Antimicrobial testinas and gas chromatoaraphic analvsis of pure oxvaenated monoterpenes 1.8-cineole, α-terpineol, terpinen-4-ol and camphor as well as target comoounds in essential oils of pine (Pinus pinaster), rosemary (Rosmarinus officinalis), tea tree (Melaleuca alternifolia)

The oxygenated monoterpenes 1,8-cineole, α-terpineol, terpinen-4-ol and camphor as well as essential oils of pine, rosemary and tea tree, rich in these volatiles, were tested for their antimicrobial activities against some different strains of yeast, Gram-(-)- and Gram-(+)-bacteria by agar diffusion and agar dilution method, respectively. The same was done using the phenolic aroma compound eugenol as a reference-substance. The monoterpene alcohols a-terpineol and terpinen-4-ol were found to be active against all strains used in a wide range. The same result was obtained for the testings of essential oils of pine, rosemary (2 samples) and tea tree. Nearly all aroma chemicals and essential oils did show a significant high antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa. For qualitative and quantitaive investigations of the key aroma compounds and the compositions of the essential oils, gas chromatographic methods (GC-FID and GCMS with columns of different polarities) were used and the results correlated with that of the antimicrobial testings. The obtained data are discussed to get more insight into the influence of pure aroma compounds on antimicrobial activities of essential oils.


Introduction
In continuation of our research work on the field of combined data interpretation of antimicrobial testings (agar diffusion and agar dilution methods) and chiral phase gas chromatographic (cyclodextrin) analysis of aroma samples, such as pure compounds, essential oils and extracts [I-61, the oxygenated monoterpenes 1,8-cineole, a-terpineol, terpinen-4-01 and camphor as well as the essential oils of pine (Pinus pinaster), rosemary (Rosmarinus officinalis, 2 samples) and tea tree (Melaleuca alternifolia), known to be rich in these aroma compounds [7-1 I], were investigated.
Although the medical, pharmaceutical, perfumistic, cosmetic and food flavouring properties [7,[8][9][10][11][12][13] as well as the compositions [14-211 of essential oils of pine, rosemary and tea tree have been reported in many papers, detailed informations about the influence of the above cited single main compounds of these essential oils on their antimicrobial activities are not available until now.
Therefore, the objectives of this research were to test these essential oils and the cited oxygenated monoterpenes by means of usual agar diffusion and agar dilution methods with various strains of microorganisms [22-401, after improvement of some parameters [4-61.Furthermore, gas chromatograpic (GC-FID and GC-MS) analyses and olfactoric evaluations of the target compounds and the essential oils could be used to get informations about the purity as well as the composition of each sample.As result of the combined data interpretation the above mentioned influence of I ,8-cineole, a-terpineol, terpinen-4-01 and camphor on the antimicrobial effects of essential oils of pine, rosemary and tea tree should be ascertained.On basis of these data, a systematic investigation of aroma compounds and odorous samples (essential oils and extracts) will be continued in a running, international project.

Results and discussion
To control the efficiency of the used microbiological testing methods, eugenol as a phenolic compound with well-known antimicrobial activity against many strains of microorganisms was tested.Eugenol shows excellent effects against all strains using both methods, agar diffusion and agar dilution.Therefore, the obtained antimicrobial data (IZ=inhibition zones and MIC=minimum inhibition concentrations) of this component were taken as references for further testings.
The commercially available essential oils did show the following antimicrobial activities (see Table 1

Compounds and Essential Oils
no inhibition observed Table 1.Antimicrobial activities of oxygenated monoterpenes, rosemary, pine and tea tree essential oils Rosemary oil: Highest effects of the sample from Spain were found against Staphylococcus aureus (IZ=19) and Pseudomonas aeruginosa (MIC=600), while the oil from Tunesia showed the best activity against Candida albicans (lZ=I4, M I C=600), Staphylococcus aureus and Escherichia coli (MI C=600, each).
Tea tree oil: The oil was most effective against Escherichia coli (12=23) and Pseudomonas aeruginosa (M IC=600).
Using gas chromatography (GC-FID and GC-MS with 2 columns of different polarity) in combination with olfactoric evaluations, the purity of the target compounds as well as the compositions of the essential oils were investigated.
In general, the purity of all tested oxygenated monoterpenes was found to be higher than 96% (calculated by %-peak area of GC with an apolar OV-5-type column) with a range from 96.3 for terpinen-4-01 upto 99.9% for 1,8-cineole.The olfactoric evaluation with characteristic attributes for each sample proves the high purity and quality of these compounds (see The gas chromatographic investigations of the essential oils gave following results (see Table 3): The pine oil is rich (higher than 3%) in the target compound aterpineol (67.3%), further terpineols (y: 8.1%, cis+: 6.4%, trans$: 5.0%) and a-2,5dimethyl styrene (3.9%).
Also in the case of the essential oils the gas chromatographic data are in accordance to that one of olfactoric evaluations (using correlations with elsewhere published odor attributes [41-451) and proves the high quality of each sample.  1 .)Eugenol as the reference component with well-known antimicrobial activities was found to possess the highest antimicrobial potential of all samples tested.This result proves that both, agar diffusion and agar dilution methods furnish reproducible antimicrobial data in testing aroma samples.
2.) The antimicrobial effects found in tests with a-terpineol are significantly compareable with these in tests with the pine oil, rich in terpineols.
3.) The same statement is possible in the case of terpinen-4-01 and tea tree oil, with a concentration of this monoterpene alcohol higher than 38.0%(further terpinenes in higher concentrations in this tea tree oil may be responsible for minor variations of the antimicrobial activity against some used microorganisms).4.) A comparison of antimicrobial effects of I ,&cineole and the rosemary oils with a higher amount of this monoterpene ether (more than 45.0%, each) shows only in tests against Staphylococcus aureus, Klebsiella pneumoniae and Candida albicans as well as partly against Pseudomonas aeruginosa similar results, while 1,8cineole, in contrary to the rosemary oils, is not active against Escherichia coli.It seems that the relatively high concentrations of the monoterpene hydrocarbons a- pinene, P-pinene, camphene and limonene are responsible for this single different result.
Further combined investigations, antimicrobial testings and GC (in combination with olfactoric evaluations), will be done to get more data for a satisfying answer of the question, how effective pure aroma chemicals in complex aroma samples (e.g.essential oils and extracts) really are.

Experimental Samples
The essential oils are products from Kurt Kitzing Co., Germany as follows: Pine oil (Pinus pinaster Aiton, Pinaceae: branch gum + oleoresin)product-no in Table 1 and 2 as well as correlated with odor impressions published elsewhere [4 1 -451.

Table 2 .
GC analyses and olfactoric evaluations of target compounds