Identifying Natural syNergist from Pongamia pinnata Using High-Speed Counter-Current Chromatography Combined with Isobolographic Analysis

For identifying the synergistic compounds from Pongamia pinnata, an approach based on high-speed counter-current chromatography (HSCCC) combined with isobolographic analysis was designed to detect the synergistic effects in the complex mixture. In the approach, the complex mixture was considered as the combination of two individual samples for isobolographic analysis: the target compound and the mixture with complete removal of the target compound (subtracted residue). The two samples were prepared by HSCCC, and were used for the calculation of the expected effect of their combination. Using this approach, three compounds representing the major peaks in the HPLC of the brine shrimp toxic extract from P. pinnata (brine shrimp lethality test (BST) LC50 36.5 μg/mL), pinnatin (1), 3,7-Dimethoxy-3′,4′-methylenedioxy flavone (2), and karanjin (3), were prepared from the extract, and were tested for their synergistic potency by BST. The two-phase solvent system containing n-hexane-ethyl acetate–MeOH–water (14:7:10:10, v/v/v/v) was selected for the one-step HSCCC separation according to the partition coefficient values (K). The extract was separated into seven fractions (Fr1–7) by HSCCC with a total mass recovery of 96.3%. Fr2, 4, and 6 were the peak fractions corresponding to compounds 3, 2, and 1, respectively. The purities and recoveries of the target compounds after the chromatographic analysis were 95.9%–97.5% and 92.2%–96.1%, respectively. The subtracted residue of each target compound was performed by recombining all HSCCC fractions except the fraction containing the target compound. Isobolographic analysis disclosed a significant synergistic effect between karanjin and its subtracted residue (potency ratio of 0.47), which gave clear evidence that the toxicity of the extract results from synergistic interactions, and karanjin was one of the synergists participating in the interaction. The other two compounds were excluded from the synergism because these two compounds showed additive effects with their subtracted residues. Karanjin was the first synergistic compound identified from P. pinnata.