Oleuropein (OE) is a secoiridoid glycoside occurring mostly in the Oleaceae family and presenting several pharmacological properties, including hypolipidemic and antioxidant properties. Based on these, several dietary supplements containing olive leaf extracts enriched with OE are commercially available in many countries. The current study aimed to examine the effect of supplementation with such an extract on the serum and urine metabolome of young healthy male athletes. For this purpose, applying a randomized, balanced, double-blind study, nine young, healthy males (physical education students) received either a commercially prepared extract or placebo for one week, followed by a two-week washout period; then, they were subsequently dosed with the alternate scheme (crossover design). Urine and serum samples were analyzed using UHPLC-HRMS, followed by evaluation with several multivariate methods of data analysis. The data were interpreted using a multilevel metabolomic approach (multilevel-sPLSDA) as it was found to be the most efficient approach for the study design. Metabolic pathway analysis of the most affected metabolites revealed that tryptophan and acylcarnitine’s biochemistries were most influenced. Furthermore, several metabolites connected to indole metabolism were detected, which may indicate enhanced serotonin turnover. Phenylethylamine and related metabolites, as well as estrone, were connected to enhanced performance. In addition, possible changes to the lipidemic profile and the blood and urine redox statuses were investigated.

Metabolic syndrome (MetS) represents a group of abnormalities that enhances the risk for cardiovascular disease, diabetes and stroke. The Mediterranean diet seems to be an important dietary pattern, which reduces the incidence of MetS. Hydroxytyrosol (HT) - a simple phenol found in olive oil - has received increased attention for its antioxidant activity. Recently, the European Foods Safety Authority (EFSA) claimed that dietary consumption of HT exhibits a protective role against cardiovascular disease. In this study, an experimental protocol has been setup, including isolated HT administration in a diet induced model of MetS in young Wistar rats, in order to find out whether HT has a protective effect against MetS. Rats were randomly divided into two groups nurtured by high-carbohydrate high-fat (H) (MetS inducing diet) and high-carbohydrate high-fat + HT (HHT). HT (20mg/kg/d oral gavage, water vehicle) was administered for 8 weeks on the basal diet. Previous pharmacological evaluation of HT showed that hepatic steatosis was reduced and the inflammatory cells into the liver were infiltrated. These indicate that HT shows bioactivity against metabolic syndrome. Therefore, the metabolomics evaluation of liver extracts would indicate the putative biochemical mechanisms of HT activity. Thus, the extracts of liver tissues were analyzed using Ultra Performance Liquid Chromatography - High Resolution Mass Spectrometry (UPLC-HRMS, Orbitrap Discovery) and Nuclear Magnetic Resonance (NMR) spectroscopy (Bruker Avance III 600MHz). Multivariate analysis was performed in order to gain insight on the metabolic effects of HT administration on the liver metabolome. Normalization employing multiple internal standards and Quality Control-based Robust LOESS (LOcally Estimated Scatterplot Smoothing) Signal Correction algorithm (QC-RLSC) was added in the processing pipeline to enhance the reliability of metabolomic analysis by reducing unwanted information. Experimentally, HHT rats were clearly distinguished from H in PLS-DA, showing differences in the liver metabolome between the groups and specific biomarkers were determined supporting the pharmacological findings. More specifically, HT has shown to be effective towards the mobilization of lipids as various lipid classes being differentially regulated between the H and HHT groups. Interestingly branched fatty acid esters of hydroxy oleic acids (OAHSA) lipids have been shown to be up regulated to the HHT group, denoting the alleviation of the MetS to the animals administered with HT.

A novel, fit-for-purpose, highly sensitive, analytical UPLC-PDA methodology was developed and fully validated, according to ICH, FDA and EMA guidelines, for the rapid and accurate quantification of trans-crocin 4 (TC4) and crocetin (CRC) in mice plasma and brain after Lp. administration. A PDA based methodology shows a wider applicability as it is cost effective and can be easily and seamlessly adopted by the pharma industry. The separation of the analytes was performed on a C18 Hypersil Gold column with 2.5 min run time, employing the internal standard (ISTD) methodology. The two methods were successfully applied for the determination of CRC and TC4 in mouse plasma and brain after Lp. administration of TC4 (50 mg/kg) in a time range of 0-240 min. Due to the selection of i.p. administration route, the first-pass metabolism and/or gastric hydrolysis were bypassed, a fact that enhanced the bioavailability of TC4. Furthermore, TC4 was found to be capable of crossing the Blood Brain Barrier (BBB) and build up levels in the mouse brain, regardless of its highly hydrophilic character. CRC was not detected in any plasma or brain sample, although it has been reported that TC4 quickly hydrolyzes to CRC after p.o. administration. Therefore i.p. administration could be used in the case of TC4 for the accurate determination of its biological role. Overall, the developed methodology offers important information about the bioavailability of TC4 in mouse plasma and for the first time, demonstrates the ability of TC4 to penetrate the BBB and localize inside the brain.

Saffron is the dried stigmas of the flower Crocus sativus L. [Iridaceae]. The main saffron constituents include crocins which are mono- and bis-esters of crocetin with glucose and/or gentiobiose [1]. Several studies indicate that the main carotenoid constituent, trans-crocin-4 (TC4) has shown remarkable activity against Alzheimer's disease [2]. Therefore, a novel UPLC-UV methodology was developed and fully validated, according to FDA and EMA guidelines, for the rapid and accurate quantification of TC4 and crocetin in mice plasma after i.p administration. The separation of the analytes was performed on a C8 Hypersil Gold column with 2.5 min run time, employing the internal standard (IS) methodology. The results show adequate linearity (r2 > 0.996) over a wide concentration range [0.01 – 6 µg/mL (n = 10)] with a Lower Limit of Quantification (LLOQ) value of 0.01 and 0.03 µg/mL for crocetin and for TC4 respectively, whereas the aforementioned methodology demonstrated adequate precision, accuracy, sensitivity and selectivity. The method was successfully applied for the determination of crocetin and TC4 in mouse plasma after 50 and 150 mg/kg TC4 i.p administration in mice. In details, 42 samples of the two administered groups (both male and female mice), covering a sampling period of 0 – 240 min were analyzed. The results showed that circulating TC4 levels were found to be statistically different in female and male population. Indicatively, for the female mice [50 mg/kg TC4], the experimentally determined plasma Cmax TC4 levels were 3304 ± 413 ng/mL (Tmax= 60 min). Interestingly, crocetin was not detected in any plasma sample, although it has been reported that TC4 quickly hydrolyzes to crocetin after p.o administration [3]. Overall, the developed methodology offers important information about the bioavailability of TC4, as well as the differences in TC4 absorption between males and females.

A highly sensitive, rapid and specific ultrahigh-performance liquid chromatography, coupled to negative electrospray ionization high-resolution tandem mass spectrometry, method was developed and validated in order to investigate the absorption of dietary oleuropein (OE) in human subjects. Serum samples were collected at predefined time points, after oral administration of an olive leaf extract enriched in OE (204.4 mg OE per capsule) to two subjects. Subsequently, samples were analyzed by the developed method after a simple solid-phase extraction step. Chromatographic separation was operated with aqueous formic acid, 0.1% (v/v), and acetonitrile following a gradient program at a flow rate of 0.45 mL/min in an RP-C18 (50 × 2.1 mm, 1.9 μm) column with a total run time of 2.7 min. The method was validated and successfully applied to the determination of OE in human serum, with the pharmacokinetic analysis of the data revealing a biphasic response.

Metabolic syndrome is a clustering of interrelated risk factors for cardiovascular disease and diabetes. The Mediterranean diet has been proposed as an important dietary pattern to confer cardioprotection by attenuating risk factors of metabolic syndrome. Hydroxytyrosol (HT) is present in olive fruit and oil, which are basic constituents of the Mediterranean diet. In this study, we have shown that treatment with HT (20mg/kg/d for 8 weeks) decreased adiposity, improved impaired glucose and insulin tolerance, improved endothelial function with lower systolic blood pressure, decreased left ventricular fibrosis and resultant diastolic stiffness and reduced markers of liver damage in a diet-induced rat model of metabolic syndrome. These results were accompanied by reduced infiltration of monocytes/macrophages into the heart with reduced biomarkers of oxidative stress. Furthermore, in an HRMS-based metabolism study of HT, we have identified 24 HT phase I and II metabolites, six of them being over-produced in high-starch, low-fat diet fed rats treated with HT compared to obese rats on high-carbohydrate, high-fat diet. These results provide direct evidence for cardioprotective effects of hydroxytyrosol by attenuation of metabolic risk factors. The implications of altered metabolism of HT in high-carbohydrate, high-fat diet fed obese rats warrant further investigation.

Hydroxytyrosol (HT), an important component of olive fruit and olive oil, improves the signs of metabolic syndrome in rats following chronic treatment. At a dose of 20mg/kg/day, HT decreased adiposity and improved cardiovascular and liver structure and function in rats fed with a high-carbohydrate, high-fat diet. An untargeted metabolomics approach has been employed using both UPLC-Orbitrap and -QqTOF methods to identify the changes induced by chronic HT administration on the plasma metabolome. 31 metabolites have been found to be differentially expressed between the examined groups. HT was shown to decrease biosynthesis of unsaturated fatty acids, fatty acid biosynthesis, and the metabolism of linoleic acid, retinol, sphingolipids and arachidonic acid, whereas glycerolipid metabolism is up-regulated. These are plausible mechanisms for the attenuation by HT of cardiovascular, liver and metabolic changes in high-carbohydrate, high-fat diet fed rats.

A multistage optimization of all the parameters affecting detection/response in an LTQ-orbitrap analyzer was performed, using a design of experiments methodology. The signal intensity, a critical issue for mass analysis, was investigated and the optimization process was completed in three successive steps, taking into account the three main regions of an orbitrap, the ion generation, the ion transmission and the ion detection regions. Oleuropein and hydroxytyrosol were selected as the model compounds. Overall, applying this methodology the sensitivity was increased more than 24%, the resolution more than 6.5%, whereas the elapsed scan time was reduced nearly to its half.A high-resolution LTQ Orbitrap Discovery mass spectrometer was used for the determination of the analytes of interest. Thus, oleuropein and hydroxytyrosol were infused via the instruments syringe pump and they were analyzed employing electrospray ionization (ES!) in the negative high-resolution full-scan ion mode. The parameters of the three main regions of the LTQ-orbitrap were independently optimized in terms of maximum sensitivity. In this context, factorial design, response surface model and Plackett-Burman experiments were performed and analysis of variance was carried out to evaluate the validity of the statistical model and to determine the most significant parameters for signal intensity.The optimum MS conditions for each analyte were summarized and the method optimum condition was achieved by maximizing the desirability function. Our observation showed good agreement between the predicted optimum response and the responses collected at the predicted optimum conditions. (C) 2015 Elsevier B.V. All rights reserved.

Opuntia ficus indica is an important food source with high nutritional value that contains also several bioactive secondary metabolites. In continuation of our research we have performed a comparative study on the chemical content and the biological activity of different tissues (cladode, fruit flesh, flower, fruit peel, and seed). Each of the plant materials was extracted with different solvents and analyzed by HPLC and then by LC-HRMS to compare relative concentrations and identify main phytochemicals. Then all extracts have been assessed for their antioxidant activity against DPPH and ABTS radicals, and for their potential skin whitening properties and toxicity.

MICROSMETICS, is an EU funded project aiming to discover and bring to development innovative anti-ageing cosmeceuticals, originating from microbial biodiversity, using emerging and state of the art technologies in the field of biotechnology, natural products chemistry and applied microbiology.

Oleuropein, a phenolic compound present in olives and extra virgin olive oils, is endowed with in vivo beneficial health effects and might be considered a functional food ingredient. Here, we investigated the health effects of neutraceutical oleuropein supplementation (0.59% w/w) on energy balance at the whole body and molecular level in mice fed a high fat diet (HFD). Oleuropein supplementation (HFD + O) prevented HFD-induced body weight gain resulting in a body weight that was comparable to that of normal fat diet (NFD)-fed mice. Furthermore, indirect calorimetric data, motoric movements, serum glucose and leptin levels, serum and hepatic lipid levels, adipocyte size and adipose tissue gene expression showed an improved health status compared to the control HFD-fed mice. In fact, it appeared indistinguishable between HFD + O-fed mice and NFD-fed mice. Initially, oleuropein might decrease intestinal energy uptake, while on the longer term weight maintenance could be related to an increased satiety signal. Our results indicate that oleuropein supplementation to a HFD can improve health by reducing adiposity.

The agricultural, food and forestry industries produce large amounts of by-products that are considered wastes. However, recent studies have shown that these by-products contain bioactive compounds with several applications in industry.

The Mediterranean diet rich in fruits, vegetables and olive oil has been related to a lower osteoporosis incidence and accordingly to a reduced fracture risk. These observations might be mediated by the active constituents of extra virgin olive oil, and especially polyphenols. In the context of exploring the features of olive oil active constituents on postmenopausal osteoporosis, an extra virgin olive oil total polyphenolic fraction (TPF) was isolated and its effect on the bone loss attenuation was investigated.

Background: Nutritional dose of oleuropein (oleu), which is the main polyphenolic constituent of olive, attenuates ischemia (isc) - reperfusion (rep) injury. Aim: We investigated the effect of pharmacological dose of oleu on infarct size, the intracellular signaling pathway changes and its potential role as postconditioning (PostC) analogue in vivo. Methods: Anesthetized male rabbits were subjected to 30-min regional myocardial isc followed by 3-hour rep. The animals were divided into 6 groups with the following additional interventions: (i) Control group: no further intervention; (ii) PostC group: 8 cycles of 30-sec isc/rep at the onset of rep; (iii) Oleu-50 group and (iv) Oleu-100 group: intravenous bolus administration of 50 and 100 mg.kg-1 oleu at the 20th min of isc, respectively; (v) Oleu-100 +L-NAME group and (vi) Oleu-100+Wortmannin group: administration of the NOS inhibitor L-NAME at a dose of 10 mg.kg-1 and of the PI3K/Akt inhibitor wortmannin at a dose of 60 μg.kg-1, respectively, one min before oleu administration. Circulating oleu plasma levels were assessed at the 1st and 20th min of rep by UHPLC-ESI-HRMS/MS. The percent infarct to area at risk ratio (%I/R) was estimated. In a second series of experiments, rabbits were subjected to the same initial interventions and then tissue samples were quickly excised from the ischemic regions of the heart at the 10th min of rep for eNOS, Akt, GSK3β and STAT3 assessment. Results: A significant %I/R reduction was observed in the Oleu-100 and PostC groups compared to the Control and Oleu-50 groups (20.0±2.5%, 26.4±0.3% vs 49.2±2.0% and 44.2±2.0 respectively, p<0.05). L-NAME and wortmannin did not abolish the cardioprotection afforded by Oleu-100 (23.8±4.42% and 24.5±2.4% respectively, p=NS vs Oleu-100 group).The phosphorylation of e-NOS, Akt, GSK3β and STAT3 was also significantly higher in the Oleu-100 and PostC groups (p<0.05 vs Control). Circulating oleu levels have been found to exhibit a 20-fold increase in the Oleu-100 group compared to the Oleu-50 group at both time points. Conclusion: Higher dose of oleu, given acutely at the index isc and prior to the onset of rep, induced a postconditioning-like effect in anesthetized rabbits, reducing the final infarct size through GSK3β inhibition and STAT3 activation, independently of eNOS and Akt. Cardioprotection from oleu is a dose-dependent effect that is possibly correlated with the increased circulating plasma levels.

It is now widely-recognized that the view that herbal remedies have no adverse effects and/or toxicity is incorrect; some traditionally-used plants can present toxicity. The well-established popular use of Ageratum conyzoides has led to its inclusion in a category of medicinal crude drugs created by the Brazilian Health Surveillance Agency. Ageratum belongs to the Eupatorieae tribe, Asteraceae, and is described as containing toxic pyrrolizidine alkaloids. Aqueous extracts of Ageratum conyzoides L. harvested in Brazil (commercial, flowering and non-flowering samples) were prepared according to the prescribed method and analyzed by HPLC-HRMS. The pyrrolizidine alkaloids lycopsamine, dihydrolycopsamine, and acetyl-lycopsamine and their N-oxides, were detected in the analyzed extracts, lycopsamine and its N-oxide being known hepatotoxins and tumorigens. Together with the pyrrolizidine alkaloids identified by HPLC-HRMS, thirteen phenolic compounds were identified, notably, methoxylated flavonoids and chromenes. Toxicological studies on A. conyzoides are necessary, as is monitoring of its clinical use. To date, there are no established safety guidelines on pyrrolizidine alkaloids-containing plants, and their use in Brazil.

Oleuropein (OE) is a secoiridoid glycoside, which occurs mostly in the Oleaceae family presenting several pharmacological properties, including antioxidant, cardio-protective, anti-atherogenic effects etc. Based on these findings OE is commercially available, as Herbal Medicinal Product (HMP), claimed for its antioxidant effects. As there are general provisions of the medicine regulating bodies e.g. European Medicines Agency, the quality of the HMP's must always be demonstrated. Therefore, a novel LC-MS methodology was developed and validated for the simultaneous quantification of OE and its main degradation product, hydroxytyrosol (HT), for the relevant OE claimed HMP's. The internal standard (IS) methodology was employed and separation of OE, HT and IS was achieved on a C18 Fused Core column with 3.1 min overall run time employing the SIM method for the analytical signal acquisition. The method was validated according to the International Conference on Harmonisation requirements and the results show adequate linearity (r2 > 0.99) over a wide concentration range [0.1–15 μg/mL (n=12)] and a LLOQ value of 0.1 μg/mL, for both OE and HT. Furthermore, as it would be beneficial to control the quality taking into account all the substances of the OE claimed HMP's; a metabolomics-like approach has been developed and applied for the total quality control of the different preparations employing UHPLC-HRMS-multivariate analysis (MVA). Four OE-claimed commercial HMP's have been randomly selected and MVA similarity-based measurements were performed. The results showed that the examined samples could also be differentiated as evidenced according to their scores plot. Batch to batch reproducibility between the samples of the same brand has also been determined and found to be acceptable. Overall, the developed combined methodology has been found to be an efficient tool for the monitoring of the HMP's total quality. Only one OE HMP has been found to be consistent to its label claim.

Metabolic syndrome is a clustering of interrelated risk factors for cardiovascular disease and diabetes. The Mediterranean diet has been proposed as an important dietary pattern to confer cardioprotection by attenuating risk factors of metabolic syndrome. Hydroxytyrosol (HT) is a single phenol present in large amount in olive byproducts and in lower amounts in olive oil and in olive fruits, which are basic constituents of the Mediterranean diet. In the context of identifying the features of HT that are responsible for its effects on the metabolic syndrome, an experimental protocol has been setup encompassing isolated HT administration in a diet induced model of metabolic syndrome in young Wistar rats. Rats were randomly divided into four groups as cornstarch (CS), cornstarch + HT (CSHT), high-carbohydrate high-fat (HF) and high-carbohydrate high-fat + HT (HFHT) (n = 6/group). HT (20 mg/kg/d oral gavage, water vehicle) was administered for 8 weeks on the basal diet (CS or HF). In order to gain insight on the metabolic effects of HT administration on the total biochemical profile (metabolome), an untargeted approach (metabolomics) has been developed and attempted on the rat plasma and urine samples from the metabolic syndrome model. The new biomarkers discovered from multivariate analysis have been identified by comparison with public domain metabolome libraries but also by confirmation using HRMS/MS, highlighting the differences in the biochemistry of HFHT animals versus controls as well as versus CSHT. Fatty acid metabolism was found to be down regulated in HFHT rats while glycerol, which releases from triacylglycerols, was the main up regulated metabolite in HFHT rats and correlates to fatty acid transportation catabolism. HT supplementation for 8 weeks reduced visceral obesity and was associated with improved left ventricular structure and function, reduced blood pressure, improved glucose disposal and reduced hepatic steatosis.

Metabolic syndrome is a clustering of interrelated risk factors for cardiovascular disease and diabetes. Hydroxytyrosol (HT) is a single phenol and it is present in large amount in byproducts from olives. An experimental protocol has been setup encompassing isolated HT administration in a diet induced rat model of metabolic syndrome. The first step was the investigation of the PK/PD of HT in plasma and tissues. As HT does not circulate anymore 30min after the administration, it was pertinent to investigate its metabolic fate. Thus, targeted identification of circulating in plasma HT metabolites was performed using the mass defect filter technique. Additionally, an untargeted approach has been attempted on the PK plasma samples and new biomarkers related to HT administration have been identified. The aforementioned metabonomic methodology has been applied to the samples from the metabolic syndrome model in order to discern differences in the biochemistry within groups. HT supplementation for 8 weeks reduced visceral obesity and was associated with improved left ventricular structure and function, reduced blood pressure, improved glucose disposal and reduced hepatic steatosis and infiltration of inflammatory cells into the liver. Data of the targeted and untargeted approaches will be presented.