Antioxidant Capacity, Antitumor Activity and Metabolomic Profile of a Beetroot Peel Flour.

In this study, a beetroot peel flour was made, and its in vitro antioxidant activity was determined in aqueous (BPFw) and ethanolic (BPFe) extracts. The influence of BPFw on breast cancer cell viability was also determined. A targeted betalain profile was obtained using high-resolution Q-Extractive Plus Orbitrap mass spectrometry (Obrtitrap-HRMS) alongside untargeted chemical profiling of BPFw using Ultra-High-Performance Liquid Chromatography with High-Resolution Mass Spectrometry (UHPLC-HRMS). BPFw and BPFe presented satisfactory antioxidant activities, with emphasis on the total phenolic compounds and ORAC results for BPFw (301.64 ± 0.20 mg GAE/100 g and 3032.78 ± 55.00 µmol T/100 g, respectively). The MCF-7 and MDA-MB-231 breast cancer cells presented reductions in viability when treated with BPFw, showing dose-dependent behavior, with MDA-MB-231 also showing time-dependent behavior. The chemical profiling of BPFw led to the identification of 9 betalains and 59 other compounds distributed amongst 28 chemical classes, with flavonoids and their derivates and coumarins being the most abundant. Three forms of betalain generated via thermal degradation were identified. However, regardless of thermal processing, the BPF still presented satisfactory antioxidant and anticancer activities, possibly due to synergism with other identified molecules with reported anticancer activities via different metabolic pathways.

Synthesis and biological activity of novel 4-aminoquinoline/1,2,3-triazole hybrids against Leishmania amazonensis.

Quinoline and 1,2,3-triazoles are well-known nitrogen-based heterocycles presenting diverse pharmacological properties, although their antileishmanial activity is still poorly exploited. As an effort to contribute with studies involving these interesting chemical groups, in the present study, a series of compounds derived from 4-aminoquinoline and 1,2,3-triazole were synthetized and biological studies using L. amazonensis species were performed. The results pointed that the derivative 4, a hybrid of 4-aminoquinoline/1,2,3-triazole exhibited the best antileishmanial action, with inhibitory concentration (IC50) values of ~1 µM against intramacrophage amastigotes of L. amazonensis , and being 16-fold more active to parasites than to the host cell. The mechanism of action of derivative 4 suggest a multi-target action on Leishmania parasites, since the treatment of L. amazonensis promastigotes caused mitochondrial membrane depolarization, accumulation of ROS products, plasma membrane permeabilization, increase in neutral lipids, exposure of phosphatidylserine to the cell surface, changes in the cell cycle and DNA fragmentation. The results suggest that the antileishmanial effect of this compound is primarily altering critical biochemical processes for the correct functioning of organelles and macromolecules of parasites, with consequent cell death by processes related to apoptosis-like and necrosis. No up-regulation of reactive oxygen and nitrogen intermediates was promoted by derivative 4 on L. amazonensis -infected macrophages, suggesting a mechanism of action independent from the activation of the host cell. In conclusion, data suggest that derivative 4 presents selective antileishmanial effect, which is associated with multi-target action, and can be considered for future studies for the treatment against disease.

Implementation and Performance of the Gas Chromatography/Combustion/Isotope Ratio Mass Spectrometry-Based Method for the Confirmatory Analysis of Endogenous Anabolic Steroids during the Rio de Janeiro Olympic and Paralympic Games 2016.

Carbon isotope ratio (CIR) confirmation is one of the most complex and delicate analyses in the doping control field, due to the nature of the molecules to be confirmed, normally present in urinary samples as a consequence of an endogenous production. The requirements for method validation established by the World Anti-Doping Agency (WADA) have been pushing the accredited laboratories to improve their methods. The choice of the method is always a cost benefit ratio involving a hard-working and time-consuming analysis and the guarantee of reporting of reliable results. This work presents the method fully validated by the Brazilian Doping Control Laboratory as part of the preparation for the Rio de Janeiro Summer Olympic and Paralympic Games 2016. Sample preparation encompassed solid-phase extraction, liquid-liquid extraction, enzymatic hydrolysis, acetylation, and purification by preparative high-performance liquid chromatography, and analyses were performed by gas chromatography/combustion/isotope ratio mass spectrometry. This proved to be a robust method to CIR confirmation in a big event, as demonstrated by the analysis of 179 samples during the Games 2016, from clearly negative results and adverse findings for testosterone (T) and related substances, boldenone and its metabolite, 19-norandrosterone and formestane. Two atypical findings were also reported for T and metabolites.

Phenolic compounds from Viscum album tinctures enhanced antitumor activity in melanoma murine cancer cells.

Cancer is one of the biggest problems in public health worldwide. Plants have been shown important role in anticancer research. Viscum album L. (Santalaceae), commonly known as mistletoe, is a semi-parasitic plant that grows on different host trees. In complementary medicine, extracts from European mistletoe (Viscum album L.) have been used in the treatment of cancer. The study was conducted to identify chemical composition and antitumor potential of Viscum album tinctures. Chemical analysis performed by high resolution chromatography equipped with high resolution mass spectrometer identified caffeic acid, chlorogenic acid, sakuranetin, isosakuranetin, syringenin 4-O-glucoside, syringenin 4-O-apiosyl-glucoside, alangilignoside C and ligalbumoside A compounds. Some of these compounds are probably responsible for the reduction of tumoral cellular growth in a dose-dependent manner. It was observed that melanoma murine cells (B16F10) were more sensitive to V. album tinctures than human leukaemic cells (K562), besides non-tumoral cells (MA-104) had a much lower cytotoxicity to them. Apoptotic-like cells were observed under light microscopy and were confirmed by a typical DNA fragmentation pattern. Additionally, flow cytometry results using Annexin-V/FITC permitted to quantify increased expression of early and late apoptotic markers on tumoral cells, confirming augmented Sub G0 population, which was probably associated with a consistent decrease in G1, and an increase in S or G2/M populations. Results indicate the chemical composition of V. album tinctures influences the mechanisms of in vitro tumoral cell death, suggesting a potential use in cancer pharmacotherapy research.

Optimization of an online heart-cutting multidimensional gas chromatography clean-up step for isotopic ratio mass spectrometry and simultaneous quadrupole mass spectrometry measurements of endogenous anabolic steroid in urine.

Measuring carbon isotope ratios (CIRs) of urinary analytes represents a cornerstone of doping control analysis and has been particularly optimized for the detection of the misuse of endogenous steroids. Isotope ratio mass spectrometry (IRMS) of appropriate quality, however, necessitates adequate purities of the investigated steroids, which requires extensive pre-analytical sample clean-up steps due to both the natural presence of the target analytes and the high complexity of the matrix. In order to accelerate the sample preparation and increase the automation of the process, the use of multidimensional gas chromatography (MDGC) prior to IRMS experiments, was investigated. A well-established instrumental configuration based on two independent GC ovens and one heart-cutting device was optimized. The first dimension (1D) separation was obtained by a non-polar column which assured high efficiency and good loading capacity, while the second dimension (2D), based on a mid-polar stationary phase, provided good selectivity. A flame ionization detector monitored the 1D, and the 2D was simultaneously recorded by isotope ratio and quadrupole mass spectrometry. The assembled MDGC set-up was applied for measuring testosterone, 5α- and 5ß-androstanediol, androsterone, and etiocholanolone as target compounds and pregnanediol as endogenous reference compound. The urine sample were pretreated by conventional sample preparation steps comprising solid-phase extraction, hydrolysis, and liquid-liquid extraction. The extract obtained was acetylated and different aliquots were injected into the MDGC system. Two high performance liquid chromatography steps, conventionally adopted prior to CIR measurements, were replaced by the MDGC approach. The obtained values were consistent with the conventional ones. Copyright © 2016 John Wiley & Sons, Ltd.

Systematic analysis of glycerol: colourimetric screening and gas chromatography-mass spectrometric confirmation.

Glycerol is a naturally occurring polyol in the human body, essential for several metabolic processes. It is widely used in the food, pharmaceutical, and medical industries and in clinical practice as a plasma volume expander (PVE). Athletes, however, may use glycerol to mask the presence of forbidden substances or to enhance performance, inclusively through hyperhydration achieved by glycerol ingestion with added fluid. These practices are considered doping, and are prohibited by the World Anti-Doping Agency (WADA). Therefore, glycerol was introduced in the prohibited list. Doping through glycerol ingestion can readily be identified by detection of elevated glycerol concentrations in urine. In this paper, a protocol for the fast detection of glycerol in urine is proposed. It consists of a previous visual colourimetric screening, followed by a quantitative/qualitative confirmation analysis by mass spectrometry. The screening procedure involves a reaction in which polyhydric alcohols are oxidized by periodate to formic acid and formaldehyde, which is detected by the addition of a fuchsin solution. For the subsequent qualitative/quantitative confirmation analysis, a gas chromatography-mass spectrometry based approach with a non-deuterated internal standard and a drying step of only 10 min is proposed. The linear correlation was demonstrated within WADA´s threshold range. The calculated RSD were 2.1% for within-day precision and 2.8% for between-day precision. The uncertainty estimation was calculated, and a value of 2.7% was obtained. The procedure may also be used for the analysis of other polyols in urine, as for example the PVE mannitol.

Analysis of synthetic 19-norsteroids trenbolone, tetrahydrogestrinone and gestrinone by gas chromatography-mass spectrometry.

Tetrahydrogestrinone, gestrinone and trenbolone are synthetic 19-norsteroids with androgenic properties sharing a labile conjugated ketotrienyl motif. Their LC-MS analyses tend to overcome classical derivatization problems, a shortcoming to the use of GC-MS. Therefore, alternative derivatization procedures were evaluated. The procedure with methoxylamine: pyridine followed by TMSImid: MSTFA gave the best results. This is attributed to the stability of the MO-TMS derivatives hindering the formation of artifacts and tautomerism. A full method is presented including SPE, hydrolysis and liquid-liquid extraction. It was possible to confirm the analytes below 2 ng/mL in urine, being the method robust and cost effective also for screening proposes.

Controle de dopagem de anabolizantes: o perfil esteroidal e suas regulações / Control of doping with anabolic agents: the steroid profile and its regulations

O conceito de perfil esteroidal é discutido neste artigo. As principais vias biossintéticas säo apresentadas. A importância do monitoramento do perfil esteroidal é demonstrada dentro da clínica médica e da medicina esportiva. Parâmetros da literatura para a identificaçäo de dopagem por esteróides endógenos säo apresentados, assim como os fatores que acarretam alterações no perfil esteroidal normal. É dada atençäo especial a essa última abordagem