Identification of Bioactive Natural Product from the Stems and Stem Barks of Cornus walteri: Benzyl Salicylate Shows Potential Anti-Inflammatory Activity in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

Abstract

Cornus walteri (Cornaceae), known as Walter's dogwood, has been used to treat dermatologic inflammation and diarrheal disease in traditional oriental medicine. As part of an ongoing research project to discover natural products with biological activities, the anti-inflammatory potential of compounds from C. walteri in lipopolysaccharide (LPS)-stimulated mouse RAW 264.7 macrophages were explored. Phytochemical analysis of the methanol extract of the stem and stem bark of C. walteri led to the isolation of 15 chemical constituents. These compounds were evaluated for their inhibitory effects on the production of the proinflammatory mediator nitric oxide (NO) in LPS-stimulated macrophages, as measured by NO assays. The molecular mechanisms underlying the anti-inflammatory activity were investigated using western blotting. Our results demonstrated that among 15 chemical constituents, lupeol and benzyl salicylate inhibited NO production in LPS-activated RAW 264.7 macrophages. Benzyl salicylate was more efficient than N^G-monomethyl-L-arginine mono-acetate salt (L-NMMA) in terms of its inhibitory effect. In addition, the mechanism of action of benzyl salicylate consisted of the inhibition of phosphorylation of IκB kinase alpha (IKKα), IκB kinase beta (IKKβ), inhibitor of kappa B alpha (IκBα), and nuclear factor kappa B (NF-κB) in LPS-stimulated macrophages. Furthermore, benzyl salicylate inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Taken together, these results suggest that benzyl salicylate present in the stem and stem bark of C. walteri has potential anti-inflammatory activity, supporting the potential application of this compound in the treatment of inflammatory diseases.

Keywords: Cornus walteri; cornaceae; cyclooxygenase-2; inducible nitric oxide synthase; inflammation; nitric oxide; nuclear factor kappa B.

Figure 1

Separation scheme of compounds 1–15. 5α-Stigmast-3,6-dione (1), 3β-sitostanol (2), 6α-hydroxy-β-sitostenone (3), 6β-hydroxysitostenone (4), norphytan (5), phytone (6), methyl 3-O-acetylbetulinate (7), 3-O-acetylbetulin (8), sitostenone (9), leucophyllone (10), lupeol (11), lupenone (12), betulinic acid (13), betulinic acid methyl ester (14), and benzyl salicylate (15). MeOH, methanol; BuOH, butanol; RP, reversed phase; HPLC, high-performance liquid chromatography.

Figure 2

Chemical structures of compounds 1–15. 5α-Stigmast-3,6-dione (1), 3β-sitostanol (2), 6α-hydroxy-β-sitostenone (3), 6β-hydroxysitostenone (4), norphytan (5), phytone (6), methyl 3-O-acetylbetulinate (7), 3-O-acetylbetulin (8), sitostenone (9), leucophyllone (10), lupeol (11), lupenone (12), betulinic acid (13), betulinic acid methyl ester (14), and benzyl salicylate (15).

Figure 3

Effects of compounds 1–15 and N^G-monomethyl-L-arginine mono-acetate salt (L-NMMA) in RAW 264.7 mouse macrophages treated with lipopolysaccharide (LPS). (A–P) The effects of compounds 1–15 and L-NMMA in RAW 264.7 mouse macrophages treated with LPS were investigated (mean ± SD, *p < 0.05 compared to group treated with 1 μg/mL LPS alone). 5α-Stigmast-3,6-dione (1), 3β-sitostanol (2), 6α-hydroxy-β-sitostenone (3), 6β-hydroxysitostenone (4), norphytan (5), phytone (6), methyl 3-O-acetylbetulinate (7), 3-O-acetylbetulin (8), sitostenone (9), leucophyllone (10), lupeol (11), lupenone (12), betulinic acid (13), betulinic acid methyl ester (14), and benzyl salicylate (15).

Figure 4

Effects of benzyl salicylate (15) on the expression of IκB kinase alpha and beta (IKKα/β), inhibitor of kappa B alpha (I-κBα), and nuclear factor kappa B (NF-κB) in RAW 264.7 mouse macrophages treated with lipopolysaccharide (LPS). (A) Representative western blots showing protein expressions of IKKα/β, phospho-IKKα/β (p-IKKα/β), I-κBα, phospho-IKKα/β (p-I-κBα), NF-κB, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (B–D) Quantitative bar chart for each protein’s expression level (mean ± SD, *p < 0.05 compared to group treated with 1 μg/mL LPS alone).

Figure 5

Effects of benzyl salicylate (15) on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in RAW 264.7 mouse macrophages treated with lipopolysaccharide (LPS). (A) Representative western blots showing protein expressions of iNOS, COX-2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (B–C) Quantitative bar chart for each protein’s expression level (mean ± SD, * p < 0.05 compared to group treated with 1 μg/mL LPS alone).

Figure 6

Schematic pathway for the potential role of benzyl salicylate (15) isolated from C. walteri in inflammatory responses. LPS, lipopolysaccharide; p, phosphorylated; IKK, IκB kinase alpha; IκBα, inhibitor of kappa B alpha; p56 and p50, cellular proteins; MAPK, mitogen-activated protein kinase; Ac, activated; TNF-α, tumor necrosis factor alpha; IL-6, interleukin 6; COX-2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase.