Quercetin and 3-O-methylquercetin in vitro skin layers permeation/retention from hydrogels: why only a methoxy group difference determines different behaviors?

OBJECTIVES: The present study was designed to verify if quercetin (QCT), a flavonoid with antioxidant and antiviral activity, and 3-O-methylquercetin (3OMQ), a quercetin C3-methoxylated derivative, present differences in their behavior against complexation with ß-cyclodextrin (ß-CD) and the corresponding permeation/retention trhough porcine ear skin, when incorporated into hydroxypropyl methylcellulose (HPMC) or chitosan (CS) hydrogels. METHODS: The influence of ß-CD on the skin permeation/retention of QCT and 3OMQ from hydrogels is comparatively evaluated for both flavonoids using porcine ear skin in Franz cells model. The properties of the two flavonoids using the semi-empirical method Recife Model was studied. KEY FINDINGS: Quercetin presented higher skin retention compared with its C3-methoxy derivative 3OMQ. The best permeation/retention of QCT was observed when it was incorporated into CS hydrogel containing 5% ß-CD, whereas, for 3OMQ, the HPMC hydrogel containing 5% ß-CD was the best formulation. The flavonoids complexation with ß-CD in water occurred preferentially with the insertion of the B ring through the secondary OH rim. CONCLUSIONS: The dynamic molecular modeling revealed that the methyl group at C3 in 3OMQ molecule determined significant difference in its complexation with ß-CD, in comparison to its analogous QCT and that difference is coincident with the permeation behavior of these flavonoids, denoting a possible relationship with their molecular dynamics.

Box Behnken design of siRNA-loaded liposomes for the treatment of a murine model of ocular keratitis caused by Acanthamoeba.

Acanthamoeba keratitis is an ophthalmic disease with no specific treatment that specially affects contact lens users. The silencing of serine phosphatase (SP) and glycogen phosphorylase (GP) proteins produced by Acanthamoeba has been shown to significantly reduce the cytopathic effect, although no vehicle was proposed yet to deliver the siRNA sequences to the trophozoites. In this study, PEGylated cationic liposomes were proposed and optimized using Box-Behnken design. The influence of DOTAP:DOPE ratio, DSPE-PEG concentration, and siRNA/DOTAP charge ratio were evaluated over both biological response and physicochemical properties of liposomes. The ratio of DOTAP:DOPE had an effect in the trophozoite activity whereas the charge ratio influenced both size and protease activity. The predicted values were very close to the observed values, yielding a formulation with good activity and toxicity profile, which was used in the following experiments. A murine model of ocular keratitis was treated with siGP + siSP-loaded liposomes, as well as their respective controls, and combined treatment of liposomes and chlorhexidine. After 15 days of eight daily administrations, the liposomal complex combined with chlorhexidine was the only treatment able to reverse the more severe lesions associated with keratitis. There was 60% complete regression in corneal damage, with histological sections demonstrating the presence of an integral epithelium, without lymphocytic infiltrate. The set of results demonstrate the efficacy of a combined therapy based on siRNA with classical drugs for a better prognosis of keratitis caused by Acanthamoeba.

siRNA-loaded liposomes: Inhibition of encystment of Acanthamoeba and toxicity on the eye surface.

Current treatments for Acanthamoeba keratitis are unspecific. Because of the presence of the resilient cyst form of the parasite, the infection is persistent. Silencing the key protein of cyst formation, glycogen phosphorylase, has shown potential for reducing encystment processes of the Acanthamoeba trophozoite. However, a suitable carrier to protect and deliver siRNA sequences is still needed. DOTAP: DOPE:DSPE-PEG liposomes were prepared by three different techniques and used to associate a therapeutic siRNA sequence. Liposomes prepared by film hydration followed by membrane extrusion were considered the most adequate ones with average size of 250 nm and zeta potential of +45 mV, being able to associate siRNA for at least 24 hr in culture medium. siRNA-liposomes could inhibit up to 66% of the encystment process. Cell viability studies demonstrated MTT reduction capacity higher than 80% after 3 hr incubation with this formulation. After 24 hr of incubation, LDH activity ranged for both the formulations from around 4% to 40%. In vivo tolerance studies in mice showed no macroscopic alteration in the eye structures up to 24 hr after eight administrations during 1 day. Histological studies showed regular tissue architecture without any morphological alteration. Overall, these results suggest that the formulations developed are a promising new strategy for the treatment of ocular keratitis caused by Acanthamoeba spp.

Nanoemulsions Containing a Coumarin-Rich Extract from Pterocaulon balansae (Asteraceae) for the Treatment of Ocular Acanthamoeba Keratitis.

This study describes the incorporation of a coumarin-rich extract from Pterocaulon balansae into nanoemulsions intended for the local treatment of ocular keratitis caused by Acanthamoeba. The n-hexane dewaxed extract of P. balansae was characterized by HPLC/PDA and UPLC/MS. The presence of four major coumarins was detected, where 5-methoxy-6,7-methylenedioxycoumarin was selected as a chemical marker. This extract was then incorporated into nanoemulsions composed of medium chain triglycerides and egg-lecithin, through spontaneous emulsification. Such a procedure yielded the formation of monodisperse nanoemulsions in a sub-300-nm range, regardless of the amount of extract incorporated (1.0-5.0 mg/mL). The amoebicidal activity against Acanthamoeba castellanii was both dose-dependent and incubation time-dependent. A reduction of 95% of trophozoite viability was detected after 24 h of incubation with a nanoemulsion at 1.25 mg/mL of coumarins, being a similar effect detected for chlorhexidine. These results suggest a potential of the formulations developed in this study as a new strategy for the treatment of ocular keratitis caused by Acanthamoeba.

Antioxidant Effect of Nanoemulsions Containing Extract of Achyrocline satureioides (Lam) D.C.-Asteraceae.

Ethanolic extracts of Achyrocline satureioides have pronounced antioxidant activity mainly due to the presence of the flavonoid quercetin. However, direct topical application of the extract is not possible due to the presence of high amounts of ethanol. In this sense, nanoemulsions arise as an alternative for topical formulation associating molecules with limited aqueous solubility. This article describes the development of topical nanoemulsions containing either A. satureioides extract or one of its most abundant flavonoid, quercetin. Nanoemulsions composed of octyldodecanol, egg lecithin, water and extract (NEE), or quercetin (NEQ) were prepared by spontaneous emulsification. This process led to monodisperse nanoemulsions presenting a mean droplet size of approximately 200-300 nm, negative zeta potential, and high association efficiency. A study of quercetin skin retention using porcine skin which was performed using a Franz diffusion cell revealed a higher accumulation of quercetin in skin for NEE when compared to NEQ. Finally, the antioxidant activity of formulations was measured by thiobarbituric acid-reactive species and the APPH model. A lower lipoperoxidation for the extract in respect to quercetin solution was observed. However, no difference between NEQ and NEE lipoperoxidation could be seen. The protection against lipoperoxidation by the formulations was also measured in the skin, where lower formation of reactive species was observed after treatment with NEE. In conclusion, this study shows the formulation effect on the physicochemical properties of nanoemulsions as well as on the skin retention and antioxidant activity of quercetin.

On the use of nanotechnology-based strategies for association of complex matrices from plant extracts

AbstractDepending on the method of extraction, plant extracts can contain an enormous variety of active molecules, such as phenolic compounds, essential oils, alkaloids, among others. In many cases, from a pharmacological point of view, it is interesting to work with crude extract or fractions instead of a single isolated compound. This could be due to multi-targeting effect of the extract, lack of knowledge of the active compounds, synergistic effect of the extract compounds, among others. In any case, in order to achieve a final product some issues must be overcome, including poor stability, solvent toxicity, and low solubility of the bioactive compound. Recently many nanotechnology-based strategies have been proposed as an alternative to solve these problems, especially liposomes, nanoemulsions and nanoparticles. In this sense, the present work aims to review the main nanotechnological approaches used for association of different plant extracts and the main achievements from using these technologies.

5-Oxoproline reduces non-enzymatic antioxidant defenses in vitro in rat brain.

5-Oxoproline (pyroglutamic acid) accumulates in glutathione synthetase deficiency, an inborn metabolic defect of the gamma-glutamyl cycle. This disorder is clinically characterized by hemolytic anemia, metabolic acidosis and severe neurological disorders. Considering that the mechanisms of brain damage in this disease are poorly known, in the present study we investigated whether oxidative stress is elicited by 5-oxoproline. The in vitro effect of (0.5-3.0 mM) 5-oxoproline was studied on various parameters of oxidative stress, such as total radical-trapping antioxidant potential, total antioxidant reactivity, chemiluminescence, thiobarbituric acid-reactive substances, sulfhydryl content, carbonyl content, and 2',7'-dichlorofluorescein fluorescence, as well as on the activities of the antioxidant enzymes catalase, superoxide dismutase and glutathione peroxidase in cerebral cortex and cerebellum of 14-day-old rats. Total radical-trapping antioxidant potential and total antioxidant reactivity were significantly reduced in both cerebral structures. Carbonyl content and 2',7'-dichlorofluorescein fluorescence were significantly enhanced, while sulfhydryl content was significantly diminished. In contrast, chemiluminescence and thiobarbituric acid-reactive substances were not affected by 5-oxoproline. The activities of catalase, superoxide dismutase and glutathione peroxidase were also not altered by 5-oxoproline. These results indicate that 5-oxoproline causes protein oxidation and reactive species production and decrease the non-enzymatic antioxidant defenses in rat brain, but does not cause lipid peroxidation. Taken together, it may be presumed that 5-oxoproline elicits oxidative stress that may represent a pathophysiological mechanism in the disorder in which this metabolite accumulates.

Citrulline and ammonia accumulating in citrullinemia reduces antioxidant capacity of rat brain in vitro.

Citrullinemia is an inborn error of the urea cycle caused by deficient argininosuccinate synthetase, which leads to accumulation of L-citrulline and ammonia in tissues and body fluids. The main symptoms include convulsions, tremor, seizures, coma, and brain edema. The pathophysiology of the neurological signs of citrullinemia remains unclear. In this context, we investigated the in vitro effects of L-citrulline and ammonia in cerebral cortex from 30-day-old rats on oxidative stress parameters, namely thiobarbituric acid-reactive substances (TBA-RS), chemiluminescence, mitochondrial membrane protein thiol content, intracellular content of hydrogen peroxide, total radical-trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR) as well as on the activities of the antioxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase). L-Citrulline significantly diminished TRAP (26%) and TAR (37%), while ammonia decreased TAR (30%). Ammonia increased SOD activity (65%) and L-citrulline did not affect the activities of any antioxidant enzymes. We also observed that L-citrulline and ammonia did not alter lipid peroxidation parameters, levels of hydrogen peroxide, and mitochondrial membrane protein thiol content. Taken together, these results may indicate that L-citrulline and ammonia decreased the antioxidant capacity of the brain, which may reflect a possible involvement of oxidative stress in the neuropathology of citrullinemia.

A chemically-induced acute model of maple syrup urine disease in rats for neurochemical studies.

We report a chemically-induced model of maple syrup urine disease (MSUD) in 10- and 30-day-old rats produced by subcutaneous administration of a branched-chain amino acids (BCAA) pool along with the analyses of plasma and brain amino acid levels by HPLC at 0-120 min after administration. We observed an increase of plasma leucine (Leu), isoleucine (Ile) and valine (Val) concentrations in both 10- and 30-day-old rats. These increases were accompanied by a concomitant reduction of plasma concentrations of methionine (Met), phenylalanine (Phe), tyrosine (Tyr), histidine (His), alanine (Ala), lysine (Lys), and ornithine (Orn) in 10-day-old rats and of Met, Phe, Tyr, tryptophan (Trp), and Orn in 30-day-old rats. These results are similar to those observed in MSUD patients during crises, when plasma levels of large neutral amino acids (LNAA) are also reduced when BCAA concentrations are increased. In the brain, increased concentrations of Leu, Ile and Val were achieved in 10-day-old rats at all times after injection. In contrast, no differences in cerebral concentrations of BCAA were observed in 30-day-old rats. In conclusion, the present MSUD model, using 10- rather than 30-day-old rats, has a similar amino acid profile to that of MSUD untreated patients and is suitable to investigate the mechanisms of brain damage characteristic of this disorder.

alpha-keto acids accumulating in maple syrup urine disease stimulate lipid peroxidation and reduce antioxidant defences in cerebral cortex from young rats.

Maple syrup urine disease (MSUD) is an inherited neurometabolic disorder caused by deficiency of branched-chain alpha-keto acid dehydrogenase complex activity which leads to tissue accumulation of the branched-chain alpha-keto acids (BCKAs) alpha-ketoisocaproic acid (KIC), alpha-ketoisovaleric acid (KIV) and alpha-keto-beta-methylvaleric acid (KMV) and their respective amino acids. Neuropathologic findings characteristic of the disease are cerebral edema and atrophy, whose pathophysiology is poorly known. In the present study, we investigated the in vitro effect of BCKAs on various parameters of oxidative stress, namely chemiluminescence (CL), thiobarbituric acid-reactive substances (TBA-RS), total radical-trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR), and the activities of the antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) in cerebral cortex of 30-day-old rats. The major effects observed were with KIC, which significantly increased CL and TBA-RS measurements, decreased TRAP and TAR values, and markedly inhibited GPx activity. KMV and KIV increased CL and decreased TRAP and TAR values. In contrast, these compounds did not affect CAT and SOD activities. Taken together, it was shown that: the BCKAs studied stimulated lipid peroxidation and reduced the brain antioxidant defences, suggesting an increased production of free radicals. In case the in vitro effects here detected also occur in vivo in MSUD, it can be presumed that oxidative stress might contribute, at least in part, to the brain damage found in the affected patients.

Evaluation of the mechanisms involved in leucine-induced oxidative damage in cerebral cortex of young rats.

Maple syrup urine disease (MSUD) is a metabolic disorder caused by the deficiency of the activity of the mitochondrial enzyme complex branched-chain L-2-keto acid dehydrogenase. The metabolic block results in tissue and body fluid accumulation of the branched-chain amino acids leucine (Leu), isoleucine and valine, as well as of their respective alpha-keto acids. Neurological sequelae are usually present in MSUD, but the pathophysiologic mechanisms of neurotoxicity are still poorly known. It was previously demonstrated that Leu elicits oxidative stress in rat brain. In the present study we investigated the possible mechanisms involved in Leu-induced oxidative damage. We observed a significant attenuation of Leu-elicited increase of thiobarbituric acid-reactive substances (TBA-RS) measurement when cortical homogenates were incubated in the presence of the free radical scavengers ascorbic acid plus trolox, dithiothreitol, glutathione, and superoxide dismutase, suggesting a probable involvement of superoxide and hydroxyl radicals in this effect. In contrast, the use of Nomega-nitro-L-arginine methyl ester or catalase (CAT) did not affect TBA-RS values. We also demonstrated an inhibitory effect of Leu on the activities of the antioxidant enzymes CAT and gluthathione peroxidase, as well as a significant reduction in the membrane-protein thiol content from mitochondrial enriched preparations. Furthermore, dichlorofluorescein levels were increased although not significantly by Leu. Taken together, our present data indicate that an unbalance between free radical formation and inhibition of critical enzyme activities may explain the mechanisms involved in the Leu-induced oxidative damage.