µ-Raman Determination of Essential Oils' Constituents from Distillates and Leaf Glands of Origanum Plants.

A novel, inexpensive and simple experimental setup for collecting µ-Raman spectra of volatile liquids in very small quantities was developed. It takes advantage of capillary forces to detain minute volatile liquid volumes. Spectra of volatile and even scattering or absorbing media can be measured more effectively. The method is used to facilitate the collection of intensity-consistent Raman spectra from a series of reference compounds present in Origanum essential oils, in order to quantify their constituents by multiple linear regression. Wild grown Origanum plants, collected from five different regions in Greece and taxonomically identified as O. onites, O. vulgare subsp. hirtum and O. vulgare subsp. vulgare, were appropriately distilled to acquire their essential oils. Comparison of the Raman results with those from headspace gas chromatography-mass spectrometry (HS GC-MS) confirmed the successful relative quantification of the most abundant essential oil constituents, highlighting the similarities and differences of the three Origanum taxa examined. Finally, it is demonstrated that directly measuring the leaf peltate glandular hairs yields exploitable results to identify the main components of the essential oil they contain, underlining the potential of in situ (field or industry) measurements utilizing microscope-equipped portable Raman spectrometers.

Unravelling the Chemotaxonomic Identity of "White" and "Black" Oregano (Origanum vulgare) in Northern Greece.

The two types of oregano used by the inhabitants of the villages of Μount Belles (GR1260001), the "white" oregano with white flowers and "black" oregano with purple flowers, were studied. The two oregano types were collected from five localities along an altitudinal gradient from 217 m up to 1299 m. "White" oregano, was found in the three lowland regions (up to 752 m) where the Pannonian-Balkanic turkey oak-sessile oak forest habitat (code 91M0) dominates. The "black" oregano was collected from the two higher altitudes, at 1177 m and 1299 m, where the Asperulo-Fagetum beech forest habitat (9130) occurs. Measurements of the density and size of peltate glandular hairs (sessile glands) on calyces, bracts, and leaves suggest that "white" oregano is distinguished by its conspicuous - apparently larger - glands. These differences were reflected in the total essential oil content, with the "white" oregano being much richer (up to 4.3 mL/100 g dry weight) than the "black" (up to 0.6%). Striking differences have also been found in the volatile fraction of their essential oil composition. The "white" oregano oils were characterized by the high content of carvacrol (up to 92.6% of identified peaks, by Headspace GC-MS). On the other hand, the two "black" oregano oils have a different aromatic profile, first reported from Greece, with main components including the sesquiterpenes ß-caryophyllene, D-germacrene, δ-cadinene and ß-bisabolene. The results so far indicate that "white" and "black" oregano, Origanum vulgare subsp. hirtum and subsp. vulgare respectively, can be clearly distinguished either by their morphological (glandular) differences or by chemical (essential oil) composition.

Diagnostic Potential of FT-IR Fingerprinting in Botanical Origin Evaluation of Laurus nobilis L. Essential Oil is Supported by GC-FID-MS Data.

The last years, non-targeted fingerprinting by Fourier-transform infrared (FT-IR) spectroscopy has gained popularity as an alternative to classical gas chromatography (GC)-based methods because it may allow fast, green, non-destructive and cost-effective assessment of quality of essential oils (EOs) from single plant species. As the relevant studies for Laurus nobilis L. (bay laurel) EO are limited, the present one aimed at exploring the diagnostic potential of FT-IR fingerprinting for the identification of its botanical integrity. A reference spectroscopic dataset of 97 bay laurel EOs containing meaningful information about the intra-species variation was developed via principal component analysis (PCA). This dataset was used to train a one-class model via soft independent modelling class analogy (SIMCA). The model was challenged against commercial bay laurel and non-bay laurel EOs of non-traceable production history. Overall, the diagnostic importance of spectral bands at 3060, 1380-1360, 1150 and 1138 cm-1 was assessed using GC-FID-MS data. The findings support the introduction of FT-IR as a green analytical technique in the quality control of these often mislabeled and/or adulterated precious products. Continuous evaluation of the model performance against newly acquired authentic EOs from all producing regions is needed to ensure validity over time.

Chemical and genetic characterization of Phlomis species and wild hybrids in Crete.

The genus Phlomis is represented in the island of Crete (Greece, Eastern Mediterranean) by three species Phlomis cretica C. Presl., Phlomis fruticosa L., the island endemic Phlomis lanata Willd. and three hybrids Phlomis x cytherea Rech.f. (P. cretica x P. fruticosa), Phlomis x commixta Rech.f. (P. cretica x P. lanata) and Phlomis x sieberi Vierh. (P. fruticosa x P. lanata). This work describes (a) the profile of hybrids and parental species concerning their volatile compounds, (b) the suitability of ribosomal nuclear (ITS region), chloroplast (trnH-psbA), and AFLP markers to identify hybrids and (c) their competence to characterize the different chemotypes of both hybrids and their parental species. The cluster analysis and PCA constructed from chemical data (volatile oils) suggest that there are three groups of taxa. Group IA includes P. cretica and P. fruticosa, group IB includes P. x cytherea, whereas group II consists of P. x commixta, P. x sieberi and P. lanata. Volatile compounds detected only in the hybrids P. x sieberi and P. x commixta correspond to the 3% of the total compounds, value that is much higher in P. x cytherea (21%). Neighbor-joining, statistical parsimony analysis and the observations drawn from ribotypes spectrum of ITS markers divided Phlomis species in two groups, P. lanata and the complex P. cretica/P. fruticosa. In contrast to the ITS region, the plastid DNA marker follows a geographically related pattern. Neighbor-Net, PCA and Bayesian assignment analysis performed for AFLP markers separated the genotypes into three groups corresponding to populations of P. cretica, P. fruticosa, and P. lanata, respectively, while populations of P. x commixta, P. x cytherea, and P. x sieberi presented admixed ancestry. Most of the P. x cytherea samples were identified as F1 hybrids by Bayesian assignment test, while those of P. x commixta and P. x sieberi were identified as F2 hybrids. Overall, high chemical differentiation is revealed in one of the three hybrids, which is likely related with niche variation. Moreover, molecular markers show potential to identify Phlomis taxa.

DNA barcoding in native plants of the Labiatae (Lamiaceae) family from Chios Island (Greece) and the adjacent Çesme-Karaburun Peninsula (Turkey).

The plant family Labiatae (Lamiaceae) is known for its fine medicinal and aromatic herbs like lavender, mint, oregano, sage and thyme and is a rich source of essential oils for the food, pharmaceutical and cosmetic industry. Besides its great economic importance, the Labiatae family contributes significantly to the endemic flora of Greece and Turkey. Owing to its economic and biological significance and to the difficult identification based on morphological characters of several of its taxa, the Labiatae family is an ideal case for developing DNA barcodes. The purpose of this study is to evaluate the utility of DNA barcoding on a local scale in discriminating Labiatae species in Chios Island (Greece) and the adjacent Çesme-Karaburun Peninsula (Turkey). We chose three cpDNA regions (matK, rbcL, trnH-psbA) that were proposed by previous studies and tested them either as single region or as multiregion barcodes based on the criteria determined by Consortium for the Barcode of Life (CBOL). Our results show that matK and trnH-psbA taken as useful in discriminating species of the Labiatae, for the species we examined, as any multiregion combination. matK and trnH-psbA could serve as single-region barcodes for Labiatae species contributing to the conservation and the trade control of valuable plant resources.

The essential oils of the Greek endemic Satureja horvatii ssp. macrophylla in relation to bioclimate.

The essential oils of the Greek endemic Satureja horvatii subsp. macrophylla collected from 36 areas (among them, 23 are included in twelve sites of the EU network NATURA 2000) belonging to different bioclimatic types were studied. The total essential-oil content is negatively related to the altitude. The variation of the essential oil composition follows a geographical pattern, which is related to the bioclimatic belts along the taxon's range. Carvacrol dominates in areas with Mediterranean and Submediterranean bioclimate (mainly in the S and C part of the taxon's distribution), linalool or trans-sabinene hydrate, and/or borneol in the Submediterranean or Temperate Axeric bioclimates (in the N part of distribution), whereas thymol is found as main oil constituent in all three bioclimates.

"Mints", smells and traditional uses in Thessaloniki (Greece) and other Mediterranean countries.

The herbs of the "mint" group traded in the herbal market of Thessaloniki include eight taxa, members of two genera, Acinos (two species) and Mentha (four species and two hybrids). The essential oil content of 72 samples examined ranged from traces up to 1.69ml/100g of dry weight. Besides three almost scentless samples, the different "mints" are distinguished according to their prominent smell differences, i.e. samples with a pungent, musty and sweet type of smell. As a result, the commercial names attributed to them correspond to a particular type of smell and not to a particular taxon. A number of 29 medicinal uses were recorded in total. In most cases uses were not associated with particular taxa but were rather determined by plant smells. A literature survey has shown that the "mints" traded in Thessaloniki are also used as herbal medicines all over the Mediterranean area, with 67 different therapeutic uses. Among them the 22 uses, already mentioned by Dioscurides, show that the utilization of "mints" as herbal medicines in the Mediterranean countries has a long tradition.

Essential oil composition is related to the natural habitats: Coridothymus capitatus and Satureja thymbra in NATURA 2000 sites of Crete.

The study of essential oils obtained from Coridothymus capitatus and Satureja thymbra collected from different natural habitat types of 11 NATURA 2000 sites scattered all over Crete has shown that they are characterized either by a high amount of carvacrol (up to 75.7%) or thymol (up to 65.6%) or by a more or less equal amount of the two phenols. The results of a discriminant analysis with predefined groups the natural habitat types wherefrom the plants were collected have shown that the oils of both species collected from the dry dwarf-shrub formations of the lowland have a high carvacrol content whereas those collected from the more mesic timber or highland formations have a high thymol content. Furthermore, the results of this study introduce the use of natural habitat unit as a tool for the assessment of essential oil variation.