A atividade microbiológica do óleo essencial de alcaravia (Carum carvi L.) de várias origens

A alcaravia e uma das plantas medicinais mais importantes cultivadas na Polonia. O oleo essencial da alcaravia tem atividade media antimicrobiana embora ele iniba o crescimento de varias bacterias e fungos. Esse estudo nao somente avalia a atividade microbiologica do oleo de alcaravia obtido da fruta oriunda de varios genotipos, mas tambem investiga a correlacao entre essa atividade e o conteudo do oleo essencial e seus componentes principais. Em experimentos realizados em 2008 e 2009, foi testado o oleo essencial de 20 genotipos selecionados, cultivar Konczewicki, oriundos de jardins botânicos europeus (17) e de nossas cepas (2). Foi analisada a Concentracao Minima Inibitoria (CMI), a qual inibiu a cepa bacteriana padrao. O valor de CMI foi recalculado para unidades antibioticas (UA) e registrou-se que a atividade microbiologica do oleo da alcaravia foi diferente. A mais forte atividade produziu oleo oriundo do genotipo Cluj (CMI = 0,16 mg mL-1; AU = 8650), enquanto o mais fraco foi derivado do oleo oriundo do genotipo Krakow (MIC = 1,75 mg mL-1; AU = 582). Uma correlacao negativa significante foi registrada entre CMI e o conteudo carvone. Todavia, a correlacao positiva foi registrada entre CMI e o conteudo limonene. O oleo essencial de alcaravia mostrou uma atividade media antimicrobiana enquanto o carvone pode ser determinado como um dos componente ativos.

Caraway essential oil performs medium antimicrobial activity.Hence it inhibits growth of many bacteria and fungi: Staphylococcus aureus, Escherichia coli, Salmonella typhi, Vibrio cholerae, Mycobacterium tuberculosis (SADOWSKA; OBIDOSKA, 1998;TOXOPEUS;BOUWMEESTER, 1992).Youself and Tawil (1980) described inhibited effect of caraway oil in concentration MIC = 190 μg mL -1 on yeast and mould growth.The investigation done by Bailer et al. (2001) and Frank et al. (2002) showed that carvone, the main compound of caraway oil, inhibited growth of some bacteria and fungi and can be used as a natural sprout suppressant during potato storage.Also research done by Hartmans et al. (1995) showed high activity of caraway oil against fungi occurred during potato storage: Fusarium sulphureum, Phoma exigua var.foveata, Helminthosporium solani.Similar effect of antifungal activity of caraway oil against Cladosporium cladosporioides, Fulvia fulvium, Alternaria alternata, Phoma macdonaldii, Phomopsis helianthi in concentration 2.5 mg mL -1 was observed by Simic et al. (2008).Caraway oil antimicrobial activity is used in medicine, food preservation, but also in cosmetic industry (DACHLER, 1998).
The aim of the study was to evaluate antibacterial activity of Caraway essential oil obtained from fruit originated from different genotypes and investigate the correlation between this activity and essential oil content and its main components (carvone and limonene).

Material and methods
In the experiments done in 2008 and 2009, essential oil of 20 selected caraway genotypes originated from: European botanical gardens (Bayreuth, Berlin, Bonn, Cluj, Göttingen, Jena, Krakow, Nantes, Poznan, Prague, Reykjawik, Riga, Salzburg, Ulm, Warsaw, Wroclaw, Zurich), cultivar 'Konczewicki' and our own breeding strains (9/1, 9/10) was tested.Two Caraway collections were established in 2007 and 2008 in Institute of Natural Fibres and Medicinal Plants in Poznan (Poland).In April each year seeds of the selected accessions were sown in greenhouse.Plants with 5-8 leaves were planted in the field at the beginning of May, 25-140 plants per accession.Mature fruit were collected from 10 plants of each accession by hand, separately from each plant in June 2008 and 2009.After harvest fruit were dried in the wellventilated, shadow place.
The content of essential oil was estimated after hydro-distillation of Caraway fruits (10 g) of each accession in two replication using Dering's apparatus following the methods recommended by Polish Pharmacopoeia VIII (2008).The hexane solution of the oil (1:10) was analyzed with gas chromatography using Perkin Elmer Clarus 500 system in the following conditions: chromatographic column Elite 1 (30 m x 0.32 mm x 0.25 μm), volume of each injected sample = 1 μL, injector temperature = 200°C, carrier gashelenium, flow = 1 mL min. - , FID detector temperature = 220°C.Time of components retention of the tested solution: carvone = 45.17 min., limonene = 15.69 min.
Antibacterial activity of caraway oil was analyzed by standard dilution in liquid media according to Cavanagh (1963).Hundred mg of essential oil was dissolved in 1 mL dimethylosulfoxide (DMSO).The range of oil dilution from 1 to 20 mg mL -1 was prepared in media Mueller-Hinton Broth of Merck.Then to the media was added 0.1 mL of 18 hours liquid culture of standard strain (Staphylococcus aureus ATCC 6538 P) diluted 1:10.000 in the same medium (number of inoculum contained 10 4 -10 5 bacterial cells in 1 mL).Incubation of the tested samples was conducted in 37°C for 18 hours.The MIC (Minimal Inhibitory Concentration) was defined as the lowest concentration of the oil completely inhibiting the growth of standard strain.These value was calculated on antibiotic units (AU), based on that value of MIC is equivalent of 1AU.The results were referenced to 1 g of oil.
The one-way analysis of variance was carried out to determinate the effect of origin on the variability of MIC, AU, essential oil content, carvone content and limonene content.Means values and coefficients of variation were calculated for each trait.The least significant differences (LSDs) for each trait were calculated and, on this basis, homogeneous groups for the analyzed traits were determined.Procedure FCORRELATION in GENSTAT Release 10.1 (2007) was used for correlation analyses.Associations among traits were studied with the help of a scatterplot matrix (CLEVELAND, 1994;KOZAK et al., 2010).

Results and discussion
Analysis of variance indicated that the main effect of plant origin was significant for all traits in the study, except for limonene content.Mean values and coefficient of variation for each origin and each trait were shown in the Table 1.Based on the minimal inhibitory concentration of the selected caraway genotypes three homogeneous groups were distinguished (Table 1).Similarly, three homogeneous groups were distinguished based on differences in antibiotic units (Table 1).
Two homogeneous groups were distinguished based on differences in content of carvone in oil (Table 1).The coefficient of variation for carvone content ranged from 1.17% (Ulm) to 25.0% (Zurich).The content of limonene was not significantly different and varied from 28% (Poznan, Prague) to 40% (Bonn, Jena, Ulm).The coefficient of variation for limonene content ranged from 4.0% (Cluj) to 41.6% (Zurich).A significant negative correlation was observed between MIC and AU (r = -0.58),MIC and carvone content (r = -0.45),carvone content and limonene content (r = -0.93)(Table 2, Figure 1).However, positive correlation was observed between MIC and limonene content (r = 0.37) (Table 2).Some of relationships are not linear (Figure 1 and Figure 2).The function defining dependence between AU and MIC were found.This function is given by AU = 1002.7MIC -1.001 and explains in 99.97 % of total variation (Figure 2).Caraway oil represents lower microbiological activity, which was confirmed by Toxopeus and Bouwmeester (1992) and Sadowska andObidoska (1998), Dachler (1998).Research done by Hołderna-Kędzia et al. (2006) revealed that the essential oils from tea tree, manuka and sandalwood had strong effect on pathogenic gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis), gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) and pathogenic fungi: yeast-like fungi (Candida albicans, Candida krusei, Geotrichum candidum), mould fungi (Aspergillus flavus), dermatophytes (Microsporum gypseum) in the concentration from 0.1 to 10.0 mg mL -1 .Singh et al. (2002) obtained the high activity of essential oil from Carum copticum fruit against bacteria grampositive and gram-negative, although the main compound of this oil was thymol (35-60%).While Simic et al. (2008) reported that MIC of caraway oil inhibited fungi growth was 2.5 mg mL -1 , however they did not analyzed the obtained results in comparison to oil composition.

Conclusion
Caraway essential oil exhibited medium antimicrobial activity and carvone can be recognized as a one of the active component.

Table 1 .
Mean values, coefficient of variation (cv) and homogeneous groups for caraway antimicrobial activity and essential oil, carvone and limonene content