Mixture of guava leaves extract and Citrus hassaku pericarp extract inhibits in vitro inflammatory biomarkers by blocking ERK, JNK, and p38 MAPK signaling and protects mice from lethal endotoxemia

Although anti-inflammatory activity of guava leaves (GLE) and fruits of Citrus hassaku Hort ex Tanaka (phalsak, PSE) has been demonstrated in cell culture system, the combination therapy of GLE and PSE with better anti-inflammatory activity and the exact mechanism remains unclear. In the present study, we set out to determine whether the anti-inflammatory effects of optimal mixture (OM) are mediated to suppress mitogen-activated protein kinases (MAPKs), nuclear factor-κB (NF-κB), and activated protein-1 (AP-1) activation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. We found that OM significantly suppressed LPS-stimulated NO production without a cytotoxic effect on RAW 264.7 cells. OM inhibited the expression of LPS-induced iNOS and COX-2 protein and their mRNA expression. Also, TNF-α, IL-6, and PGE₂ secretion were decreased by OM in LPS-stimulated macrophages. As a result, OM inhibited pro-inflammatory markers such as TNF-α, IL-6, and PGE₂, which is hypothesized as being due to the suppression of LPS-induced ERK, p38, and JNK signaling pathway, but not NF-κB and AP-1. Moreover, OM improved the survival rate during lethal endotoxemia by inhibiting the production of TNF-α and IL-6 in an animal model.