Comparative analysis of ursolic acid in Hawthorn leaves by HPLC

AIM: To develop a simple HPLC for the determination of ursolic acid in Hawthorn leaves, and to compare ursolic acid content in Hawthorn leaves of different species, locations and growth stages, so as to supply some evidences for the exploitation and utilization of Hawthorn leaves reasonably. METHODS: By high-performance liquid chromatography method. Lichrospher C18 column (250×4.6 mm I.D. 5 μm); mobile phase, acetonitrile-water-orthophosphoric acid (85∶14.95∶0.05) with a flow-rate of 1.00 ml/min; column temperature at ~30 °C ; injection volume, 5 μl; UV detector at 210 nm. RESULTS: The detection limit (S/N=3) was less than ~4.024 μg/ml and the limit of quantification(S/N=10) was less than 12.05 μg/ml. The calibration curve showed good linear regression(r=0.9999) within measurement ranges(16.09-1030 μg/ml). The intra-day and inter-day variation were 0.71% and 6.15%, respectively. The recoveries at low to high concentration were 89%-105%. Under these conditions, the ursolic acid content in different Hawthorn leaves were determined:1.90%-~1.95% in C. scabrifolia (Franch.) Rehd, 1.00%-1.45% in C. cuneata Sieb. Zucc, 0.45%-0.65% in C. pinnatifida Bge. var. major N.E.Br.; In differnet growth stages of C. pinnatifida Bge. var. major N.E.Br., the young leaves contain higher content of ursolic acid. CONCLUSION:The method is successfully applied to quantify ursolic acid in Hawthorn leaves. And the ursolic acid contents in Hawthorn leaves of differnent species are very different; C. scabrifolia (Franch.) Rehd contains the highest ursolic acid content in them. However, there is a little difference among different locations and growth stages for same species.