Polyphenolic and molecular variation in Thymus species using HPLC and SRAP analyses.

In the present research, inter and intra genetic variability of 77 accessions belonging to 11 Thymus species were assessed using eight SRAP primer combinations. High polymorphism (98.3%) was observed in the studied species. The cluster analysis classified Thymus species into five main groups. According to molecular variance (AMOVA) analysis, 63.14% of total genetic variation was obtained within the species, while 36.86% of variation was observed among species. STRUCTURE analysis was also performed to estimate the admixture of species. For instance, T. carmanicus and T. transcaspicus revealed high admixtures. HPLC analysis also demonstrated the presence of rosmarinic acid (32.3-150.7 mg/100 g DW), salvianolic acid (8-90 mg/100 g DW), and cinnamic acid (1.7-32.3 mg/100 g DW) as major phenolic acids, as well as apigenin, epicatechin, and naringenin as the major flavonoids. The highest phenolic and flavonoid contents were detected in T. transcaspicus (37.62 mg gallic acid equivalents (GAE) g-1 DW) and T. vulgaris (8.72 mg quercetin equivalents (QE) g-1 DW), respectively. The antioxidant properties and total phenolic of Thymus species were examined using DPPH and ß-carotene-linoleic acid model systems and consequently T. vulgaris and T. pubescens were detected with the highest and the lowest antioxidant activities respectively. Cluster and principal Components Analysis (PCA) of the components classified the species in to three groups. Finally, similarity within some species was observed comparing molecular and phytochemical markers. For instance, T. vulgaris separated from other species according to major polyphenolic profiles and molecular analyses, as well as T. transcaspicus, T. carmanicus, and T. fedtschenkoi that were clustered in the same groups.

Transcriptome Landscape Variation in the Genus Thymus.

Among the Lamiaceae family, the genus Thymus is an economically important genera due to its medicinal and aromatic properties. Most Thymus molecular research has focused on the determining the phylogenetic relationships between different species, but no published work has focused on the evolution of the transcriptome across the genus to elucidate genes involved in terpenoid biosynthesis. Hence, in this study, the transcriptomes of five different Thymus species were generated and analyzed to mine putative genes involved in thymol and carvacrol biosynthesis. High-throughput sequencing produced ~43 million high-quality reads per sample, which were assembled de novo using several tools, then further subjected to a quality evaluation. The best assembly for each species was used as queries to search within the UniProt, KEGG (Kyoto Encyclopedia of Genes and Genomes), COG (Clusters of Orthologous Groups) and TF (Transcription Factors) databases. Mining the transcriptomes resulted in the identification of 592 single-copy orthogroups used for phylogenetic analysis. The data showed strongly support a close genetic relationship between Thymus vulgaris and Thymus daenensis. Additionally, this study dates the speciation events between 1.5-2.1 and 9-10.2 MYA according to different methodologies. Our study provides a global overview of genes related to the terpenoid pathway in Thymus, and can help establish an understanding of the relationship that exists among Thymus species.

Evaluation of oxidative stability of mayonnaise containing poly ε- caprolactone nanoparticles loaded with thyme essential oil

Suspensions of poly ε-caprolactone (PCL) nanoparticles loaded with thyme essential oil were prepared as a natural antioxidant in mayonnaise. Mean particle size was 204.9 ± 2.7 and 240.0 ± 5.5 nm respectively for nanoparticles prepared with PCL alone (NP-C) and for those loaded with thyme essential oil (NP-T). The polydispersity index indicated a homogeneous distribution of all particles, with no significant difference between NP-C and NP-T samples. The nanoparticles showed a large negative charge evidenced by zeta potential rates, indicating high physical stability. The use of PCL as a polymer provided high encapsulation efficiency for thyme essential oil (91.15 ± 2.12 %). DPPH (2,2-diphenyl-1-picrylhydrazyl) method determined IC50 rates were 476.4 ± 33.6 and 483.5 ± 20.4 µg mL-1respectively for unencapsulated oil and for NP-T, evidencing pronounced antioxidant activity. NP-C, NP-T and synthetic antioxidant butylhydroxytoluene (BHT) were applied to samples of mayonnaise and their oxidative stability evaluated for eight days in an oven at 63 ± 3ºC. Results of hydroperoxide value (HP) and thiobarbituric acid reactive substances (TBARS) showed that NP-T had a similar performance as synthetic antioxidant BHT in the prevention of mayonnaise lipid oxidation

Essential oil composition, total phenolic, flavonoid contents, and antioxidant activity of Thymus species collected from different regions of Iran.

In this study, the essential oil (EO) composition, flavonoid and phenolic contents, and antioxidant activities of fourteen Thymus accessions belonging to ten species were evaluated. Gas chromatography-mass spectrometry (GC-MS) analysis revealed the presence of 38 compounds with the major constituents including thymol (12.4-79.74%), carvacrol (4.37-42.14%), geraniol (0.3-22.44%), and p-cymene (0.8-12.86%). Cluster analysis identified three groups of high thymol, geraniol/linalool, and high carvacrol. The highest phenolic and flavonoid contents were detected in T. daenensis-1 (70.6mg tannic acid equivalents (TAE) g-1 DW) and T. vulgaris (8.55mg quercetin equivalents (QE) g-1 DW), respectively. The antioxidant activities of the samples were determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and reducing power assay. The results demonstrated that T. daenensis-3 (IC50=273.36), T. vulgaris (IC50=289.3), and T. fedtschenkoi-3 (IC50=339.22) possessed higher antioxidant activities than the others. Finally, the Thymus species with high bioactive compounds may be recommended for further food applications.

Composição química e concentração mínima bactericida de dezesseis óleos essenciais sobre Escherichia coli enterotoxigênica / Determination of minimum bactericidal concentration of sixteen essential oils on enterotoxigenic Escherichia coli

RESUMO Este trabalho teve por objetivo avaliar o efeito bactericida in vitro de dezesseis óleos essenciais sobre Escherichia coli enterotoxigênica (ETEC). Dentre os óleos essenciais estudados, três foram extraídos in situ por arraste a vapor e treze foram adquiridos comercialmente. Todos os óleos foram analisados por CG-EM e CG-DIC. A atividade bactericida foi avaliada pelo método de microdiluição utilizando-se caldo triptona de soja e microplacas de poliestireno de 96 poços, com posterior plaqueamento das culturas em ágar triptona de soja. Os óleos essenciais de Cinnamomum cassia e de Thymus vulgaris apresentaram concentração mínima bactericida (CMB) de 0,12% e 0,25%, respectivamente. Já os óleos comerciais de Syzygium aromaticum e Origanum vulgare apresentaram ambos CMB de 0,50% e os óleos extraídos in situ de Cymbopogon citratus e Origanum vulgare apresentaram ambos CMB de 1,00%. Os dezesseis óleos essenciais apresentaram composição química qualitativa e quantitativa distintas. As análises químicas dos óleos essenciais de Cinnamomum cassia e de Thymus vulgaris tiveram a presença majoritária de E-cinamaldeído (84,52%) e timol (50,89%). Conclui-se que os óleos de C. cassia e T. vulgaris foram os mais eficazes na inibição do crescimento in vitro dessa bactéria, a qual possui diferentes níveis de sensibilidade dependendo da composição química do óleo.

ABSTRACT This study aimed to evaluate the bactericidal effect in vitro of sixteen essential oils on enterotoxigenic Escherichia coli (ETEC). Among the essential oils, three were extracted in situ by steam distillation and thirteen were purchased commercially. All oils were analyzed by GC-MS and GC-FID. The bactericidal activity was evaluated by the microdilution method using tryptone soy broth, and 96-well polystyrene microplates with subsequent plating of the cultures in tryptone soy agar. Cinnamomum cassia and Thymus vulgaris essential oils showed minimal bactericidal concentration (MBC) 0.12% and 0.25%, respectively. Both commercial oils of Syzygium aromaticum and Origanum vulgare showed MBC of 0.50% and the oils extracted in situ Origanum vulgare and Cymbopogon citratus showed both MBC of 1.00%. The sixteen essential oils pointed out distinct qualitative and quantitative chemical composition. Chemical analysis of Cinnamomum cassia and Thymus vulgaris oils had the predominant presence of E-cinnamaldehyde (84.52% ± 0.07%) and thymol (50.89% ± 0.31%). In conclusion, T. vulgaris and C. cassia oils were the most effective in inhibiting in vitro growth of this bacterium, which has different sensitivity levels depending on the chemical composition of the oil.

Plants belonging to the genus Thymus as antibacterial agents: from farm to pharmacy.

In traditional medicine, plants have been used since ancient times for the prevention and/or protection against infectious diseases. In recent years, the use of herbal medicines and food supplements containing botanical ingredients, as alternative therapy for infectious diseases, has been intensified due to their high content of antimicrobial agents such as polyphenols, i.e. flavonoids, tannins, and alkaloids. Plants from the genus Thymus are important medicinal herbs, which are known to contain antimicrobial agents, and are rich in different active substances such as thymol, carvacrol, p-cymene and terpinene. In this review, we summarise the available literature data about the in vitro antibacterial effects of the main plants belonging to the genus Thymus. We also provide information about cultivation, chemical composition of the essential oils obtained from these plants, and their use for medicinal purposes.

Genetic diversity and chemical polymorphism of some Thymus species.

To ascertain whether there are chemical and genetic relationships among some Thymus species and also to determine correlation between these two sets of data, the essential-oil composition and genetic variability of six populations of Thymus including: T. daenensis CELAK. (two populations), T. fallax FISCH. & C.A.MEY., T. fedtschenkoi RONNIGER, T. migricus KLOKOV & DES.-SHOST., and T. vulgaris L. were analyzed by GC and GC/MS, and also by randomly amplified polymorphic DNA (RAPD). Thus, 27 individuals were analyzed using 16 RAPD primers, which generated 264 polymorphic scorable bands and volatiles isolated by distillation extraction were subjected to GC and GC/MS analyses. The yields of oils ranged from 2.1 to 3.8% (v/w), and 34 components were identified, amounting to a total percentage of 97.8-99.9%. RAPD Markers allowed a perfect distinction between the different species based on their distinctive genetic background. However, they did not show identical clustering with the volatile-oil profiles.

Molluscicidal and Mosquitocidal Activities of the Essential oils of Thymus capitatus Hoff. et Link. and Marrubium vulgare L. / Atividades moluscicida e mosquitocida de óleos essenciais de Thymus capitatus Hoff. et Link. e de Marrubium vulgare L

Steam distillation of essential oils of aerial parts of Thymus capitatus and Marrubium vulgare L. collected at North cost of Egypt yielded 0.5% and 0.2%, respectively. Results of Gas chromatography-mass spectrometry analyses of the two samples identified 96.27% and 90.19% of the total oil composition for T. capitatus and M. vulgare, respectively. The two oil samples appeared dominated by the oxygenated constituents (88.22% for T. capitatus and 57.50% for M. vulgare), composed of phenols, mainly carvacrol (32.98%) and thymol (32.82%) in essential oil of T. capitatus, and thymol (34.55%) in essential oil of M. vulgare. It was evaluated the molluscicidal activity of T. capitatus and M. vulgare essential oils on adult and eggs of Biomphalaria alexandrina as well as their mosquitocidal activity on Culex pipiens. The LC50 and LC90 of T. capitatus essential oil against adult snails was 200 and 400 ppm/3hrs, respectively, while for M. vulgare it was 50 and 100 ppm/3hrs, respectively. Moreover, M. vulgare showed LC100 ovicidal activity at 200 ppm/24 hrs while T. capitatus oil showed no ovicidal activity. It was verified mosquitocidal activity, with LC50 and LC90 of 100 and 200 ppm/12hrs respectively for larvae, and 200 and 400 ppm/12hrs respectively for pupae of C. pipiens.

A destilação por arraste a vapor dos óleos essenciais de partes aéreas de Thymus capitatus Hoff. et Link. e de Marrubium vulgare L. coletadas na costa norte do Egito resultaram em rendimento de 0,5% e 0,2%, respectivamente. Resultados de análises por cromatografia gasosa acoplada à espectrometria de massas de ambas as amostras possibilitaram a identificação de 96,27% e 90,19% dos constituintes químicos respectivamente de T. capitatus e M. vulgare. Verificou-se predomínio de constituintes oxigenados (88,22% para T. capitatus e 57,50% para M. vulgare, principalmente fenóis, como carvacrol (32,98%) e timol (32,82%) no óleo essencial de T. capitatus, e timol (34,55%) no óleo essencial de M. vulgare. Avaliou-se a atividade dos óleos essenciais obtidos contra adultos e ovos de Biomphalaria alexandrina, bem como em larvas e pupas de Culex pipiens. A CL50 e CL90 do óleo essencial de T. capitatus em moluscos adultos foi respectivamente 200 e 400 ppm/3hrs, enquanto para o óles essencial de M. vulgare verificou-se CL50 e CL90 de 50 e 100 ppm/3hrs, respectivamente. Além disso, M. vulgare apresentou atividade ovicida, com CL 100 de 200 ppm/24 horas, enquanto o óleo essencial de T. capitatus não demonstrou atividade ovicida. Verificou-se ainda atividade mosquitocida, com CL50 e CL90 de 100 e 200 ppm/12hrs respectivamente para larvas, e 200 e 400 ppm/12hrs contra pupas de C. pipiens.

Chemical composition, olfactory analysis and antibacterial activity of Thymus vulgaris chemotypes geraniol, 4-thujanol/terpinen-4-ol, thymol and linalool cultivated in southern France.

The essential oils of four chemotypes of Thymus vulgaris L. (Lamiaceae) were analyzed for their composition and antibacterial activity to assess their different properties. GC-MS and GC-FID analyses revealed that the essentials oils can be classified into the chemotypes thymol (41.0% thymol), geraniol (26.4% geraniol), linalool (72.5% linalool) and 4-thujanol/terpinen-4-ol (42.2% cis- and 7.3% trans-sabinene hydrate, 6.5 % terpinen-4-ol). The olfactory examination confirmed the explicit differences between these chemotypes. Furthermore, antibacterial activity was investigated against several strains of two Gram-positive (Brochothrix thermosphacta and Staphylococcus aureus) and four Gram-negative food-borne bacteria (Escherichia coli, Salmonella abony, Pseudomonas aeruginosa and P. fragi). All essential oil samples were demonstrated to be highly effective against Gram-positive strains, whereas the impact on Gram-negative microorganisms was significantly smaller, but still considerable. The results obtained indicate that, despite their different properties, the essential oils of selected T. vulgaris chemotypes are potent antimicrobials to be employed as useful additives in food products as well as for therapeutic applications.

Molluscicidal and Mosquitocidal activities of the essential oils of Thymus capitatus Hoff. et Link. and Marrubium vulgare L.

Steam distillation of essential oils of aerial parts of Thymus capitatus and Marrubium vulgare L. collected at North cost of Egypt yielded 0.5% and 0.2%, respectively. Results of Gas chromatography-mass spectrometry analyses of the two samples identified 96.27% and 90.19% of the total oil composition for T. capitatus and M. vulgare, respectively. The two oil samples appeared dominated by the oxygenated constituents (88.22% for T. capitatus and 57.50% for M. vulgare), composed of phenols, mainly carvacrol (32.98%) and thymol (32.82%) in essential oil of T. capitatus, and thymol (34.55%) in essential oil of M. vulgare. It was evaluated the molluscicidal activity of T. capitatus and M. vulgare essential oils on adult and eggs of Biomphalaria alexandrina as well as their mosquitocidal activity on Culex pipiens. The LC50 and LC90 of T. capitatus essential oil against adult snails was 200 and 400 ppm/3hrs, respectively, while for M. vulgare it was 50 and 100 ppm/3hrs, respectively. Moreover, M. vulgare showed LC100 ovicidal activity at 200 ppm/24 hrs while T. capitatus oil showed no ovicidal activity. It was verified mosquitocidal activity, with LC50 and LC90 of 100 and 200 ppm/12hrs respectively for larvae, and 200 and 400 ppm/12hrs respectively for pupae of C. pipiens.

Concentration levels of selected metals in the leaves of different species of thyme (T. schimperi and T. vulgaris) grown in Ethiopia.

The contents of some selected metals Ca, Mg, Fe, Mn, Co, Cu, Zn, Ni, and Cd in different thyme leaf samples widely consumed in Ethiopia were determined by flame atomic absorption spectroscopy (FAAS) after acid digestion with 1:1 HNO(3)/HClO(4) for 3 h at a temperature of 240°C by a Kjeldahl apparatus hot plate digester. The level of the nutrients in the four samples ranged from 1,239-2,517 µg/g, Ca; 1,524-1,786 µg/g, Mg; 728-2,517 µg/g, Fe; 37.7-114 µg/g, Mn; 2.59-4.3 µg/g, Co; 7.69-9.3 µg/g, Cu; 8.7-52 µg/g, Zn; and 9.83-14.2 µg/g, Ni; respectively. While the level of toxic metal Cd in the four samples ranged from 0.87-1.3 µg/g. The concentration of Ca was higher than the other metals in the three samples and Cd was the least of all the metals in the analyzed samples. The overall reproducibility of the method obtained from spiking experiment was within the range ±10%. This result will complement available data on food composition in Ethiopia.

Volatiles from Thymbra and Thymus species of the western Mediterranean basin, Portugal and Macaronesia.

Thyme is the common name of many taxa belonging to the Thymbra and Thymus genera. Given the economic importance of thyme oils, many thyme species have been studied and their essential oils and other volatile-containing extracts chemically characterized. Thymbra and Thymus species are frequent in the west Mediterranean region, considered to be the centre of origin of the genus Thymus, and extend further westwards in the Iberian Peninsula and northwest Africa, to the Macaronesian region in the Atlantic Ocean. The present work gives an overview of the chemical composition of the volatiles from the taxa of these two genera occurring in the above geographic area.

Variation of the chemical composition of essential oils in Tunisian populations of Thymus algeriensis Boiss. et Reut. (Lamiaceae) and implication for conservation.

The variation of the essential-oil composition among 14 Tunisian natural populations of Thymus algeriensis Boiss. et Reut. (=Thymus hirtus Willd. ssp. algeriensis Boiss. et Reut.) was assessed by GC (RI) and GC/MS. The populations were collected from different geographical regions belonging to the sub-humid, upper semi-arid, mean semi-arid, lower semi-arid, and upper-arid bioclimates. A total of 47 constituents, representing 81.0 to 96.5% of the total oil, were identified. The main volatiles at the species level were 1,8-cineole (17.7%), alpha-pinene (15.5%), and camphor (8.2%). A high variation among populations for the majority of the compounds was shown. Camphor (0.2-14.0%), linalool (0.2-22.4%), borneol (<0.01-24.3%), caryophyllene oxide (<0.01-18.8%), thymol (<0.01-54.9%), gamma-terpinene (0.4-6.5%), alpha-copaene (0.4-7.6%), linalyl acetate (<0.01-6.4%), and methyl eugenol (<0.01-6.9%) were the main constituents differentiating the populations. The chemical differentiation among populations, assessed by principal component analysis (PCA) and a UPGMA (unweighted pair-group method with averaging) cluster analysis performed on all populations and compounds, was high. Six chemotypes according to the main compounds have been distinguished, i.e., caryophyllene oxide/1,8-cineole/alpha-pinene, 1,8-cineole/alpha-pinene, 1,8-cineole/alpha-pinene/camphor, borneol/1,8-cineole/alpha-pinene, linalool, and thymol chemotypes. The spatial chemotype distribution was linked to the geographic distance among populations rather than to bioclimates, indicating that local selective environmental factors act on the chemotype diversity. The high chemical variation among populations according to their geographical and bioclimatic distribution imposes that conservation strategies of populations should be made appropriately, taking into account these factors. The in situ and ex situ conservation strategies should concern all populations representing the different chemotypes.

Genetic variation for sensitivity to a thyme monoterpene in associated plant species.

Recent studies have shown that plant allelochemicals can have profound effects on the performance of associated species, such that plants with a history of co-existence with "chemical neighbour" plants perform better in their presence compared to naïve plants. This has cast new light on the complexity of plant-plant interactions and plant communities and has led to debates on whether plant communities are more co-evolved than traditionally thought. In order to determine whether plants may indeed evolve in response to other plants' allelochemicals it is crucial to determine the presence of genetic variation for performance under the influence of specific allelochemicals and show that natural selection indeed operates on this variation. We studied the effect of the monoterpene carvacrol-a dominant compound in the essential oil of Thymus pulegioides-on three associated plant species originating from sites where thyme is either present or absent. We found the presence of genetic variation in both naïve and experienced populations for performance under the influence of the allelochemical but the response varied among naïve and experienced plant. Plants from experienced populations performed better than naïve plants on carvacrol soil and contained significantly more seed families with an adaptive response to carvacrol than naïve populations. This suggests that the presence of T. pulegioides can act as a selective agent on associated species, by favouring genotypes which perform best in the presence of its allelochemicals. The response to the thyme allelochemical varied from negative to neutral to positive among the species. The different responses within a species suggest that plant-plant interactions can evolve; this has implications for community dynamics and stability.

Genetic variability and geographic differentiation in Thymus daenensis subsp. daenensis, an endangered medicinal plant, as revealed by inter simple sequence repeat (ISSR) markers.

Thymus daenensis is an aromatic medicinal plant endemic to Iran. We used inter simple sequence repeat (ISSR) markers to detect genetic polymorphism in this herb using 17 T. daenensis accessions collected from different geographic regions in Iran. The 15 primers chosen for analysis revealed 256 bands, of which 228 (88.9%) were polymorphic. Jaccard's similarity indices based on ISSR profiles were subjected to UPGMA cluster analysis. The generated dendrogram revealed two major groups. The Tc group included the accessions collected from the center of the Zagros Mountains, and the Te group was collected from the extremes of the Zagros range. A principal coordinate analysis confirmed the results of clustering. The results showed that the divergence of accessions based on the Zagros Mountains is more logical in comparison with classification on the basis of provincial borders. Gene diversity and expected heterozygosity were greater in the Tc group than in the Te group, suggesting that the germplasm collected from the center of the Zagros Mountains is more variable.

Application and exploration of fast gas chromatography-surface acoustic wave sensor to the analysis of thymus species.

Fast gas chromatography combined with surface acoustic wave sensor (GC/SAW) has been applied for the detection of volatile aroma compounds emanated from thymus medicinal plants such as T. quinquecostotus (Jeju and Mt. Gaya in South Korea), T. quinquecostotus var. japonica (Ulreung island in South Korea), T. mongolicus (Northeastern Asia), and T. serpyllum (Europe). The GC/SAW involving the fragrance pattern analysis provides a novel analytical method with a very fast separation and characterization of aromas caused by the delicate difference of chemical composition according to botanical and geographical origin. On the comparison of experiments, the characteristic components and analytical tendency for air-dried thymus species detected by GC/SAW appear to be quite similar to those obtained by headspace solid-phase microextraction (HS-SPME)-GC-MS, but the abundance ratios between these two methods are different. In addition to that, the discrimination of various thymus species by using VaporPrint image based on GC/SAW provides a quite reliable result. On the basis of principal component analysis (PCA) results, the ability for classification among species of completely different chemotypes by HS-SPME-GC-MS is good enough, but the classification of same chemotypes species which are from different geographical origin in same country, original species and its variety, an air-drying term for 13 days and 16 months appear much lower than GC/SAW. Interestingly, the present experiment reveals that the air-drying term influences the aroma composition: the concentration of the pharmacologically active species, monoterpene phenol (thymol), reaches its highest concentrations after it was dried for 5 days or 13 days, which is much higher than in fresh or over-dried for a long times.

Comparison of the volatile composition in thyme honeys from several origins in Greece.

Thyme honey is the most appreciated unifloral Greek honey in Greece as well as around the world. In an effort to investigate the headspace composition of this type of honey, 28 samples were analyzed by means of solid-phase microextraction coupled to a gas chromatography-mass spectrometry system. The botanical origin of the samples was ascertained by pollen analysis, and samples displayed relative frequencies of thyme pollen between 18 and 41%. A total of 62 compounds were isolated, and phenylacetaldehyde was the most abundant (32.9% of the total peak area). Possible botanical markers are 1-phenyl-2,3-butanedione (13.4%), 3-hydroxy-4-phenyl-2-butanone, 3-hydroxy-1-phenyl-2-butanone (14.7%), phenylacetonitrile (4.8%), and carvacrol (0.9%), since these compounds are found only in thyme honey. Additionally, high proportions of phenylacetaldehyde are also characteristic ( F = 12.282, p < 0.001). The average concentrations of seven compounds were significantly different ( p < 0.05), namely phenylacetaldehyde, acetophenone, octanoic acid, carvacrol, phenylethyl alcohol, nonanal, and hexadecane. Applying principal component analysis to the data, six components were extracted, explaining 85.4% of the total variance. The first component explained 46.2% of the total variance and was positively correlated to phenylacetaldehyde, nonanoic acid, acetophenone, decanoic acid, benzaldehyde, phenylacetonitrile, isophorone, and nonanal. The extracted components were used as variables to the discriminant analysis, which showed good discrimination, especially for samples from Crete. A leave-one-out classification showed 85.7% of cross-validated grouped cases correctly classified. These results are promising to establish a discrimination model for these geographical regions. This is crucial for local beekeeper corporations on their effort to produce honey with geographical origin label.