In Silico and Chromatographic Methods for Analysis of Biotransformation of Prospective Neuroprotective Pyrrole-Based Hydrazone in Isolated Rat Hepatocytes.

In the current study, chromatographic and in silico techniques were applied to investigate the biotransformation of ethyl 5-(4-bromophenyl)-1-(2-(2-(2-hydroxybenzylidene) hydrazinyl)-2-oxoethyl)-2-methyl-1H-pyrrole-3-carboxylate (11b) in hepatocytic media. The initial chromatographic procedure was based on the employment of the conventional octadecyl stationary phase method for estimation of the chemical stability. Subsequently, a novel and rapid chromatographic approach based on a phenyl-hexyl column was developed, aiming to separate the possible metabolites. Both methods were performed on a Dionex 3000 ThermoScientific (ACM 2, Sofia, Bulgaria) device equipped with a diode array detector set up at 272 and 279 nm for analytes detection. An acetonitrile: phosphate buffer of pH 3.5: methanol (17:30:53 v/v/v) was eluted isocratically as a mobile phase with a 1 mL/min flow rate. A preliminary purification from the biological media was achieved by protein precipitation with methanol. A validation procedure was carried out, where the method was found to correspond to all ICH (Q2) and M10 set criteria. Additionally, an in silico-based approach with the online server BioTransformer 3.0 was applied in an attempt to predict the possible metabolites of the title compound 11b. It was hypothesized that four CYP450 isoforms (1A2, 2C9, 3A4, and 2C8) were involved in the phase I metabolism, resulting in the formation of 12 metabolites. Moreover, docking studies were conducted to evaluate the formation of stable complexes between 11b and the aforementioned isoforms. The obtained data indicated three metabolites as the most probable products, two of which (M9_11b and M10_11b) were synthesized by a classical approach for verification. Finally, liquid chromatography with a mass detector was implemented for comprehensive and summarized analysis, and the obtained results revealed that the metabolism of the 11b proceeds possibly with the formation of glucuronide and glycine conjugate of M11_11b.

Hepatoprotective and antioxidant effects of alcesefoliside from Astragalus monspessulanus

Abstract The hepatoprotective potential of alcesefoliside (AF) from Astragalus monspessulanus was investigated. Iron sulphate/ascorbic acid (Fe2+/AA) lipid peroxidation was induced in rat liver microsomes and pre-incubated with AF and silybin (100, 10 and 1 µmol). Pronounced effects were observed in 100 µmol. In vivo experiments were carried out on rats, challenged orally with carbon tetrachloride (CCl4) alone and after pre-treatment and followed by curative treatment with AF (10 mg/kg). The activity of the serum and antioxidant enzymes, together with reduced glutathione (GSH) levels and malonedialdehyde (MDA) quantity were measured. Microsomal incubation with Fe2+/AA increased MDA production. The pre-incubation with AF reduced the formation of MDA, comparable to silybin. These findings were supported by the in vivo study where CCl4-induced liver damage was discerned by significant increase in serum enzymes and in MDA production as well as by GSH depletion and reduced antioxidant enzymes activity. The AF pre-treatment and consecutive curative treatment normalizes the activity of the serum and antioxidant enzymes alike, as well as the levels of GSH and MDA. Histological examination of AF-treated livers showed a decrease in the abnormal accumulation of lipids in hepatocytes as well as reduced alterative changes in their structure in a model of CCl4-induced toxicity.

Neuroprotective and MAOB inhibitory effects of a series of caffeine-8-thioglycolic acid amides

The effects of new derivatives of caffeine-8-thioglycolic acid (100 µM) on isolated rat brain synaptosomes, human neuroblastoma cell line SH-SY5Y and human recombinant MAOB enzyme (hMAOB) (1 µM) were evaluated. Most of the compounds, administered alone, didn't show statistically significant neurotoxic effects on SH-SY5Y, when compared to the control (non-treated cells). Of all studied structures JTA-2Ox, JTA-11, JTA-12 and JTA-13 decreased cell viability. In combination with 6-hydroxydopamine (6-OHDA) (100 µM), only JTA-1 and JTA-2 revealed neuroprotective effects, stronger than those of caffeine. All compounds administered alone revealed, neurotoxic effects on synaptosomes, as compared to non-treated synaptosomes. JTA-1, JTA-2 and JTA-3 showed lowest neurotoxic effects and were investigated in a model of 6-OHDA-induced oxidative stress. In this model of neurotoxicity, only JTA-1 and JTA-2 showed statistically significant neuroprotective effect, by preserving the synaptosomal viability and the level of reduced glutathione. Inhibition of hMAOB, was revealed by JTA-1 and JTA-2. They inhibited the enzyme by 23% and 25% respectively, thus approaching the selegiline activity, which was 42%. The possible mechanisms of neuroprotection of JTA-1 and JTA-2 might be a result from the inhibition of hMAOB, which catalyze the production of neurotoxic p-quinone from 6-OHDA.

Micellar propolis nanoformulation of high antioxidant and hepatoprotective activity

ABSTRACT The present study reports a promising antioxidant protection by a recently developed micellar propolis formulation, against oxidative stress in in vitro and in vivo models of toxicity. The formulation, based on poplar propolis encapsulated in poly(ethylene oxide)-β-poly(propylene oxide)-β-poly(ethylene oxide) triblock copolymer (PEO26-PPO40-PEO26) micelles is characterized by small size (D h = 20 nm), enhances aqueous solubility and good colloidal stability. In vitro, propolis-loaded PEO26-PPO40-PEO26 micelles (20-100 µg/ml) significantly increased the cell viability of human hepatoma HepG2 cells, subjected to H2O2-induced cell injury (0.1 mM, 1 h). Antioxidant activity and protection of the micellar propolis were evaluated in a model of carbon tetrachloride-induced hepatotoxicity in rats (10% CCl4 solution, 1.25 ml/kg, p.o.) by measurement of non-enzyme (malondialdehyde and glutathione) and enzyme (catalase and superoxide dismutase) biomarkers of oxidative stress. Clinic observations, hematological, biochemical parameters and histological analysis were also performed. In vivo, micellar propolis (20 mg/kg b.w., p.o., 14 days) ameliorated CCl4-induced acute liver injury in rats. The oral administration of micellar propolis significantly prevented serum transaminase increases, as well as brought the levels of malondialdehyde, glutathione, and antioxidant enzymes catalase and superoxide dismutase toward the controls levels. Therefore, PEO26-PPO40-PEO26 micelles could be considered as a promising oral delivery system of propolis against oxidative stress injury in liver cells.

Alcesefoliside protects against oxidative brain injury in rats

ABSTRACT This study investigated the possible antioxidant and neuroprotective effects of alcesefoliside, isolated from Astragalus monspessulanus L., Fabaceae, against carbon tetrachloride (CCl4)-induced brain injury in Wistar rats. Iron sulphate/ascorbic acid lipid peroxidation was induced in rat brain microsomes and pre-incubated with alcesefoliside and silybin. Male rats were treated in vivo with alcesefoliside and with silymarin alone; animals challenged with CCl4; and pre-treated with alcesefoliside or silymarin in respective doses for 7 days, challenged with CCl4, followed by curative treatment (additional 14 days). The activity of acetylcholine esterase and the antioxidant enzymes: superoxide-dismutase, catalase, glutathione-peroxidase, glutathione reductase and glutathione-S-transferase as well as the biomarkers of oxidative stress malondialdehyde and reduced glutathione were measured. The alcesefoliside pre-treatment and consecutive curative treatment normalizes the activity of the antioxidant enzymes as well as levels of malondialdehyde and reduced glutathione. The observed effects on tissue level correlate with the histopathological observations of the brain. They were comparable to the effects of silymarin, used as a positive control. The results showed that alcesefoliside has a neuroprotective effect against CCl4-induced brain toxicity in rats.