Ribemansides A and B, TRPC6 Inhibitors from Ribes manshuricum That Suppress TGF-β1-Induced Fibrogenesis in HK-2 Cells

Article abstract of DOI:10.1021/acs.jnatprod.7b01037

Two new acylated β-hydroxynitrile glycosides, ribemansides A (1) and B (2), were isolated from the aerial parts of Ribes manshuricum. Their structures were elucidated by comprehensive spectroscopic analysis. Ribemansides A and B inhibited transforming growth factor β1 (TGF-β1)-induced expression of α-smooth muscle actin, fibronectin release, and changes in cell morphology in the human proximal tubular epithelial cell line (human kidney-2, HK-2). Further biological evaluation demonstrated that both 1 and 2 inhibit the activity of canonical transient receptor potential cation channel 6 (TRPC6), with IC₅₀ values of 24.5 and 25.6 μM, respectively. The antifibrogenic effect of these compounds appears to be mediated through TRPC6 inhibition, since the TRPC6 inhibitor, SAR7334, also suppressed TGF-β1-induced fibrogenesis in HK-2 cells.

Full text of DOI:10.1021/acs.jnatprod.7b01037

Article
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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Ribemansides A and B, TRPC6 Inhibitors from Ribes manshuricum
That Suppress TGF-β1-Induced Fibrogenesis in HK‑2 Cells
Baoping Zhou,§ Yange Wang,§ Chunlei Zhang,§ Guolin Yang, Fan Zhang, Boyang Yu, Chengzhi Chai,*
and Zhengyu Cao*
Department of TCM Pharmacology, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu
211198, People’s Republic of China
S
* Supporting Information
ABSTRACT: Two new acylated β-hydroxynitrile glycosides, ribemansides A (1) and B (2), were isolated from the aerial parts of
Ribes manshuricum. Their structures were elucidated by comprehensive spectroscopic analysis. Ribemansides A and B inhibited
transforming growth factor β1 (TGF-β1)-induced expression of α-smooth muscle actin, fibronectin release, and changes in cell
morphology in the human proximal tubular epithelial cell line (human kidney-2, HK-2). Further biological evaluation
demonstrated that both 1 and 2 inhibit the activity of canonical transient receptor potential cation channel 6 (TRPC6), with IC50
values of 24.5 and 25.6 μM, respectively. The antifibrogenic effect of these compounds appears to be mediated through TRPC6
inhibition, since the TRPC6 inhibitor, SAR7334, also suppressed TGF-β1-induced fibrogenesis in HK-2 cells.
the antifibrogenic activities of these two purified compounds
were evaluated using HK-2 cells. The two new isolated
compounds were identified as ribemanside A [2β-D-(6′-O-p-
hydroxybenzoyl)glucopyranosyloxymethy-4-hydroxy-2(E)-bu-
tenenitrile] and ribemanside B [2β-D-(6′-O-vanilloyl)-
glucopyranosyloxymethy-4-hydroxy-2(E)-butenenitrile].
he genus of Saxifragaceae Ribes is distributed worldwide
1
Tincluding Asia and North and South America. Due to
their perceived beneficial health effects, the seeds and fruits of
Ribes species have attracted considerable attention2 and have
been reported to be a rich source of unsaturated fatty acids,
phenolic acids, anthocyanins and other flavonoids, vitamins,
and amino acids.3−5 An aqueous extract of Ribes diacanthum is
used in Mongolian folk medicine to treat urinary complaints
such as bladder infections, cystitis, and edema, exerts diuretic
effects,6 and has also been found to protect against cisplatin-
induced kidney injury, possibly through anti-inflammatory
effects.4
Ribes manshuricum (Maxim.) Kom., a deciduous shrub, is
distributed mainly in the northeast area of the Inner Mongolia
Autonomous Region in the People’s Republic of China. When
compared to the relatively extensive research on other species
of the genus, little is known regarding the chemical constituents
and the biological activity of this species. Unpublished pilot
studies found that an ethanolic extract of R. manshuricum could
protect against unilateral ureteral occlusion (UUO)-induced
kidney injury in mice and inhibit transforming growth factor β1
(TGF-β1)-induced fibrogenesis in a human proximal tubular
epithelial cell line (human kidney-2, HK-2). In the present
study, two new acylated β-hydroxynitrile glycosides (1 and 2)
were isolated from the ethanolic extract of R. manshuricum, and
RESULTS AND DISCUSSION
The HRESIMS of ribemanside A (1) yielded a [M + H] ion
with m/z 396.1287, consistent with a molecular formula of
C18H21NO9, with nine degrees of unsaturation. The character-
istic UV absorptions at 206 and 258 nm suggested the presence
of a phenyl ring. The IR absorptions at 3394, 1693, and 2226
cm−1 indicated the presence of hydroxy, carbonyl, and cyano
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Received: December 10, 2017
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b01037
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
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1
1
Table 1. H NMR and 13C NMR Spectroscopic Data for
groups, respectively. The H NMR spectrum showed a typical
a
AA′BB′ system at δH 7.98 (2H, d, J = 8.5 Hz) and 6.90 (2H, d,
J = 8.5 Hz), indicating the presence of a p-substituted phenyl
ring. An olefinic proton signal at δH 6.71 (1H, t, J = 6.5 Hz)
suggested a trisubstituted double bond to be present in the
molecule. An anomeric proton signal occurring at δH 4.45 (1H,
d, J = 8.5 Hz) implied a β-linkage of the sugar unit to the
aglycon. The 13C NMR spectrum showed a total of 18 carbon
signals comprising an ester carbonyl, six aromatic carbons, two
olefinic carbons, a cyano carbon, and eight oxygenated aliphatic
carbons. Among them, seven signals at δC 168.1, 163.6, 132.9
(2C), 122.2, and 116.3 (2C) supported the presence of a p-
hydroxybenzoyl moiety. Six signals at δC 104.2, 77.9, 75.7, 75.0,
71.7, and 64.7 gave evidence of a sugar unit being present. The
downfield chemical shift of the tertiary olefinic carbon signal at
δC 144.6 suggested the presence of an electrophilic group
connected to a double bond. The carbon signal at δC 118.2 was
assigned to the cyano group, which was supported by the
chemical formula of 1, which contained a nitrogen atom.7,8 The
structure of the aglycon was determined as 2-hydroxymethyl-4-
hydroxy-2-butenenitrile by 2D NMR spectroscopic analysis
(Figure 1). The NOE correlation between H-5 (δH 4.59) and
Compounds 1 and 2
1
2
position
δH
δC
δH
δC
1
118.2
116.7
144.6
63.2
118.2
116.0
144.5
63.1
2
3
6.71, t (6.5)
4.17, d (6.5)
4.59, s
6.73, t (6.5)
4.20, d (6.5)
4.61, m
4
5
68.2
68.2
1′
2′
3′
4′
5′
6′
4.45, d (8.0)
3.32, m
104.2
75.0
4.47, d (7.5)
3.33, m
104.2
74.9
3.48, m
77.9
3.50, m
77.8
3.48, m
71.7
3.50, m
71.7
3.66, m
75.7
3.70, m
75.7
4.72, m
64.7
4.74, m
64.8
4.45, m
4.50, m
1″
2″
3″
4″
5″
6″
7″
−OCH3
122.2
132.9
116.3
163.6
116.3
132.9
168.1
122.5
113.7
148.8
152.9
116.0
125.3
168.0
56.5
7.98, d (8.5)
6.90, d (8.5)
7.66, d (2.0)
6.90, d (8.5)
7.98, d (8.5)
6.94, d (8.0)
7.69, dd (8.0, 2.0)
3.99 s
a
1H NMR data (δ) were measured in CD3OD at 500 MHz; 13C NMR
data (δ) were measured in CD3OD at 125 MHz.
myofibroblasts. Injured epithelial cells have been reported to
serve as the primary source for myofibroblasts through a
process known as epithelial mesenchymal transition
(EMT).13−15 During this so-called trans-differentiation process,
the cells acquire a spindle-like shape and start producing α-
SMA (a marker of myofibroblasts in the kidney) and matrix
proteins and show enhanced cell migration and invasion.15 Our
unpublished preliminary data demonstrated that the ethanolic
extract of the aerial parts of R. manshuricum protected mice
from UUO-induced kidney injury. We therefore tested whether
ribemansides A (1) and B (2) are capable of mitigating EMT-
mediated fibrogenesis in HK-2 cells. TGF-β1 exposure (5 ng/
mL, 36 h) induced a spindle-like morphology of HK-2 cells
(Figure 2A). The ratio of length/width was increased
significantly from 1.63 0.04 to 2.56 0.05 (p < 0.01, n =
40) (Figure 2B). Pretreatment with 1 or 2 (30 μM)
significantly reversed the HK-2 cell morphological changes
(Figure 2B). Consistent with the previous study, the expression
level of α-SMA was significantly increased by 1.82-fold (p <
0.01, n = 4) (Figure 2C and D). Compounds 1 and 2 (30 μM)
both inhibited TGF-β1-induced increase of α-SMA expression
(p < 0.01, n = 4) (Figure 2C and D). Similarly, 1 and 2 both
inhibited TGF-β1-induced fibronectin release (p < 0.01, n = 4)
(Figure 2E).
Figure 1. Selected H−H COSY (−), HMBC (→), and NOESY (↔)
correlations of ribemanside A (1).
H-3 (δH 6.71) suggested an E-configuration of the Δ2(3)-double
bond. To determine the structure of the sugar moiety,
ribemanside A (1) was hydrolyzed with acid, and the sugar
was found to be D-glucose by comparing the optical rotation
with an authentic sample. HMBC correlations between H-1′
(δH 4.45) and C-5 (δC 68.2) and between H-5 (δH 4.59) and C-
1′ (δC 104.2) demonstrated that the D-glucose unit is linked to
C-5. In turn, HMBC correlations of H-6′ (δH 4.72 and 4.45)
with C-7″ (δC 168.1), coupled with the downfield chemical
shift of C-6′, demonstrated that the p-hydroxybenzoyl moiety is
connected to C-6′. Consequently, the structure of ribemanside
A (1) was determined to be 2β-D-(6′-O-p-hydroxybenzoyl)-
glucopyranosyloxymethy-4-hydroxy-2(E)-butenenitrile.
The HRESIMS of ribemanside B (2) gave a [M + H] + mass
at m/z 426.1375, consistent with a molecular formula of
1
C19H23NO10. The H NMR data of 2 (Table 1) resembled
those of compound 1, but contained an extra methoxy group
signal at δH 3.99 (3H, s) and a typical ABX-coupling system
comprising three aromatic protons resonating at δH 7.69 (1H,
dd, J = 8.0, 1.5 Hz), 7.66 (1H, d, J = 2.0 Hz), and 6.94 (1H, d, J
TRPC6 channels are Ca2+-selective members of the TRPC
subfamily.16,17 Mutations in TRPC6 have been demonstrated
to be associated with kidney dysfunction. A single point gain-of-
function mutation (P112Q) in TRPC6 is sufficient to cause
focal segmental glomerular sclerosis,18 and increased TRPC6
expression has been reported in several proteinuric kidney
diseases in humans.19 A more recent proof-of-concept study
further demonstrated that UUO-induced kidney injury resulted
in increased TRPC6 expression, while TRPC6 knockout
ameliorated UUO-induced kidney fibrosis in tubular epithelia
cells in mice.20 We therefore tested the effects of 1 and 2 on
1
= 8.0 Hz). The H NMR data together with the 13C NMR
(Table 1) spectrum indicated the presence of a vanilloyl group,
attached to the C-6′ position, based on HMBC correlations of
H-6′ (δH 4.76, 4.52) with the ester carbonyl at δC 168.0.
Consequently, ribemanside B (2) was identified as 2β-D-(6′-O-
vanilloyl)glucopyranosyloxymethy-4-hydroxy-2(E)-buteneni-
trile.
Fibrosis is a key step of renal repair following injury or in
response to cytokines and other factors9−12 and is characterized
by the proliferation of fibroblasts and their transformation into
B
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Figure 2. Ribemansides A (1) and B (2) suppress TGF-β1-induced fibrogenesis in HK-2 cells. (A) Representative images for TGF-β1-induced HK-2
cellular morphological changes in the presence and absence of 1 or 2. (B) Quantification of the ratio of HK-2 cell length/width in the presence and
absence of 1 or 2 after TGF-β1 exposure. (C) Representative Western blot for TGF-β1-induced α-SMA expression in HK-2 cells in the presence and
absence of 1 or 2. (D) Quantification of α-SMA expression in the presence and absence of 1 or 2 after TGF-β1 exposure. (E) Ribemansides A (1)
and B (2) inhibit the TGF-β1-induced secretion of fibronectin. The experiments were performed twice each in duplicates (**, p < 0.01, TGF-β1 vs
vehicle (Veh); ##, p < 0.01, TGF-β1 + 1 or 2 vs TGF-β1).
measured on a UV−vis spectrophotometer (UV-2550, Shimadzu). IR
spectra were recorded on an FT-IR microscope spectrometer (Nicolet
5700). NMR spectra were recorded on a Bruker-500 spectrometer.
HRESIMS data were recorded on an Accurate-Mass Q-TOF LC/MS
spectrometer (Agilent Technologies 6520). RP-HPLC separations
were performed using a Welch Ultimate XB-C18 column (250 × 10
mm, 5 μm) on an Agilent 1260 instrument coupled to a VWD
detector. Sephadex LH-20 (Amersham Pharmacia Biotech AB,
Sweden), ODS (45−70 μm, Welch, Shanghai, People’s Republic of
China), and silica gel (200−300 mesh, Qingdao Marine Chemical Ltd.,
Qingdao, People’s Republic of China) were used for column
chromatography. Thin-layer chromatography (TLC) was carried out
on precoated GF254 silica gel plates (Qingdao Marine Chemical Ltd.).
Plant Material. The aerial parts of Ribes manshuricum were
acquired in September 2015 from the Changbai Mountain area of Jilin
Province, People’s Republic of China, and were identified by one of
the authors (B.Y.). A voucher specimen (RM-201509) was deposited
at the School of Traditional Chinese Pharmacy, China Pharmaceutical
University.
Extraction and Isolation. The dried aerial parts of R.
manshuricum (50 kg) were extracted three times with 95% EtOH (5
× 50 L) heated at reflux for 4 h each. The solvent was evaporated
under reduced pressure to give a crude extract (1570 g), which was
suspended in 5 L of water and first extracted with petroleum ether (15
L), then ethyl acetate (12 L) and then n-butanol (8 L). The n-butanol
extract (320 g) was applied to a D101 macroporous resin column (160
× 12 cm) eluted with 0%, 20%, 30%, 40%, and 60% EtOH in H2O,
successively, to give five fractions (I−V). Fraction II (50 g) was
subjected to reversed-phase C18 silica gel column chromatography (60
× 4 cm), conducted with a stepwise gradient of MeOH in H2O (5%,
20%, and 40%), to afford three subfractions (IIa−IIc). Fraction IIc was
TRPC6 channel activity. Both 1 and 2 suppressed the TRPC6
agonist M08521 and induced Ca2+ influx in HEK-293 cells
expressing TRPC6 at micromolar concentrations (Figure 3A
and B). The IC50 values for 1 and 2 in terms of suppressing
TRPC6 activity were 24.5 and 25.6 μM, respectively (Figure
3C).
In order to determine whether inhibition of TRPC6
contributed to the antifibrogenic effect of 1 and 2, the effect
of SAR7334, a TRPC6 inhibitor, was evaluated on TGF-β1-
stimulated fibrogenesis in HK-2 cells. SAR7334 (1 μM) alone
had no effect on the cellular morphological changes (Figure
S19A and B, Supporting Information). However, SAR7334 did
reverse TGF-β1-induced spindle-like morphology in a concen-
tration-dependent manner (Figure S19A and B, Supporting
Information). SAR7334 also concentration-dependently de-
creased the TGF-β1-induced increase of α-SMA expression
without any effects on basal α-SMA expression (Figure S19C
and D, Supporting Information). Similar to 1 and 2, SAR7334
also concentration-dependently reduced TGF-β1-induced
secretion of fibronectin (Figure S19E, Supporting Information).
Taken together, these data suggest that TRPC6 channels play a
significant role in fibrogenesis in HK-2 cells and that 1 and 2
may be valuable as lead compounds to design novel TRPC6
inhibitors for the treatment of renal fibrosis.
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were taken
on an Autopol-IV automatic digital polarimeter. UV spectra were
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measuring their optical rotations and by co-TLC with an authentic
sample.22
HK-2 Cell Culture. The HK-2 cell line was generously provided by
Professor Bicheng Liu (Zhongda Hospital, Southeast University,
Nanjing, People’s Republic of China) and grown in RPMI 1640
medium containing 2 mg/mL NaHCO3, 10% fetal bovine serum
(FBS) (Sigma-Aldrich, St. Louis, MO, USA), 10 mM HEPES, 100 U/
mL penicillin, and 0.1 mg/mL streptomycin (ThermoFisher Scientific,
Waltham, MA, USA). The cells at approximately 80% confluency were
digested with 0.05% trypsin−EDTA (ThermoFisher Scientific) and
seeded in 96-well or 12-well plates (Corning; Corning, NY, USA) at
densities of 6000 cells/well or 40 000 cells/well. The cells were then
cultured in serum-free medium and stimulated with TGF-β1 (5 ng/
mL, R&D Systems, Minneapolis, MN, USA) in the absence and
presence of ribemanside A (1) or B (2) or SAR7334 for 36 h.
TRPC6-HEK-293 Cell Culture. HEK-293 cells stably expressing
mouse TRPC6 were provided by Professor Michael X. Zhu at the
University of Texas Health Science Center at Houston and cultured as
described previously.21 Cells at approximately 80% confluency were
digested with 0.05% trypsin−EDTA and seeded in poly-D-lysine-
coated 96-well plates at a density of ∼20 000 cells/well. The cells were
cultured for 6 h before use.
Intracellular Ca2+ Concentration Determination. The intra-
cellular Ca2+ concentration was determined as described previously.21
Briefly, after incubation with 4 μM Fluo-8/AM (TEFlabs, Austin, TX,
USA) for 45 min, the TRPC6-HEK-293 cells were gently washed four
times and loaded into the chamber of a fluorescent imaging plate
reader (FLIPRTetra; Molecular Devices, Sunnyvale, CA, USA). Basal
fluorescence units (F0) were recorded for 30 s followed by the addition
of vehicle or compounds, and the fluorescent signals (F) were
recorded for an additional 5 min before addition of M085 (1 μM)
(ethyl 4-(3-(4-fluorophenyl)-7-hydroxy-2-methylpyrazolo[1,5-a]-
pyrimidin-5-yl)piperidine-1 carboxylate).21 Data are presented as F/
F0. To analyze the concentration−response relationship, the area
under the curve (AUC) was calculated from a time period of 300 s
right after addition of M085.
Western Blotting. The Western blotting experiments were
performed as described previously.23 Equal amounts (30 μg) of
protein were mixed with loading buffer, and the samples were loaded
onto a 12% SDS-PAGE gel. After electrophoresis, proteins were
transferred to a nitrocellulose membrane by electroblotting.
Membranes were blocked with 5% skimmed milk in phosphate-
buffered saline for 1 h at room temperature and then were incubated
overnight at 4 °C with anti-α-SMA (1:1500) (Abcam, Cambridge, MA,
USA) and anti-tubulin (1:5000) (Bioworld, Shanghai, People’s
Republic of China) antibodies. After washing, the blots were incubated
with the IRDye (680RD or 800CW)-labeled secondary antibodies
(1:10 000) for 1 h at room temperature and then were scanned with
the LI-COR Odyssey infrared imaging system (LI-COR Biotechnol-
ogy, Lincoln, NE, USA). Densitometry was performed using the LI-
COR Odyssey infrared imaging system application software (version
2.1).
Figure 3. Ribemansides A (1) and B (2) suppress the TRPC6 agonist
(M085)-induced Ca2+ response in HEK-293 cells expressing TRPC6.
(A) Representative traces for 1 suppressing the M085-induced Ca2+
response in HEK-293 cells, with TRPC6 expressed as a function of
time. (B) Representative traces for 2 suppressing the M085-induced
Ca2+ response in HEK-293 cells, with TRPC6 expressed as a function
of time. (C) Concentration−response relationships for 1 and 2 in
suppressing TRPC6 activity. The experiments were performed twice,
with each in triplicate.
further separated by HPLC [CH3CN: 0.1% TFA−H2O (14:86, v/v, 2
mL/min)] to yield 1 (tR = 14.1 min, 16.0 mg) and 2 (tR = 29.4 min,
14.7 mg).
Ribemanside A (1): brown paste; [α]2D0 −11.3 (c 0.18, MeOH); UV
(MeOH) λmax (log ε) 258 (3.89) nm; IR νmax 3394, 2911, 2226, 1693,
1608, 1515, 1280, 1168, 1066 cm−1; 1H NMR and 13C NMR data, see
Table 1; HRESIMS m/z 396.1287 [M + H]+ (calcd for 396.1289,
C18H22NO9).
Measurement of Fibronectin. Fibronectin secretion was
determined by a commercial ELISA kit (Jin-Yi-Bai Biological
Technology Co. Ltd., Nanjing, People’s Republic of China) according
to the instructions of the manufacturer. The optical density value was
detected using a Tecan Infinite 200 Pro microplate reader (Tecan
Ribemanside B (2): brown paste; [α]2D0 −7.1 (c 0.15, MeOH); UV
(MeOH) λmax (log ε) 263 (3.83), 288 (3.65) nm; IR νmax 3390, 2924,
1
2225, 1696, 1598, 1515, 1287, 1222, 1080 cm−1; H NMR and 13C
NMR data, see Table 1; HRESIMS m/z 426.1375 [M + H]+ (calcd for
426.1370, C19H24NO10).
Trading AG, Mannedorf, Switzerland) at a wavelength of 450 nm.
̈
Data Analysis. Data plotting and statistical analysis were
performed with GraphPad Prism software (version 5.0, GraphPad
Software Inc., San Diego, CA, USA). Concentration response curves
were fit by nonlinear regression using a three-parameter logistic
equation. Statistical significance between groups was calculated using
ANOVA and, where appropriate, a Dunnett’s multiple comparison
test; p values of less than 0.05 were considered statistically significant.
Acid Hydrolysis. An aliquot of 6 mg of ribemanside A (1) or
ribemanside B (2) was refluxed in 6% HCl (4.0 mL) at 80 °C for 2 h.
The reaction mixture was extracted with CHCl3 (3 × 5 mL), and the
aqueous layer was dried by a N2 stream. The residue was loaded onto a
silica gel column and eluted with EtOAc−EtOH−H2O (7:4:1) to yield
D-glucose (1.7 mg) from 1, [α]2D0 +47 (c 0.09, H2O), and D-glucose
(1.5 mg) from 2, [α]2D0 +42 (c 0.08, H2O), respectively, identified by
D
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(19) Moeller, C. C.; Wei, C.; Altintas, M. M.; Li, J.; Greka, A.; Ohse,
T.; Pippin, J. W.; Rastaldi, M. P.; Wawersik, S.; Schiavi, S.; Henger, A.;
Kretzler, M.; Shankland, S. J.; Reiser, J. J. Am. Soc. Nephrol. 2007, 18,
29−36.
(20) Wu, Y. L.; Xie, J.; An, S. W.; Oliver, N.; Barrezueta, N. X.; Lin,
M. H.; Birnbaumer, L.; Huang, C. L. Kidney Int. 2017, 91, 830−841.
(21) Qu, C. R.; Ding, M. M.; Zhu, Y. M.; Lu, Y. G.; Du, J.; Miller, M.;
Tian, J. B.; Zhu, J. M.; Xui, J.; Wen, M.; Er-Bu, A.; Wan, J. L.; Xiao, Y.
L.; Wu, M.; McManus, O. B.; Li, M.; Wu, J. L.; Luo, H. R.; Cao, Z. Y.;
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.7b01037.
■
S
1D and 2D NMR, UV, IR, and mass spectra for
compounds 1 and 2 (PDF)
AUTHOR INFORMATION
Corresponding Authors
*Tel/Fax: +86 25 86185158. E-mail: chaichengzhi@126.com
(C. Z. Chai).
*Tel/Fax: +86 25 86185158. E-mail: zycao1999@hotmail.com
(Z. Y. Cao).
■
(22) Liu, Y. F.; Shi, G. R.; Wang, X.; Zhang, C. L.; Wang, Y.; Chen,
R. Y.; Yu, D. Q. J. Nat. Prod. 2016, 79, 428−433.
(23) Cao, Z. Y.; George, J.; Baden, D. G.; Murray, T. F. Brain Res.
2007, 1184, 17−27.
ORCID
Zhengyu Cao: 0000-0002-2692-2949
Author Contributions
§B. P. Zhou, Y. G. Wang, and C. L. Zhang contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by National Natural Science
Foundation of the People’s Republic of China (81603389,
21777192, and 81473539), the Innovative Drug Development
Program from Ministry of Science and Technology
(2017ZX09101003-004-002), and the Natural Science Founda-
tion of Jiangsu Province (CN) (BK20160764, BK20160754).
We thank Prof. H. Wulff at UCDavis for English editing.
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