Accepted Manuscript
Phytochemical constituents from Scutellaria baicalensis in solubleepoxide hydrolase inhibition: Kinetics and interaction mechanismmerged with simulations
Zhong-Bo Liu, Cheng-Peng Sun, Jian-Xia Xu, ChristopheMorisseau, Bruce D. Hammock, Feng Qiu
PII: S0141-8130(18)37341-0DOI: https://doi.org/10.1016/j.ijbiomac.2019.04.055Reference: BIOMAC 12131
To appear in: International Journal of Biological Macromolecules
Received date: 8 February 2019Revised date: 23 March 2019Accepted date: 9 April 2019
Please cite this article as: Z.-B. Liu, C.-P. Sun, J.-X. Xu, et al., Phytochemical constituentsfrom Scutellaria baicalensis in soluble epoxide hydrolase inhibition: Kinetics andinteraction mechanism merged with simulations, International Journal of BiologicalMacromolecules, https://doi.org/10.1016/j.ijbiomac.2019.04.055
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Phytochemical constituents from Scutellaria baicalensis in
soluble epoxide hydrolase inhibition: Kinetics and interaction
mechanism merged with simulations
Zhong-Bo Liua,b
, Cheng-Peng Sunc, Jian-Xia Xu
a, Christophe Morisseau
d, Bruce D.
Hammockd*
, and Feng Qiua,b*
a Tianjin State Key Laboratory of Modern Chinese Medicine and School of Chinese
Materia Medica, Tianjin University of Traditional Chinese Medicine, 312 Anshanxi
Road, Nankai District, Tianjin, China.
b School of Pharmacy,Shenyang Pharmaceutical University, Shenyang, China.
c College of Pharmacy, College (Institute) of Integrative Medicine, The National &
Local Joint Engineering Research Center for Drug Development of
Neurodegenerative Disease, Dalian Medical University, Dalian, China.
d Department of Entomology and Nematology, UC Davis Comprehensive Cancer
Center, University of California, Davis, CA 95616.
Corresponding authors at:
Feng Qiu:Tianjin State Key Laboratory of Modern Chinese Medicine and School of
Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin,
China. E-mail: [email protected];
Bruce D. Hammock: Department of Entomology and Nematology, UC Davis
Comprehensive Cancer Center, University of California, Davis, CA 95616.
E-mail: [email protected].
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ABSTRACT: In our search for soluble epoxide hydrolase (sEH) inhibitors from
plants, we found that water extracts of Scutellaria baicalensis Georgi displayed
significant inhibitory activity against sEH in vitro. Extracts of S. baicalensis were
separated, resulting in the isolation of thirty compounds (1-30), including six
lignins (1-6), sixteen flavones (7-22), and five amides (23-27). Their structures
were determined on the basis of 1H and
13C NMR and MS spectra. Compounds
1-6 were first reported in the genus Scutellaria. All the isolated compounds were
assayed for their inhibitory activities against sEH. Compounds 25-27 showed
significant inhibitory activities agaisnt sEH with IC50 values of 6.06 0.12, 7.83
0.52, and 6.32 0.31 µM, respectively, and compounds 3-6, 12, 18, and 22 displayed
moderate inhibitory actitivities against sEH with IC50 values from 20.82 0.78 µM to
56.61 0.98 µM. The inhibition kinetic analysis results indicated that compounds
25-27 were all uncompetitive. Molecular docking studies were performed to get
insights into inhibition mechanisms of compounds 25-27 against sEH.
Keywords: Scutellaria baicalensis; phytochemical constituents; soluble epoxide
hydrolase
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1. Introduction
Soluble epoxide hydrolase (sEH, EC:3.3.2.10) belonging to the α/β hydrolase fold
super family is a 120 kD homodimer enzyme with a C-terminal hydrolase and
N-terminal phosphatase [1, 2]. It is widely distributed throughout the body with the
most concentrated expression in the liver, kidney, intestine, vasculature, and brain in
mammals [3-6]. The N-terminal domain in charge of hydrolyzing phosphorylated
lipids, whereas the C-terminal domain hydrolyzes the epoxides by the catalytic triad
Asp335-Asp496-His524 to their corresponding diols [7-9]. It catalyzes the hydrolysis
of epoxy eicosatrienoic acids (EETs). Epoxy eicosatrienoic acids (EETs) as bioactive
metabolites of arachidonic acid, such as 5,6-EET, 8,9-EET, 11,12-EET, and
14,15-EET [10, 11], can be catalyzed by sEH into the corresponding dihydroxy
eicosatrienoic acids (DHETs), and exert anti-inflammatory effects in many diseases,
including inflammatory bowel disease, chronic peptic ulcer, arthritis, osteoporosis,
chronic periodontitis, sepsis, and cardiovascular disease [10, 12-14]. Therefore, sEH
has become an important target of inflammatory diseases, and searching for potential
sEH inhibitors has also attracted more and more attention [15, 16].
The genus Scutellaria belongs to a member of the Labiatae family comprising
approximately 350 species [17], and is widespreadly distributed in the tropical regions,
including Asia, Europe, and North America [18-20]. Scutellaria baicalensis Georgi,
known as “Huang qin” in China, is first recorded in Shennong Materia Medica that is
the earliest Chinese pharmacopoeia, and possesses clearing heat, purging fire, and
detoxification effects [19]. Recent pharmacological studies revealed that it had
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attracted attention in the treatment of fever, cancer, diarrhea, and hepatitis, especially
inflammation [18]. Phytochemical investigations have indicated that flavones are the
major constituents of S. baicalensis [18, 19], such as wogonin, norwogonin, baicalein,
and baicalin, and some of them displayed remarkable anti-inflammatory [21, 22],
anticancer [23], and hepatoprotective activities [24-26]. Accordingly, as part of our
ongoing research to discover of natural sEH inhibitors and anti-inflammatory drugs
from traditional Chinese medicines [27-32], water extracts of roots of S. baicalensis
were investigated and led to the isolation of thirty compounds (1-30, Fig. 1), including
six lignins (1-6), sixteen flavones (7-22), and five amides (23-27). Their structures
were elucidated according to 1H and
13C NMR and MS spectra. We evaluated the sEH
inhibitory activities of all the isolated compounds and enzyme kinetics in vitro. The
potential interactions between inhibitors and sEH were investigated by molecular
docking.
2. Material and methods
2.1 General Experimental Procedures.
Perkin-Elmer 241 polarimeter was used to record optical rotations. UV spectra were
recorded on a Shimadzu UV 2201 spectrophotometer. Bruker AV-400 spectrometer
was used in the NMR experiments. Chemical shift values were expressed in (ppm)
using the peak signals of the solvent DMSO-d6 (H 2.50 and C 39.5) or CDCl3 (H
7.27 and C 77.2) as a reference. Preparative HPLC was performed on an Elite P2300
instrument with an Elite UV2300 detector (Dalian, China) and a YMC C18 column
(250 mm × 10 mm, 5 m). All solvents were obtained from Tianjin Kemiou
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Chemical Reagent Company (Tianjing, China), MeOH for HPLC analysis was
chromatographic grade (Merck, Darmstadt, Germany). Silica gel (200−300 mesh) was
purchased from Qingdao Marine Chemical Factory (Qingdao, China). sEH protein
was provided from Prof. Bruce D. Hammock in Department of Entomology and
Nematology, UC Davis Comprehensive Cancer Center, University of California.
(E)-4-[(2-methypropyl)amino]-4-oxo-2-butenoic acid (23), pellitorine (24),
piperlonguminine (25), dihydropiperlonguminine (26), futoamide (27),
(+)-crotepoxide (28), syringlaldehyde (29), and vanillin (30) were previously isolated
from S. baicalensis [33], and their purities were more than 98% analyzed by HPLC.
2.2 Plant material
Dried roots of S. baicalensis were purchased in July 2013 from Chengdafangyuan
Co., Ltd., China, and identified by Prof. Jing-Ming Jia, Shenyang Pharmaceutical
University. A voucher specimen (SB201307) has been deposited in the herbarium of
the Department of Natural Products Chemistry, Shenyang Pharmaceutical University.
2.3 Extraction and isolation
Dried roots of S. baicalensis (5 kg) were extracted with water (2 × 2 h × 50 L),
and then was subjected to a D101 macroporous resin column eluted with EtOH-H2O
(10%-100%) and a polyamide column eluted with EtOH-H2O (0%-90%) to afford
twelve fractions A1-A12. Fraction A1 (4 g) was separated by silica gel column
Chromatography (CC) eluted with petroleum ether-EtOAc (100:0 to 0:100), resulting
in the isolation of ten subfractions A1.1-A1.10. Subfraction A1.5 (1 g) was purified
by Sephadex LH-20 CC (CH2Cl2-MeOH, 1:1) and preparative HPLC (MeOH-H2O,
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70%) to afford compound 4 (200.0 mg). Compound 5 (26.0 mg) was separated from
subfraction A1.6 (842 mg) by Sephadex LH-20 CC (CH2Cl2-MeOH, 1:1) and
preparative HPLC (MeOH-H2O, 70%). Fraction A2 (5 g) was purified by silica gel
CC eluted with petroleum ether-EtOAc (100:0 to 0:100) and preparative HPLC eluted
with MeOH-H2O (70%) to afford compounds 1 (10.0 mg), 2 (16.0 mg), 3 (26.0 mg),
and 5 (28.0 mg). Fraction A3 (10 g) was purified through a silica gel column
(petroleum ether-EtOAc,100:0 to 0:100) and preparative HPLC (MeOH-H2O,
55%-65%), yielding compound 7 (7.0 mg).
Fraction A7 (4 g) was separated by a polyamide column and eluted with
CH2Cl2-MeOH (100:0 to 0:100) to afford four subfractions A7.1-A7.4. Purification of
subfraction A7.1 (1.2 g) by Sephadex LH-20 CC (CH2Cl2-MeOH, 1:1) produced
compound 8 (20.0 mg). Subfration A7.3 (887.0 mg) was separated by preparative
HPLC (MeOH-H2O, 70%) to afford compounds 11 (9.0 mg), 12 (23.0 mg), 13 (5.9
mg), 14 (17.5 mg). Separation of fraction A8 (15 g) by polyamide CC
(CH2Cl2-MeOH, 100:0 to 0:100) yielded compounds 16 (26.0 mg), 21 (532.0 mg),
and an impure subfraction, which was further purified by preparative HPLC
(MeOH-H2O, 52%) to afford compounds 17 (11.0 mg), 18 (8.3 mg), 19 (20.4 mg),
and 20 (10.2 mg). Finally, fraction A10 (15 g) was subjected to ODS CC
(MeOH-H2O, 0:100 to 100:0), Sephadex LH-20 CC (MeOH-H2O, 1:1) and polyamide
CC (CH2Cl2-MeOH, 100:0 to 0:100), yielding compounds 9 (15.1 mg), 10 (15.3 mg),
15 (5.9 mg), 22 (56.2 mg).
2.4 sEH inhibitory activity
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All the isolated compounds 1-30 were assayed for inhibitory activities against sEH
as our pervious method [15, 16]. Compounds 1-30 were dissolved in DMSO and
diluted to final concentrations from 0.1 µM to 100.0 µM. All compounds were
hydrolyzed by sEH at 37 ℃ with the probe substrate PHOME (Cayman Chemical,
Ann Arbor, MI) in a 96-well plate, then the fluorescence signal was detected at 465
nm. The probe substrate groups (without evaluated compounds) were used as control.
N-[1-(1-Oxopropyl)-4-piperidinyl]-N'-[4-(trifluoromethoxy)phenyl]urea (TPPU) was
used as a control drug.
2.5 Inhibitory kinetic analysis
Kinetic analysis of sEH enzyme for compounds 25-27 was conducted by using the
Lineweaver-Burk analyses. The experiment was conducted at different PHOME
concentrations from 1 µM to 30 µM in the absence and presence of compounds 25-27
(1-24 µM).
2.6 Molecular docking
The X-ray crystal structure of soluble epoxide hydrolase (sEH) with its cognate
ligand (1-[1-(2-methylpropanoyl)piperidin-4-yl]-3-[4-(trifluoromethyl)phenyl]urea)
was downloaded from the Protein Data Bank (PDB)
(http://www.rcsb.org/pdb/home/home.do, PDB ID: 4OCZ). The 3D structure of
compounds 25-27 were built and minimized using CHARMm force field in Discovery
Studio 3.5 (BIOVIA Inc., San Diego, CA, USA). Then, the cognate ligand and
compounds were optimized according to the “Prepare Ligands” function at pH 6.8 in
Discovery Studio 3.5 which was consistent with the experiment conditions used in
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this study. The receptor structure was prepared by “Prepare Protein” function at pH
6.8 and the ligand and water molecules on the protein were removed manually in
Discovery Studio 3.5. Subsequently, the CDOCKER protocol of Discovery Studio 3.5
was used for the receptor and ligand docking with CHARMm force field. The binding
mode for 1-[1-(2-methylpropanoyl)piperidin-4-yl]-3-[4-(trifluoromethyl)phenyl]urea
to sEH was investigated by CDOCKER protocol to ensure the RMSD were normal
(RMSD < 2.0) [7, 8].
3. Results and discussion
3.1. Structural identification
The investigation of S. baicalensis resulted in the isolation of thirty compounds,
including veraguensin (1) [34], galgravin (2) [35], denudanolide B (3) [36], denudatin
B (4) [37],
(2R,3R,3aS)-5-allyl-2-(3,4-dimethoxy-phenyl)-3a-methoxy-3-methyl-3,3a-dihydroben
zofuran-6(2H)-one (5) [38], eupomatenoid-7 (6) [39], velutin (7) [40],
dihydrooroxylin A (8) [41], 3,5,7,2',6'-pentahydroxy flavanone (9) [42],
5,7,2′-trihydroxy-8,6′-dimethoxy flavone (10) [43], skullcapflavone Ⅱ (11) [44],
oxylin A (12) [45], chrysin (13) [46], wogonin (14) [47], norwogonin (15) [48],
5,6,7-trihydroxy-8-methoxy flavone (16) [49], 5,7,2′-trihydroxy-6′-dimethoxy flavone
(17) [50], scutevulin (18) [51], tenaxin Ⅱ (19) [50], 2′-hydroxy chrysin (20) [50],
baicalein (21) [52], and baicalin (22) [25]. Compounds 1-6 were first reported in the
genus Scutellaria. Their structures were determined by comparison of their 1H and
13C NMR data with literatures, and their
1H and
13C NMR data were listed in Tables
S1-S22.
3.2 sEH inhibitory effects
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Compounds 1-30 were assayed for their inhibitor activities against sEH in vitro
using a fluorescence method on the basis of hydrolysis of the substrate PHOME by
sEH. The amount of 6-methoxy-2-naphthaldehyde converted from PHOME was
quantified in the presence or absence of compounds 1-30 using a microplate reader at
excitation and emission wavelengths of 330 and 465 nm, respectively. TPPU was used
as a positive control with IC50 value of 0.05 0.01 µM. Compounds 1-30 had
inhibitory ratios ranging from 5.87 1.05% to 93.12 1.58% at 100 µM (Table 1),
and compounds that displayed more than 50% inhibition in a concentration-dependent
manner had IC50 values ranging from 6.06 0.12 to 93.68 4.52 µM. Compounds
25-27 showed significant inhibitory activities against sEH with IC50 values of 6.06
0.12, 7.83 0.52, and 6.32 0.31 µM (Fig. 2A-2C), respectively, whereas
compounds 3-6, 12, 18, and 22 displayed moderate inhibitory activities against sEH
with IC50 values of 37.64 1.19, 56.61 0.98, 26.16 0.87, 20.82 0.78, 44.82
0.73, 33.91 1.02, and 33.89 1.11 µM (Table 1), respectively.
3.3 Enzyme kinetics
In order to investigate the binding mechanisms of compounds 25-27, kinetic
analyses were performed in the presence of compounds 25-27 (3-24 µM) at various
substrate concentrations (1-30 µM), and then inhibition kinetics results of compounds
25-27 against sEH were conducted by using Lineweaver-Burk plots and GraphPad
Prsim software to determine their inhibition type and kinetics parameters, and the
results were listed in Fig. 2 and Table 2. As shown in Fig. 2G-2I, Lineweaver-Burk
plots of compounds 25-27 displayed a series of parallel lines, and Km and Vmax were
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all decreased with increasing concentration of compounds 25-27, indicating that their
inhibition types were all uncompetitive type with Ki values of 2.90, 4.35, and 1.58
µM, respectively [7, 8].
3.4 Molecular docking
Molecular docking was performed to discover the active site or predict binding site
of compounds 25-27 with sEH. The structure of sEH (PDB: 4OCZ) from Protein Data
Bank was used to calculated with the Discovery Studio 3.5 according to their
inhibition kinetic results. As shown in Fig. 3 and 4, compounds 25-27 could be well
docked into the catalytic site cavity of sEH. The nitrogen atom of an amide group in
compounds 25 and 26 could formed one hydrogen bond with the catalytic site residue
Asp335 that was in charge of ring-opening the epoxide, and the oxygen atom of an
amide group in compounds 25 and 26 could also formed two hydrogen bonds with
two tyrosine residues (Tyr383 and Tyr466) of the epoxide hydrolase catalytic pocket
[53], respectively, and compound 26 possessed an extra hydrogen bond with Trp525.
However, compound 27 has an interaction with sEH through a hydrogen bond
between Asp335 and the nitrogen atom of the amide group. Previous investigations
have indicated that urea compounds, as sEH inhibitors, have interactions with Asp335,
Tyr383, and Tyr466 by hydrogen bonds [54]. Compounds 25-27 were all amide type
compounds, and possessed the similar structure with urea compounds, such as TPPU,
1-[1-(2-methylpropanoyl)piperidin-4-yl]-3-[4-(trifluoromethyl)phenyl]urea.
Furthermore, compounds 25-27 displayed interactions with Asp335, Tyr383, Tyr466,
and Trp525, respectively, which was similar to urea compounds, indicating that
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compounds 25-27 could be regarded as potential sEH inhibitors.
4. Conclusions
In this study, the investigation of S. baicalensis led to the isolation of thirty
compounds 1-30. Compounds 1-6 were first reported in the genus Scutellaria.
All the isolated compounds were evaluated for their inhibitory activities against sEH.
Compounds 25-27 displayed significant sEH inhibitory activities with IC50 values of
6.06 0.12, 7.83 0.52, and 6.32 0.31 µM, respectively. According to their
inhibition kinetic results, compounds 25-27 were uncompetitive inhibitors with Ki
values of 2.90, 4.35, and 1.58 µM, respectively. The potential interaction mechanisms
of compounds 25-27 with sEH were also analysed by molecular docking, which
results indicated that the amide group of compounds 25-27 had interactions with
Asp335, Tyr383, and Tyr466, respectively.
CONFLICT OF INTEREST
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by the State Key Program of National Natural Science
Foundation of China (No. 81430095).
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Fig. 1. Chemical constituents isolated from S. baicalensis
Fig. 2. (A-C) Compounds 25-27 displayed concentration-dependent inhibitory
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activities against sEH. (D-F) Michealis-Menten plots of compounds 25-27 against
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sEH
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Fig. 4. Interaction mechanisms of compounds 25-27 (A-C) with sEH
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Table 1. Inhibitory effects of compounds 1-30 against sEH.
Compounds Inhibition of compounds against sEH
100 µM (%) IC50 (µM)
1 15.21 5.81 100
2 7.12 1.89 100
3 75.56 4.48 37.64 1.19
4 76.89 4.59 56.61 0.98
5 86.57 5.48 26.16 0.87
6 82.46 2.57 20.82 0.78
7 25.64 3.48 100
8 68.98 4.15 76.19 3.59
9 15.47 1.76 100
10 12.34 1.99 100
11 20.83 2.62 100
12 75.88 2.31 44.82 0.73
13 24.33 2.49 100
14 20.88 2.45 100
15 15.96 1.81 100
16 35.58 1.69 100
17 65.12 2.85 80.08 6.47
18 76.33 3.72 33.91 1.02
19 34.82 2.49 100
20 34.96 2.59 100
21 36.86 1.99 100
22 75.86 2.54 33.89 1.11
23 25.86 3.12 100
24 52.12 1.89 93.68 4.52
25 86.54 1.23 6.06 0.12
26 93.12 1.58 7.83 0.52
27 88.58 1.36 6.32 0.31
28 5.87 1.05 100
29 13.48 2.67 100
30 19.25 2.42 100
TPPUa -
b 0.05 0.01
aPositive control.
bNo tested.
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Table 2. Kinetic parameters of compounds 25-27 against sEH.
Compounds Inhibition type Vmax (mM/min/ng) Km (µM) Ki (µM)
25 uncompetitive 64.07 18.04 2.90
26 uncompetitive 66.78 20.18 4.35
27 uncompetitive 66.70 19.94 1.58
Table 3. Interaction information of compounds 25-27 with sEH.
Compounds Interaction amino acids Hydrogen
bonds
Lowest
binding
energy
(kcal/mol)
25
Asp335, Trp336, Met339, Thr360,
Phe381, Typ383, Gln384, Leu408,
Arg410, Leu417, Ser418, Leu428,
Tyr466, Val498, Leu499, His524, Trp525
Asp335,
Tyr383,
Tyr466
-37.92
26
Asp335, Trp336, Phe381, Tyr383,
Leu408, Leu417, Tyr466, Leu499,
His524, Trp525
Asp335,
Tyr383,
Tyr466,
Trp525
-40.45
27 Phe267, Asp335, Met339, Ile363,
Tyr383, Trp525 Asp335 -40.48
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Phytochemical constituents from Scutellaria baicalensis in
soluble epoxide hydrolase inhibition: Kinetics and interaction
mechanism merged with simulations
Zhong-Bo Liua,b
, Jian-Xia Xua, Cheng-Peng Sun
c , Christophe Morisseau
d, Bruce D. Hammock
d*,
and Feng Qiua,b,*
1. Thirty compounds 1-30 were isolated from S. baicalensis.
2. All the isolated compounds were assayed for their inhibitory activities against sEH.
3. The inhibition kinetics of compounds 25-27 against sEH were studied in vitro.
4. Interaction mechanisms of compounds 25-27 against sEH were analysed by
molecular docking.
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