Chemical composition of volatiles in Sardinian myrtle (Myrtus communis L.) alcoholic extracts and essential oils

The chemical composition of the volatile fraction of myrtle (Myrtus communis L.) alcoholic extracts and essential oils from leaves and berries collected in different places in Sardinia (Italy) was studied. A simple and rapid liquid-liquid extraction method was used to isolate volatile compounds from myrtle alcoholic extracts followed by GC and GC-MS analysis allowing the detection of 24 compounds. The volatile fraction was characterized by the terpenes fraction corresponding to that of the essential oils and by a fatty acid ethyl esters fraction. The variation during extraction of the volatile fraction in alcoholic extracts of berries and leaves was evaluated. Essential oils were obtained by hydrodistillation, and the yields were on average 0.52 +/- 0.03% (v/w dried weight) and 0.02 +/- 0.00% for leaves and berries, respectively. The essential oils were analyzed by GC and GC-MS, and a total of 27 components were detected, accounting for 90.6-98.7% of the total essential oil composition. Strong chemical variability depending on the origin of the samples was observed. The major compounds in the essential oils were alpha-pinene (30.0 and 28.5%), 1,8-cineole (28.8 and 15.3%), and limonene (17.5 and 24.1%) in leaves and berries, respectively, and were characterized by the lack of myrtenyl acetate.     

Genetic Diversity and Structure of Wild Tunisian Myrtus communis L. (Myrtaceae) Populations

Myrtus communis L. (Myrtaceae), in Tunisia, is closely associated with Quercus suber L. forest which stretched continuously from the North to parts of Cap Bon and Tunisian Dorsal. The destruction of the primary oak forests associated to an over-exploitation of Myrtus for its essential oil quality had led to discontinuous populations exhibiting various levels of degradation. Using starch gel electrophoresis, we analyzed the polymorphism of nine isozymes in order to assess genetic diversity and structuring of 17 natural populations prospected in the three geographical regions and coinciding with subhumid, humid inferior and semi-arid superior bioclimates. The analysis of the level and the distribution of the genetic diversity in this species might help in its conservation. Out of the 18 loci detected for all populations and isozymes analyzed, 12 loci were polymorphic. Allelic frequencies differed according to populations and particular alleles characterized ecological groups. A high level of genetic variation within populations was observed. The mean number of alleles per polymorphic locus was Ap = 1.67, the percentage of polymorphic loci was P = 60.3% and the observed (Ho) and the expected (He) heterozygosities were respectively 0.144 and 0.215. Populations belonging to subhumid (Cap Bon) and semi-arid superior (Tunisian Dorsal) climates, located in degraded sites exhibited the highest level of inbreeding (0.425 < F_IS < 0.450). A high level of differentiation (F_ST = 0.396) and a low gene flow (Nm = 0.337) among populations, as a result of habitat intermediate destruction, were revealed. The differentiation of populations within the same bioclimate (or geographic) group was substantial and relatively higher for semi-arid superior populations (F_ST = 0.262), which were more distant. The three ecological groups exhibited a high level of structuring (F_ST = 0.401). These differentiations might be due to geographic distances and to the variations of ecological factors between sites, including human activities and environmental factors. Nei’s (1972) genetic distances calculated between pairs of populations were globally low (0.006 < D < 0.367) with a mean of 0.15. They indicate a high level of similarity between populations. UPGMA clustering, established through Nei’s genetic distances, showed three population aggregates according to their geographic/bioclimatic appartenances. The high differentiation between populations and the low level of their genetic divergence indicated their recent isolation under anthropic pressures. The species conservation (in situ or ex situ) strategies should take into account the genetic diversity level within populations and its variation between geographic groups.     

Anthocyanins from bay (Laurus nobilis L.) berries

Anthocyanin composition in the berries of Laurus nobilis L., a perennial tree or shrub typical of the Mediterranean region, was determined for the first time. The pigments were extracted from the berries with 0.1% HCl in methanol, purified on a C-18 solid-phase cartridge, and characterized by means of high-performance liquid chromatography (HPLC)-diode array detection (DAD)-mass spectrometry (MS) analysis. The major anthocyanins were characterized as cyanidin 3-O-glucoside (41%) and cyanidin 3-O-rutinoside (53%). Furthermore, two minor anthocyanins were detected and identified as 3-O-glucoside and 3-O-rutinoside derivatives of peonidin (5%). The two major pigments were also isolated by preparative HPLC and characterized by H1 nuclear magnetic resonance (NMR) spectroscopy. The attractive color and the great abundance of the plant in the south of Italy make Laurus nobilis berries a new and very good source of natural pigments.     

Lipoxygenase distribution in coffee (Coffea arabica L.) berries

In this paper lipoxygenase (LOX) presence was investigated in coffee berries to determine its involvement in lipid degradative metabolism of plants grown in organic and conventional cultivations. An immunochemical analysis has evidenced a ca. 80 kDa protein, cross-reacting with an anti-LOX antibody, only in the pulp fraction of berries obtained from plants of both cultivations. LOX activity in this fraction could be monitored either as conjugated diene formation or reaction products (determined by HPLC) and was mainly associated with a heavy membrane fraction (HMF, enriched in tonoplast, endoplasmic reticulum, plasma membrane, and mitochondria) and a light membrane fraction (LMF, enriched in plasma membrane and endoplasmic reticulum, with low levels of tonoplast and mitochondria). The LOX activity of LMF from berries of both cultivations showed an optimum at pH 8.0. The HMF exhibited a different activity peak in samples from conventional (pH 8.0) and organic (pH 5.5) cultures, suggesting the presence of different isoenzymes. These findings were also confirmed by variation of the ratio of 9- and 13-hydroperoxides in organic (1:1) and conventional cultivations (1:10), indicating that the organic one was subjected to an oxidative stress in the coffee pulp fraction leading to the expression of an acidic LOX. Such de novo synthesized LOX activity could be responsible for the production of secondary metabolites, which may interfere with the organoleptic profile of coffee.     

Effect of centrifugal ultrafiltration on the composition of aqueous extracts of saffron spice (Crocus sativus L.)

The purpose of this research was to study the effect of centrifugal ultrafiltration (UF) on the composition of aqueous extracts of saffron spice. The contents of seven crocetin esters, picrocrocin, and two kaempferol glycosides were analyzed by UV-vis and HPLC in the filtrate and retentate fractions from 16 centrifugal filter devices with regenerated cellulose (RC) and polyethersulfone (PES) membranes ranging from 1-100 kDa nominal molecular weight cutoff (MWCO). The separation of crocetin esters from picrocrocin and their concentration with centrifugal UF have been demonstrated. A great heterogeneity of results regarding devices with equal MWCO was found that could not be related to the membrane material or manufacturer. Four devices of 5 and 10 kDa MWCO, three of which had RC membranes, showed the best results. The device having the lowest MWCO also showed a potential to obtain picrocrocin without crocetin esters and could be considered in successive UF steps. The less polar crocetin esters were rejected better than the others.     

Chemical composition of Juniperus communis L. fruits supercritical CO2 extracts: dependence on pressure and extraction time

Ground fruits of the common juniper (Juniperus communis L.), with a particle size range from 0.250-0.400 mm, forming a bed of around 20.00 +/- 0.05 g, were extracted with supercritical CO(2) at pressures of 80, 90, and 100 bars and at a temperature of 40 degrees C. The total amount of extractable substances or global yield (mass of extract/mass of raw material) for the supercritical fluid extraction process varied from 0.65 to 4.00% (wt). At each investigated pressure, supercritical CO(2) extract fractions collected in successive time intervals over the course of the extraction were analyzed by capillary gas chromatography, using flame ionization (GC-FID) and mass spectrometric detection (GC-MS). More than 200 constituents were detected in the extracts, and the contents of 50 compounds were reported in the work. Dependence of the percentage yields of monoterpene, sesquiterpene, oxygenated monoterpene, and oxygenated sesquiterpene hydrocarbon groups on the extraction time was investigated, and conditions that favored the yielding of each terpene groups were emphasized. At all pressures, monoterpene hydrocarbons were almost completely extracted from the berries in the first 0.6 h. It was possible to extract oxygenated monoterpenes at 100 bar in 0.5 h and at 90 bar in 1.2 h. Contrary to that, during an extraction period of 4 h at 80 bar, it was possible to extract only 75% of the maximum yielded value of oxygenated monoterpene at 100 bar. Intensive extraction of sesquiterpenes could be by no means avoided at any pressure, but at the beginning of the process (the first 0.5 h) at 80 bar, they were extracted about 8 and 3 times slower than at 100 and 90 bar, respectively. Oxygenated sesquiterpenes were yielded at fast, constant extraction rates at 100 and 90 bar in 1.2 and 3 h, respectively. This initial fast extraction period was consequently followed by much slower extraction of oxygenated sesquiterpenes.     

Tocopherols and tocotrienols in sea buckthorn (Hippophae rhamnoides L.) berries during ripening

Sea buckthorn berries ( Hippophae rhamnoides L.) are used in foods, cosmetics, and pharmaceutical products. They are of particular interest for their high content of healthy phytochemicals, including vitamin E-related compounds (tocopherols and tocotrienols). This study investigated the content of tocopherols and tocotrienols during ripening in berries from four cultivars of sea buckthorn over a three-year period. The results showed large variations in tocopherols and tocotrienols depending on harvest date, cultivar, and year. Levels of alpha-tocopherol were higher early in the ripening period, while at later dates, delta-tocopherol levels increased. Great differences in amounts and composition of tocopherols and tocotrienols were observed between cultivars. Tocopherol levels were positively correlated with daily temperature, but this trend varied between years. Variations in tocopherols and tocotrienol levels in sea buckthorn berries due to cultivar, year, and ripening stage should therefore be considered in the production of nutritional products.     

Dietary fiber from Tunisian common date cultivars (Phoenix dactylifera L.): chemical composition, functional properties, and antioxidant capacity

The dietary fibers (DF) of 10 date varieties from Tunisian oases have been investigated. Further knowledge on the content, composition, and technological applications of those fibers could support their genetic variability and promote the socioeconomical development of growing areas. The composition, water- and oil-holding capacities, solubility, and antiradical activity have been determined. The DF content ranged from 4.7% (Matteta, Rochdi) to >7% (Deglé Nour, Garen Gaze, Smeti). Composition varied significantly among cultivars, and the results evidenced that uronic acids and lignin determine to a great extent the organoleptic quality of dates. Many of the varieties that have been studied (Garen Gaze, Matteta, Kenta, Rochdi, Mermella, Korkobbi, Eguwa) were selected because of great interest from technological and functional points of view. Among their physicochemical characteristics, these samples presented water- and oil-holding capacities of higher than 17 and 4 mL/g fiber, respectively, which make them suitable for use as additives in fiber-enriched foods. Also, DF of Garen Gaze, Smeti, Mermella, and Eguwa had a high antiradical capacity (>230 Trolox equiv/kg fiber). It was concluded that some of these varieties could be grown as potential sources of DF, which could be included in the formulation of fiber- and antioxidant-enriched foods.     

Assessment of anthocyanins in grape (Vitis vinifera L.) berries using a noninvasive chlorophyll fluorescence method

Anthocyanins (Anths) in grape (Vitis vinifera L.) berries harvested at véraison from Pinot Noir and Pinot Meunier cultivars were assessed nondestructively by measuring chlorophyll fluorescence (ChlF) excitation spectra. With increasing Anth content, less excitation light was transmitted to the deeper Chl layers, and thus the ChlF signal decreased proportionally. By applying Beer-Lambert's law, the logarithm of the ratio between the fluorescence excitation spectra (log FER) from a green and a red berry gave the in vivo absorption spectrum of Anths, which peaked at about 540 nm. Absolute quantitative nondestructive determination of Anths for each berry was obtained by the log FER calculated for two excitation wavelengths, 540 and 635 nm (absorbed and not-absorbed by Anths, respectively) of ChlF at 685 nm. Over a range of skin colors going from green to purple, the relationship between the log [ChlF(635)/ChlF(540)] and the Anth concentration of berry extracts was fairly well fitted (r 2 = 0.92) using a power function. Reflectance spectra on the same berry samples were also measured, and Anth reflectance indices, which were originally developed for apples and table grapes, were derived. The log FER Anth index was superior to the reflectance-ratio-based index, but was as good as the color index for red grapes (CIRG) calculated from the whole visible reflectance spectrum. The proposed log FER method, applied by means of suitable portable devices, may represent a new, rapid, and noninvasive tool for the assessment of grape phenolic maturity in vineyards.     

Comparison of yield, composition, and antioxidant activity of turmeric (Curcuma longa L.) extracts obtained using various techniques

Turmeric extracts were obtained from two lots of raw material (M and S) using various techniques: hydrodistillation, low pressure solvent extraction, Soxhlet, and supercritical extraction using carbon dioxide and cosolvents. The solvents and cosolvents tested were ethanol, isopropyl alcohol, and their mixture in equal proportions. The composition of the extracts was determined by gas chromatography-flame ionization detection (GC-FID) and UV. The largest yield (27%, weight) was obtained in the Soxhlet extraction (turmeric (S), ethanol = 1:100); the lowest yield was detected in the hydrodistillation process (2.1%). For the supercritical extraction, the best cosolvent was a mixture of ethanol and isopropyl alcohol. Sixty percent of the light fraction of the extracts consisted of ar-turmerone, (Z)-gamma-atlantone, and (E)-gamma-atlantone, except for the Soxhlet extracts (1:100, ethanol), for which only ar-turmeronol and (Z)-alpha-atlantone were detected. The maximum amount of curcuminoids (8.43%) was obtained using Soxhlet extraction (ethanol/isopropyl alcohol). The Soxhlet and low pressure extract exhibited the strongest antioxidant activities.     

Chemical composition of the volatile extract and antioxidant activities of the volatile and nonvolatile extracts of Egyptian corn silk (Zea mays L.)

A total of 36 compounds, which comprised 99.4% of the extract, were identified by gas chromatography and mass spectrometry (GC-MS) in the volatile dichloromethane extract obtained from Egyptian corn silk. The main constituents of the volatile extract were cis-alpha-terpineol (24.22%), 6,11-oxidoacor-4-ene (18.06%), citronellol (16.18%), trans-pinocamphone (5.86%), eugenol (4.37%), neo-iso-3-thujanol (2.59%), and cis-sabinene hydrate (2.28%). Dried Egyptian corn silk was also directly extracted with petroleum ether, ethanol, and water. All extracts from solvent extraction and the volatile extract described above exhibited clear antioxidant activities at levels of 50-400 microg/mL in the 2,2-diphenyl-1-picrylhydrazyl (DPPH)/linoleic acid assay. The ethanol extract inhibited DPPH activity by 84% at a level of 400 microg/mL. All samples tested via the beta-carotene bleaching assay also exhibited satisfactory antioxidant activity with clear dose responses. This study indicates that corn silk could be used to produce novel natural antioxidants as well as a flavoring agent in various food products.     

Regiospecific analysis of diricinoleoylacylglycerols in castor (Ricinus communis L.) oil by electrospray ionization-mass spectrometry

HPLC fractions of diricinoleoylacylglycerols containing one non-ricinoleoyl chain from castor oil were used to identify the regiospecific location of this non-ricinoleoyl chain on the glycerol backbone using electrospray ionization-MS3 of lithium adducts. The regiospecific ions used were from the loss of alpha,beta-unsaturated fatty acid specific at the sn-2 position. The content of 1,3-diricinoleoyl-2-oleoyl-sn-glycerols (ROR) among the three stereospecific isomers, RRO, ROR and ORR, was about 91%. The contents of other 1,3-diricinoleoyl-2-acyl-glycerols among the three stereospecific isomers were as follows: 1,3-diricinoleoyl-2-linoleoyl-sn-glycerol, 95%; 1,3-diricinoleoyl-2-linolenoyl-sn-glycerol, 96%; 1,3-diricinoleoyl-2-stearoyl-sn-glycerol, 96%; 1,3-diricinoleoyl-2-palmitoyl-sn-glycerol, 78%; and 1,3-diricinoleoyl-2-lesqueroloyl-sn-glycerol, 31%. These non-hydroxyl fatty acids were mostly at the sn-2 position of triacylglycerols in castor oil. These results suggest that phospholipase A2 hydrolysis of phosphatidylcholine (PC) containing non-hydroxyl fatty acid at the sn-2 position is either blocked or partially blocked in vivo. Phospholipase A2 hydrolysis of 2-lesqueroloyl-PC is not blocked and is similar to that of 2-ricinoleoyl-PC. Transgenic inhibition of phospholipase C hydrolysis of PC might be used to block the incorporation of non-hydroxyl fatty acids into triacylglycerols, thus increasing the content of ricinoleate in seed oil.     

Genetic Diversity of Tunisian Olive Tree (Olea europaea L.) Cultivars Assessed by AFLP Markers

About 29 olive (Olea europaea L.) cultivars including oil and table olive cultivars originating from Tunisia and other Mediterranean countries, were genotyped using amplified fragment length polymorphism (AFLP) DNA markers. This technique is a rapid and efficient method for producing DNA fingerprints. Using nine AFLP primer combinations, we produced a total of 410 AFLP markers, among which 172 revealed polymorphism. The results demonstrated a high degree of polymorphism in the olive germplasm we examined with an average of 39%. These AFLP markers were analyzed to estimate genetic distances between pairs of cultivars using Jaccard’s similarity coefficient. Furthermore, cluster and principal component analyses were performed in order to identify the genetic variation patterns. Two main groups were obtained: one comprising primarily small-fruited cultivars grown mainly for oil production and the other comprising large fruited cultivars (regardless of their end-use). Our results show no evidence of clustering of olive cultivars according to their geographic origin.     

Effectiveness of Amaranthus retroflexus L. aqueous extract in preventing iron chlorosis of pear trees (Pyrus communis L.)

The aim of this study was to evaluate the effectiveness of soil-applied aqueous extract of Amaranthus retroflexus L. in preventing lime-induced iron (Fe) chlorosis of pear trees (Pyrus communis L.). Tree growth, nutritional status, yield and fruit quality were also assessed. The aqueous extract was obtained by soaking dried and ground canopy (epigeal part) of spontaneous A. retroflexus plants in tap water. A. retroflexus extract was chosen because of its ability to solubilize Fe from calcareous soil, which was found to be 100-fold higher than deionized water alone. Two experiments were carried out (controlled environment and commercial field conditions) where soil-applied aqueous extract of A. retroflexus alone or mixed with iron sulfate (FeSO₄) was compared with synthetic Fe-chelate and an untreated control. Soil-applied aqueous extract of A. retroflexus increased shoot length, leaf SPAD and total plant biomass in controlled environment. In the commercial orchard control trees showed severe leaf Fe-chlorosis symptoms effectively prevented by Fe-chelate. The supply of A. retroflexus aqueous extract improved Fe nutrition of trees, particularly when enriched with FeSO₄. Fe-chelate increased tree yield but decreased fruit weight, leaf potassium (K) and manganese (Mn) concentration. At harvest, all strategies raised fruit soluble solid concentration compared to the untreated control. Results showed that soil-applied A. retroflexus aqueous extract improved Fe nutritional status of pear trees, probably because of the natural Fe chelating capacity of the compounds released by its tissues.     

Chemical composition of volatile extract and biological activities of volatile and less-volatile extracts of juniper berry (Juniperus drupacea L.) fruit

Volatile chemicals in a dichloromethane extract from a steam distillate of juniper berry fruit (Juniperus drupacea L.) and its two column chromatographic fractions (eluted with hexane and ethyl ether) were analyzed by gas chromatography/mass spectrometry. The major compounds in the dichloromethane extract were alpha-pinene (23.73%), thymol methyl ether (17.32%), and camphor (10.12%). A fraction eluted with hexane contained alpha-pinene (44.24%) as the major constituent. A fraction eluted with ethyl ether had thymol methyl ether (22.27%) and camphor (19.65%) as the main components. Three samples prepared from the distillate and two additional samples prepared by petroleum ether and ethanol extraction directly from juniper berry fruits exhibited clear antioxidant activities with dose response in both 1,2-diphenyl picrylhydrazyl and beta-carotene assays. All samples except the hexane fraction showed comparable activities to that of the synthetic antioxidant t-butyl hydroquinone at a level of 200 microg/mL in the two testing systems. The extracts of dichloromethane, petroleum ether, and ethanol exhibited appreciable antimicrobial activities against six microorganisms with minimum inhibitory concentrations ranging from 0.5 mg/mL (volatile extract against Candida albicans ) to 1.2 mg/mL (ethanol extract against Aspergillus niger ). The results of the present study suggest that this fruit could be a natural antioxidant supplement for foods and beverages.     

Proanthocyanidin composition in the seed coat of lentils (Lens culinaris L.)

Lentils (Lens culinaris L.) are a popular food in many countries. However, little is known about their phenolic composition. Because polyphenols in lentils are located essentially in their seed coat, the objective of this work was to study the composition of proanthocyanidins, the major group of polyphenols, in this part of the tissue. The use of C(18) Sep-Pak cartridges permitted the fractionation of lentil seed coat extract into monomer, oligomer, and polymer proanthocyanidin fractions. Subsequent thiolysis of oligomer and polymer fractions followed by HPLC analysis allowed the mean degree of polymerization (mDP) and the structural composition of proanthocyanidins to be determined. A fractionation of lentil seed coat extracts on a polyamide column followed by HPLC and HPLC-DAD-MS analyses was used to identify the individual proanthocyanidins. The results showed that the major monomeric flavan-3-ol was (+) catechin-3-glucose, with lesser amounts of (+)-catechin and (-)-epicatechin. In the oligomer fraction, various dimer, trimer, and tetramer proanthocyanidins constituted of catechin, gallocatechin, and catechin gallate units were identified, and several procyanidins and prodelphinidins from pentamers to nonamers constitute the polymer fraction. The most abundant proanthocyanidins in the seed coat of lentils are the polymers (65-75%), with a mDP of 7-9, followed by the oligomers (20-30%), with a mDP of 4-5.     

Genotype and climate conditions influence the drop off of flowers and premature berries of blackcurrant (Ribes nigrum L.)

Abstract

The aim of the present research was to study the extent of the drop off of blackcurrant flowers and young berries in the conditions of northern Europe, depending on particular genotypes and weather conditions. The study was carried out in southern Estonia in 2004–2007. The experiment involved nine recently selected genotypes from the blackcurrant breeding program of the Estonian University of Life Sciences Institute of Agricultural and Environmental Sciences Polli Horticultural Research Centre and 10 parent genotypes. Drop off is considerably influenced by the weather conditions during flowering (temperature, precipitation) and the particular genotype. Drop off is lower in genotypes ‘Ats’, 6-90-5, 1-90-15 and ‘Pilenai’, whereas it is high in genotypes ‘Zagadka’ and ‘Karri’. There is an average positive correlation between the cluster length, the number of flower buds in cluster and the extent of drop off. A strong negative correlation was found between the intra-flower fertilization ability of the genotype and drop off. There may be specimens with both similar as well as a very different extent of drop off among the genotypes originating from the same crossing combination. Drop off was lower among the genotypes where cultivar ‘Öjebyn’ was one of the parents.     

Genetic variation in Tunisian melon (Cucumis melo L.) germplasm as assessed by morphological traits

The morphological variation and the systematical status of 28 traditional melon including two ancient varieties collected from different geographical and bioclimatic areas of Tunisia were assessed based on 34 qualitative and quantitative traits. Yellow Canary was used as a reference variety. Significant differences among accessions within and among sites of collection were revealed for the majority of traits. The accessions from the Sahel were more polymorphic than those from Tozeur’s oasis. However, the classification of the local accessions according to Munger and Robinson’s varietal groups did not allow clear segregation of several of them because of their high heterogeneity. The principal component analysis (PCA) performed on all measured traits, allowed a distinction among the introduced varieties clustered according to their varietal group, and the local accessions grouped separately according to their geographical origin and bioclimatic zone. Mahalanobis distances among PCA’s groups were significant. The correlation found between Mahalanobis distance matrix and geographical distance matrix among origins corroborated isolation per distance. Conservation strategies should be made appropriately according to origins. Accessions from the Sahel showing the highest polymorphism within and among sites of collection should be firstly preserved.