Development of new isolation technique and validated HPLC method development for khellin- A major constituent of Ammi visnaga Lam. fruits

Abstract

Ammi visnaga Lam. (Family-Apiaceae) commonly known as khella or picktooth fruit is used for skeletal muscle relaxant activity. In this study, furanochromone khellin was isolated from its fruits by solvent extraction followed by partitioning of solvents. The isolated compound was identified by UV visible spectroscopy, IR, MASS, 1H NMR, 13C NMR and TLC. Reverse phase high performance liquid chromatography (RP-HPLC) method has been developed for quantification of khellin by using C-18 column (250 x 4.6, 5-μm) with an isocratic mobile phase, acetonitrile and water with 0.1 % ortho phosphoric acid (40:60 v/v) at a flow rate of 1.0 mL/min. The wavelength of maximum absorption (λmax) selected for quantification of khellin was 338 nm. The amount of khellin present in fruits was found to be 1.06 % w/w. The developed method has been validated for accuracy, precision and specificity and successfully applied for quantification of isolated compound in fruits. Thus, isolated khellin could be used as phytochemical markers for the quality control of this plant as well as other formulation containing khella fruits.