Chiral resolution and neuroprotective activities of enantiomeric 8-O-4′ neolignans from the fruits of Crataegus pinnatifida Bge

Article abstract of DOI:10.1016/j.fitote.2019.05.003

Five pairs of enantiomeric 8-O-4′ neolignans (1a/1b-5a/5b), including seven new compounds (1a/1b, 2a, 3a, 4a/4b and 5b), were obtained from the fruits of Crataegus pinnatifida Bge. Their structures were determined by comprehensive spectroscopic analyses. The absolute configurations of the enantiomers were determined by comparison of the experimental ECD with the calculated data. Additionally, all the enantiomeric neolignans were evaluated for their neuroprotective activity against H₂O₂-induced cell injury in human neuroblastoma SH-SY5Y cells, and most of them showed potent selective neuroprotective activity. Especially, 3b (72.98%)showed the best protective effect, better than 3a (63.49%)and trolox (62.86%)at 25 μM.

Full text of DOI:10.1016/j.fitote.2019.05.003

Fitoterapia136(2019)104164
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Chiral resolution and neuroprotective activities of enantiomeric 8-O-4′
neolignans from the fruits of Crataegus pinnatifida Bge
Peng Zhaoa, Hao Zhanga, Feng-Ying Hana, Rui Guoa, Shun-Wang Huangb, Bin Linc,
⁎
Xiao-Xiao Huanga,d, , Shao-Jiang Songa
a School of Traditional Chinese Materia Medica, Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical
University, Shenyang 110016, People's Republic of China
b Hefei Wisdom Pharma-Tech Co., Ltd, Hefei 230088, People's Republic of China
c School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, People's Republic of China
d Chinese People's Liberation Army 210 Hospital, Dalian 116021, 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:
Five pairs of enantiomeric 8-O-4′ neolignans (1a/1b-5a/5b), including seven new compounds (1a/1b, 2a, 3a,
4a/4b and 5b), were obtained from the fruits of Crataegus pinnatifida Bge. Their structures were determined by
comprehensive spectroscopic analyses. The absolute configurations of the enantiomers were determined by
comparison of the experimental ECD with the calculated data. Additionally, all the enantiomeric neolignans
were evaluated for their neuroprotective activity against H2O2-induced cell injury in human neuroblastoma SH-
SY5Y cells, and most of them showed potent selective neuroprotective activity. Especially, 3b (72.98%) showed
the best protective effect, better than 3a (63.49%) and trolox (62.86%) at 25 μM.
Crataegus pinnatifida
8-O-4′ neolignans
Chiral resolution
ECD calculation
Neuroprotective activities
1. Introduction
metabolites from the fruits of C. pinnatifida, five pairs of 8-O-4′ types of
neolignans (1a/1b-5a/5b) including seven new compounds were iso-
There has been an increase in the global prevalence of neurode-
generative diseases such as Parkinson's, Alzheimer's (AD), and stroke.
No effective treatment has yet been developed for these neurodegen-
erative diseases [1,2]. Oxidative stress is proposed to be one of the key
factors closely associated with the etiology of these neurodegenerative
diseases [3]. With the increased oxidative damage and the weakened
activities of antioxidant defense system in the brain of neurodegen-
erative diseases, pharmacological approach to attenuate oxidative
stress may be one of the promising therapeutic strategies [4].
Crataegus pinnatifida, belonging to the family of Rosaceae, is planted
as a fruit crop widely distributed in China, Korea and Siberia [5,6]. It
has attracted increasing attention in the application in foodstuff and
leechdom because of its amazing health benefits [7–9]. Previous phy-
tochemical investigations on C. pinnatifida led to the isolation and
identification of a variety of secondary metabolites, which mainly in-
clude flavonoids, triterpenoids, phenylpropanoids and organic acids
[10,11].
lated [15]. The enantioseparations of these compounds were achieved
by chiral chromatographic column, and their structures including the
absolute configurations were elucidated by extensive spectroscopic
analyses and ECD calculations. In addition, the neuroprotective activ-
ities of these enantiomers against H2O2-induced oxidative injury in
human neuroblastoma SH-SY5Y cells were investigated.
2. Materials and methods
2.1. General experimental procedures
The UV spectra were measured on a Shimadzu UV-1700 spectro-
meter (Shimadzu, Japan). Optical rotations were recorded by using a
JASCO DIP-370 digital polarimeter. Electronic circular dichroism (ECD)
experiments were performed on a MOS 450 spectrometer (Bio-Logic
Science Instruments, Seyssinet-Pariset, France). The 1H NMR, 13C NMR,
HMBC, HSQC, 1H-1H COSY spectra were obtained on Bruker AVIII-600
(Bruker Corporation, Bremen, Germany) spectrometers with TMS as an
internal standard. HRESIMS data were measured on an Agilent G6520
Q-TOF spectrometer (Agilent Technologies Inc., Santa Clara, USA).
Preparative high performance liquid chromatography (HPLC) was
Earlier investigations on the biological activities of C. pinnatifida
showed that they displayed anti-inflammatory, anti-oxidative, anti-
neurodegenerative and anti-cancer activities [12–14]. In the course of
our continuing research for biologically active and structurally diverse
⁎ Corresponding author.
E-mail address: xiaoxiao270@163.com (X.-X. Huang).
https://doi.org/10.1016/j.fitote.2019.05.003
Received 8 April 2019; Received in revised form 30 April 2019; Accepted 5 May 2019
Availableonline06May2019
0367-326X/©2019ElsevierB.V.Allrightsreserved.

P. Zhao, et al.
Fitoterapia136(2019)104164
performed on a Shimadzu liquid chromatograph with SPD-20A UV–vis
detector and LC-6AD high-pressure pump using YMC reversed-phase
column (20 × 250 mm, 5 μm). Chiral HPLC separation was performed
by Daicel Chiralpak AD-H chiral column (4.6 × 250 mm, 5 μm, Daicel
Polymer Ltd., Tokyo, Japan). The chromatographic silica gel (200–300
meshes) was purchased from Qingdao Marine Chemical Factory. ODS
(50 μm) was produced by YMC Co., Ltd. All solvents for extraction and
chromatography were commercially purchased and routinely distilled
prior to use.
1738, 1591, 1512, 1462, 1384, 1273, 1154, 1122, 1033, 874, 820,
698 cm−1 1H and 13C NMR data see Tables 1 and 2; HRESIMS: m/z
;
431.1659 [M + Na]+ (calcd for C21H28NaO8, 431.1676).
“(+)-7S,8R-4,7,9,9′-tetrahydroxy-3,3′,5′-trimethoxy-8-O-4′-neo-
lignan (2a): [α]D20+20.5 (c 0.10, MeOH); ECD (MeOH) λmax (Δε) 228
(−0.36), 245 (+2.38), 279 (+0.76) nm.
(−)-7R,8S-4,7,9,9′-tetrahydroxy-3,3′,5′-trimethoxy-8-O-4′-neo-
lignan (2b): [α]D20–22.0 (c 0.10, MeOH); ECD (MeOH) λmax (Δε) 227
(+0.72), 243 (−1.89), 281(−0.69) nm.”
Erythro
lignan (3): Colorless gum, [α]D20+1.2 (c 0.10, MeOH); UV (MeOH)
max (log ε) 218 (2.58), 258 (0.23) nm; IR (KBr) νmax 3421, 2922, 2851,
1614, 1518, 1462, 1383, 1278, 1212, 1141, 1041, 954, 908, 884,
4,7,9,9′-tetrahydroxy-3,5,3′,5′-tetramethoxy-8-O-4′-neo-
2.2. Plant material
λ
The fruits of Crataegus pinnatifida were collected in August 2017
from Hebei province, China. Professor Jin-Cai Lu identified this plant
material (School of Traditional Chinese Materia Medica, Shenyang
Pharmaceutical University). A voucher specimen (No. 20180604) has
been deposited in the Herbarium of Shenyang Pharmaceutical
University.
706 cm−1 1H and 13C NMR data see Tables 1 and 2; HRESIMS: m/z
;
461.1785 [M + Na]+ (calcd for C22H30NaO9, 461.1782).
(+)-7R,8S-4,7,9,9′-tetrahydroxy-3,5,3′,5′-tetramethoxy-8-O-4′-neo-
lignan (3a): [α]D20+32.0 (c 0.10, MeOH); ECD (MeOH) λmax (Δε) 238
(+0.25), 253 (+2.06), 276 (+2.42) nm.
(−)-7S,8R-4,7,9,9′-tetrahydroxy-3,5,3′,5′-tetramethoxy-8-O-4′-neo-
lignan (3b): [α]D20–28.2 (c 0.10, MeOH); ECD (MeOH) λmax (Δε) 238
(−0.01), 255 (−2.02), 276 (−2.99) nm.
2.3. Extraction and isolation
The air-dried fruits of C. pinnatifida (50 kg) were extracted with 70%
EtOH under reflux three times. The EtOH extract (3600 g) was parti-
tioned sequentially with ethyl acetate and n-BuOH. The EtOAc extract
(500 g) was subjected to a silica gel column and eluted with CH2Cl2-
MeOH (100:1–1:1, v/v) of increasing polarity to produce 4 fractions
(Fr.A-D). Fr·B was subjected to column chromatography on HP-20
macroporous resin with H2O, 30%, 60% and 90% EtOH to provide three
fractions (Fr.A1, Fr.A2 and Fr.A3). Fr.A1 (30 g), Fr.A2 (90 g) and Fr. A3
(40 g) were separated by ODS column chromatography, eluted with a
gradient of MeOH-H2O from 20:80 to 90:10, respectively, and then
redistributed in four fractions (Fr. 1–4) on the basis of silica gel TLC
analysis. Fr. 4 (27 g) was further purified by a silica gel CC using pet-
roleum ether/EtOAc (v/v, 100:1 to 0:1) and CH2Cl2/MeOH (v/v, 30: 1
to 1:1) to yield Fr.4.1–4.9. Fr. 4.6.1–4.6.6 was obtained from Fr.4.6 by
Threo guaiacylglycerol-8-acetovanillone ether (4): Colorless gum,
[α]D20+0.3 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 227 (1.00), 247
(0.22), 277 (0.62) nm; IR (KBr) νmax 3421, 2922, 2851, 1658, 1592,
1518, 1463, 1383, 1272, 1198, 1111, 1031, 920, 869, 818, 722 cm−1
;
1H and 13C NMR data see Tables 1 and 2; HRESIMS: m/z 385.1245
[M + Na]+ (calcd for C19H22O7Na, 385.1258).
(+)-7S,8S-guaiacylglycerol-8-acetovanillone
ether
(4a):
[α]D20+17.2 (c 0.10, MeOH); ECD (MeOH) λmax (Δε) 233 (−0.59), 274
(−0.35), 300 (−0.29) nm.
(−)-7R,8R-guaiacylglycerol-8-acetovanillone
ether
(4b):
[α]D20–19.0 (c 0.10, MeOH); ECD (MeOH) λmax (Δε) 232 (+0.51), 266
(+0.14), 298 (+0.01) nm.
Threo guaiacylglycerol 8-vanillin ether (5): Colorless gum,
[α]D20+0.4 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 230 (1.24), 249
(0.18), 278 (0.83) nm; IR (KBr) νmax 3417, 2924, 2851, 1676, 1588,
preparative HPLC (MeOH/H2O, 70:30). Compound
1 (5.0 mg, tR
46.8 min) was obtained from fraction 4.6.1 by semipreparative HPLC
eluted with CH3CN-H2O (34:66). Fraction 4.6.2 was purified in the
same way as Fr. 4.6.3 to obtain 2 (50.8 mg, tR 45.3 min) and 3 (8.6 mg,
tR 32.1 min). In a similar manner, compound 4 (4.0 mg, tR 52.0 min)
and 5 (4.7 mg, tR 49.7 min) was isolated from Fr. 4.6.5 by RP-HPLC
(MeOH/ H2O, 42:58). Compounds 1a (1.1 mg) and 1b (1.0 mg) were
obtained on a Daicel Chiralpak AD-H chiral column, with n-hexane/
isopropanol (2:1, v/v) at the flow rate of 0.5 mL/min. Compounds 2a
(2.8 mg) and 2b (3.1 mg), 3a (2.8 mg) and 3b (2.1 mg) were obtained
by using a Daicel Chiralpak AD-H chiral column and an elution mixture
of n-hexane/isopropanol (1:1, v/v) at the flow rate of 0.5 mL/min.
Compounds 4a (1.8 mg) and 4b (1.6 mg), 5a (1.2 mg) and 5b (1.5 mg)
were obtained by using a Daicel Chiralpak AD-H chiral column with an
elution mixture of n-hexane/isopropanol (1:1, v/v) at the flow rate of
0.5 mL/min.
1506, 1464, 1338, 1268, 1158, 1131, 1028, 862, 814, 782, 731 cm−1
;
1H and 13C NMR data see Tables 1 and 2; HRESIMS: m/z 371.1112
[M + Na]+ (calcd for C18H20O7Na, 371.1101).
(+)-7S,8S-guaiacylglycerol 8-vanillin ether (5a): [α]D20+26.0 (c
0.10, MeOH); ECD (MeOH) λmax (Δε) 274 (−0.36), 298 (−0.04), 321
(−0.19) nm.
(−)-7R,8R-guaiacylglycerol 8-vanillin ether (5b): [α]D20–24.5 (c
0.10, MeOH); ECD (MeOH) λmax (Δε) 276 (+0.49), 301 (+0.29), 319
(+0.29) nm.
2.4. Neuroprotective activities assay
The ability of compounds 1–5 to protect human neuroblastoma SH-
SY5Y cells against oxidative stress induced by H2O2 was evaluated by
the MTT assay. And trolox was used as the positive control. The human
neuroblastoma SH-SY5Y cells (1.2 × 104 cells/well) were grown in 96-
well plates for 12 h. At the end of incubation period, the SH-SY5Y cells
were incubated with or without tested compounds (12.5, 25 and 50 μM)
for 1 h. In order to measure their cytoprotective effect, 200 μM newly-
prepared H2O2 was added to the cells for 3 h. After then, the drug so-
lutions in the wells were discarded and 20 μL MTT dissolved by phos-
phate-buffered saline medium was added in each well. The plates were
gently shaken and incubated for another 4 h at 37 °C in 5% CO2 at-
mosphere. Then, the medium was cleared and 150 μL of DMSO was
added to cells to dissolve the formazan. The results were obtained in
absorbance at 490 nm using a Thermo microplate reader and the cell
viability of each group was expressed as a percentage relative to the
value of the control group (100%).
Threo 4,7,9,9′-tetrahydroxy-3,3′,5′-trimethoxy-8-O-4′-neolignan (1):
Colorless gum, [α]D20–1.5 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 205
(4.00), 251 (0.18), 278 (0.31) nm; IR (KBr) νmax 3440, 2930, 2850,
1739, 1606, 1518, 1463, 1383, 1275, 1210, 1140, 852, 770 cm−1 1H
;
and 13C NMR data see Tables 1 and 2; HRESIMS: m/z 431.1674
[M + Na]+ (calcd for C21H28NaO8, 431.1676).
(+)-7S,8S-4,7,9,9′-tetrahydroxy-3,3′,5′-trimethoxy-8-O-4′-neo-
lignan (1a): [α]D20+18.0 (c 0.10, MeOH); ECD (MeOH) λmax (Δε) 201
(+1.26), 211 (−1.02), 239 (−1.12) nm.
(−)-7R,8R-4,7,9,9′-tetrahydroxy-3,3′,5′-trimethoxy-8-O-4′-neo-
lignan (1b): [α]D20–20.0 (c 0.10, MeOH); ECD (MeOH) λmax (Δε) 207
(−1.73), 218 (+1.19), 237 (+1.40) nm.
Erythro 4,7,9,9′-tetrahydroxy-3,3′,5′-trimethoxy-8-O-4′-neolignan
(2): Colorless gum, [α]D20–0.8 (c 0.10, MeOH); UV (MeOH) λmax (log ε)
204 (1.80), 254 (0.13), 279 (0.40) nm; IR (KBr) νmax 3422, 2922, 2852,
2

P. Zhao, et al.
Fitoterapia136(2019)104164
Table 1
1H NMR spectroscopic data for compounds 1a/1b–5a/5b in CDCl3.
Position
1a/1b
2a/2b
3a/3b
4a/4b
5a/5b
1
2
3
4
5
6
7
8
9
–
–
–
–
–
6.97, d (1.7)
–
–
6.88, d (7.9)
6.96, dd (7.9, 1.7)
5.01, d (8.8)
3.86, overlapped
3.56, m
3.31, m
–
6.95, d (1.9)
–
–
6.84, d (8.2)
6.74, dd (8.2, 1.9)
4.99, d (2.6)
4.10, m
3.47, m
3.23, m
–
6.58, s
–
–
–
6.58, s
4.99, d (3.3)
4.10, dt (6.7, 3.3)
3.45, m
6.97, d (1.6)
–
–
6.90, overlapped
6.91, overlapped
4.97, d (7.7)
4.22, ddd (7.7, 4.6, 3.4)
3.67, dd (12.4, 3.4)
3.60, dd (12.4, 4.6)
–
6.96, d (1.5)
–
–
6.90, overlapped
6.91, overlapped
4.97, d (7.6)
4.29, ddd (7.6, 4.7, 3.5)
3.69, dd (12.4, 3.5)
3.62, dd (12.4, 4.7)
–
3.16, m
–
1′
2′
3′
6.47, s
–
6.47, s
–
6.49, s
–
7.58, d (2.0)
–
7.46, d (1.9)
–
4′
–
–
–
–
–
5′
–
–
–
7.15, d (8.3)
7.23, d (8.1)
6′
7′
8′
9′
6.47, s
2.68, m
1.88, m
3.70, t (6.3)
3.89, s
–
6.47, s
2.68, m
1.90, m
3.69, t (6.4)
3.87, s
–
6.49, s
7.54, d (8.3, 2.0)
–
2.58, s
–
3.89, s
–
3.97, s
–
7.44, d (8.1, 1.9)
9.88, s
–
–
3.88, s
–
3.96, s
–
2.70, m
1.91, dt (13.9, 6.4)
3.71, t (6.4)
3.87, s
3.87, s
3.87, s
3-OCH3
5-OCH3
3′-OCH3
5′-OCH3
3.89, s
3.89, s
3.85, s
3.85, s
3.87, s
Table 2
was used to determine significant differences via ANOVA followed by
13C NMR data for compounds 1–5 in CDCl3.
Student's t-test. The data were expressed as means
P < .05 were considered as statistically significant.
SD, and values
Position
1
2
3
4
5
1
2
3
4
5
6
7
8
9
1′
2′
3′
4′
5′
6′
7′
132.3
110.0
146.6
145.5
114.4
120.6
74.3
133.0
108.6
146.8
144.9
114.3
118.9
72.5
130.6
102.6
147.2
134.0
147.2
102.6
72.7
131.0
109.2
146.7
145.7
114.4
120.1
74.2
88.8
61.6
132.8
111.1
150.9
152.2
118.6
123.2
196.7
26.5
–
131.3
109.3
146.9
145.9
114.6
120.2
74.2
3. Results and discussion
Compound 1 (1a/1b) was obtained as a colorless gum. The mole-
cular formula of 1 was established as C21H28O8 based on the HRESIMS
quasi molecular ion peak [M + Na]+ at m/z 431.1674 (calcd for
C
21H28NaO8, 431.1676) and 13C NMR data. The IR spectra exhibited
89.2
60.8
87.1
60.7
87.2
60.8
88.3
61.6
the absorptions of hydroxy group (3440 cm−1) and aromatic func-
tionality (1606, 1518 cm−1). Its 1H NMR spectrum (Table 1) displayed
a 1,3,4-trisubstituted aromatic moieties at δH 6.97 (H, d, J = 1.7 Hz, H-
2), 6.88 (H, d, J = 7.9 Hz, H-5) and 6.96 (H, d, J = 7.9, 1.7 Hz, H-6), a
1,3,4,5-tetrasubstituted benzene ring at δH 6.47 (2H, s, H-2′,6′), two
oxygenated methines δH 5.01 (1H, d, J = 8.8 Hz, H-7), 3.86 (1H,
overlapped, H-8), four methylenes including two oxidated methylenes
138.8
105.6
153.0
133.6
153.0
105.6
32.7
138.8
105.4
153.2
133.0
153.2
105.4
32.7
138.9
105.4
153.3
133.1
153.3
105.4
32.8
132.1
110.3
151.4
153.6
118.4
126.5
191.0
–
8′
9′
34.4
62.3
34.3
62.1
34.4
62.3
δ
H 3.56 (1H, m, H-9a), 3.31 (1H, m, H-9b) and 3.70 (2H, t, J = 6.3 Hz,
H-9′), and another two methylene singlets at δH 2.68 (2H, m, H-7′) and
1.88 (2H, m, H-8′), and three methoxy groups at δH 3.89 (9H, s). The
13C NMR spectrum and HSQC spectra revealed 21 carbons, corre-
sponding to six aliphatic carbons, three methoxy groups and 12 aro-
matic carbons. In the HMBC spectrum, the correlations from H-7 to C-9,
C-1 and C-2/6, H-8 to C-1 and C-4′ and the characteristic signal at δH
5.01 (1H, d, J = 8.8 Hz) confirmed that the basic skeleton of compound
1 was 8-O-4′-type neolignan (Fig. 2). The three methoxy groups were
assigned at C-3, C-3′ and C-5′, respectively, on the basis of the HMBC
correlations of δH 3.89 (9H, s) to δC 146.6 (C-3) and 153.0 (C-3′/5′), and
δH 2.68 (2H, m) to δC 138.8 (C-1′) and 105.6 (C-2′/6′). The relative
stereochemistry of H-7 and H-8 was established as a threo by comparing
the large J7,8 coupling constant (8.8 Hz in CDCl3) with J7,8 coupling
constant (2.6 Hz in CDCl3) of compound 2 [21,22].
The absence of Cotton effect in the ECD spectrum and specific ro-
tation indicated that compound 1 was likely to be a racemic mixture.
The subsequent chiral resolution led to the isolation of the enantiomers
1a and 1b in a ratio of 1:1 by using Chiralpak AD-H column (see
Supporting Information), which showed anticipated mirror image-like
ECD curves and specific rotations (1a: [α]D20+18.0; 1b: [α]D20−20.0).
In order to determine the absolute configurations of 1a/1b, the ECD
spectra were measured and compared with the calculated ECD spectra
(Fig. 3). The experimental ECD spectrum of 1b was consistent with the
3-OCH3
5-OCH3
3′-OCH3
5′-OCH3
56.1
–
56.3
56.3
56.1
–
56.2
56.2
56.5
56.5
56.3
56.3
56.0
–
56.1
–
56.1
–
56.2
–
2.5. ECD calculations
Conformational analysis of all the possible conformers of com-
pounds 1–5 were performed by using the MMFF94 force field in
CONFLEX software [16,17]. All the conformers obtained were screened
based on the energy of optimized structures at the B3LYP/6-31G(d)
level in an energy window of 3 kcal/mol in the Gaussian 09 software
[18]. Then, the ECD data of all the selected conformers were calculated
with the time-dependent density functional theory (TDDFT) method at
the B3LYP/6-311++G(2d, p) levels with the CPCM model in methanol
solution [19]. Finally, the Boltzmann-averaged ECD curve was gener-
ated using SpecDis 1.51 [20].
2.6. Statistical analysis
All results and data were confirmed in at least three separate ex-
periments. SPSS (Statistical Package for the Social Sciences) software
3

P. Zhao, et al.
Fitoterapia136(2019)104164
Fig. 1. The structures of compounds 1a/1b-5a/5b.
Fig. 2. Key HMBC correlations of compounds 1, 3–5.
4

P. Zhao, et al.
Fitoterapia136(2019)104164
Fig. 3. Calculated and experimental ECD spectra of 1a/1b-5a/5b.
calculated ECD spectrum of (7R,8R)-1. Thus, 1a and 1b were assigned
as 7S,8S and 7R,8R-4,7,9,9′-tetrahydroxy-3,3′,5′-trimethoxy-8-O-4′-
neolignan, respectively (Fig. 1).
Compound 2 (2a/2b), obtained as a colorless gum, possesses the
same molecular formula (C21H28O8) as compound 1, which was de-
termined from 13C NMR data and an HRESIMS [M + Na]+ molecular
ion peak at m/z 431.1659 (calcd for C21H28NaO8, 431.1676). A com-
parison of the NMR spectroscopic data of 2 with those of 1 (Tables 1
and 2) revealed that the only difference was the small change in cou-
pling constant between H-7 and H-8 [δH 5.01 (1H, d, J = 8.8 Hz, H-7) in
1; δH 4.99 (1H, d, J = 2.6 Hz, H-7) in 2]. These findings, the small
coupling constant between H-7 and H-8 (J = 2.6 Hz), implied that 2
was a stereoisomer of 1 with an erythro configuration [23,24]. Re-
garding their absolute configurations, compound 2 showed weak
Cotton effects in its ECD spectrum and unmeasurable optical rotations,
and its racemic nature was confirmed by Chiralpak AD-H column in the
5

P. Zhao, et al.
Fitoterapia136(2019)104164
(J7,8 = 7.7 Hz). Subsequently, 4a/4b were also isolated as optically
pure compounds and showed two peaks in the chiral HPLC using a
Chiralpak AD-H column. The absolute configuration of enantiomers 4b
was identified as 7R, 8R by comparing their calculated ECD and ex-
perimental ECD (Fig. 3). Therefore, compounds 4a and 4b were es-
tablished as 7S,8S-guaiacylglycerol-8-acetovanillone ether and 7R,8R-
guaiacylglycerol-8-acetovanillone ether, respectively.
Compound 5 (5a/5b) possesses the molecular formula C18H20O7, as
determined from a quasi-molecular ion peak in the positive HRESIMS at
m/z 371.1112 [M + Na]+ (calcd for C18H20NaO7, 371.1101) combined
with its 13C NMR data. A comparison of the 1H and 13C NMR spectro-
scopic properties showed that 5 was quite similar to that of 4 and the
only difference was the absence of a methyl group in 5 (Tables 1 and 2).
The planar structure of 5 was further confirmed by HSQC, HMBC and
COSY (Fig. 2). Besides, the relative configuration of H-7 and H-8 of 5
was identified as a threo configuration based on the larger coupling
constant (J7,8 = 7.6 Hz). Considering the partial racemic nature of 4,
compound 5 was separated by Chiralpak AD-H column to afford en-
antiomers (+)-5a and (−)-5b, showing typical antipodal ECD curves.
To determine their absolute configurations, the ECD spectra of 5a and
5b were compared with the calculated ECD spectra of the enantiomers.
The experimental ECD spectrum of 5b was consistent with the calcu-
lated ECD spectrum of 7R,8R-5 (Fig. 3). Thus, 5a and 5b were eluci-
dated as 7S,8S-guaiacylglycerol 8-vanillin ether and 7R,8R-guaia-
cylglycerol 8-vanillin ether, respectively.
Fig. 4. Neuroprotective effects of compounds 1a/1b-5a/5b against H2O2-in-
duced cell growth inhibition of SH-SY5Y cells. In the presence or absence of the
tested compounds at different concentrations (12.5, 25, and 50 μM), the MTT
assay was used to examine the cell viability after H2O2 (200 μM) treatment for
4 h. ###p < .001 vs. control group. ⁎⁎p < .01, ⁎⁎⁎p < .001 vs. the H2O2
treated group.
same manner as that of 1, affording compounds 2a and 2b. By com-
paring their calculated ECD and experimental ECD, the absolute con-
figuration of 2a and 2b were determined to be 7R,8S and 7S,8R, re-
spectively (Fig. 3).
All isolated compounds (1a/1b-5a/5b) were tested for their neu-
roprotective activities against human neuroblastoma SH-SY5Y cell da-
mage induced by H2O2 using MTT assay. As depicted in Fig. 4, most of
the compounds showed significant neuroprotective activity, which
were more potent than the positive control. Among all tested com-
pounds, the most actives were compound 2 and 3. They present four
methoxy groups at C-3/5/3′/5′ position, suggesting that methoxy
groups are associated with improvements on the neuroprotective ac-
tivity. In the concentration of 25 μM, compounds 3b showed more re-
markable neuroprotective activity than 3a, the cell viability of them
were 63.94% and 72.98%, respectively. These results suggested that the
absolute configuration is also crucial to obtain compounds possessing
good neuroprotective activities.
Compound 3 (3a/3b) was obtained as a colorless gum. The
HRESIMS ion at m/z 461.1785 [M + Na]+ (calcd for C22H30NaO9,
461.1782) and the 13C NMR data established its molecular formula as
C
22H30O9. The UV, IR and NMR spectroscopic data of 3 were similar to
those of 1 and 2, except for the typical NMR signals of two symmetric
1,3,4,5-tetrasubstituted phenyl moieties in 3 rather than the 1,3,4-tri-
substituted phenyl groups in 1 and 2 (Tables 1 and 2). These findings,
along with the presence of an additional methoxy group, suggested that
3 was a 5′-methoxy derivative of 1, which was further supported by the
HMBC correlations from H-2/H-6 to C-4 and C-7 (Fig. 2). Subsequently,
the chiral HPLC purification of 3 afforded the enantiomers (+)-3a and
(−)-3b in a ratio of 1:1 with Chiralpak AD-H column. Because of the
smaller coupling constant (J7,8 = 3.3 Hz) of H-7 and H-8, the relative
configuration of H-7 and H-8 of 3 was identified as a erythro config-
uration. The absolute configurations of 3a/3b were also determined by
comparison of experimental ECD with corresponding ECD calculations.
The ECD spectra of 3a and 3b were measured in MeOH and compared
with the calculated ECD spectra of the enantiomers (Fig. 3). Thus, their
absolute configurations were assigned as 7R, 8S and 7S, 8R. Finally, 3a
and 3b were established as 7R,8S and 7S,8R-4,7,9,9′-tetrahydroxy-
3,5,3′,5′-tetramethoxy-8-O-4′-neolignan, respectively.
Compound 4 was obtained as a colorless gum. Its molecular formula
was determined to be C19H22O7, as elucidated by the HRESIMS m/z
385.1245 [M + Na]+ (calcd for C19H22NaO7, 385.1258) and 13C NMR
data. Its 1H NMR spectrum (Table 1) displayed two sets of aromatic
protons [δH 6.97 (1H, d, J = 1.6 Hz, H-2), 6.90 (1H, overlapped, H-5),
6.91 (1H, overlapped, H-6)] and [δH 7.58 (1H, d, J = 2.0 Hz, H-2′), 7.15
(1H, d, J = 8.3 Hz, H-5′), 7.54 (1H, dd, J = 8.3, 2.0 Hz, H-6′)] arising
from two 1,3,4-trisubstituted aromatic rings system, respectively, a
methyl group [δH 2.58, (3H, s)], two methoxy groups [δH 3.89 (3H, s),
3.97 (3H, s)]. There were also typical proton signals at δH 4.97 (1H, d,
J = 7.7 Hz, H-7), 4.22 (1H, ddd, J = 7.7, 4.6, 3.4 Hz, H-8), 3.67 (1H,
dd, J = 12.4, 3.4 Hz, H-9a), 3.60 (1H, dd, J = 12.4, 4.6 Hz, H-9a) that
accounted for the CH(O)-CH(O)-CH2(O) unit. The 13C NMR and HSQC
spectra revealed 19 carbons, corresponding to a methyl, a methylene,
two methines, a carbonyl and two benzene rings (Table 2). The HMBC
correlations, H-7 to C-8, C-9, C-1 and C-2/6, H-8 to C-1 and C-9, H-7′ to
C-2′and C-6′, confirmed that 4 was an 8-O-4′-neolignan with the ab-
sence of C-9′ carbon (Fig. 2). The relative configuration of H-7 and H-8
was identified as a threo configuration due to its large coupling constant
4. Conclusion
In the present study, five pairs of 8-O-4′ neolignans (1a/1b-5a/5b)
including seven new compounds (1a/1b, 2a, 3a, 4a/4b and 5b) were
isolated from the fruits of C. pinnatifida. The enantioseparations of these
compounds were achieved using Chiralpak AD-H column and the ab-
solute configurations of the enantiomers were firmly determined by
comparing experimental ECD with the calculated data. In addition, the
neuroprotective activities of these enantiomers against H2O2-induced
oxidative injury in human neuroblastoma SH-SY5Y cells were in-
vestigated. The results indicated that 3b (72.98%) exhibited best neu-
roprotective effects against SH-SY5Y cell at 25 μM, suggesting that the
methoxy groups and its absolute configuration may be associated with
improvements on the neuroprotective activity.
Acknowledgments
This work was supported by Career Development Support Plan for
Young and Middle-aged Teachers in Shenyang Pharmaceutical
University (ZQN2018006) and the Project of Innovation Team
Foundation (LT2015027).
Conflict of interest
The authors declare that there is no conflict of interest.
6

P. Zhao, et al.
Fitoterapia136(2019)104164
Appendix A. Supplementary data
physiological activities of Chinese hawthorn extracts, J. Agr. Chem. Soc. 70 (2006)
2948–2956.
[14] C. Li, Antioxidant activity of hawthorn gruit in vitro, J. Appl. Biol. Chem. 53 (2010)
8–12.
[15] X.X. Huang, Q. Ren, X.Y. Song, L. Zhou, G.D. Yao, X.B. Wang, S.J. Song, Seven new
sesquineolignans isolated from the seeds of hawthorn and their neuroprotective
activities, Fitoterapia. 125 (2018) 6–12.
[16] 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.
[17] 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.
[18] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman,
G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato,
X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada,
M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda,
O. Kitao, H. Nakai, T. Vreven, J.A. Montgomery Jr., J.E. Peralta, F. Ogliaro,
M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, T. Keith,
R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar,
J. Tomasi, M. Cossi, N. Rega, J.M. Millam, M. Klene, J.E. Knox, J.B. Cross,
V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev,
A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma,
V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich,
A.D. Daniels, O. Farkas, J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox, Gaussian
09, Revision D.01, Gaussian, Inc, Wallingford CT, 2013.
[19] X.L. Qi, Y.Y. Zhang, P. Zhao, L. Zhou, X.B. Wang, X.X. Huang, B. Lin, S.J. Song, Ent-
kaurane diterpenoids with neuroprotective properties from Corn Silk (Zea mays), J.
Nat. Prod. 81 (2018) 1225–1234.
[20] 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.
[21] Y.Y. Lu, Y.B. Xue, J.J. Liu, G.M. Yao, D.Y. Li, B. Sun, J.W. Zhang, Y.F. Liu, C.X. Qi,
M. Xiang, Z.W. Luo, G. Du, Y.H. Zhang, ( )-Acortatarinowins A-F, norlignan,
neolignan, and lignan enantiomers from Acorus tatarinowii, J. Nat. Prod. 78 (2015)
2205–2214.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.fitote.2019.05.003.
References
[1] K.J. Barnham, C.L. Masters, A.I. Bush, Neurodegenerative diseases and oxidative
stress, Nat. Rev. Drug Discov. 3 (2004) 205–214.
[2] J. Everse, P.W. Coates, Neurodegeneration and peroxidases, Neurobiol. Aging 30
(2009) 1011–1025.
[3] M.H. Yan, X. Wang, X. Zhu, Mitochondrial defects and oxidative stress in Alzheimer
disease and Parkinson disease, Free. Radical Bio. Med. 62 (2013) 90–101.
[4] X.J. Wang, L.Y. Wang, Y. Fu, J. Wu, X.C. Tang, W.M. Zhao, H.Y. Zhang, Promising
effects on ameliorating mitochondrial function and enhancing Akt signaling in SH-
SY5Y cells by (M)-bicelaphanol A, a novel dimeric podocarpane type trinordi-
terpene isolated from Celastrus orbiculatus, Phytomedicine. 20 (2013) 1064–1070.
[5] J.Q. Wu, W. Peng, R.X. Qin, H. Zhou, Crataegus pinnatifida: chemical constituents,
pharmacology, and potential applications, Molecules 19 (2014) 1685–1712.
[6] P.C. Zhang, S.X. Xu, Flavonoid ketohexosefuranosides from the leaves of Crataegus
pinnatifida Bge. var. major N.E.Br, Phytochemistry 57 (2001) 1249–1253.
[7] N. Hellenbrand, J. Sendker, M. Lechtenberg, F. Petereit, A. Hensel, Isolation and
quantification of oligomeric and polymeric procyanidins in leaves and flowers of
hawthorn (Crataegus spp.), Fitoterapia 104 (2015) 14–22.
[8] E. Hwang, S.Y. Park, S.Y. Chang, H.T. Kim, M.K. Yong, T.H. Yi, Antiaging effects of
the mixture of Panax ginseng and Crataegus pinnatifida in human dermal fibroblasts
and healthy human skin, J. Gins. Res. 41 (2017) 69–77.
[9] Q. Chang, Z. Zuo, M.S.S. Chow, W.K.K. Ho, Effect of storage temperature on phe-
nolics stability in hawthorn (Crataegus pinnatifida var. major) fruits and a hawthorn
drink, Food Chem. 98 (2006) 426–430.
[10] X.X. Ying, R.X. Wang, J. Xu, W.J. Zhang, H.B. Li, C.S. Zhang, F.M. Li, HPLC de-
termination of eight polyphenols in the leaves of Crataegus pinnatifida Bge. var.
major, J. Chromatogr. Sci. 47 (2009) 201–205.
[22] X.W. Yang, P.J. Zhao, Y.L. Ma, H.T. Xiao, Y.Q. Zuo, H.P. He, L. Li, X.J. Hao, Mixed
lignan-neolignans from Tarenna attenuata, J. Nat. Prod. 70 (2007) 521–525.
[23] C. Zhao, J. Chen, J. Shao, J. Shen, K. Li, W. Gu, S. Li, J. Fan, Neolignan constituents
with potential beneficial effects in prevention of type 2 diabetes from Viburnum
fordiae Hance fruits, J. Agric. Food Chem. 66 (2018) 10421–10430.
[24] M.L. Gan, Y.L. Zhang, S. Lin, M.T. Liu, W.X. Song, J.C. Zi, Y.C. Yang, X.N. Fan,
J.G. Shi, J.F. Hu, J.D. Sun, N.H. Chen, Glycosides from the root of Iodes cirrhosa, J.
Nat. Prod. 71 (2008) 647–654.
[11] J. Tunde, S. Jiri, R. Otakar, M. Jiri, B. Stefan, S. Ladislav, A. Vojtech, K. Rene,
Polyphenolic profile and biological activity of Chinese hawthorn (Crataegus pinna-
tifida BUNGE) fruits, Molecules. 17 (2012) 14490–14509.
[12] P. Liu, B. Yang, H. Kallio, Characterization of phenolic compounds in Chinese
hawthorn (Crataegus pinnatifida Bge. var. major) fruit by high performance liquid
chromatography–electrospray ionization mass spectrometry, Food Chem. 121
(2010) 1188–1197.
[13] C. Tong, N. Kozo, T. Su, K. Hiroshi, T. Yi-Ling, Polyphenolic content and
7