Soluble Epoxide Hydrolase Inhibitory Constituents from the Heartwood of Toxicodendron vernicifluum: Isolation, Kinetic Characterization, Molecular Modeling, and Quantitative Analysis

Soluble Epoxide Hydrolase Inhibitory Constituents from the Heartwood of Toxicodendron vernicifluum: Isolation, Kinetic Characterization, Molecular Modeling, and Quantitative Analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Soluble Epoxide Hydrolase Inhibitory Constituents from the Heartwood of Toxicodendron vernicifluum: Isolation, Kinetic Characterization, Molecular Modeling, and Quantitative Analysis Jang Hoon Jang, Jae-Young Cheon, Jin Yu, Yong-Goo Kim, Sung Yeon Kim, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8274523/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Soluble epoxide hydrolase (sEH) is a therapeutic target for managing inflammation by preserving anti-inflammatory epoxy fatty acids (EpFAs). This study investigates the sEH inhibitory potential of secondary metabolites isolated from the heartwood of Toxicodendron vernicifluum (Stokes) F.A. Barkley. Bioactivity-guided fractionation of the ethanol extract revealed that the ethyl acetate-soluble fraction possessed significant sEH inhibitory activity (~ 60% at 50 µg/mL). Subsequent purification yielded 11 polyphenolic compounds, which were identified via spectroscopic methods and quantified using a validated Ultra-High-Performance Liquid Chromatography (UPLC) protocol. Among these, the aurone sulfuretin ( 7 ) and the flavonol fisetin ( 5 ) exhibited potent competitive inhibition with IC 50 values of 8.8 ± 0.3 µM and 9.6 ± 0.8 µM, respectively. The chalcone butein ( 9 ) demonstrated strong non-competitive inhibition (IC 50 = 21.4 ± 1.5 µM). Molecular docking and 100 ns molecular dynamics (MD) simulations revealed that sulfuretin forms a stable high-affinity complex within the sEH catalytic pocket, anchored by persistent hydrogen bonds with Asp335 and Tyr383. Conversely, butein interacts with a peripheral allosteric site. These findings highlight T. vernicifluum heartwood as a source of diverse sEH inhibitors with potential for development as anti-inflammatory agents. Biological sciences/Biochemistry Biological sciences/Chemical biology Physical sciences/Chemistry Biological sciences/Drug discovery Toxicodendron vernicifluum flavonoid soluble epoxide hydrolase competitive inhibitor molecular docking Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The regulation of inflammation and vascular tone relies heavily on the metabolism of polyunsaturated fatty acids. Soluble epoxide hydrolase (sEH, EC 3.3.2.10) plays a pivotal role in this process by hydrolyzing epoxy fatty acids (EpFAs), such as epoxyeicosatrienoic acids (EETs) and epoxydocosapentaenoic acids (EDPs), into their corresponding diols [ 1 , 2 ]. Since EpFAs possess potent anti-inflammatory and vasodilatory properties, their rapid degradation by sEH attenuates the body's natural protective responses [ 1 ]. Consequently, the inhibition of sEH has emerged as a promising therapeutic strategy for treating various inflammatory conditions, including cardiovascular, neurological, and renal disorders [ 3 ]. By preventing the hydrolysis of EpFAs, sEH inhibitors stabilize endogenous levels of these beneficial lipids, thereby enhancing their protective and anti-fibrotic effects [ 4 , 5 ]. While synthetic sEH inhibitors have demonstrated efficacy in preclinical models [ 4 ], and candidates such as EC5026 have progressed to clinical trials [ 6 ], concerns regarding pharmacokinetic limitations and potential adverse effects persist [ 7 ]. These challenges have necessitated the search for alternative inhibitory scaffolds from natural sources. Natural products, characterized by their immense structural diversity, represent a compelling reservoir for novel therapeutic agents. Although natural inhibitors may sometimes exhibit lower potency compared to optimized synthetic analogues, they often offer distinct advantages in terms of safety profiles and accessibility [ 4 ]. Therefore, exploring plant-derived compounds, particularly those with a history of ethnopharmacological use, offers a strategic avenue to discover novel sEH modulators that bridge traditional medicine and modern pharmacology [ 8 ]. Toxicodendron vernicifluum (Stokes) F.A. Barkley, commonly known as the Chinese or Japanese lacquer tree, is a deciduous species of the Anacardiaceae family native to East Asia [ 9 ]. While the tree is widely cultivated for the industrial application of its sap in lacquerware, it also holds significant value in traditional East Asian medicine. Various parts of the plant, including the heartwood, leaves, and seeds, have been historically utilized to treat ailments ranging from internal parasites and hemorrhage to inflammatory conditions [ 10 , 11 ]. Modern phytochemical investigations have revealed a complex matrix of bioactive constituents in T. vernicifluum , including flavonoids, polyphenols, triterpenes, and urushiols [ 11 ]. However, despite well-documented evidence of the plant's anti-inflammatory properties, the specific molecular mechanisms underlying these effects, specifically its potential modulation of the arachidonic acid pathway via sEH inhibition, remain largely unexplored. We hypothesized that the anti-inflammatory efficacy of T. vernicifluum heartwood is mediated, at least in part, by the inhibition of sEH. Therefore, the primary objective of this study was to isolate and characterize the active sEH inhibitory flavonoid compounds from the ethanol extract of T. vernicifluum heartwood using a bioactivity-guided isolation strategy. Furthermore, we aimed to employ a multidisciplinary approach, including enzyme kinetic assays, molecular docking, and molecular dynamics (MD) simulations, to provide a structural basis for the observed sEH inhibitory activity. Finally, we used Ultra-High-Performance Liquid Chromatography (UPLC) to quantify these active principles, linking the level of the specific flavonoids to the overall sEH inhibitory potential of the heartwood extract. Ultimately, this comprehensive profiling seeks to highlight the potential of these specific flavonoids as lead compounds for novel anti-inflammatory therapeutics. Materials and Methods Chemicals and reagents The solvents used for separation (methanol, n -hexane, chloroform, and ethyl acetate) were purchased from Duksan General Science (Seoul, Republic of Korea). Chromatographic separations were performed via thin layer chromatography (TLC) using glass plates pre-coated with silica gel 60 F 254 and silica gel RP-18 F 254 (20 × \(\:\:\) 20 cm; Merck, Darmstadt, Germany). Materials were visualized under ultraviolet light at 254 nm and 365 nm, respectively. The TLC color reagent was 10% ethanol–sulfuric acid. Column chromatography employed 230–400-mesh silica gel 60 (Merck) and 12-nm ODS-A (YMC, Kyoto, Japan) columns. Nuclear magnetic resonance (NMR) data were acquired using a Bruker Avance Ⅲ 300-MHz platform (Bruker, Billerica, MA, USA). Reference standards (ethyl gallate, fustin, garbanzol, fisetin, sulfuretin, and butein; all > 98% purity) were utilized. High-performance liquid chromatography (HPLC)-grade acetonitrile, methanol, and water were from J.T. Baker; analytical grade formic acid was purchased from Merck (St. Louis, MO, USA). Human recombinant sEH (10011669), PHOME (10009134), and AUDA (10007972) were from Cayman Chemical (Ann Arbor, MI, USA). Plant material T. vernicifluum heartwood was obtained from a herbal medicinal company of the Republic of Korea in August 2023. The plant was identified by Dr. J.H. Kim. A voucher specimen (TV 23) has been deposited in the herbarium of the Department of Herbal Crop Research, National Institute of Horticultural and Herbal Science, Republic of Korea. T. vernicifluum heartwood was obtained from a herbal medicinal company of the Republic of Korea in August 2023. The plant was identified by Dr. J.H. Kim. A voucher specimen (TV 23) has been deposited in the herbarium of the Department of Herbal Crop Research, National Institute of Horticultural and Herbal Science, Republic of Korea. Experimental research and field studies on plants, including the collection of plant material, complied with relevant institutional, national, and international guidelines and legislation, including the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. Extraction and isolation T. vernicifluum heartwood (5 kg) was extracted twice with 95% ethanol (36 L) in a double bath at 60℃ for 2 days. The solution that passed through filter paper was evaporated under reduced pressure to yield an ethanol extract (208 g). The extract was suspended in 7 L distilled water and materials therein successively extracted into n -hexane (45 g), ethyl acetate (106 g, EA), and water layers (52 g), respectively. The EA extract was subjected to silica gel column chromatography using a gradient solvent system [CHCl 3 :MeOH (10:0.5 → 1:1)] to obtain four fractions (EA1–EA4). EA2 was subjected to C-18 column chromatography using a gradient solvent system [H 2 O:MeOH (1:0 → 1:5)] to yield six fractions (EA21–EA26). E22 was subjected to C-18 column chromatography using a gradient solvent system [H 2 O:MeOH (2:1 → 1:2)] to yield compound 2 (21 mg). E23 was chromatographed on C-18 column chromatography using a gradient solvent system [H 2 O:MeOH (3:2 → 1:2)] to gain compound 4 (15 mg). EC24 was subjected to C-18 column chromatography using a gradient solvent system [H 2 O:MeOH (1:0 → 1:5)] to yield compound 6 (6 mg). Compound 8 (25.5 mg) was isolated from E25 via C-18 column chromatography using a gradient solvent system [H 2 O:MeOH (3:2 → 1:2)]. Compound 7 (76 mg) and EA261 fraction were purified from EA26 via Sephadex LH-20 column chromatography using methanol as the eluent. Compound 9 (32.3 mg) was purified from EA261 fraction via C-18 column chromatography using a gradient solvent system [H 2 O:MeOH (3:1 → 1:3)]. EA3 was subjected to C-18 column chromatography using a gradient solvent system [H 2 O:MeOH (10:0.5 → 1:5)] to yield compounds 1 (820 mg) and 3 (18.3 mg), and four further fractions (EA31–EA34). Compound 5 (130 mg) was isolated from E34 via C-18 column chromatography using a gradient solvent system [H 2 O:MeOH (1:1 → 1:3)]. E4 was subjected to C-18 column chromatography using a gradient solvent system [H 2 O:MeOH (1:0 → 1:3)] to yield compound 10 (35 mg) and three further fractions (EA41–EA43). EA43 was subjected to Sephadex LH-20 column chromatography with methanol as the eluent; this yielded compound 11 (17 mg). sEH inhibitory assay The in vitro sEH assay was performed as previously described [ 12 ]. Briefly, 130 µL of sEH in 25 mM bis-tris-HCl buffer (pH 7.0, with 0.1% BSA) was mixed with 20 µL of each inhibitor (0.032 to 1 mM) dissolved in MeOH. Each mixture was then added to 50 µL of PHOME at 37℃. Excitation (330 nm) and emission (465 nm) were monitored for 40 min. The inhibition rate was calculated using the following equation: Inhibition activity (%) = ( Δ C – Δ I)/ Δ C × 100 (1) Where methanol (C) and inhibitor (I) are the intensity of the C and I, respectively, after 40 min. 50 (%) = (a × x)/(b + x) + y 0 (2) Where x is the IC 50 values, y is the y 0 intercept value, a is the difference between the maximum and minimum values, and b refers to the value of x at 0.5 × a value. Molecular docking of fisetin (5), sulfuretin (7) and butein (9) into sEH A three-dimensional (3D) structure of each fisetin ( 5 ), sulfuretin ( 7 ), and butein ( 9 ) were built and minimized by MM2 running the Chem3D program (CambridgeSoft, Cambridge, MA, USA). A 3D structure of sEH (coded in 3ANS) was obtained from the RCSB protein data bank. The enzyme with water and the 4-cyano- N -[(1 S ,2 R )-2-phenylcyclopropyl]benzamide removed was hydrogenated by the Autodock tool and the Gasteiger charge then applied. Each ligand was formulated as a torsion tree with a focus on the torsion root and the rotatable bonds. The grid box sizes were 60 × 60 × 60 (compounds 5 and 7 ) at 0.375 Å and 126 × 126 × 126 (compound 9 ). Molecular docking employed a Lamarckian genetic algorithm running the maximum number of evaluations [ 13 ]. Graphics were created as Ligplot (Cambridge, UK) and chimera (San Francisco, CA, USA). Molecular dynamics of fisetin (5), sulfuretin (7) and butein (9) into sEH To explore the interactions between sEH and two inhibitors (fisetin and butein), sEH-inhibitor complexes were subjected to molecular dynamics simulations using the Gromacs 4.6.5 package. The ligand topology was generated employing the Gromacs G54A7FF All-Atom package of the Automated Topology Builder (ATB) and the Repository. sEH was charged by the GROMOS96 54a7 force field. The corresponding products were dissolved in water in a cubic box of the default value using the simple point charge water model with six Cl – ions. The complex was minimized by a maximal force of 10 kJ/mol via the steepest descent method. This sequentially simulated constant temperature/ constant volume (NVT) equilibration at 300K, constant temperature/constant pressure (NPT) equilibration with the particle Mesh-Ewald long-range electrostatics at 1 bar, and the molecular dynamics over 100 ns, respectively. Graphics and movies were created using chimera (San Francisco, CA, USA). Preparation of sample and standard solutions T. vernicifluum heartwood (16 g) underwent sonication-assisted ethanol (0.8 L) extraction three times at 45°C for 2 h. The resulting solution was filtered and then evaporated under reduced pressure at 50°C to yield an extract powder (0.81 g, 4.94% yield). Five milligrams of this extract was dissolved in 1 mL methanol and filtered through a 0.45-µm pore-sized syringe filter. Stock solutions of the six reference standards (1 mg/mL) were prepared in HPLC-grade methanol and stored below 4°C. Working solutions were prepared via serial dilution of these stock solutions in methanol. Validation of the method The UPLC method was validated in terms of linearity, the limit of detection (LOD), the limit of quantification (LOQ), precision, and accuracy. Linearity was established using six concentrations of standard solutions each analyzed in triplicate. The calibration curves were fitted via linear regression. LOD and LOQ values were determined based on signal-to-noise ratios of 3 and 10, respectively. Precision was evaluated by analyzing a standard solution five times within a single day (intra-day precision) and on three consecutive days (inter-day precision); the results were expressed as relative standard deviations (RSDs). Accuracy was ascertained via recovery tests. Varying amounts (low, medium, and high) of standards were added to samples with known quantities of materials and the percentage recoveries calculated. Instrumentation and the chromatographic conditions Analysis employed an Agilent 1290 Infinity UPLC system with a binary pump, a degasser, an auto-sampler, a column compartment, and a diode array detector. Chromatographic separation was achieved on a Halo C18 column (100 × 4.6 mm, 2.7 µm) at 25°C. The mobile phases were 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B). Gradient elution proceeded at a flow rate of 1.0 mL/min as follows: 0–5 min, 10% A; 5–15 min, 10–20% A; 15–23 min, 20–22% A; 23–30 min, 22–25% A; 30–35 min, 25–40% A; 35–37 min, 40–100% A; and 37–40 min, 100% A. Before each injection, the column was re-equilibrated with 10% A (90% B) for 5 min. The injection volume was 5 µL, and detection proceeded at 254 nm. Data processing employed Agilent ChemStation software. Results Bioactivity-guided fractionation and isolation To identify the active constituents, the crude ethanol extract of T. vernicifluum heartwood was suspended in water and subjected to successive liquid-liquid partitioning with n -hexane and ethyl acetate (EtOAc). The resulting fractions were screened for sEH inhibitory activity at a concentration of 50 µg/mL. The n -hexane fraction, which typically contains lipophilic components such as urushiols and lipids, showed negligible activity. In contrast, the EtOAc-soluble fraction exhibited significant inhibition (57.6 ± 1.4%) (Fig. 1 A). Based on this bioactivity profile, the EtOAc fraction was selected for further phytochemical investigation. Repeated chromatographic separation of the EtOAc fraction using silica gel, C-18 reversed-phase silica, and Sephadex LH-20 columns resulted in the isolation of 11 compounds. Structural elucidation was performed by analyzing 1 H and 13 C NMR spectroscopic data and comparing them with reported literature values. The isolated compounds were identified as fustin ( 1 ) [ 15 ], ethyl gallate ( 2 ) [ 16 ], taxifolin ( 3 ) [ 15 ], garbanzol ( 4 ) [ 15 ], fisetin ( 5 ) [ 15 ], butin ( 6 ) [ 17 ], sulfuretin ( 7 ) [ 15 ], eriodictyol ( 8 ) [ 17 ], butein ( 9 ) [ 15 ], gallic acid ( 10 ) [ 15 ], and fisetinidol-4α-ol ( 11 ) [ 15 ] (Fig. 1 B and Figure S1 –S11). This collection represents a diverse array of structural subclasses, including dihydroflavonols ( 1 , 3 , 6 , 11 ), flavonols ( 4 , 5 ), aurones ( 7 ), and chalcones ( 9 ), as well as simple phenolic acids ( 2 , 10 ). sEH inhibitory activities of compounds 1–11 from T. vernicifluum Compounds 1 – 11 were evaluated for their inhibitory potential against sEH in vitro (Fig. 2 A and B). The modes of inhibition were determined using Lineweaver–Burk (Fig. 2 C– 2 E) and Dixon (Fig. 2 F– 2 H) plots. The results are summarized in Table 1 . The initial screening employed sEH at 100 µM using 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), a recognized sEH inhibitor, as the positive control. Of the tested compounds, sulfuretin ( 7 ) exhibited the highest inhibitory activity (98.24% at 100 µM) (Eq. 1) and an IC₅₀ of 8.8 ± 0.3 µM (Eq. 2). Kinetic analysis revealed that sulfuretin ( 7 ) acted as a competitive inhibitor with a K i value of 1.4 µM. Fisetin ( 5 ) also potently inhibited sEH with an IC 50 of 9.6 ± 0.8 µM via a competitive binding mode ( K i = 5.8 µM) although the inhibitory rate at 100 µM was lower (> 100%). Butein ( 9 ) also exhibited strong inhibition (85.07% at 100 µM). The IC₅₀ was 21.4 ± 1.5 µM, and inhibition was non-competitive ( K i = 19.5 µM). Taxifolin ( 3 ), garbanzol ( 4 ), butin ( 6 ), and eriodictyol ( 8 ) exhibited moderate inhibition, ranging from 35 to 46%, but fustin ( 1 ), ethyl gallate ( 2 ), and gallic acid ( 10 ) were less active. The least active compound was fisetiniol-4α-ol ( 11 ) (13.86% inhibition at 100 µM). These findings suggested that several flavonoids, particularly fisetin ( 5 ), sulfuretin ( 7 ), and butein ( 9 ), exhibited significant sEH inhibitory activity and may therefore serve as promising lead compounds for the development of anti-inflammatory agents targeting the sEH pathway. Table 1 sEH inhibitory activities, IC 50 values, and kinetic parameters of compounds 1 – 11 . Compound 100 µM (%) IC 50 (µM) a Binding mode ( K i , µM) 1 Fustin 27.52 - b - 2 Ethyl gallate 18.60 - - 3 Txifolin 46.26 - - 4 Garbanzol 39.69 - - 5 Fisetin > 100 9.6 ± 0.8 Competitive (5.8) 6 Butin 35.15 - - 7 Sulfuretin 98.24 8.8 ± 0.3 Competitive (1.4) 8 Eriodictyol 42.45 - - 9 Butein 85.07 21.4 ± 1.5 Non-competitive (19.5) 10 Gallic acid 27.75 - - 11 Fisetiniol-4-ol 13.86 - - AUDA c 92.52 15.0 ± 0.7 nM a IC 50 values are expressed as the mean ± SD of three independent experiments b Not determined due to low inhibitory activity c AUDA was used as a positive control Molecular docking of fisetin (5), sulfuretin (7) and butein (9) into sEH To elucidate the molecular interactions underlying the inhibitory effects of selected sEH inhibitors, sEH molecular docking simulations were conducted using fisetin, sulfuretin, and butein. This revealed the binding modes and affinities of the compounds to/for the active site of sEH. Figure 3 A– 3 D and Table 2 show that fisetin ( 5 ) exhibited a docking score of − 7.06 kcal/mol and formed multiple hydrogen bonds with key active site residues, including Pro371 (2.64, 3.39 Å), Gln383 (3.16 Å), Tyr383 (2.89 Å), and Tyr466 (2.95 Å). Sulfuretin ( 7 ), which exhibited the highest level of in vitro inhibition, evidenced a stronger docking score of − 7.67 kcal/mol and hydrogen bond interactions with Pro371 (2.92 Å), Tyr383 (2.61 Å), Tyr466 (2.89, 3.01 Å), and Asp335 (2.72 Å), supporting the suggestion that competitive inhibition was in play. Butein ( 9 ), a non-competitive inhibitor, exhibited the strongest binding affinity (docking score − 8.33 kcal/mol), forming hydrogen bonds with Phe267 (2.74 Å), Asp335 (2.82, 2.96 Å), Tyr466 (2.83 Å), Phe497 (2.67 Å), and Lys495 (2.94, 3.46 Å). The interactions with sites outside the catalytic domain supported the non-competitive nature of the inhibition. Figure 2 shows that the spatial configurations and hydrogen-bonding profiles of the inhibitors supported their observed enzyme inhibition profiles. The docking results corroborated the kinetic data, and further suggested that the binding affinities and interaction patterns of the flavonoids to/with sEH may indicate that the compounds could serve as useful leads for anti-inflammatory drug development. Table 2 Molecular docking scores and hydrogen bond interactions of fisetin ( 5 ), sulfuretin ( 7 ), and butein ( 9 ) with sEH. Autodock score(kcal/mol) Hydrogen bonds(Å) 5 −7.06 Pro371(2.64, 3.39), Gln383(3.16), Tyr383(2.89), Tyr466(2.95) 7 −7.67 Pro371(2.92), Tyr383(2.61), Tyr466(2.89, 3.01), Asp335(2.72) 9 −8.33 Phe267(2.74), Asp335(2.82, 2.96), Tyr466(2.83), Phe497(2.67), Lys495(2.94, 3.46) Molecular dynamics simulations of fisetin (5) and sulfuretin (7) with sEH To elucidate the dynamic behavior of the ligand-protein interactions in a fluid state, we performed 100-ns molecular dynamics (MD) simulations for the sEH complexes with the two most potent inhibitors, fisetin ( 5 ) and sulfuretin ( 7 ). Visual inspection of the simulation trajectories confirmed that both compounds maintained stable binding poses within the active site throughout the simulation period. As illustrated in the superposition of snapshots, fisetin ( 5 ) was stably accommodated in the binding pocket adjacent to the Asp335–Trp342 loop (Fig. 4 A), while sulfuretin ( 7 ) remained tightly bound near the Asn359–Phe387 active site loop (Fig. 4 B), suggesting high affinity for the catalytic pocket. The global stability of these complexes was quantitatively assessed using Root Mean Square Deviation (RMSD) analysis. As shown in Fig. 5 A, the backbone RMSD values for both sEH–ligand complexes stabilized at approximately 3.0 Å after an initial equilibration phase, indicating that the ligands achieved a stable equilibrium within the binding pocket. The thermodynamic stability of these systems was further corroborated by the total potential energy, which averaged approximately − 540 × 10³ kJ/mol for both complexes (Fig. 5 B). To investigate local protein flexibility upon ligand binding, we analyzed the Root Mean Square Fluctuation (RMSF) per residue (Fig. 5 C). While the overall globular structure remained rigid, distinct fluctuation patterns were observed in specific loop regions; the Pro367–Val367 loop exhibited significant perturbation in the presence of fisetin ( 5 ), whereas the sEH–sulfuretin ( 7 ) complex displayed increased flexibility in the Glu414–Gly426 loop (RMSF ~ 4.0 Å). Finally, we analyzed the intermolecular contacts to define the structural basis of inhibition. Hydrogen bond analysis revealed a clear distinction in binding modes; sulfuretin ( 7 ) consistently maintained a higher number of hydrogen bonds (4–6 bonds) compared to fisetin ( 5 ) (1–3 bonds) throughout the trajectory (Fig. 6 A and 6 B). This observation was supported by distance measurements between key pharmacophores and active site residues. For fisetin ( 5 ), the hydroxyl groups remained within the hydrogen-bonding threshold (< 3.5 Å) of Asp335 and Ile363 (Fig. 6 C and 6 E). In contrast, sulfuretin ( 7 ) established a more extensive network of persistent interactions, with its A and B ring hydroxyl groups positioned within 3.5 Å of Asp335, Thr360, Asn378, and Gln384 (Fig. 6 D and 6 F– 6 H). These results suggest that sulfuretin ( 7 ) is effectively anchored in the catalytic center through multiple stable hydrogen bonds, which may contribute to its potent inhibitory activity. UPLC optimization and peak identification To correlate the biological activity with the chemical composition of the extract, a robust UPLC-DAD method was developed and validated. Optimal chromatographic conditions were established using the Halo C18 column, gradient elution employing acetonitrile and water (both with 0.1% formic acid), a column temperature of 25°C, a flow rate of 1.0 mL/min, and detection at 254 nm. Under these conditions, the six reference standards were well-resolved within 40 min (Fig. 7 A). When the retention times and ultraviolet spectra of materials in the T. vernicifluum heartwood extract were compared to those of the standards, the major peaks in the chromatogram (Fig. 7 B) were identified as ethyl gallate (peak 1), fustin (peak 2), garbanzol (peak 3), fisetin (peak 4), sulfuretin (peak 5), and butein (peak 6). Method validation The UPLC method was carefully validated. The calibration curves for all six analytes exhibited good linearity, with all correlation coefficients (r²) ≥ 0.9995 within the tested concentration ranges. The LODs determined at a signal-to-noise ratio of 3 ranged from 0.92 to 21.86 µg/mL. The LOQs determined at a signal-to-noise ratio of 10 ranged from 0.31 to 7.21 µg/mL (Table 3 ). Intra- and inter-day precisions were assessed using standard solutions at various concentrations. The RSD values for intra-day precision ranged from 0.13 to 2.15%. In terms of inter-day precision, the RSD values ranged from 0.23 to 2.69% (Table 4 ). The low RSD values indicate that the analytical method was very precise. Accuracy was evaluated via recovery tests. The extract was spiked with known quantities of the standards. The average recoveries of the six targeted compounds ranged from 96.88 to 104.96%, with all RSD values below 3.97% (Table 5 ). The UPLC method was accurate. Table 3 Calibration curve, linearity, LOD, and LOQ for six reference standards ( n = 3) Compound Regression equation a Linear range (µg/mL) Correlation coefficient ( r 2 ) LOD b (µg/mL) LOQ c (µg/mL) Ethyl gallate y = 4.414x + 0.7516 6.25–200 0.9999 4.33 1.43 Fustin y = 2.4936x + 46.592 125–4000 0.9999 21.86 7.21 Ethyl gallate y = 4.414x + 0.7516 6.25–200 0.9999 4.33 1.43 Garbanzol y = 5.3794x + 1.1065 3.125–100 0.9996 0.92 0.31 Fisetin y = 13.778x − 16.769 7.812–250 0.9999 6.52 2.15 Sulfuretin y = 7.8088x + 11.033 15.625–500 0.9999 11.09 3.66 Butein y = 8.0222x − 2.4665 3.125–100 0.9995 0.92 0.31 a y, peak area of compound; x, concentration (µg/mL) of compound b LOD, limit of detection, S/N = 3 c LOQ, limit of quantification, S/N = 10 Table 4 Precision (intra- and inter-day) of six reference standards ( n = 3) Compound Analyte concentration (µg/mL) Intra-day Inter-day Detected concentration (µg/mL) RSD (%) Detected concentration (µg/mL) RSD (%) Ethyl gallate 100 100.42 ± 0.95 0.94 100.07 ± 1.09 1.09 50 50.09 ± 0.36 0.73 50.18 ± 0.26 0.51 25 24.90 ± 0.14 0.57 24.30 ± 0.66 2.69 Fustin 2000 1971.14 ± 23.11 1.17 1970.62 ± 13.41 0.68 1000 120.18 ± 0.16 0.13 120.11 ± 0.28 0.23 500 160.11 ± 0.74 0.47 160.04 ± 0.43 0.31 Garbanzol 50 51.00 ± 0.52 1.03 50.94 ± 0.44 0.86 25 24.90 ± 0.13 0.54 24.82 ± 0.18 0.70 12.5 12.22 ± 0.81 1.59 11.73 ± 0.05 0.41 Fisetin 62.5 61.74 ± 1.33 2.15 62.13 ± 1.56 2.51 31.25 31.81 ± 0.27 0.85 30.74 ± 0.62 2.02 15.625 16.67 ± 0.05 0.30 14.95 ± 0.05 0.34 Sulfuretin 125 124.81 ± 1.28 1.03 127.76 ± 1.83 1.43 62.5 63.54 ± 1.15 1.75 63.65 ± 0.46 0.73 31.25 31.81 ± 0.20 0.62 31.23 ± 0.38 1.22 Butein 50 49.37 ± 0.13 0.27 49.80 ± 1.25 2.51 25 24.84 ± 0.28 1.11 24.64 ± 0.36 1.46 12.5 13.52 ± 0.09 0.69 13.78 ± 0.34 2.48 Table 5 Recovery of six reference standards ( n = 3) Compound Spiked concentration (µg/mL) Detected concentration (µg/mL) Recovery (%) RSD (%) Ethyl gallate 100 101.04 ± 2.73 101.04 2.70 50 50.29 ± 0.65 100.58 1.30 25 24.22 ± 0.84 96.88 3.47 Fustin 2000 2061.21 ± 14.34 103.06 0.70 1000 973.73 ± 19.55 97.37 2.01 500 519.22 ± 6.33 103.84 1.22 Garbanzol 50 51.38 ± 0.45 102.77 0.87 25 24.59 ± 0.25 98.37 1.02 12.5 12.51 ± 0.10 100.09 0.80 Fisetin 62.5 61.67 ± 1.02 98.67 1.65 31.25 31.69 ± 0.34 101.40 1.06 15.625 16.40 ± 0.42 104.96 2.54 Sulfuretin 125 128.42 ± 2.55 102.74 1.99 62.5 61.00 ± 0.51 97.60 0.84 31.25 30.45 ± 1.21 97.45 3.97 Butein 50 50.07 ± 0.58 100.15 1.16 25 26.19 ± 0.31 104.77 1.17 12.5 13.02 ± 0.23 104.19 1.79 Quantitative analysis of the extract The validated UPLC method was used to quantify the six compounds (in mg/g) in the ethanol extract of T. vernicifluum heartwood. The analysis was performed in triplicate, and the contents of the compounds were expressed as mg/g of extract (Table 6 ). Fustin was the most abundant component (262.10 ± 1.90 mg/g, 26.21% of the extract), followed by sulfuretin (23.47 ± 0.25 mg/g) and fisetin (14.20 ± 0.10 mg/g). Ethyl gallate (9.70 ± 0.08 mg/g), butein (5.93 ± 0.52 mg/g), and garbanzol (4.16 ± 0.05 mg/g) were present in smaller quantities. Table 6 Contents of six compounds in the T. vernicifluum heartwood extract Compound Content (Mean ± SD, n = 3) mg/g % Ethyl gallate 9.70 ± 0.10 0.97 Fustin 262.10 ± 0.02 26.21 Garbanzol 4.16 ± 0.04 0.42 Fisetin 14.20 ± 0.02 1.42 Sulfuretin 23.47 ± 0.02 2.35 Butein 5.93 ± 0.04 0.59 Discussion This comprehensive study employed UPLC to quantitate key flavonoids in a heartwood extract of T. vernicifluum , followed by evaluation of their sEH inhibitory capacities. Fustin was the most abundant compound (262.10 ± 1.90 mg/g) but exhibited relatively weak sEH inhibition. This underscores the principle that phytochemical concentration alone does not dictate biological potency; rather, the structural features of a compound and its precise molecular interactions with the target enzyme are paramount. While fustin, a dihydroflavonol, is known to exhibit other biological activities, including antioxidant and neuroprotective effects [ 18 ], its direct contribution to the overall sEH inhibition of the crude extract appears to be limited. In contrast, sulfuretin, fisetin, and butein were identified as potent sEH inhibitors, despite being present in lower quantities (23.47, 14.20, and 5.93 mg/g, respectively). The aurone sulfuretin ( 7 ) and the flavonol fisetin ( 5 ) both exhibited competitive inhibition with IC 50 values of 8.8 and 9.6 µM, respectively. This suggests that both compounds directly compete with natural enzyme substrates for binding at the catalytic site. Molecular docking results supported this, indicating interactions with key sEH active site residues such as Tyr383 and Tyr466 [ 16 ]. These residues are crucial for sEH hydrolase activity, and their engagement by inhibitors often mimics the binding of natural substrates or urea-based synthetic inhibitors [ 19 ]. To complement the static docking models, molecular dynamics simulations provided a time-resolved view of the ligand-enzyme interactions for the competitive inhibitors. The simulations confirmed that both fisetin and sulfuretin form stable complexes with sEH, as evidenced by steady RMSD values. A key finding was the differential modulation of loop flexibility; fisetin significantly perturbed the Pro367–Val367 loop, whereas sulfuretin increased fluctuations in the Glu414–Gly426 region. Furthermore, hydrogen bond analysis over the simulation trajectory reinforced the superior potency of sulfuretin, which maintained a higher number of stable contacts with catalytic residues, particularly Asp335, compared to fisetin. This dynamic stability serves as strong validation for the potential of sulfuretin as a robust sEH inhibitor. Distinct from the competitive inhibitors, the chalcone butein ( 9 ) exhibited strong non-competitive sEH inhibition (IC 50 = 21.4 µM). This indicates that butein likely binds to an allosteric site on sEH rather than the substrate-binding pocket, inducing a conformational change that attenuates enzyme activity. Earlier studies on butein derivatives, such as butein-4'- O -glucoside, have similarly reported non-competitive sEH inhibition and described interactions with residues consistent with allosteric binding [ 20 ]. The distinct modes of inhibition observed, competitive for sulfuretin and fisetin, and non-competitive for butein, suggest that synergistic effects may be possible if these compounds are used in combination. The ability to distinguish these individual contributions was made possible by the development and validation of a robust UPLC method. This analytical approach ensured reliable quantitative data, which is essential for quality control and for linking chemical composition to biological activity in natural product research [ 21 ]. The precise quantification allowed us to determine that while fustin is the major constituent, the biological activity is driven by the less abundant, high-potency flavonoids. The inhibition of sEH is a well-recognized strategy for mitigating inflammation and related pathologies, as it prevents the degradation of anti-inflammatory EpFAs like EETs [ 22 ]. The potent sEH inhibition demonstrated by sulfuretin, fisetin, and butein may partially explain the traditional use of T. vernicifluum in East Asia for treating inflammatory conditions, pain, and gastric ailments [ 23 ]. Moreover, these flavonoids are known to exert anti-inflammatory effects via additional pathways, notably by modulating NF-κB and MAPK signaling cascades [ 18 ]. This multi-targeting capability, simultaneous sEH inhibition and signaling pathway modulation, represents a major advantage of natural product-derived therapies over single-target synthetic drugs. This distinct advantage of natural product-derived polypharmacology is particularly relevant given the significant hurdles encountered in the clinical development of synthetic sEH inhibitors [ 24 ]. While synthetic analogues often demonstrate high potency, their translation into approved therapeutics has been frequently impeded by pharmacokinetic limitations, including poor absorption, short biological half-lives, low bioavailability, and potential off-target toxicities [ 25 ]. In contrast, natural products occupy a vast and biologically relevant chemical space, offering immense structural diversity that has evolved to interact with biological macromolecules [ 4 ]. This structural complexity may provide molecules with superior physicochemical properties, better safety profiles, and enhanced oral bioavailability compared to synthetic libraries. Consequently, the flavonoids identified in T. vernicifluum not only serve as promising leads that circumvent the common pitfalls of synthetic inhibitors but also offer a strategic scaffold for developing novel, safe, and effective anti-inflammatory agents. However, translating these promising in vitro results into clinical therapeutics requires addressing specific challenges. A primary concern is the presence of urushiols in T. vernicifluum , which are known to cause severe contact dermatitis [ 26 ]. Consequently, future development strategies must prioritize the rigorous purification of active flavonoids or the implementation of validated detoxification protocols to eliminate allergenic risks. Furthermore, comprehensive in vivo studies are essential to verify the anti-inflammatory efficacy, safety, and pharmacokinetic profiles of these compounds in physiological systems. Looking forward, the identification of both competitive and non-competitive inhibitors within the same source offers a unique opportunity to explore synergistic combination therapies. Ultimately, this study establishes a scientific basis for valorizing T. vernicifluum heartwood not merely as a traditional remedy, but as a refined source of potent, multi-targeted sEH inhibitors. Conclusion This study successfully identified and characterized several secondary metabolites from the heartwood of T. vernicifluum that effectively inhibited sEH. A validated UPLC analysis method revealed that fustin was the most abundant of all measured flavonoids, but only weakly inhibited sEH. In contrast, sulfuretin, fisetin, and butein, all of which were present in lower but still significant quantities, were more potent inhibitors, with IC 50 values in the low micromolar range. Kinetic studies, supported by molecular docking tests, revealed that fisetin and sulfuretin were competitive inhibitors and butein a non-competitive inhibitor. These findings, emphasized by the precise quantitative data derived via UPLC analysis, highlight the fact that specific T. vernicifluum heartwood constituents may serve as valuable leads for the development of novel, natural product-based anti-inflammatory agents that target the sEH pathway. Further in vivo studies are essential to comprehensively evaluate the therapeutic efficacies and safeties of such promising compounds. Declarations Conflict of interest The authors declare that there are no conflicts of interest. Additional information The online version contains supplementary information available at ~ . Funding This research was supported by the Cooperative Research Program of the Rural Development Administration (RS-2022-RD010239) and a grant from the Korea Institute of Oriental Medicine (KSN2224020), Republic of Korea. Author Contribution Jang Hoon Kim: Writing – original draft, Conceptualization, Data curation, Resources, Investigation, Methodology, Software, Visualization. Jae-Young Cheon: Data curation, Investigation, Methodology, Software, Visualization, Validation. Jin Yu: Investigation, Methodology. Yong-Goo Kim: Investigation, Methodology. Sung Yeon Kim: Investigation, Methodology. Kyong-Hwan Bang: Investigation, Methodology. Jin Tae Jeong: Investigation, Methodology. Hyun-Ju Jung: Writing – review and editing, Conceptualization. Ik Soo Lee: Writing – original draft, review, and editing, Data curation. Data Availability The datasets used and/or analyzed during the current study are available from the corresponding author, Dr. Ik Soo Lee ( [email protected] ), on reasonable request. References Wagner, K. M., McReynolds, C. B., Schmidt, W. K. & Hammock, B. D. Soluble epoxide hydrolase as a therapeutic target for pain, inflammatory and neurodegenerative diseases. Pharmacol. Ther. 180 , 62–76 (2017). Zhang, G., Kodani, S. & Hammock, B. D. Stabilized epoxygenated fatty acids regulate inflammation, pain, angiogenesis and cancer. 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Screening of soluble epoxide hydrolase inhibitory ingredients from traditional Chinese medicines for anti-inflammatory use. J. Ethnopharmacol. 194 , 475–482 (2016). Dong, H., Wang, C., Gong, K. & Zhang, F. Advances in the research on the chemical constituents and comprehensive utilization of Rhus verniciflua Stokes. Chem. Ind. Prod. 29 , 225–232 (2009). Li, M. C. et al. Traditional uses, phytochemistry, and pharmacology of Toxicodendron vernicifluum (Stokes) F.A. Barkley - A review. J. Ethnopharmacol. 267 , 113476 (2021). Li, M. C. et al. Chemical constituents from the heartwood of Toxicodendron vernicifluum (Stokes) F.A. Barkley. Biochem. Syst. Ecol. 90 , 104017 (2020). Kim, J. H. et al. In vitro and in silico investigation of anthocyanin derivatives as soluble epoxide hydrolase inhibitors. Int. J. Biol. Macromol. 112, 961–967 (2018). Kim, J. H. et al. Soluble epoxide hydrolase inhibitory constituents from Selaginella tamariscina . Bull. Korean Chem. Soc. 36 , 300–304 (2015). Jo, A. R. et al. Soluble epoxide hydrolase inhibitory components from Rheum undulatum and in silico approach. J. Enzyme Inhib. Med. Chem. 31 , 70–78 (2016). Hashida, K., Tabata, M. & Kuroda, K. Phenolic extractives in the trunk of Toxicodendron vernicifluum : Chemical characteristics, contents and radial distribution. J. Wood Sci. 60 , 160–168 (2014). Wang, D., Mu, Y. & Dong, H. Chemical constituents of the ethyl acetate extract from Diaphragma juglandis fructus and their inhibitory activity on nitric oxide production in vitro . Molecules 23 , 72 (2017). Lee, M. H., Lin, Y. P., Hsu, F. L., Zhan, G. R. & Yen, K. Y. Bioactive constituents of Spatholobus suberectus in regulating tyrosinase-related proteins and mRNA in HEMn cells. Phytochemistry 67 , 1262–1270 (2006). Bawadood, A. S. et al. Fustin alleviates lipopolysaccharide-induced anxiety-depression-like performances by modulation of oxidative stress/neuroinflammatory markers/ NF-κB/caspase-3/BDNF expression in rodents. Eur. Rev. Med. Pharmacol. Sci. 28 , 419–432 (2024). Barbosa-Sicard, E. et al. Inhibition of the soluble epoxide hydrolase by tyrosine nitration. J. Biol. Chem. 284 , 28156–28163 (2009). Kim, J. H. et al. Inhibitory activity of glycosides from Elsholtzia ciliata against soluble epoxide hydrolase and cytokines in RAW264.7 Cells. J. Microbiol. Biotechnol. 35 , e2410011 (2024). Gao, G. et al. UPLC-ESI-MS/MS based characterization of active flavonoids from Apocynum spp. and anti-bacteria assay. Antioxid. (Basel) . 10 , 1901 (2021). Hashimoto, K. Role of soluble epoxide hydrolase in metabolism of PUFAs in psychiatric and neurological disorders. Front. Pharmacol. 10 , 36 (2019). Li, M. C. et al. Chemical constituents from the heartwood of Toxicodendron vernicifluum (Stokes) F.A. Barkley. Biochem. Syst. Ecol. 90 , 104017 (2020). Sun, C. P. et al. Discovery of soluble epoxide hydrolase inhibitors from chemical synthesis and natural products. J. Med. Chem. 64 , 184–215 (2021). Lee, K. S. S. et al. Optimized inhibitors of soluble epoxide hydrolase improve in vitro target residence time and in vivo efficacy. J. Med. Chem. 57 , 7016–7030 (2014). Bai, G. et al. The chromosome-level genome for Toxicodendron vernicifluum provides crucial insights into Anacardiaceae evolution and urushiol biosynthesis. iScience 25 , 104512 (2022). Additional Declarations No competing interests reported. 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09:32:07","extension":"xml","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":127909,"visible":true,"origin":"","legend":"","description":"","filename":"69e7fc9f93ea47eeaeaeb84bfa7cab5e1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8274523/v1/a3242e811d7db7fee078b66e.xml"},{"id":98210676,"identity":"6fd0c8c1-a748-4045-9919-fa8d2bdfe204","added_by":"auto","created_at":"2025-12-15 09:32:07","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":141700,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8274523/v1/bff58ca232ebec83c3159de7.html"},{"id":98433425,"identity":"9151c984-e53c-4e18-a7a2-aed6bc9b0e14","added_by":"auto","created_at":"2025-12-17 16:50:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":206193,"visible":true,"origin":"","legend":"\u003cp\u003esEH inhibitory activity of \u003cem\u003eT. vernicifluum\u003c/em\u003e fractions and chemical structures of isolated compounds. (A) Inhibitory activity of the crude ethanol extract (EtOH) and its solvent-partitioned fractions against sEH at a concentration of 50 µg/mL. The dashed line represents 50% inhibition. (B) Chemical structures of the constituents (\u003cstrong\u003e1\u003c/strong\u003e–\u003cstrong\u003e11\u003c/strong\u003e) isolated from the active ethyl acetate fraction.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8274523/v1/4304596d6527ca363ffc86a4.png"},{"id":98210658,"identity":"46802583-32df-4458-8a39-2f076c32992b","added_by":"auto","created_at":"2025-12-15 09:32:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":474817,"visible":true,"origin":"","legend":"\u003cp\u003esEH inhibitory activity and kinetic analysis of isolated compounds. (A) Inhibitory activity of compounds \u003cstrong\u003e1\u003c/strong\u003e–\u003cstrong\u003e11\u003c/strong\u003e against sEH at a concentration of 50 µM. (B) Dose-response inhibition curves for fisetin (\u003cstrong\u003e5\u003c/strong\u003e), sulfuretin (\u003cstrong\u003e7\u003c/strong\u003e), and butein (\u003cstrong\u003e9\u003c/strong\u003e). (C–E) Lineweaver–Burk plots for sEH inhibition by compounds \u003cstrong\u003e5\u003c/strong\u003e (C), \u003cstrong\u003e7\u003c/strong\u003e (D), and \u003cstrong\u003e9\u003c/strong\u003e (E). (F–H) Dixon plots for the determination of inhibition constants (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ei\u003c/sub\u003e) for compounds \u003cstrong\u003e5\u003c/strong\u003e (F), \u003cstrong\u003e7\u003c/strong\u003e (G), and \u003cstrong\u003e9\u003c/strong\u003e (H).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8274523/v1/8ff0b916b7b6293bd708f0eb.png"},{"id":98431491,"identity":"27e1c396-461b-4c47-ae2c-7814ff61deea","added_by":"auto","created_at":"2025-12-17 16:47:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":425559,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking analysis of compounds \u003cstrong\u003e5\u003c/strong\u003e, \u003cstrong\u003e7\u003c/strong\u003e, and \u003cstrong\u003e9\u003c/strong\u003e with sEH. (A) Superimposed 3D binding poses of fisetin (\u003cstrong\u003e5\u003c/strong\u003e), sulfuretin (\u003cstrong\u003e7\u003c/strong\u003e), and butein (\u003cstrong\u003e9\u003c/strong\u003e) in the active site of sEH. (B–D) 2D ligand-interaction diagrams of fisetin (\u003cstrong\u003e5\u003c/strong\u003e) (B), sulfuretin (\u003cstrong\u003e7\u003c/strong\u003e) (C), and butein (\u003cstrong\u003e9\u003c/strong\u003e) (D). Key amino acid residues participating in hydrogen bond interactions are indicated by green dashed lines with bond distances in Angstroms (Å), and hydrophobic interactions are represented by red arcs.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8274523/v1/8b627c686f0f29448200ca32.png"},{"id":98431295,"identity":"6934d1f4-6d09-42f7-9f23-f5902e583dc9","added_by":"auto","created_at":"2025-12-17 16:47:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":294035,"visible":true,"origin":"","legend":"\u003cp\u003eSuperimposed binding modes of fisetin (\u003cstrong\u003e5\u003c/strong\u003e) (A) and sulfuretin (\u003cstrong\u003e7\u003c/strong\u003e) (B) in the active site of sEH obtained from MD simulations (red : 0 ns, orange: 10 ns, yellow: 20 ns, green 30 ns, cyan: 40 ns, blue: 50 ns, conflower blue: 60 ns, purple: 70 ns, hot pink: 80 ns, meganta: 90 ns, black: 100 ns).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8274523/v1/d48d9ee79cade5f0c5209d34.png"},{"id":98210659,"identity":"4678bf43-eacf-4231-8eea-1b7368ef478d","added_by":"auto","created_at":"2025-12-15 09:32:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":199121,"visible":true,"origin":"","legend":"\u003cp\u003eGlobal stability and fluctuation analysis of sEH in complex with fisetin (\u003cstrong\u003e5\u003c/strong\u003e) and sulfuretin (\u003cstrong\u003e7\u003c/strong\u003e) during 100-ns MD simulations. (A) Time-dependent RMSD of the protein backbone atoms. (B) Total potential energy of the systems over the simulation time. (C) RMSF of sEH residues.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8274523/v1/2e75f948198b51452c31462d.png"},{"id":98210664,"identity":"9876b0b9-e5cf-42d4-a96d-92e8ab31be29","added_by":"auto","created_at":"2025-12-15 09:32:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":513662,"visible":true,"origin":"","legend":"\u003cp\u003eIntermolecular interaction profiling of fisetin (\u003cstrong\u003e5\u003c/strong\u003e) and sulfuretin (\u003cstrong\u003e7\u003c/strong\u003e) with sEH active site residues. (A, B) Time-evolution of the number of hydrogen bonds formed between sEH and fisetin (\u003cstrong\u003e5\u003c/strong\u003e) (A) and sulfuretin (\u003cstrong\u003e7\u003c/strong\u003e) (B). (C, D) Representative 3D snapshots highlighting key binding residues. Fisetin (\u003cstrong\u003e5\u003c/strong\u003e) interacts with Asp335 and Ile363 (C), while sulfuretin (\u003cstrong\u003e7\u003c/strong\u003e) interacts with Asp335, Thr360, Asn378, and Gln384 (D). (E–H) Distance analysis of key hydrogen bonding interactions over the 100-ns trajectory. Panel (E) monitors the distance between fisetin and residues Asp335/Ile363. Panels (F–H) monitor the distances between sulfuretin and residues Asp335 (F), Thr360/Asn378 (G), and Gln384 (H).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8274523/v1/bc646ee74a287560f6a6f8ba.png"},{"id":98210661,"identity":"5dd5c854-ec6b-4ca5-95ed-80f39d0ebd4d","added_by":"auto","created_at":"2025-12-15 09:32:06","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":174683,"visible":true,"origin":"","legend":"\u003cp\u003eUPLC Chromatograms of standard mixtures (A) and \u003cem\u003eT. vernicifluum\u003c/em\u003e heartwood extract (B). Peak identification: 1, ethyl gallate; 2, fustin; 3, garbanzol; 4, fisetin; 5, sulfuretin; 6, butein. Chromatographic conditions are described in the text. Detection was at 254 nm.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8274523/v1/1c7e899e31992a21dd02ad23.png"},{"id":101151636,"identity":"0ce1cec8-b516-4ae9-a695-a49ffa582c5f","added_by":"auto","created_at":"2026-01-26 15:59:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3657882,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8274523/v1/fe00ff99-80e4-4fed-b699-e6e801a248b1.pdf"},{"id":98433092,"identity":"61e53b4c-a9bb-495e-9a33-9ea159cbdf7d","added_by":"auto","created_at":"2025-12-17 16:50:15","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1223513,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterialr1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8274523/v1/762675faf347894dae107ceb.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Soluble Epoxide Hydrolase Inhibitory Constituents from the Heartwood of Toxicodendron vernicifluum: Isolation, Kinetic Characterization, Molecular Modeling, and Quantitative Analysis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe regulation of inflammation and vascular tone relies heavily on the metabolism of polyunsaturated fatty acids. Soluble epoxide hydrolase (sEH, EC 3.3.2.10) plays a pivotal role in this process by hydrolyzing epoxy fatty acids (EpFAs), such as epoxyeicosatrienoic acids (EETs) and epoxydocosapentaenoic acids (EDPs), into their corresponding diols [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Since EpFAs possess potent anti-inflammatory and vasodilatory properties, their rapid degradation by sEH attenuates the body's natural protective responses [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Consequently, the inhibition of sEH has emerged as a promising therapeutic strategy for treating various inflammatory conditions, including cardiovascular, neurological, and renal disorders [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. By preventing the hydrolysis of EpFAs, sEH inhibitors stabilize endogenous levels of these beneficial lipids, thereby enhancing their protective and anti-fibrotic effects [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhile synthetic sEH inhibitors have demonstrated efficacy in preclinical models [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and candidates such as EC5026 have progressed to clinical trials [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], concerns regarding pharmacokinetic limitations and potential adverse effects persist [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These challenges have necessitated the search for alternative inhibitory scaffolds from natural sources. Natural products, characterized by their immense structural diversity, represent a compelling reservoir for novel therapeutic agents. Although natural inhibitors may sometimes exhibit lower potency compared to optimized synthetic analogues, they often offer distinct advantages in terms of safety profiles and accessibility [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, exploring plant-derived compounds, particularly those with a history of ethnopharmacological use, offers a strategic avenue to discover novel sEH modulators that bridge traditional medicine and modern pharmacology [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eToxicodendron vernicifluum\u003c/em\u003e (Stokes) F.A. Barkley, commonly known as the Chinese or Japanese lacquer tree, is a deciduous species of the Anacardiaceae family native to East Asia [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. While the tree is widely cultivated for the industrial application of its sap in lacquerware, it also holds significant value in traditional East Asian medicine. Various parts of the plant, including the heartwood, leaves, and seeds, have been historically utilized to treat ailments ranging from internal parasites and hemorrhage to inflammatory conditions [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Modern phytochemical investigations have revealed a complex matrix of bioactive constituents in \u003cem\u003eT. vernicifluum\u003c/em\u003e, including flavonoids, polyphenols, triterpenes, and urushiols [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, despite well-documented evidence of the plant's anti-inflammatory properties, the specific molecular mechanisms underlying these effects, specifically its potential modulation of the arachidonic acid pathway via sEH inhibition, remain largely unexplored.\u003c/p\u003e\u003cp\u003eWe hypothesized that the anti-inflammatory efficacy of \u003cem\u003eT. vernicifluum\u003c/em\u003e heartwood is mediated, at least in part, by the inhibition of sEH. Therefore, the primary objective of this study was to isolate and characterize the active sEH inhibitory flavonoid compounds from the ethanol extract of \u003cem\u003eT. vernicifluum\u003c/em\u003e heartwood using a bioactivity-guided isolation strategy. Furthermore, we aimed to employ a multidisciplinary approach, including enzyme kinetic assays, molecular docking, and molecular dynamics (MD) simulations, to provide a structural basis for the observed sEH inhibitory activity. Finally, we used Ultra-High-Performance Liquid Chromatography (UPLC) to quantify these active principles, linking the level of the specific flavonoids to the overall sEH inhibitory potential of the heartwood extract. Ultimately, this comprehensive profiling seeks to highlight the potential of these specific flavonoids as lead compounds for novel anti-inflammatory therapeutics.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eChemicals and reagents\u003c/h2\u003e\u003cp\u003eThe solvents used for separation (methanol, \u003cem\u003en\u003c/em\u003e-hexane, chloroform, and ethyl acetate) were purchased from Duksan General Science (Seoul, Republic of Korea). Chromatographic separations were performed via thin layer chromatography (TLC) using glass plates pre-coated with silica gel 60 F\u003csub\u003e254\u003c/sub\u003e and silica gel RP-18 F\u003csub\u003e254\u003c/sub\u003e (20 \u0026times;\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\)\u003c/span\u003e\u003c/span\u003e20 cm; Merck, Darmstadt, Germany). Materials were visualized under ultraviolet light at 254 nm and 365 nm, respectively. The TLC color reagent was 10% ethanol\u0026ndash;sulfuric acid. Column chromatography employed 230\u0026ndash;400-mesh silica gel 60 (Merck) and 12-nm ODS-A (YMC, Kyoto, Japan) columns. Nuclear magnetic resonance (NMR) data were acquired using a Bruker Avance Ⅲ 300-MHz platform (Bruker, Billerica, MA, USA). Reference standards (ethyl gallate, fustin, garbanzol, fisetin, sulfuretin, and butein; all \u0026gt;\u0026thinsp;98% purity) were utilized. High-performance liquid chromatography (HPLC)-grade acetonitrile, methanol, and water were from J.T. Baker; analytical grade formic acid was purchased from Merck (St. Louis, MO, USA). Human recombinant sEH (10011669), PHOME (10009134), and AUDA (10007972) were from Cayman Chemical (Ann Arbor, MI, USA).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePlant material\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eT. vernicifluum\u003c/em\u003e heartwood was obtained from a herbal medicinal company of the Republic of Korea in August 2023. The plant was identified by Dr. J.H. Kim. A voucher specimen (TV 23) has been deposited in the herbarium of the Department of Herbal Crop Research, National Institute of Horticultural and Herbal Science, Republic of Korea.\u003c/p\u003e\u003cp\u003e\u003cem\u003eT. vernicifluum\u003c/em\u003e heartwood was obtained from a herbal medicinal company of the Republic of Korea in August 2023. The plant was identified by Dr. J.H. Kim. A voucher specimen (TV 23) has been deposited in the herbarium of the Department of Herbal Crop Research, National Institute of Horticultural and Herbal Science, Republic of Korea. Experimental research and field studies on plants, including the collection of plant material, complied with relevant institutional, national, and international guidelines and legislation, including the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.\u003c/p\u003e\n\u003ch3\u003eExtraction and isolation\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eT. vernicifluum\u003c/em\u003e heartwood (5 kg) was extracted twice with 95% ethanol (36 L) in a double bath at 60℃ for 2 days. The solution that passed through filter paper was evaporated under reduced pressure to yield an ethanol extract (208 g). The extract was suspended in 7 L distilled water and materials therein successively extracted into \u003cem\u003en\u003c/em\u003e-hexane (45 g), ethyl acetate (106 g, EA), and water layers (52 g), respectively. The EA extract was subjected to silica gel column chromatography using a gradient solvent system [CHCl\u003csub\u003e3\u003c/sub\u003e:MeOH (10:0.5 \u0026rarr; 1:1)] to obtain four fractions (EA1\u0026ndash;EA4). EA2 was subjected to C-18 column chromatography using a gradient solvent system [H\u003csub\u003e2\u003c/sub\u003eO:MeOH (1:0 \u0026rarr; 1:5)] to yield six fractions (EA21\u0026ndash;EA26). E22 was subjected to C-18 column chromatography using a gradient solvent system [H\u003csub\u003e2\u003c/sub\u003eO:MeOH (2:1 \u0026rarr; 1:2)] to yield compound \u003cb\u003e2\u003c/b\u003e (21 mg). E23 was chromatographed on C-18 column chromatography using a gradient solvent system [H\u003csub\u003e2\u003c/sub\u003eO:MeOH (3:2 \u0026rarr; 1:2)] to gain compound \u003cb\u003e4\u003c/b\u003e (15 mg). EC24 was subjected to C-18 column chromatography using a gradient solvent system [H\u003csub\u003e2\u003c/sub\u003eO:MeOH (1:0 \u0026rarr; 1:5)] to yield compound \u003cb\u003e6\u003c/b\u003e (6 mg). Compound \u003cb\u003e8\u003c/b\u003e (25.5 mg) was isolated from E25 via C-18 column chromatography using a gradient solvent system [H\u003csub\u003e2\u003c/sub\u003eO:MeOH (3:2 \u0026rarr; 1:2)]. Compound \u003cb\u003e7\u003c/b\u003e (76 mg) and EA261 fraction were purified from EA26 via Sephadex LH-20 column chromatography using methanol as the eluent. Compound \u003cb\u003e9\u003c/b\u003e (32.3 mg) was purified from EA261 fraction via C-18 column chromatography using a gradient solvent system [H\u003csub\u003e2\u003c/sub\u003eO:MeOH (3:1 \u0026rarr; 1:3)]. EA3 was subjected to C-18 column chromatography using a gradient solvent system [H\u003csub\u003e2\u003c/sub\u003eO:MeOH (10:0.5 \u0026rarr; 1:5)] to yield compounds \u003cb\u003e1\u003c/b\u003e (820 mg) and \u003cb\u003e3\u003c/b\u003e (18.3 mg), and four further fractions (EA31\u0026ndash;EA34). Compound \u003cb\u003e5\u003c/b\u003e (130 mg) was isolated from E34 via C-18 column chromatography using a gradient solvent system [H\u003csub\u003e2\u003c/sub\u003eO:MeOH (1:1 \u0026rarr; 1:3)]. E4 was subjected to C-18 column chromatography using a gradient solvent system [H\u003csub\u003e2\u003c/sub\u003eO:MeOH (1:0 \u0026rarr; 1:3)] to yield compound \u003cb\u003e10\u003c/b\u003e (35 mg) and three further fractions (EA41\u0026ndash;EA43). EA43 was subjected to Sephadex LH-20 column chromatography with methanol as the eluent; this yielded compound \u003cb\u003e11\u003c/b\u003e (17 mg).\u003c/p\u003e\n\u003ch3\u003esEH inhibitory assay\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e sEH assay was performed as previously described [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Briefly, 130 \u0026micro;L of sEH in 25 mM bis-tris-HCl buffer (pH 7.0, with 0.1% BSA) was mixed with 20 \u0026micro;L of each inhibitor (0.032 to 1 mM) dissolved in MeOH. Each mixture was then added to 50 \u0026micro;L of PHOME at 37℃. Excitation (330 nm) and emission (465 nm) were monitored for 40 min. The inhibition rate was calculated using the following equation:\u003c/p\u003e\u003cp\u003eInhibition activity (%) = (\u003cem\u003eΔ\u003c/em\u003eC \u0026ndash; \u003cem\u003eΔ\u003c/em\u003eI)/\u003cem\u003eΔ\u003c/em\u003eC \u0026times; 100 (1)\u003c/p\u003e\u003cp\u003eWhere methanol (C) and inhibitor (I) are the intensity of the C and I, respectively, after 40 min.\u003c/p\u003e\u003cp\u003e50 (%) = (a \u0026times; x)/(b\u0026thinsp;+\u0026thinsp;x)\u0026thinsp;+\u0026thinsp;y\u003csub\u003e0\u003c/sub\u003e (2)\u003c/p\u003e\u003cp\u003eWhere x is the IC\u003csub\u003e50\u003c/sub\u003e values, y is the y\u003csub\u003e0\u003c/sub\u003e intercept value, a is the difference between the maximum and minimum values, and b refers to the value of x at 0.5 \u0026times; a value.\u003c/p\u003e\n\u003ch3\u003eMolecular docking of fisetin (5), sulfuretin (7) and butein (9) into sEH\u003c/h3\u003e\n\u003cp\u003eA three-dimensional (3D) structure of each fisetin (\u003cb\u003e5\u003c/b\u003e), sulfuretin (\u003cb\u003e7\u003c/b\u003e), and butein (\u003cb\u003e9\u003c/b\u003e) were built and minimized by MM2 running the Chem3D program (CambridgeSoft, Cambridge, MA, USA). A 3D structure of sEH (coded in 3ANS) was obtained from the RCSB protein data bank. The enzyme with water and the 4-cyano-\u003cem\u003eN\u003c/em\u003e-[(1\u003cem\u003eS\u003c/em\u003e,2\u003cem\u003eR\u003c/em\u003e)-2-phenylcyclopropyl]benzamide removed was hydrogenated by the Autodock tool and the Gasteiger charge then applied. Each ligand was formulated as a torsion tree with a focus on the torsion root and the rotatable bonds. The grid box sizes were 60 \u0026times; 60 \u0026times; 60 (compounds \u003cb\u003e5\u003c/b\u003e and \u003cb\u003e7\u003c/b\u003e) at 0.375 \u0026Aring; and 126 \u0026times; 126 \u0026times; 126 (compound \u003cb\u003e9\u003c/b\u003e). Molecular docking employed a Lamarckian genetic algorithm running the maximum number of evaluations [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Graphics were created as Ligplot (Cambridge, UK) and chimera (San Francisco, CA, USA).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eMolecular dynamics of fisetin (5), sulfuretin (7) and butein (9) into sEH\u003c/h2\u003e\u003cp\u003eTo explore the interactions between sEH and two inhibitors (fisetin and butein), sEH-inhibitor complexes were subjected to molecular dynamics simulations using the Gromacs 4.6.5 package. The ligand topology was generated employing the Gromacs G54A7FF All-Atom package of the Automated Topology Builder (ATB) and the Repository. sEH was charged by the GROMOS96 54a7 force field. The corresponding products were dissolved in water in a cubic box of the default value using the simple point charge water model with six Cl\u003csup\u003e\u0026ndash;\u003c/sup\u003e ions. The complex was minimized by a maximal force of 10 kJ/mol via the steepest descent method. This sequentially simulated constant temperature/ constant volume (NVT) equilibration at 300K, constant temperature/constant pressure (NPT) equilibration with the particle Mesh-Ewald long-range electrostatics at 1 bar, and the molecular dynamics over 100 ns, respectively. Graphics and movies were created using chimera (San Francisco, CA, USA).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePreparation of sample and standard solutions\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eT. vernicifluum\u003c/em\u003e heartwood (16 g) underwent sonication-assisted ethanol (0.8 L) extraction three times at 45\u0026deg;C for 2 h. The resulting solution was filtered and then evaporated under reduced pressure at 50\u0026deg;C to yield an extract powder (0.81 g, 4.94% yield). Five milligrams of this extract was dissolved in 1 mL methanol and filtered through a 0.45-\u0026micro;m pore-sized syringe filter. Stock solutions of the six reference standards (1 mg/mL) were prepared in HPLC-grade methanol and stored below 4\u0026deg;C. Working solutions were prepared via serial dilution of these stock solutions in methanol.\u003c/p\u003e\n\u003ch3\u003eValidation of the method\u003c/h3\u003e\n\u003cp\u003eThe UPLC method was validated in terms of linearity, the limit of detection (LOD), the limit of quantification (LOQ), precision, and accuracy. Linearity was established using six concentrations of standard solutions each analyzed in triplicate. The calibration curves were fitted via linear regression. LOD and LOQ values were determined based on signal-to-noise ratios of 3 and 10, respectively. Precision was evaluated by analyzing a standard solution five times within a single day (intra-day precision) and on three consecutive days (inter-day precision); the results were expressed as relative standard deviations (RSDs). Accuracy was ascertained via recovery tests. Varying amounts (low, medium, and high) of standards were added to samples with known quantities of materials and the percentage recoveries calculated.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eInstrumentation and the chromatographic conditions\u003c/h2\u003e\u003cp\u003eAnalysis employed an Agilent 1290 Infinity UPLC system with a binary pump, a degasser, an auto-sampler, a column compartment, and a diode array detector. Chromatographic separation was achieved on a Halo C18 column (100 \u0026times; 4.6 mm, 2.7 \u0026micro;m) at 25\u0026deg;C. The mobile phases were 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B). Gradient elution proceeded at a flow rate of 1.0 mL/min as follows: 0\u0026ndash;5 min, 10% A; 5\u0026ndash;15 min, 10\u0026ndash;20% A; 15\u0026ndash;23 min, 20\u0026ndash;22% A; 23\u0026ndash;30 min, 22\u0026ndash;25% A; 30\u0026ndash;35 min, 25\u0026ndash;40% A; 35\u0026ndash;37 min, 40\u0026ndash;100% A; and 37\u0026ndash;40 min, 100% A. Before each injection, the column was re-equilibrated with 10% A (90% B) for 5 min. The injection volume was 5 \u0026micro;L, and detection proceeded at 254 nm. Data processing employed Agilent ChemStation software.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eBioactivity-guided fractionation and isolation\u003c/h2\u003e\u003cp\u003eTo identify the active constituents, the crude ethanol extract of \u003cem\u003eT. vernicifluum\u003c/em\u003e heartwood was suspended in water and subjected to successive liquid-liquid partitioning with \u003cem\u003en\u003c/em\u003e-hexane and ethyl acetate (EtOAc). The resulting fractions were screened for sEH inhibitory activity at a concentration of 50 \u0026micro;g/mL. The \u003cem\u003en\u003c/em\u003e-hexane fraction, which typically contains lipophilic components such as urushiols and lipids, showed negligible activity. In contrast, the EtOAc-soluble fraction exhibited significant inhibition (57.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Based on this bioactivity profile, the EtOAc fraction was selected for further phytochemical investigation.\u003c/p\u003e\u003cp\u003eRepeated chromatographic separation of the EtOAc fraction using silica gel, C-18 reversed-phase silica, and Sephadex LH-20 columns resulted in the isolation of 11 compounds. Structural elucidation was performed by analyzing \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR spectroscopic data and comparing them with reported literature values. The isolated compounds were identified as fustin (\u003cb\u003e1\u003c/b\u003e) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], ethyl gallate (\u003cb\u003e2\u003c/b\u003e) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], taxifolin (\u003cb\u003e3\u003c/b\u003e) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], garbanzol (\u003cb\u003e4\u003c/b\u003e) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], fisetin (\u003cb\u003e5\u003c/b\u003e) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], butin (\u003cb\u003e6\u003c/b\u003e) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], sulfuretin (\u003cb\u003e7\u003c/b\u003e) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], eriodictyol (\u003cb\u003e8\u003c/b\u003e) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], butein (\u003cb\u003e9\u003c/b\u003e) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], gallic acid (\u003cb\u003e10\u003c/b\u003e) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and fisetinidol-4α-ol (\u003cb\u003e11\u003c/b\u003e) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u0026ndash;S11). This collection represents a diverse array of structural subclasses, including dihydroflavonols (\u003cb\u003e1\u003c/b\u003e, \u003cb\u003e3\u003c/b\u003e, \u003cb\u003e6\u003c/b\u003e, \u003cb\u003e11\u003c/b\u003e), flavonols (\u003cb\u003e4\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e), aurones (\u003cb\u003e7\u003c/b\u003e), and chalcones (\u003cb\u003e9\u003c/b\u003e), as well as simple phenolic acids (\u003cb\u003e2\u003c/b\u003e, \u003cb\u003e10\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003esEH inhibitory activities of compounds 1\u0026ndash;11 from T. vernicifluum\u003c/h2\u003e\u003cp\u003eCompounds \u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e11\u003c/b\u003e were evaluated for their inhibitory potential against sEH \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B). The modes of inhibition were determined using Lineweaver\u0026ndash;Burk (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u0026ndash;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) and Dixon (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF\u0026ndash;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH) plots. The results are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The initial screening employed sEH at 100 \u0026micro;M using 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), a recognized sEH inhibitor, as the positive control. Of the tested compounds, sulfuretin (\u003cb\u003e7\u003c/b\u003e) exhibited the highest inhibitory activity (98.24% at 100 \u0026micro;M) (Eq.\u0026nbsp;1) and an IC₅₀ of 8.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 \u0026micro;M (Eq.\u0026nbsp;2). Kinetic analysis revealed that sulfuretin (\u003cb\u003e7\u003c/b\u003e) acted as a competitive inhibitor with a \u003cem\u003eK\u003c/em\u003e\u003csub\u003ei\u003c/sub\u003e value of 1.4 \u0026micro;M. Fisetin (\u003cb\u003e5\u003c/b\u003e) also potently inhibited sEH with an IC\u003csub\u003e50\u003c/sub\u003e of 9.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 \u0026micro;M via a competitive binding mode (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ei\u003c/sub\u003e = 5.8 \u0026micro;M) although the inhibitory rate at 100 \u0026micro;M was lower (\u0026gt;\u0026thinsp;100%). Butein (\u003cb\u003e9\u003c/b\u003e) also exhibited strong inhibition (85.07% at 100 \u0026micro;M). The IC₅₀ was 21.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 \u0026micro;M, and inhibition was non-competitive (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ei\u003c/sub\u003e = 19.5 \u0026micro;M). Taxifolin (\u003cb\u003e3\u003c/b\u003e), garbanzol (\u003cb\u003e4\u003c/b\u003e), butin (\u003cb\u003e6\u003c/b\u003e), and eriodictyol (\u003cb\u003e8\u003c/b\u003e) exhibited moderate inhibition, ranging from 35 to 46%, but fustin (\u003cb\u003e1\u003c/b\u003e), ethyl gallate (\u003cb\u003e2\u003c/b\u003e), and gallic acid (\u003cb\u003e10\u003c/b\u003e) were less active. The least active compound was fisetiniol-4α-ol (\u003cb\u003e11\u003c/b\u003e) (13.86% inhibition at 100 \u0026micro;M). These findings suggested that several flavonoids, particularly fisetin (\u003cb\u003e5\u003c/b\u003e), sulfuretin (\u003cb\u003e7\u003c/b\u003e), and butein (\u003cb\u003e9\u003c/b\u003e), exhibited significant sEH inhibitory activity and may therefore serve as promising lead compounds for the development of anti-inflammatory agents targeting the sEH pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003esEH inhibitory activities, IC\u003csub\u003e50\u003c/sub\u003e values, and kinetic parameters of compounds \u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e11\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCompound\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100 \u0026micro;M (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;M)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eBinding mode (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ei\u003c/sub\u003e, \u0026micro;M)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFustin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEthyl gallate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTxifolin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e46.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGarbanzol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e39.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFisetin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCompetitive (5.8)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eButin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSulfuretin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e98.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCompetitive (1.4)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEriodictyol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e42.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eButein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e85.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e21.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNon-competitive (19.5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGallic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e11\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFisetiniol-4-ol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAUDA\u003c/b\u003e\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e92.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 nM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003ea\u003c/sup\u003eIC\u003csub\u003e50\u003c/sub\u003e values are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three independent experiments\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003eb\u003c/sup\u003eNot determined due to low inhibitory activity\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003ec\u003c/sup\u003eAUDA was used as a positive control\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eMolecular docking of fisetin (5), sulfuretin (7) and butein (9) into sEH\u003c/h2\u003e\u003cp\u003eTo elucidate the molecular interactions underlying the inhibitory effects of selected sEH inhibitors, sEH molecular docking simulations were conducted using fisetin, sulfuretin, and butein. This revealed the binding modes and affinities of the compounds to/for the active site of sEH. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e show that fisetin (\u003cb\u003e5\u003c/b\u003e) exhibited a docking score of \u0026minus;\u0026thinsp;7.06 kcal/mol and formed multiple hydrogen bonds with key active site residues, including Pro371 (2.64, 3.39 \u0026Aring;), Gln383 (3.16 \u0026Aring;), Tyr383 (2.89 \u0026Aring;), and Tyr466 (2.95 \u0026Aring;). Sulfuretin (\u003cb\u003e7\u003c/b\u003e), which exhibited the highest level of \u003cem\u003ein vitro\u003c/em\u003e inhibition, evidenced a stronger docking score of \u0026minus;\u0026thinsp;7.67 kcal/mol and hydrogen bond interactions with Pro371 (2.92 \u0026Aring;), Tyr383 (2.61 \u0026Aring;), Tyr466 (2.89, 3.01 \u0026Aring;), and Asp335 (2.72 \u0026Aring;), supporting the suggestion that competitive inhibition was in play. Butein (\u003cb\u003e9\u003c/b\u003e), a non-competitive inhibitor, exhibited the strongest binding affinity (docking score \u0026minus;\u0026thinsp;8.33 kcal/mol), forming hydrogen bonds with Phe267 (2.74 \u0026Aring;), Asp335 (2.82, 2.96 \u0026Aring;), Tyr466 (2.83 \u0026Aring;), Phe497 (2.67 \u0026Aring;), and Lys495 (2.94, 3.46 \u0026Aring;). The interactions with sites outside the catalytic domain supported the non-competitive nature of the inhibition. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows that the spatial configurations and hydrogen-bonding profiles of the inhibitors supported their observed enzyme inhibition profiles. The docking results corroborated the kinetic data, and further suggested that the binding affinities and interaction patterns of the flavonoids to/with sEH may indicate that the compounds could serve as useful leads for anti-inflammatory drug development.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMolecular docking scores and hydrogen bond interactions of fisetin (\u003cb\u003e5\u003c/b\u003e), sulfuretin (\u003cb\u003e7\u003c/b\u003e), and butein (\u003cb\u003e9\u003c/b\u003e) with sEH.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAutodock score(kcal/mol)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHydrogen bonds(\u0026Aring;)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026minus;7.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePro371(2.64, 3.39), Gln383(3.16), Tyr383(2.89), Tyr466(2.95)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026minus;7.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePro371(2.92), Tyr383(2.61), Tyr466(2.89, 3.01), Asp335(2.72)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026minus;8.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePhe267(2.74), Asp335(2.82, 2.96), Tyr466(2.83), Phe497(2.67), Lys495(2.94, 3.46)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eMolecular dynamics simulations of fisetin (5) and sulfuretin (7) with sEH\u003c/h2\u003e\u003cp\u003eTo elucidate the dynamic behavior of the ligand-protein interactions in a fluid state, we performed 100-ns molecular dynamics (MD) simulations for the sEH complexes with the two most potent inhibitors, fisetin (\u003cb\u003e5\u003c/b\u003e) and sulfuretin (\u003cb\u003e7\u003c/b\u003e). Visual inspection of the simulation trajectories confirmed that both compounds maintained stable binding poses within the active site throughout the simulation period. As illustrated in the superposition of snapshots, fisetin (\u003cb\u003e5\u003c/b\u003e) was stably accommodated in the binding pocket adjacent to the Asp335\u0026ndash;Trp342 loop (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), while sulfuretin (\u003cb\u003e7\u003c/b\u003e) remained tightly bound near the Asn359\u0026ndash;Phe387 active site loop (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), suggesting high affinity for the catalytic pocket.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe global stability of these complexes was quantitatively assessed using Root Mean Square Deviation (RMSD) analysis. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, the backbone RMSD values for both sEH\u0026ndash;ligand complexes stabilized at approximately 3.0 \u0026Aring; after an initial equilibration phase, indicating that the ligands achieved a stable equilibrium within the binding pocket. The thermodynamic stability of these systems was further corroborated by the total potential energy, which averaged approximately \u0026minus;\u0026thinsp;540 \u0026times; 10\u0026sup3; kJ/mol for both complexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). To investigate local protein flexibility upon ligand binding, we analyzed the Root Mean Square Fluctuation (RMSF) per residue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). While the overall globular structure remained rigid, distinct fluctuation patterns were observed in specific loop regions; the Pro367\u0026ndash;Val367 loop exhibited significant perturbation in the presence of fisetin (\u003cb\u003e5\u003c/b\u003e), whereas the sEH\u0026ndash;sulfuretin (\u003cb\u003e7\u003c/b\u003e) complex displayed increased flexibility in the Glu414\u0026ndash;Gly426 loop (RMSF\u0026thinsp;~\u0026thinsp;4.0 \u0026Aring;).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFinally, we analyzed the intermolecular contacts to define the structural basis of inhibition. Hydrogen bond analysis revealed a clear distinction in binding modes; sulfuretin (\u003cb\u003e7\u003c/b\u003e) consistently maintained a higher number of hydrogen bonds (4\u0026ndash;6 bonds) compared to fisetin (\u003cb\u003e5\u003c/b\u003e) (1\u0026ndash;3 bonds) throughout the trajectory (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). This observation was supported by distance measurements between key pharmacophores and active site residues. For fisetin (\u003cb\u003e5\u003c/b\u003e), the hydroxyl groups remained within the hydrogen-bonding threshold (\u0026lt;\u0026thinsp;3.5 \u0026Aring;) of Asp335 and Ile363 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). In contrast, sulfuretin (\u003cb\u003e7\u003c/b\u003e) established a more extensive network of persistent interactions, with its A and B ring hydroxyl groups positioned within 3.5 \u0026Aring; of Asp335, Thr360, Asn378, and Gln384 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF\u0026ndash;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). These results suggest that sulfuretin (\u003cb\u003e7\u003c/b\u003e) is effectively anchored in the catalytic center through multiple stable hydrogen bonds, which may contribute to its potent inhibitory activity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eUPLC optimization and peak identification\u003c/h2\u003e\u003cp\u003eTo correlate the biological activity with the chemical composition of the extract, a robust UPLC-DAD method was developed and validated. Optimal chromatographic conditions were established using the Halo C18 column, gradient elution employing acetonitrile and water (both with 0.1% formic acid), a column temperature of 25\u0026deg;C, a flow rate of 1.0 mL/min, and detection at 254 nm. Under these conditions, the six reference standards were well-resolved within 40 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). When the retention times and ultraviolet spectra of materials in the \u003cem\u003eT. vernicifluum\u003c/em\u003e heartwood extract were compared to those of the standards, the major peaks in the chromatogram (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) were identified as ethyl gallate (peak 1), fustin (peak 2), garbanzol (peak 3), fisetin (peak 4), sulfuretin (peak 5), and butein (peak 6).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eMethod validation\u003c/h2\u003e\u003cp\u003eThe UPLC method was carefully validated. The calibration curves for all six analytes exhibited good linearity, with all correlation coefficients (r\u0026sup2;)\u0026thinsp;\u0026ge;\u0026thinsp;0.9995 within the tested concentration ranges. The LODs determined at a signal-to-noise ratio of 3 ranged from 0.92 to 21.86 \u0026micro;g/mL. The LOQs determined at a signal-to-noise ratio of 10 ranged from 0.31 to 7.21 \u0026micro;g/mL (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Intra- and inter-day precisions were assessed using standard solutions at various concentrations. The RSD values for intra-day precision ranged from 0.13 to 2.15%. In terms of inter-day precision, the RSD values ranged from 0.23 to 2.69% (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The low RSD values indicate that the analytical method was very precise. Accuracy was evaluated via recovery tests. The extract was spiked with known quantities of the standards. The average recoveries of the six targeted compounds ranged from 96.88 to 104.96%, with all RSD values below 3.97% (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The UPLC method was accurate.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCalibration curve, linearity, LOD, and LOQ for six reference standards (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCompound\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRegression equation\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLinear range (\u0026micro;g/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCorrelation\u003c/p\u003e\u003cp\u003ecoefficient (\u003cem\u003er\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLOD\u003csup\u003eb\u003c/sup\u003e (\u0026micro;g/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLOQ\u003csup\u003ec\u003c/sup\u003e (\u0026micro;g/mL)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEthyl gallate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ey\u0026thinsp;=\u0026thinsp;4.414x\u0026thinsp;+\u0026thinsp;0.7516\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.25\u0026ndash;200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.9999\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFustin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ey\u0026thinsp;=\u0026thinsp;2.4936x\u0026thinsp;+\u0026thinsp;46.592\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e125\u0026ndash;4000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.9999\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e21.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e7.21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEthyl gallate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ey\u0026thinsp;=\u0026thinsp;4.414x\u0026thinsp;+\u0026thinsp;0.7516\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.25\u0026ndash;200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.9999\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGarbanzol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ey\u0026thinsp;=\u0026thinsp;5.3794x\u0026thinsp;+\u0026thinsp;1.1065\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.125\u0026ndash;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.9996\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFisetin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ey\u0026thinsp;=\u0026thinsp;13.778x \u0026minus;\u0026thinsp;16.769\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.812\u0026ndash;250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.9999\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSulfuretin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ey\u0026thinsp;=\u0026thinsp;7.8088x\u0026thinsp;+\u0026thinsp;11.033\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15.625\u0026ndash;500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.9999\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e11.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3.66\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eButein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ey\u0026thinsp;=\u0026thinsp;8.0222x \u0026minus;\u0026thinsp;2.4665\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.125\u0026ndash;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.9995\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003ea\u003c/sup\u003ey, peak area of compound; x, concentration (\u0026micro;g/mL) of compound\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003eb\u003c/sup\u003eLOD, limit of detection, S/N\u0026thinsp;=\u0026thinsp;3\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003ec\u003c/sup\u003eLOQ, limit of quantification, S/N\u0026thinsp;=\u0026thinsp;10\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrecision (intra- and inter-day) of six reference standards (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCompound\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c3\" namest=\"c2\" rowspan=\"2\"\u003e\u003cp\u003eAnalyte concentration (\u0026micro;g/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e\u003cp\u003eIntra-day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u003cp\u003eInter-day\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eDetected concentration (\u0026micro;g/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eRSD (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003eDetected concentration (\u0026micro;g/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eRSD (%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"2\" nameend=\"c2\" namest=\"c1\" rowspan=\"3\"\u003e\u003cp\u003eEthyl gallate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e0.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e100.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.09\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e50.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e0.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e50.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.51\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e24.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e0.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e24.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e2.69\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"2\" nameend=\"c2\" namest=\"c1\" rowspan=\"3\"\u003e\u003cp\u003eFustin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1971.14\u0026thinsp;\u0026plusmn;\u0026thinsp;23.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e1.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e1970.62\u0026thinsp;\u0026plusmn;\u0026thinsp;13.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.68\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e120.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e120.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.23\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e160.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e0.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e160.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"2\" nameend=\"c2\" namest=\"c1\" rowspan=\"3\"\u003e\u003cp\u003eGarbanzol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e51.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e1.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e50.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.86\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e24.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e0.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e24.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.70\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e1.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e11.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"2\" nameend=\"c2\" namest=\"c1\" rowspan=\"3\"\u003e\u003cp\u003eFisetin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e62.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e61.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e2.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e62.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e2.51\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e31.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e0.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e30.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e2.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15.625\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e0.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e14.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"2\" nameend=\"c2\" namest=\"c1\" rowspan=\"3\"\u003e\u003cp\u003eSulfuretin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e124.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e1.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e127.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e62.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e63.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e1.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e63.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.73\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e31.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e0.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e31.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.22\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"2\" nameend=\"c2\" namest=\"c1\" rowspan=\"3\"\u003e\u003cp\u003eButein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e49.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e49.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e2.51\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e24.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e1.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e24.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.46\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e0.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e13.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e2.48\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eRecovery of six reference standards (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCompound\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSpiked concentration (\u0026micro;g/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDetected concentration (\u0026micro;g/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRecovery (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eRSD (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eEthyl gallate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e101.04\u0026thinsp;\u0026plusmn;\u0026thinsp;2.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e101.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.70\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e24.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e96.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.47\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eFustin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2061.21\u0026thinsp;\u0026plusmn;\u0026thinsp;14.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e103.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.70\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e973.73\u0026thinsp;\u0026plusmn;\u0026thinsp;19.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e97.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e519.22\u0026thinsp;\u0026plusmn;\u0026thinsp;6.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e103.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.22\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eGarbanzol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e51.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e102.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.87\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e24.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e98.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.80\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eFisetin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e62.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e61.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e98.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.65\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e31.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e101.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.06\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15.625\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e104.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.54\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eSulfuretin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e128.42\u0026thinsp;\u0026plusmn;\u0026thinsp;2.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e102.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e62.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e61.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e97.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.84\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e31.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e97.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.97\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eButein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e26.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e104.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e104.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.79\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eQuantitative analysis of the extract\u003c/h2\u003e\u003cp\u003eThe validated UPLC method was used to quantify the six compounds (in mg/g) in the ethanol extract of \u003cem\u003eT. vernicifluum\u003c/em\u003e heartwood. The analysis was performed in triplicate, and the contents of the compounds were expressed as mg/g of extract (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Fustin was the most abundant component (262.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.90 mg/g, 26.21% of the extract), followed by sulfuretin (23.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 mg/g) and fisetin (14.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 mg/g). Ethyl gallate (9.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 mg/g), butein (5.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52 mg/g), and garbanzol (4.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 mg/g) were present in smaller quantities.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eContents of six compounds in the \u003cem\u003eT. vernicifluum\u003c/em\u003e heartwood extract\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCompound\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eContent (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003emg/g\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e%\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEthyl gallate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.97\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFustin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e262.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e26.21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGarbanzol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.42\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFisetin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.42\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSulfuretin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e23.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eButein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.59\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis comprehensive study employed UPLC to quantitate key flavonoids in a heartwood extract of \u003cem\u003eT. vernicifluum\u003c/em\u003e, followed by evaluation of their sEH inhibitory capacities. Fustin was the most abundant compound (262.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.90 mg/g) but exhibited relatively weak sEH inhibition. This underscores the principle that phytochemical concentration alone does not dictate biological potency; rather, the structural features of a compound and its precise molecular interactions with the target enzyme are paramount. While fustin, a dihydroflavonol, is known to exhibit other biological activities, including antioxidant and neuroprotective effects [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], its direct contribution to the overall sEH inhibition of the crude extract appears to be limited.\u003c/p\u003e\u003cp\u003eIn contrast, sulfuretin, fisetin, and butein were identified as potent sEH inhibitors, despite being present in lower quantities (23.47, 14.20, and 5.93 mg/g, respectively). The aurone sulfuretin (\u003cb\u003e7\u003c/b\u003e) and the flavonol fisetin (\u003cb\u003e5\u003c/b\u003e) both exhibited competitive inhibition with IC\u003csub\u003e50\u003c/sub\u003e values of 8.8 and 9.6 \u0026micro;M, respectively. This suggests that both compounds directly compete with natural enzyme substrates for binding at the catalytic site. Molecular docking results supported this, indicating interactions with key sEH active site residues such as Tyr383 and Tyr466 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These residues are crucial for sEH hydrolase activity, and their engagement by inhibitors often mimics the binding of natural substrates or urea-based synthetic inhibitors [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo complement the static docking models, molecular dynamics simulations provided a time-resolved view of the ligand-enzyme interactions for the competitive inhibitors. The simulations confirmed that both fisetin and sulfuretin form stable complexes with sEH, as evidenced by steady RMSD values. A key finding was the differential modulation of loop flexibility; fisetin significantly perturbed the Pro367\u0026ndash;Val367 loop, whereas sulfuretin increased fluctuations in the Glu414\u0026ndash;Gly426 region. Furthermore, hydrogen bond analysis over the simulation trajectory reinforced the superior potency of sulfuretin, which maintained a higher number of stable contacts with catalytic residues, particularly Asp335, compared to fisetin. This dynamic stability serves as strong validation for the potential of sulfuretin as a robust sEH inhibitor.\u003c/p\u003e\u003cp\u003eDistinct from the competitive inhibitors, the chalcone butein (\u003cb\u003e9\u003c/b\u003e) exhibited strong non-competitive sEH inhibition (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;21.4 \u0026micro;M). This indicates that butein likely binds to an allosteric site on sEH rather than the substrate-binding pocket, inducing a conformational change that attenuates enzyme activity. Earlier studies on butein derivatives, such as butein-4'-\u003cem\u003eO\u003c/em\u003e-glucoside, have similarly reported non-competitive sEH inhibition and described interactions with residues consistent with allosteric binding [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The distinct modes of inhibition observed, competitive for sulfuretin and fisetin, and non-competitive for butein, suggest that synergistic effects may be possible if these compounds are used in combination.\u003c/p\u003e\u003cp\u003eThe ability to distinguish these individual contributions was made possible by the development and validation of a robust UPLC method. This analytical approach ensured reliable quantitative data, which is essential for quality control and for linking chemical composition to biological activity in natural product research [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The precise quantification allowed us to determine that while fustin is the major constituent, the biological activity is driven by the less abundant, high-potency flavonoids.\u003c/p\u003e\u003cp\u003eThe inhibition of sEH is a well-recognized strategy for mitigating inflammation and related pathologies, as it prevents the degradation of anti-inflammatory EpFAs like EETs [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The potent sEH inhibition demonstrated by sulfuretin, fisetin, and butein may partially explain the traditional use of \u003cem\u003eT. vernicifluum\u003c/em\u003e in East Asia for treating inflammatory conditions, pain, and gastric ailments [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Moreover, these flavonoids are known to exert anti-inflammatory effects via additional pathways, notably by modulating NF-κB and MAPK signaling cascades [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This multi-targeting capability, simultaneous sEH inhibition and signaling pathway modulation, represents a major advantage of natural product-derived therapies over single-target synthetic drugs.\u003c/p\u003e\u003cp\u003eThis distinct advantage of natural product-derived polypharmacology is particularly relevant given the significant hurdles encountered in the clinical development of synthetic sEH inhibitors [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. While synthetic analogues often demonstrate high potency, their translation into approved therapeutics has been frequently impeded by pharmacokinetic limitations, including poor absorption, short biological half-lives, low bioavailability, and potential off-target toxicities [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In contrast, natural products occupy a vast and biologically relevant chemical space, offering immense structural diversity that has evolved to interact with biological macromolecules [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This structural complexity may provide molecules with superior physicochemical properties, better safety profiles, and enhanced oral bioavailability compared to synthetic libraries. Consequently, the flavonoids identified in \u003cem\u003eT. vernicifluum\u003c/em\u003e not only serve as promising leads that circumvent the common pitfalls of synthetic inhibitors but also offer a strategic scaffold for developing novel, safe, and effective anti-inflammatory agents.\u003c/p\u003e\u003cp\u003eHowever, translating these promising \u003cem\u003ein vitro\u003c/em\u003e results into clinical therapeutics requires addressing specific challenges. A primary concern is the presence of urushiols in \u003cem\u003eT. vernicifluum\u003c/em\u003e, which are known to cause severe contact dermatitis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Consequently, future development strategies must prioritize the rigorous purification of active flavonoids or the implementation of validated detoxification protocols to eliminate allergenic risks. Furthermore, comprehensive \u003cem\u003ein vivo\u003c/em\u003e studies are essential to verify the anti-inflammatory efficacy, safety, and pharmacokinetic profiles of these compounds in physiological systems. Looking forward, the identification of both competitive and non-competitive inhibitors within the same source offers a unique opportunity to explore synergistic combination therapies. Ultimately, this study establishes a scientific basis for valorizing \u003cem\u003eT. vernicifluum\u003c/em\u003e heartwood not merely as a traditional remedy, but as a refined source of potent, multi-targeted sEH inhibitors.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study successfully identified and characterized several secondary metabolites from the heartwood of \u003cem\u003eT. vernicifluum\u003c/em\u003e that effectively inhibited sEH. A validated UPLC analysis method revealed that fustin was the most abundant of all measured flavonoids, but only weakly inhibited sEH. In contrast, sulfuretin, fisetin, and butein, all of which were present in lower but still significant quantities, were more potent inhibitors, with IC\u003csub\u003e50\u003c/sub\u003e values in the low micromolar range. Kinetic studies, supported by molecular docking tests, revealed that fisetin and sulfuretin were competitive inhibitors and butein a non-competitive inhibitor. These findings, emphasized by the precise quantitative data derived via UPLC analysis, highlight the fact that specific \u003cem\u003eT. vernicifluum\u003c/em\u003e heartwood constituents may serve as valuable leads for the development of novel, natural product-based anti-inflammatory agents that target the sEH pathway. Further \u003cem\u003ein vivo\u003c/em\u003e studies are essential to comprehensively evaluate the therapeutic efficacies and safeties of such promising compounds.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of interest\u003c/h2\u003e\u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eAdditional information\u003c/h2\u003e\u003cp\u003eThe online version contains supplementary information available at ~ .\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research was supported by the Cooperative Research Program of the Rural Development Administration (RS-2022-RD010239) and a grant from the Korea Institute of Oriental Medicine (KSN2224020), Republic of Korea.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJang Hoon Kim: Writing \u0026ndash; original draft, Conceptualization, Data curation, Resources, Investigation, Methodology, Software, Visualization. Jae-Young Cheon: Data curation, Investigation, Methodology, Software, Visualization, Validation. Jin Yu: Investigation, Methodology. Yong-Goo Kim: Investigation, Methodology. Sung Yeon Kim: Investigation, Methodology. Kyong-Hwan Bang: Investigation, Methodology. Jin Tae Jeong: Investigation, Methodology. Hyun-Ju Jung: Writing \u0026ndash; review and editing, Conceptualization. Ik Soo Lee: Writing \u0026ndash; original draft, review, and editing, Data curation.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author, Dr. Ik Soo Lee ([email protected]), on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWagner, K. M., McReynolds, C. B., Schmidt, W. K. \u0026amp; Hammock, B. D. Soluble epoxide hydrolase as a therapeutic target for pain, inflammatory and neurodegenerative diseases. \u003cem\u003ePharmacol. 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The chromosome-level genome for \u003cem\u003eToxicodendron vernicifluum\u003c/em\u003e provides crucial insights into Anacardiaceae evolution and urushiol biosynthesis. \u003cem\u003eiScience\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e, 104512 (2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Toxicodendron vernicifluum, flavonoid, soluble epoxide hydrolase, competitive inhibitor, molecular docking","lastPublishedDoi":"10.21203/rs.3.rs-8274523/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8274523/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSoluble epoxide hydrolase (sEH) is a therapeutic target for managing inflammation by preserving anti-inflammatory epoxy fatty acids (EpFAs). This study investigates the sEH inhibitory potential of secondary metabolites isolated from the heartwood of \u003cem\u003eToxicodendron vernicifluum\u003c/em\u003e (Stokes) F.A. Barkley. Bioactivity-guided fractionation of the ethanol extract revealed that the ethyl acetate-soluble fraction possessed significant sEH inhibitory activity (~\u0026thinsp;60% at 50 \u0026micro;g/mL). Subsequent purification yielded 11 polyphenolic compounds, which were identified via spectroscopic methods and quantified using a validated Ultra-High-Performance Liquid Chromatography (UPLC) protocol. Among these, the aurone sulfuretin (\u003cb\u003e7\u003c/b\u003e) and the flavonol fisetin (\u003cb\u003e5\u003c/b\u003e) exhibited potent competitive inhibition with IC\u003csub\u003e50\u003c/sub\u003e values of 8.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 \u0026micro;M and 9.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 \u0026micro;M, respectively. The chalcone butein (\u003cb\u003e9\u003c/b\u003e) demonstrated strong non-competitive inhibition (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;21.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 \u0026micro;M). Molecular docking and 100 ns molecular dynamics (MD) simulations revealed that sulfuretin forms a stable high-affinity complex within the sEH catalytic pocket, anchored by persistent hydrogen bonds with Asp335 and Tyr383. Conversely, butein interacts with a peripheral allosteric site. These findings highlight \u003cem\u003eT. vernicifluum\u003c/em\u003e heartwood as a source of diverse sEH inhibitors with potential for development as anti-inflammatory agents.\u003c/p\u003e","manuscriptTitle":"Soluble Epoxide Hydrolase Inhibitory Constituents from the Heartwood of Toxicodendron vernicifluum: Isolation, Kinetic Characterization, Molecular Modeling, and Quantitative Analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-15 09:32:02","doi":"10.21203/rs.3.rs-8274523/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-22T07:02:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-20T05:37:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-13T20:50:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"292303280070643269237424882846838427739","date":"2025-12-10T15:33:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"192952979110838295560975086404920179330","date":"2025-12-10T12:07:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"174446029327601617155925544522054725764","date":"2025-12-10T03:42:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"55624028022745259168633720598758222072","date":"2025-12-09T23:57:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-09T13:44:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-09T13:36:13+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-09T11:25:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-09T01:13:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-12-09T01:07:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6dd8003f-3c85-48d1-a209-2889ee0bcc5c","owner":[],"postedDate":"December 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":59609360,"name":"Biological sciences/Biochemistry"},{"id":59609361,"name":"Biological sciences/Chemical biology"},{"id":59609362,"name":"Physical sciences/Chemistry"},{"id":59609363,"name":"Biological sciences/Drug discovery"}],"tags":[],"updatedAt":"2026-01-26T15:59:29+00:00","versionOfRecord":{"articleIdentity":"rs-8274523","link":"https://doi.org/10.1038/s41598-026-36728-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-20 15:56:53","publishedOnDateReadable":"January 20th, 2026"},"versionCreatedAt":"2025-12-15 09:32:02","video":"","vorDoi":"10.1038/s41598-026-36728-3","vorDoiUrl":"https://doi.org/10.1038/s41598-026-36728-3","workflowStages":[]},"version":"v1","identity":"rs-8274523","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8274523","identity":"rs-8274523","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}