Identification and Quantitation of Ursolic and Oleanolic Acids in Ilex aquifolium L. Leaf Extracts Using 13C and 1H-NMR Spectroscopy.

Leaves of Ilex aquifolium L. have been used for their therapeutic properties. In previous studies, components contained in the leaves were first isolated by various chromatographic techniques. Then, quantitation of oleanolic and ursolic acids, which are responsible for the biological and therapeutic activities of the plant, was performed by HPLC, HPTLC, and somewhat by GC-MS. Our objective was to develop a simple method that allows the identification of compounds contained in the leaves of Corsican I. aquifolium and to quantify ursolic and oleanolic acids. Leaves were successively extracted with hexane and dichloromethane. The extracts were chromatographed on silica gel and the fractions of column chromatography submitted to ¹³C-NMR analysis, following a computerized method developed in the laboratory. ¹³C-NMR allowed the identification of various triterpenes including ursolic acid and oleanolic acid. Quantitation of both acids was achieved, for the first time, by ¹H-NMR after validation of the method (accuracy, precision, linearity, limit of detection and limit of quantitation). Ursolic and oleanolic acids accounted for 55.3% and 20.8% of the dichloromethane extract, respectively. This represents 1.3% and 0.5% of the mass of dried leaves. ¹H-NMR spectroscopy appeared as a powerful tool for a rapid quantitation of biologically active compounds from I. aquifolium.