Abstract:

Centella asiatica (L.) Urban is a tropical medicinal plant with a long history of therapeutic uses. Madecassic acid and terminolic acid, which are a pair of structural isomers, are two constituents of Centella asiatica. A method using reversed-phase high performance liquid chromatography in which β-cyclodextrin (β-CD) was the additive in mobile phase has been developed for separation of the structural isomers and determination of madecassic acid. The two compounds can be isolated with high resolution on a C18 reversed-phase column with the addition of β-CD in the mobile phase. The separation mechanism of the isomers was discussed. It was assumed that the separation of the isomers might have been resulted from different inclusion forces of complexes with β-CD. The effects of β-CD concentration and the pH of mobile phase on resolution were investigated. It was found that the resolution of the isomers increased with the increase of β-CD concentration when the mobile phase consisted of methanol-water (65∶35, v/v) at pH 4. The correlation coefficient (r2) of the linear calibration curve between peak area and concentration of madecassic acid was 0.9989 in the range of 0.1-5.0 g/L. This method was successfully used to determine the madecassic acid in triterpenic genins of Centella asiatica.

Key words: Centella asiatica , isomer, madecassic acid, terminolic acid, β-cyclodextrin, reversed-phase high performance liquid chromatography (RP-HPLC)