Efficient method for screening and identification of radical scavengers in the leaves of Olea europaea L

In this article, an efficient method was developed to screen, isolate and identify the major radical scavengers in the leaves of Olea europaea L. by DPPH‐HPLC‐DAD, HSCCC and NMR. The method of DPPH‐HPLC‐DAD was used to screen the major radical scavengers. It was found that three major constituents (A, B, C) in the extract of the leaves of O. europaea L. possessed potential antioxidant activities. In order to identify the chemical structures of those compounds, the HSCCC method with a two‐phase solvent system composed of petroleum ether–ethyl acetate–water at an optimized volume ratio of 6:600:700 (v/v/v) together with column chromatography was developed to isolate and purify the active compounds. Pure compounds A (225 mg), B (10 mg) and C (12 mg) with purities 92.6, 95.1 and 96.4%, respectively, were obtained from the crude sample (500 mg). Their structures were identified as oleuropein (A), luteolin‐7‐O‐glucoside (B) and verbascoside (C) by ¹H‐NMR and ¹³C‐NMR. Copyright © 2010 John Wiley & Sons, Ltd.     

Extraction,purification procedures and HPLC-RI analysis of carbohydrates in olive (Olea europaea L.) plants

Summary Qualitative and quantitative analyses of carbohydrates in olive (Olea europaea L.) tissues have been carried out by HPLC-RI. Sample purification was by two successive solid-liquid extractions to remove completely plant phenolics and pigments. Five carbohydrate peaks; sucrose (stachyose + raffinose + sucrose), glucose, galactose, fructose (fructose + myo-inositol) and mannitol were detected when plant extracts were run on a Sugar SC 1011 column operating at 75°C, using water as eluent 0.5 ml min⁻¹. The use of two serial Sugar SC 1011 columns operating at 90°C and eluting the plant extracts with H₂O−CH₃CN (95/5 v/v) enables identification and quantification of nine carbohydrates, including tetra and tri-saccharides.     

Analysis and quantification of flavonoidic compounds from Portuguese olive (Olea europaea L.) leaf cultivars

Twenty three samples of 18 Portuguese olive leaf cultivars were analysed by a reversed-phase HPLC/DAD procedure and eight flavonoidic compounds were identified and quantified (luteolin 7,4'-O-diglucoside, luteolin 7-O-glucoside, rutin, apigenin 7-O-rutinoside, luteolin 4'-O-glucoside, luteolin, apigenin and diosmetin). Luteolin 7,4'-O-diglucoside and luteolin 4'-O-glucoside were identified by HPLC/DAD/MS/MS - ESI. The studied olive leaf samples showed a common phenolic pattern, in which luteolin 4'-O-glucoside was almost always the major compound.     

Optimisation of the extraction of olive (Olea europaea) leaf phenolics using water/ethanol-based solvent systems and response surface methodology

An experimental setup based on a 2³ full-factorial, central-composite design was implemented with the aim of optimising the recovery of polyphenols from olive leaves by employing reusable and nontoxic solutions composed of water/ethanol/citric acid as extracting media. The factors considered were (i) the pH of the medium, (ii) the extraction time and (iii) the ethanol concentration. The model obtained produced a satisfactory fit to the data with regard to total polyphenol extraction (R ² = 0.91, p = 0.0139), but not for the antiradical activity of the extracts (R ² = 0.67, p = 0.3734). The second-order polynomial equation obtained after analysing the experimental data indicated that ethanol concentration and time mostly affected the extraction yield, but that increased pH values were unfavourable in this regard. The maximum theoretical yield was calculated to be 250.2 ± 76.8 mg gallic acid equivalent per g of dry, chlorophyll-free tissue under optimal conditions (60% EtOH, pH 2 and 5 h). Liquid chromatography–electrospray ionisation mass spectrometry of the optimally obtained extract revealed that the principal phytochemicals recovered were luteolin 7-O-glucoside, apigenin 7-O-rutinoside and oleuropein, accompanied by smaller amounts of luteolin 3′,7-O-diglucoside, quercetin 3-O-rutinoside (rutin), luteolin 7-O-rutinoside and luteolin 3′-O-glucoside. Simple linear regression analysis between the total polyphenol and antiradical activity values gave a low and statistically insignificant correlation (R ² = 0.273, p > 0.05), suggesting that it is not the sheer amount of polyphenols that provides high antioxidant potency; instead, this potency is probably achieved through interactions among the various phenolic constituents.     

Direct chemical profiling of olive (Olea europaea) fruit epicuticular waxes by direct electrospray‐ultrahigh resolution mass spectrometry

In the present paper, an electrospray ionization (ESI)‐Orbitrap method is proposed for the direct chemical profiling of epicuticular wax (EW) from Olea europaea fruit. It constitutes a rapid and efficient tool suitable for a wide‐ranging screening of a large number of samples. In a few minutes, the method provides a comprehensive characterization of total EW extracts, based on the molecular formula of their components. Accurate mass measurements are obtained by ultrahigh resolution mass spectrometry, and compositional restrictions are set on the basis of the information available from previous studies of olive EW. By alternating positive and negative ESI modes within the same analysis, complementary results are obtained and a wide range of chemical species is covered. This provides a detailed compositional overview that otherwise would only be available by applying multiple analytical techniques. Copyright © 2015 John Wiley & Sons, Ltd.     

Comparative study on fatty acid composition of olive (Olea europaea L.), with emphasis on phytosterol contents

The present study was designed to determine the fatty acid composition and phytosterol contents of Turkish native olive cultivars, namely Kilis Yağlık and Nizip Yağlık cv. In this context, olive fruits from 34 locations were sampled and then screened for their components in comparison. Fifteen different fatty acids were found in both olive oils. In the order of abundance, the most important ones were oleic acid (18:1) > palmitic acid (16:0) > linoleic acid (18:2) > stearic acid (18:0). Significant differences were observed in the contents of oleic acid (18:1), palmitic acid (16:0), linoleic acid (18:2) but not for stearic acid content in comparison both oils (p < 0.01). There were significant differences in terms of unsaturated fatty acids, saturated fatty acids and polyunsaturated fatty acids (p < 0.01). The seven phytosterols – cholesterol, campesterol, stigmasterol, β ‐sitosterol, Δ‐5‐avenasterol, Δ‐7‐stigmastenol and Δ‐7‐avenasterol – were studied in both oil sources. The predominant sterols were β ‐sitosterol, Δ5‐avenasterol and campesterol in the samples analysed. However, no significant differences were found in the levels of the phytosterols between the two olive cultivars.     

Separation and purification of hydroxytysol and oleuropein from Olea europaea L. (olive) leaves using macroporous resins and a novel solvent system

The separation and purification of hydroxytysol and oleuropein from Olea europaea L. (olive) using a macroporous resin with a novel solvent system was systematically investigated. Static adsorption experiments with BMKX–4 resin revealed that the experimental data of both hydroxytysol and oleuropein fitted best to the pseudo‐second‐order kinetic and Freundlich isotherm models. The thermodynamic parameters indicated spontaneous and exothermic adsorption processes. The novel solvent system, composed of n–hexane:ethyl acetate:methanol:water in a (v/v/v/v) ratio of 1:9:1:9, had two phases (upper and lower). The separation and purification parameters of hydroxytysol and oleuropein were optimized using dynamic adsorption/desorption on a column packed with BMKX–4 resin. The effects of flow rates and volumes of the upper and lower phases on the separation efficiency were systematically studied. Under optimal conditions, the fraction of hydroxytysol in the final product increased by 6.34‐fold from 0.46 to 2.96%, with a yield rate of 88.58% w/w, while that of oleuropein increased 4.17‐fold from 11.40 to 47.59%, with a 93.31% w/w yield rate. These results may be help in selecting a suitable eluent for improved separation of macroporous adsorption resins.     

13C nuclear magnetic resonance spectroscopy for determining the different components of epicuticular waxes of olive fruit (Olea europaea) Dritta cultivar

¹³C nuclear magnetic resonance spectroscopy (NMR) was applied to determine the different components of apolar and polar fractions which were isolated by column chromatography from the crude chloroform-soluble waxes of olive fruits (Olea europaea) Dritta cultivar.¹³C NMR enabled the determination in the wax apolar fraction, of aliphatic aldehydes, and of benzyl, alkyl and glyceryl esters. In particular, the fatty acid composition of alkyl esters, comprising saturated and unsaturated oleic and linoleic acids, was determined. Acyl chain composition and the chain composition of 1,3- and 2-glycerol positions were also determined for triacylglycerols of olive fruit waxes.Oleanolic and maslinic acids were confirmed to be the major components of wax polar fraction. Complete assignments of ¹³C NMR chemical shifts of oleanolic and maslinic acids as a mixture were achieved by using homonuclear correlation spectroscopy with gradient (g-COSY), attached proton test (APT), inverse-detected heteronuclear single-quantum coherence with gradient (g-HSQC), high-resolution heteronuclear correlation spectroscopy (HETCOR) for C-H directly attached pairs and C-H long-range-coupled experiments.     

Antioxidant Activities of Polyphenols Extracted from Olive (Olea europaea) of Chamlal Variety

The antioxidant properties of phenolic compounds from olive pulp (PCO) of chamlal variety and those of individual phenolic compounds were evaluated and compared with that of vitamin C (Vit C). The antioxidant activity was measured by the tests of iron reduction and scavenging hydrogen peroxide (H₂O₂). Results showed that all the substances tested exhibit a reducing power. The PCO present activities of iron reduction and H₂O₂ scavenging higher than those of Vit C. The protective effect of PCO against oxidation of lipids and proteins from erythrocyte membranes was studied. The measurement of malondialdehyde generated under oxidative stress conditions induced by hydroxyl radicals generating system revealed that PCO have the most significant protective activity against lipid peroxidation (IC₅₀?=?49.27?±?1.91 μg mL^?1). Paradoxically, Vit C revealed a pro-oxidant effect. Proteins oxidation was evaluated using the H₂O₂/FeSO₄ system and electrophoresis. In the presence of PCO at 1 mg mL^?1, proteins of erythrocyte membranes were protected contrary to those treated with Vit C at the same concentration.     

Development of an ultra-high performance liquid chromatography analytical methodology for the profiling of olive (Olea europaea L.) pulp proteins

Ultra-high performance liquid chromatography (UHPLC) constitutes an interesting proposal to speed protein separations but it is almost not explored. In this work UHPLC is proposed, for the first time, to separate olive pulp proteins. An important difficulty in the analysis of proteins is related to their extraction. The difficulty in the extraction of proteins from the olive pulp is derived from its high content in lipids and phenolic compounds. Eight different methods for the extraction of pulp proteins were designed and evaluated. The optimized extraction procedure consisted of a cleaning step to remove interfering compounds, followed by the extraction of proteins with a Tris–HCl buffer containing sodium dodecyl sulphate (SDS) and dithiothreitol (DTT), precipitation of proteins with acetone, and solubilization in the Tris–HCl buffer. This methodology yielded the most successful isolation of pulp proteins and enabled the optimization of a UHPLC methodology for their separation. The method was applied to the profiling of olive pulp proteins from different olive cultivars observing in all cases a protein that had never been described before.     

Effectiveness of dairy treated wastewater and different irrigation systems on the growth,biomass and fruiting of a Tunisian olive orchard (Olea europaea L., cv Chemlali)

Abstract

The effectiveness of dairy treated wastewater (TWW) was evaluated, in comparison with tap water (TW), on a Tunisian olive orchard (Olea europea, cv Chemlali), irrigated manually (MI) and by surface dripping (SDI). To this purpose, tree growth, biomass and fruiting were monthly tested for a one-year period. Similar trunk diameters, nodes/tree, root lengths were obtained, independently of source and system of irrigation. Also, comparable tree length and leaf area, shoots/tree and biomass, were observed between TWW and TW plants. However, such parameters improved under SDI rather than MI. Fruiting occurred only in TWW and TW trees treated by MI. Concerning growth, biomass and fruiting, TWW represented a valid alternative source for the irrigation of olive orchard, especially in Tunisia, already facing the freshwater scarcity. Monitoring of soil, TWW, fruits and oil will be required to validate the use of such effluent for olive orchard irrigation.     

Ethanol production from pretreated olive tree wood and sunflower stalks by an SSF process

Olive tree wood and sunflower stalks are agricultural residues largely available at low cost in Mediterranean countries. As renewable lignocellulosic materials, their bioconversion may allow both obtaining a value-added product, for fuel ethanol, and facilitating their elimination. In this work, the ethanol production from olive tree wood and sunflower stalks by a simultaneous saccharification and fermentation (SSF) process is studied. As a pretreatment, steam explosion at different temperatures was applied. The water insoluble fractions of steam-pretreated sunflower stalks and steamed, delignified olive tree wood were used as substrates at 10% w/v concentration for an SSF process by a cellulolytic commercial complex and Saccharomyces cerevisiae. After 72-h fermentation, ethanol concentrations up to 30 g/L were obtained in delignified steam-pretreated olive tree wood at 230°C and 5 min. Sunflower stalks pretretated at 220°C and 5 min gave maximum ethanol concentrations of 21 g/L in SSF experiments.     

Processing Effect and Characterization of Olive Oils from Spanish Wild Olive Trees (Olea europaea var. sylvestris)

Wild olive trees have important potential, but, to date, the oil from wild olives has not been studied significantly, especially from an analytical point of view. In Spain, the wild olive tree is called “Acebuche” and its fruit “Acebuchina”. The objective of this work is to optimize the olive oil production process from the Acebuchina cultivar and characterize the oil, which could be marketed as healthy and functional food. A Box–Behnken experimental design with five central points was used, along with the Response Surface Methodology to obtain a mathematical experimental model. The oils from the Acebuchina cultivar meet the requirements for human consumption and have a good balance of fatty acids. In addition, the oils are rich in antioxidants and volatile compounds. The highest extraction yield, 12.0 g oil/100 g paste, was obtained at 90.0 min and the highest yield of phenolic compounds, 870.0 mg/kg, was achieved at 40.0 °C, and 90.0 min; but the maximum content of volatile compounds, 26.9 mg/kg, was obtained at 20 °C and 30.0 min. The oil yield is lower than that of commercial cultivars, but the contents of volatile and phenolic compounds is higher.     

Phytochemical Characterization of Olea europaea L. Cultivars of Cilento National Park (South Italy) through NMR-Based Metabolomics

Olea europaea germplasm is constituted by a huge number of cultivars, each one characterized by specific features. In this context, endemic cultivars evolved for a very long period in a precise local area, developing very specific traits. These characteristics include the production and accumulation of phytochemicals, many of which are also responsible for the nutraceutical value of the drupes and of the oils therefrom. With the aim of obtaining information on the phytochemical profile of drupes of autochthonous cultivars of Cilento, Vallo di Diano and Alburni National Park, a metabolomics-based study was carried out on 19 selected cultivars. Multivariate data analysis of ¹H-NMR data and 2D NMR analyses allowed the rapid identification of metabolites that were qualitatively and/or quantitatively varying among the cultivars. This study allowed to identify the cultivars Racioppella, Guglia, Pizzulella, Oliva amara, and Racioppa as the richest in health-promoting phenolic compounds. Furthermore, it showed a significant variability among the different cultivars, suggesting the possibility of using metabolic fingerprinting approaches for cultivar differentiation, once that further studies aimed at assessing the influence of growing conditions and environmental factors on the chemical profiles of single cultivars are carried out.     

Synthesis of deuterium-labelled substrates for the study of oleuropein biosynthesis in Olea europaea callus cultures

We propose the cell culture approach to investigate oleuropein (1) biogenesis in Olea europaea L. We suggest employing olive callus cultures to identify the iridoidic precursor of oleuropein. In fact, we confirmed that callus cells from olive shoot explants are able to produce key secoiridoid as 1. To enable this approach, we synthesised and characterised deuterium-labelled iridoidic precursors belonging both to the loganin and the 8-epiloganin series. These iridoids are [7,8-²H₂]-7-deoxy-8-epi-loganin (2^D), [8,10-²H₂]-8-epi-loganin (4^D) and [7,8-²H₂]-7-deoxy-loganin (3^D).     

Oxygen scavengers and sensitizers for reduced oxygen inhibition in radical photopolymerization

Oxygen inhibition in the free‐radical photopolymerization of (meth)acrylates is one of the most challenging problems in thin film application. Apart from the utilization of an inert gas atmosphere, additives reducing oxygen inhibition due to production of new propagating centers are used. In the present study, a more straightforward approach to reduce oxygen inhibition by photosensitized generation of singlet oxygen and subsequent scavenging of these species by selective singlet oxygen trappers was investigated. The potential of 1,2‐dions conventionally used as type‐II photoinitiators for visible light polymerization to function as singlet oxygen generators was verified in sensitized steady state photooxidation experiments in solution. A set of furan and anthracene derivatives were tested as oxygen scavengers and their corresponding relative reaction rates were determined. The ability of these sensitizer/scavenger systems to reduce oxygen inhibition in practical applications was studied in photo‐DSC‐experiments. In thin film polymerization (investigated by ATR‐FTIR), the formation of insufficiently cured surfaces could be prevented by the usage of singlet oxygen trappers. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 6916–6927, 2008     

Electrochemical behaviour of a Cu/CuSe microelectrode and its application in detecting temporal and spatial localisation of copper(II) fluxes along Olea europaea roots

The electrochemical behaviour of a Cu/CuSe electrode was studied in order to define its selectivity towards cupric ions, Nerstian response, limit of detection and response time. The chalcogenide electrode was prepared by cathodic deposition of Se and subsequent formation of a thin layer of CuSe on a copper substrate. A Cu/CuSe microelectrode was prepared using copper wire 75 μm in diameter. The dimensions and response time (<0.5 s) allowed use of this electrode in the “vibrating probe method” with the aim of measuring net influxes as well as effluxes of copper(II) ions in Olea europaea roots. The electrode potential was measured along the root at a distance of 5 μm from the surface for 5 s, and then again for 5 s at a distance of 55 μm, moving the microelectrode with respect to the root surface by steps with a frequency of 0.1 Hz. The potentials measured at the two extremes of vibration were then converted to copper(II) concentrations. Substitution of these values in Fick's law yields the flux, assuming the diffusion constant D for copper ions in aqueous solutions. The results enabled us to detect copper(II) fluxes as small as 0.05 pmol cm⁻² s⁻¹. Copper(II) influx showed marked spatial and temporal features: it was highest at about 1.5 mm from the root apex and exhibited an oscillatory pattern in time. Received: 29 September 1999 / Accepted: 11 January 2000     

Measurement of tritium in tree rings: Isolation of α-cellulose from wood chips using explosive depressurization

Explosive depressurization offered an effective pre-treatment for isolating -cellulose from wood chips using the common chlorite method. This operation shortened the time required for isolation of -cellulose of relatively high purity by up to 6 h, and raised the relative crystallinity of the product to 65–72% from 50% or below for the unexploded samples. The steam-explosion was carried out by combining 2 min of pre-hydrolysis at a pressure of 3.55 MPa with a rapid decompression. This was found to be the best operating condition for pine wood chips of 1 mm thickness to analyze the tritium content in tree rings accurately.