Hplc-based activity profiling for hERG channel inhibitors from Galenia africana and Gnidia polycephala, and counter-current chromatographic isolation of antimicrobials from Colophospermum mopane
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Date
2015-01
Authors
Du, Kun
Journal Title
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Publisher
University of the Free State
Abstract
The 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 Several drugs have been withdrawn from the market or received severe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.
Description
Keywords
Natural products, hERG channel inhibitors, Antimicrobials, HPLC-based activity profiling, High-speed counter-current chromatography, Medicinal plants, Galenia africana (Aizoaceae), Gnidia polycephala (Thymelaeaceae), Colophospermum mopane (Fabaceae), Drug discovery, Thesis (Ph.D. (Chemistry))--University of the Free State, 2015, Chromatographic analysis., Drugs -- Analysis