The Biological Activities of 20 Nature Identical Essential Oil Constituents

Agar diffusion techniques are used widely to assay plant extracts for antimicrobial activity, but there are problems associated with this technique. A micro-dilution technique was developed using 96-well microplates and tetrazolium salts to indicate bacterial growth. p-Iodonitrotetrazolium violet [0.2 mg/ml] gave better results than tetrazolium red or thiazolyl blue. The method is quick, worked well with Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, and Escherichia coli and with non-aqueous extracts from many different plants. The method gave reproducible results; required only 10-25 microliters of extract to determine minimal inhibitory concentrations, distinguished between microcidal and microstatic effects, and provided a permanent record of the results. Using S. aureus, and a Combretum molle extract, the technique was 32 times more sensitive than agar diffusion techniques and was not sensitive to culture age of the test organism up to 24 hours. The S. aureus culture could be stored up to 10 days in a cold room with little effect on the assay results. This method was useful in screening plants for antimicrobial activity and for the bioassay-guided isolation of antimicrobial compounds from plants. MIC values determined for sulfisoxazole, norfloxacin, gentamicin, and nitrofuratoin were similar to values indicated in the literature but values obtained with trimethroprim and ampicillin were higher with some bacteria.

A rapid, semiautomated microdilution method was developed for measuring the activity of potential antimalarial drugs against cultured intraerythrocytic asexual forms of the human malaria parasite Plasmodium falciparum. Microtitration plates were used to prepare serial dilutions of the compounds to be tested. Parasites, obtained from continuous stock cultures, were subcultured in these plates for 42 h. Inhibition of uptake of a radiolabeled nucleic acid precursor by the parasites served as the indicator of antimalarial activity. Results of repeated measurements of activity with chloroquine, quinine, and the investigational new drug mefloquine demonstrated that the method is sensitive and precise. Several additional antimalarial drugs and compounds of interest were tested in vitro, and the results were consistent with available in vivo data. The use of P. falciparum isolates with known susceptibility to antimalarial drugs also permitted evaluation of the cross-resistance potential of each compound tested. The applications and expectations of this new test system within a drug development program are discussed.

The natural antimicrobial compound carvacrol shows a high preference for hydrophobic phases. The partition coefficients of carvacrol in both octanol-water and liposome-buffer phases were determined (3.64 and 3.26, respectively). Addition of carvacrol to a liposomal suspension resulted in an expansion of the liposomal membrane. Maximum expansion was observed after the addition of 0.50 micromol of carvacrol/mg of L-alpha-phosphatidylethanolamine. Cymene, a biological precursor of carvacrol which lacks a hydroxyl group, was found to have a higher preference for liposomal membranes, thereby causing more expansion. The effect of cymene on the membrane potential was less pronounced than the effect of carvacrol. The pH gradient and ATP pools were not affected by cymene. Measurement of the antimicrobial activities of compounds similar to carvacrol (e.g., thymol, cymene, menthol, and carvacrol methyl ester) showed that the hydroxyl group of this compound and the presence of a system of delocalized electrons are important for the antimicrobial activity of carvacrol. Based on this study, we hypothesize that carvacrol destabilizes the cytoplasmic membrane and, in addition, acts as a proton exchanger, thereby reducing the pH gradient across the cytoplasmic membrane. The resulting collapse of the proton motive force and depletion of the ATP pool eventually lead to cell death.