Inhibitory Activity of Glycosides from Elsholtzia ciliata against Soluble Epoxide Hydrolase and Cytokines in RAW264.7 Cells

Abstract

Soluble epoxide hydrolase (sEH) and pro-inflammatory cytokines are associated with the development of inhibitors for cardiovascular and inflammatory diseases. Here, we report on four natural sEH inhibitors isolated from the aerial parts of Elsholtzia ciliata (Thunb.) Hyl.. The four compounds, 1-4, were identified as luteolin-7-O-glucoside (1), yuanhuanin (2), apigenin-7-O-glucoside (3), and butein-4'-O-glucoside (4). Among them, compounds 2 and 4 are reported for the first time from this plant. In vitro and in silico, they showed inhibitory activity towards sEH at micromole concentrations. Moreover, they suppressed pro-inflammatory cytokines in polyinosinic:polycytidylic acid (poly(I:C))-stimulated RAW264.7 cells. Notably, 4 significantly downregulated the sEH catalytic reaction, NO and PGE2 production, and the expression levels of iNOS, COX-2, IL-6 mRNA, and sEH mRNA. Therefore, butein-4'-O-glucoside (4) is a potential sEH inhibitor that may be suitable for treating inflammation and cardiovascular diseases caused by infection.

Keywords: Elsholtzia ciliata; Lamiaceae; cytokines; glycoside; soluble epoxide hydrolase.

Fig. 1. The structure of compounds 1–4 derived from the aerial parts of E. ciliata.

Fig. 2. The inhibitory activity (A), Lineweaver-burk (B–E) and Dixon (F–I) plots of compounds 1–4 toward sEH.

Fig. 3. The hydrogen bonds of inhibitors 1–4 (A–D) with sEH.

The overlapped pose of inhibitor 4 (E) with sEH for 100 ns (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, menganta: 80 ns, white: 90 ns, black: 100 ns). RMSD (F), RMSF (G), the potential energy (H), and hydrogen bonds (I) of the simulation.

Fig. 4. Cell viability of inhibitors on RAW264.7 cells (A).

The inhibitory activity of inhibitors 1–4 on NO (B) and PGE₂ (C) production in poly (I.C.)-stimulated RAW264.7 cells. iNOS and COX-2 protein levels (D) in western blot and relative densities iNOS (E) and COX-2 (F) of by inhibitors. IL-6 (G) protein levels, and IL-6 (H) and sEH (I) mRNA expression levels.