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

This is a PDF file of an unedited manuscript that has been accepted for publication. Asa service to our customers we are providing this early version of the manuscript. Themanuscript will undergo copyediting, typesetting, and review of the resulting proof beforeit is published in its final form. Please note that during the production process errors maybe discovered which could affect the content, and all legal disclaimers that apply to thejournal pertain.

ACC

EPTE

D M

ANU

SCR

IPT

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: fengqiu20070118@163.com;

Bruce D. Hammock: Department of Entomology and Nematology, UC Davis

Comprehensive Cancer Center, University of California, Davis, CA 95616.

E-mail: bdhammock@ucdavis.edu.

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

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

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

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

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

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

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

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,

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

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

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

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

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

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

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

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

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

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

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

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).

REFERENCES

[1] J.K. Beetham, T. Tian, B.D. Hammock, cDNA cloning and expression of a soluble

epoxide hydrolase from human liver, Arch. Biochem. Biophys. 305(1) (1993)

197-201.

[2] D.F. Grant, D.H. Storms, B.D. Hammock, Molecular cloning and expression of

murine liver soluble epoxide hydrolase, J. Biol. Chem. 268(23) (1993) 17628-33.

[3] S.D. Kodani, S. Bhakta, S.H. Hwang, S. Pakhomova, M.E. Newcomer, C.

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

Morisseau, B.D. Hammock, Identification and optimization of soluble epoxide

hydrolase inhibitors with dual potency towards fatty acid amide hydrolase, Bioorg.

Med. Chem. Lett. 28(4) (2018) 762-768.

[4] T.R. Harris, B.D. Hammock, Soluble epoxide hydrolase: gene structure,

expression and deletion, Gene 526(2) (2013) 61-74.

[5] W. Swardfager, M. Hennebelle, D. Yu, B.D. Hammock, A.J. Levitt, K. Hashimoto,

A.Y. Taha, Metabolic/inflammatory/vascular comorbidity in psychiatric disorders;

soluble epoxide hydrolase (sEH) as a possible new target, Neurosci. Biobehav. Rev.

87 (2018) 56-66.

[6] X. Qin, Q. Wu, L. Lin, A. Sun, S. Liu, X. Li, X. Cao, T. Gao, P. Luo, X. Zhu, X.

Wang, Soluble Epoxide Hydrolase Deficiency or Inhibition Attenuates MPTP-Induced

Parkinsonism, Mol. Neurobiol. 52(1) (2015) 187-95.

[7] J.H. Kim, I.S. Cho, J. Ryu, J.S. Lee, J.S. Kang, S.Y. Kang, Y.H. Kim, In vitro and

in silico investigation of anthocyanin derivatives as soluble epoxide hydrolase

inhibitors, Int. J. Biol. Macromol. 112 (2018) 961-967.

[8] H.H. Leem, G.Y. Lee, J.S. Lee, H. Lee, J.H. Kim, Y.H. Kim, Soluble epoxide

hydrolase inhibitory activity of components from Leonurus japonicus, Int. J. Biol.

Macromol. 103 (2017) 451-457.

[9] A. Oguro, S. Imaoka, Lysophosphatidic acids are new substrates for the

phosphatase domain of soluble epoxide hydrolase, J. Lipid Res. 53(3) (2012) 505-12.

[10] K.M. Wagner, C.B. McReynolds, W.K. Schmidt, B.D. Hammock, Soluble

epoxide hydrolase as a therapeutic target for pain, inflammatory and

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

neurodegenerative diseases, Pharmacol. Ther. 180 (2017) 62-76.

[11] Q. Ren, M. Ma, J. Yang, R. Nonaka, A. Yamaguchi, K. Ishikawa, K. Kobayashi,

S. Murayama, S.H. Hwang, S. Saiki, W. Akamatsu, N. Hattori, B.D. Hammock, K.

Hashimoto, Soluble epoxide hydrolase plays a key role in the pathogenesis of

Parkinson's disease, Proc. Natl. Acad. Sci. U. S. A. 115(25) (2018) E5815-E5823.

[12] L.J. Zhong, J.T. Zhou, D.W. Wang, X.J. Zou, Y.X. Lou, D. Liu, B. Yang, Y. Zhu,

X.X. Li, Proteomics and bioinformatics analysis of mouse hypothalamic neurogenesis

with or without EPHX2 gene deletion, Int. J. Clin. Exp. Pathol. 8(10) (2015)

12634-12645.

[13] W.H. Schunck, A. Konkel, R. Fischer, K.H. Weylandt, Therapeutic potential of

omega-3 fatty acid-derived epoxyeicosanoids in cardiovascular and inflammatory

diseases, Pharmacol. Ther. 183 (2018) 177-204.

[14] A.I. Ostermann, M. Reutzel, N. Hartung, N. Franke, L. Kutzner, K. Schoenfeld,

K.H. Weylandt, G.P. Eckert, N.H. Schebb, A diet rich in omega-3 fatty acids enhances

expression of soluble epoxide hydrolase in murine brain, Prostaglandins Other Lipid

Mediat. 133 (2017) 79-87.

[15] N.M. Wolf, C. Morisseau, P.D. Jones, B. Hock, B.D. Hammock, Development of

a high-throughput screen for soluble epoxide hydrolase inhibition, Anal. Biochem.

355(1) (2006) 71-80.

[16] V. Burmistrov, C. Morisseau, T.R. Harris, G. Butov, B.D. Hammock, Effects of

adamantane alterations on soluble epoxide hydrolase inhibition potency, physical

properties and metabolic stability, Bioorg. Chem. 76 (2018) 510-527.

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

[17] T. Wu, Q. Wang, C. Jiang, S.L. Morris-Natschke, H. Cui, Y. Wang, Y. Yan, J. Xu,

K.H. Lee, Q. Gu, Neo-clerodane diterpenoids from Scutellaria barbata with activity

against Epstein-Barr virus lytic replication, J. Nat. Prod. 78(3) (2015) 500-9.

[18] S. EghbaliFeriz, A. Taleghani, Z. Tayarani-Najaran, Central nervous system

diseases and Scutellaria: a review of current mechanism studies, Biomed.

Pharmacother. 102 (2018) 185-195.

[19] X. Jintao, Y. Quanwei, L. Chunyan, J. Yun, W. Shuangxi, Z. Mingxiang, L. Peng,

Rapid and Simultaneous Determination of Three Active Components in Raw and

Processed Root Samples of Scutellaria baicalensis by Near-infrared Spectroscopy,

Planta Med. (2018).

[20] H.W. Lee, H.W. Ryu, M.G. Kang, D. Park, H. Lee, H.M. Shin, S.R. Oh, H. Kim,

Potent inhibition of monoamine oxidase A by decursin from Angelica gigas Nakai and

by wogonin from Scutellaria baicalensis Georgi, Int. J. Biol. Macromol. 97 (2017)

598-605.

[21] T. Shimizu, N. Shibuya, Y. Narukawa, N. Oshima, N. Hada, F. Kiuchi,

Synergistic effect of baicalein, wogonin and oroxylin A mixture: multistep inhibition

of the NF-kappaB signalling pathway contributes to an anti-inflammatory effect of

Scutellaria root flavonoids, J. Nat. Med. 72(1) (2018) 181-191.

[22] Y.J. Kim, H.J. Kim, J.Y. Lee, D.H. Kim, M.S. Kang, W. Park, Anti-Inflammatory

Effect of Baicalein on Polyinosinic(-)Polycytidylic Acid-Induced RAW 264.7 Mouse

Macrophages, Viruses 10(5) (2018) 224.

[23] X. Yu, Y. Liu, Y. Wang, X. Mao, Y. Zhang, J. Xia, Baicalein induces cervical

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

cancer apoptosis through the NF-kappaB signaling pathway, Mol Med Rep 17(4)

(2018) 5088-5094.

[24] S.I. Jang, H.J. Kim, K.M. Hwang, S.J. Jekal, H.O. Pae, B.M. Choi, Y.G. Yun, T.O.

Kwon, H.T. Chung, Y.C. Kim, Hepatoprotective effect of baicalin, a major flavone

from Scutellaria radix, on acetaminophen-induced liver injury in mice,

Immunopharmacol. Immunotoxicol. 25(4) (2003) 585-94.

[25] J. Zhang, H. Zhang, X. Deng, N. Zhang, B. Liu, S. Xin, G. Li, K. Xu, Baicalin

attenuates non-alcoholic steatohepatitis by suppressing key regulators of lipid

metabolism, inflammation and fibrosis in mice, Life Sci. 192 (2018) 46-54.

[26] Q. Dong, F. Chu, C. Wu, Q. Huo, H. Gan, X. Li, H. Liu, Scutellaria baicalensis

Georgi extract protects against alcoholinduced acute liver injury in mice and affects

the mechanism of ER stress, Mol Med Rep 13(4) (2016) 3052-62.

[27] C.P. Sun, A.G. Kutateladze, F. Zhao, L.X. Chen, F. Qiu, A novel withanolide with

an unprecedented carbon skeleton from Physalis angulata, Org. Biomol. Chem. 15(5)

(2017) 1110-1114.

[28] C.P. Sun, C.Y. Qiu, T. Yuan, X.F. Nie, H.X. Sun, Q. Zhang, H.X. Li, L.Q. Ding, F.

Zhao, L.X. Chen, F. Qiu, Antiproliferative and Anti-inflammatory Withanolides from

Physalis angulata, J. Nat. Prod. 79(6) (2016) 1586-97.

[29] C.P. Sun, C.Y. Qiu, F. Zhao, N. Kang, L.X. Chen, F. Qiu, Physalins V-IX,

16,24-cyclo-13,14-seco withanolides from Physalis angulata and their

antiproliferative and anti-inflammatory activities, Sci. Rep. 7(1) (2017) 4057.

[30] C. Wang, X.K. Huo, Z.L. Luan, F. Cao, X.G. Tian, X.Y. Zhao, C.P. Sun, L. Feng,

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

J. Ning, B.J. Zhang, X.C. Ma, Alismanin A, a Triterpenoid with a C34 Skeleton from

Alisma orientale as a Natural Agonist of Human Pregnane X Receptor, Org. Lett.

19(20) (2017) 5645-5648.

[31] Y.L. Lan, J.J. Zhou, J. Liu, X.K. Huo, Y.L. Wang, J.H. Liang, J.C. Zhao, C.P. Sun,

Z.L. Yu, L.L. Fang, X.G. Tian, L. Feng, J. Ning, B.J. Zhang, C. Wang, X.Y. Zhao, X.C.

Ma, Uncaria rhynchophylla Ameliorates Parkinson's Disease by Inhibiting HSP90

Expression: Insights from Quantitative Proteomics, Cell. Physiol. Biochem. 47(4)

(2018) 1453-1464.

[32] C.P. Sun, M.B. Oppong, F. Zhao, L.X. Chen, F. Qiu, Unprecedented 22,26-seco

physalins from Physalis angulata and their anti-inflammatory potential, Org. Biomol.

Chem. 15(41) (2017) 8700-8704.

[33] J.X. Xu, L.Q. Ding, M.M. Jiang, F. Qiu, Non-flavonoid constituents from the

roots of Scutellaria baicalensis Georgi, Chinese Journal of Medicinal Chemistry 26

(2016) 480-484

[34] E.C. Costa, T.B. Cassamale, D.B. Carvalho, L.S. Bosquiroli, M. Ojeda, T.V.

Ximenes, M.F. Matos, M.C. Kadri, A.C. Baroni, C.C. Arruda, Antileishmanial

Activity and Structure-Activity Relationship of Triazolic Compounds Derived from

the Neolignans Grandisin, Veraguensin, and Machilin G, Molecules 21(6) (2016).

[35] L.J. Xu, H.T. Liu, Y. Peng, W. Xiao, S.L. Chen, S.B. Chen, P.G. Xiao, Chemical

constituents from stems of Schisandra propinqua, China Journal of Chinese Materia

Medica 33(5) (2008) 521-3.

[36] M. Kuroyanagi, K. Yoshida, A. Yamamoto, M. Miwa, Bicyclo[3.2.1]octane and

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

6-oxabicyclo[3.2.2]nonane type neolignans from Magnolia denudata, Chem. Pharm.

Bull. (Tokyo) 48(6) (2000) 832-7.

[37] I. Toshiyuki, I. Kazuhiko, I. Kazuo, Neolignans from Magnolia denudata,

Phytochemistry 21(12) (1980) 2939-2941.

[38] A.K. Prasad, O.D. Tyagi, J. Wengel, P.M. Boll, C.E. Olsen, K.S. Bisht, A. Singh,

A. Sarangi, R. Kumar, S.C. Jain, V.S. Parmar, Neolignans and a lignan from Piper

clarkii, Phytochemistry 39(3) (1995) 655-658.

[39] M.D.P. Pereira, T. da Silva, A.C.C. Aguiar, G. Oliva, R.V.C. Guido, J.K.U.

Yokoyama-Yasunaka, S.R.B. Uliana, L.M.X. Lopes, Chemical Composition,

Antiprotozoal and Cytotoxic Activities of Indole Alkaloids and Benzofuran Neolignan

of Aristolochia cordigera, Planta Med. 83(11) (2017) 912-920.

[40] C. Formisano, D. Rigano, F. Senatore, S. Bancheva, A. Maggio, S. Rosselli, M.

Bruno, Flavonoids in subtribe Centaureinae (Cass.) Dumort. (tribe Cardueae,

Asteraceae): distribution and (13)C-NMR spectral data, Chem. Biodivers. 9(10) (2012)

2096-158.

[41] J.Z. Yang, J.H. Jiang, G.L. Huang, B. Liu, Y. Liu, R. Zhan, Y.G. Chen, Two new

flavanone glycosides from Sunipia scariosa, Biochem. Syst. Ecol. 57 (2014) 317-321.

[42] Y. H.M., P. T., H. D.Q., L. X.N., L. B.Q., L. Z.Q., Y. J., H. G.M., L. Y.C.,

Chemical constituents from the ethyl acetate extract parts of QinzhuSiwu Tang,

Chinese Joumal of Experimental Traditional Medical Fomulae 19(3) (2013) 136-139.

[43] T. Tomimori, Y. Miyaichi, Y. Imoto, Y. Tanabe, Studies on the constituents of

Scutellaria Species. IV. On the flavonoid constituents of the root of Scutellaria

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

baicalensis Georgi, Yakugaku Zasshi 104(5) (1984) 529-534.

[44] Y. Q.W., R.J. Zhong, G.P. Zhou, H.Z. Fu, T.X. Zhang, Chemical constituents

from Camellia oleifera stem, Journal of Chinese Medicinal Materials 38

(2015) 2102-2104

[45] M.R. Marques, C. Stuker, N. Kichik, T. Tarrago, E. Giralt, A.F. Morel, I.I. Dalcol,

Flavonoids with prolyl oligopeptidase inhibitory activity isolated from Scutellaria

racemosa Pers, Fitoterapia 81(6) (2010) 552-556.

[46] T.K.P. Nguyen, K.P.P. Nguyen, F.S. Kamounah, W. Zhang, P.E. Hansen, NMR of

a series of novel hydroxyflavothiones, Magn. Reson. Chem. 47(12) (2009)

1043-1054.

[47] C. Formisano, D. Rigano, F. Senatore, S. Bancheva, A. Maggio, S. Rosselli, M.

Bruno, Flavonoids in Subtribe Centaureinae (Cass.) Dumort. (Tribe Cardueae,

Asteraceae): Distribution and 13C-NMR Spectral Data, Chem. Biodivers. 9(10) (2012)

2096-2158.

[48] L.L. Jing, X.F. Fan, Z.P. Jia, P.C. Fan, H.P. Ma, Convergent Synthesis of

Moslosooflavone, Isowogonin and Norwogonin from Chrysin, Nat. Prod. Commun.

10(3) (2015) 387-388.

[49] L.L. Jing, X.F. Fan, H.P. Ma, P.C. Fan, Z.P. Jia, Expeditious synthesis of A-ring

of polyoxygenated flavones from chrysin, Chemical Reagents 35(9) (2012) 782-784.

[50] V.M. Malikov, M.P. Yuldashev, Phenolic Compounds of plants of the Scutellaria

L. genus. Distribution, structure, and properties, Chem. Nat. Compd. 38(5) (2002)

358-406.

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

[51] Y. Miyaichi, E. Hanamitsu, H. Kizu, T. Tomimori, Studies on the constituents of

Scutellaria species (XXII). Constituents of the roots of Scutellaria amabilis HARA,

Chem. Pharm. Bull. (Tokyo) 54(4) (2006) 435-441.

[52] Q. Liu, K.R. Markham, P.W. Paré, R.A. Dixon, T.J. Mabry, Flavonoids from

elicitor-treated cell suspension cultures of Cephalocereus senilis, Phytochemistry,

32(4) (1993) 925-928.

[53] A.B. Gurung, B. Mayengbam, A. Bhattacharjee, Discovery of novel drug

candidates for inhibition of soluble epoxide hydrolase of arachidonic acid cascade

pathway implicated in atherosclerosis, Comput. Biol. Chem. 74 (2018) 1-11.

[54] K.S.S. Lee, J.Y. Liu, K.M. Wagner, S. Pakhomova, H. Dong, C. Morisseau, S.H.

Fu, J. Yang, P. Wang, A. Ulu, C.A. Mate, L.V. Nguyen, S.H. Hwang, M.L. Edin, A.A.

Mara, H. Wulff, M.E. Newcomer, D.C. Zeldin, B.D. Hammock, Optimized Inhibitors

of Soluble Epoxide Hydrolase Improve in Vitro Target Residence Time and in Vivo

Efficacy, J. Med. Chem. 57(16) (2014) 7016-7030.

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

Fig. 1. Chemical constituents isolated from S. baicalensis

Fig. 2. (A-C) Compounds 25-27 displayed concentration-dependent inhibitory

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

activities against sEH. (D-F) Michealis-Menten plots of compounds 25-27 against

sEH. (G-I) Lineweaver-Burk plots of compounds 25-27 against sEH.

Fig. 3. Catalytic cavities (A-C) and 3D structures (D-F) of compounds 25-27 with

sEH

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

Fig. 4. Interaction mechanisms of compounds 25-27 (A-C) with sEH

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

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.

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

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

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

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.

ACCEPTED MANUSCRIPT