Browsing by Author "Zonyane, Samkele"
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- ItemThe antimicrobial interactions of Agathosma crenulata, Dodonaea viscosa and Eucalyptus globulus combination and their chemical profiling(Stellenbosch : Stellenbosch University, 2013-12) Zonyane, Samkele; Makunga, N. P.; Van Vuuren, S. F.; Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology.ENGLISH ABSTRACT: In traditional medicine, there is a long-standing culture of combining herbal drugs to increase the therapeutic efficacy. The improved medical action is thought to be due to synergistic interactions between different plant bioactive components. The aim of this study was to test the pharmacological interactions in a medicinal plant combination which consisted of Agathosma crenulata, Dodonaea viscosa and Eucalyptus globulus. The rationale for the analysis of this particular mixture is that it had noteworthy antibacterial activity and exhibited the highest activity out of seven medicinal plant mixtures previously investigated. Using chromatographic analysis, the phytochemistry of the plants was also assessed. The chloroform: methanol (1:1; v/v) extracts or hydo-distilled essential oils (A. crenulata and E. globulus) were screened individually and in combinations (double and triple plant combination) for activity against five respiratory pathogens using a microdilution assay. The antimicrobial interactions in combinations were assessed with the fractional inhibitory concentration (FIC) and the isobolograms. The organic extracts generally showed the highest antimicrobial activity with E. globulus having the highest activity with MIC values below 1 mg ml-1 representing noteworthy activity. The overall activity of the aqueous extracts was poor. The essential oil activity of E. globulus was mostly noteworthy (0.5 to 2 mg ml-1) while A. crenulata essential oil displayed moderate activity (1 to 4 mg ml-1). The ΣFIC values for double combinations (1:1) of A. crenulata with D. viscosa, A. crenulata with E. globulus and D. viscosa with E. globulus were calculated from the minimum inhibitory concentration (MIC) data and the interactions were classified as synergistic, additive, indifferent and antagonistic. The highest synergistic interactions observed were for a 1:1 combination of A. crenulata with E. globulus against K. pneumoniae, S. aureus and B. subtilis with ΣFIC values of 0.07. There was only one incident of antagonism noted in the study for D. viscosa with E. globulus (1:1) against C. neoformans with ΣFIC value of 4.25. The double combinations against selective pathogens (K. pneumoniae, S. aureus and E. coli) were further analysed for interactions using isobolograms. Mostly, the antimicrobial interactions as presented by the isobolograms were congruent with FIC results which further validated the occurrence of relevant antimicrobial interactions in those combinations. The ΣFIC values for triple combinations (1:1:1) revealed mostly synergistic interactions. When the triple combinations were analysed further against certain pathogens based on the predictions of the Design of Experiments software program (MODDE 9.1®), the MIC values remained the same despite the different combinations that were tested Thin layer chromatography (TLC) was used for a quick chemical fingerprinting of the plant extracts. This was followed by a bio-autographic assay. The chemical profiles of the organic extracts and essential oils from two of the study aromatic plants (A. crenulata and E. globulus) were further analysed with liquid chromatography mass spectrometry (LC-MS) and gas chromatography mass spectrometry (GC-MS) respectively. For combined plant extracts, a multivariate data analysis using principal component analysis (PCA) and hierarchical clustering analysis (HCA) was used to determine the relationship of the chemical make-up of combinations with that of individual plant extracts. According to the TLC analysis, E. globulus extracts had more compounds than the other two plants in the study. For the bio-autographic assay, E. globulus and combinations that included this plant showed greater inhibition zones than A. crenulata and D. viscosa. For the LC-MS analysis, PCA and HCA showed a close relationship between A. crenulata with D. viscosa, D. viscosa with E. globulus and the triple combination. Twenty one components were identified in the essential oil of A. crenulata representing 88.83% of the total oil composition. The oil was dominated by oxygen-containing monoterpenes (46.25%). In the essential oil of E. globulus, twenty six compounds were identified making up to 95.62% of the oil composition. Oxygen-containing monoterpenes (32.98%) also dominated the E. globulus essential oil. There was no great variation in essential oil metabolites of the individual plants and their combination as shown by both PCA and HCA. The enhanced in vitro antimicrobial activity and pharmacological interactions (synergy and additivity) in some of the combinations (double and triple) that were tested in this study adds scientific support to the use of medicinal plant combinations in Western Cape traditional medicine. The metabolic profiles of plants in combination might be unique due to interaction of the different plant bioactive molecules and thus result into defined antimicrobial activity.
- ItemComparative cardio and developmental toxicity induced by the popular medicinal extract of Sutherlandia frutescens (L.) R.Br. detected using a zebrafish Tuebingen embryo model(BMC (part of Springer Nature), 2018-10-05) Chen, Longsheng; Xu, Minjie; Gong, Zhunan; Zonyane, Samkele; Xu, Shuwen; Makunga, Nokwanda P.Background: Sutherlandia frutescens is one of the most promising commercialized, indigenous and medicinal plants of South Africa that is used as an immune-booster, and a traditional treatment for cancer. However, few studies report on its toxicology and dosage in vivo. There is still room to better understand its cytotoxicity effects in animal systems. Methods: We prepared two extracts, one with 80% (v/v) ethanol, and the other, with water. Both were studied to determine the maximum tolerable concentration when extracts were applied at 0 to 200 μg/ml to a Tuebingen zebrafish embryo line. The development of zebrafish embryos after 24 h post fertilization (hpf) was studied. A concentration range of 5 μg/ml to 50 μg/ml was then chosen to monitor the ontological development of cultured embryos. A liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method was used to study the differences of the two experimental extracts. Chemical variation between the extracts was illustrated using chemometrics. Results: Both extracts led to bleeding and pericardial cyst formation when applied at high concentrations to the zebrafish embryo culture. Chronic teratogenic toxicities, leading to pericardial edema, yolk sac swelling, and other abnormal developmental characteristics, were detected. The aqueous extracts of S. frutescens were less toxic to the larvae than the ethanol extracts, validating preference for aqueous preparations when used in traditional medicine. Chemical differences between the water extracts and alcoholic extracts were analysed using LC-MS/MS. A supervised metabolomics approach, targeting the sutherlandiosides and sutherlandins using orthogonal partial least squares-discriminant analysis (OPLS-DA), illustrated that sutherlandiosides were the main chemical features that can be used to distinguish between the two extracts, despite the extracts being highly similar in their chemical constituents. Conclusion: The water extract caused less cytotoxic and abnormal developmental effects compared to the ethanolic extract, and, this is likely due to differences in concentrations of extracted chemicals rather than the chemical profile per se. This study provides more evidence of cytotoxicity effects linked to S. frutescens using the zebrafish embryo bioassay as a study tool.
- ItemThe implication of chemotypic variation on the anti-oxidant and anti-cancer activities of sutherlandia frutescens (L.) R.Br. (Fabaceae) from different geographic locations(MDPI, 2020) Zonyane, Samkele; Fawole, Olaniyi A.; La Grange, Chris; Stander, Maria A.; Opara, Umezuruike L.; Makunga, Nokwanda P.Extracts of Sutherlandia frutescens (cancer bush) exhibit considerable qualitative and quantitative chemical variability depending on their natural wild origins. The purpose of this study was thus to determine bioactivity of extracts from different regions using in vitro antioxidant and anti-cancer assays. Extracts of the species are complex and are predominantly composed of a species-specific set of triterpene saponins (cycloartanol glycosides), the sutherlandiosides, and flavonoids (quercetin and kaempferol glycosides), the sutherlandins. For the Folin-Ciocalteu phenolics test values of 93.311 to 125.330 mg GAE/g DE were obtained. The flavonoids ranged from 54.831 to 66.073 mg CE/g DE using the aluminum chloride assay. Extracts from different sites were also assayed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical scavenging method and ferric reducing anti-oxidant power (FRAP) methods. This was followed by an in vitro Cell Titer-Glo viability assay of various ecotypes using the DLD-1 colon cancer cell line. All test extracts displayed anti-oxidant activity through the DPPH• radical scavenging mechanism, with IC50 values ranging from 3.171 to 7.707 µg·mL−1. However, the degree of anti-oxidant effects differed on a chemotypic basis with coastal plants from Gansbaai and Pearly Beach (Western Cape) exhibiting superior activity whereas the Victoria West inland group from the Northern Cape, consistently showed the weakest anti-oxidant activity for both the DPPH• and FRAP methods. All extracts showed cytotoxicity on DLD-1 colon cancer cells at the test concentration of 200 µg·mL−1 but Sutherlandia plants from Colesburg (Northern Cape) exhibited the highest anti-cancer activity. These findings confirm that S. frutescens specimens display variability in their bioactive capacities based on their natural location, illustrating the importance of choosing relevant ecotypes for medicinal purposes.