Separation of six compounds from pigeon pea leaves by elution–extrusion counter‐current chromatography

A valid and reliable method was established to separate six compounds from pigeon pea leaves via elution‐extrusion counter‐current chromatography. A solvent system composed of n‐hexane/methanol/formic acid aqueous solution with pH = 3 (10:6:4, v/v) was screened to achieve satisfactory isolation from the ethanol extract of pigeon pea leaves. Four compounds, 9.2 mg of compound 1 (96.8%), 3.2 mg of 2 (88.0%), 6.2 mg of 4 (94.2%) and 25.2 mg of 5 (94.2%), were obtained by conventional elution from 100 mg of the precipitation fraction, respectively. Two compounds, 14.4 mg of 3 (96.3%) and 28.1 mg of 6 (96.6%), with high K values were obtained by the subsequent extrusion procedure. The compounds 1–6 were identified as 3‐methoxy‐5‐(2‐phenylethenyl)‐phenol, pinostrobin chalcone, pinostrobin, 2‐hydroxy‐4‐methoxy‐6‐(2‐phenylvinyl)‐benzoic acid, longistylin C and cajaninstilbene acid by quadrupole time‐of‐flight mass spectrometry, and 1H and 13C NMR spectroscopy. The in vitro antiproliferation activities of compounds 1, 5 and 6 against human hepatoma cell were evaluated and the half‐maximum inhibitory concentrations were acquired.