Nikolaos Lemonakis

Pyrrolizidine alkaloids in medicinal tea of Ageratum conyzoides

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.

Oral administration of chios mastic gum or extracts in mice: quantification of triterpenic acids by liquid chromatography-tandem mass spectrometry.

Chios mastic gum, the resin obtained as an exudate from the trunk and branches of Pistacia lentiscus L var. chia, is used extensively as a constituent of herbal drugs or functional foods. The oral absorption of its major constituents still remains unclear. In the context of identifying the features of mastic gum that are responsible for either therapeutic effects or effects of nutritional value, a methodology based on high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (MS/MS) was developed and applied for the quantification of mastic gum triterpenic acids, 24Z-isomasticadienonic acid (IMNA), and 24Z-isomasticadienolic acid (IMLA) in mouse plasma. The specific compounds were selected based on their biological activity and potential against Helicobacter pylori. Concentrations were determined simultaneously in mouse plasma after oral administration of mastic gum or total mastic extract without polymer (TMEWP) in order to evaluate the role of the natural polymer, poly-β-myrcene, in the absorption process. Following TMEWP administration in mice, circulating IMNA and IMLA plasma levels were significantly higher (approximately 10-fold) in comparison to IMNA and IMLA plasma levels following total mastic gum administration (CMG), suggesting that the polymer plays a critical role in the absorption process. More specifically following TMEWP administration, Cmax plasma values were 3300 ± 859 ng/mL for IMNA and 163 ± 58 ng/mL for IMLA. In comparison, following CMG administration, Cmax plasma values were 329 ± 57 ng/mL for IMNA and 28 ± 8 ng/mL for IMLA. The methodological approaches presented in this study, along with the findings, offer valuable information on the availability of bioactive components following ingestion of mastic and facilitate the uses of mastic either as an ingredient of functional foods or as a herbal drug.

Hydroxytyrosol ameliorates metabolic, cardiovascular and liver changes in a rat model of diet-induced metabolic syndrome: pharmacological and metabolism-based investigation.

Graphical abstract Figure. No Caption available. Abstract 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 (20 mg/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.

Development and Validation of a Combined Methodology for Assessing the Total Quality Control of Herbal Medicinal Products – Application to Oleuropein Preparations

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.

The LC-MS-based metabolomics of hydroxytyrosol administration in rats reveals amelioration of the metabolic syndrome

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.

Optimization of parameters affecting signal intensity in an LTQ-orbitrap in negative ion mode: A design of experiments approach

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 (ESI) 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.

Alteration in the liver metabolome of rats with metabolic syndrome after treatment with Hydroxytyrosol. A Mass Spectrometry And Nuclear Magnetic Resonance - based metabolomics study

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 bioanalytical method based on UHPLC-HRMS/MS for the quantification of oleuropein in human serum. Application to a pharmacokinetic study.

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.

Trans-crocin 4 is not hydrolyzed to crocetin following i.p. administration in mice, while it shows penetration through the blood brain barrier

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 i.p. 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 i.p. 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.