A New Ceanothane-Type Triterpenoid Saponin Isolated from Gouania leptostachya DC. var. tonkinensis Pit. and Its Underlying Anti-Inflammatory Effects

Abstract

Metabolites from medicinal plants continue to hold significant value in the exploration and advancement of novel pharmaceuticals. In the search for plants containing compounds with anti-inflammatory effects, we observed that the ethanol (EtOH) extract obtained from the aerial components of Gouania leptostachya DC. var. tonkinensis Pit. exhibited substantial suppression of nitric oxide (NO) in vitro. In a phytochemical study on an EtOH extract of G. leptostachya, 11 compounds were purified, including one unreported compound namely gouanioside A (1). Their chemical structures were unambiguously determined through the use of various spectroscopic techniques, such as 1 and 2D NMR, IR, and HR-ESI-MS, and by producing derivatives via chemical reactions. The EtOH extract, fractions, and a new compound exerted inflammatory effects by altering NO synthesis in murine RAW264.7 macrophage cells stimulated with lipopolysaccharide. The underlying inflammatory mechanism of the new compound 1 was also explored through various in vitro experiments. The results of this study indicate the potential usefulness of new compound 1 from G. leptostachya as a treatment for inflammatory diseases.

Keywords: Gouania leptostachya; anti-inflammatory effect; gouanioside A; saponin.

Fig. 1. Structures of the isolated compound (1-11) purified from G. leptostachya aerial parts.

Fig. 2

(A) Connectivity deduced by the COSY (bold), DEPT, and HSQC spectra, and key HMBC correlations (blue arrows) of 1. (B) Significant NOESY (→) correlations of aglycon of 1. After energy minimization, the threedimensional conformation of substance 1 was generated using the Macromodel program (version 12.5, Schrodinger LLC).

Fig. 3

(A) Cytotoxic effects of ethanol (EtOH) extract (1, 5, and 25 μg) and fractions on RAW 264.7 cells over 3 days. GLT (EtOH extract), GLH (n-hexane fraction), GLB (BuOH fraction), GLE (EtOAc fraction), and GLW (water fraction), respectively. The positive control utilized in the experiment was Dexamethasone (Dexa). (B) Inhibition of the activation of RAW 264.7 cells by the EtOH extract and fractions in vitro. Different concentrations of the treatment were administered to the cells (1, 5, and 25 μg) of extract and fractions, or with Dexa as the positive control. The levels of nitric oxide (NO) in the culture supernatants were determined using the Griess method, **p < 0.01, ***p < 0.001. Dexamethasone was shown to have stable activity in the experiment.

Fig. 4

Inhibitory effects of new compound 1 (GLB5) on lipopolysaccharide (LPS)-stimulated (A) NO, (B) PGE₂, and (C) TNF-α production in RAW264.7 macrophages, respectively. RAW264.7 cells (2 × 10⁶ cell/well) were pretreated with compound 1 (GLB5) for 1 h before being stimulated for 12 h with LPS. The NO, PGE₂, and TNF-α levels were determined by the Griess method and an enzyme linked immunosorbent assay kit. Data represent five independent experiments and are expressed as the mean ± the standard error of the mean (SEM). Dexamethasone (dexa) was used as the positive control. The experiments were repeated five times. (**p < 0.01, ***p < 0.001). Dexamethasone was shown to have stable activity in the experiment.

Fig. 5

(A) Effects of compound 1 (GLB5, 10 μM) with the capacity for influencing the expression of COX-2 mRNA using the RT-PCR technique, (B) COX-2 gene activity through luciferase gene assay, and (C) evaluating COX-2 protein expression through Western blot analysis, respectively. β-actin as a loading control. The intensity of the bands was measured using ImageJ software. **p < 0.01 versus the LPS alone group.

Fig. 6. Molecular docking results of compound 1 with the COX-2 protein (PDB: 1PXX).

(A) three-dimensional (3D) docking image of compound 1 was created by Pymol 2.5. (B) two-dimensional (2D) docking image of 1 was created by LigPlot+ 2.2.