Olea europaea L. Flowers as a new promising anticancer natural product: phenolic composition, antiproliferative activity and apoptosis induction.

The aim of this study was to characterise phenolic compounds of olive flower obtained from Olive tree cultivar Chemlali and to investigate their anticancer effect on MCF-7 cells. Phenolic characterisation was determined using LC/MS-MS. Cytotoxicity of the extract was determined using MTT. Biochemical markers of apoptosis were evaluated by immunoblotting. Our results showed that olive flower contained significant amounts of phenolic compounds mainly flavonoids, secoiridoids and simple phenols. Furthermore, the phenolic extract exerted a significant reduction in MCF-7 cell viability (EC50 values equal to 220.8 µg/ml). Western blot analysis revealed the presence of the cleaved forms of Parp-1. The DAPI staining analysis demonstrated a significant reduction in the number of cells and a considerable change in the morphology of the treated cells. In conclusion, Olea europaea. L flower contained great amounts of different bio-phenols able to reduce the proliferative activity of breast cancer MCF-7 cells by the induction of apoptosis.

Olive Leaf Addition Increases Olive Oil Nutraceutical Properties.

The aim of the present research was to study the effects of olive leaf addition (0 and 3%) on the major antioxidants and the antioxidant activity of Neb Jmel and Oueslati olive oils. Olives and leaves of the two Tunisian varieties were harvested during the 2016/2017 crop season. Both leaves and oils were characterised for their concentrations in phenolics, tocopherols and antioxidant power. Other parameters such as free acidity, peroxide value, chlorophyll and carotenoid concentrations were also taken into consideration. Compared to Oueslati, the Neb Jmel oil showed a lower free acidity (50%) and peroxide value (5.6-fold), and higher chlorophyll (1.6-fold), total phenolics (1.3-fold), flavonoid (3-fold) and oleuropein derivative (1.5-fold) concentrations, in addition to an increased antioxidant activity (1.6-fold). Leaf addition promoted a significant increment in total chlorophyll, α-tocopherol and phenolics in both varieties, above all in Oueslati oil, due to a higher abundance of bioactive constituents in the corresponding leaves. In particular, chlorophyll and carotenoid concentrations reached values twice higher than in Neb Jmel leaves, and flavonoids and oleouperin derivatives were three-fold higher. This prevented the oxidation and the formation of peroxides, reducing the peroxide value of the fortified oil to the half. The results provide evidence on the performance of the Tunisian Neb Jmel and Oueslati varieties, showing that their oils present a chemical profile corresponding to the extra virgin olive oil category and that, after leaf addition, their nutritional value was improved.

Physical activity and Mediterranean diet based on olive tree phenolic compounds from two different geographical areas have protective effects on early osteoarthritis, muscle atrophy and hepatic steatosis.

PURPOSE: Osteoarthitis (OA) leads to progressive loss of articular cartilage, pain and joint disability. An acute injury constitutes an important risk factor for early OA, determining an inflammatory process responsible of cartilage degeneration and muscle atrophy, due to the joint pain and immobility. The study aims to assess the effects of conjugation of physical activity and diet enriched by olive tree compounds [extra virgin olive oil (EVOO) and olive leaf extract (OLE)], on the musculoskeletal system in OA rat model. METHODS: OA was induced by anterior cruciate ligament transection and confirmed by Mankin and OARSI scores. Rats were subjected to physical activity on treadmill 5 days a week for 10 min daily and fed with experimental diets (standard diet enriched with Sicilian EVOO, Tunisian EVOO and Tunisian EVOO-OLE) for 12 weeks. Immunohistochemistry was used to evaluate IL-6 and lubricin expression in cartilage tissue and ELISA was used to quantify these proteins in serum at different time points. Histology and histomorphometry analysis were done to valuate liver steatosis, muscle atrophy and cartilage pathological changes. RESULTS: Compared to the OA group, the experimental groups showed general increased lubricin and decreased IL-6 expression, significant muscle hypertrophy and no signs of liver steatosis, suggesting the beneficial effects of physical activity coupled with EVOO-enriched diets on rat articular cartilage. Interestingly, the best result was shown for Sicilian EVOO-enriched diet. CONCLUSION: In conclusion, the conjugation of physical activity and EVOO-enriched diet determines a significant articular cartilage recovery process in early OA.

Use of Tunisian flavored olive oil as anisakicidal agent in industrial anchovy marinating process.

BACKGROUND: Natural compounds are more frequently used against Anisakis, responsible for the important fish-borne disease anisakidosis. The aim of the study was to evaluate the effectiveness of enriched Tunisian olive oil with different spices (cumin, turmeric, clove, thyme, and lemon) against Anisakis larvae type 1. RESULTS: In vitro experiment: larvae were submerged separately in the aforementioned oils and then examined to check viability. For each oil, LT50 and LT100 were calculated. Turmeric and cumin oils are the most effective against the parasites; followed by lemon, thyme and clove oils. For the in vivo experiment, turmeric and cumin oils were tested in anchovy fillets previously artificially parasitized with L3 larvae. Cumin was the most effective against parasites (dead after 5 days) compared with turmeric (8 days). For the two oils, the resulting odor was pleasant, as was the taste, while changes in color were much more evident in turmeric fillets. CONCLUSION: All the flavored oils demonstrated a good nematodical action against Anisakis. Cumin oil was the most effective against encysted larvae. Turmeric oil showed the best activity in the in vitro experiment. The use of flavored oils in the marinating process could represent an efficient strategy to devitalize Anisakis. © 2017 Society of Chemical Industry.

Olive oil polyphenols extracts inhibit inflammatory markers in J774A.1 murine macrophages and scavenge free radicals.

Here we evaluate the olive oil antiradical and anti-inflammatory potential through its polyphenols extracts and examine the influence of olive maturity on olive oil quality properties, polyphenols composition and biological potentials. Samples have been obtained from minor Tunisian olive cultivars (Chemchali, Fouji and Zarrazi) at different maturity indices. Principal quality properties were evaluated and polyphenols analysis was carried out by Folin Ciocalteu reagent and HPLC-UV-MS. Antiradical activity was examined by DPPH and FRAP scavenging assays while J774A.1 murine macrophages were used to evaluate anti-inflammatory potential by analyzing NO production with Griess reagent method and iNOS and COX-2 expression by cytofluorimetric analysis. Our results revealed that quality characteristics, total phenol content, as well as phenolic compound concentrations were significantly affected by the olive maturity levels. On the other hand, the polyphenols extracts showed an interesting radical scavenging capacity and a potential ability to inhibit inflammatory markers at 90% for NO release and 75% for iNOS expression. Thus, our study establishes that olive oil through its polyphenols extracts has a substantial antiradical and anti-inflammatory potential. Likewise a lot of attention should be attributed to olive ripening level in order to decide the optimum harvesting time.

Pedologic Factors Affecting Virgin Olive Oil Quality of "Chemlali" Olive Trees (Olea europaea L.).

The aim of this study examined the characterization of extra virgin olive oil samples from the main cultivar Chemlali, grown in five olive orchards with different soil type (Sandy, Clay, Stony, Brown, Limestone and Gypsum). Volatile compounds were studied using headspace-solid phase micro-extraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) technics. Moreover, the sterol profile was established using gas chromatography-mass spectrometry. 35 different volatile compounds were identified: alcohols, esters, aldehydes, ketones and hydrocarbons. The chemical composition of the volatile fraction was characterized by the preeminence of 2-hexenal (32.75%) and 1-hexanol (31.88%). Three sterols were identified and characterized. For all olive oil samples, ß-sitosterol (302.25 mg/kg) was the most abundant sterol. Interestingly, our results showed significant qualitative and quantitative differences in the levels of the volatile compounds and sterols from oils obtained from olive trees grown in different soil type.

Drought priming improves subsequent more severe drought in a drought-sensitive cultivar of olive cv. Chétoui.

Drought is a major factor limiting crop production worldwide. The objective of this study was to test whether pre-exposure to drought can enhance the subsequent drought response of a drought-sensitive variety of olive cv. Chétoui. Seven-months old olive plants were grown in a controlled conditions and divided into control plants (irrigated daily), primed plants (PP, primed by exposure to drought for 21 days, re-watered for 60 days and then exposed to water depletion for 30 days) and non-primed plants (NPP, well watered for 81 days and immediately followed by intermediate drought as PP). Compared to the non-primed plants, primed plants showed an improvement in biomass production and healthy values of photosynthesis parameters with a higher accumulation of photosynthetic pigments. Additionally, the data of chlorophyll fluorescence were significantly similar to those of control, implying that no photodamage was occurred. Moreover, primed plants exhibited high accumulation of total sugar and proline which lead to the better water status maintenance. The lower level of oxidative status measured in term of hydrogen peroxide (H2O2), malondiadehyde (MDA) and electrolyte leakage (EC) in primed plants confirmed the alleviation of oxidative stress. Furthermore, the primed plants possessed more effective oxygen scavenging systems as exemplified by the increased activities of CAT, SOD, GP and high accumulation of polyphenols, resulting in a better maintenance in homeostasis of ROS production. Our investigation is indicative of the result of the benefit memory effects caused by stress pre-exposure in young olive plants cv.'Chétoui' to overcome subsequent stress.

Differential metabolomic analysis of the potential antiproliferative mechanism of olive leaf extract on the JIMT-1 breast cancer cell line.

A new differential metabolomic approach has been developed to identify the phenolic cellular metabolites derived from breast cancer cells treated with a supercritical fluid extracted (SFE) olive leaf extract. The SFE extract was previously shown to have significant antiproliferative activity relative to several other olive leaf extracts examined in the same model. Upon SFE extract incubation of JIMT-1 human breast cancer cells, major metabolites were identified by using HPLC coupled to electrospray ionization quadrupole-time-of-flight mass spectrometry (ESI-Q-TOF-MS). After treatment, diosmetin was the most abundant intracellular metabolite, and it was accompanied by minor quantities of apigenin and luteolin. To identify the putative antiproliferative mechanism, the major metabolites and the complete extract were assayed for cell cycle, MAPK and PI3K proliferation pathways modulation. Incubation with only luteolin showed a significant effect in cell survival. Luteolin induced apoptosis, whereas the whole olive leaf extract incubation led to a significant cell cycle arrest at the G1 phase. The antiproliferative activity of both pure luteolin and olive leaf extract was mediated by the inactivation of the MAPK-proliferation pathway at the extracellular signal-related kinase (ERK1/2). However, the flavone concentration of the olive leaf extract did not fully explain the strong antiproliferative activity of the extract. Therefore, the effects of other compounds in the extract, probably at the membrane level, must be considered. The potential synergistic effects of the extract also deserve further attention. Our differential metabolomics approach identified the putative intracellular metabolites from a botanical extract that have antiproliferative effects, and this metabolomics approach can be expanded to other herbal extracts or pharmacological complex mixtures.

Triacylglycerols composition and volatile compounds of virgin olive oil from Chemlali cultivar: comparison among different planting densities.

The present study focused on the comparison the chemical composition of virgin olive oil samples obtained from fruits of the main Tunisian olive cultivar (Chemlali) grown in four planting densities (156, 100, 69, and 51 trees ha(-1)). Despite the variability in the triacylglycerols and volatile compounds composition, the quality indices (free fatty acids, peroxide value, and spectrophotometric indices K(232) and K(270)) all of the virgin olive oils samples studied met the commercial standards. Decanal was the major constituent, accounting for about 30% of the whole volatiles. Moreover, the chemical composition of the volatile fraction of the oil from fruits of trees grown at the planting density of 156, 100, and 51 trees ha(-1) was also characterised by the preeminence of 1-hexanol, while oils from fruits of trees grown at the planting density of 69 trees ha(-1) had higher content of (E)-2-hexenal (20.3%). Our results confirm that planting density is a crucial parameter that may influence the quality of olive oils.

Use of advanced techniques for the extraction of phenolic compounds from Tunisian olive leaves: phenolic composition and cytotoxicity against human breast cancer cells.

A comparison among different advanced extraction techniques such as microwave-assisted extraction (MAE), supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE), together with traditional solid-liquid extraction, was performed to test their efficiency towards the extraction of phenolic compounds from leaves of six Tunisian olive varieties. Extractions were carried out at the best selected conditions for each technique; the obtained extracts were chemically characterized using high-performance liquid chromatography (HPLC) coupled to electrospray time-of-flight mass spectrometry (ESI-TOF-MS) and electrospray ion trap tandem mass spectrometry (ESI-IT-MS(2)). As expected, higher extraction yields were obtained for PLE while phenolic profiles were mainly influenced by the solvent used as optimum in the different extraction methods. A larger number of phenolic compounds, mostly of a polar character, were found in the extracts obtained by using MAE. Best extraction yields do not correlate with highest cytotoxic activity against breast cancer cells, indicating that cytotoxicity is highly dependent on the presence of certain compounds in the extracts, although not exclusively on a single compound. Therefore, a multifactorial behavior is proposed for the anticancer activity of olive leaf compounds.

The occurrence and bioactivity of polyphenols in Tunisian olive products and by-products: a review.

Polyphenols have become a subject of intense research because of their perceived beneficial effects on health due to their anticarcinogenic, antiatherogenic, anti-inflammatory, and antimicrobial activities. It is well known that olives and their derivatives are rich in phenolic substances with pharmaceutical properties, some of which exert important antioxidant effects. The characterization and quantification of their polyphenol composition is one of the first steps to be taken in any evaluation of the putative contribution of the olive to human health. This review is concerned with polyphenols in Tunisian olive (Olea europaea L.) products (fruit and oil) and some by-products (leaves and olive-mill wastewater) with an emphasis on the analytical methods used, as well as the biological activities described in recent years.

Hydroxytyrosol induces vascular smooth muscle cells apoptosis through NO production and PP2A activation with subsequent inactivation of Akt.

Olive oil has been shown to exhibit beneficial effects in the prevention of cardiovascular diseases although its molecular mechanism still remains unclear. In the present study, we investigated the effect of hydroxytyrosol (HT), a major phenolic component in olive oil and leaves from OLEA EUROPAEA L. (Oleaceae family), on vascular smooth muscle cells (VSMCs) survival, migration, and apoptosis. HT treatment resulted in a dose-dependent decrease of cell survival and migration in the presence or absence of platelet-derived growth factor (PDGF) by inducing apoptosis of VSMCs. HT enhanced nitric oxide (NO) production in a dose-dependent manner, and the NO synthase inhibitor L-NMMA blocked HT-mediated effects on VSMCs survival. HT as well as the NO donor SNAP reduced the phosphorylation levels of Akt, suggesting that HT inactivates Akt via NO production with subsequent apoptosis of VSMCs. Moreover, HT-dependent apoptosis and reduction in the phosphorylation level of Akt were suppressed by okadaic acid, an inhibitor of protein phosphatase 2A (PP2A) that dephosphorylates Akt. In contrast, the phosphorylation of phosphoinositide-dependent protein kinase 1 (PDK1), an upstream activator of Akt, was not affected by HT. Together, these findings indicate that HT could induce VSMCs apoptosis through NO production and PP2A activation followed by inactivation of Akt signaling pathway.

Effects of the planting density on virgin olive oil quality of "Chemlali" olive trees (Olea europaea L.).

Here, we report the characterization of virgin olive oil samples obtained from fruits of the main Tunisian olive cultivar (Chemlali) grown in four planting densities (156, 100, 69, and 51 trees ha(-1)). Olive oil samples obtained from fruits of trees grown at 100 trees ha(-1) had a higher content of oleic acid (65.5%), a higher content of chlorophyll and carotenoids, and a higher content in total phenols (1059.08 mg/kg). Interestingly, olives grown at the two highest planting densities yielded more stable oils than olives grown at the two lowest ones. Thus planting density is found to be a key factor for the quality of olive oils in arid regions.

Microwave heating effects on the chemical composition and the antioxidant capacity of tataouine virgin olive oils from Tunisia.

Four Tunisian virgin olive oils (VOOs), derived from varieties (Chemlali Tataouine, Zarrazi Douirat, Fakhari Douirat, and Dhokar Douirat) grown in the harsh pedoclimatic conditions of the region of Tataouine, were evaluated for their responses to microwave heating. Aside from fatty acid composition, all other evaluated parameters were affected by microwave heating, and their variations depend on the genetic factor. Chemlali Tataouine VOO exhibited the slowest biophenol degradation rate and the least diminution in oxidative stability and consequently, its total fraction and both lipidic and methanolic fractions remained unchanged with an exceptional antioxidant potential. In the remaining studied VOOs, the biophenol contents, the oxidative stability, and the antioxidant potential underwent gradual decreases; nevertheless, their levels at the longer treatment time are close to some fresh VOOs. These results should be taken into consideration when Tataouine VOOs are recommended for microwave heating.

Effect of olive ripeness on chemical properties and phenolic composition of Chétoui virgin olive oil.

BACKGROUND: The aim of the present work was to investigate the influence of fruit ripening on oil quality in an attempt to establish an optimum harvesting time for Chétoui olives, the second main olive variety cultivated in Tunisia. RESULTS: Our results showed that many analytical parameters, i.e., peroxide value, UV absorbance at 232 and 270 nm, chlorophyll pigments, carotenoids and oleic acid contents decreased during ripening, whilst oil content and linolenic acid increased. Free acidity remained practically stable with a very slight rise at the highest maturity index. The trend of oxidative stability, total phenols and o-diphenols, showed an increase at the early stages followed by a reduction at more advanced stages of maturity. The major phenolic compounds, such as hydroxytyrosol, ligstroside aglycon, elenolic acid, acetoxy-pinoresinol and oleuropein aglycon, seemed to have the same behaviour. In the case of tyrosol, a strong decrease was observed directly related with the ripening progress. CONCLUSION: On the basis of the evolution of the analytical parameters studied, the best stage of Chétoui olive fruits for oil processing seems to be at ripeness index higher than 2.0 and lower than 3.0.

Characterization of volatiles in virgin olive oil produced in the Tunisian area of Tataouine.

The Tataouine province in southern Tunisia is well known for its severe pedoclimatic conditions. Using solid phase microextraction (SPME) and gas chromatography (GC), coupled to flame ionization and mass spectrometer detectors, we characterized virgin olive oils from Chemlali Tataouine, Fakhari Douirat, Zarrazi Douirat, and Dhokar Douirat varieties, which grow in the harsh arid region of Tataouine. Significant differences in the proportions of volatile constituents from oils of different varieties were detected. The results showed that lipoxygenase products were generally the major metabolites of the volatile fraction, and (E)-Hex-2-enal was the principal compound characterizing the olive oil headspace for most samples, though the absolute levels varied greatly, never exceeding 76.45 and 32.16%, respectively. The C5 compounds were unusually abundant, comprising 42.97% of the total lipoxygenase products and a remarkably high level of penten dimers. Each autochthonous variety could thus be differentiated according to the percentage of each metabolite.

Sterolic composition of Chétoui virgin olive oil: Influence of geographical origin.

The sterol profile of Tunisian virgin olive oils produced from Chétoui cultivar, the second main variety cultivated in the north of the country, grown under different environmental conditions, was established by gas chromatography using a flame ionisation detector. More than ten compounds were identified and characterised. As expected for virgin olive oil, the main sterols found in all Chétoui olive oils were ß-sitosterol, Δ5-avenasterol, campesterol and stigmasterol. Cholesterol, 24-methylenecholesterol, clerosterol, campestanol, sitostanol, Δ7-stigmastenol, Δ5,24-stigmastadienol, and Δ7-avenasterol were also found in all samples, but in lower amounts. Most of these compounds are significantly affected by the geographical origin. The majority of the Chétoui virgin olive oils analysed respected EC Regulation No. 2568, and in all cases total sterol amounts were higher than the minimum limit set by legislation, ranging from 1017 to 1522mg/kg. Two triterpenic dialcohols (erythrodiol and uvaol), were also detected besides the sterolic components. Their content was below the upper legal limit of 4% in all analysed samples, with a range from 1.2% to 3.2%. These results suggest that, besides the genetic factor, environmental conditions influence the sterolic fraction.

Induction of growth inhibition and differentiation of human leukemia HL-60 cells by a Tunisian gerboui olive leaf extract.

Cancer protection associated with the consumption of olive products is well established, but not for leukemia. The protective effects of olive (Olea europaea L.) leaves were investigated by incubating human promyelocytic leukemia HL-60 cells with olive leaf extracts (OLEs) from seven principal Tunisian olive varieties, namely, Chemchali, Chemlali, Chétoui, Gerboui, Sayali, Zalmati and Zarrazi. The results showed significant growth inhibition of HL-60 cells incubated for 48 h with a 100-fold dilution of each OLE which had been obtained by incubating 10 g of dried leaves in 100 ml of 70% ethanol for one week with subsequent ultrafiltration. DNA fragmentation was observed in the cells incubated for 19 h with a 100-fold dilution of the Chemchali, Chemlali and Zalmati extracts. The results of a nitroblue tetrazolium (NBT) assay revealed NBT reduction, a differentiation marker, by the OLE-treated cells after an overnight incubation. The Gerboui extract showed the highest NBT reduction ability at more than 90%. An HPLC analysis revealed the presence of apigenin 7-glucoside in the extract, which was found in subsequent experiments to be responsible for the Gerboui extract-mediated cell differentiation.

Changes in content and fatty acid profiles of total lipids of two halophytes: Sesuvium portulacastrum and Mesembryanthemum crystallinum under cadmium stress.

Changes in lipid content and fatty acid composition were determined in leaves of two halophytes: Sesuvium portulacastrum and Mesembryanthemum crystallinum exposed to cadmium (Cd). Experiments were carried out using young small-sized plants grown hydroponically (S. portulacastrum) or aseptically germinated seeds (M. crystallinum). Cd treatment was applied at different concentrations (0, 50, 100 and 200microM) for 30 days. At high cadmium doses (200microM), contents of total lipids (TL) and lipid fractions including galactolipids (GL), phospholipids (PL) and neutral lipids (NL) decreased more in M. crystallinum leaves than in S. portulacastrum leaves. Moreover, there were no significant changes in the total fatty acid composition of S. portulacastrum leaves during metal treatment. In contrast, M. crystallinum leaves showed a decrease in the percentage of the tri-unsaturated fatty acid (C18:3), and a corresponding increase in the percentage of di-unsaturated fatty acid (C18:2). These different responses suggested that S. portulacastrum seems to be more feasible for phytoremediation.

Agronomic and molecular analyses for the characterisation of accessions in Tunisian olive germplasm collections

In this study, a total of 14 agronomic traits, five AFLP primer combinations and ten SSR loci were used to describe and to classify a group of Tunisian olive genotypes into groups based on molecular profiles and agronomic traits. The analysis of variance of the agronomical data revealed significant differences among accessions for all measured traits. The mean phenotypic dissimilarity (0.34 with a range of 0.08-0.6) was low in comparison to dissimilarity calculated using AFLP (0.50 with a range of 0.16-0.70) and SSR markers (0.76 with a range 0.35-0.94). The correlation between the agronomical dissimilarity matrix and the matrices of genetic dissimilarity based on SSR and AFLP markers was very weak: 0.156 (p = 0.05) and 0.185 (p = 0.05), respectively. The SSR-AFLP dendrogram based on unweighted pair-group cluster analysis using Jaccard's index revealed that the genetic diversity was predominantly structured according to fruit size. A trend of clustering together of accessions originating from the same or adjacent regions was also observed. The data obtained can be used for the varietal survey and construction of a database of all olive varieties grown in Tunisia and providing also additional information that could form the basis for the rational design of breeding programs.