Hplc-based activity profiling for hERG channel inhibitors from Galenia africana and Gnidia polycephala, and counter-current chromatographic isolation of antimicrobials from Colophospermum mopane

[摘要] The human ether-a-go-go-related gene (hERG) channel plays a critical role in cardiac actionpotential 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 insafety pharmacology. Several drugs have been withdrawn from the market or received severerestrictions on use for this reason, and numerous compounds have been blocked from progressingfurther into phases of clinical development because of hERG channel inhibition. In contrast to theroutine screening in industry for potential hERG liabilities of drug leads, comparably little is knownabout hERG inhibitors in medicinal plants (viz. phytomedicine). These are widely used ascomplementary medicines and continue to increase in popularity. There is an urgent need tocritically assess the potential risks of these botanical products.A library of 700 extracts from different parts of 142 South African medicinal plants has beenscreened by our research group for the potential of hERG channel inhibition with a Xenopus laevisoocytes 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 moleculesresponsible for the blocking activity were investigated with the HPLC-based activity profilingapproach. 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 somestructure-activity related information. Structures were elucidated by a combination of advancedoff-line analytical methods including MS and highly sensitive microprobe NMR. The absoluteconfigurations were determined by single-crystal X-ray diffraction with Cu Kα radiation or bycomparison of their experimental and calculated ECD spectra.HPLC-based activity profiling of Galenia africana enabled the identification of nine flavonoids inthe active time windows. However, the hERG-channel inhibition of isolated compounds was lesspronounced than that of extract and active microfractions (hERG inhibition between 10.1 ± 5% and14.1 ± 1.6% at 100 M). The two major constituents, 7,8-methylenedioxyflavone and7,8-dimethoxyflavone were quantified (4.3% and 9.4%, respectively) in the extract. Further hERGinhibition 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 adetailed phytochemical profiling of the active extract, a total of 20 phenolic compounds, includingsix new ones were isolated and characterized.HPLC-based activity profiling of Gnidia polycephala enabled the identification of threedaphnane-type diterpenoid orthoesters (DDOs) as the hERG channel inhibitors with inhibition of55.4 ± 7.0% (n = 4); 42.5 ± 16.0% (n = 3) and 51.3 ± 9.4% (n = 4) respectively at 100 M. DDOshave demonstrated remarkable bioactivities. This is the first report of DDOs as hERG channelinhibitors and they represent a new scaffold for hERG channel inhibition. In a detailedphytochemical profiling of the active extract, a total of sixteen compounds, including two newDDOs, two new guaiane sesquiterpenoids, polycephalone A and B with an unprecedented carbonskeleton and ten known compounds were isolated and characterized.New antimicrobials need to be discovered and developed urgently due to the constant evolution ofresistant microorganism phenotypes, the emergence of new diseases, and toxicity associated withcurrent drugs. A preliminary screen of the lipophilic extracts (DCM) of seeds, leaves and hulls ofColophospermum mopane (Fabaceae) had shown positive antimicrobial activities. Ourantimicrobial bioassay requires relatively a larger quantity of sample (2 to 5 mg for purecompounds), thus an efficient preparative isolation of the secondary metabolites in the mixture wasneeded. High-speed counter-current chromatography (HSCCC) an all-liquid technique with anunique separation mechanism was employed. Three new and two known labdane diterpenoids, onenew 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 withspectroscopic (1D and 2D NMR) and spectrometric methods (MS). The absolute configurations ofcompounds with a crystalline form were determined by single-crystal X-ray diffraction with Cu Kαradiation. The absolute configuration of a labdane diterpenoid and an isolabdane diterpenoidwithout a crystalline form was established by modified the Mosher's method, and corroborated bycomparison of experimental and calculated ECD spectra of their 3-p-bromobenzoate derivatives.The compounds were evaluated for antimicrobial activities. A clerodane diterpenoid was the mostactive 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.

[发布机构] University of the Free State