Quantitative HPLC Assay for the Phthalides in Celery Products
Celery Apium graveolens has been an important culinary plant whose medicinal properties have been used in homeopathic medicine for treatment as an anti-inflammatory, anti-pyretic, diuretic, and sedative. In the celery "sweetening" process for desired flavors in foods, the bitter portion is separated out. Analysis of this bitter portion has revealed two major phthalide compounds called sedanenolide and 3-n-butyl phthalide. 3-nbutyl phthalide has been scientifically proven to induce detoxifying enzymes that can prevent cancer. Sedanenolide which constitutes 70-80% of the bitter portion of Indian celery has not had much documentation. The purpose of this experiment is to isolate and quantify sedanenolide and the phthalides in celery products by HPLC assay. Bitter fractions from the celery "sweetening" process are taken through solvent extractions to remove solids and other impurities. Using AgN03 -10% on silica and a solvent of 75% hexane/25% acetone in column chromatography, HPLC analysis of column separations gave peak area percentages of 93.4% sedanenolide and 6.06% 3-n-butyl phthalide. Pure isolation of sedanenolide was not possible with the column chromatographies performed. Using a standard 3-n-butyl phthalide, peak areas for the compound were plotted as a function for the amount of sample injected into the HPLC to give a standard calibration curve. Since sedanenolide accounts for the rest of the composition in celery bitter products, its weight was deduced from the amount of sample injected into the HPLC minus 3-n-butyl phthalide. A calibration curve was then computed for sedanenolide. Relative amounts of each compound in a celery product could then be quantified. For an oleoresin, results of 3.8 ± 0.2% sedanenolide and 2.3 ± 0.1% 3-n-butyl phthalide were obtained. Percent recovery of the HPLC procedure were 99 % for sedanenolide and 100% for 3-n-butyl phthalide.