Soluble Epoxide Hydrolase Inhibitory Activity of Selaginellin Derivatives from Selaginella tamariscina

Selaginellin derivatives 1–3 isolated from Selaginella tamariscina were evaluated for their inhibition of soluble epoxide hydrolase (sEH) to demonstrate their potential for the treatment of cardiovascular disease. All selaginellin derivatives (1–3) inhibited sEH enzymatic activity and PHOME hydrolysis, in a dose-dependent manner, with IC50 values of 3.1 ± 0.1, 8.2 ± 2.2, and 4.2 ± 0.2 μM, respectively. We further determined that the derivatives function as non-competitive inhibitors. Moreover, the predicted that binding sites and interaction between 1–3 and sEH were solved by docking simulations. According to quantitative analysis, 1–3 were confirmed to have high content in the roots of S. tamariscina; among them, selaginellin 3 exhibited the highest content of 189.3 ± 0.0 μg/g.


Introduction
Selaginella tamariscina, belonging to the genus Selaginellaceae, is used as a Chinese and Korean herbal medicine to promote blood circulation and for the treatment of cancer, hepatitis, diabetes, and skin diseases [1]. Fifteen derivatives, selaginellin A-N, have been isolated from Selaginella since 2007 [2]. Their rare structure includes a carbon scaffold containing pigments similar to the alkynylphenol and para-quinone methide skeleton [1]. Due to characteristic features of this structure, their color changes from red to pink within a pH range of 1.5 to 1, and from red to purple within a pH range of 7.5 to 8.0. In addition, their UV-Vis absorption intensities change according to the pH [2]. In biosynthesis pathways, selaginellin derivatives are constructed from the basic structure of orsellinic acid, and produced by type III polyketide synthases (type III PKS) using four malonyl-CoA [3]. Their inhibitory effects on the expression of HeLa cells, MCF-7, and free radical scavenging, as well as their protective effects against cytotoxicity and apoptosis, have been shown in differentiated PC12 cells [1,2,4].
Soluble epoxide hydrolase (sEH) is widely present in mammalian tissues. sEH consists of a homodimer of~62-kDa monomeric units, with a C-terminal domain of~35 kDa, which provides its epoxide hydrolase activity; this is responsible for the hydrolysis function that converts the epoxide to the corresponding diol [5,10]. For example, 14,15-EETs are metabolized as corresponding 14,15-dihydroxyeicosatrienoic acids (DHETs) by sEH [5]. Recent efforts have attempted to develop an sEH inhibitor for the treatment of cardiovascular disease. A previous study found that urea-type inhibitors, such as AUDA and t-AUCB, exhibit inhibitory activity on sEH within the nanomolar range [5,11]. However, some limitations of these inhibitors include their low solubility and increased metabolism [12]. The research described herein focuses on efforts to develop a new sEH inhibitor from S. tamariscina. The inhibitory activity of isolated selaginellin derivatives 1-3 on sEH was examined through enzyme kinetics and docking simulations to provide insight into the interaction between receptor and ligand, as well as a quantitative analysis by comparison with methanol extracts from both the whole plants and roots.
Soluble epoxide hydrolase (sEH) is widely present in mammalian tissues. sEH consists of a homodimer of ~62-kDa monomeric units, with a C-terminal domain of ~35 kDa, which provides its epoxide hydrolase activity; this is responsible for the hydrolysis function that converts the epoxide to the corresponding diol [5,10]. For example, 14,15-EETs are metabolized as corresponding 14,15-dihydroxyeicosatrienoic acids (DHETs) by sEH [5]. Recent efforts have attempted to develop an sEH inhibitor for the treatment of cardiovascular disease. A previous study found that urea-type inhibitors, such as AUDA and t-AUCB, exhibit inhibitory activity on sEH within the nanomolar range [5,11]. However, some limitations of these inhibitors include their low solubility and increased metabolism [12]. The research described herein focuses on efforts to develop a new sEH inhibitor from S. tamariscina. The inhibitory activity of isolated selaginellin derivatives 1-3 on sEH was examined through enzyme kinetics and docking simulations to provide insight into the interaction between receptor and ligand, as well as a quantitative analysis by comparison with methanol extracts from both the whole plants and roots.

Docking Calculation
For insight into the interaction between molecule and ligand, a docking simulation was performed using the AutoDock software (version 4.2) from The Scripps Research Institute [16,17]. On the basis of a non-competitive mechanism, the docking simulation was tested in the blind docking of

Docking Calculation
For insight into the interaction between molecule and ligand, a docking simulation was performed using the AutoDock software (version 4.2) from The Scripps Research Institute [16,17].
On the basis of a non-competitive mechanism, the docking simulation was tested in the blind docking of each condition on the free enzyme or enzyme-substrate complex (PDB ID: 3ANS from RCSB) with the three-dimensional structure of the ligand, which was sketched and structurally optimized by minimization with minimun RMS gradient (0.01) implemented in Chem3D 12.0 (ChembridgeSoft). The predicted binding site was selected so that the favorable cluster possessed the lowest energy and the largest number of superpositions of the ligand on the enzyme. A cluster analysis was performed by 2.0 Å rmsd from 50 docking runs (Supporting information, Figure S11). Figure 3A-F displays the interactions between selaginellin A (1), selaginellin B (2), and selaginellin (3) with the free enzyme and the enzyme-substrate complex. The ligand from the original protein database was suggested as a substrate due to the binding form in the active site. These results revealed differences in the predicted binding sites of 1-3 in two situations: the docked pose of 1 with free enzyme (binding energy´10.81 kcal/mol) showed that the three hydroxyl groups and one ketone group were bound by hydrogen to ASN472 (2.  Table 2). These bound to predicted binding sites including a portion of the active site, with free enzyme exhibiting the best pose at pocket A, which was located far from the active site within the enzyme-substrate complex ( Figure S12).
Molecules 2015, 20, page-page 4 each condition on the free enzyme or enzyme-substrate complex (PDB ID: 3ANS from RCSB) with the three-dimensional structure of the ligand, which was sketched and structurally optimized by minimization with minimun RMS gradient (0.01) implemented in Chem3D 12.0 (ChembridgeSoft). The predicted binding site was selected so that the favorable cluster possessed the lowest energy and the largest number of superpositions of the ligand on the enzyme. A cluster analysis was performed by 2.0 Å rmsd from 50 docking runs (Supporting information, Figure S11). Figure 3A-F displays the interactions between selaginellin A (1), selaginellin B (2), and selaginellin (3) Table 2). These bound to predicted binding sites including a portion of the active site, with free enzyme exhibiting the best pose at pocket A, which was located far from the active site within the enzyme-substrate complex ( Figure S12).

HPLC Analysis
High-performance liquid chromatography (HPLC) was used to separate selaginellin derivatives 1-3 and compare the quantitative content between methanol extracts of whole plant and roots of S. tamariscina. Mixed standards (1-3) were measured by HPLC at 280 nm ( Figure 4). Peak retention times were as follows: compound 3, Rt = 24.6 min; compound 1, Rt = 31.0 min; compound 2, Rt = 32.6 min. Profiles of extracts 1-3 were confirmed by comparing their peak retention times with those of the standards (Figure 4). Their contents were calculated as shown in Table 3. The selaginellin B (2) and selaginellin (3) contents in the whole S. tamariscina plant were similar to those reported previously [18]. The contents of the individual compounds differed markedly depending on whether they were extracted from the whole plants or roots. The contents of 1-3 were low in whole methanol extracts. The total amount of selaginellin 3 was three-and six-fold higher than that of selaginellin A (1) and B (2), respectively. However, the contents of 1-3 from root extracts were three-to four-fold higher than those from whole methanol extracts. Specifically, selaginellin 3 was confirmed, with a content of 189.3 ± 0.0 μg/g in the dried roots of S. tamariscina, which was revaluated as a high precious part to obtain selaginellin 3 ( Table 3). The coefficient of correlation (R 2 ) > 0.99 of the three standards indicated good linearity. Limits of detection (LODs) of the method ranged from 0.1-0.2 μg/mL, and limits of

HPLC Analysis
High-performance liquid chromatography (HPLC) was used to separate selaginellin derivatives 1-3 and compare the quantitative content between methanol extracts of whole plant and roots of S. tamariscina. Mixed standards (1-3) were measured by HPLC at 280 nm ( Figure 4). Peak retention times were as follows: compound 3, Rt = 24.6 min; compound 1, Rt = 31.0 min; compound 2, Rt = 32.6 min. Profiles of extracts 1-3 were confirmed by comparing their peak retention times with those of the standards (Figure 4). Their contents were calculated as shown in Table 3. The selaginellin B (2) and selaginellin (3) contents in the whole S. tamariscina plant were similar to those reported previously [18]. The contents of the individual compounds differed markedly depending on whether they were extracted from the whole plants or roots. The contents of 1-3 were low in whole methanol extracts. The total amount of selaginellin 3 was three-and six-fold higher than that of selaginellin A (1) and B (2), respectively. However, the contents of 1-3 from root extracts were three-to four-fold higher than those from whole methanol extracts. Specifically, selaginellin 3 was confirmed, with a content of 189.3˘0.0 µg/g in the dried roots of S. tamariscina, which was revaluated as a high precious part to obtain selaginellin 3 ( Table 3). The coefficient of correlation (R 2 ) > 0.99 of the three standards indicated good linearity. Limits of detection (LODs) of the method ranged from 0.1-0.2 µg/mL, and limits of quantitation (LOQs) ranged from 0.3-0.6 µg/mL (Table S1). Furthermore, the precision values (% relative standard deviation [RSD]) were 0.3%-2.1% for intra-day and 0.5%-1.6% for inter-day. However, the accuracy (%) was 98.2%-102.5% for intra-day and 96.1%-105.4% for inter-day. Thus, the newly developed method was validated in terms of its LOD, LOQ, linearity, precision (intra-and inter-day), and accuracy (intra-and inter-day) (Table S2).

General Experimental Procedures
NMR spectra was obtained on Bruker Advance 600 and 300 MHz (Bruker; Rheinstetten, Germany) spectrometer. Column chromatography was performed using silica gel (Kieselgel 60; 70-230; and 230-400 mesh; Merck; Darmstadt, Germany) and YMC RP-18 resins. Thin layer chromatography (TLC) was performed using pre-coated silica-gel 60 F254 and RP-18 F254S plates (both 0.25 mm; Merck; Darmstadt, Germany). Spots were visualized by spraying with 10% aqueous H2SO4 solution followed by heating. Methanol; acetonitrile (Sigma-Aldrich; St. Louis, MO, USA) and trifluoroacetic acid (Alfa Aesar; Ward Hill, MA, USA) used in this study were of HPLC grade and the distilled water was prepared by the ultra-pure water manufacturing device (Optimos; Sinhan Science Tech, Daejeon, Korea). The chromatographic system was Shimazdu prominence system equipped with a SPD-20A dual UV-Vis detector and installed with a Shimadzu LC solution software (Ver. 1.25; Shimadzu; Kyoto, Japan). The quantitative analysis for three selaginellins was carried out on Optimapak C18

Extraction and Isolation
Dried whole plant (2.4 kg) was extracted three times with 95% methanol (36 L) at 50˝C for 5 h. Concentrated methanol extract (270 g) was suspended in distilled water (2.5 L) and successively partitioned with n-hexane, chloroform, ethyl acetate, and buthanol. Ethyl acetate layer (21 g) was chromatographed with silica gel column chromatography using the gradient system of chloroform and methanol (from 100:1 to 1:1) to obtain seven fractions (2A to 2G). Fraction 2D was subjected to silica gel column chromatography with gradient solvent system of chloroform and methanol (from 50:1 to 0.5:1) to achieve eight fractions (2DA to 2DH). Fraction 2DF was divided by RP-18 column chromatography using the isocratic solvent system of 60% methanol, and then pure compound, selaginellin (3) was gained. Fraction 2DE was chromatographed with RP-18 column chromatography using the gradient solvent system of distilled water and methanol (from 100:1 to 0.2:1) to achieve selaginellin A (1) and B (2).

Enzymatic Assay
Briefly, 130 µL of sEH (62.5 ng/mL) in 25 mM bis-Tris-HCl buffer (pH 7.0) containing 0.1% BSA and 20 µL of various concentrations of the inhibitors dissolved in MeOH were added in a 96 well plate and then mixed with 50 µL of 40 µM PHOME at 37˝C. The mixture was recorded at excitation and emission of 330 and 465 nm for one hour [15]. The inhibitory ratio was calculated according to the following equation: Inhibitory activity rate p%q " 100´rpS 60´S0 q{pC 60´C0 qsˆ100 (1) where C 60 and S 60 were the fluorescence of control and inhibitor after 60 min, S 0 and C 0 were the fluorescence of control and inhibitor at zero min.

sEH Kinetic Analysis
To analyze the non-competitive inhibition, the Lineweaver-Burk equation can be written in double reciprocal form: Secondary plots can be constructed from

Docking Calculation
The 3D structure of the ligand was modeled and minimized by MM2 using the Chem3D Pro (version: 12.0). The flexible bonds of ligand were assigned with AutoDockTools. The protein structure (PDB ID: 3ANS) was downloaded from RCSB (protein data bank), after which the water, or the water and inhibitor suggested as substrate were removed from receptor complexes using Chimera. All hydrogen atoms and gasteiger charges in two protein structures were added. Simulation studies were performed using the AutoDock program (version 4.2). Briefly, to perform blind docking in AutoDock 4.2, the grid dimensions were established using grid enzyme center, number of points (X: 126, Y: 126, Z: 126), and spacing (0.375 Å). Docking simulation of protein structures and ligand were performed using the Lamarckian Genetic Algorithm (Runs 50 and the maximum number of evaluations was set as long). The docking simulation results were prepared using Chimera [16,17].

HPLC Analysis Condition
The quantitative analysis for three selaginellin derivatives 1-3 from each sample solution was performed on a Shimadzu Prominence system. All samples were analyzed on an Optimapak ODS C18 column by mobile phase constituted with (A) 0.1% trifluoroacetic acid aqueous solution and (B) acetonitrile containing with 0.1% trifluoroacetic acid. The elution conditions applied were: 5%-75% B for 40 min, 75% B for 5 min, 75%-5% B for 1 min, and 5% B for 9 min, successively. The flow rate and injection volume were 1 mL/min and 20 µL, respectively. This system was operated at 30˝C. All peaks were detected at wavelength of 280 nm. Finally, total run time of quantitative analysis was 55 min.

Statistical Analysis
All activity tests in the presence of inhibitors were performed in triplicate and results are presented as the means˘standard error of the mean (SEM). The results were subjected to analysis using Sigma Plot (SPP Inc., Chicago, IL, USA).

Conclusions
Selaginellin derivatives (1-3) generated with a rare carbon scaffold were isolated from S. tamariscina using silica gel, C-18, and Sephadex LH-20 column chromatography. The derivatives exhibited low IC 50 values of 3.1˘0.1 (1), 8.2˘2.2 (2), and 4.2˘0.2 µM (3) against sEH. Regarding enzyme kinetics, Lineweaver-Burk plots indicated that lines generating using various inhibitor concentrations crossed the equal x-intercept on the abscissa, and Equation (3) of secondary plots yielded the following Ki values: 1, 2.9˘1.2; 2, 6.8˘0.5; 3, 1.8˘1.5 µM. The derivatives were confirmed to interact with sEH as non-competitive inhibitors. Therefore, the enzyme is involved in the reaction in two of four states: free enzyme, enzyme-substrate complex, enzyme-inhibitor complex, or enzyme-substrate-inhibitor complex. Selaginellin derivatives 1-3 likely bind with the enzyme via two routes, the free enzyme or enzyme-substrate complex. Therefore, compounds 1-3 were subjected to a docking simulation with free enzyme and the enzyme-substrate complex, respectively. In the former route, all derivatives occupied pocket A and a small portion of the substrate in the free enzyme, but only pocket A of the enzyme-substrate complex in the latter route. This study suggests that compounds 1-3 occupy pocket A and a small portion of the substrate when bound to free enzymes. Furthermore, when the substrate was added to the active site, the compounds could fully slide into pocket A simultaneously. Regarding the second route, 1-3 may directly bind to pocket A when bound to the enzyme-substrate complex. Additionally, according to these docking simulation results, ASN 472 in pocket A, which is the positively charged amino acid, plays the common catalytic residues relating to the binding with all selaginellin derivatives (1-3). These proposals may be a clue for the development of new sEH inhibitor instead of urea-type inhibitors. Furthermore, an HPLC analysis of selaginellin derivative (1-3) contents in whole plants and roots of S. tamariscina was developed and validated for the first time. The contents of compounds 1-3 were higher in the root extract than in whole plants. Among them, selaginellin 3 exhibited the highest content in dried roots (189.3˘0.0 µg per 1 g). This study was the first to analyze selaginellins in the roots and whole plants of S. tamariscina, and included evaluation of their sEH inhibitory activity, enzyme kinetics, and docking simulations. Overall, we conclude that selaginellin derivatives 1-3 are a promising lead compounds for development of inhibitor targeting sEH.