The pharmaceutical potential of compounds from Tasmanian Clematis species

Aims The aim of the study was to investigate the antitumour, antibacterial and antiinflammatory activities of some Tasmanian native Clematis spp. and to isolate and identify the potential pharmaceutical constituents. Methods The antitumour activities, antibacterial activities and anti-inflammatory effects of leaf material of Clematis spp. were screened by P388 cytotoxic assay, minimum inhibition concentrations (MICs) and inhibitory NO production by lipopolysaccharides (LPS) stimulated Raw 264.7 cells, respectively. The bioactiveguided fractionation methodologies, including cartridge fractionation, HPLC columns fractionation and Sephadex LH-20 column purification, were employed to isolate active constituents. The chemical profiles of active constituents were determined by ELSD, UV, LC-MS and GC-MS. Scanning electron microscopy (SEM), Gram stain, antibiotic interaction with chequerboard, deoxycholate-induced lysis and the antibiotic resistant mechanism study of P. aeruginosa were employed to study the antibacterial mechanism of the active constituent. The anti-inflammatory activity was also investigated by basal (unstimulated) and LPS- and phytohaemagglutinin A (PHA)-stimulated cytokine release from peripheral blood mononuclear cells (PBMC). Some novel compounds in Tasmanian native Clematis spp. were determined by HPLC, LC-MS and LTQ-Orbitrap-MS. Results and discussion Ten out of eleven investigated Clematis spp. showed cytotoxic activities against P388 cells with different IC50 values (0.084-3.106 mg/ml). Ranunculin and its isomer were determined as the antitumour constituents. These compounds could be hydrolysed by the cell medium to produce protoanemonin. The antitumour study determined that ranunculin was a pro-cytotoxin with the antitumour activity derived from protoanemonin. Eight investigated Clematis spp. showed different antibacterial effects against E. coli (MIC=0.39-3.13 mg/ml) and P. aeruginosa (MIC=0.31-6.25 mg/ml). Ranunculin, the antibacterial constituent in Clematis spp., was selective against Gram negative bacteria. In particular, it had a stronger antibacterial effect than gentamicin against clinically isolated multi-drug resistant P. aeruginosa. The change in ranunculin treated sensitive P. aeruginosa was elongation and cell lysis in multi-drug resistant P. aeruginosa observed by SEM and obtained by deoxycholateinduced lysis study. These results imply that the antibacterial mechanism of ranunculin against sensitive and multi-drug resistance P. aeruginosa may be different. The antibacterial effect of ranunculin might be still contributed to by protoanemonin. Eleven investigated Clematis spp. showed varied inhibition of NO production. Ranunculin and its isomers were determined as one of the anti-inflammatory constituents in Tasmanian Clematis spp. For the cytokine study, in the presence of LPS and PHA, C. aristata-L leaf at 10 ˜í¬¿g/ml significantly decreased the release of IL-1˜í‚⧠(P<0.01) and TNF-˜í¬± (P<0.05) compared with LPS and PHA alone. These results provide experimental data of the anti-inflammatory activities of Tasmanian Clematis spp. Flavonoids and hydroxycinnamate esters were obtained in these investigated Clematis spp. The amounts of nine hydroxycinnamate esters were varied in each Clematis leaf material. This would be the first time to discover these hydroxycinnamte esters in Tasmanian Clematis spp. Conclusion The study demonstrated the antitumour, antibacterial and anti-inflammatory activities of Tasmanian native Clematis spp. Ranunculin was discovered to be a pro-cytotoxin of protoanemonin, which was one of the active constituents. This study was the first to investigate the therapeutic value of Tasmanian native Clematis spp. and discover the antibacterial value of protoanemonin in multi-drug resistant P. aeruginosa and its complicated antibacterial mechanism against sensitive and multidrug resistant P. aeruginosa. The anti-inflammatory activities may be the most distinguishing therapeutic value of Tasmanian Clematis spp. Furthermore, study of the chemical constituents suggested that Tasmanian Clematis spp. contained phenolic compounds. Although this study only provided the basic and preliminary experimental data on the biological, potential pharmaceutical constituents and some novel compounds of Tasmanian Clematis spp., further investigation into usage and identification of effective pharmaceutical constituents would be worthy.