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.

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.

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.

Changes in phenolic composition in olive tree parts according to development stage.

The aim of this study was to investigate the changes in the phenolic profile in different organs (buds, flowers, fruits and leaves) from Chemlali olive variety cultivated in the Center of Tunisia according to development stage. Phenolics recovery was carried out using aqueous methanol from freeze-dried powdered tissues. The extracts were then analyzed by using high performance liquid chromatography coupled to time of flight and ion trap mass spectrometry detectors. Qualitatively, secoiridoids, flavonoids, simple phenols, cinnamic acid derivatives and lignans were identified in the analyzed extracts. Quantitatively, floral buds showed the highest phenolic contents compared to the other tissues under study. The highest content of secoiridoids was observed for unripe fruit extract, whereas the highest content of flavonoids was registered for floral buds.

LC-MS-based metabolite profiling of methanolic extracts from the medicinal and aromatic species Mentha pulegium and Origanum majorana.

INTRODUCTION: There has been increasing interest dedicated to the phenolic compounds with a view to their antioxidant and healthy properties. Recent studies have focused on plants from the Lamiaceae family with special interest in phenolic compounds antioxidant potential. OBJECTIVE: The metabolite profile of methanolic extracts from two Lamiacea medicinal plants was investigated. MATERIALS AND METHODS: Mentha pulegium and Origanum majorana methanolic extracts were analysed using reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) coupled to electrospray ionisation quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS) detection in the negative ion mode. RESULTS: A total of 85 metabolites were characterised from different families, such as organic acids and derivatives, amino acids and derivatives, nucleosides, phenolic compounds as well as other polar metabolites, by using the MS and MS/MS information provided by the QTOF-MS. However, the total phenols and flavonoids were also quantified spectrophotometrically and they registered higher amounts in Mentha pulegium than in Origanum majorana extract. Gallocatechin was the major compound in M. pulegium extract whereas quercetin dimethyl ether, jaceidin and dihydrokaempferide were the major ones in O. majorana extract. CONCLUSION: The distribution of phenolic compounds in the methanolic extract showed a variation among studied plants. Mentha pulegium can be considered as a source of gallocatechin.

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.

UPLC-QTOF/MS for a rapid characterisation of phenolic compounds from leaves of Myrtus communis L.

INTRODUCTION: Although continuous research has been conducted on the biological activities of myrtle and the characterisation of its essential oil, few studies have focused on its phenolic composition despite major beneficial properties. OBJECTIVE: To carry out a comprehensive characterisation of infusion and methanolic extract from myrtle leaves by UPLC-QTOF/MS. METHODS: Myrtle-leaf infusions, prepared using deionised water, and the methanolic extracts were analysed by reversed-phase ultra-performance liquid chromatography (UPLC) coupled to electrospray ionisation quadrupole time-of-flight mass spectrometry (ESI/QTOF/MS). The MS and MS/MS experiments were conducted using the negative-ionisation mode, in order to provide molecular-mass information and production spectra of the compounds for structural elucidation. RESULTS: The analytical method applied enabled the characterisation of several compounds such as gallic acid and galloyl derivatives, ellagic acid and derivatives, hexahydroxydiphenolyl and derivatives, flavonoids, lignans and gallomyrtucommulones. Flavonoids, ellagic acid and its derivatives and gallic acid and its derivatives formed the major fractions. CONCLUSION: UPLC combined with QTOF/MS is a powerful analytical method for characterising infusions and alcoholic extracts from myrtle leaves.

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.

Classification of 'Chemlali' accessions according to the geographical area using chemometric methods of phenolic profiles analysed by HPLC-ESI-TOF-MS.

The present work describes a classification method of Tunisian 'Chemlali' olive oils based on their phenolic composition and geographical area. For this purpose, the data obtained by HPLC-ESI-TOF-MS from 13 samples of extra virgin olive oils, obtained from different production area throughout the country, were used for this study focusing in 23 phenolics compounds detected. The quantitative results showed a significant variability among the analysed oil samples. Factor analysis method using principal component was applied to the data in order to reduce the number of factors which explain the variability of the selected compounds. The data matrix constructed was subjected to a canonical discriminant analysis (CDA) in order to classify the oil samples. These results showed that 100% of cross-validated original group cases were correctly classified, which proves the usefulness of the selected variables.

Optimization of microwave-assisted extraction for the characterization of olive leaf phenolic compounds by using HPLC-ESI-TOF-MS/IT-MS(2).

In the present work, a simple and rapid method for the extraction of phenolic compounds from olive leaves, using microwave-assisted extraction (MAE) technique, has been developed. The experimental variables that affect the MAE process, such as the solvent type and composition, microwave temperature, and extraction time, were optimized using a univariate method. The obtained extracts were analyzed by 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)) to prove the MAE extraction efficiency. The optimal MAE conditions were methanol:water (80:20, v/v) as extracting solvent, at a temperature equal to 80 °C for 6 min. Under these conditions, several phenolic compounds could be characterized by HPLC-ESI-MS/MS(2). As compared to the conventional method, MAE can be used as an alternative extraction method for the characterization of phenolic compounds from olive leaves due to its efficiency and speed.

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.

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.

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.

Exploratory characterization of the unsaponifiable fraction of tunisian virgin olive oils by a global approach with HPLC-APCI-IT MS/MS analysis.

The unsaponifiable fraction of six Tunisian monovarietal virgin olive oils from the region of Medenine was evaluated within a single chromatographic run by using HPLC-APCI-tandem MS. Separation of the compounds under study was achieved by the RP-LC method, giving a reasonable analysis time and good resolution. Detection was done by an ion trap (working alternatively in MS and MS/MS modes), the fact which made our method suitable to unequivocally identify a high number of compounds belonging to different families of the unsaponifiable fraction of oil and to carry out their reliable and sensitive quantification. A great amount of qualitative information was generated in every analysis, although we focused on the quantification of sterols, tocopherols, and triterpenic dialcohols since their standards were commercially available. The limits of detections achieved were within the range of 1.21 and 10.31 microg/kg for sitostanol and beta-sitosterol, respectively. Significant differences were observed in the composition of the studied olive cultivars. Jemri Ben Guerdane oil was the richest one in terms of all of the sterols under study. alpha-Tocopherol was the main vitamin E isomer in all samples, ranging from 70.14 to 130.72 mg/kg. Principal component analysis (PCA) and cluster analysis were applied to the whole data set in order to explore the distribution of the olive cultivars according to their oil composition.

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.

Multi-component analysis (sterols, tocopherols and triterpenic dialcohols) of the unsaponifiable fraction of vegetable oils by liquid chromatography-atmospheric pressure chemical ionization-ion trap mass spectrometry.

A simple and sensitive method for the analysis of sterols, tocopherols and triterpenic dialcohols from the unsaponifiable fraction from oil samples in a single analytical run using liquid chromatography coupled to mass spectrometry was developed. With this method, the compounds could be detected directly after dissolving the unsaponifiable fraction in acetonitrile without necessity of time-consuming sample pre-treatment or derivatization. Separation of the analytes was carried out at room temperature, by using a C18 column (5 microm i.d. 3.0 mm x 250 mm) with a linear gradient of acetonitrile/water (0.01% acetic acid) at a flow rate of 1.5 mL/min. The full scan mass spectra of the investigated compounds were measured by an ion trap mass spectrometer equipped with an APCI ion source. The optimized methodology was suitable for the identification of 23 compounds belonging to different families present in olive oil and other kinds of oils, as well as for the quantification of 15 analytes (vs. their commercial standards).