Simultaneous analysis of three bioactive compounds in Artemisia annua hairy root cultures by reversed-phase high-performance liquid chromatography-diode array detector.

ABSTRACT

INTRODUCTION: Artemisia annua is a rich source of biologically active substances such as terpenoids, coumarins and polyacetylenes. These chemicals have been reported to show beneficial pharmacological properties such as antitumor and antibacterial activities. In genetically transformed root cultures of A. annua, three bioactive metabolites, namely, ponticaepoxide (an insecticidal polyacetylene, 1), drimartol A (an anticancer sesquiterpene coumarin, 2) and (Z)-7-acetoxy-methyl-11-methyl-3-methylene-dodeca-1,6,10-triene (a new anticancer sesquiterpene, 3) were isolated and identified in our recent work. However, no quantitative analysis methods for any of them are yet available, nor for their simultaneous analysis. OBJECTIVE: To develop an HPLC-PAD method for simultaneous determination of 1, 2 and 3 in hairy root cultures of A. annua. METHODOLOGY: HPLC operating conditions were optimised and the chromatographic separation was performed on a C(18) column with a gradient acetonitrile water as mobile phase. RESULTS: Linear relationships within the range of investigated concentrations were observed for the three metabolites with their correlation coefficients greater than 0.997. The method was validated for repeatability (RSD <3.59%) and intra- and inter-day precision (RSD <3.1%) with recovery between 94.8 and 107.6% and the RSD less than 3.40%. The method was successfully applied to the time-course of accumulation of the bioactive compounds in genetically transformed root cultures of A. annua. CONCLUSION: The HPLC-PAD method developed for the simultaneous determination of three bioactive metabolites 1, 2 and 3 was simple, reproducible and sensitive.