1′ to C-3 (δC 145.7) indicated that the glucosyl moiety was located to
C-3. The correlations from H-6′ to C-5′ (δC 73.8) and C-1″ (δC 172.7)
established a bridge between a hexose ring and an alkyl chain. Acid
moderate effects against H2O2-induced injury in SH-SY5Y cells at the
concentration of 12.5 and 25.0 μm.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgments
HPLC-OR analysis (Fig. S30D).-glucose, as determined by LCNetII/ADC
hydrolysis of 10 afforded
Known compounds 3β-glucopyranosyloxy-β-ionone (3) [19], blu-
menol C glucoside (4) [20], byzantionoside B (5) [20], 4β,10β-ar-
omadendranediol (8) [21], 4-epi-isodauc-6-ene-10β,14-diol (9) [22],
and erigeroside (11) [23] were identified by comparing their experi-
mental and reported spectroscopic data.
This study was financially supported by the National Nature Science
Foundation (81673324) of the People's Republic of China and the in-
novative experiment projects of undergraduate (201910163113) of
Shenyang Pharmaceutical University.
All compounds (1–11) were tested for neuroprotective effects
against H2O2-induced damage in human neuroblastoma SH-SY5Y cells
at the concentration of 12.5, 25.0 and 50.0 μm, with Trolox used as a
positive control, using a MTT assay (Fig. 6) [16]. As a result, com-
pounds 1 and 2 had moderate neuroprotective effects at concentrations
of 12.5 and 50.0 μM, which improved cell viabilities by > 10% com-
pared with the model group (Table S1). Compounds 3 and 4 had weak
neuroprotective effects. Furthermore, compounds 10 and 11 with
pyrone glycosides exhibited no neuroprotective effects at the tested
concentrations.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.fitote.2019.104288.
References
[1] J. Yang, G.H. Zhang, J. Zhang, H. Liu, W. Chen, X. Wang, Y.H. Li, Y. Dong,
S.H. Yang, Hybrid de novo genome assembly of the Chinese herbal fleabane Erigeron
breviscapus, GigaScience 6 (2017), https://doi.org/10.1093/gigascience/gix028.
[2] J.L. Gao, G. Chen, H.Q. He, C. Liu, X.J. Xiong, J. Li, J. Wang, Therapeutic effects of
breviscapine in cardiovascular diseases: a review, Front. Pharmacol. 8 (2017),
https://doi.org/10.3389/fphar.2017.00289.
4. Conclusion
In summary, five novel metabolites including two sesquiterpenoid
glycosides (1–2), two sesquiterpenes (6–7) and one pyranone glycoside
(10) were obtained from the whole plant of E. breviscapus. Compounds
1 and 2 were a pair of diastereomer separated by chiral-phase column
chromatography. All compounds (1–11) were evaluated for their neu-
roprotective activities in vitro, and compounds 1 and 2 showed
[3] J. Qu, Y.M. Wang, G.A. Luo, Z.P. Wu, Identification and determination of glucur-
onides and their aglycones in Erigeron breviscapus by liquid chromatography-tandem
mass spectrometry, J. Chromatogr. A 928 (2001) 155–162, https://doi.org/10.
1016/s0021-9673(01)01111-6.
[4] X. Li, K.X. Song, J.B. Yang, T.S. Yi, Isolation and characterization of 11 new mi-
crosatellite loci in Erigeron breviscapus (Asteraceae), an important Chinese tradi-
tional herb, Int. J. Mol. Sci. 12 (2011) 7265–7270, https://doi.org/10.3390/
7

S. Han, et al.
ijms12107265.
Fitoterapia138(2019)104288
using the coulomb-attenuating method (CAM-B3LYP), Chem. Phys. Lett. 393 (2004)
[5] H. Sun, A drug for treating cardio-cerebrovascular diseases-phenolic compounds of
Erigeron breviscapus, Prog. Chem. 21 (2009) 77–83.
51–57, https://doi.org/10.1016/j.cplett.2004.06.011.
[15] X.W. Hou, S. Han, Y.Y. Zhang, H.B. Su, P.Y. Gao, L.Z. Li, S.J. Song, Neogenkwanine I
from the flower buds of Daphne genkwa with its stereostructure confirmation using
quantum calculation profiles and antitumor evaluation, Nat. Prod. Res. (2018) 1–8,
https://doi.org/10.1080/14786419.2018.1536133.
[6] M. Karamese, H.S. Erol, M. Albayrak, G. Findik Guvendi, E. Aydin, S. Aksak
Karamese, Anti-oxidant and anti-inflammatory effects of apigenin in a rat model of
sepsis: an immunological, biochemical, and histopathological study,
Immunopharmacol. Immunotoxicol. 38 (2016) 228–237, https://doi.org/10.3109/
08923973.2016.1173058.
[16] Q. Ren, W.Y. Zhao, S.C. Shi, F.Y. Han, Y.Y. Zhang, Q.B. Liu, G.D. Yao, B. Lin,
X.X. Huang, S.J. Song, Guaiane-type sesquiterpenoids from the roots of Daphne
genkwa and evaluation of their neuroprotective effects, J. Nat. Prod. 82 (2019)
1510–1517, https://doi.org/10.1021/acs.jnatprod.8b01049.
[7] Q.C. Chu, T. Wu, L. Fu, J.N. Ye, Simultaneous determination of active ingredients in
Erigeron breviscapus (Vant.) Hand-Mazz. by capillary electrophoresis with electro-
chemical detection, J. Pharmaceut. Biomed. 37 (2005) 535–541, https://doi.org/
10.1016/j.jpba.2004.11.018.
[17] Y. Tang, Y.B. Xue, G. Du, J.P. Wang, J.J. Liu, B. Sun, X.N. Li, G.M. Yao, Z.W. Luo,
Y.H. Zhang, Structural revisions of a class of natural products: scaffolds of aglycon
analogues of fusicoccins and cotylenins isolated from fungi, Angew. Chem. 128
(2016) 4137–4141, https://doi.org/10.1002/ange.201600313.
[8] S.Y. Chen, M. Li, Y.T. Li, H.J. Hu, Y. Li, Y. Huang, Y. Zheng, Y. Lu, J. Hu, Y.Y. Lan,
A.M. Wang, Y.J. Li, Z.P. Gong, Y.L. Wang, A UPLC-ESI-MS/MS method for si-
multaneous quantitation of chlorogenic acid, scutellarin, and scutellarein in rat
plasma: application to a comparative pharmacokinetic study in sham-operated and
MCAO rats after oral administration of Erigeron breviscapus extract, Molecules 23
(2018) 1808, https://doi.org/10.3390/molecules23071808.
[18] C.Y. Zhang, L. Luo, J. Xia, Y.N. Song, L.J. Zhang, M. Zhang, K. Rahman, Y. Ye,
H. Zhang, J.Y. Zhu, Sesquiterpenes and lignans from the flower buds of Daphne
genkwa and their nitric oxide inhibitory activities, Nat. Prod. Res. (2017) 1–7,
https://doi.org/10.1080/14786419.2017.1389937.
[9] W.Y. Zhao, X.Y. Song, L. Zhao, C.X. Zou, W.Y. Zhou, B. Lin, G.D. Yao, X.X. Huang,
S.J. Song, Quassinoids from Picrasma quassioides and their neuroprotective effects,
J. Nat. Prod. 82 (2019) 714–723, https://doi.org/10.1021/acs.jnatprod.8b00470.
[10] C. Zhang, J. Li, J. Wang, X.L. Song, J. Zhang, S. Wu, J.J. Zhang, S. Wu, C.L. Hu,
Z.Y. Gong, L. Jia, Antihyperlipidaemic and hepatoprotective activities of acidic and
enzymatic hydrolysis exopolysaccharides from Pleurotus eryngii SI-04, BMC
Complem. Altern. M. 17 (2017) 403, https://doi.org/10.1186/s12906-017-1892-z.
[11] J. Bulusu, R. Surasani Sumathi, R.P. Thangavelu, Conformational preferences of
1,4,7-trithiacyclononane: a molecular mechanics and density functional theory
study, J. Mol. Model. 10 (2004) 55–59, https://doi.org/10.1007/s00894-003-
0166-5.
[19] A. Fiorentino, B. D’Abrosca, S. Pacifico, C. Mastellone, V. Piscopo, P. Monaco,
Spectroscopic identification and antioxidant activity of glucosylated carotenoid
metabolites from Cydonia vulgaris Fruits, J. Agric. Food Chem. 54 (2006)
9592–9597, https://doi.org/10.1021/jf062125e.
[20] K. M atsunami, H. Otsuka, Y. Takeda, Structural revisions of blumenol C glucoside
and byzantionoside B, Chem. Pharm. Bull. 58 (2010) 438–441, https://doi.org/10.
1248/cpb.58.438.
[21] I.C. Moreira, J.H.G. Lago, M.C.M. Young, N.F. Roque, Antifungal aromadendrane
sesquiterpenoids from the leaves of Xylopia brasiliensis, J. Braz. Chem. Soc. 14
(2003) 828–831, https://doi.org/10.1590/s0103-50532003000500020.
[22] X.M. Zhou, C.J. Zheng, Y.Q. Zhang, X.P. Zhang, X.P. Song, W. Xu, G.Y. Chen,
Guaiane-type sesquiterpenoids from Fissistigma oldhamii inhibit the proliferation of
synoviocytes, Planta Med. 83 (2016) 217–223, https://doi.org/10.1055/s-0042-
111440.
[12] H. Goto, E. Osawa, Corner flapping: a simple and fast algorithm for exhaustive
generation of ring conformations, J. Am. Chem. Soc. 111 (1989) 8950–8951,
https://doi.org/10.1021/ja0020 (6a046).
[13] T. Bruhn, A. Schaumlöffel, Y. Hemberger, G. Bringmann, SpecDis: quantifying the
comparison of calculated and experimental electronic circular dichroism spectra,
Chirality. 25 (2013) 243–249, https://doi.org/10.1002/chir.22138.
[14] T. Yanai, D.P. Tew, N.C. Handy, A new hybrid exchange-correlation functional
[23] M. Tafazzoli, M. Ghiasi, M. Moridi, Dynamic stereochemistry of erigeroside by
measurement of 1H-1H and 13C-1H coupling constants, Spectrochim. Acta A 70
(2008) 350–357, https://doi.org/10.1016/j.saa.2007.10.033.
8