*3.5. High Performance Centrifugal Partition Chromatography (HPCPC or CPC)*

The solvent system containing CHCl3:MeOH:H2O–0.1% HCO2H (7:13:8) was mixed vigorously in a separating funnel and allowed to reach hydrostatic equilibration [3,12]. Following the separation of the two-immiscible phase solvent systems, both phases were degassed using a sonicator-degasser (Soniclean Pty Ltd. Adelaide, SA Australia). Then the rotor column of HPCPC™, CPC240 (Ever Seiko Corporation, Tokyo, Japan) was filled with the stationary phase (the aqueous upper phase) in the descending mode at a flow rate of 5 mL min−<sup>1</sup> by Dual Pump model 214 (Tokyo, Japan), with a revolution speed of 300 rpm. The lower mobile phase was pumped in the descending mode at a flow rate of 1.2 mL min−<sup>1</sup> with a rotation speed of 900 rpm within 2 h. One hundred and twenty milligrams of isobutanol-enriched saponins mixture was injected into the machine in the descending mode. The chromatogram was developed for 3 hours at 1.2 mL min−<sup>1</sup> and 900 rpm using the Variable Wavelength UV-VIS Detector S-3702 (Soma optics, Ltd. Tokyo, Japan) and chart recorder (Ross Recorders, Model 202, Topac Inc. Cohasset, MA, USA). The fractions were collected in 3 mL tubes using a Fraction collector. At Fraction 54, the elution mode was switched to ascending mode and the aqueous upper phase was pumped at the same flow rate for 3 h to recover saponins. Fractions were monitored by TLC as described above. Monitoring of the fractions was necessary, as most of the saponins could

not be detected by UV due to the lack of a chromophore structure. Fractions were concentrated with nitrogen gas.

### *3.6. Mass Spectrometry*

The resultant HPCPC purified polar extracts were further analyzed by MALDI- and ESI-MS to elucidate and characterize the molecular structures of compounds.
