Cinar, Dursun (2009) Purification and antimicrobial properties of oleuropein. Doctoral thesis, University of West London.
Dursan_Cinar_-_PhD.pdf - Accepted Version
Olive leaves contain substantial amounts of phenolic substances, including the polyphenol oleuropein. This compound has been reported to possess antimicrobial properties. The purpose of this study was to develop an improved method for the extraction and purification of oleuropein from olive leaves and characterise its activity as well as the mode of action against a range of bacteria.
Phenolic compounds from olive leaves were extracted in methanol and oleuropein was separated from the mixture by countercurrent chromatography (CCC). Separation was confirmed by high performance liquid chromatography. CCC processing resulted in a purity of oleuropein of 60% and this was later improved to 90% (compared to 83% in a commercially available oleuropein product). Flash chromatography was successfully introduced as an additional purification step and this eliminated some of the surfactants in the extract.
Fifteen strains of bacteria and one yeast, including species commonly associated with hospital infections, were tested for their sensitivity to oleuropein in agar supplemented with oleuropein and by disc diffusion on agar media. Most of the bacteria used in this study were inhibited by oleuropein but the amount of oleuropein required for inhibition varied from 0.25 to 3.0%. The two most sensitive strains were Enterobacter faecalis and one strain of group A Streptococci in agar supplemented with oleuropein. Staphylococcus spp. were inhibited by oleuropein concentrations of 0.5 to 1.5%. Gram-negative species, such as Escherichia coli and Pseudomonas aeruginosa, the Gram-positive Micrococcus Iuteus and Bacillus subtilis as well as the fungus Candida albicans were not inhibited in the same assay. In the disc diffusion test, 10% oleuropein inhibited Gram-negatives (4-15 mm) whereas 5 and 10% oleuropein resulted in inhibition zones from 12 to 30 mm in Gram-positives and C. albicans. Four strains of S. aureus were subjected to further studies. In bacterial-time kill assays, exposure to 2% oleuropein resulted in reductions of up to 6 log cfu mL-1 in 4 hours and 6 hours for two methicillin resistant and two methicillin susceptible strains, respectively.
A methicillin susceptible and a methicillin resistant S. aureus were investigated using transmission electron microscopy following exposure to 2% oleuropein. Cells of both types showed leakage of cell contents and ultimately lysis within two and four hours of exposure. Further work on leakage of cell constituents based on absorbance measurements was inconclusive due to interference by coloured compounds formed by the oxidation of oleuropein.
Leakage of amino acids from cells treated with oleuropein was investigated using ninhydrin and Bradford assays. It was observed that 12 to 38% of amino acids leaked from S. aureus treated with oleuropein. The results were confirmed by sodium dodecyl acrylamide electrophoresis where several bands were absent from treated cell extracts. In addition, fluorescent microscopy of lectin labelled S. aureus cells was attempted to investigate damage of glycoproteins attached on the extracellular cell wall. Lectin binding was unsuccessful and was replaced by fluorescein isocyanate, which selectively binds to lysine groups; the latter indicated reduced fluorescence in treated cells.
In conclusion this work demonstrated the application of a novel purification method based on countercurrent chromatography to obtain oleuropein with improved purity. The antimicrobial studies showed that oleuropein has the potential to eliminate bacteria. The mode of action studies showed that denaturation of proteins by oleuropein occurred, resulting in irreversible cell degradation. Oleuropein might be contemplated as a cleaning agent in envirnoments where strong acids and bases are harmful for equipment.
|Item Type:||Thesis (Doctoral)|
|Uncontrolled Keywords:||Olive leaves; Oleuropein; Extraction; Purification|
|Subjects:||Medicine and health > Microbiology|
|Depositing User:||Rod Pow|
|Date Deposited:||22 Aug 2012 13:20|
|Last Modified:||30 Mar 2017 12:04|
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