Van der Westhuizen, JanHamburger, MatthiasDu, Kun2016-02-122016-02-122015-01http://hdl.handle.net/11660/2286The human ether-a-go-go-related gene (hERG) channel plays a critical role in cardiac action potential repolarization. Blocking of the channel by drugs may lead to a fatal type of arrhythmia, named “torsades de pointes” (TdP). The hERG channel is thus considered as a primary antitarget in safety pharmacology. Several drugs have been withdrawn from the market or received severe restrictions on use for this reason, and numerous compounds have been blocked from progressing further into phases of clinical development because of hERG channel inhibition. In contrast to the routine screening in industry for potential hERG liabilities of drug leads, comparably little is known about hERG inhibitors in medicinal plants (viz. phytomedicine). These are widely used as complementary medicines and continue to increase in popularity. There is an urgent need to critically assess the potential risks of these botanical products. A library of 700 extracts from different parts of 142 South African medicinal plants has been screened by our research group for the potential of hERG channel inhibition with a Xenopus laevis oocytes based bioassay. The DCM extracts from stems and leaves of Galenia africana (Aizoaceae) and from roots of Gnidia polycephala (Thymelaeaceae) showed the strongest inhibition [50.3 ± 5.4% (n = 3) and 58.8 ± 13.4% (n = 3) respectively at a concentration of 100 g/mL]. The molecules responsible for the blocking activity were investigated with the HPLC-based activity profiling approach. Compounds in the active time window were isolated for further pharmacological testing. We also isolated structurally related compounds in the inactive fractions in view of deriving some structure-activity related information. Structures were elucidated by a combination of advanced off-line analytical methods including MS and highly sensitive microprobe NMR. The absolute configurations were determined by single-crystal X-ray diffraction with Cu Kα radiation or by comparison of their experimental and calculated ECD spectra. HPLC-based activity profiling of Galenia africana enabled the identification of nine flavonoids in the active time windows. However, the hERG-channel inhibition of isolated compounds was less pronounced than that of extract and active microfractions (hERG inhibition between 10.1 ± 5% and 14.1 ± 1.6% at 100 M). The two major constituents, 7,8-methylenedioxyflavone and 7,8-dimethoxyflavone were quantified (4.3% and 9.4%, respectively) in the extract. Further hERG inhibition tests for 7,8-methylenedioxyflavone and 7,8-dimethoxyflavone at 300 M showed a concentration-dependent inhibitory activity (33.2 ± 12.4% and 30.0 ± 7.4%, respectively). In a detailed phytochemical profiling of the active extract, a total of 20 phenolic compounds, including six new ones were isolated and characterized. HPLC-based activity profiling of Gnidia polycephala enabled the identification of three daphnane-type diterpenoid orthoesters (DDOs) as the hERG channel inhibitors with inhibition of 55.4 ± 7.0% (n = 4); 42.5 ± 16.0% (n = 3) and 51.3 ± 9.4% (n = 4) respectively at 100 M. DDOs have demonstrated remarkable bioactivities. This is the first report of DDOs as hERG channel inhibitors and they represent a new scaffold for hERG channel inhibition. In a detailed phytochemical profiling of the active extract, a total of sixteen compounds, including two new DDOs, two new guaiane sesquiterpenoids, polycephalone A and B with an unprecedented carbon skeleton and ten known compounds were isolated and characterized. New antimicrobials need to be discovered and developed urgently due to the constant evolution of resistant microorganism phenotypes, the emergence of new diseases, and toxicity associated with current drugs. A preliminary screen of the lipophilic extracts (DCM) of seeds, leaves and hulls of Colophospermum mopane (Fabaceae) had shown positive antimicrobial activities. Our antimicrobial bioassay requires relatively a larger quantity of sample (2 to 5 mg for pure compounds), thus an efficient preparative isolation of the secondary metabolites in the mixture was needed. High-speed counter-current chromatography (HSCCC) an all-liquid technique with an unique separation mechanism was employed. Three new and two known labdane diterpenoids, one new isolabdane, three new and two known clerodane diterpenoids, were isolated from the seeds, husks and leaves of Colophospermum mopane. The structures of the isolates were elucidated with spectroscopic (1D and 2D NMR) and spectrometric methods (MS). The absolute configurations of compounds with a crystalline form were determined by single-crystal X-ray diffraction with Cu Kα radiation. The absolute configuration of a labdane diterpenoid and an isolabdane diterpenoid without a crystalline form was established by modified the Mosher’s method, and corroborated by comparison of experimental and calculated ECD spectra of their 3-p-bromobenzoate derivatives. The compounds were evaluated for antimicrobial activities. A clerodane diterpenoid was the most active with MIC values as low as 51.3 μM, 51.3 μM and 102.9 μM against Escherichia coli, Staphylococcus aureus and Enterococcus faecalis, respectively.enNatural productshERG channel inhibitorsAntimicrobialsHPLC-based activity profilingHigh-speed counter-current chromatographyMedicinal plantsGalenia africana (Aizoaceae)Gnidia polycephala (Thymelaeaceae)Colophospermum mopane (Fabaceae)Drug discoveryThesis (Ph.D. (Chemistry))--University of the Free State, 2015Chromatographic analysis.Drugs -- AnalysisThesisUniversity of the Free State