Ethanol extract of lymphanax with gypenoside 17 and ginsenoside Re exerts anti-inflammatory properties by targeting the AKT/NF-κB pathway

Background: Ginseng is processed into several types such as white ginseng, red ginseng, and black ginseng, according to the processing methods such as drying, steaming, and heating. These processing conditions can change the portion of the useful ingredients. Recently, new processing method was established to develop 'lymphanax', an aged fresh white ginseng prepared under anaerobic condition. This aging process was revealed to increase the content of gypenoside 17 (Gyp17) as well as ginsenoside Re, known to have anti-inflammatory effects. As the next step, therefore, we aimed to investigate the anti-inflammatory activity of lymphanax using its ethanol extract of lymphanax (Lymphanax-EE). Methods: LC-MS/MS identified the ginsenoside content of lymphanax-EE. A nitric oxide (NO) assay revealed the anti-inflammatory activity of lymphanax-EE. Pro-inflammatory gene expression was analyzed by quantitative PCR. Finally, we identified the underlying mechanism for the anti-inflammatory activity of lymphanax-EE through luciferase analysis, Western blotting, and CETSA. Results: The LC-MS/MS analysis revealed lymphanax-EE to contain more protopanaxatriol-type ginsenosides, and Gyp17 than fresh ginseng. Lymphanax-EE (0-200 mu g/ml) suppressed NO release and mRNA levels of pro- inflammatory cytokines such as iNOS and COX-2 in LPS-treated RAW264.7 cells. Moreover, lymphanax-EE (200 mu g/ml) reduced the activity of NF-kappa B and phosphorylation of NF-kappa B signal proteins such as p65, p50, I kappa B alpha, and IKK alpha/ . Finally, lymphanax-EE (200 mu g/ml) decreased the phosphorylation of IKK alpha/ induced by AKT overexpression. Among the components of lymphanax-EE, ginsenoside Re and Gyp17 were found to suppress AKT1 activity. Conclusions: Lymphanax-EE-containing ginsenosides and Gyp17 with anti-inflammatory properties suppressed LPS-induced inflammation by reducing the NF-kappa B signal.