Hydroxychavicol‐Rich Piper betle Extracts Attenuate TNFα‐Driven Endothelial Inflammatory Response by Modulating Adhesion Molecules and Cytokine Release

Atherosclerosis is a chronic inflammatory disorder that involves endothelial dysfunction and accumulation of inflammatory cells in the vessel intima. Approximately 32% of global mortality is attributed to cardiovascular diseases, with atherosclerosis playing a major role. Endothelial dysfunction is a key hallmark of atherosclerosis, with key mediator tumor necrosis factor‐α (TNF‐α) driving endothelial–monocyte interactions that further lead to adverse cardiovascular events. Hydroxychavicol, a phenolic compound enriched in Piper betle (PB), methanolic and chloroform extracts, has demonstrated anti‐inflammatory activity, yet its effects on endothelial activation remain insufficiently characterized. Therefore, this study aimed to investigate the endothelial‐protective effects of hydroxychavicol‐rich PB methanol (PB‐M) and chloroform (PB‐C) extracts by evaluating their impact on the expression of adhesion molecules and proinflammatory cytokines in TNF‐α‐induced human umbilical vein endothelial cells (HUVECs). HPLC was performed to confirm the presence and determine the amount of hydroxychavicol in both PB‐M and PB‐C. The cytotoxicity effects of both extracts were first assessed by MTT assay. HUVECs were pretreated with NF‐κB inhibitor BAY11‐7082, PB‐M, or PB‐C before being exposed to TNF‐α for 4 h. Adhesion molecules and cytokines expression were measured by Western blot and ELISA, respectively. Both PB‐M and PB‐C were shown to contain hydroxychavicol, yet PB‐C has about 3.8 times higher levels. MTT assays yielded IC ₅₀ values of 128.9 µg/mL for PB‐M and 18.59 µg/mL for PB‐C, while HUVEC morphology remained normal at 100 µg/mL PB‐M and 20 µg/mL PB‐C. Both extracts displayed dose‐dependent suppressing effects on TNF‐α‐induced E‐selectin, VCAM‐1, and ICAM‐1 expression. PB‐M resulted in a pronounced inhibition of IL‐6, IL‐8, and MCP‐1, while PB‐C displayed cytokine‐selective suppressive effects. In conclusion, both PB‐M and PB‐C attenuated TNF‐α‐induced endothelial inflammation, with PB‐C acting at lower effective doses but PB‐M providing broader anti‐inflammatory suppression, suggesting that their different phytochemical compositions and possible synergistic interactions beyond hydroxychavicol may contribute to their distinct bioactivities, warranting further investigations.