Repressing effect of transformed ginsenoside Rg3-mix against LPS-induced inflammation in RAW264.7 macrophage cells

BackgroundRg3-ginsenoside, a protopanaxadiol saponin, is a well-known adaptogen used for the prevention of cancer and inflammation. However, despite its distinct biological activity, the concentration of Rg3 in the total ginseng extract is insufficient for therapeutic applications. This study aims to convert PPD-class of major ginsenosides into a mixture of minor ginsenoside, to analyze its immune-regulatory role in macrophage cells.ResultsUsing heat and organic acid treatment, three major ginsenosides, Rc, Rd, and Rb1, were converted into a mixture of minor ginsenosides, GRg3-mix [Rg3(S), Rg3(R), Rg5, and Rk1]. Purity and content analysis of the transformed compound were performed using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), compared with their standards. Preceding with the anti-inflammatory activity of GRg3-mix, lipopolysaccharide (LPS)-stimulated murine RAW264.7 macrophage cells were treated with various concentrations of GRg3-mix (6.25, 12.5, 25, and 50 mu g/mL). The cell viability assay revealed that the level of cell proliferation was increased, while the nitric oxide (NO) assay showed that NO production decreased dose-dependently in activated RAW264.7 cells. The obtained results were compared to those of pure Rg3(S) >= 98% (6.25, 12.5, and 25 mu g/mL). Preliminary analysis of the CCK-8 and NO assay demonstrated that GRg3-mix can be used as an anti-inflammatory mediator, but mRNA and protein expression levels were evaluated for further confirmation. The doses of GRg3-mix significantly suppressed the initially upregulated mRNA and protein expression of inflammation-related enzymes and cytokines, namely inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear transcription factor kappa B (NF-kappa B), tumor necrosis factor (TNF-alpha), and interleukins (IL-6 and IL1B), as measured by reverse transcription-polymerase chain reaction and western blotting.ConclusionsOur pilot data confirmed that the mixture of minor ginsenosides, namely GRg3-mix, has high anti-inflammatory activity and has an easy production procedure.