HPLC analysis of alizarin and purpurin produced by Rubia tinctorum L. hairy root cultures

Abstract

We have determined the quantities of the anthraquinones alizarin and purpurin, with especial regard to their effective antigenotoxic activity, in genetically transformed hairy root cultures of Rubia tinctorum L. Hairy roots were cultured on solid and in liquid Gamborg B5 and 1/2 NMS media in a shaking cabinet and in a bioreactor. Methanolic extracts of lyophilized hairy roots were hydrolysed, and then purified by solid phase extraction with good recovery. A new HPLC method was developed for the determination of alizarin and purpurin. The analysis was performed on a Luna C8 RP column using a 45:55 (v/v) mixture of acetonitrile:20 mM ammonium formate-formic acid buffer (pH 3.00) as eluent. Peaks were identified by addition of standards and by diode-array detection. External standardization allowed the determination of alizarin and purpurin with good sensitivity and reliability. The maximum purpurin content was observed in cultures cultivated on solid B5 medium (5.94 mg g(-1)). The highest alizarin content was measured in cultures cultivated on solid 1/2 NMS medium (2.14 mg g(-1)).