Cycloartane Saponins from Astragalus glycyphyllos and Their In Vitro Neuroprotective, Antioxidant, and hMAO-B-Inhibiting Effects.

Astragalus glycyphyllos (Fabaceae) is used in the traditional medicine of many countries against hepatic and cardiac disorders. The plant contains mainly flavonoids and saponins. From a defatted methanol extract from its overground parts, a new triterpenoid saponin, 3-O-[α-L-rhamnopyranosyl-(1→2)]-ß-D-xylopyranosyl]-24-O-α-L-arabinopyranosyl-3ß,6α,16ß,24(R),25-pentahydroxy-20R-cycloartane, together with the rare saponin astrachrysoside A, were isolated using various chromatography methods. The compounds were identified via extensive high resolution electrospray ionisation mass spectrometry (HRESIMS) and NMR analyses. Both saponins were examined for their possible antioxidant and neuroprotective activity in three different in vitro models. Rat brain synaptosomes, mitochondria, and microsomes were isolated via centrifugation using Percoll gradient. They were treated with the compounds in three different concentrations alone, and in combination with 6-hydroxydopamine or tert-butyl hydroperoxide as toxic agents. It was found that the compounds had statistically significant dose-dependent in vitro protective activity on the sub-cellular fractions. The compounds exhibited a weak inhibitory effect on the enzyme activity of human recombinant monoamine oxidase type B (hMAO-B), compared to selegiline.

The dopamine, serotonin and norepinephrine releasing activities of a series of methcathinone analogs in male rat brain synaptosomes.

RATIONALE: Novel synthetic "bath salt" cathinones continue to appear on the street as abused and addictive drugs. The range of subjective experiences produced by different cathinones suggests that some compounds have primarily dopaminergic activity (possible stimulants) while others have primarily serotonergic activity (possible empathogenics). An understanding of the structure activity relationships (SARs) of these compounds will help in assessing the likely behavioral effects of future novel structures, and to define potential therapeutic strategies to reverse any reinforcing effects. OBJECTIVES: A series of methcathinone analogs was systematically studied for their activity at the dopamine and serotonin transporters. Compound structures varied at the aromatic group, either by substituent or by replacement of the phenyl ring with a naphthalene or indole ring. METHODS: A novel, high-yielding synthesis of methcathinone hydrochlorides was developed which avoids isolation of the unstable free bases. Neurotransmitter transporter release activity was determined in rat brain synaptosomes as previously reported. Compounds were also screened for activity at the norepinephrine transporter. RESULTS: Twenty-eight methcathinone analogs were analyzed and fully characterized in dopamine and serotonin transporter release assays. Compounds substituted at the 2-position (ortho) were primarily dopaminergic. Compounds substituted at the 3-position (meta) were found to be much less dopaminergic, with some substituents favoring serotonergic activity. Compounds substituted at the 4-position (para) were found to be far more serotonergic, as were disubstituted compounds and other large aromatic groups. One exception was the fluoro-substituted analogs which seem to favor the dopamine transporter. CONCLUSIONS: The dopaminergic to serotonergic ratio can be manipulated by choice of substituent and location on the aromatic ring. It is therefore likely possible to tweak the subjective and reinforcing effects of these compounds by adjusting their structure. Certain substituents like a fluoro group tend to favor the dopamine transporter, while others like a trifluoromethyl group favor the serotonin transporter.

Thallium-Induced Toxicity in Rat Brain Crude Synaptosomal/Mitochondrial Fractions is Sensitive to Anti-excitatory and Antioxidant Agents.

The mechanisms by which the heavy metal thallium (Tl+) produces toxicity in the brain remain unclear. Herein, isolated synaptosomal/mitochondrial P2 crude fractions from adult rat brains were exposed to Tl+ (5-250 µM) for 30 min. Three toxic endpoints were evaluated: mitochondrial dysfunction, lipid peroxidation, and Na+/K+-ATPase activity inhibition. Concentration-response curves for two of these endpoints revealed the optimum concentration of Tl+ to induce damage in this preparation, 5 µM. Toxic markers were also estimated in preconditioned synaptosomes incubated in the presence of the N-methyl-D-aspartate receptor antagonist kynurenic acid (KYNA, 50 µM), the cannabinoid receptor agonist WIN 55,212-2 (1 µM), or the antioxidant S-allyl-L-cysteine (SAC, 100 µM). All these agents prevented Tl+ toxicity, though SAC did it with lower efficacy. Our results suggest that energy depletion, oxidative damage, and Na+/K+-ATPase activity inhibition account for the toxic pattern elicited by Tl+ in nerve terminals. In addition, the efficacy of the drugs employed against Tl+ toxicity supports an active role of excitatory/cannabinoid and oxidative components in the toxic pattern elicited by the metal.

Controversial alkoxyl and peroxyl radical scavenging activity of the tryptophan metabolite 3-hydroxy-anthranilic acid.

3-Hydroxy-anthranilic acid (3-OHAA), a tryptophan metabolite produced in the kynurenine pathway, is an efficient antioxidant towards peroxyl radicals (ROO) derived from the AAPH (2,2'-azobis(2-amidinopropane) dihydrochloride) thermolysis. However, self-reactions of ROO can give rise to alkoxyl radicals (RO), which could strongly affect the fate of scavenging reactions. In the present work, we studied the influence of RO in the scavenging activity of 3-OHAA in three different systems: i) Monitoring of the direct reaction between 3-OHAA and AAPH-derived free radicals (kinetic studies); ii) Evaluation of the protective effect of 3-OHAA on the AAPH-induced consumption of fluorescein; and, iii) Inhibition, given by 3-OHAA, of the AAPH-initiated lipid peroxidation of both, rat brain synaptosomes and homogenate preparations (assessed by chemiluminescence). For such purposes, the fraction of free radicals (f) trapped per 3-OHAA molecule was determined in each system. Kinetic results show that the oxidation of 3-OHAA follows a process dominated by ROO with a zero order kinetic limit in 3-OHAA, and a fraction (fri) equal to 0.88. From the induction times, elicited by 3-OHAA in the kinetic profiles of fluorescein consumption, a fraction (fT) of 0.28 was determined. 3-OHAA also generated induction times in the kinetic profiles of light emission during the AAPH-initiated lipid peroxidation of rat brain synaptosomes and homogenates. From such induction times, fractions of 0.61 and 0.63 were determined for rat brain synaptosomes (fsyn) and homogenates (fhom), respectively. These results show that during the incubation of 3-OHAA and AAPH, a low fraction of ROO self-reacts to generate RO. Nevertheless, when 3-OHAA is employed to protect particular targets, such as fluorescein, rat brain synaptosomes and homogenates, reactions of ROO and/or RO should be considered.

Curcumin delivery from poly(acrylic acid-co-methyl methacrylate) hollow microparticles prevents dopamine-induced toxicity in rat brain synaptosomes.

The potential of poly(methyl methacrylate-co-acrylic acid) (PMMA-AA) copolymers to form hollow particles and their further formulation as curcumin delivery system have been explored. The particles were functionalized by crosslinking the acrylic acid groups via bis-amide formation with either cystamine (CYS) or 3,3'-dithiodipropionic acid dihydrazide (DTP) which simultaneously incorporated reversibility due to the presence of disulfide bonds within the crosslinker. Optical micrographs showed the formation of spherical hollow microparticles with a size ranging from 1 to 7 µm. Curcumin was loaded by incubation of its ethanol solution with aqueous dispersions of the cross-linked particles and subsequent evaporation of the ethanol. Higher loading was observed in the microparticles with higher content of hydrophobic PMMA units indicating its influence upon the loading of hydrophobic molecules such as curcumin. The in vitro release studies in a phosphate buffer showed no initial burst effect and sustained release of curcumin that correlated with the swelling of the particles under these conditions. The capacity of encapsulated and free curcumin to protect rat brain synaptosomes against dopamine-induced neurotoxicity was examined. The encapsulated curcumin showed greater protective effects in rat brain synaptosomes as measured by synaptosomal viability and increased intracellular levels of glutathione.