Panos V. Petrakis

Volatile constituents of needles of five Pinus species grown in Greece

Abstract The chemical composition of the volatile metabolites from needles of Pinus halepensis Miller, P. brutia Tenore, P. nigra Arnold, P. pinea Linnaeus and P. canariensis Sweet and Sprengel, grown in natural habitats in Attiki, Greece, was analysed. The variability and chemotaxonomic importance of the terpenoid constituents are discussed.

Geographical Characterization of Greek Virgin Olive Oils (Cv. Koroneiki) Using 1H and 31P NMR Fingerprinting with Canonical Discriminant Analysis and Classification Binary Trees

This work deals with the prediction of the geographical origin of monovarietal virgin olive oil (cv. Koroneiki) samples from three regions of southern Greece, namely, Peloponnesus, Crete, and Zakynthos, and collected in five harvesting years (2001-2006). All samples were chemically analyzed by means of 1H and 31P NMR spectroscopy and characterized according to their content in fatty acids, phenolics, diacylglycerols, total free sterols, free acidity, and iodine number. Biostatistical analysis showed that the fruiting pattern of the olive tree complicates the geographical separation of oil samples and the selection of significant chemical compounds. In this way the inclusion of the harvesting year improved the classification of samples, but increased the dimensionality of the data. Discriminant analysis showed that the geographical prediction at the level of three regions is very high (87%) and becomes (74%) when we pass to the thinner level of six sites (Chania, Sitia, and Heraklion in Crete; Lakonia and Messinia in Peloponnesus; Zakynthos). The use of classification and binary trees made possible the construction of a geographical prediction algorithm for unknown samples in a self-improvement fashion, which can be readily extended to other varieties and areas.

Detection of refined olive oil adulteration with refined hazelnut oil by employing NMR spectroscopy and multivariate statistical analysis

NMR spectroscopy was employed for the detection of adulteration of refined olive oil with refined hazelnut oil. Fatty acids and iodine number were determined by (1)H NMR, whereas (31)P NMR was used for the quantification of minor compounds including phenolic compounds, diacylglycerols, sterols, and free fatty acids (free acidity). Classification of the refined oils based on their fatty acids content and the concentration of their minor compounds was achieved by using the forward stepwise canonical discriminant analysis (CDA) and the classification binary trees (CBTs). Both methods provided good discrimination between the refined hazelnut and olive oils. Different admixtures of refined olive oils with refined hazelnut oils were prepared and analyzed by (1)H NMR and (31)P NMR spectroscopy. Subsequent application of CDA to the NMR data allowed the detection of the presence of refined hazelnut oils in refined olive oils at percentages higher than 5%. Application of the non-linear classification method of the binary trees offered better possibilities of measuring adulteration of the refined olive oils at a lower limit of detection than that obtained by the CDA method.

Influence of harvest year, cultivar and geographical origin on Greek extra virgin olive oils composition: a study by NMR spectroscopy and biometric analysis.

Two hundred twenty-one extra virgin olive oils (EVOO) were extracted from four olive mono-cultivars (Koroneiki, Tsounati, Adramitini, and Throubolia) originated from four divisions of Greece (Peloponnesus, Crete, Zakynthos, and Lesvos) and collected in five harvesting periods (2002-2006 and 2007-2008). All samples were chemically analysed by means of (1)H and (31)P NMR spectroscopy and characterised according to their content in fatty acids, phenolic compounds, diacylglycerols, total free sterols, free acidity, and iodine number. The influence of cultivars on the compositional data of the EVOO samples according to harvest year and geographical origin was examined by means of the forward stepwise canonical discriminant analysis (CDA) and classification binary trees (CBT). The CDA, when the a priori grouping was in accordance with harvest, was high (94%), whereas the classification in terms of groups formed by inclusions of geographical origin was reduced to 85%. Inclusion of both the harvesting year and geographical origin in the CDA analysis resulted in a high classification (90%) for the EVOO samples grouped into the four cultivars. The variables that most satisfactorily classified the Greek olive oils were the phenolics p-coumaric acid, pinoresinol, 1-acetoxypinoresinol, syringaresinol, luteolin, apigenin, and the hydrolysis products of oleuropein expressed collectively by the concentration of total hydroxytyrosol. Amongst the fatty acids, linoleic acid was the predictor with the highest discriminatory power. Finally, the phylogenetic significance of the olive oil compounds as determined by NMR was investigated by estimating their support to monophyly of cultivars.

Iridoid glucosides with insecticidal activity from Galium melanantherum.

The insecticidal activity of the endemic species Galium melanantherum was evaluated against Crematogaster scutellaris ants and Kalotermes flavicollis termites. Iridoid glucosides 1-7 were isolated for the first time as metabolites of the investigated plant, along with the coumarin scopolin. The main components of the extract were found to be the non-acetylated iridoids: geniposidic acid (1), 10-hydroxyloganin (2), deacetyldaphylloside (3), monotropein (4), deacetylasperulosidic acid (5) and scandoside (6), while asperulosidic acid (7) was present only in minute quantities. All isolated metabolites were identified on the basis of their spectral data. Laboratory bioassays revealed significant levels of toxicity for 1-4 against Kalotermes flavicollis termites and Crematogaster scutellaris ants

Seasonal Variation of Oleoresin Terpenoids from Pinus halepensis and Pinus pinea and Host Selection of the Scale Insect Marchalina hellenica (Homoptera, Coccoidea, Margarodidae, Coelostonidiinae)

Summary Seasonal variation of the volatile terpenoids of Pinus pinea Ten and Pinus halepensis Mill, infested and uninfested by the caterpillar Marchalina hellenica, was followed by GC and GC-MS analyses of the pines cortical oleoresin. α-Pinene was found to be the dominant monoterpene in P. halepensis, while in P. pinea limonene was the most abundant compound. A significant decrease in the number of identified volatiles from winter to summer was observed and was more pronounced in the minor metabolites. Variation of the terpenoids according to the depth of the draining (drilling) holes in the trees was also determined. In addition, terpenoids were correlated with the results of cross feeding experiments designed for the study of the host preference of M. hellenica. All results revealed the dependence of M. hellenica on the secondary chemistry of the host. P. pinea, having a high content of limonene, appears to be more resistant to the caterpillar. Canonical discriminant analysis, in the discriminant space of the relative contribution of the ten major terpenes, separated P. pinea from P. halepensis, and infested from uninfested P. halepensis trees.

Biological control of mosquito populations: An applied aspect of pest control by means of natural enemies

Mosquitoes were recognized as a health and nuisance problem only in the last century. Since mosquito oviposition sites were initially associated with the expansion of human settlements, which as a rule is done by building actions of poor economies, the biological control became a necessity. Even with the development of chemical industry, the preparation of synthetic formulations of insecticides is environmentally hostile and ecologically unsafe since the main side effect of the application is the extinction of natural enemies of mosquitoes such as odonates, beetles, fishes and hemipterans in water pools apart from the induced resistance in a short time. The advancement of biological knowledge made available many new controlling methods of mosquito populations though substantially more expensive than synthetic insecticides. The most important of them is the set of semiochemicals (natural products) associated with the classical biological control by means of predators. Many investigations have proven that predators are able to control mosquitoes in ecosystems of variable size, nutrients and prey densities. Semiochemicals are employed in many aspects predation. Prey detection, oviposition site selection, chemical crypsis, kairomonal confusion is among the ways that predators and their prey mosquitoes are using to affect the outcome of predation. Microbials as killing agents, are extensively used in projects for mosquito control in order to minimize the environmental side effects of insecticides. However, they fail to eradicate pest populations. In the present report is described an integrated system for the control of mosquitoes from experience gained in Mediterranean areas of application. The control system is able to incorporate any future developments in mosquito population management such as new introductions or releases from rearing of predators, new genetic methods or inexpensive repellents and oviposition deterrents.

MONOTERPENOID DIVERSITY IN RELATION TO MORPHOLOGY OF PINUS BRUTIA AND PINUS HALEPENSIS IN AN EAST MEDITERRANEAN AREA (ATTIKI, GREECE): IMPLICATIONS FOR PINE EVOLUTION

Pinus halepensis and P. brutia hybridize extensively. The analysis of monoterpenoid and morphological variability of 122 pine trees at five sites showed that the within-site heterogeneity is high while the entire provenance diversity is comparable with that of a circum-Mediterranean scale. Four morphotypes and four chemotypes were recognized. The congruence between morphological and monoterpenoid classification of pines was high (coefficient of contingency 89%) while thirteen family groups of genetically closely related trees were found by means of a specifically designed sampling scheme of continuous classification – identification and re-sampling. Putative hybrids contributed substantially to the observed diversity and there is considerable heterogeneity between sites. Low-altitude sites, presumably susceptible to sea-level fluctuations, alluvial deposition and human influence, are in general more diverse at both levels of genetic complexity than inland sites. The hybrid phenotypes were not merely intermediate forms of parental taxa, and in a multivariate sense they are located on a parabola in the space of morphological properties. The arrangement of hybrids in the monoterpenoid space did not reveal any clear-cut pattern except that three major composite axes can summarize 98.9% of the entire monoterpenoid variation. The observed pattern of variation was interpreted as a result of waves of introgressive influence of eastern P. brutia population on P. halepensis , which was caused by marine transgressions in Pliocene and eustatic sea-level fluctuations in Pleistocene. The human influence on this introgression accelerated the process, and amplified the resulted pattern, although it blurred in many instances the biogeographic routes of germplasm mixing. Its bearing on the evolution of pines is discussed, and it was found that the ‘Eocene refugia hypothesis’ of C.I. Millar holds by analogy also in central Aegean pines.

Foliar and cortex oleoresin variability of silver fir (Abies alba Mill.) in Albania.

Terpene composition of needle and cortical oleoresin from lateral shoots were analyzed by GC/MS for four Silver fir (Abies alba Mill.) populations scattered in natural species range in Albania. More than sixty compounds were detected in the needle oleoresin, which was characterized by a high content of α-pinene, camphene, β-pinene, limonene and bornyl acetate. Three monoterpenes, α-pinene, β-pinene and limonene, and two sesquiterpenes, β-caryophyllene and germacrene D, comprised the majority of cortical oleoresin. The terpene composition differences among the populations that led to the recognition of two chemo-types. The needle oleoresin from the provinces of Puka, Bulqiza and Llogara were characterised by high amounts of β-pinene, camphene and α-pinene and low amounts of limonene, while that from Drenova had high amounts of β-pinene and limonene. A similar pattern was found in the cortical oleoresin with the exception of camphene that was a minor contributor. Geographical and seasonal variation between the populations was, also, investigated. Multivariate analysis of both needle and cortical oleoresin separated Drenova (southeastern population) from the other sites. When both major monoterpenes and sesquiterpenes were considered four chemical profiles could be attributed. Based on their chemical profiles, the populations can be divided into two groups: Populations with high content of β-pinene and α-pinene but a low content of limonene (Puka, Bulqiza and Llogara), typical of most of A. alba populations in all its distribution range. Population with a high content of limonene and a moderate content of β-pinene and α-pinene (Drenova)

Secondary Metabolites and Insecticidal Activity of Anemone pavonina

The insecticidal properties of the crude extracts of the leaves and flowers of Anemone pavonina were evaluated on Pheidole pallidula ants and showed significant levels of activity. Bioassay-guided fractionations led to the isolation of the butenolide ranunculin (1) as the active principle. Chemical investigations of the extracts showed them to contain as major components the sitosterol glycopyranoside lipids 2-5 and the glycerides 6-8. The structures of the metabolites were elucidated, following acetylation and hydrolysis of the natural products, by interpretation of their NMR and mass spectral data. The uncommon lipid metabolites 2-8 were isolated for the first time from the genus Anemone and this is the first report of insecticidal activity of the Anemone metabolite ranunculin against ants.