Development of a UHPLC-PDA method for the simultaneous quantification of 4-phenylcoumarins and chlorogenic acid in Exostema caribaeum stem bark.

Potential toxic effects in mice of an infusion prepared from the stem bark of Exostema caribaeum was assessed by means of the Lorke procedure. The preparation was not found to be toxic, with the LD50 value estimated to be more than 5 g/kg. This preparation at 100, 300, and 500 mg/kg also caused a significant hypoglycemic effect and a reduction in the postprandial glycemia peak in both normal and nicotinamide/streptozotocin (NA/STZ)-diabetic mice in an oral sucrose tolerance test. Phytochemical analysis of the infusion revealed that the major active principles are 4-phenylcoumarins (2-8) and chlorogenic acid (1). During this process, a new 4-phenylcoumarin was isolated along with several known analogues. The structure of the new compound was established as 5-O-[β-D-xylopyranosyl-(1→6)-β-D-glucopyranosyl]-7,3',4'-trihydroxy-4-phenylcoumarin (2) by spectroscopic means. A simple, efficient, fast, and reliable UHPLC-PDA analytical method for quantifying 4-phenylcoumarins and chlorogenic acid (1) was developed and validated. Parameters assessed for the method validation were selectivity, linearity, the limits of detection (LOD) and quantification (LOQ), precision, and accuracy. It was found that all calibration curves showed good linearity (R(2) > 0.9931), within the range of concentrations tested.