Saponin of Momordica cymbalaria exhibits anti-inflammatory activity by suppressing the expression of inflammatory mediators in lipopolysaccharide-stimulated RAW264.7 macrophages

Abstract

Background: Inflammation is an intricate biological process that commonly occurs in response to pathologic stimuli, and natural products have potential in healing inflammation. However, the anti-inflammatory of Momordica cymbalaria is not evaluated yet. Objectives: The anti-inflammatory mechanism of saponin of M. cymbalaria (SMC) was investigated in bacterial lipopolysaccharide (LPS)-stimulated RAW264.7 mouse macrophage cell line. Methods: The cytotoxicity of SMC on RAW264.7 cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay at 500, 250, 125, 62.5, 31.25, 15.625, 7.812, 3.906, and 1.953 μg/mL. For anti-inflammatory activity, RAW264.7 cells were stimulated with Escherichia coli LPS (1 μg/ml) in the presence or absence of SMC (50 μg/ml) for 16–24 h. Western blotting was carried to comprehend the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide (NO) synthase (iNOS) whereas expressions of the pro-inflammatory cytokines (interleukin [IL]-6, IL-1β, and tumor necrosis factor-alpha [TNF-α]) and prostaglandin E2 (PGE2) were studied by enzyme-linked immunosorbent assay (ELISA). NO production was estimated by Griess s method. Salicylic acid, a nonsteroidal anti-inflammatory drug, was used as a standard. Results: The saponin did not exert significant cytotoxicity on RAW264.7 cells. Western blot analysis revealed reduction in COX-2 and iNOS expression on SMC treatment. Production of PGE2, IL-6, IL-1β, and TNF-α was also found to be reduced when analyzed by ELISA. NO levels were also lowered. Conclusions: The findings suggest that the SMC possesses potential anti-inflammatory activity by suppressing the expression of inflammatory mediators, COX-2, iNOS, PGE2, and NO, and the cytokines in LPS-stimulated RAW264.7 cells. Abbreviations Used: IL-6: Interleukin-6; IL-1β: Interleukin-1 beta; TNF-α: Tumor necrosis factor-alpha; NO: Nitric oxide; COX-2: Cycloxygenase-2; LPS: Lipopolysaccharide; HPTLC: High-performance thin layer chromatography; TLC: Thin layer chromatography; HPLC: High-performance liquid chromatography; SMC: Saponin of Momordica cymbalaria; DMSO: Dimethylsulfoxide; EDTA: Ethylenediaminetetraacetate; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TBST: Tris buffer saline tween; IgG-HRP: Immunoglobulin G-horse radish peroxidase; H2O2: Hydrogen peroxide; ELISA: Enzyme-linked immunosorbent assay.