Bioassay Guided Fractionation Protocol for Determining Novel Active Compounds in Selected Australian Flora

Abstract

A large variety of unique and distinct flora of Australia have developed exceptional survival methods and phytochemicals and hence may provide a significant avenue for new drug discovery. This study proposes a bioassay guided fractionation protocol that maybe robust and efficient in screening plants with potential bioactive properties and isolating lead novel compounds. Hence, five native Australian plants were selected for this screening process, namely Pittosporum angustifolium (Gumbi gumbi), Terminalia ferdinandiana (Kakadu plum, seeds (KPS), and flesh (KPF)), Cupaniopsis anacardioides (Tuckeroo, seeds (TKS) and flesh (TKF)), Podocarpus elatus (Illawarra plum, seeds (IPS) and flesh (IPF)) and Pleiogynium timoriense (Burdekin plum, seeds (BPS) and flesh (BPF)). The methanolic extracts of the plants samples were analysed for Total phenolic content (TPC) and antioxidant capacity measure by FRAP. The highest values were found in the KPF which were 12,442 ± 1355 mg GAE/ 100 g TPC and 16,670 ± 2275 mg TXE/100 g antioxidant capacity. Extracts of GGL was deemed to be most potent with complete cell inhibition in HeLa and HT29, and about 95% inhibition in HuH7 cells. Comparative activity was also seen for KPS extract, where more than 80% cell inhibition occurred in all tested cell lines. Dose-dependent studies showed higher SI values (0.72–1.02) in KPS extracts than GGL (0.5–0.73). Microbial assays of the crude extracts were also performed against five bacterial strains commonly associated with causing food poisoning diseases were selected (Gram positive—Staphylococcus aureus and Gram negative—Escherichia coli, Salmonella typhi and Pseudomonas aeruginosa bacteria). KPF extracts were effective in suppressing microbial growth of all tested bacterial strains except for P. aeruginosa, while TKS and TKF were only slightly effective against S. aureus. Due to the potential of the GGL crude extract to completely inhibit the cells compared to KPS, it was further fractionated and tested against the cell lines. HPLC phenolic profiling of the crude extracts were performed, and numerous peak overlaps were evident in the fruit extracts. The KPF extracts demonstrated the strongest peaks which was coherent with the fact that it had the highest TPC and antioxidant capacity values. A high occurrence of t-ferulic acid in the GGL extracts was found which may explain the cytotoxic activity of GGL extracts. Peaks in KPS and KPF extracts were tentatively identified as gallic acid, protocatechuic acid, 4-hydroxybenzoic acid and syringic acid and possibly ellagic acid. HPLC time-based fractionation of the GGL extract (F1–F5) was performed and Dose dependent cytotoxic effects were determined. It was construed that F1, having the highest SI value for HeLa, HT29 and HuH7 (1.60, 1.41 and 1.67, respectively) would be promising for further fractionation and isolation process.

Bioassay-Guided Fractionation of Pittosporum angustifolium and Terminalia ferdinandiana with Liquid Chromatography Mass Spectroscopy and Gas Chromatography Mass Spectroscopy Exploratory Study

“…Fraction 2 demonstrated the highest activity and was the only fraction that inhibited the growth of all tested bacterial strains compared to the other fractions, possibly because it was the most concentrated fraction. Overall, even though the flesh extracts of T. ferdinandiana fruit have previously demonstrated antibacterial activity in several studies [ 11 , 44 , 45 ], including our previous studies [ 14 ], the low activity of the fractions suggests that the bioactive compounds in the lyophilized extract may work synergistically to produce its antibacterial property.…”

“…While the subfractions of P. angustifolium Fraction 1 did not demonstrate significant cytotoxic properties or contain compounds of interest, Fraction 1 was found to be effective in reducing cell viability in the tested cancer cell lines and had a higher selective index, suggesting its effectiveness in killing cancer cells while minimizing harm to normal cells compared to the other fractions [ 14 ]. Moreover, given the possibility of a synergistic effect at play in dictating the cytotoxic behavior of P. angustifolium Fraction 1, characterizing the potential phenolic metabolites in the fraction was considered a valuable pursuit.…”

“…Previous studies have also identified chlorogenic acid in P. angustifolium leaves [ 17 , 18 , 19 ]. Additionally, our previous work has suggested the presence of chlorogenic acid in crude MeOH extracts of P. angustifolium leaves [ 14 , 20 ].…”

“…Considering the promising antibacterial activity demonstrated by the crude flesh extract of T. ferdinandiana [ 14 ], the fractions were also evaluated against four bacterial strains. The results are presented in Table 4 .…”

“…Based on the findings from our previous study [ 14 ], the objective of this study was to further fractionate Fraction 1 of P. angustifolium methanolic crude extracts into subfractions, utilizing phase 3 of the proposed bioassay-guided fractionation protocol design ( Figure 1 ), and to perform LC-MS and GC-MS analysis for compound characterization. This is among the first/few studies investigating the bioactivity of fractions from P. angustifolium.…”