Determination of bioactive coumarins in Radix angelicae pubescentis by HPLC

Radix angelicae pubescentis (Duhuo in Chinese) was collected in Chinese Pharmacopoeia 2010 and widely used to treat thrombosis, arthritic disease, and anti-inflammatory. Pharmacological studies and clinical practice have demonstrated that Radix angelicae pubescentis possesses bioactive coumarins, including osthole, O-acetyl-columbianetin, and columbianadin.

The HPLC method for determination of O-acetyl-columbianetin, osthol and columbianadin in radix angelicae pubescentis has been developed in this paper. HPLC separations were carried out with a Diamonsil C₁₈ column (250mm×4.6mm, 5µm). The mobile phase was consisted of water and acetonitrile. The gradient elution was as follows: 0 to15min, isocratic (52:48, v/v); 15 to 45min linear gradient (52:48 to 42:58, v/v). The column temperature operated at 40°C. The detection wavelength was 322nm. The system suitability tests, including the linearity, limit of detection, limit of quantification, precision, repeatability and accuracy have been made. The developed method provides high selectivity and sensitivity with good accuracy and reproducibility.

The established method was applied to simultaneous determination of the three analytes in seven samples of radix angelicae pubescentis obtained from different companies and batches. The content ranges of three bioactive coumarins are 3.60 to 1.36mg/g for O-acetyl-columbianetin, 6.03 to 4.44mg/g for osthol, and 4.04 to 0.94mg/g for columbianadin. The results demonstrated the influence of the treatment method, and the storage time on the contents of O-acetyl-columbianetin, osthole, and columbianadin in Radix angelicae pubescentis.

Acknowledgement: This research work was supported by the Research Council of Zhejiang University and Skyherb Ingredients.