Filipe Miguel Areias

Phenolic fingerprint of peppermint leaves

A reversed-phase high-performance liquid chromatography procedure is proposed for the determination of 10 phenolic compounds (eriodictyol 7-O-rutinoside, eriodictyol 7-O-glucoside, luteolin 7-O-rutinoside, luteolin 7-O-glucoside, hesperetin 7-O-rutinoside, apigenin 7-O-rutinoside, rosmarinic acid, 5,6-dihydroxy-7,8,3′,4′-tetramethoxyflavone, pebrellin and gardenin B) in peppermint. The chromatographic separation was achieved using a reversed-phase Spherisorb ODS 2 (5 μm particle size; 25.0×0.46 cm) column. Of the several extractive solvents tried, ethanol was the best for qualitative and quantitative analysis. Best resolution was obtained using a gradient of water-phosphoric acid (999:1) and acetonitrile. Fourteen samples were subjected to quantification and showed a common composition pattern.

Simultaneous Determination of Flavonoids, Phenolic Acids, and Coumarins in Seven Medicinal Species by HPLC/DIODE-Array Detector

Abstract A simple and accurate reversed phase HPLC procedure is proposed for the determination of 19 phenolic compounds (flavonoids, phenolic acids, and coumarins) in seven medicinal species. The sample preparation involved extraction, alkaline and acid hydrolysis, and purification through a Bond-Elut C18 column. The chromatographic separation was achieved using a reversed phase column Spherisorb ODS2 (5μm; 25.0 × 0.46 cm). Gradient elution was carried out using water-formic acid (19:1) (A) and methanol (B). A diode-array detector monitored the effluent and chromatograms were recorded at 280, 320, and 350 nm. The optimised methodology seems to be useful for the phytochemical analysis of plant extracts. A close correlation between the phenolic compound patterns and the botanical origin of plants was found.

HPLC/DAD analysis of phenolic compounds from lavender and its application to quality control

A reversed phase HPLC procedure is proposed for the determination of eight phenolic compounds (2-O-glucosilcoumaric acid, o-coumaric acid, rosmarinic acid, apigenin-7-O-glucoside, coumarin, herniarin, luteolin, and apigenin) in lavender. The chromatographic separation was achieved using a reversed-phase Spherisorb ODS 2 (5 μm particle size: 25.0 × 0.46 cm) column. From the several extractive solvents assayed, only ethanol was able to extract all the mentioned compounds. Best resolution was obtained using a gradient of water-formic acid (19:1) and acetonitrile. Ten samples were subjected to quantification, all of them showing a common composition pattern.

Distinct glycolysis inhibitors determine retinal cell sensitivity to glutamate-mediated injury.

In this study, we analyzed how distinct glycolysis inhibitors influenced the redox status of retinal cells, used as a neuronal model. Three different approaches were used to inhibit glycolysis: the cells were submitted to iodoacetic acid (IAA), an inhibitor of glyceraldehyde 3-phosphate dehydrogenase, to 2-deoxy-glucose (DG) in glucose-free medium, which was used as a substitute of glucose, or in the absence of glucose. The redox status of the cells was evaluated by determining the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). By the analysis of dose-response curves of MTT reduction, IAA showed values of IC50 = 7.02 × 10−5 M, whereas DG showed values of IC50 = 7.42 × 10−4 M. Upon 30 min-incubation, glucose deprivation, per se, did not significantly affect MTT reduction. We also evaluated the reduction of MTT as an indicator of cell injury by exposing the cells to 100 μM glutamate during the decrement of glycolysis function. In the presence of glutamate, for 2 h, there was a decrease in MTT reduction, which was potentiated in the presence of DG (10-20% decrease), in the presence of IAA (about 30% decrease) or in glucose-free medium (about 30% decrease). Major changes observed by the MTT assay, upon exposure to glutamate, indicative of changes in the redox status of retinal cells, were concomitant with variations in intracellular ATP. Under glucose deprivation, endogenous ATP decreased significantly from 38.9 ± 4.4 to 13.3 ± 0.7 nmol/mg protein after exposure to 100 μM glutamate. The results support a different vulnerability of retinal cells after being exposed to distinct forms of glycolysis inhibition.

Tetraoxygenated xanthones from centaurium erythraea

Abstract Three tetraoxygenated xanthones, 1,6-dihydroxy-3,5-dimethoxyxanthone, 3-hydroxy-1,5,6-trimethoxyxanthone and 1,3,5-trihydroxy-2-methoxyxanthone, were isolated for the first time from the aerial parts of C. erythraea. Their structures were established by spectroscopic means and by comparison with literature references.

Glutamate regulates the viability of retinal cells in culture

In this study, we show that glutamate regulates the viability of cultured retinal cells upon transient glucose deprivation. At low concentrations (10-100 microM) glutamate decreased MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction to about 50% of control and decreased intracellular ATP levels (about 4-fold) after transient glucose removal. Under these conditions, the decrease in MTT reduction was associated with the activation of NMDA (N-methyl-D-aspartate) receptors. Upon exposure to high (10 mM) glutamate and transient glucose deprivation, the intracellular levels of glutamate increased. High glutamate significantly counteracted the decrease in MTT reduction and ATP production observed in the presence of low glutamate concentrations. AOAA (aminooxyacetic acid), a non-specific inhibitor of mitochondrial transaminases, enhanced the intracellular glutamate levels, but did not largely affect glutamate-mediated changes in MTT reduction or ATP production. Furthermore, the intracellular levels of pyruvate were not significantly altered, suggesting that changes in ATP production were not due to an increase in glycolysis. Thus, the recovery from glucose deprivation seems to be facilitated in retinal neuronal cells that had been exposed to high glutamate, in comparison with low glutamate, suggesting a role for high glutamate and glucose in maintaining retinal cell function following conditions of glucose scarcity.