Pinus halepensis, Pinus pinaster, Pinus pinea and Pinus sylvestris Essential Oils Chemotypes and Monoterpene Hydrocarbon Enantiomers, before and after Inoculation with the Pinewood Nematode Bursaphelenchus xylophilus.

Pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease, a serious threat to global forest populations of conifers, especially Pinus spp. A time-course study of the essential oils (EOs) of 2-year-old Pinus halepensis, Pinus pinaster, Pinus pinea and Pinus sylvestris following inoculation with the PWN was performed. The constitutive and nematode inoculation induced EOs components were analyzed at both the wounding or inoculation areas and at the whole plant level. The enantiomeric ratio of optically active main EOs components was also evaluated. External symptoms of infection were observed only in P. pinaster and P. sylvestris 21 and 15 days after inoculation, respectively. The EO composition analysis of uninoculated and unwounded plants revealed the occurrence of chemotypes for P. pinaster, P. halepensis and P. sylvestris, whereas P. pinea showed a homogenous EO composition. When whole plants were evaluated for EO and monoterpene hydrocarbon enantiomeric chemical composition, no relevant qualitative and quantitative differences were found. Instead, EO analysis of inoculated and uninoculated wounded areas revealed an increase of sesquiterpenes and diterpenic compounds, especially in P. pinea and P. halepensis, comparatively to healthy whole plants EOs.

Volatile-oils composition, and bioactivity of the essential oils of Plectranthus barbatus, P. neochilus, and P. ornatus grown in Portugal.

Volatile-oils chemical composition and bioactivity of the essentail oils from Plectranthus barbatus, P. neochilus, and P. ornatus (Lamiaceae) were assessed. Aerial parts from these three related Plectranthus species were collected from cultivated plants grown in Portugal, during vegetative and flowering phases. Volatiles, isolated by distillation-extraction, were analyzed by GC and GC/MS. Monoterpene hydrocarbons (12-74%) and sesquiterpene hydrocarbons (4-45%) constituted the main fractions in all volatiles. α-Pinene (3; 12-67%), oct-1-en-3-ol (6; traces-28%), ß-pinene (7; 0.1-22%), and ß-caryophyllene (50; 7-12%) dominated P. barbatus volatiles. P. neochilus major volatile components were α-terpenyl acetate (41; traces-48%), α-thujone (2; 2-28%), ß-caryophyllene (50; 2-28%), ß-pinene (7; 1-25%), and α-pinene (3; 1-19%). Oct-1-en-3-ol (6; 13-31%), ß-pinene (7; 11-24%), α-pinene (3; 11-19%), and ß-caryophyllene (50; traces-11%) were the main constituents from P. ornatus volatiles. These chemical compositions were rather different from those previously found for specimens harvested in Africa and Brazil. Moreover, the volatiles from the flowers are herewith reported for the first time. Essential oils, isolated by hydrodistillation from leaves and stems, showed a yellowish color and unpleasant odor, with yields ranging from 0.08% to 0.84% (v/dry weight). Antioxidant and antimicrobial activities of the essential oils were evaluated by DPPH· and TBARS assays, and agar disc-diffusion method, respectively. Results showed low or moderate antioxidant capacity and significant antimicrobial activity against Gram-positive bacteria.

Genomic characterization, molecular cloning and expression analysis of two terpene synthases from Thymus caespititius (Lamiaceae).

The identification, isolation and functional characterization of two genes encoding two monoterpene synthases-γ-terpinene synthase (Tctps2) and α-terpineol synthase (Tctps5)-from three chemically distinct Thymus caespititius (Lamiaceae) genotypes were performed. Genomic exon-intron structure was also determined for both terpene synthase genes, revealing an organization with seven exons and six introns. The cDNA of Tctps2 was 2,308 bp long and had an open reading frame of 1,794 bp encoding for a protein with 598 amino acids. Tctps5 was longer, mainly due to intron sequences, and presented high intraspecific variability on the plants analyzed. It encoded for a protein of 602 amino acids from an open reading frame of 1,806 bp comprising a total of 2,507 bp genomic sequence. The amino acid sequence of these two active Tctps genes shared 74 % pairwise identity, ranging between 42 and 94 % similarity with about 50 known terpene synthases of other Lamiaceae species. Gene expression revealed a multi-product Tctps2 and Tctps5 enzymes, producing γ-terpinene and α-terpineol as major components, respectively. These enzymatic results were consistent with the monoterpene profile present in T. caespititius field plants, suggesting a transcriptional regulation in leaves. Herewith reported for the first time for this species, these two newly characterized Tctps genes improve the understanding of the molecular mechanisms of reaction responsible for terpene biosynthesis and chemical diversity found in T. caespititius.

Bioassays against pinewood nematode: assessment of a suitable dilution agent and screening for bioactive essential oils.

Acetone was investigated and found to be an appropriate alternative to Triton X-100 as a solvent of essential oils in bioassays aimed to investigate their effects on pinewood nematode (Bursaphelenchus xylophilus) mortality. Therefore it was used as dilution agent to screen the effectiveness of fifty two essential oils against this pest. Thirteen essential oils were highly effective, resulting in more than 90% pinewood nematode mortality at 2 mg/mL, with six of them resulting in 100% mortality. LC100 values ranged between 0.50 mg/mL and 0.83 mg/mL for the essential oils of Origanum vulgare and Satureja montana, respectively. Essential oils were submitted to gas chromatography and gas chromatography-mass spectrometry analysis and their chemical composition established. Data from essential oils with 100% mortality at 2 mg/mL and other essential oils previously found to have LC100 ≤ 2 mg/mL was combined, their chemical profiles investigated by correspondences analysis plus automatic classification.

Composition and antioxidant activity of Thymus vulgaris volatiles: comparison between supercritical fluid extraction and hydrodistillation.

Supercritical fluid extraction (SFE) of the volatile oil from Thymus vulgaris L. aerial flowering parts was performed under different conditions of pressure, temperature, mean particle size and CO(2) flow rate and the correspondent yield and composition were compared with those of the essential oil isolated by hydrodistillation (HD). Both the oils were analyzed by GC and GC-MS and 52 components were identified. The main volatile components obtained were p-cymene (10.0-42.6% for SFE and 28.9-34.8% for HD), gamma-terpinene (0.8-6.9% for SFE and 5.1-7.0% for HD), linalool (2.3-5.3% for SFE and 2.8-3.1% for HD), thymol (19.5-40.8% for SFE and 35.4-41.6% for HD), and carvacrol (1.4-3.1% for SFE and 2.6-3.1% for HD). The main difference was found to be the relative percentage of thymoquinone (not found in the essential oil) and carvacryl methyl ether (1.0-1.2% for HD versus t-0.4 for SFE) which can explain the higher antioxidant activity, assessed by Rancimat test, of the SFE volatiles when compared with HD. Thymoquinone is considered a strong antioxidant compound.

Effect of Monochamus galloprovincialis feeding on Pinus pinaster and Pinus pinea, oleoresin and insect volatiles.

In Portugal, the pine black sawyer Monochamus galloprovincialis is the principal vector of the pinewood nematode, Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), a lethal phyopathogen with major ecological and economic consequences to European forestry. The aim of this study was to determine the influence of M. galloprovincialis feeding on the volatiles emitted by pine trees. This study focused on the pine species which are most relevant to Portugal, that is, Pinus pinaster (maritime pine) and Pinus pinea (stone or umbrella pine), assessing to what extent pine chemotypes might influence feeding by the insect vector. Preliminary evaluation of each maritime pine essential oil allowed recognizing the existence of two main chemotypes (C1 and C2) and absence of chemical variability in P. pinea. Emission of volatiles from pine trees was evaluated before and during 24 h of feeding by a mixed-sex pair of newly emerged, unfed M. galloprovincialis. Volatiles were also collected from the oleoresin released from the feeding wounds as well as from the insects after feeding. Pine volatiles were collected by solid phase microextraction (SPME) and insect volatiles extracted with pentane, and all analysed by gas chromatography-mass spectrometry (GC-MS) and by GC for component identification and quantification, respectively. Of the seventeen emitted volatiles detected in SPME analyses of P. pinaster, ß-pinene, α-pinene, ß-caryophyllene, and germacrene D showed the highest average fold increases as a result of M. galloprovincialis feeding. When grouped by P. pinaster chemotype, C1 and C2 groups of trees showed different patterns of responses. ß-Caryophyllene and germacrene D showed the highest fold increase in C1 trees, whereas ß-pinene and α-pinene clearly dominated in C2 trees. Likewise, the oleoresin volatiles from C1 trees were dominated by δ-3-carene and/or ß-pinene, whereas α-pinene and ß-pinene were the main volatile components from oleoresin of C2 trees. Nine components were detected in P. pinea volatiles, of which limonene showed the highest fold increase as a result of insect feeding. The volatiles collected from the insects after they had fed on P. pinaster included α-pinene, ß-pinene, and abietic acid, and by the straight-chain n-alkanes n-C27, n-C29, and n-C25, together with the methyl-branched hydrocarbons 3-meC29, 2-meC28, and 3-meC27. A better understanding of the responses of different P. pinaster chemotypes to feeding by M. galloprovincialis may be helpful in the development of new lures to improve pine sawyer trapping in integrated pest management for control of PWD.

Enrichment of the thymoquinone content in volatile oil from Satureja montana using supercritical fluid extraction.

Supercritical fluid extraction (SFE) of the volatile oil from Satureja montana L. was performed under different conditions of pressure (90 and 100 bar), temperature (40 and 50 degrees C), mean particle sizes (0.4, 0.6 and 0.8 mm) and CO(2) flow rate (0.8, 1.1 and 1.3 kg/h) to understand the influence of these parameters on the composition and yield of this oil. The results were compared with those obtained for the essential oil isolated by hydrodistillation (HD). The volatile and the essential oil were analysed by GC and GC-MS. The main compounds are carvacrol (52.2-62.0% for HD vs. 41.7-64.5% for SFE), thymol (8.6-11.0% for HD vs. 6.0-11.3% for SFE), p-cymene (6.9-12.8% for HD vs. 6.0-17.8% for SFE), gamma-terpinene (6.4-9.4% for HD vs. 2.3-6.0% for SFE) and beta-bisabolene (2.0-2.7% for HD vs. 2.2-3.5% for SFE). The major difference between SFE and HD was the relative amount of thymoquinone, an oxygenated monoterpene with important biological activities, which can be ten-fold higher in volatile oil (1.6-3.0 for SFE vs. 0.2% for HD). The morphology of the glandular trichomes of S. montana and the effect of the grinding process on them was also evaluated by SEM.

Supercritical fluid extraction of the volatile oil from Santolina chamaecyparissus.

Supercritical fluid extraction (SFE) of the volatile oil from Santolina chamaecyparissus L. flower heads was performed under different conditions of pressure, temperature, mean particle size and CO(2) flow rate. This oil was compared with the essential oil isolated by hydrodistillation (HD). The SFE volatile and essential oils were analysed by GC and GC-MS. The range of the main volatile components obtained with HD and SFE were, respectively: 1,8-cineole (25-30% and 7-48%), camphor (7-9% and 8-14%), borneol (7-8% and 2-11%), terpinen-4-ol (6-7% and 1-4%), terpinolene (1-4% and 1-7%) and isobornyl acetate (1-2% and 1-11%). The chemical composition of the extracts was greatly influenced by the conditions of pressure and temperature used. In fact, it was possible to enrich the sesquiterpene fraction by increasing the pressure from 8 to 9 MPa, while changing the temperature from 50 to 40 degrees C at 9 MPa enriched the volatiles in n-alkanes [corrected].

Biotransformation of menthol and geraniol by hairy root cultures of Anethum graveolens: effect on growth and volatile components.

Two oxygen-containing monoterpene substrates, menthol or geraniol (25 mg l(-1)), were added to Anethum graveolens hairy root cultures to evaluate the influence of the biotransformation capacity on growth and production of volatile compounds. Growth was assessed by the dissimilation method and by fresh and dry weight measurement. The volatiles were analyzed by GC and GC-MS. The total constitutive volatile component was composed, in more than 50%, by falcarinol (17-52%), apiole (11-24%), palmitic acid (7-16%), linoleic acid (4-9%), myristicin (4-8%) and n-octanal (2-5%). Substrate addition had no negative influence on growth. The relative amount of menthol quickly decreased 48 h after addition, and the biotransformation product menthyl acetate was concomitantly formed. Likewise, the added geraniol quickly decreased over 48 h alongside with the production of the biotransformation products. The added geraniol was biotransformed in 10 new products, the alcohols linalool, alpha-terpineol and citronellol, the aldehydes neral and geranial, the esters citronellyl, neryl and geranyl acetates and linalool and nerol oxides.

Simple gas chromatographic method for the stereodifferentiation of methyl nilate, a chiral alpha-methyl-beta-hydroxy ester.

The four stereoisomers of methyl nilate, methyl 3-hydroxy-2-methylbutanoate, have been separated and analyzed by the direct simultaneous achiral-chiral high-resolution gas chromatography (HRGC) and also by the chiral high-resolution gas chromatography-time-of-flight mass spectrometry (HRGC-TOF-MS). The method involved the use of DB-5 and CP Chirasil-Dex CB columns. The elution sequences on the two columns were determined and are reported. The one-step GC method allows the identification by retention data and simultaneously to determine unequivocally the absolute configuration of nilic acid moieties contained in minor complex natural products without previous isolation, as part of a general strategy for the structure determination of the compounds.

Constituents of the essential oil of sea fennel (Crithmum maritimum L.) growing wild in Turkey.

The essential oils isolated by hydrodistillation from freshly obtained aerial parts of sea fennel (Crithmum maritimum L.) were found to be a yellow liquid, obtained in a yield of 0.2% (vol/wt) based on wet weight. The essential oils obtained from young leaves and branches of C. maritimum, collected at two different locations in Turkey (Sipahili and Yesilovacik), were analyzed by gas chromatography-mass spectrometry. Twenty-three components were identified accounting for more than 99% of the total oils. The oils contained mainly monoterpenes, amounting to 89.0% and 99.6%, respectively, of the oils, with gamma-terpinene (36% and 32%), beta-phellandrene (21% and 22%), and sabinene (13% and 9%) as the main components.

Antibacterial and antioxidant activities of essential oils isolated from Thymbra capitata L. (Cav.) andOriganum vulgare L.

Antilisterial activities of Thymbra capitata and Origanum vulgare essential oils were tested against 41 strains of Listeria monocytogenes. The oil of T. capitata was mainly constituted by one component, carvacrol (79%), whereas for O. vulgare three components constituted 70% of the oil, namely, thymol (33%), gamma-terpinene (26%), and p-cymene (11%). T. capitata essential oil had a significantly higher antilisterial activity in comparison to O. vulgare oil and chloramphenicol. No significant differences in L. monocytogenes susceptibilities to the essential oils tested were registered. The minimum inhibitory concentration values of T. capitata essential oil and of carvacrol were quite similar, ranging between 0.05 and 0.2 microL/mL. Antioxidant activity was also tested, the essential oil of T. capitata showing significantly higher antioxidant activity than that of O. vulgare. Use of T. capitata and O. vulgare essential oils can constitute a powerful tool in the control of L. monocytogenes in food and other industries.

Identification and characterization of a second isogene encoding γ-terpinene synthase in Thymus caespititius.

Thymus caespititius Brot. is an Iberian endemic species, whose essential oils possess high polymorphism. They consist mostly of mono- and sesquiterpene, some of them with interest for the pharmaceutical and food industries. The search for terpene synthase genes was performed in three in vitro T. caespititius genotypes. For these plants, the expression of a previously described γ-terpinene synthase gene, Tctps2, was confirmed, occurring concomitantly with a new gene encoding an enzyme with similar activity, named Thymus caespititius terpene synthase 4 (Tctps4). The two isogenes were isolated and functionally characterized in the three plant genotypes. Alignment of the two Tctps revealed a transit peptide much shorter in Tctps4 than in Tctps2 (3-4 amino acids instead of 47). The Tctps4 open reading frame is shorter than Tctps2 (1665 bp versus 1794 bp). The amino acid sequence of both γ-terpinene synthases shared an 88% pairwise identity. The fact that T. caespititius carries two isogenes for γ-terpinene synthases, suggests gene duplication along the evolutionary process, followed by mutations leading to the differentiation of both genes. These mutations didn't compromise protein activity. A high accumulation of transcripts from both genes was found in shoots of in vitro plantlets, while in roots they could not be detected. Still, γ-terpinene levels in aerial parts were reduced, probably due to fast conversion into carvacrol and thymol, the main components from T. caespititius essential oils. This study is a contribution to the identification of terpene synthase genes in Lamiaceae.

Intraspecific variability of the essential oil of Cladanthus mixtus from Morocco.

Air-dried aerial parts of wild Cladanthus mixtus were collected from two different regions of Morocco, Bouznika and Oujda, during 2011 and 2012. Forty individual plant samples were hydrodistilled using a Clevenger apparatus and the obtained essential oils were analyzed by GC and GC/MS. The yield obtained varies greatly with a range of 0.3 to 0.8%. The chemical composition of C. mixtus oils changes from one region to another. A total of 53 constituents were identified. To the best of our knowledge, two chemotypes were defined for the first time for this species in the regions studied. 2-Methyl-2-trans-butenyl methacrylate (32.8-35.2%) / ar-curcumene (13-14%) characterize the chemotypeof the C. mixtus plants from Bouznika, and trans-beta-farnesene (35.5-50.3%) the chemotype from Oujda.

Propolis volatiles characterisation from acaricide-treated and -untreated beehives maintained at Algarve (Portugal).

The variability of the volatile profile of 70 propolis samples from acaricide-treated and -untreated beehives maintained at Algarve (Portugal) was evaluated. Propolis samples were collected in three regions of Algarve at three different periods. Cluster analysis based on the propolis volatiles' chemical composition defined two main clusters, not related to the time of year, collection site, altitude, temperature or humidity ranges, and was based mainly on the relative amounts of viridiflorol, n-tricosane and n-nonadecane for cluster I. Cluster II was mainly characterised by the high thymol content, followed by viridiflorol, n-tricosane and n-nonadecane. The presence of higher thymol levels in propolis samples from cluster II may reflect the long use of an acaricide with thymol as main active ingredient. All samples showed an intense rock-rose aroma supported by the presence of characteristic Cistus and labdanum oil volatile components. Given the nowadays frequent propolis household use, volatiles thorough characterisation may assist in its quality assessment.

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.

Herbicidal activity of volatiles from coriander, winter savory, cotton lavender, and thyme isolated by hydrodistillation and supercritical fluid extraction.

The volatiles from Coriandrum sativum L., Satureja montana L., Santolina chamaecyparissus L., and Thymus vulgaris L. were isolated by hydrodistillation (essential oil) and supercritical fluid extraction (volatile oil). Their effect on seed germination and root and shoot growth of the surviving seedlings of four crops ( Zea mays L., Triticum durum L., Pisum sativum L., and Lactuca sativa L.) and two weeds ( Portulaca oleracea L. and Vicia sativa L.) was investigated and compared with those of two synthetic herbicides, Agrocide and Prowl. The volatile oils of thyme and cotton lavender seemed to be promising alternatives to the synthetic herbicides because they were the least injurious to the crop species. The essential oil of winter savory, on the other hand, affected both crop and weeds and can be appropriate for uncultivated fields.

Foeniculum vulgare essential oils: chemical composition, antioxidant and antimicrobial activities.

The essential oils from Foeniculum vulgare commercial aerial parts and fruits were isolated by hydrodistillation, with different distillation times (30 min, 1 h, 2 h and 3 h), and analyzed by GC and GC-MS. The antioxidant ability was estimated using four distinct methods. Antibacterial activity was determined by the agar diffusion method. Remarkable differences, and worrying from the quality and safety point of view, were detected in the essential oils. trans-Anethole (31-36%), alpha-pinene (14-20%) and limonene (11-13%) were the main components of the essentials oil isolated from F. vulgare dried aerial parts, whereas methyl chavicol (= estragole) (79-88%) was dominant in the fruit oils. With the DPPH method the plant oils showed better antioxidant activity than the fruits oils. With the TBARS method and at higher concentrations, fennel essential oils showed a pro-oxidant activity. None of the oils showed a hydroxyl radical scavenging capacity > 50%, but they showed an ability to inhibit 5-lipoxygenase. The essential oils showed a very low antimicrobial activity. In general, the essential oils isolated during 2 h were as effective, from the biological activity point of view, as those isolated during 3 h.

Menthol and geraniol biotransformation and glycosylation capacity of Levisticum officinale hairy roots.

The biotransformation capacity of Levisticum officinale W.D.J. Koch hairy root cultures was studied by evaluating the effect of the addition of 25 mg/L menthol or geraniol on morphology, growth, and volatiles production. L. officinale hairy root cultures were maintained for 7 weeks in SH medium, in darkness at 24 degrees C and 80 r.p.m., and the substrates were added 15 days after inoculation. Growth was evaluated by measuring fresh and dry weight and by using the dissimilation method. Volatiles composition was analyzed by GC and GC-MS. Hairy roots morphology and growth were not influenced by substrate addition. No new volatiles were detected after menthol addition and, as was also the case with the control cultures, volatiles of these hairy roots were dominated by (Z)-falcarinol (1-45%), N-octanal (3-8%), palmitic acid (3-10%), and (Z)-ligustilide (2-9%). The addition of geraniol induced the production of six new volatiles: nerol/citronellol/neral (traces-15%), alpha-terpineol (0.2-3%), linalool (0.1-1.2%), and geranyl acetate (traces-2%). The relative amounts of the substrates and some of their biotransformation products decreased during the course of the experiment. Following the addition of beta-glycosidase to the remaining distillation water, analysis of the extracted volatiles showed that lovage hairy roots were able to convert both substrates and their biotransformation products into glycosidic forms. GC:gas chromatography GC-MS:gas chromatography-mass spectrometry SH:Schenk and Hildebrandt (1972) culture medium.

Nitrogen stress induction on Levisticum officinale hairy roots grown in darkness and under photoperiod conditions: effect on growth and volatile components.

Six-year-old Levisticum officinale (lovage) hairy root cultures were used to study the effect of eight different NH(4) (+):NO(3) (-) ratios on their growth and volatile components. All cultures were kept at 24 degrees C on orbital shakers at 80 rpm, in darkness or in a 16 h light/8 h dark photoperiod. Growth was evaluated by dry and fresh weight determination. The volatiles were isolated by distillation-extraction and analysed by GC and GC-MS. Greater growth was attained in darkness with 10:90 (control, SH medium), 50:50 and 25:75 NH(4) (+):NO(3) (-) ratios, and also with SH control medium under the photoperiod condition, with a 10, 14, 12.5 and 12.5 fold increase of biomass in terms of dry weight, respectively, at the end of 42 days of growth. UPGMA cluster analysis of the mixtures of volatiles isolated from the hairy roots grown with different NH(4) (+):NO(3) (-) ratios confirmed their chemical variability. Although no particular grouping was detected in relation to the NH(4) (+):NO(3) (-) ratios or light conditions studied, most of the mixtures of volatiles isolated from the hairy roots were either dominated by n-octanal, (Z)-falcarinol or both components in about the same relative amounts.