Mustafa Din, Wardah
(2014)
A phytochemical and pharmacological study of acalypha wilkesiana var. macafeana hort. (euphorbiaceae juss.): antioxidant and antibacterial analyses.
PhD thesis, University of Nottingham.
Abstract
A tropical shrub from Euphorbiaceae (Juss.) family, namely Acalypha wilkesiana var. macafeana hort. was investigated for its antioxidant and antibacterial properties. The antioxidant properties were assayed by Ferric Reducing Antioxidant Power (FRAP) assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and β-carotene bleaching assay. Assessment of its antibacterial properties was conducted with pour plate disc diffusion assay and dilution methods. The plant was collected, dried, grinded and soaked continuously in four different solvent starting from non-polar to polar solvent: hexane, ethyl acetate, ethanol and water. The crude extracts were concentrated under pressure and kept in -20 °C prior to investigation.
The ethanol extract of A. wilkesiana var. macafeana hort. exhibited good antioxidant and antibacterial activities with results more potent than the standards used. To further locate the bioactive constituents of the plant, we fractionated the ethanol extract leading to five fractions, namely F1, F2, F3, F4 and F5. Both antioxidant and antibacterial assays were conducted upon all the five fractions. Results showed profound activity from F5 of both antioxidant and antibacterial properties which warrants further investigation.
To shed light on the active constituents of F5, identification was done with high performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LCMS) and nuclear magnetic resonance (NMR). Separation was obtained with reversed phase HPLC which showed one major compound and 6 minor compounds. The major compound was collected with a fraction collector and identified as geraniin via interpretation and comparison of its NMR shifts, while the other 3 compounds were identified by fragmentations of LC-MS. The compounds identified are β-glucogallin, potentillin and sanguiin H-6. All identified compounds are ellagitannins, except for β-glucogallin which is a gallotannin.
The in vitro cell-based assay was performed to HepG2 cells to assess the ability of antioxidants like ellagitannins to protect cells against oxidative insults, and F5 was observed to be able to protect cells against cell death induced by t-BHP insults in a dose-dependent manner. F5 was also found non-toxic in the concentration needed to protect the cells, which is 100 µg/mL.
We then explored the synergistic property of the tannin fraction, F5 with commercial antibiotics and observed F5 and ampicillin inhibit the growth of Staphylococcus aureus synergistically. Field Emission Scanning Electron Microscopy (FESEM) analysis was able to demonstrate that the bactericidal mechanism of F5 involves cell wall lysis as the result illustrates indentation of the cell surface and some showed total collapse of the cells. To explore its ability to be formulated and used as a topical agent for treating bacterial infections, a preliminary formulation was made incorporating F5, and formulated with 3 different bases. The formulation made with the paraffin base was observed able to exert the antibacterial property of F5 against Staphylococcus aureus in the in-vitro assay. In vivo animal study on guinea pigs with an incised cut infected with Staphylococcus aureus and treated with the formulation showed that the closure and healing of the wound was faster than Burnol®.
Our results indicate possible use of ellagitannins from A. wilkesiana var. macafeana hort. with ampicillin to treat Staphylococcus aureus infections as it is bactericidal via a mechanism involving cell lysis. It also illustrates the possibility to be used as a topical wound healing agent with respect to its antibacterial and antioxidant properties. Ellagitannins from A. wilkesiana var. macafeana hort. can be viewed as a possible bactericidal agent that can contribute to the development of topical antibacterial drug or cosmetics in tropical countries.
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