Spectrophotometric Quantification of Flavonoids in Herbal Material, Crude Extract, and Fractions from Leaves of Eugenia uniflora Linn

Background: The traditional use of Eugenia uniflora L. (“Pitanga”) is reported due to several properties, which have often been related to its flavonoid content. Objective: The aim was to evaluate analytical procedures for quantification of total flavonoids content (TFCs) by ultraviolet-visible (UV-Vis) spectrophotometry in the herbal material (HM), crude extract (CE), and fractions from leaves of E. uniflora. Materials and Methods: The method for quantification of flavonoids after complexation with aluminum chloride (AlCl₃) was evaluated: amount of sample (0.25–1.5 g); solvent (40%–80% ethanol); reaction time and AlCl₃concentration (2.5%–7.5%). The procedures by direct dilution (DD) and after acid hydrolysis (AH) were used and validated for HM and CE and applied to the aqueous fraction (AqF), hexane fraction, and ethyl acetate fractions (EAF). Results: The ideal conditions of analysis were ethanol 80% as solvent; 0.5 g of sample; λmax of 408 (DD) and 425 nm (AH); 25 min after addition of AlCl₃ 5%. The procedures validated for standards and samples showed linearity (R² > 0.99) with limit of detection and limit of quantification between 0.01 and 0.17 mg/mL (rutin and quercetin); and 0.03 and 0.09 mg/mL (quercetin), for DD and AH, respectively. The procedures were accurate (detect, practice, and repair <5% and recovery >90%), and stable under robustness conditions (luminosity, storage, reagents, and equipment). The TFCs in AqF and EAF were 0.65 g% and 17.72 g%, calculated as rutin. Conclusions: UV-Vis methods for quantification of TFC in HM, CE, and fractions from leaves of E. uniflora were suitably validated. Regarding the analysis of fractions, the EAF achieved enrichment of about nine times in the content of flavonoids.