A new phenolic derivative with soluble epoxide hydrolase and nuclear factor-kappaB inhibitory activity from the aqueous extract of Acacia catechu

Abstract One novel phenolic compound, (4S,5R)-4-(3,4-dihydroxyphenyl)-5-(3-oxobutyl)dihydrofuran-2(3H)-one (1), as well as 12 known compounds (2–13) was obtained from the aqueous extract of Acacia catechu and their chemical structures were determined by spectroscopic analysis. Compounds 8 and 9 exhibited significant soluble epoxide hydrolase (sEH) inhibitory activities with IC50 values of 26.6 ± 0.5 and 24.4 ± 5.6 μM, respectively. Compounds 7–10 showed significant inhibitory effects on TNFα-induced nuclear factor kappa B (NF-κB) transcriptional activity in a dose-dependent manner, with IC50 values ranging from 11.15 to 19.45 μM. Graphical abstract


Results and discussion
The inhibitory activity of isolated compounds was first evaluated against sEH at 100 μM. Compounds 8-10 showed significant inhibitory effects, with inhibition rates of 73.6-90.8%. Various concentrations (12.5, 25, 50 and 100 μM) of these compounds were then tested, and compounds 8 and 9 were found to exert potent activities, with IC 50 values of 24.4 ± 5.6 μM and 26.6 ± 0.5 μM, respectively (Table 1). Compound 10 showed moderate effects with an IC 50 value of 62.4 ± 0.5 μM.
All compounds were tested for cytotoxic activity against HepG2 cells. And no significant cytotoxicity was observed at the tested concentrations (data shown in supporting information). Compounds 1-13 were pretreated with transfected HepG2 cells at various concentrations (0.1, 1 and 10 μM), stimulating with TNF-α. The results showed that compounds 1-10 significantly inhibited TNFα-induced NF-κB transcriptional activity in a dose-dependent manner, with IC 50 values ranging from 11.15 to 32.07 μM. Compounds 11 and 13 had weak effects, with IC 50 values of 40.48 and 49.60 μM, respectively (Table 2). However, compound 12 was inactive (IC 50 > 50 μM) at the tested concentrations.
Based on the structure-activity relationship of the isolated compounds, the results showed that flavonoids (8-10), especially flavonols (8 and 9) are primary bioactive constituents against sEH. Conversely, phenols and flavonoids (1-10) exerted significant NF-κB inhibitory activities. These results indicated that phenols and flavonoid derivative components of A. catechu may play key functional roles in anti-inflammatory activity.

Plant material
The aqueous extracts of A. catechu were purchased from the herbal company, Naemome dah, ulsan, Korea in 2013, and were identified by Prof. Young Ho Kim, Chungnam National university. A voucher specimen (CNu13107) was deposited at herbarium, College of Pharmacy, Chungnam National university, Korea.

sEH assay
The sEH inhibition assays were performed as described in the previous study. A solution of recombinant sEH from human (the enzymes were purchased from Cayman Chemical Company) in buffer (Bis-Tris-HCl, 25 mM, pH 7.0, containing 0.1 mg/ml BSA) was incubated with an inhibitor at room temperature for 30 min. To the resultant solution, cyano(6-methoxynaphthalen-2-yl)methyl trans-[(3-phenyloxiran-2-yl)methyl] carbonate (purchased from Cayman Chemical Company) was added and incubated at room temperature for 20-45 min. ZnSO 4 was added and the resultant solution of fluorescence intensity (excitation filter 330 nm, emission filter 465 nm) was measured.
Briefly, 50 μL of sEH and 20 μL of various concentrations of the compounds dissolved in MeOH were added in 96-well plate containing 80 μL of 25 mM bis-tris-HCl buffer, mixed with 50 μL of 40 μM PHOME. After starting the enzyme reaction at 37 °C, products by hydrolysis of the substrate were monitored at excitation and emission of 330 and 465 nm during 1 h.
Enzyme activity (%) = [S 60 − S 0 ∕C 60 − C 0 ] × 100 where C 60 and S 60 were the fluorescence of control and test samples after 60 min, S 0 and C 0 is the fluorescence of control at 0 min.

Cell viability assay
A CCK-8 cell-counting kit (dojindo, Kumamoto, Japan) was used to analyse the cell toxicity of the compounds according to the manufacturer's instructions. HepG2 cells were cultured overnight in a 96-well plate (about 1 × 10 4 cells/well). After 24 h, each compound was added in a dose-dependent manner. After 24 h of treatment, 10 μL of the CCK-8 solution was added to triplicate wells and incubated for 1 h. The absorbance at 450 nm was measured to determine the viable cell numbers.

NF-κB luciferase assay
The luciferase vector was first transfected into human hepatocarcinoma HepG2 cells. HepG2 cells were transfected with NF-κB luciferase reporter (3Enhancer-ConA) with the Neon R Transfection system (Invitrogen) according to the manufacturer's instructions. After a limited amount of time, the cells were lysed, and luciferin, the substrate of luciferase, was introduced into the cellular extract along with Mg 2+ and an excess of ATP. under these conditions, luciferase enzymes expressed by the reporter vector could catalyse the oxidative carboxylation of luciferin. Cells were seeded at 2 × 10 5 cells per well in 12-well plates and grown. After 24 h, cells were transfected with inducible NF-κB luciferase reporter and constitutively expressing Renilla reporter. After 24 h of transfection, the medium was changed to assay medium (Opti-MEM + 0.5% FBS + 0.1 mM NEAA + 1 mM sodium pyruvate + 100 units/mL penicillin + 10 μg/mL streptomycin) and cells were pretreated for 1 h with either vehicle (dMSO) and compounds, followed by 1 h of treatment with 10 ng/mL TNF-α for 20 h. unstimulated cells were used as a negative control (−), apigenin was used as a positive control. dual luciferase assay was performed 48 h after transfection, and promoter activity values are expressed as arbitrary units using a Renilla reporter for internal normalisation.

Conclusion
In this study, 13 phenolic compounds (1-13) were isolated from the aqueous extract of A. catechu . Compounds 1, 3, 6, 11 and 13 were isolated from the Leguminosae family for the first time. Moreover, this study is the first to show that phenolic components from A. catechu inhibit sEH and TNFα-induced NF-κB transcriptional activity. The results presented herein suggest that the phenolic components in A. catechu contribute to its observed anti-inflammatory effects.

Disclosure statement
No potential conflict of interest was reported by the authors.