Eleni Melliou

Direct Measurement of Oleocanthal and Oleacein Levels in Olive Oil by Quantitative 1H NMR. Establishment of a New Index for the Characterization of Extra Virgin Olive Oils

A new method for direct measurement of the oleocanthal and oleacein levels in olive oil by quantitative (1)H NMR was developed. The method was applied to the study of 175 monovarietal commercial Greek and California olive oil samples. The main findings were as follows: (1) There was a significant variation concerning the concentrations of oleocanthal and oleacein among the studied samples. Their concentrations ranged from nondetectable to 355 mg/kg and their sum (index D1) from 0 to 501 mg/kg. (2) There are olive varieties that independent of geographic origin and harvest time produce oil that contains both compounds in low levels. (3) There is a positive correlation of a high level of oleocanthal and oleacein in olive oils with the early time of harvest. Although there is a need for more extensive study, a new index for the characterization of extra virgin olive oils, which is a combination of D1 = oleocanthal + oleacein level and D2 = oleocanthal/oleacein ratio, seems to be very useful.

Composition and enantiomeric analysis of the essential oil of the fruits and the leaves of Pistacia vera from Greece.

The essential oils of the fruits and the leaves of pistachio (Pistacia vera L.) were analyzed by GC and GC/MS. Fresh unripe pistachio fruits were richer in essential oil (0.5%, w/w) than the leaves (0.1%, w/w). Twenty one compounds were identified in the essential oil of the fruits and the major components were (+)-alpha-pinene (54.6%) and terpinolene (31.2%). The enantiomeric ratio of the major constituents of the essential oil of the fruits was determined using chiral GC/MS and it was found that the (+)/(-)-alpha-pinene ratio was 99.5:0.5, (+)/(-)-limonene 80:20, (+)/(-)-beta-pinene 96:4, and (+)/(-)-alpha-terpineol 0:100. Thirty three compounds were identified in the essential oil of the leaves and the major components were found to be alpha-pinene (30.0%), terpinolene (17.6%) and bornyl acetate (11.3%).

Quantitative measurement of major secoiridoid derivatives in olive oil using qNMR. Proof of the artificial formation of aldehydic oleuropein and ligstroside aglycon isomers.

A previously developed method for measurement of oleocanthal and oleacein in olive oil by quantitative (1)H NMR was expanded to include the measurement of the monoaldehydic forms of oleuropein and ligstroside aglycons. The method was validated and applied to the study of 340 monovarietal Greek and Californian olive oils from 23 varieties and for a 3-year period. A wide variation concerning the concentrations of all four secoiridoids was recorded. The concentration of each one ranged from nondetectable to 711 mg/kg and the sum of the four major secoiridoids (named as D3) ranged from nondetectable to 1534 mg/kg. Examination of the NMR profile of the olive oil extract before and after contact with normal or reversed stationary chromatography phase proved the artificial formation of the 5S,8S,9S aldehydic forms of oleuropein and ligstroside aglycon isomers during chromatography. Finally, methyl elenolate was identified for the first time as a minor constituent of olive oil.

Fatty Acids Derived from Royal Jelly Are Modulators of Estrogen Receptor Functions

Royal jelly (RJ) excreted by honeybees and used as a nutritional and medicinal agent has estrogen-like effects, yet the compounds mediating these effects remain unidentified. The possible effects of three RJ fatty acids (FAs) (10-hydroxy-2-decenoic-10H2DA, 3,10-dihydroxydecanoic-3,10DDA, sebacic acid-SA) on estrogen signaling was investigated in various cellular systems. In MCF-7 cells, FAs, in absence of estradiol (E2), modulated the estrogen receptor (ER) recruitment to the pS2 promoter and pS2 mRNA levels via only ERβ but not ERα, while in presence of E2 FAs modulated both ERβ and ERα. Moreover, in presence of FAs, the E2-induced recruitment of the EAB1 co-activator peptide to ERα is masked and the E2-induced estrogen response element (ERE)-mediated transactivation is inhibited. In HeLa cells, in absence of E2, FAs inhibited the ERE-mediated transactivation by ERβ but not ERα, while in presence of E2, FAs inhibited ERE-activity by both ERβ and ERα. Molecular modeling revealed favorable binding of FAs to ERα at the co-activator-binding site, while binding assays showed that FAs did not bind to the ligand-binding pocket of ERα or ERβ. In KS483 osteoblasts, FAs, like E2, induced mineralization via an ER-dependent way. Our data propose a possible molecular mechanism for the estrogenic activities of RJs components which, although structurally entirely different from E2, mediate estrogen signaling, at least in part, by modulating the recruitment of ERα, ERβ and co-activators to target genes.

Antiproliferative Activity of Greek Propolis

The butanolic extract and the isolated chemical constituents, mainly diterpenes and flavonoids, from Greek propolis have been tested for their cytostatic activities against human malignant and normal cell strains. The extract and the diterpenes were found to be the most active against HT-29 human colon adenocarcinoma cells, without affecting normal human cells. Manool, a diterpene isolated for the first time from Greek propolis, was the most active compound, arresting the cancer cells at the G(2)/M phase of the cell cycle.

Chemical constituents of selected unifloral Greek bee-honeys with antimicrobial activity

(±)-3-Hydroxy-4-phenyl-2-butanone (1) and (+)-8-hydroxylinalool (2) were isolated from Greek honey samples and their structures were determined by spectroscopic and mass spectrometry methods. The enantiomeric ratio of 1 and 2 was determined using chiral GC-MS. Compound 1 has recently been proposed as a possible chemical marker of thyme honey, but although 1, in our hands, was identified only in monofloral thyme honey amongst the honey samples, it was not found in all studied thyme honey samples and additionally its origin was not directly associated with thyme, proving that compound 1 should not be considered as an unambiguous marker of thyme honey. Compound 2 was isolated and identified as the (+)-6S isomer only in monofloral citrus honey. The combination of (+)-8-hydroxylinalool with methyl anthranilate and caffeine could be proposed as a fingerprint marker for the description of citrus honey. All studied honey extracts and the isolated compounds were also tested for their antimicrobial activity.

Oleuropein as a bioactive constituent added in milk and yogurt

Oleuropein is a bioactive natural product from olives known to display a broad variety of health beneficial properties. However its presence in most edible olives is lowered due to debittering. In this respect, we envisaged the incorporation of oleuropein into dairy products (cows milk and yogurt) aiming to produce novel functional foods. Additionally, an analytical method for the monitoring of oleuropein in milk and yogurt was also developed and validated. Oleuropein was not affected during heat treatment of milk, while during the milk fermentation process it was not hydrolysed by the produced acids. Oleuropein was not metabolised by lactic acid bacteria, did not inhibit their growth and its stability in the final products was proven. The novel products displayed same taste, colour and texture as the conventional ones. Results herein indicate that oleuropein can be added as an active ingredient in milk and yogurt preparations to provide two novel functional dairy products.

Ultrahigh-Pressure Liquid Chromatography Triple-Quadrupole Tandem Mass Spectrometry Quantitation of Polyphenols and Secoiridoids in California-Style Black Ripe Olives and Dry Salt-Cured Olives

The chemical composition of finished table olive products is influenced by the olive variety and the processing method used to debitter or cure table olives. Herein, a rapid ultrahigh-pressure liquid chromatography triple-quadrupole tandem mass spectrometry method, using dynamic multiple reaction monitoring, was developed for the quantitation of 12 predominant phenolic and secoiridoid compounds in olive fruit, including hydroxytyrosol, oleuropein, hydroxytyrosol-4-O-glucoside, luteolin-7-O-glucoside, rutin, verbascoside, oleoside-11-methyl ester, 2,6-dimethoxy-p-benzoquinone, phenolic acids (chlorogenic and o-coumaric acids), oleuropein aglycone, and ligstroside aglycone. Levels of these compounds were measured in fresh and California-style black ripe processed Manzanilla olives and two dry salt-cured olive varieties (Mission from California and Throuba Thassos from Greece). Results indicate that the variety and debittering processing method have strong impact on the profile of phenolic and secoiridoid compounds in table olives. The dry salt-cured olives contained higher amounts of most compounds studied, especially oleuropein (1459.5 ± 100.1 μg/g), whereas California-style black ripe olives had a significant reduction or loss of these bioactive compounds (e.g., oleuropein level at 36.7 ± 3.1 μg/g).

Quantitation of oleuropein and related metabolites in decoctions of Olea europaea leaves from ten Greek cultivated varieties by HPLC with diode array detection (HPLC-DAD)

Abstract An extraction procedure and chromatographic methodology for the simultaneous quantitation of four major constituents in the boiling water extracts (decoctions) of Olea europaea leaves has been developed. The four studied constituents were oleuropein, elenolic acid, hydroxytyrosol, and tyrosol. The quantitation was performed using HPLC‐DAD, whereas qualitative data were acquired using LC‐MS. The developed methodology was applied in the study of ten Olea europaea varieties commonly cultivated in Greece. The chromatographic analysis revealed important differences among the varieties. The decoction of variety gaidouroelia was identified as the best source of oleuropein, but it was completely lacking of elenolic acid. The decoction of variety koronaiiki was the best source of hydroxytyrosol, whereas the variety mastoides was the best source of tyrosol and elenolic acid. In addition, the methanol and acetone extracts of one of the studied varieties (koranaiiki) were investigated, in order to compare the concentration of oleuropein in the extracts and the decoction. Interestingly, only a very low percent of the total oleuropein is present in the traditionally prepared decoction, while elenolic acid, which is a minor constituent of the extracts, was found to be one of the major constituents of the decoction.

Chemical constituents of Lavatera trimestris L.--antioxidant and antimicrobial activities.

Nine phenolic compounds, such as cis-/trans-p-coumaric acid, cis-/trans-p-coumaric acid methyl ester, glucose ester of cis-/trans-p-coumaric acid, caffeic acid methyl ester, kaempferol 7-O-β-d-glucoside and kaempferol 3-O-β-d-glucoside, were isolated from Lavatera trimestris flowers by chromatographic techniques and their structures were elucidated by spectral means (NMR). All compounds were tested for their antioxidant activity, while the methanolic extract was tested also for its antimicrobial activity. Also several non-polar constituents have been identified using GC and GC/MS methods. This is the first time that phenolic esters and non-polar constituents were identified in the flowers of L. trimestris L.