DEVELOPMENT AND VALIDATION OF A HPLC METHOD FOR QUANTIFICATION OF URSOLIC ACID IN SOLID DISPERSIONS

DEVELOPMENT AND VALIDATION OF A HPLC METHOD FOR QUANTIFICATION OF URSOLIC ACID IN SOLID DISPERSIONS. Ursolic acid is a natural molecule that presents several pharmacological properties. In this work, an analytical method by RP-HPLC has been developed and validated for quantification of this drug in the solid dispersions, using PEG 6000 and Poloxamer 407 as polymers. The method was specific, linear in the range of 1.0-50.0 mu g mL(-1) (r<0.99), precise (CV < 5% for both inter-and intra-assays), accurate (maximum deviation of +/- 13%), and robust to the parameters evaluated. This method has proved to be simple and useful for ursolic acid determination in solid dispersions, enabling its determination in pharmaceutical dosage form.

Synthesis and Antiplasmodial Activity of Betulinic Acid and Ursolic Acid Analogues

More than 40% of the World population is at risk of contracting malaria, which affects primarily poor populations in tropical and subtropical areas. Antimalarial pharmacotherapy has utilised plant-derived products such as quinine and artemisinin as well as their derivatives. However, worldwide use of these antimalarials has caused the spread of resistant parasites, resulting in increased malaria morbidity and mortality. Considering that the literature has demonstrated the antimalarial potential of triterpenes, specially betulinic acid (1) and ursolic acid (2), this study investigated the antimalarial activity against P. falciparum chloroquine-sensitive 3D7 strain of some new derivatives of 1 and 2 with modifications at C-3 and C-28. The antiplasmodial study employed flow cytometry and spectrofluorimetric analyses using YOYO-1, dihydroethidium and Fluo4/AM for staining. Among the six analogues obtained, compounds 1c and 2c showed excellent activity (IC50 = 220 and 175 nM, respectively) while 1a and b demonstrated good activity ( IC50 = 4 and 5 mu M, respectively). After cytotoxicity evaluation against HEK293T cells, 1a was not toxic, while 1c and 2c showed IC50 of 4 mu M and a selectivity index (SI) value of 18 and 23, respectively. Moreover, compound 2c, which presents the best antiplasmodial activity, is involved in the calcium-regulated pathway(s).

Neurotoxicity of Anhydroecgonine Methyl Ester, a Crack Cocaine Pyrolysis Product

Smoking crack cocaine involves the inhalation of cocaine and its pyrolysis product, anhydroecgonine methyl ester (AEME). Although there is evidence that cocaine is neurotoxic, the neurotoxicity of AEME has never been evaluated. AEME seems to have cholinergic agonist properties in the cardiovascular system; however, there are no reports on its effects in the central nervous system. The aim of this study was to investigate the neurotoxicity of AEME and its possible cholinergic effects in rat primary hippocampal cell cultures that were exposed to different concentrations of AEME, cocaine, and a cocaineAEME combination. We also evaluated the involvement of muscarinic cholinergic receptors in the neuronal death induced by these treatments using concomitant incubation of the cells with atropine. Neuronal injury was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. The results of the viability assays showed that AEME is a neurotoxic agent that has greater neurotoxic potential than cocaine after 24 and 48 h of exposure. We also showed that incubation for 48 h with a combination of both compounds in equipotent concentrations had an additive neurotoxic effect. Although both substances decreased cell viability in the MTT assay, only cocaine increased LDH release. Caspase-3 activity was increased after 3 and 6 h of incubation with 1mM cocaine and after 6 h of 0.1 and 1.0mM AEME exposure. Atropine prevented the AEME-induced neurotoxicity, which suggests that muscarinic cholinergic receptors are involved in AEME's effects. In addition, binding experiments confirmed that AEME has an affinity for muscarinic cholinergic receptors. Nevertheless, atropine was not able to prevent the neurotoxicity produced by cocaine and the cocaineAEME combination, suggesting that these treatments activated other neuronal death pathways. Our results suggest a higher risk for neurotoxicity after smoking crack cocaine than after cocaine use alone.

Solid-phase microextraction combined with comprehensive two-dimensional gas chromatography for fatty acid profiling of cell wall phospholipids

The combination of solid-phase microextraction (SPME) with comprehensive two-dimensional gas chromatography is evaluated here for fatty acid (FA) profiling of the glycerophospholipid fraction from human buccal mucosal cells. A base-catalyzed derivatization reaction selective for polar lipids such as glycerophospholipid was adopted. SPME is compared to a miniaturized liquidliquid extraction procedure for the isolation of FA methyl esters produced in the derivatization step. The limits of detection and limits of quantitation were calculated for each sample preparation method. Because of its lower values of limits of detection and quantitation, SPME was adopted. The extracted analytes were separated, detected, and quantified by comprehensive two-dimensional gas chromatography with flame ionization detection (FID). The combination of SPME and comprehensive two-dimensional gas chromatography with FID, using a selective derivatization reaction in the preliminary steps, proved to be a simple and fast procedure for FA profiling, and was successfully applied to the analysis of adult human buccal mucosal cells.

Ursolic acid directly promotes protein accretion in myotubes but does not affect myoblast proliferation

Ursolic acid (UA) has been recently proposed as a potential candidate for the treatment of muscle wasting conditions because of its protein sparring/anabolic effects. Despite this finding, it is unknown whether this response is the consequence of a direct effect on the muscle fibre or if it is mediated by neural or other systemic factors. In the present study, we sought to determine if UA has direct effects in skeletal muscle cells, whether it can increase myoblast proliferation and whether UA can become myotoxic at higher doses. Our results demonstrate that UA directly promoted protein accretion in cultured myotubes but did not modulate myoblast proliferation. At higher doses, UA compromised cell viability in both myoblasts and myotubes. We conclude that the anabolic properties of UA seen in vivo and in vitro are likely a direct effect on the muscle cell, but at higher doses, the benefits decline in favour of a myotoxic outcome. Copyright (C) 2012 John Wiley & Sons, Ltd.

Caffeic acid phenethyl ester reduces the activation of the nuclear factor kappa B pathway by high-fat diet-induced obesity in mice

Objective. The aim of this study was to investigate the effect of CAPE on the insulin signaling and inflammatory pathway in the liver of mice with high fat diet induced obesity. Material/Methods. Swiss mice were fed with standard chow or high-fat diet for 12-week. After the eighth week, animals in the HFD group with serum glucose levels higher than 200 mg/dL were divided into two groups, HFD and HFD receiving 30 mg/kg of CAPE for 4 weeks. After 12 weeks, the blood samples could be collected and liver tissue extracted for hormonal and biochemical measurements, and insulin signaling and inflammatory pathway analyzes. Results. The high-fat diet group exhibited more weight gain, glucose intolerance, and hepatic steatosis compared with standard diet group. The CAPE treatment showed improvement in glucose sensitivity characterized by an area under glucose curve similar to the control group in an oral glucose tolerance test Furthermore, CAPE treatment promoted amelioration in hepatic steatosis compared with the high-fat diet group. The increase in glucose sensitivity was associated with the improvement in insulin-stimulated phosphorylation of the insulin receptor substrate-2, followed by an increase in Akt phosphorylation. In addition, it was observed that CAPE reduced the induction of the inflammatory pathway, c-jun-N- terminal kinase, the nuclear factor kappa B, and cyclooxygenase-2 expression, respectively. Conclusions. Overall, these findings indicate that CAPE exhibited anti-inflammatory activity that partly restores normal metabolism, reduces the molecular changes observed in obesity and insulin resistance, and therefore has a potential as a therapeutic agent in obesity. (C) 2012 Elsevier Inc. All rights reserved.

Solid Dispersion of Ursolic Acid in Gelucire 50/13: a Strategy to Enhance Drug Release and Trypanocidal Activity

Solid dispersions (SDs) are an approach to increasing the water solubility and bioavailability of lipophilic drugs such as ursolic acid (UA), a triterpenoid with trypanocidal activity. In this work, Gelucire 50/13, a surfactant compound with permeability-enhancing properties, and silicon dioxide, a drying adjuvant, were employed to produce SDs with UA. SDs and physical mixtures (PMs) in different drug/carrier ratios were characterized and compared using differential scanning calorimetry, hot stage microscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), particle size, water solubility values, and dissolution profiles. Moreover, LLC-MK2 fibroblast cytotoxicity and trypanocidal activity evaluation were performed to determine the potential of SD as a strategy to improve UA efficacy against Chagas disease. The results demonstrated the conversion of UA from the crystalline to the amorphous state through XRD. FTIR experiments provided evidence of intermolecular interactions among the drug and carriers through carbonyl peak broadening in the SDs. These findings helped explain the enhancement of water solubility from 75.98 mu g/mL in PMs to 293.43 mu g/mL in SDs and the faster drug release into aqueous media compared with pure UA or PMs, which was maintained after 6 months at room temperature. Importantly, improved SD dissolution was accompanied by higher UA activity against trypomastigote forms of Trypanosoma cruzi, but not against mammalian fibroblasts, enhancing the potential of UA for Chagas disease treatment.

Isolation, selection and characterization of root-associated growth promoting bacteria in Brazil Pine (Araucaria angustifolia)

Araucaria angustifolia, a unique species of this genus that occurs naturally in Brazil, has a high socio-economic and environmental value and is critically endangered of extinction, since it has been submitted to intense predatory exploitation during the last century. Root-associated bacteria from A. angustifolia were isolated, selected and characterized for their biotechnological potential of growth promotion and biocontrol of plant pathogenic fungi. Ninety-seven strains were isolated and subjected to chemical tests. All isolates presented at least one positive feature, characterizing them as potential PGPR. Eighteen isolates produced indole-3-acetic acid (IAA), 27 were able to solubilize inorganic phosphate, 21 isolates were presumable diazotrophs, with pellicle formation in nitrogen-free culture medium, 83 were phosphatases producers, 37 were positive for siderophores and 45 endospore-forming isolates were antagonistic to Fusarium oxysporum, a pathogen of conifers. We also observed the presence of bacterial strains with multiple beneficial mechanisms of action. Analyzing the fatty acid methyl ester (FAME) and partial sequencing of the 16S rRNA gene of these isolates, it was possible to characterize the most effective isolates as belonging to Bacillaceae (9 isolates), Enterobacteriaceae (11) and Pseudomonadaceae (1). As far as we know, this is the first study to include the species Ewingella americana as a PGPR. (C) 2011 Elsevier GmbH. All rights reserved.

Synthesis and Solar Cell Application of New Alternating Donor-Acceptor Copolymers Based on Variable Units of Fluorene, Thiophene, and Phenylene

A new series of donor acceptor copolymers were synthesized via the Witting route and applied as an active layer in organic thin-films solar cells. These copolymers are composed of fluorene thiophene and phenylene thiophene units. The ratio between those was systematically varied, and copolymers containing 0%, 50%, and 75% of phenylene thiophene were characterized and evaluated when used in photovoltaic devices. The copolymers' composition, photophysical, electrical, and morphological properties are addressed and correlated with device performance. The 50% copolymer ratio was found to be the best copolymer of the series, yielding a power conversion efficiency (PCE) under air mass (AM) 1.5 conditions of 2.4% in the bilayer heterojunction with the C-60 molecule. Aiming at flexible electronics applications, solutions based on the heterojunction of this copolymer with PCBM (6,6-phenyl-C-61-butyric acid methyl ester) were also successfully deposited using an inkjet printing method and used as an active layer in solar cells.

Synthesis and photovoltaic performance of a fluorene-bithiophene copolymer

We present the synthesis of a copolymer structure, poly(9,9′-n-di-hexyl-2,7-fluorene-alt-2,5- bithiophene), referred to herein as LaPPS43, and its physico-chemical characterization. Thin films of this polymer mixed with phenyl-C61-butyric acid methyl ester (PCBM) were used as the active layer in photovoltaic devices using the ITO/PEDOT:PSS/LaPPS43: PCBM/Ca/Al bulk heterojunction structure. The devices of different active layer thicknesses were electrically studied using J-V curves and the Photo-Celiv technique. The obtained results show that LaPPS43 combined with PCBM is a promising system for photovoltaic devices. Device performance is discussed in terms of the mean drift distance x for charge carriers. Photophysical data showed that the excitonic species are all localized in the aggregated forms. The mechanism of exciton formation and dissociation is also discussed.

Microcystis aeruginosa lipids as feedstock for biodiesel synthesis by enzymatic route

The cyanobacterium Microcystis aeruginosa strain NPCD-1, isolated from sewage treatment plant and characterized as a non-microcystin producer by mass spectrometry and molecular analysis, was found to be a source of lipid when cultivated in ASM-1 medium at 25 degrees C under constant white fluorescent illumination (109 mu mol photon m(-2) s(-1)). In these conditions, biomass productivity of 46.92 +/- 3.84 mg L-1 day(-1) and lipid content of 28.10 +/- 1.47% were obtained. Quantitative analysis of fatty acid methyl esters demonstrated high concentration of saturated fatty acids (50%), palmitic (24.34%) and lauric (13.21%) acids being the major components. The remaining 50% constituting unsaturated fatty acids showed higher concentrations of oleic (26.88%) and linoleic (12.53%) acids. The feasibility to produce biodiesel from this cyanobacterial lipid was demonstrated by running enzymatic transesterification reactions catalyzed by Novozym (R) 435 and using palm oil as feedstock control. Batch experiments were carried out using tert-butanol and iso-octane as solvent. Results showed similarity on the main ethyl esters formed for both feedstocks. The highest ethyl ester concentration was related to palmitate and oleate esters followed by laurate and linoleate esters. However, both reaction rates and ester yields were dependent on the solvent tested. Total ethyl ester concentrations varied in the range of 44.24-67.84 wt%, corresponding to ester yields from 80 to 100%. Iso-octane provided better solubility and miscibility, with ester yield of 98.10% obtained at 48 h for reaction using the cyanobacterium lipid, while full conversion was achieved in 12 h for reaction carried out with palm oil. These results demonstrated that cyanobacterial lipids from M. aeruginosa NPCD-1 have interesting properties for biofuel production. (c) 2012 Elsevier B.V. All rights reserved.

THE NEGATIVE EFFECTS OF ALCOHOL HANGOVER ON HIGH-ANXIETY PHENOTYPE RATS ARE INFLUENCED BY THE GLUTAMATE RECEPTORS OF THE DORSAL MIDBRAIN

Alcoholism is a chronic disorder characterized by the appearance of a withdrawal syndrome following the abrupt cessation of alcohol intake that includes symptoms of physical and emotional disturbances, anxiety being the most prevalent symptom. In humans, it was shown that anxiety may increase the probability of relapse. In laboratory animals, however, the use of anxiety to predict alcohol preference has remained difficult. Excitatory amino acids as glutamate have been implicated in alcohol hangover and may be responsible for the seizures and anxiety observed during withdrawal. The dorsal periaqueductal gray (DPAG) is a midbrain region critical for the modulation/expression of anxiety- and fear-related behaviors and the propagation of seizures induced by alcohol withdrawal, the glutamate neurotransmission being one of the most affected. The present study was designed to evaluate whether low- (LA) and high-anxiety rats (HA), tested during the alcohol hangover phase, in which anxiety is the most prevalent symptom, are more sensitive to the reinforcing effects of alcohol when tested in a voluntary alcohol drinking procedure. Additionally, we were interested in investigating the main effects of reducing the excitatory tonus of the dorsal midbrain, after the blockade of the ionotropic glutamate receptors into the DPAG, on the voluntary alcohol intake of HA and LA motivated rats that were made previously experienced with the free operant response of alcohol drinking. For this purpose, we used local infusions of the N-metil D-Aspartato (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-kainate receptors antagonist DL-2-Amino-7-phosphonoheptanoic acid - DL-AP7 (10 nmol/0.2 mu l) and L-glutamic acid diethyl ester - GDEE (160 nmol/0.2 mu l) respectively. Alcohol intoxication was produced by 10 daily bolus intraperitonial (IP) injections of alcohol (2.0 g/kg). Peak-blood alcohol levels were determined by gas-chromatography analysis in order to assess blood-alcohol content. Unconditioned and conditioned anxiety-like behavior was assessed by the use of the fear-potentiated startle procedure (FPS). Data collected showed that anxiety and alcohol drinking in HA animals are positively correlated in animals that were made previously familiarized with the anxiolytic effects of alcohol. In addition, anxiety-like behavior induced during alcohol hangover seems to be an effect of changes in glutamatergic neurotransmission into DPAG possibly involving AMPA/kainate and NMDA receptors, among others. (C) 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Two series of new semisynthetic triterpene derivatives: differences in anti-malarial activity, cytotoxicity and mechanism of action

Background The discovery and development of anti-malarial compounds of plant origin and semisynthetic derivatives thereof, such as quinine (QN) and chloroquine (CQ), has highlighted the importance of these compounds in the treatment of malaria. Ursolic acid analogues bearing an acetyl group at C-3 have demonstrated significant anti-malarial activity. With this in mind, two new series of betulinic acid (BA) and ursolic acid (UA) derivatives with ester groups at C-3 were synthesized in an attempt to improve anti-malarial activity, reduce cytotoxicity, and search for new targets. In vitro activity against CQ-sensitive Plasmodium falciparum 3D7 and an evaluation of cytotoxicity in a mammalian cell line (HEK293T) are reported. Furthermore, two possible mechanisms of action of anti-malarial compounds have been evaluated: effects on mitochondrial membrane potential (ΔΨm) and inhibition of β-haematin formation. Results Among the 18 derivatives synthesized, those having shorter side chains were most effective against CQ-sensitive P. falciparum 3D7, and were non-cytotoxic. These derivatives were three to five times more active than BA and UA. A DiOC6(3) ΔΨm assay showed that mitochondria are not involved in their mechanism of action. Inhibition of β-haematin formation by the active derivatives was weaker than with CQ. Compounds of the BA series were generally more active against P. falciparum 3D7 than those of the UA series. Conclusions Three new anti-malarial prototypes were obtained from natural sources through an easy and relatively inexpensive synthesis. They represent an alternative for new lead compounds for anti-malarial chemotherapy.

Comparison of extraction and transesterification methods on the determination of the fatty acid contents of three Brazilian seaweed species

Seaweeds are photosynthetic organisms important to their ecosystem and constitute a source of compounds with several different applications in the pharmaceutical, cosmetic and biotechnology industries, such as triacylglycerols, which can be converted to fatty acid methyl esters that make up biodiesel, an alternative source of fuel applied in economic important areas. This study evaluates the fatty acid profiles and concentrations of three Brazilian seaweed species, Hypnea musciformis (Wulfen) J.V. Lamouroux (Rhodophya), Sargassum cymosum C. Agardh (Heterokontophyta), and Ulva lactuca L. (Chlorophyta), comparing three extraction methods (Bligh & Dyer - B&D; AOAC Official Methods - AOM; and extraction with methanol and ultrasound - EMU) and two transesterification methods (7% BF3 in methanol - BF3; and 5% HCl in methanol - HCl). The fatty acid contents of the three species of seaweeds were significantly different when extracted and transesterified by the different methods. Moreover, the best method for one species was not the same for the other species. The best extraction and transesterification methods for H. musciformis, S. cymosum and U. lactuca were, respectively, AOM-HCl, B&D-BF3 and B&D-BF3/B&D-HCl. These results point to a matrix effect and the method used for the analysis of the fatty acid content of different organisms should be selected carefully.

Inhibition of human platelet aggregation by eosinophils

AIMS: The relationship between the activity of eosinophils and platelets has been observed in recent decades by many scientists. These observations include increased numbers of eosinophils associated with platelet disorders, including changes in the coagulation cascade and platelet aggregation. Based on these observations, the interaction between eosinophils and platelets in platelet aggregation was analyze. MAIN METHODS: Human platelets were incubated with eosinophil cytosolic fraction, promyelocytic human HL-60 clone 15 cell lineage, and eosinophil cationic protein (ECP). Platelet rich plasma (PRP) aggregation was induced by adenosine diphosphate, platelet activating factor, arachidonic acid, and collagen, and washed platelets (WP) were activated by thrombin. KEY FINDINGS: Aggregation induced by all agonists was dose dependently inhibited by eosinophil cytosolic fraction. This inhibition was only partially reversed by previous incubation of the eosinophils with l-Nitro-Arginine-Methyl-Ester (l-NAME). Previous incubation with indomethacin did not prevent the cytosolic fraction induced inhibition. The separation of eosinophil cytosolic fraction by gel filtration on Sephadex G-75 showed that the inhibitory activity was concentrated in the lower molecular weight fraction. HL-60 clone 15 cells differentiated into eosinophils for 5 and 7 day were able to inhibit platelet aggregation. The ECP protein inhibited the platelet aggregation on PRP and WP. This inhibition was more evident in WP, and the citotoxicity MTT assay proved the viability of tested platelets, showing that the observed inhibition by the ECP protein does not occur simply by cell death. SIGNIFICANCE: Our results indicate that eosinophils play a fundamental role in platelet aggregation inhibition