Systematic analysis of glucoiridoids from Penstemon serulatus Menz by high-performance liquid chromatography with pre-column solid-phase extraction

Samples (122) of crude ethanol-extracts of callus tissues from Penstemon serrulatus Menz. were used to develope a solid-phase extraction (SPE) clean-up procedure using the octadecylsilica packed cartridge for removing a complex mixture of free phenolic acids and anthocyanine-like colour substances, for the isolation of a sufficiently clean glucoiridoid fraction (GF). An additional SPE sample pretreatment step of the crude extracts enabled the enhancement of selectivity and sensitivity on applied high-performance liquid chromatography (HPLC) for the identification and quantitation of the iridoid constituents of the GF fraction. In particular, the presence of five valeriana-type esterified glucoiridoids which consist of penstemide, serrulatoloside, 8-epi-valerosidate, 7-desoxy-8-epi-valerosidate and serrulatoside in prepared GF fractions were determined by isocratic HPLC measurements. During a single HPLC separation, the traces of four non-esterified glucoiridoids, i.e. harpagide, aucuboside, loganine and plantarenaloside, were satisfactorily resolved and detected on the registered HPLC chromatograms of investigated GF fractions. The HPLC analyses were carried out on an octadecylsilica column (25 X 0.4 cm I.D.) using methanol-water (30:70, v/v) as the mobile phase with a flow-rate of 2 ml/min and the absorbance was monitored at 220 nm using an UV detector. The described chromatographic assay for penstemide, which exhibits potential antitumor activity against the P-388 lymphocytic leukemia cells, was applied to the monitoring and standarization of growth conditions for the callus cultures of P. serrulatus. Penstemide contents were found to range from 0.05% to 2.7% of the fresh weight of the investigated callus samples. Multivariate statistical methods (principal components analysis) was applied to demonstrate the influence of a variety of compositions of growth media (especially the type and concentration of synthetic growth regulators, e.g. 3-indolylacetic acid, 2,4-dichlorophenoxyacetic acid or 1-naphthylacetic acid, on the formation of different profiles of glucoiridoids in the callus cultures of P. serrulatus. Calculated principal component values were found to be useful for the explanation of variations in the penstemide/serrulatoloside ratio in the investigated samples and for determining the most favourable growth conditions in plants leading to optimal glucoiridoid biosynthesis.