Pharmacological and phytochemical evaluation of seven plants used for microbial-related ailments in South African traditional medicine.
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Since antiquity, man has always believed in the healing properties of plants. The age-old practice of phytotherapy is now justified by numerous phytochemical and pharmacological studies which substantiate the presence of biologically active compound(s) in some medicinal plants. Quite often, ethnopharmacological studies provide important leads for the development of different types of plant-based therapeutic drugs. New effective antibiotics are urgently needed to combat multiple and extensively drug-resistant bacterial strains that are currently threatening public health globally. The rich floral diversity in southern Africa and the resultant extensive chemical diversity provide encouraging prospects for discovering novel pharmacologically important phytocompounds within this region. Against this background, the current study was designed to evaluate the in vitro antibacterial properties, phenolic profiles and mutagenic potentials of extracts obtained from seven South African plants used traditionally to manage bacterial infections and where the active principles were unknown. The study also aimed at identifying the principal antibacterial compound(s) in selected plants that demonstrated potent and broad-spectrum antibacterial properties. Aqueous and organic solvent extracts of the seven selected plants namely Bolusanthus speciosus, Cucumis myriocarpus, Ekebergia capensis, Protea caffra, Prunus africana, Searsia lancea and Solanum panduriforme were screened for antibacterial (minimum inhibitory concentration, MIC) properties against a panel of seven bacterial strains (Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, multiple drug-resistant (MDR) E. coli, MDR K. pneumoniae, drug-sensitive Staphylococcus aureus and penicillin-resistant S. aureus) using the microdilution technique. The extracts were also screened for antigonococcal properties using microdilution and agar disk-diffusion techniques. In addition, combinations of the different plant extracts, as well as plant extracts with four antibiotics (ampicillin, cefotaxime, chloramphenicol and penicillin) were evaluated for antibacterial synergistic interactions against MDR Gram-negative bacterial strains (E. coli and K. pneumoniae) using the checkerboard titration and time-kill bio-assays. Preliminary phytochemical analyses for phenolics in aqueous methanol (50% MeOH) plant extracts were conducted using spectrophotometric methods. In addition, specific phenolic acids in 80% MeOH extracts of the plants were quantified with the use of ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The mutagenic properties of plant extracts that demonstrated noteworthy antibacterial activities (MIC<1 mg/ml) were assessed using the Ames Salmonella/Microsome assay involving two tester strains, TA98 and TA102 (without S9 metabolic activation). The most potent antibacterial activities were exhibited by the MeOH bark extracts of B. speciosus which yielded MIC values of 0.039 and 0.078 mg/ml against S. aureus (penicillin-resistant and drug-sensitive strains, respectively). Dichloromethane (DCM) leaf extracts of S. lancea yielded an MIC value of 0.63 mg/ml against five of the seven test bacterial strains including MDR E. coli and MDR K. pneumoniae. B. speciosus bark (MeOH and DCM), P. caffra seeds (DCM) and twigs (MeOH) also demonstrated broad-spectrum antibacterial activities. B. speciosus MeOH bark extracts, as well as the DCM leaf extracts of P. africana and S. lancea demonstrated moderate antigonococcal properties (MIC range: 0.31-0.63 mg/ml). The checkerboard assay detected antibacterial synergistic interactions in combinations of chloramphenicol with each of B. specious leaf MeOH extracts, P. africana leaf MeOH and DCM extracts against MDR E. coli (fractional inhibitory concentration index ≤ 0.5). However, the time-kill assay did not detect any significant synergistic interactions in any of these three aforementioned combinations. Total phenolic content in the plant extracts investigated ranged from 2.38 to 62.73 mg gallic acid equivalents (GAE)/g dry matter. Variations in the quantities of flavonoids, condensed tannins, hydroxybenzoic and hydroxycinnamic acids among these plant extracts were also observed. In generally, higher levels of phenolic compounds were detected in S. lancea (leaves), P. africana (leaves) and P. caffra (leaves, twigs), while lower levels were detected in B. speciosus (barks), C. myriocarpus (fruits) and S. panduriforme (fruits, leaves, roots, stem). Based on the Ames test, all test plant extracts were non-mutagenic against Salmonella typhimurium tester strain TA98 and TA102 (without S9 metabolic activation). S. lancea leaves and P. caffra twigs demonstrated broad-spectrum antibacterial properties and as a result, a detailed analysis of their phytochemical constituencies was conducted. Antibacterial activity directed fractionation led to the isolation of two compounds from the aqueous (80%) MeOH leaf extracts of S. lancea. The isolated compounds were characterised using NMR data and both of them demonstrated noteworthy antibacterial activities (MIC range: 0.016-0.25 mg/ml) against E. faecalis and S. aureus. 1-Tetracosanol (43.98%), 1-nonadecanol (37.5%), eicosane (7.67%), 1,7-di-iso-propylnaphthalene (4.23%), 1-pentadecanol (3.04%), 2,6-di-iso-propylnaphthalene (1.96%) 1,3-iso-propylnaphthalene (1.62 %) were identified by GC-MS in an active S. lancea leaf ethyl acetate sub-fraction. The sub-fraction inhibited the growth of both E. faecalis and S. aureus at an MIC value of 0.25 mg/ml. 1-Tetracosanol and 1-nonadecanol, which accounted for more than 80% of the total phytochemical constituents of the fraction, are known antibacterial compounds which most likely contributed to the antibacterial properties of S. lancea leaves. Gas chromatography-mass spectrometry (GC-MS) analysis also revealed the presence of 15 compounds in three fractions obtained from a methanolic P. caffra twig extract. Two of the identified compounds, 1-adamantanecarboxylic acid and levoglucosan, are often incorporated into antimicrobial moieties to improve the efficacy of the therapeutic molecule, or as carbon skeletons. Two known antibacterial compounds, namely 1-heptacosanol and 1-nonadecanol were also among the compounds detected in the methanolic P. caffra twig extract. Overall, the present study reaffirmed that botanical medicines can potentially be used to manage drug-sensitive and drug-resistant bacterial infections often prevalent in humans. The study also provided the scientific rationale for the use of P. caffra, C. myriocarpus and S. panduriforme in South African folk medicine.