Antimicrobial and efflux inhibiting activity of natural products from Swazi medicinal plants

Plant-derived medicines have played a major role in the history of man, especially as anti-infective agents. In the wake of the emergence of multidrug-resistant microbial infections, natural products are still a potential source of drug leads since they contain a wide array of structurally diverse compounds. Alongside the classical approach of finding anti-infective agents, this study also sought to investigate the potential of Swazi medicinal plants as efflux inhibitors in bacterial cell systems. This is because bacteria utilise the efflux system to avert the inhibitory action of antibiotics which may lead to multidrug resistance. The development of efflux inhibitors is one of the methods being employed to counter multidrug resistance in bacteria. Seven plant species used as traditional medicines in Swaziland for the treatment of infections were identified and collected with the assistance of traditional healers or local people who had knowledge of medicinal plants. The plants were Breonadia salicina, Ozoroa sphaerocarpa, Dioscorea sylvatica, Dioscorea cotinifolia, Syzygium cordatum, Ehretia rigida and Helichrysum acutatum. After preliminary screening of the plant extracts for antimicrobial activity using various Staphylococcus aureus strains and Escherichia coli; as well as for antibiotic potentiating activity using S. aureus strains over-expressing MDR efflux proteins (SA-1199B (NorA) and XU212 (TetK) pump), a diverse range of phytochemicals were isolated and characterised from the plants using chromatographic and spectroscopic (IR, UV, MS, NMR) techniques. Some cardanols and anacardic acids from Ozoroa sphaerocarpa exhibited antimicrobial activity against a range of microorganisms (MIC 8 – 512 µg/mL) whilst dioscin, from Dioscorea sylvatica, displayed anti-Candida activity (MIC 1 µg/mL). None of the plant extracts, nor the isolated compounds, had antimycobacterial activity against Mycobacterium smegmatis and M. aurum. At a sub-inhibitory concentration, (Z)-3-(heptadec-8-en-1-yl)phenol, from O. sphaerocarpa and ursolic acid, from B. salicina, potentiated the activity of tetracycline and norfloxacin against the S. aureus XU212 and SA-1199B strains, respectively. Their activity resulted in a 512-fold reduction in the minimum inhibitory concentration (MIC) of both antibiotics on the S. aureus strains. Some cinnamic acid derivatives isolated from D. sylvatica and D. cotinifolia also potentiated the activity of tetracycline and norfloxacin. The efflux pump inhibitory activity of the compounds was investigated using the ethidium bromide accumulation assay and a bibenzyl, from D. sylvatica and some diaryl nonadiones and a ω-hydroxy acid ester from D. cotinifolia, resulted in an increase in intracellular ethidium bromide accumulation in the SA-1199B strain, suggesting NorA pump inhibition. Ethidium bromide was not found to be a strong substrate for the TetK pump in the XU212 strain. Apart from moronic acid, isolated from O. sphaerocarpa, which showed some comparable efflux pump inhibition to reserpine, all the compounds which exhibited tetracycline-potentiating activity did not show a corresponding TetK pump inhibition activity. This suggested that some other mechanism of antibiotic potentiation activity was a possibility. Of the isolated compounds, dioscin was the most selective towards fungal cells compared to the human dermal cells; whilst ursolic acid was the least selective, showing toxicity to the human dermal cells at its active concentration. The anacardic acids and moronic acid also showed some degree of selectivity. In conclusion, the results of the study showed the potential of Swazi medicinal plants as a source of antimicrobial drug leads; as well as their potential use in combination therapy for the management of bacterial resistance

Attenuation of oxidative stress in U937 cells by polyphenolic-rich bark fractions of Burkea africana and Syzygium cordatum

BACKGROUND: Oxidative stress has been implicated in the progression of various diseases, which may result in the depletion of endogenous antioxidants. Exogenous supplementation with antioxidants could result in increased protection against oxidative stress. As concerns have been raised regarding synthetic antioxidant usage, the identification of alternative treatments is justified. The aim of the present study was to determine the antioxidant efficacy of Burkea africana and Syzygium cordatum bark extracts in an in vitro oxidative stress model. METHODS: Cytotoxicity of crude aqueous and methanolic extracts, as well as polyphenolic-rich fractions, was determined in C2C12 myoblasts, 3T3-L1 pre-adipocytes, normal human dermal fibroblasts and U937 macrophage-like cells using the neutral red uptake assay. Polyphenolic content was determined using the Folin-Ciocalteau and aluminium trichloride assays, and antioxidant activity using the Trolox Equivalence Antioxidant Capacity and DPPH assays. The extracts efficacy against oxidative stress in AAPH-exposed U937 cells was assessed with regards to reactive oxygen species generation, cytotoxicity, apoptosis, lipid peroxidation and reduced glutathione depletion. RESULTS: B. africana and S. cordatum showed enrichment of polyphenols from the aqueous extract, to methanolic extract, to polyphenolic-rich fractions. Antioxidant activity followed the same trend, which correlated well with the increased concentration of polyphenols, and was between two- to three-fold stronger than the Trolox antioxidant control. Both plants had superior activity compared to ascorbic acid in the DPPH assay. Polyphenolic-rich fractions were most toxic to the 3T3-L1 (IC(50)’s between 13 and 21 μg/ml) and C2C12 (IC(50)’s approximately 25 μg/ml) cell lines, but were not cytotoxic in the U937 and normal human dermal fibroblasts cultures. Free radical-induced generation of reactive oxygen species (up to 80%), cytotoxicity (up to 20%), lipid peroxidation (up to 200%) and apoptosis (up to 60%) was successfully reduced by crude extracts of B. africana and the polyphenolic-rich fractions of both plants. The crude extracts of S. cordatum were not as effective in reducing cytotoxic parameters. CONCLUSION: Although oxidative stress was attenuated in U937 cells, cytotoxicity was observed in the 3T3-L1 and C2C12 cell lines. Further isolation and purification of polyphenolic-fractions could increase the potential use of these extracts as supplements by decreasing cytotoxicity and maintaining antioxidant quality