Development of a UHPLC-PDA Method for the Simultaneous Quantification of 4-Phenylcoumarins and Chlorogenic Acid in Exostema caribaeum Stem Bark

Abstract

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.

Molecular Markers Associated With Chemical Analysis: A Powerful Tool for Quality Control Assessment of Copalchi Medicinal Plant Complex

“…In order to identify the diagnostic compounds in the leaves of the Rubiaceae species, the chromatographic profiles of the infusions were compared with the retention time and UV spectra and by spiking by triplicate with standards (10 μL of standard stock solution) separated under the same analytical conditions for the following compounds: chlorogenic acid ( 1 ), 5- O -β-D-glucopyranosyl-7,3′,4′-trihydroxy-4-phenylcoumarin ( 2 ), 5- O -[β-D-xylopyranosyl-(1 → 6)-β-D-glucopyranosyl]-7-methoxy-3′,4′-dihydroxy-4-phenylcoumarin ( 3 ), 5- O -[β-D-apiofuranosyl-(1 → 6)-β-D-glucopyranosyl]-7-methoxy-3′,4′-dihydroxy-4-phenylcoumarin ( 4 ), 5- O -β-D-galactopyranosyl-7-methoxy-3′,4′-dihydroxy-4-phenylcoumarin ( 5 ), 5- O -β-D-glucopyranosyl-7-methoxy-3′,4′-dihydroxy-4-phenylcoumarin ( 6 ), 6′′- O -acetyl-5- O- β-D-galactopyranosyl-7,4′-dihydroxy-4-phenylcoumarin ( 7 ), 6′′- O -acetyl-5- O- β-D-galactopyranosyl-7,3′,4′-trihydroxy-4-phenylcoumarin ( 8 ), 6′′- O -acetyl-5- O- β-D-galactopyranosyl-7-methoxy-3′,4′-dihydroxy-4-phenylcoumarin ( 9 ), and 5- O -[β-D-xylopyranosyl-(1 → 6)-β-D-glucopyranosyl]-7,4′-dimethoxy-4-phenylcoumarin ( 10 ) (Table 1 ). The stock standard solutions were prepared separately by accurately weighing 10 mg, pouring into 10 mL volumetric flasks, and dissolving in CH 3 CN-H 2 O (1:3) (Mata et al, 1990 , 2008 ; Guerrero-Analco et al, 2007 ; Cristians et al, 2009 , 2014 ; Pérez-Vásquez et al, 2014 ).…”

Development of a HPLC-DAD Method for the Simultaneous Quantification of 5-O-β-D-galactopyranosyl-7-methoxy-3’,4’-dihydroxy-4-phenylcoumarin and its Aglycone in Feces

“…Since the previous analytical methods for quantification of 4-phenylcoumarins [27][28][29] did not work in a biological matrix base analysis, different mobile phases (acetonitrile and TFA acidified water 50:50 Vol/Vol; 20:80 Vol/Vol; 24:76 Vol/Vol; 15:85 Vol/Vol; 10:90 Vol/Vol), flow rates (1 mL/min; 1.2 mL/min; 1.5 mL/min), columns (Symmetry C-18, Waters; Synergi C-18, Phenomenex; Eclipse C-8, Agilent; Zorbax C-18, Agilent) and column temperature were tested in order to obtain chromatographic resolution. The detection wavelength was set at 328nm in an HPLC-DAD 1100 (Agilent Technologies, Palo alto, CA, USA).…”

“…The method development and optimization were established empirically for the best solubilization conditions for both 4-PC and aglycone; also, the former analytical methods reported for 4-phenylcoumarins analysis [27][28][29] were considered for those goals ( Table 1). The development of this method was focused to the column, mobile phase and flow rate, in order to achieve the simultaneous detection and quantification of the bioactive compounds in feces in the less analytical time.…”

“…The situation of 4-phenylcoumarins is no exception, the analytical methods generated were fixed on quantify these compounds as active markers of the stem-bark infusions of H. latiflora and H. standleyana crude drugs, using HPLC-UV detector [27], identify the 4-phenylcoumarins as chemical constituents of the leaves infusions of both Hintonia species by HPLC method [28]. Recently, the simultaneous quantification of both 4-phenylcoumarins and chlorogenic acid in Exostema caribaeum stem-bark aqueous extract by UPLC-PDA method has also been reported [29]; nevertheless, there is no records of analytical methods aimed to detect and quantify the 4-phenylcoumarins and their metabolites, the aglycones, in biological matrices as a part of pharmacokinetics studies. The goal of this investigation was to develop and validate an HPLC-DAD method for the quantification of 4-phenylcoumarin and its aglycone in biological fluids, such as feces in order to apply it in preclinical pharmacokinetic studies.…”

Safety and Efficacy of Banaba–Moringa oleifera–Green Coffee Bean Extracts and Vitamin D3 in a Sustained Release Weight Management Supplement

“…The average daily intake of chlorogenic acid primarily from coffee consumption has been estimated to be about 150 mg per day, depending upon the kind of coffee and the amount consumed (Ludwig et al, 2014;Grosso et al, 2014). Chlorogenic acid appears to be exceedingly safe with an oral LD50 in mice greater than 2 g/kg (Perez-Vasquez et al, 2014).…”