Abstract
The presence of Securinega-type alkaloids in the biomass of Phyllanthus glaucus Wall. ex Müll. Arg. (Euphorbiaceae) obtained in vitro and ex vitro was revealed. To elucidate their structures, the isolation from the preliminary purified 0.5 M HCl extract was performed using the SPE method with a silica gel-C6H5 column and preparative TLC with Si60 F254 plates as a stationary phase and a mixture of chloroform: methanol: 25% NH4OH (10:1:0.01; v/v/v) as the mobile phase. On the basis of their 1D- and 2D-NMR spectra, the alkaloids were recognized as securinine and allosecurinine.
The quantitative analysis of the alkaloids in the P. glaucus biomasses was performed using RP-HPLC with UV detection on monolithic column and a linear gradient from 20 to 50% CH3CN in water. The method was validated in terms of selectivity, linearity, repeatability, intra- and interday precision, LOD, LOQ, and recovery. The leaves of the plants obtained under the ex vitro conditions contained over 3.5 times higher concentration of securinine (13.02 mg/g d.w.) than shoots harvested in vitro (3.56 mg/g d.w.).