Activation and Stabilization of Olive Recombinant 13-Hydroperoxide Lyase Using Selected Additives.

The stabilization of olive recombinant hydroperoxide lyases (rHPLs) was investigated using selected chemical additives. Two rHPLs were studied: HPL full-length and HPL with its chloroplast transit peptide deleted (matured HPL). Both olive rHPLs are relatively stable at 4 °C, and enzyme activity can be preserved (about 100% of the rHPL activities are maintained) during 5 weeks of storage at -20 or at -80 °C in the presence of glycerol (10%, v/v). Among the additives used in this study, glycine (2.5% w/v), NaCl (0.5 M), and Na2SO4 (0.25 M) provided the highest activation of HPL full-length activity, while the best matured HPL activity was obtained with Na2SO4 (0.25 M) and NaCl (1 M). Although the inactivation rate constants (k) showed that these additives inactivate both rHPLs, their use is still relevant as they strongly increase HPL activity. Results of C6-aldehyde production assays also showed that glycine, NaCl, and Na2SO4 are appropriate additives and that NaCl appears to be the best additive, at least for hexanal production.

Chemical Diversity and Antimicrobial Activity of Volatile Compounds from Zanthoxylum zanthoxyloides Lam. according to Compound Classes, Plant Organs and Senegalese Sample Locations.

The chemical diversity of Zanthoxylum zanthoxyloides growing wild in Senegal was studied according to volatile compound classes, plant organs and sample locations. The composition of fruit essential oil was investigated using an original targeted approach based on the combination of gas chromatography (GC) and liquid chromatography (LC) both coupled with mass spectrometry (MS). The volatile composition of Z. zanthoxyloides fruits exhibited relative high amounts of hydrocarbon monoterpenes (24.3 - 55.8%) and non-terpenic oxygenated compounds (34.5 - 63.1%). The main components were (E)-ß-ocimene (12.1 - 39%), octyl acetate (11.6 - 21.8%) and decanol (9.7 - 15.4%). The GC and GC/MS profiling of fruit essential oils showed a chemical variability according to geographical locations of plant material. The LC/MS/MS analysis of fruit oils allowed the detection of seven coumarins in trace content. The chemical composition of fruit essential oils was compared with volatile fractions of leaves and barks (root and trunk) from the same plant station. Hexadecanoic acid, germacrene D and decanal were identified as the major constituents of leaves whereas the barks (root and trunk) were dominated by pellitorine (85.8% and 57%, respectively), an atypic linear compound with amide group. The fruit essential oil exhibited interesting antimicrobial activities against Staphylococcus aureus and Candida albicans, particularly the alcohol fraction of the oil.

Chemical Variability and Biological Activities of Essential Oils of Micromeria inodora (Desf.) Benth. from Algeria.

The chemical composition of the essential oils isolated from the aerial parts of Micromeria inodora (Desf.) Benth. collected in 24 Algerian localities was investigated from the first time using GC-FID, GC/MS and 13 C-NMR. Altogether, 83 components which accounted for 94.7% of the total oil composition were identified. The main compounds were trans-sesquisabinene hydrate (1; 20.9%), α-terpinyl acetate (2; 19.8%), globulol (3; 4.9%), caryophyllene oxide (4; 4.3%), ß-bisabolol (5; 2.9%) and trans-7-epi-sesquisabinene hydrate (6; 2.6%). Comparison with the literature highlighted the originality of the Algerian M. inodora oil and indicated that 1 might be used as taxonomical marker. The study of the chemical variability allowed the discrimination of two main clusters confirming that there is a relation between the essential-oil compositions and the soil nature of the harvest locations. Biological activity of M. inodora essential oil was assessed against fourteen species of microorganisms involved in nosocomial infections using paper disc diffusion and dilution agar assays. The in vitro study demonstrated a good activity against Gram-positive strains such as Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, and Enterococcus faecalis, and moderate activity against Candida albicans. These results might be useful for the future commercial valorization of M. inodora essential oil as a promising source of natural products with potential against various nosocomial community and toxinic infections.

Olive Recombinant Hydroperoxide Lyase, an Efficient Biocatalyst for Synthesis of Green Leaf Volatiles.

Volatile C6-aldehydes are the main contributors to the characteristic odor of plants known as "green note" and are widely used by the flavor industry. Biotechnological processes were developed to fulfill the high demand in C6-aldehydes in natural flavorants and odorants. Recombinant hydroperoxide lyases (HPLs) constitute an interesting alternative to overcome drawbacks arising from the use of HPL from plant extracts. Thus, olive recombinant 13-HPL was assayed as biocatalysts to produce C6-aldehydes. Firstly, a cDNA encoding for olive HPL of Leccino variety was isolated and cloned in pQE-30 expression vector. In order to improve the enzyme solubility, its chloroplast transit peptide was deleted. Both enzymes (HPL wild type and HPL deleted) were expressed into Escherichia coli strain M15, purified, characterized, and then used for bioconversion of 13-hydroperoxides of linoleic and linolenic acids. Aldehydes produced were extracted, then identified and quantified using gas chromatography and mass spectrometry. Recombinant HPL wild type (HPLwt) allowed producing 5.61 mM of hexanal and 4.39 mM of 3Z-hexenal, corresponding to high conversion yields of 93.5 and 73 %, respectively. Using HPL deleted (HPLdel) instead of HPLwt failed to obtain greater quantities of hexanal or 3Z-hexenal. No undesirable products were formed, and no isomerization of 3Z-hexenal in 2E-hexenal occurred. The olive recombinant HPLwt appears to be a promising efficient biocatalyst for the production of C6-aldehydes.

Chemical variability and antioxidant activity of Limbarda crithmoides L. essential oil from Corsica.

The chemical compositions of 25 Corsican Limbarda crithmoides ssp. longifolia essential oils were investigated for the first time using GC-FID, GC/MS, and NMR analyses. Altogether, 65 compounds were identified, accounting for 90.0-99.3% of the total oil compositions. The main components were p-cymene (1; 15.1-34.6%), 3-methoxy-p-cymenene (4; 11.8-28.5%), 2,5-dimethoxy-p-cymenene (5; 5.9-16.4%), thymol methyl ether (6; 1.3-14.9%), α-phellandrene (2; 0.9-11.9%), and α-pinene (3; 0.2-13.4%). The chemical variability of the Corsican oil samples was studied using multivariate statistical analysis, which allowed the discrimination of two main clusters. A direct correlation between the water salinities of the plant locations and the chemical compositions of the L. crithmoides essential oils was evidenced. Indeed, essential oils rich in 1 (30.4-34.6%) were found in samples growing in the wetlands of the southern oriental coast, which exhibit high salinity levels (24.4±0.2-33.9±0.2 ppt), and essential oils with lower contents of 1 (15.1-27.3%) were isolated form samples growing in the wetlands of northern Corsica, which exhibit lower salinity levels (10.90±0.20-15.47±0.15 ppt). The antioxidant potential of L. crithmoides essential oil was also investigated, by assessing the DPPH(.) - and ABTS(.+) -scavenging activities and the reducing power of ferric ions, and was found to be interesting. Moreover, using bioassay-guided fractionation of the essential oil, a higher antioxidant activity was obtained for the oxygenated fraction and both ester and alcohol subfractions.

Bicyclo[4.4.0]decane oxygenated sesquiterpenes from Eryngium maritimum essential oil.

Investigation of the essential oil of the aerial parts of Eryngium maritimum L. from Corsica led to the isolation of one known sesquiterpene (1) and three new oxygenated sesquiterpenes with a muurolane or cadinane skeleton (2-4). Structure assignments of 4 ßH -muurol-9-en-15-al (1), 4 ßH -cadin-9-en-15-al (2), 4 ßH -muurol-9-en-15-ol (3) and 4 ßH -cadin-9-en-15-ol (4) were carried out by GC/MS (EI and CI) and comprehensive 1D and 2D NMR spectroscopy. Antibacterial activity of Eryngium maritimum L. oil and column chromatography fractions have been investigated for the first time. The oxygenated fraction, which contains the four sesquiterpenes, was efficient against Listeria monocytogenes and Echerichia coli.

Chemical and genetic differentiation of Corsican subspecies of Teucrium flavum L.

Corsica Island exhibited the particularity to display Teucrium flavum subsp. glaucum and subsp. flavum on the same territory with the same bioclimatic conditions. For the first time, volatile components extracted from aerial parts and genetic diversity of both Corsican T. flavum L. subspecies have been investigated through (i) the characterization of the chemical composition of essential oils and (ii) the study of three polymorphic genetic markers. Chemical analysis were performed using combination of capillary GC/RI, GC-MS after fractionation on column chromatography and the definition of the genetic structure were carried out using two chlororoplast markers (RPL32-TRNL and TRNL-F) and ribosomal nuclear markers (ITS region). According to statistical analysis, both subspecies were clearly distinguished by the chemical and genetic studies. Chemical compositions of oils from both subspecies were qualitatively similar but they differed by the normalized% abundances of their major components; oils from subsp. flavum were dominated by large amounts of hydrocarbon monoterpenes while oils obtained from subsp. glaucum were characterized by higher amounts of oxygenated compounds. The genetic analysis divided T. flavum L. populations in two groups, the first displayed subsp. glaucum populations and the latter group exhibited subsp. flavum populations. The presence of two groups is weakly consistent with chemical differentiation. These data suggest that the differences in the volatile composition of the two T. flavum subspecies depends more on the genetic background and less on environmental factors.

Chemical composition and antioxidant activity of essential oils and solvent extracts of Ptychotis verticillata from Morocco.

The objective of this study was to characterize the chemical composition of the essential oil and extracts of Ptychotis verticillata. The antioxidative activities of this species were also evaluated to suggest it as a new potential source of natural antioxidants. Analysis of the chemical composition of P. verticillata essential oil from Morocco was carried out using GC and GC-MS. The oil was dominated by phenolic compounds (48.0%) with carvacrol (44.6%) and thymol (3.4%) as the main compounds. Plant phenolics constitute one of the major groups of components that act as primary antioxidant free radical terminators. The amounts of total phenolics and flavonoids in the solvent extracts (diethyl ether and ethyl acetate) were determined spectrometrically. Furthermore, the antioxidant activities of the essential oil and extracts were determined using a DPPH test system. The DPPH scavenging activity of extracts increased in the order ethyl acetate>ascorbic acid>diethyl ether>essential oil. Finally, a relationship was observed between the antioxidant activity potential and total phenolic and flavonoid levels of the extract.

Characterization of volatile compounds of Daucus crinitus Desf. Headspace Solid Phase Microextraction as alternative technique to Hydrodistillation.

BACKGROUND: Traditionally, the essential oil of aromatic herbs is obtained using hydrodistillation (HD). Because the emitted volatile fraction plays a fundamental role in a plant's life, various novel techniques have been developed for its extraction from plants. Among these, headspace solid phase microextraction (HS-SPME) can be used to obtain a rapid fingerprint of a plant's headspace. Daucus crinitus Desf. is a wild plant that grows along the west coast of Algeria. Only a single study has dealt with the chemical composition of the aerial part oils of Algerian D. crinitus, in which isochavicol isobutyrate (39.0%), octyl acetate (12.3%), and ß-caryophyllene (5.4%) were identified. Using GC-RI and GC-MS analysis, the essential oils and the volatiles extracted from separated organs of D. crinitus Desf. were studied using HS-SPME. RESULTS: GC-RI and GC-MS analysis identified 72 and 79 components in oils extracted using HD and in the volatile fractions extracted using SPME, respectively. Two types of essential oils were produced by the plant: the root oils had aliphatic compounds as the main component (87.0%-90.1%), and the aerial part oils had phenylpropanoids as the main component (43.1%-88.6%). HS-SPME analysis showed a more precise distribution of compounds in the organs studied: oxygenated aliphatic compounds were well represented in the roots (44.3%-84.0%), hydrocarbon aliphatic compounds were in the leaves and stems (22.2%-87.9%), and phenylpropanoids were in the flowers and umbels (47.9%-64.2%). Moreover, HS-SPME allowed the occurrence of isochavicol (29.6 - 34.7%) as main component in D. crinitus leaves, but it was not detected in the oils, probably because of its solubility in water. CONCLUSIONS: This study demonstrates that HD and HS-SPME modes could be complimentary extraction techniques in order to obtain the complete characterization of plant volatiles.

Chemical composition of fatty acid and unsaponifiable fractions of leaves, stems and roots of Arbutus unedo and in vitro antimicrobial activity of unsaponifiable extracts.

The chemical composition of the fatty acid and unsaponifiable fractions of the leaves, stems and roots of Arbutus unedo L. were determined using gas chromatography and gas chromatography-mass spectrometry. The fatty acid fractions of the leaves, stems and roots contained 38.5%, 31.3% and 14.1% palmitic acid, respectively, along with other long-chain fatty acids (up to C22). The chemical composition of the unsaponifiable fractions differed: the leaf and stem fractions contained high levels of aliphatic (32.1% and 62.6%, respectively) and terpenic compounds (49.6% and 25.7%, respectively), and the root fraction mainly contained esters, of which the most abundant was benzyl cinnamate (36.6%). The antimicrobial activities of the unsaponifiable fractions against nine species of microorganisms were assessed. The unsaponifiable leaf and stem extracts inhibited the growth of Klebsiella pneumoniae, Enterococcus faecalis and Candida albicans.

Chemotaxonomic investigations of peel and petitgrain essential oils from 17 citron cultivars.

Chemical compositions of essential oils from 17 citron cultivars were studied using GC and GC/MS. To the best of our knowledge, chemical compositions of peel and petitgrain oils from seven of them were reported for the first time. Combined analysis of peel and petitgrain essential oils led to the identification of 37 components (amounting to 98.2-99.9% of the total oil) and 42 components (97.0-99.9%), respectively. Statistical analysis was applied to identify possible relationships between citron cultivars. The levels of seven components, i.e., limonene, beta-pinene, gamma-terpinene, neral, geranial, nerol, and geraniol, indicated that the cultivars could be classified in four main chemotypes for peel and petitgrain oils. Chemotaxonomic investigations were carried out to establish relations between the morphological characteristics of citron cultivars and their corresponding oil compositions.

Morphological, chemical and genetic differentiation of two subspecies of Cistus creticus L. (C. creticus subsp. eriocephalus and C. creticus subsp. corsicus).

Cistus creticus L., an aromatic species from the Mediterranean area, contains various diterpenes bearing the labdane skeleton. The production of essential oil from this species has potential economic value, but so far, it has not been optimized. In order to contribute to a better knowledge of this species and to its differentiation, the morphological characters, volatile chemical composition and genetic data of two subspecies (C. creticus subsp. eriocephalus and C. creticus subsp. corsicus) were investigated. The leaf trichomes were studied using scanning electron microscopy. The chemical composition of Corsican essential oil (C. creticus subsp. corsicus) has been reported using GC, GC/MS and 13C NMR; the main constituents were oxygenated labdane diterpenes (33.9%) such as 13-epi-manoyl oxide (18.5%). Using plant material (54 samples) collected from 18 geographically distinct areas of the islands of Corsica and Sardinia, the basis of variation in the headspace solid-phase microextraction volatile fraction and an inter-simple sequence repeat genetic analysis were also examined. It was shown that the two subspecies of C. creticus differed in morphology, essential oil production, volatile fraction composition and genetic data.

Identification of chrysanthenyl esters from the essential oil of Anthemis maritima L. investigated by GC/RI, GC-MS (EI and CI) and 13C-NMR spectroscopy: chemical composition and variability.

INTRODUCTION: Anthemis maritima L. (Asteraceae) is a wild plant growing in Corsica and Sardinia. No previous studies of the chemical composition of A. maritima essential oil have been found. The oil contains two series of cis- and trans-chrysanthenyl esters for which the MS data are not present in available libraries and the 13C-NMR data are not reported in the literature. The determination of these compounds is a challenging problem since some of these esters are present in low concentrations and they exhibit very similar EI-MS. OBJECTIVE: To develop comprehensive strategies involving integrated techniques including CC, GC/RI, GC-MS (EI and CI), 1H- and 13C-NMR and hemi-synthesis for the identification of both series of chrysanthenyl esters in A. maritima oils. METHODOLOGY: Seventeen oil samples were prepared by hydrodistillation. A fine analysis of a pool of Corsican oils was conducted after repeated CC. A selected Sardinian oil was used as source of product for the hemi-synthesis of chrysanthenyl esters. The identification of these compounds was based on joint information from GC-MS (EI and CI), 13C-NMR and hemi-synthesis. RESULTS: Integrated analysis of Corsican and Sardinian oils allowed the identification of 124 components accounting for 83.3-91.6% of the total oil. With the aid of principal component analysis, oil samples could be divided in two groups. Among 16 chrysanthenyl esters identified, 12 were laboratory-synthesised and their 13C-NMR data are reported for the first time. CONCLUSIONS: The study illustrates the contribution of integrated techniques for the characterisation of unusual essential oils.

Spectral data of two new asymmetric sesquiterpene alcohols: (14R)-beta-oplopenol and (14S)-beta-oplopenol.

The epimeric sesquiterpene alcohols (14R)-beta-oplopenol and (14S)-beta-oplopenolwere obtained by LiAlH(4) reduction of beta-oplopenone. The complete (1)H- and (13)C-NMR assignments of these two new sesquiterpene alcohols have been made using 1D and 2D NMR techniques, including COSY, NOESY, HSQC, HMBC experiments.

Comparison of liquid-liquid extraction with headspace methods for the characterization of volatile fractions of commercial hydrolats from typically Mediterranean species.

Chemical composition of volatile fractions of nine commercial hydrolats and corresponding essential oils obtained using an industrial process were studied. The hydrolat volatile fractions were reported for the first time. A comparative study of those obtained, on the one hand, by liquid-liquid extraction (LLE) and, on the other hand, using five solid-phase microextraction (SPME) fibers and also purge-and-trap-automatic thermal desorption (P&T-ATD) was conducted with analysis performed by GC and GC/MS. The use of various techniques has resulted in a change of chromatographic profile of the hydrolat volatile fractions. Quantitative differences were established between chemical compositions of headspace and those obtained by a conventional method (LLE). Statistical analyses were carried out to summarize the results.

Analysis of volatile constituents isolated by hydrodistillation and headspace solid-phase microextraction from Adenostyles briquetii Gamisans.

The volatile fraction of the whole plant and separated organs of Adenostyles briquetii Gamisans (syn. Cacalia briquetii; family Asteraceae), an endemic species from Corsica, has been studied by headspace solid-phase microextraction (HS-SPME), GC and GC-MS(EI and CI). A total of 141 components were identified, representing 93% of the entire amount. The volatile fraction was characterised by sesquiterpene hydrocarbons (52.8%) and oxygenated sesquiterpenes (25.9%). The major components were germacrene D (18.5%), zingiberene (12.9%) and beta-oplopenone (10.8%). The influence of HS-SPME parameters on the extraction of family components is reported for the first time.

(E)-Methylisoeugenol and elemicin: antibacterial components of Daucus carota L. essential oil against Campylobacter jejuni.

The essential oil of wild Daucus carota L. obtained from aerial parts at the end of the flowering stage (DCEO) was reported as antimicrobial against the human enteropathogen Campylobacter jejuni. The aim of the present study was to extend this analysis to other Campylobacter species and to identify the active compounds of the essential oil, subjected to GC, GC-MS, and (13)C NMR analysis. A minimum inhibitory concentration assay was used to quantify the antimicrobial activity of DCEO and the major components, isolated on column chromatography. Growth of all the C. jejuni, Campylobacter coli, and Campylobacter lari strains tested, including one multidrug resistant C. jejuni, was inhibited to the same extent by DCEO. Molecules that were responsible for the antibacterial activity were identified as (E)-methylisoeugenol and elemicin. Moreover, the use of structural analogues of these compounds allowed us to identify important features that may account for the activity.

Variations in the concentration of phenolic compounds in the seagrass Posidonia oceanica under conditions of competition.

The concentration of phenolic compound was measured in the seagrass Posidonia oceanica when interacting with two Bryopsidophyceae, Caulerpa taxifolia and Caulerpa racemosa, between May 1999 and May 2000. These measurements were performed on adult and intermediate leaves and in sheaths of the seagrass. Sampling was carried out at three stations subject to increasing levels of interaction with Caulerpa. The number of tannin cells was also analysed. Five phenolic compounds were identified in P. oceanica, with a predominance of caffeic acid in the adult and intermediate leaves. For a given level of interaction (and for both caulerpa sp.), a significant seasonal variation in phenolic compounds was shown in the adult leaves (higher in November and lower in September and March for example for the interaction with C. taxifolia). Only for two compounds (corresponding to a mixture containing ferulic acid and the ester methyl 12-acetoxyricinoleate) were significant differences observed as a function of the level of interaction with C. taxifblia, and only in the adult leaves: higher concentrations of phenols were observed with increasing level of interaction. Thus,adult leaves gave values of 55.5 +/- 14.1 microg g(-1) dm without interaction (OCt) and 94.9 +/- 23.4 microg g(-l) dm with high interaction (2Ct),corresponding to an increase of 70%. No significant difference was observed with intermediate leaves and sheaths, or for interaction with C. racemosa. The number of tannin cells (supposed to produce the phenolic compounds) largely increased in the adult and intermediate leaves when the degree of interaction with C. taxifolia increased: 90 mm above the base of the sheath (in adult leaves), 16.7 +/- 10.6 tannin cells cm(-2) were found without interaction (OCt), and 57.8 + 21.2 tannin cells cm(-2) with high interaction (2Ct). No significant difference was found for C. racemosa interaction. It thus appears that when the seagrass P. oceanica is in interaction with C. taxifolia, it accelerates its production of secondary metabolites so as to limit invasion of the beds.