Inhibition of Soluble Epoxide Hydrolase Activity by Components of Glycyrrhiza uralensis

Soluble epoxide hydrolase (sEH) is a target enzyme for the treatment of inflammation and cardiovascular disease. A Glycyrrhiza uralensis extract exhibited ~50% inhibition of sEH at 100 μg/mL, and column chromatography yielded compounds 1–11. Inhibitors 1, 4–6, 9, and 11 were non-competitive; inhibitors 3, 7, 8, and 10 were competitive. The IC50 value of inhibitor 10 was below 2 μM. Molecular simulation was used to identify the sEH binding site. Glycycoumarin (10) requires further evaluation in cells and animals.

In particular, sEH is an enzyme mainly expressed in the liver, and alcohol-induced inflammation, injury, and steatosis were reduced in hepatic sEH-knockout mice [10]. Moreover, an sEH inhibitor, PTUPB, inhibited the expression of TNF-a, MCP-1, and IL-6 in non-alcoholic fatty liver disease mice [11]. Therefore, sEH inhibition is known as a target enzyme to reduce the inflammatory response in alcohol-or non-alcohol-induced inflammation in the liver [10,11].
Licorice (Gancao) prepared from the roots of Glycyrrhiza species (Fabaceae) is widely used to treat various human ailments [12]. The genus Glycyrrhiza contains approximately 30 species of perennial herbs, principally derived from Asia, Europe, North and South America, and Australia [13]. Three species, Glycyrrhiza glabra L., G. inflata Bat., and G. uralensis Fisch., are recognized by the Chinese Pharmacopoeia as sources of licorice [14]. During our continuing efforts to identify effective natural sEH inhibitors, we found that G. uralensis root extracts exhibited significant anti-sEH effects, and we isolated eleven active flavonoids. We here report the isolation of these materials, and their sEH inhibitory effects.

Extract and Isolation of Materials
An ethanol extract of G. uralensis roots that significantly inhibited sEH activity (50.6 ± 0.8% inhibition at 100 µg/mL) was suspended in water and successively partitioned using CHCl 3 and EtOAc. These fractions were subjected to a series of chromatographic steps to isolate flavonones 1-3 [15], chalcones 4-7 [16], and an isoflavonoid 8 [15] from the EtOAc-soluble fraction, and coumestan 9 [16], prenyl-coumarine 10 [16], and prenyl-isoflavonoid 11 [16] from the CHCl 3 -soluble fraction. The chemical structures of these isolates were identified by comparing their physicochemical and spectral data to those of the literature (Figures 1 and S1-S11) Licorice (Gancao) prepared from the roots of Glycyrrhiza species (Fabaceae) is widely used to treat various human ailments [12]. The genus Glycyrrhiza contains approximately 30 species of perennial herbs, principally derived from Asia, Europe, North and South America, and Australia [13]. Three species, Glycyrrhiza glabra L., G. inflata Bat., and G. uralensis Fisch., are recognized by the Chinese Pharmacopoeia as sources of licorice [14]. During our continuing efforts to identify effective natural sEH inhibitors, we found that G. uralensis root extracts exhibited significant anti-sEH effects, and we isolated eleven active flavonoids. We here report the isolation of these materials, and their sEH inhibitory effects.

Inhibition of sEH by the Isolated Compounds
The inhibitory effects of the isolated compounds 1-11 against sEH in vitro were determined using a modification of an earlier method, employing commercially available AUDA, a known inhibitor of sEH, as the reference standard. Although the sEH inhibitory activities of the isolates were less potent than those of AUDA (IC50, 22 ± 0.8 nM), all tested compounds except 2 exhibited a considerable inhibition (Equation (1)) of sEH activity, with IC50 values (Equation (2)) from 1.9 ± 0.2 to 85.7 ± 1.2 μM (Figure 2A,B, Table 1).

Inhibition of sEH by the Isolated Compounds
The inhibitory effects of the isolated compounds 1-11 against sEH in vitro were determined using a modification of an earlier method, employing commercially available AUDA, a known inhibitor of sEH, as the reference standard. Although the sEH inhibitory activities of the isolates were less potent than those of AUDA (IC 50 , 22 ± 0.8 nM), all tested compounds except 2 exhibited a considerable inhibition (Equation (1)) of sEH activity, with IC 50 values (Equation (2)) from 1.9 ± 0.2 to 85.7 ± 1.2 µM (Figure 2A,B, Table 1).   To further characterize the inhibitory behaviors, enzyme kinetics were studied in the presence of various concentrations of 10 active compounds (1 and 3-11). As shown by the Lineweaver-Burk plot ( Figure   To further characterize the inhibitory behaviors, enzyme kinetics were studied in the presence of various concentrations of 10 active compounds (1 and 3-11). As shown by the Lineweaver-Burk plot ( Figure 2C-L), the x-intercept (−1/Km) was unaffected by increasing concentrations of 1, 4-6, 9, and 11, but 1/Vmax gradually increased, indicating that these compounds were noncompetitive inhibitors. On the other hand, compounds 3, 7, 8, and 10 were competitive inhibitors, with different Km values. Dixon plot analysis indicated that the inhibition constants (k i values) of compounds 1 and 3-11 ranged from 2.0 to 89.1 µM ( Figure 3A-J, Table 1).

Molecular Docking
To predict the binding of the sEH inhibitors 1 and 3-11 to sEH, we performed molecular docking analyses guided by the enzyme kinetic data. The noncompetitive inhibitors 1, 4-6, 9, and 11 were subjected to blind docking, and the competitive inhibitors 3, 7, 8, and 10 were subjected to docking at the active site. As indicated in Figure 4 and Table 2, compounds 1 and 3-11 bound stably to she, with autodock scores of −8.25, −9.38, −8.51, −10.06, −9.15, −7.94, −9.22, −9.40, −8.57, and −10.38 kcal/mol, respectively. All the compounds formed hydrogen bonds with amino acid residues of she, both in and around the binding pocket. The results are described in detail in Figure 4 and Table 2.

Molecular Dynamics
A dynamic study was performed, based on the docking results, to predict the binding of 10 to sEH. As shown in Figure 5A, the complex of compound 10 and sEH exhibited stable fluidic motion. The inhibitor initially bound to the active site, but towards the right pocket (Pro371-Met469), commencing at 3 ns. Compound 10 evidenced root mean square deviation (RMSD) values of about 3 Å, and a potential energy of approximately −2.7 × 106 kJ/mol across the duration of the simulation ( Figure 5B,C). The enzyme residues affected by inhibitor 10 showed fluidities below 3 Å of the root mean square fluctuations (RMSFs) ( Figure 5D), which were maintained principally by one, but occasionally two to four, hydrogen bonds ( Figure 5E). Inhibitor 10 maintained a distance within 4 Å from the active site, and could thus bind Phe371 and Met469 ( Figure 5F-H).  Table 1).

Molecular Dynamics
A dynamic study was performed, based on the docking results, to predict the binding of 10 to sEH. As shown in Figure 5A, the complex of compound 10 and sEH exhibited stable fluidic motion. The inhibitor initially bound to the active site, but towards the right pocket (Pro371-Met469), commencing at 3 ns. Compound 10 evidenced root mean square deviation (RMSD) values of about 3 Å, and a potential energy of approximately −2.7 × 106 kJ/mol across the duration of the simulation ( Figure 5B,C). The enzyme residues affected by inhibitor 10 showed fluidities below 3 Å of the root mean square fluctuations (RMSFs) ( Figure 5D), which were maintained principally by one, but occasionally two to four, hydrogen bonds ( Figure 5E). Inhibitor 10 maintained a distance within 4 Å from the active site, and could thus bind Phe371 and Met469 ( Figure 5F-H).

Discussion
sEH inhibition elevates EET levels, which would be expected to elicit a variety of beneficial biological effects [17] that effectively treat atherosclerosis, diabetes, hypertension, lung disease, pain, inflammation, immune disorders, and other diseases [18]. Thus, sEH is a potential pharmaceutical target. Secondary metabolites of medicinal plants play important roles in drug discovery by providing lead scaffolds that can then be optimized

Discussion
sEH inhibition elevates EET levels, which would be expected to elicit a variety of beneficial biological effects [17] that effectively treat atherosclerosis, diabetes, hypertension, lung disease, pain, inflammation, immune disorders, and other diseases [18]. Thus, sEH is a potential pharmaceutical target. Secondary metabolites of medicinal plants play important roles in drug discovery by providing lead scaffolds that can then be optimized by synthetic and medicinal chemists. So far, research has been conducted to develop sEH inhibitors from natural products, and, as a result, flavonoids [19], triterpenoids [20], and macamides [21] are representative compounds.
During our continuous efforts to identify potent sEH inhibitors in medicinal plants, we focused on the polyphenols of G. uralensis roots; these evidence diverse biological activities. G. uralensis is one of the most popular Chinese medicinal herbs, and has been shown to exhibit various pharmacological activities, including antidiabetic, anti-inflammatory, antioxidant, antiviral, cytotoxic, skin-whitening, hepatoprotective, and cholinergic properties [22][23][24]. The roots of this plant contain various secondary metabolites, and include different classes of phenolic compounds, such as flavonoids, chalcones, coumarins, and triterpenoid saponins [25]. Of these, flavonoids have frequently been reported to show major anti-inflammatory, antibacterial, antimicrobial, antioxidant, and cytotoxic activities [25]. We thus screened a licorice ethanol extract in terms of sEH inhibition, which showed 50.6 ± 0.8% inhibition at 100 µg/mL. However, the value for glycyrrhizin, the main compound of G. uralensis, was only 18.4 ± 0.5% at 100 µM; we isolated eleven polyphenol components 1-11 from G. uralensis in a search for potential inhibitors. Except for coumarin 10, chalcone compounds 4-7 exhibited higher efficacies than flavonoids 1-3, isoflavonoids 8 and 11, and coumestan 9. In particular, compounds 1, 4, and 7, in the form of aglycones, showed better efficacies than glycosides 2, 3, 5, and 6. In addition, glycosides with two sugars (3 and 6) were more effective than those with one sugar (2 and 5). When comparing the biological activities of 4-7 to 1-3, as well as the activity of 10 to 9, inhibitors 4-7 and 10, with flexible carbon-carbon bonds, showed better inhibitory activities. In particular, the potential inhibitor 10 remained~3.5 Å distant from Pro371 and Met469 over the simulation time. This inhibitor remained bound in a mobile fashion to the active site and the adjacent pocket. Flexible compounds would be expected to more easily bind to sEH than inflexible materials.

sEH Assay
To evaluate inhibitory activities, 130 µL of an sEH solution in 25 mM bis-Tris-HCl buffer (pH 7.0), with 0.1% BSA, was added to 20 µL of a putative inhibitor dissolved in MeOH. Next, 50 µL of a substrate was added, and the mixture was incubated at 37 • C to allow for sEH hydrolysis. Product development was monitored (330 nm excitation wavelength and 465 nm emission wavelength) for approximately 40 min. The inhibitory activity was calculated as follows: Inhibitory activity (%) = [(∆C − ∆I)/∆C] × 100 (1) where ∆C and ∆I are the optical densities of the control and inhibitor tubes, respectively, after 40 min, and: where y0 is the minimum y-axis value, a is the difference between the maximum and minimum values, and b is the x value at 50% of the a value.

Molecular Docking
To dock ligands to receptors, two three-dimensional (3D) ligand structures were prepared and minimized using Chem3D Pro (CambridgeSoft, Cambridge, MA, USA). The receptor protein structure coded in 3ANS was downloaded from the RCSB data bank. Only the A-chain of the enzyme is involved in docking; we did not evaluate the B-chain. Water and 4-cyano-N-[(1S,2R)-2-phenylcyclopropyl]-benzamide were excluded from the A-chain. The revised A-chain was hydrogenated using AutoDockTools (Scripps Research, La Jolla, CA, USA); the Gasteiger charge model was then applied. Flexible ligand docking was evaluated using a torsion tree that detected torsion roots and rotatable bonds. The grid box sizes were 126 × 126 × 126 at 0.375 Å and 60 × 60 × 60 (center grid box: x, 24.612; y, 26.057; z, 117.11) for blinded docking and ligand docking to the active site, respectively. Molecular docking was evaluated using a Lamarckian algorithm and the maximum possible evaluation number. The resulting values were calculated and visualized using AutoDockTools, Chimera ver. 1.14 (San Francisco, CA, USA), and LIGPLOT (European Bioinformatics Institute, Hinxton, UK).

Molecular Dynamics
3D ligand structures were built using the GlycoBioChem server. The sEH Gro was developed using the GROMOS96 45a3 force field. All complexes were surrounded by water molecules (cubic size 12 × 12 × 12) and six Cl anions. The energy level attained 10.0 kJ/mol via steepest descent minimization. Each inhibitor-sEH complex was sequentially subjected to NVT equilibration at 300 K, NPT and Particle Mesh Ewald evaluation of long-range electrostatics at 1 bar, and MD simulation for 30 ns.

Statistical Analysis
All measurements were performed in triplicate (three independent experiments) and the results are means ± standard errors of the means (SEMs). The results were compared using Sigma Plot 10.0 (Systat Software Inc., San Jose, CA, USA).

Conclusions
Bioactivity screening revealed that an ethanol extract of G. uralensis roots inhibited sEH, but the main compound, glycyrrhizin, lacked such an effect. In this study, eleven polyphenol compounds, 1-11, were isolated from G. uralensis. Among them, compounds 1, 3-9, and 11 were found to exert moderate inhibitory effects, and inhibitor 10 showed a potent effect, with an IC 50 value of 1.9 ± 0.2 µM. Molecular simulation was used to study the binding of 1, and 3-11 to sEH and it was found that the potential inhibitor 10 maintained a stable fluidic bond with the enzyme. Therefore, of the polyphenols, compound 10 was the strongest sEH inhibitor.