Centrifugal partition chromatography elution gradient for isolation of sesquiterpene lactones and flavonoids from Anvillea radiata.

An innovative procedure coupling pressurized solvent extraction and centrifugal partition chromatography (CPC) used in linear gradient elution mode was developed to isolate two pure germacranolides (9α-hydroxyparthenolide and 9β-hydroxyparthenolide) and to separate flavonoids (nepetin, isorhamnetin and jaceosidin) and chlorophyll pigments from aerial parts of Anvillea radiata (Coss.&Durieu). The two main germacranolides recovered using this method represent 2 and 5% of the dried plant material respectively. These molecules were extracted using accelerated solvent extraction with chloroform. After optimization of the CPC method, a two-phase solvent system composed of heptane/ethyl acetate/methanol/water (1:5:1:5 v/v/v/v) was employed in descending mode to isolate the germacranolides. Then the lower phase of a heptane/ethyl acetate/methanol/water (6:5:6:5 v/v/v/v) system was pumped in descending mode to generate a linear elution gradient, progressively decreasing the mobile phase polarity, that enabled the flavonoid compounds to be separated in the same run. The efficiency of the preparative separation was controlled through RP-HPLC analysis of the obtained fractions using UV, evaporative light scattering and mass spectrometry detection. The structural identification of the two germacranolides purified over 99% was established by (1)H NMR and (13)C NMR. The least abundant flavonoids were identified by mass spectrometry.