Quantitative analysis of anti-inflammatory and radical scavenging triterpenoid esters in evening primrose oil

Cold pressed, nonraffinated evening primrose oil (EPO) was recently found to contain lipophilic triterpenoidal esters with radical scavenging and anti-inflammatory properties. A simple and robust method for the quantitative analysis of these 3-O-trans-caffeoyl derivatives of betulinic, morolic, and oleanolic acid was developed and validated. Separation was achieved by normal phase chromatography on a Diol column and with hexane/ethyl acetate (50:50) as eluent. The analytes could be determined directly in the oil matrix, without need of a previous removal of the triglycerides. Normal phase LC ESI-MS with a makeup flow of polar modifier was used for checking the identity and purity of analyte peaks. Samples from 22 commercially available EPOs were analyzed. The average caffeoyl ester contents were 58 mg/100 g in cold pressed oils and 4.7 mg/100 g in partially raffinated oils. In fully raffinated EPO samples, the concentration was below the limit of detection. The influence of extraction temperature on the content of caffeoyl esters in nonraffinated EPO was investigated with seeds of Oenothera biennis and Oenothera lamarckiana, respectively. With O. lamarckiana, the concentration of caffeoyl esters in the oil increased with rising pressure and temperature, whereas no such dependency was found with O. biennis. Microscopic analysis revealed some differences in the histology of the seed testa, which may explain in part the differing behaviors in the extraction experiments. There was a difference between O. biennis and O. lamarckiana oils with respect to the relative amounts of the three esters. The temperature of the extraction process had no effect on the ratio of the compounds.