A liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis method was developed to determine the contents of esculetin in medicinal plants. The analysis was performed using multiple reaction monitoring (MRM) in negative mode, and an XBridgeT...
A liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis method was developed to determine the contents of esculetin in medicinal plants. The analysis was performed using multiple reaction monitoring (MRM) in negative mode, and an XBridgeTM C18 column (2.1×100 mm, 3.5 m) was used. Methanol and 0.1% formic acid were used for gradient analysis. The calibration curve showed good linearity (r2>0.9993). The limit of detection (LOD) and limit of quantitation (LOQ) were 0.02 and 0.07 ng/ml respectively. The intra- and inter-day precisions were 1.5-6.8% and 2.0-5.3%, respectively, and the accuracy was 102.0-110.2%. The contents of esculetin in 35 different plants were determined, and Fraxini cortex showed the highest content of esculetin (761.5-5,475.0 mg/kg). In Mori folium and Artemisiae capillaris herba, 5.2-21.5 mg/kg and 7.0-17.6 mg/kg of esculetin were found, respectively. In other medicinal plants, no esculetin was detected, or it was present at a concentration less than 10 mg/kg. The analysis method appears to be simple, sensitive, and reproducible. The pharmacological effects of esculetin isolated from medicinal plants were investigated as part of the new medicines development. Esculetin is known to inhibit the proliferation of vascular smooth muscle cells (VSMC). However, the signaling pathway by which esculetin mediates its molecular effects in VSMC remains to be identified. The present results suggest an unexpected role of the p38 MAPK signaling pathway in esculetin-induced inhibition of VSMC growth. Treatment of VSMC with esculetin resulted in significant growth inhibition and G1-phase cell cycle arrest, which was followed by down-regulation of cyclins and cyclin-dependent kinase (CDK) expression. This G1-phase cell-cycle arrest was due to up-regulation of p21WAF1 expression. In addition, esculetin treatment activated p38 MAPK and ERK1/2. Pretreatment with SB203580, which is a p38 MAPK specific inhibitor, or expression of the dominant negative p38 MAPK (DN p38 MAPK) gene blocked esculetin-induced p38 MAPK activation and p21WAF1 expression. Finally, both the growth inhibition and the down-regulation of CDKs induced by esculetin were suppressed by either SB203580 or the DN p38 MAPK mutant gene. In conclusion, these results demonstrate that activation of p38 MAPK contributes to esculetin-induced p21WAF1 expression in VSMC by decreasing both the cyclin D1/CDK4 and cyclin E/CDK2 complexes.