Antioxidant Assay-Guided Purification and LC Determination of Ellagic Acid in Pomegranate Peel

On the basis of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay-guided purification, ellagic acid was isolated from the methanol extract of pomegranate fruit peel by liquid-liquid extraction and chromatographic techniques. A reversed-phase high-performance liquid chromatography was described for determination of ellagic acid in pomegranate fruit peel extract. The method involved the use of a TSK-gel ODS-80Tm column with a mixture of 2% aqueous acetic acid and methanol (gradient elution mode: 0-15 min, 40-60% v/v methanol and 15-20 min, 60% v/v methanol) as the mobile phase and detection at 254 nm. The parameters of linearity, repeatability, reproducibility, accuracy, and specificity of the method were evaluated. The recovery of the method was 98.5% and linearity (r(2) > 0.9995) was obtained for ellagic acid. A high degree of specificity as well as repeatability and reproducibility (relative standard deviation values less than 5%) were also achieved. The limits of detection and quantification were 1.00 and 2.50 microg/mL, respectively. The solvent for extraction of ellagic acid from pomegranate fruit peel was examined in order to maximize the ellagic acid content of the extract. A solution of 10% v/v water in methanol was capable of increasing the ellagic acid content in the extract up to 7.66% w/w. The ellagic acid content and antioxidant activity of the ethyl acetate fraction separated from the crude extract using water and ethyl acetate partition was higher than that of the crude extract.