Planta Med 2013; 79 - PN56
DOI: 10.1055/s-0033-1348737

HPLC-DAD Method for Quantification of the Flavonoids with Antiradicalar Activity in the Hydroethanolic Extract from Tonina fluviatilis aubl. (Eriocaulaceae)

MR de Amorim 1, D Rinaldo 2, FP do Amaral 3, MA Galvão Magenta 4, W Vilegas 3 L Campaner dos Santos 1*
  • 1Department of Organic Chemistry, Institute of Chemistry, UNESP – Sao Paulo State University, Araraquara CEP 14800 – 900, Sao Paulo, Brazil
  • 2UNESP, São Paulo State University, Faculty of Sciences, Chemistry Department, CEP 17033 – 360, Bauru, São Paulo, Brazil
  • 3Experimental Campus of Sao Vicente, UNESP – Sao Paulo State University, Sao Vicente, CEP 11350 – 000, Sao Paulo, Brazil
  • 4Unisanta University – Bertioga – Sao Paulo State – CEP 11045 – 907, Sao Paulo, Brazil

This work describes the isolation of flavonoids by preparative chromatography, and identification by NMR experiments, mass spectrometry (ESI-ITMS) and the development and validation for simultaneous quantification based on HPLC-DAD method for this flavonoids in the hydroethanolic extract of the aerial-parts from Tonina fluviatilis. The flavonoids identified and quantified in the extract were: 6,7-dimethoxyquercetin-3-O-β-D-glicopyranoside (1), 6-hidroxy-7-methoxyquercetin-3-O-β-D-glicopyranoside (2) and 6-methoxyquercetin-3-O-β-D-glicopyranoside (3). The HPLC-DAD analyses were performed using a C18 column and gradient programs with water and methanol, both acidified with 0.1% TFA, as mobile phase. The analysis of the samples spiked with known amounts of (1 – 3) demonstrated that the response was proportional to the concentrations of the samples with the determination coefficient being r2= 0.9989 (1) and r2= 0.9992 (2 and 3) for the linear range of the analytical calibration curves (10, 25, 50, 75, 100 and 250 µg.mL-1).The method is sensitive enough for the analysis of the three flavonoids quercetin derivatives, with (LOD within 8.90 and 10.64 µg.mL-1; LOQ within 26.99 and 32.22 µg.mL-1). Also, good results were obtained with respect to repeatability (coefficients of variation – CV < 2%) and recovery (higher than 90%) in three different concentrations (1, 20 and 40 µg.mL-1). The antiradicar activity (DPPH) for hydroethanolic extract was (29.1%) and substances 1 (39.5%) and 2-3 (87.7%) in the kidnapping concentration of 200 µg.mL-1 compared to positive controls (quercetin and gallic acid).

(Granted by FAPESP, CNPq).