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Charred areca nut (CAN) is used to treat dyspepsia and abdominal distension in children. However, reports revealed that arecoline, the most important active constituent of CAN, possesses potential toxicities. This study was designed to investigate the optimum arecoline content in CAN, using the “target constituent removal combined with bioactivity assay” strategy. Based on PTLC method, we prepared CAN lacking all arecoline (WAC-100R) and a series of different ratios of arecoline-removed CAN samples (WAC-Rx). MTT and acute toxicity assays indicated that decreasing content by 50% decreased CAN toxicity significantly. Animal results revealed arecoline contents over 50% could guarantee the beneficial effects of CAN on gastrointestinal tract. Additionally, decreasing arecoline content in CAN by 50% decreased its pro-apoptotic effects significantly. Furthermore, decreasing 50% arecoline content in CAN down-regulated the expressions of Cleaved-Caspase-3, c- jun , c- fos , COX-2, PGE2, and IL-1α. Thus, our results revealed that CAN with 50% arecoline content (WAC-50R) has similar beneficial effects on the gastrointestinal tract to CAN, whereas its toxicity was decreased significantly. Collectively, our study suggested that the strategy of “target constituent removal combined with bioactivity assay” is a promising method to identify the optimum arecoline content in CAN, which is approximately 0.12%.

Using the “target constituent removal combined with bioactivity assay” strategy to investigate the optimum arecoline content in charred areca nut