Characterization of Olive-Leaf Phenolics by ESI-MS and Evaluation of their Antioxidant Capacities by the CAT Assay

Olive leaves are a very abundant vegetable material containing various phenolic compounds, such as secoiridoids and flavonoids, that are expected to exert strong antioxidant capacity. However, little is known about the variation of olive-leaf phenolic composition during maturation and its influence on antioxidant capacity. To answer this question, young and mature Olea Europaea L. leaves were submitted to successive extraction with dichloromethane, ethyl acetate, and methanol, then characterized by ESI-MS. It appeared that mature olive-leaf extracts contained higher levels of verbascoside isomers and glucosylated forms of luteolin, while young ones presented higher contents of oleuropein, ligstroside, and flavonoid aglycones. Moreover, antioxidant capacity evaluation using our newly developed conjugated autoxidizable triene assay showed that, in a lipid-based emulsified system, this phenolic composition variation leads to a change in the ability of extracts to counteract lipid oxidation. Mature olive-leaf extracts exhibit higher antioxidant capacity than young olive-leaf extracts. This result enables us to hypothesize that two main bioconversion scenarios may occur during maturation of olive leaves, which could explain changes observed in antioxidant capacity: (1) a bioconversion of oleuropein and ligstroside into verbascoside isomers and oleuroside, and (2) a bioconversion of flavonoid aglycones into glucosylated forms of luteolin. Finally, this study leads to a better understanding of the relationship between phenolic profile and antioxidant capacity of olive leaves.     

Isolation of polyphenols from the extracts of olive leaves (Olea europaea L.) by adsorption on silk fibroin

General objective of the present work was to assess the isolation of polyphenols from olive leaves. The effects of extraction conditions on the total phenol content and antioxidant activity of olive leaf extract (OLE) were investigated. An extract with good antioxidant activity (7.52 mmol of Trolox equivalent antioxidant capacity (TEAC)/g olive leaf extract), and a high content of oleuropein (13.4%) and rutin (0.18%) could be obtained using 70% ethanol as extraction solvent. There was a good correlation between the antioxidant activity and the total phenol content. Furthermore, silk fibroin was used as a novel adsorbent to recover the polyphenols from the olive leaf extracts. The adsorbed amounts of rutin and oleuropein were 15 mg rutin/g silk fibroin and 96 mg oleuropein/g silk fibroin. Fraction consisting of mainly oleuropein and fraction rich in rutin, luteolin-7-glucoside, verbascoside, apigenin-7-glucoside were obtained by using silk fibroin filled column. Silk fibroin was found to be a promising adsorbent for the purification of oleuropein and rutin from olive leaf extracts.     

Investigation of the Effects of Virgin Olive Oil Cleaning Systems on the Secoiridoid Aglycone Content Using High Performance Liquid Chromatography–Mass Spectrometry

Phenolic compounds are useful markers to control olive oil technological processes, including the virgin olive oil (VOO)/water separation after olive oil extraction. In this investigation, VOO extracted from olives of cv. Coratina using a mild oil/water separator called the hydrocyclone sedimentation system (Hydroil) was compared with VOO obtained using a conventional vertical centrifuge separator (Cenoil), which is mostly used in the modern olive oil industry. Secoiridoid aglycones were selected, among phenolic compounds, as markers and analyzed using reversed‐phase liquid chromatography coupled to linear quadrupole ion‐trap mass spectrometry with electrospray ionization in the negative mode. VOO samples obtained using the Hydroil system were found to contain significantly higher levels of secoiridoid aglycones, compared to the Cenoyl‐type samples. In particular, the total content of the aglycones of decarboxymethyl oleuropein, decarboxymethyl ligstroside, ligstroside, and oleuropein, expressed in terms of oleuropein, was estimated as 35.40 ± 0.80 mg kg^?1, compared to 8.06 ± 0.41 mg kg^?1 in the Cenoil samples (n = 3). Since no significant difference in residual water (P < 0.05) was found between the two types of VOO samples, the higher amount of secoiridoids obtained for Hydroil‐type ones was explained by the lower extent of oxidation occurring during the mild oil/water separation achieved using the Hydroil system.     

Phenolic Composition and Antioxidant Activity of Monovarietal and Commercial Portuguese Olive Oils

Olive oil composition has been investigated using chemical approaches, since the composition has a direct impact on its quality and safety and it may be used for certification purposes. In this paper, eleven monovarietal and twelve commercial Portuguese olive oils were analyzed to determine spectrophotometrically their total polyphenol content, ortho-diphenols and antioxidant activity. The phenolic profiles of these olive oils were also studied by high performance liquid chromatography. The lowest phenolic content and antioxidant activity were observed for monovarietal olive oils, however, among these group, ‘Cobrançosa’ and ‘Redondil’ cultivars showed the highest values of these two chemical parameters. In commercial olive oils, the concentration of polyphenols, determined according to the Folin–Ciocalteu method, and the antioxidant activity (ABTS method) ranged from 97.37 ± 1.10 to 219.7 ± 1.50 mg GAE/kg of oil and from 387.2 ± 20.00 to 997.5 ± 30.90 µmol Trolox/kg, respectively. The study of the phenolic profile demonstrated that the highest concentrations of the most abundant compounds in olive oil (tyrosol, hydroxytyrosol and oleuropein) are present in commercial olive oils. The correlation coefficient between total phenolics and antioxidant activity was statistically significant (r = 0.95, p < 0.0001). The same was observed for ortho-diphenol content and antioxidant capacity (r = 0.94, p < 0.0001).     

Influence of Water Deficit in Bioactive Compounds of Olive Paste and Oil Content

The main objective of this study was to evaluate the effect of different deficit irrigation treatments (control, regulated deficit irrigation [RDI]‐1, RDI‐2, and RDI‐3) on the phenolic profile of the olive paste and oil content. Irrigation treatments with more stress water led to a considerable increase in the phenolic compounds of olive paste, especially in oleuropein (60.24%), hydroxytyrosol (82%), tyrosol (195%), and verbascoside (223%) compared to control. A significant increase in the content of total flavonoids and phenolic acids was also observed for these samples. In virgin olive oils (VOO) elaborated from the most stressed olive trees (RDI‐2 and RDI‐3), a noticeable increase in phenolic substances with antioxidant properties (oleuropein, hydroxytyrosol, tyrosol, secoiridoid derivatives, and o‐vanillin) was observed. Consequently, water stress conditions improved antioxidant activity of VOO.     

Phenolic Compounds and Antioxidant Capacity of Monovarietal Olive Oils Produced in Argentina

Virgin olive oil has high levels of phenolic compounds that are highly bioavailable; these compounds are receiving considerable attention for their antioxidant activity, closely related to the prevention of non‐communicable chronic diseases. The aim of this work was to characterize the phenolic profile and antioxidant capacity of monovarietal olive oils cvs. Arauco, Arbequina, Farga and Empeltre produced in Argentina. This study focused on the relationship between the single molecules or classes of molecules quantified by SPE‐CZE, the corresponding Folin‐Ciocalteu results, and antioxidant capacity using three different tests. Fifteen compounds were simultaneously determined: tyrosol, vinylphenol, oleuropein, hydroxytyrosol, rutin, catechin, naringenin, cinnamic acid, chlorogenic acid, syringic acid, luteolin, apigenin, vanillin acid, quercetin, and caffeic acid. The phenolic contents of the monovarietal olive oils show significant differences between different varieties (p < 0.05), with positive and significant Pearson's correlation found between Folin–Ciocalteu and CZE. Besides, the correlation between the content of total polyphenols and antioxidant capacity was high for all the antioxidant assays performed. When analyzing the correlation coefficients of the different families of phenolic compounds studied, simple phenols and cinnamic acid derivatives show a higher correlation with antioxidant capacity. Thus, findings obtained in this study demonstrated that Arauco olive oil, autochthonous for Argentina, possesses the highest antioxidant/free‐radical scavenging properties, which are very likely due to the presence of high contents of phenolic compounds.     

Extra Virgin Olive Oil Components and Oxidative Stability from Olives Grown in Tunisia

The effects of the contents of lipids, pigments, α-tocopherol and phenols were studied in relation to the antioxidant capacity of five virgin olive oils obtained from five olive cultivars planted in Tunisia (Arbequina, Koroneiki, Leccino, Oueslati and Chemchali). The antioxidant capacities were evaluated by two different radical scavenging activities: radical scavenging activity by the DPPH assay (RSA-DPPH) and total antioxidant status by the ABTS test (TAA-ABTS). The highest contents of antioxidant compounds (75.96, 10.34, 6.32, 15.39 and 241.52 mg kg⁻¹ for oleic acid, O/L ratio, carotenes, chlorophylls and total phenols, respectively) were found for the Koroneiki cultivar except for α-tocopherol and o-diphenols, which had the highest contents (369 and 160.7 mg kg⁻¹, respectively) in the Leccino and Chemchali cultivars (cvs). Furthermore, the highest antioxidant capacity in virgin olive oil was observed in the Koroneiki cultivar (0.24 mmol TE kg⁻¹) followed by the Chemchali and Leccino cvs (0.22 and 0.13 mmol TE kg⁻¹) for the TAA-ABTS test. However, the RSA-DPPH activity was higher for the Chemchali cultivar (19.9%) than for the Koroneiki and Leccino cvs (18.4 and 13.5%, respectively). Correlation between these capacities and the oil composition revealed that they were mainly influenced by the carotene content, followed by chlorophyll and phenolic contents where the ABTS test was more pronounced. Then, the antioxidant capacity of the virgin olive oils was correlated with polar components and the lipid profile which are important for its shelf life.     

Changes in Quality Indices,Phenolic Content and Antioxidant Activity of Flavored Olive Oils during Storage

The chemical characteristics, phenolic content and antioxidant activity of olive oils flavored with garlic, lemon, oregano, hot pepper, and rosemary were evaluated during 9 months of storage. At the end of the storage period, the unflavored and the garlic-flavored oils maintained their chemical parameters within the limits fixed for extra-virgin olive oils. After 9 months of storage, a noticeable decrease in phenolic content was observed in all the oils. The highest (35.0 ± 3.9 mg/kg oil) and the lowest (6.3 ± 0.4 mg/kg) phenolic contents were detected in the unflavored and garlic-flavored oils, respectively. Compounds such as 3,4-DHPEA-EDA (3,4-dihydroxyphenylethyl 4-formyl-3-formylmethyl-4-hexenoate, the dialdehydic form of decarboxymethyl elenolic acid linked to hydroxytyrosol) and p-HPEA-EDA (dialdehydic form of the decarboxymethyl elenolic acid linked to tyrosol) were the most abundant in both unflavored and lemon-flavored oils up till 6 months of storage. At the end of the storage period, increases in 3,4-DHPEA (hydroxytyrosol) and p-HPEA (tyrosol) were measured in almost all the oils. During storage, the antioxidant activity coefficients of the phenolic extracts, calculated according to the β-carotene bleaching assay, significantly decreased and, after 9 months, were in a decreasing order: rosemary (51.3 ± 4.2), hot pepper, lemon, oregano, unflavored, and garlic (8.5 ± 0.7).     

The Effect of Harvesting Time on Olive Fruits and Oils Quality Parameters of Tortiglione and Dritta Olive Cultivars

Despite the fact that Italy holds the most important olives heritage in the world, with about 800 cultivars, most of them are still underestimated, in particular those from Abruzzo, a region located in the center of the peninsula. The aim of this work is to study the changes in quality parameters of olive fruits and related oils of two autochthonous Abruzzo olive cultivars, Tortiglione and Dritta during ripening (from September to November 2017). Both cultivar and ripening time affect the chemical parameters of olive fruits. Results highlight an increasing trend of the oil content with final values, based on fresh matter, of 38.7 ± 0.3% and 38.1 ± 0.9% for Tortiglione and Dritta, respectively. Olive oils chemical composition is also affected by ripening time and cultivar, with Tortiglione oils resulting generally richer than Dritta oils; on the first sampling time (30th of October) values for total phenolic content, antioxidant activity, and chlorophylls are 803.8 ± 68.2 mg gallic acid equivalent kg⁻¹, 2.7 ± 0.5 mmol trolox equivalent kg⁻¹, and 30.8 ± 1.6 mg pheophytin a kg⁻¹, respectively. Tocopherols seem to be more affected by ripening time than by cultivar, in particular for Dritta. Practical Application : The results on Abruzzo minor olive cultivars indicate that olive fruits and olive oil composition are strongly influenced by both cultivar and ripening time, giving rational indications about the optimal cultivar specific harvesting time and opening interesting opportunities for olive oil producers in a perspective of sustainable production to obtain high quality fruits and oils. The research provides detailed information about Tortiglione and Dritta olive cultivar, useful in the global context of revaluation of Italian minor olive varieties.     

Quality Characteristics and Antioxidant Activity during Fruit Ripening of Three Monovarietal Olive Oils Cultivated in China

Numerous olive cultivars have been planted in China, mainly to reap the health benefits of olive oil. This study characterized the quality and investigated the antioxidant activity of olives harvested at three different maturation stages, defined by the skin color of the olives (black, purple, and green), from three newly introduced olive cultivars (Barnea, Manzanilla and Kadesh) grown in China. The oleic acid content of olives from all three olive cultivars decreased significantly during maturation, whereas the linoleic acid content increased. The highest content of total phenols was recorded in Manzanilla cultivar (284.94 mg kg⁻¹), whereas the lowest was recorded in Kadesh cultivar (134.82 mg kg⁻¹). In addition, a total of 13 individual phenolic compounds were obtained, and their concentrations were significantly influenced by the cultivar and maturity (P < 0.05). Secoiridoids were the main group of phenolic compounds, but their quantity decreased during maturation. The content of secoiridoids in the Manzanilla cultivar was significantly higher than that of the Barnea and Kadesh cultivars. In general, a significant decrease (P < 0.05) in the values of antioxidant activity and the contents of pigments, α-tocopherol, and squalene occurred during maturation. Furthermore, principal component analysis was used to classify the nine olive oil samples according to the cultivar and ripening degree.     

Investigation of Oleuropein Content in Olive Leaf Extract Obtained by Supercritical Fluid Extraction and Soxhlet Methods

The separation processes of the phenolic compounds from solid plant matrixes are of great importance. In the scope of developing more efficient methods to separate olive leaf extract, dried and ground olive tree leaves from Aegean region of Turkey were extracted by means of Soxhlet and supercritical fluid extraction (SFE) methods. In the Soxhlet method, different types of solvents (hexane, water, ethanol, methanol, and methanol/hexane (3:2, v/v) mixture) were used to determine the effect of the solvent type on the extraction performance. In the SFE method, the effect of pressure (100–300 bar), temperature (50 and 100°C), and type of co-solvent on the amount of both extract and oleuropein were investigated. Ethanol, methanol and water were selected as co-solvent in 20% (v/v) amount. Quantitative analysis was performed by using a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) technique. The results of SFE were compared with those obtained by the Soxhlet method. Whereas the highest oleuropein yield was achieved via the Soxhlet method through methanol with the value of 37.84 mg/g dried leaf, the best oleuropein yield was achieved with the value of 14.26 mg/g dried leaf by using CO₂ modified by methanol at 300 bar and 100°C in the SFE method.     

Use of Novel Drying Technologies to Improve the Retention of Infused Olive Leaf Polyphenols

The infusion of phenolic extracts in dried fruits constitutes an interesting means of improving their nutritional content. However, drying can affect the further process of impregnation. In this work, different drying treatments (air temperature and ultrasound application) were applied to apple samples and impregnated with olive leaf extract. The application of ultrasound during drying did not significantly (p < 0.05) affect the infusion capacity of samples, but the ultrasonically assisted dried samples showed a greater antioxidant capacity than those conventionally dried. The highest content of oleuropein and verbascoside was found in samples dried at low temperature using ultrasound.     

Effect of extraction systems on the phenolic composition of virgin olive oils

The effect of extraction systems on the phenolic composition of virgin olive oils obtained from two different Italian cultivars (Coratina and Oliarola) was determined. The oils extracted using two-phase centrifugation showed in all cases higher phenolic concentration in comparison to oils obtained from three-phase centrifugation. In particular, the highest differences were observed for aglykone derivatives of oleuropein (3,4-DHPEA-EDA and 3,4-DHPEA-EA) that are the most concentrated antioxidant phenolic compounds of virgin olive oil. These results were confirmed by the autoxidation stability of the oils examined.     

Selective adsorption of oleuropein from olive (Olea europaea) leaf extract using macroporous resin

Extraction, optimization, and adsorption of oleuropein from olive (Olea europaea) leaves were carried out, respectively. Face-centered composite design model was chosen for designing the experimental conditions for extraction of olive leaves through response surface methodology. Olive leaf extract obtained under the optimum conditions was concentrated by several macroporous resins (Amberlites XAD 2, XAD 4, XAD 7HP, and XAD 16). The crude and purified extracts were evaluated according to their total phenolic material (TPM) and oleuropein concentration. XAD 7HP showed the best performance regarding adsorption (91%) and desorption ratio (97%) for oleuropein. Pseudo-first- and second-order and Elovich kinetic models were efficient to represent the experimental data for the adsorption of TPM and oleuropein with high correlation coefficients. Equilibrium data were fitted to Langmuir and Freundlich isotherms at four different temperature values. The antioxidant capacity of the extracts was evaluated with several assays such as Cupric ion reducing antioxidant capacity, 2,2-diphenyl-1-picrylhydrazyl, and 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt.     

Modification of Phenolic Compounds and Volatile Profiles of Chemlali Variety Olive Oil in Response to Foliar Biofertilization

The main objective of this work was to study the effects of foliar biofertilizers on individual volatile profiles and phenolic compounds of olive oil (Olea europaea L. cv. Chemlali). Three foliar biofertilizers were used in two successive application seasons: T1 (rich in nitrogen, phosphorus and potassium); T2 (rich in calcium); and T3 (application of both T1 and T2). Results showed that foliar fertilization with T2 increased the phenolic compound contents (e.g., oleuropein aglycone and decarboxymethyl ligstroside aglycone) of Chemlali olive oil. It also enhanced the levels of many volatile compounds responsible for the good flavor of olive oil such as hexanal. However, T1-tested fertilizer led to a significant decrease in the content of phenolic compounds, although they seemed to improve significantly the levels of the majority of volatile compounds, especially hexanal. Based on these results, a significant relationship between plant nutrition and quality of oil was observed. Our results demonstrated a potential positive influence on the concentration of sensory quality compounds under T2 (Ca²⁺-based fertilizer). This result should be considered in the design of foliar nutrient application management strategies for olive trees.     

Enhancement of Bioactive Phenols and Quality Values of Olive Oil by Recycling Olive Mill Waste Water

Mature ‘Chondrolia Chalkidikis’ olives were processed in an industrial olive oil mill equipped with a three‐phase decanter. Water was added to the decanter at a 1:2 water‐to‐paste ratio. Olive mill waste water (ΟΜWW) was used to replace the added water at a rate of 50 or 100%. Following the final separation, the obtained oil was used for chemical analysis and sensory evaluation. All oils had similar acidity, peroxide and Κ values. OMWW‐treated olive oils presented higher total phenolic content and higher antioxidant activity based on DPPH and oven tests, but lower chlorophyll and carotenoids content. However, there was no significant difference between the 50 and 100% replacement. The phenolic profile of the treated olive oils analyzed by quantitative ¹Η NMR revealed more than twofold oleocanthal and oleacein as well as oleuropein and ligstroside aglycone contents than in the control. Sensory evaluation of treated oils also showed an enhancement of fruity, bitter and pungent attributes compared to the control.     

Molecular Imprinted Solid-Phase Extraction System for the Selective Separation of Oleuropein from Olive Leaf

Oleuropein has many antimicrobial, antiviral, and anticancer features found in olive leaf. Therefore, its isolation from olive leaf is very important in such kinds of applications. In this study, a solid-phase extraction system based on the molecularly imprinted polymer (MIP) was proposed for the selective separation of oleuropein from olive leaf. First, oleuropein imprinted polymer has been prepared by the suspension polymerization using methacrylolamidoantiprine–iron (III) metal-chelate monomers. After that, the oleuropein adsorption capacity and selectivity of the prepared imprinted polymer has been determined. The maximum adsorption capacity of oleuropein has found to be 140 mg g^?1. Finally, MIP has been used as a sorbent in the solid-phase extraction for the separation of oleuropein from crude extract of olive leaves. The oleuropein analyses have been realized by high performance liquid chromatography. The obtained results indicated that the prepared molecularly imprinted sorbent could be used for at least 10 times for purification of oleuropein from olive leaf. The application of the proposed system in the real sample showed that 24.2 mg pure oleuropein could be obtained from 1.0 g of crude olive leaf extract. As a result, the low cost, simple, and selective adsorbent has been developed for oleuropein adsorption. Supplemental materials are available for this article. Go to the publisher's online edition of Separation Science & Technology to view the supplemental file.     

Stability of refined olive oil and olive‐pomace oil added by phenolic compounds from olive leaves

Refined olive oil and olive‐pomace oil were enriched with olive leaf phenolic compounds in order to enhance its quality and bring it closer to virgin olive oil. The changes that occurred in the concentrations of pure oleuropein, oleuropein aglycone, hydroxytyrosol acetyl and α‐tocopherol at 400 µg/kg of oil during the storage of refined olive oil and olive‐pomace oil under accelerated conditions (50 °C) were investigated. In a period of 4 months, α‐tocopherol decomposed by 75% whereas less than 40% of the phenols were lost. During storage, enzymatic olive leaf extract hydrolysate that contains two major compounds, hydroxytyrosol and oleuropein aglycone showed the highest antioxidant activity and the lowest detected stability, followed by oleuropein. The oleuropein in olive leaf extracts exhibited similar degradation profiles, reducing by 60–50% and 80% for the olive oil and olive‐pomace oil in 6 months, respectively. The acetylated extract, however, displayed a loss of 10 and 5% in olive oil and olive‐pomace oil, respectively. In the fatty acid composition, an increase in oleic acid and a decrease in linoleic acid were observed. The antiradical activities of the olive oil and olive‐pomace oil enriched with olive leaf phenolic compounds at 400 ppm showed that enzymatic hydrolysate extract had the highest protective effect against oil oxidation. Based on the Rancimat method, the oils with added leaf enzymatic hydrolysate extract had the lowest peroxide value and the highest stability. After 6 months of storage and at 120 °C, the oxidative resistance of refined olive oil and olive‐pomace oil reached 0.71 and 0.89 h, respectively, whereas that of the non‐enriched samples fell to zero.     

Characterization of Aegean Olive Oils by Their Minor Compounds

This study presents a combined approach of establishing cultivar differences between Aegean olive oils, obtained from economically important olive oil producing cultivars (cv. Ayvalik and Memecik), based on chemometric evaluation of their content and in particular composition of the minor compounds. Evaluation of minor compounds with principal component analysis and linear discriminant analysis (LDA) indicated differentiation according to the cultivars. LDA produced a 100% correct group classification. Moreover, stigmasterol, apparent β-sitosterol and total sterols were found to have the highest discriminating power. Memecik oils were characterized by the highest content of antioxidant compounds (α-tocopherol, phenolic compounds and total phenolic compounds). On the other hand, Ayvalik oil had the highest level of total sterols. The data were analyzed statistically to evaluate the differences according to variety and crop season. The minor compounds of Ayvalik and Memecik oils presented statistically significant differences (p < 0.01) according to variety, except for the hydroxytyrosol and clerosterol content. The amount of α-tocopherol, total phenolic compounds, apparent β-sitosterol and total sterols varied with respect to crop season. A good correlation was observed between the amount of α-tocopherol, total phenolic compounds, apparent β-sitosterol and total sterols and some climatic variables.     

Postharvest Heat Treatment for Olive Oil Debittering at the Industrial Scale

To enhance the debittering of olive oil, 500-kg olive fruit (Olea europaea L.) samples in duplicate from different olive cultivars and orchard locations in Spain (Manzanilla olive fruits from Villarrasa during the 2002/2003, 2004/2005 and 2005/2006 seasons, or from Dos Hermanas during the 2004/2005 and 2005/2006 seasons, Picual olive fruits from Cabra during the 2004/2005 season and Verdial olives from Villarrasa during the 2004/2005 and 2005/2006 seasons) were treated by dipping in hot water under different conditions (50–68 °C for 3 or 5 min), which had been previously determined based on laboratory-scale experiments, and subsequently processed for virgin olive oil extraction. Heat treatment produced a change in the intensity of the oil bitterness in all cases, increased the pigment content, decreased stability and reduced the sensory freshness of the oil. Although heat treatment reduced the phenolic content of the oil, this effect was not uniform among the different phenolic compounds and depended on the crop season and olive variety. Therefore, the determination of debittering conditions will require a series of preliminary laboratory-scale experiments.