Mestrado em Ciências Farmacêuticas

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Avaliação do potencial quimioprotetor de nanodispersão e extrato etanólico de Mikania glomerata Sprengel (asteraceae)
(Universidade Federal do Espírito Santo, 2016-02-04) Santana, Esdras Andrade; Batitucci, Maria do Carmo Pimentel; Matsumoto, Silvia Tamie; Giuberti, Cristiane dos Santos
Mikania glomerata is a medicinal plant having the coumarin as a chemical tracer, and due to bronchodilatory properties is widely used in traditional medicine in Brazil. The main objective of this study was to evaluate the potential of ethanol extract (EE) and nanodispersion (ND) of M. glomerata against cytotoxic and mutagenic effects induced by chemotherapy cyclophosphamide (CPA). This assessment was performed using micronucleus assay in bone marrow erythrocytes of Swiss albino mice (Mus musculus) in vivo through the simultaneous treatment protocol. The treatments using EE were performed with the 1.0; 0.5; 0.25; 0.008 e 0.004 mg.kg-1 concentrations of coumarin and ND in 0.008 e 0.004 mg.kg-1 concentrations of coumarin. The results showed that most of treatments reduced the cytotoxic damage caused by CPA. Only the treatment with EE 0.004 mg.kg-1 of coumarin did not show this effect. In the evaluation of antimutagenicity, the treatments with 1.0; 0.5; 0.25 mg.kg-1 concentrations of coumarin and ND (0.008 e 0.004 mg.kg-1 of coumarin), demonstrated to be effective in reducing the micronucleus frequency, which means they are antimutagenic agents. The treatment using ND (0.008 mg.kg-1 of coumarin) showed a 60.38% damage reduction, indicating greater effectiveness among the treated groups. Overall, treatments with nanodispersions were more efficient in chemoprotective action, although its low coumarin content in the experimental conditions. The results reinforce the importance of using nanotechnology to transport of natural compounds and the potential of compounds extraction technology through nanodispersions.
Avaliação do perfil químico e atividades biológicas de Myrcíaria Strigipes O. Berg (Myrtaceae)
(Universidade Federal do Espírito Santo, 2016-03-17) Faitanin, Rafael Destefani; Kitagawa, Rodrigo Rezende; Jamal, Claudia Masrouah; Gonçalves, Rita de Cássia Ribeiro; Silveira, Dâmaris
Brazil is the owner of the largest and richest genetic diversity of plant species, however, most of them don’t have chemical-biologial studies. Myrciaria strigipes O. Berg (Myrtaceae) is a native species known popularly as "cambucá da praia" and "cabeludinha da praia" and it’s used in traditional medicine for cramps, edema and abdominal pain. The aim of this study was to evaluate the chemical profile, the toxicity and the antimicrobial, antioxidant, thrombolytic activities and α-amylase, α- glucosidase and tyrosinase enzymes inhibition activities of M. strigipes. The chemical profile was characterized from phytochemical screening, High Performance Liquid Chromatography analysis and the fractionation/purification techniques. The toxicity was determined against larvae of Artemia salina Leach. and the antimicrobial activity against Staphylococcus aureus, Escherichia coli and Candida albicans. The antioxidant activity was measured using the synthetic radicals DPPH and ABTS. The in vitro thrombolytic activity was evaluated through the extracts capacity to cause lysis in human blood clot. The inhibitory activities of α-amylase, α-glucosidase and tyrosinase enzymes were determined using in vitro spectrophotometric methods. Preliminary phytochemical analysis of ethanolic extracts of leaves (EEF) and branches (EEG) indicated the presence of flavonoids, coumarins, alkaloids, saponins, tannins, triterpenes, steroids and anthraquinones, yet this was only found in the EEF. They were isolated and/or identified of the EEF five compounds of terpenes class, four pentacyclic triterpenes (friedelin, 28-hydroxyfriedelin, glutinol and ursolic acid) and a tetracyclic steroid (β-sitosterol), and three phenolic compounds (isoquercitrin, hyperoside and ellagic acid). The EEF showed a little toxicity (LD50 = 648.17 µg/mL) and only the EEG showed antimicrobial activity, inhibiting the growth of S. aureus. The EEF and EEG showed antioxidant capacity and promising inhibitory activity on α-glucosidase, EC50 of 40,66 e 18,52 µg/mL, respectively, while the positive control showed EC50 of 37,30 µg/mL. This study reported, for the first time, chemical aspects and biological activities of M. strigipes species, and the results demonstrate the need for more studies to further chemical knowledge and understanding about the mechanism(s) and substance(s) responsible for activities shown by this species.
Caracterização de implantes poliméricos biodegradáveis contendo sirolimus e avaliação de sua estabilidade em condições de estresse químico
(Universidade Federal do Espírito Santo, 2016-03-18) Campos, Michele Soares Tacchi; Oliveira, Marcelo Antônio de; Jamal, Claudia Masrouah; Nogueira, Fernando Henrique Andrade
The use of sirolimus and its analogs has been evaluated in studies aimed at combating several types of cancer; however, because of the limited bioavailability of the drug, the search for new forms of administration is required. Biodegradable polymeric implants containing sirolimus were assessed as an alternative method of drug administration. Distinct implants containing 25% (w/w) sirolimus were prepared employing the polymer matrices chitosan, polycaprolactone and Poly(lactic-co-glycolic acid) (PLGA) in two proportions: PLGA 50:50 and PLGA 75:25. Thermal analysis techniques such as thermogravimetry and differential scanning calorimetry, combined with x-ray diffraction and microscopy were used to characterize and evaluate the compatibility of the constituents of the formulation, no incompatibilities were found between the components, but drug amorphization was observed in all samples. Sirolimus was unstable when exposed to conditions: heat, neutral and basic hydrolysis, at the analysis by high-performance liquid chromatography (HPLC ) were detected two degradation products after exposure to heat and detected one degradation product after basic hydrolysis and neutral hydrolysis, the degradations product showed similar UV spectrum to the drug’s spectrum. Through the isothermal kinetic study on solid media, it was observed that the drug molecule degrades second diffusive three-dimensional model, with validity time to 6 years. HPLC analysis showed that the lyophilized powder for elaboration of the implant prepared with PLGA 75:25 did not present degradation products and maintained its appropriate drug content of 24,6%, after analysis in solution by HPLC, and represents the most suitable polymer for use in developed by biodegradable implants containing Sirolimus for the treatment of malignant solid tumors, however, is still necessary to further study the drug effects after amorphization of the crystal and also stability and solubility analysis
Avaliação da eficácia de atropina e pralidoxima na intoxicação aguda por clorpirifós e desenvolvimento de metodologia analítica para identificação e quantificação de organofosforados e seus antídotos por HPLC-MS/MS
(Universidade Federal do Espírito Santo, 2016-03-22) Marques, Graziany Leite Moreira; Pelição, Fabrício Souza; Sampaio, Karla Nívea; Pires, Rita Gomes Wanderley; Martinis, Bruno Spinosa de
Organophosphorus insecticides (OP) are probably the most widely used pesticides in the world. In Brazil, these compounds are considered one of the main responsible for poisoning. Chlorpyrifos (CPF), an OP compound highly used, promotes, similarly to other OP, inhibition of the cholinesterases (ChEs) leading to the accumulation of acetylcholine in the central and peripheral cholinergic synapses. The treatment for OP poisoning involves a combination of muscarinic antagonists, such as atropine (ATR); oximes for reactivating the ChEs, mainly represented by the pralidoxime (2-PAM) and benzodiazepines, generally diazepam, for treatment of occasional seizures. However, the effectiveness of oximes in humans is still doubtful, mainly due to the polarity of these compounds, which leads to poor penetration of these drugs into the central nervous system (CNS). Therefore, this study aims to investigate the efficacy of the antidotes adopted in the OP poisoning, over the clinical signs and on the reactivation of the ChEs inhibition induced by the acute poisoning with CPF, as well as validate analytical method for identifying and quantifying various OPs, ATR and 2-PAM, using high-performance liquid chromatography coupled to mass spectrometry (HPLCMS/MS). Adult male rats (n=280) were divided into 8 groups: saline (SAL), CPF, SAL+ATR, CPF+ATR, SAL+2-PAM, CPF+2-PAM, SAL+ATR+2-PAM and CPF+ATR+2-PAM. The antidotes treatment with ATR (10 mg/kg); 2-PAM (40 mg/kg) or ATR (10mg/kg) +2-PAM (40 mg/kg) was delivered by intraperitoneal injections (i.p.) one hour after the administration of CPF (30 mg/kg, i.p.) or SAL (0.9%, i.p.) and the animals were observed for acute toxicity signs for up to 5 hours. In periods of 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours and 24 hours after the antidotes treatment administration, the animals were decapitated for collecting blood and brain (except cerebellum) samples for measurements of ChEs activity, as well as for CPF and CPF-oxon analysis by HPLC-MS/MS. The method developed for the OPs and antidotes analysis proved to be efficient and fast, attending the validation criteria stated in the RDC nº 27 of ANVISA (BRASIL, 2012). The analysis demonstrates that the CPF blood concentration significantly decreased over the first 24 hours after administration. The CPF caused inhibition of plasma ChE and treatment with 2-PAM was capable of reversing this inhibition for up to 1 hour after administration. However, when considering the brain AChE the 2-PAM only induced a small reactivation of the enzyme from the second hour of the treatment administration. Although not fully effective, the treatments with ATR, 2-PAM or ATR+2-PAM employed prevented or reverted, at different levels, the typical cholinergic symptoms of the OP poisoning.
Avaliação in vitro da atividade anti-Helicobacter pylori e potencial antioxidante de extratos e frações de Baccharis trimera (Less.) DC
(Universidade Federal do Espírito Santo, 2016-03-22) Nunes, Otalibio Castiglioni; Gonçalves, Rita de Cássia Ribeiro; Vellosa, José Carlos Rebuglio; Borges, Warley de Souza
One of the major risk factors for the development of ulcers and gastric cancer is the infection by Helicobacter pylori, in which there is a considerable oxidative stress. In Brazil, the plant Baccharis trimera (Less) DC, known as "Carqueja", is commonly used to treat gastrointestinal and liver disorders. Tannins, flavonoids, saponins, diterpene lactones, sesquiterpenes, phenolic compounds and volatile oils were identified in this specie. Thus, the goal of this study was to evaluate the antiHelicobacter pylori activity of the aqueous (AqE), ethanolic (EE), and hydroalcoholic (HE) extracts of B. trimera, as well as the aqueous (AqF), hexane (HxF), and acetonitrile / chloroform (ACF) fractions obtained from the HE. In addition, it aims to analyze the phytochemical composition and its effects on free radicals and biological oxidants. For all extracts and fractions, it was performed a phytochemical screening and the determination of the content of polyphenol, flavonoid and total tannins. The anti- Helicobacter pylori activity was evaluated by microdilution broth assay and urease inhibitory capacity. Bacterial morphology, after sample exposure, was evaluated by scanning electron microscope (SEM) using the extracts and fractions that reached the minimum inhibitory concentration (CIM90). Screening of antioxidant activity for all samples was determined by DPPH and ABTS radical scavenging activities. Assays using the oxidizing biological agents, hydrogen peroxide (H2O2), superoxide anion (O2 •- ), hypochlorous acid (HOCl), hydroxyl radical (HO• ), and nitric oxide (NO• ) were performed with HE and its fractions due the best results in the antiH. pylori and initial screening antioxidant assays, except for the HxF because of its poor performance in the antioxidant screening. The highest concentration of polyphenols was observed in the HE, flavonoids in the ACF, and tannins in the AqF. Anti-H pylori assay presented a CIM90 of 512 μg/mL for HE, and 1024 μg/mL for ACF, the latter being bactericidal. SEM showed morphological changes such as stretching and cell lysis in the samples tested, and may suggest some modifications in the wall, such as alterations in the peptidoglycan synthesis. In the enzyme urease inhibition assay, the most significant result was obtained by the ACF, which inhibited 36.24% in the highest concentration tested. The best result among the extracts in the initial antioxidant screening was achieved by the HE, which obtained the following EC50: DPPH, 17.40 ± 0.52 and ABTS, 9.99 ± 1.21. The best EC50 for the fractions were: DPPH, AqF, 27.41 ± 1.65, and ABTS, AqF, 10.80 ± 1.90. In the O2 •- scavenging activity, AqF showed EC50 of 5.85 ± 0.86 μg/mL. In the inhibition assay of HOCl, all samples tested were able to inhibit greater than 50%, except AqF. In the inhibition assay of HO• , NO• e H2O2, the results were better for ACF and its EC50 was 2.90 ± 0.48, 132.13 ± 7.38 e 66.70 ± 2.30 μg/mL, respectively. The analyzes indicate that B. trimera, in particular the HE and its fractions, AqF and ACF, may exhibit promising compounds for the prevention and treatment of diseases caused by H. pylori. These results were based on the relationship between the presence of phenolic compounds and the inhibition of oxidants, as well as changes in the bacterial membrane.
Estudo de solubilidade e das atividades antioxidante e anti-Helicobacter pylori da isocumarina paepalantina obtida de Paepalanthus latipes Silv.
(Universidade Federal do Espírito Santo, 2016-03-24) Damasceno, João Paulo Loureiro; Giuberti, Cristiane dos Santos; Kitagawa, Rodrigo Rezende; Schuenck, Ricardo Pinto; Khalil, Najeh Maissar
Helicobacter pylori is a Gram-negative bacteria that infects the gastric mucosa, causing disorders like gastritis, peptic ulcers and stomach cancer, and which eradication is difficult. The standard treatment is not always successful because of its side effects, high costs and poor patient compliance, leading to the search for new drugs. In this context, the isocoumarin paepalantine, 9,10-dihydroxy-5,7-dimethoxy1H-naphtho (2,3c) pyran-1-one, isolated from the capitula of Paepalanthus bromelioides, has demonstrated a broad range of biological activities such as antibacterial, antioxidant, anti-inflammatory and cytotoxic. Among these, the antimicrobial and antioxidant activities stand out and allow the evaluation of the effects of H. pylori infection and the modulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) involved in this process. The molecule is poorly studied and there are few data in the literature on its solubility, including a viable alternative to the use of DMSO as a solvent, considering its toxicity in cell culture and interference in the antioxidant activity tests. Thus, the goal of this study was to evaluate alternative solvents to DMSO and validate an analytical spectrophotometric method using a suitable solvent for the antioxidant assays of radical scavenging activity (DPPH and ABTS), ROS (HOCl, OH● , O2 -● , H2O2), RNS (NO● ), and anti-H. pylori activity. Among the evaluated solvents, propylene glycol at pH 7.5 was the solvent of choice. The results suggest less interference in the assays compared to DMSO and that it is a viable alternative from a technological point of view. The results show a strong antioxidant activity of paepalantine in comparison to Trolox®. In addition, it was observed a significant effect on H. pylori culture with MIC of 128 μg/ml, MBC of 256 μg/ml, and a synergism of its sub-MIC doses with amoxicillin and metronidazole. This demonstrates a possible action on the permeability of bacterial membrane by inhibition of Penicillin-Bindind Proteins (PBPs), as observed through morphological changes using scanning electron microscopy. Therefore, paepalantine is promising for the development of drugs to combat H. pylori and its associated disorders.
Influência da suplementação com ácido ascórbico e zinco na morfologia testicular de ratos Wistar expostos ao arsenito de sódio
(Universidade Federal do Espírito Santo, 2016-03-30) Altoé, Luciana Schulthais; Pirovani, Juliana Castro Monteiro; Pancoto, João Alexandre Trés; Gomes, Marcos de Lucca Moreira
Several studies has shown that exposure to heavy metal arsenic, an environmental contaminant, may result in toxic effects, acute or chronic, causing different disorders, including potentially leading to damage to male fertility. Substances with potential protective action has been investigated. These include zinc and vitamin C, substances with known antioxidant action, are cofactors of cell division and playing an important role in reproduction. The aim of this study was to evaluate testicular function in rats exposed to arsenic in the form of sodium arsenite, and the possible protective role of co-administration of zinc or vitamin C by seminal, biometric and morphological (morphometry and stereology) analysis of the testis. Male Wistar rats (60 days old) were divided into six groups of six animals each: (1) control (distilled water), (2) sodium arsenite (5 mg/kg), (3) vitamin C (100 mg/kg) (4) zinc chloride (20 mg/kg), (5) sodium arsenite and vitamin C and (6) sodium arsenite and zinc chloride. The dosages were administered daily by gavage for 60 days. At the end of the treatment period, all animals were sacrificed and testis and accessory organs were removed and weighed. Testis fragments were processed for analysis by light microscopy and one centimeter of the deferens duct was removed for seminal analysis. There were no significant changes in the biometrics in any groups, but changes in the Leydig cells and in the seminiferous tubules were showed. Arsenic reduces the tubular diameter and the germinal epithelium height, resulting in a lower number of spermatids per testicle gram. Vitamin C and zinc were able to protect the Leydig cells and the proportion of normal spermatozoa of arsenic effects. It can be concluded that chronic exposure to sodium arsenite alter the spermatogenic process, reducing the total number of normal sperm, which can lead reduce fertility in male rats, and that coadministration of vitamin C or zinc although not able to neutralize the numerical damage can increase the proportion of normal spermatozoa.
Efeito de concentração subterapêutica de vancomicina sobre Staphylococcus aureus, no contexto do tratamento de bacteremias no paciente em hemodiálise
(Universidade Federal do Espírito Santo, 2016-06-20) Chagas, Rodrigo Altoé; Santos, Kênia Valéria dos; Schuenck, Ricardo Pinto; Silva, Vânia Lúcia da
In most patients undergoing hemodialysis treatment, due to the high rate of Staphylococcus aureus resistant to methicillin (MRSA) in bloodstream infections (BSI), vancomycin has been the antibiotic of choice for the empirical treatment of these infections. Failure in dose adjustment and drug dosing interval makes these patients often display a below therapeutic level (<10μg/mL) serum concentrations of vancomycin. The objective of this study was to investigate the effects of vancomycin subtherapeutic dose in some aspects of physiology and virulence factors of S. aureus. Strains of S. aureus with different susceptibility to vancomycin were exposed to subtherapeutic dose of the drug (<10mg / ml) to kill curve experiment. Then, the bacterial strains were cultured in the presence of drug for 5 consecutive days on Müller Hinton agar containing (0 a 10µg/mL), simulating the pattern empiric therapy of BSI in hemodialysis patients. Control cultures (without drug exposure) of the same bacterial strains were included in all experiments. At the end of the fifth day, the cultures were evaluated (i) for the minimum inhibitory concentration of vancomycin, daptomycin and oxacillin; (ii) biofilm production capacity and (iii) resistance to bactericidal activity of whole blood. In conclusion we reported that vancomycin overdose was not able to eliminat S. aureus and had limited effect against strains in exponential phase of growth. However, overdose of vancomycin has led to changes in the physiology of the microorganism as: (i) reduced susceptibility to vancomycin and daptomycin; (ii) increased resistance to the bactericidal action of the immune system and (iii) induction of a different response biofilm production among clinical isolates of S. aureus. These findings are consistent with therapeutic failure reports associated with underdosing vancomycin documented in the literature.
Estudo de polimorfismos da região 3' não traduzida do gene HLA-G em pacientes portadores de hepatite B
(Universidade Federal do Espírito Santo, 2016-10-07) Fontes, André Martins; Farro, Ana Paula Cazerta; Pancoto, João AlexandreTrés; Guimarães do Bem, Daniela Amorim Melgaço; Palomino, Gustavo Martelli
The World Health Organization (WHO) estimates that about 2 billion people worldwide have had contact with the hepatitis B virus (HBV), and 240 million to become chronic carriers. Hepatitis B is a type of disease in which the pathogenetic mechanisms are not yet fully understood, and it can evolve into chronic form and promote clinical manifestations such as cirrhosis and hepatocellular carcinoma, leading to a large number of deaths annually. Considering the changes in the immune system promoted by HBV, we use a data processing through geographic information system (GIS) technology and epidemiological analysis, in the cities of the northern region of the Espírito Santo State, with the initial objective to characterize the number of individuals infected with HBV. In addition, we used this initial analysis as a basis for evaluation of gene polymorphisms that may be associated with the pathogenesis of the disease, including situated genes within the MHC as the 3 'untranslated region of the HLA-G (presence or absence of 14 pairs base, + 3001 C/T, + 3003 C/T, + 3010C/G, + 3027 A/C, + 3035 C/T, + 3142 C/G, + 3187 A/G e + 3196 C/T). A GIS and epidemiological analysis showed that São Mateus had the largest number of individuals infected with HBV, followed by the cities of Pedro Canario and Jaguaré. The population aged 20-59 years, female sex, were the most affected by the disease, where the main forms of transmission occurred through the use of injectable drugs and dental care. The main drugs used in the treatment were Tenofovir and Entecavir. Among the polymorphic sites studied in individuals of the northern state Espírito Santo and the state of Mato Grosso, only the frequency allelic +3196G [p <0.0130; (OR = 1.540; 95% CI = 1.097 to 2.162)] and frequency genotypic Del/Ins 14-bp [p <0.0194; (OR = 1.645; 95% CI = 1.101 to 2.458)] were more frequent in patients with hepatitis B compared to controls, which may be related to the encouragement of viral escape and progression of infection. Regarding the genotypes +3003C/T, +3003 TT and +3196C/C, these were more frequent in controls compared to patients and are usually associated as microRNAs target (mirRNA) being related to the degradation of mRNA, resulting in low production of soluble and membrane HLA-G isoforms. Together, considering the results found in our study, it is observed that in the north there are many patients infected with HBV, and most of the female, which may be associated with poor adherence to treatment and due to low socio-economic level, sexual transmission, and dental/medical/surgical failures by the standards of biosecurity procedures. Evaluation of genetic polymorphisms in the population suggests that they have genotypes favorable to high production of HLA-G and thus this immunoregulatory molecule could be influencing the development of the chronic form of hepatitis B and the emergence of possible complications of this disease such as liver cirrhosis and hepatocellular carcinoma.
Participação de receptores noradrenérgicos na substância cinzenta periaquedutal dorsal na modulação de comportamentos defensivos relacionados à ansiedade
(Universidade Federal do Espírito Santo, 2016-11-04) Souza, Dayane de Oliveira; Martins, Juliano Manvailer; Bortoli, Valquíria Camin de; Harres, Vanessa Beijamini; Bittencourt, Ana Paula Santana de Vasconcellos
The role of the dorsal periaqueductal gray matter (DPAG) in fear and anxiety has been studied over the last decades. The evidence regarding the involvement of the noradrenergic system in anxiety is conflicting, depending on brain structure studied. Thus, in the present study we tested the hypothesis that noradrenaline injected directly into the DPAG of rats has anxiolytic-like effect in the light-dark transition test. The lightdark transition test is an ethological model of anxiety, quick and easy to perform, that does not require animal training. For this, Wistar rats were given intra-DPAG administration of noradrenaline (30, 60 or 90 nmol) or saline and tested in light-dark test. In addition, we investigated the effect of pre-treatment with intra-DPAG nonselective antagonists of alpha and beta-adrenergic receptors, phentolamine and propranolol, respectively, in the effect of noradrenaline injection in the same structure. After the tests, the animals were placed for five minutes in the open field, for measurement of locomotor activity. Our results show that noradrenaline (30 and 60 nmols) administered directly into the SCPD increased time spent in the light compartment and the number of transitions of the animals submitted to the light-dark transition test, suggesting an anxiolytic-like effect. In addition, intra-SCPD injection of norepinephrine did not change locomotor activity of animals in the open field test. The results also show that pre-treatment with intra-SCPD propranolol or phentolamine attenuated anxiolytic-like effect of norepinephrine when analyzing the time spent in the light compartment of the model. Thus, this work suggests an involvement of noradrenergic neurotransmission in the SCPD, via alpha and beta-adrenergics receptors in defensive reactions associated with generalized anxiety disorder in animals in the light-dark transition model.
Avaliação das atividades gastroprotetora, anti-Helicobacter pylori, imunomoduladora e antioxidante dos "boldos" de interesse ao SUS : Plectranthus barbatus Andrews (Lamiaceae) e Vernonia condensata Baker (Asteraceae)
(Universidade Federal do Espírito Santo, 2017-02-02) Borges, Augusto Santos; Kitagawa, Rodrigo Rezende; Beltrame, Flávio Luís; Fronza, Marcio
Helicobacter pylori is a Gram-negative bacteria capable to survive the gastric environment and has a great influence in the development of many gastric diseases, like gastritis, peptic ulcers and even the gastric cancer. Plectranthus barbatus (Falso-boldo) and Vernonia condensata (Boldo-baiano) are species of medicinal plants with a wide traditional use in Brazil for the treatment of gastric injuries, however, none scientific reports have been described about the activity against H. pylori. Thus, this study aimed to determine the anti-H. pylori activity, gastroprotection activity, immunomodulation activity, antioxidant activity and phytochemical profile of extracts and fractions of these species. The phytochemical profile was evaluated through preliminary phytochemical assay, polyphenols and flavonoid content assays and mass spectrometry. The gastroprotective was measured by in vivo ulcer model induced by ethanol. The anti-H. pylori activity was made by minimal inhibitory and bactericidal concentration (MIC and MBC) and urease inhibition activity. The immunomodulatory activity was measured by detection of nitric oxide and cytokines (TNF-α, IL-1β and IL-6) released from LPS stimulated macrophages. Furthermore, was made the inhibitory potential against the artificial radicals (DPPH and ABTS•+) and oxygen and nitrogen reactive species (O2 - , HOCl, H2O2 and NO). The oral treatment with the extracts from both species shows gastroprotection ranging 58 – 78%. The anti-H. pylori results was significant only for the P. barbatus species, which showed an MIC of 256 µg/mL for the ethyl acetate fraction (AcOEt) and 512 µg/mL for the aqueous extract. Furthermore, the AcOEt fraction showed MBC of 1024 µg/mL. The results of the antioxidant capacity were significant for both species against the artificial radicals as well to the reactive oxygen species: O2 - (IC50: 32 – 72 µg/mL) and HOCl (IC50: 53 – 85 µg/mL). For the results of immunomodulatory activity, was possible to realize anti-inflammatory capacity in both species, but, the V. condensata showed greater potential and managed to decrease the productions of NO and cytokines, in a dose-response way, in the LPS stimulated macrophages (28 – 88 % of inhibition). Both species showed significant amounts of flavonoids and polyphenols. In addition, P. barbatus presented different diterpenes such as plectrin, hydrolyzed abietane, barbatusin, 3β- hydroxy-3deoxybarbatusin and cyclobutatusin as well the polyphenols coleoside B and rosmarinic acid. Altogether, the P. barbatus species, in special your AcOEt fraction, proved to be promising for future use in the prevention and treatment of H. pylori infections, because in addition to the inhibition of growth of H. pylori capacity, it also showed promising gastroprotective, immunomodulatory and antioxidant activity.
Investigação de parâmetros de inflamação e estresse oxidativo em pacientes com diabetes mellitus tipo 2 usuários e não usuários de insulinoterapia
(Universidade Federal do Espírito Santo, 2017-02-22) Paiva, Kainá Kiffer; Gonçalves, Rita de Cássia Ribeiro; Guimarães do Bem, Daniela Amorim Melgaço; Rodrigues, Lívia Carla de Melo; Borges, Karina Braga Gomes
Type 2 diabetes mellitus (DM2) is a metabolic disorder resulting from hyperglycemia and insulin resistance which is characterized by increased production of reactive species leading to oxidative stress and inflammatory changes. The goal of this study was to investigate whether the use of insulin therapy in patients with Type 2 Diabetes Mellitus influences levels of inflammatory markers and oxidative stress. It is a clinical study in which 80 patients diagnosed with T2DM (40 in insulin therapy and 40 without insulin therapy) and 40 in the control group (without DM2) were selected. Adherence to treatment was assessed by the Morisky-Green test. Metabolic control was evaluated through tests of fasting glycemia, glycated hemoglobin and lipid profile. Oxidative stress was investigated through levels of nitric oxide (NO), lipid peroxidation (Malondialdehyde - MDA) and superoxide dismutase (SOD). The inflammation evaluation was performed through the markers: tumor necrosis factor alpha (TNFα), ultra sensitive C reactive protein (CRP) and fibrinogen. In addition, the -308 G / A polymorphism was investigated in the promoter region of the TNFα gene and its correlation with its plasma levels. In this study, an inadequate glycemic control was found in patients with DM2 who were on insulin therapy and a low adherence to treatment in both DM2 groups (approximately 50% of the patients). The DM2 group with insulin therapy had lower levels of NO and SOD, higher levels of fibrinogen and TNF-α, and lower levels of CRP than the DM2 group without insulin therapy. In lipid peroxidation both groups of patients with DM2 had higher levels of MDA than controls. There was no correlation of the -308 G / A polymorphism in the promoter region of the TNF-α gene with its plasma levels in patients with T2DM. In conclusion, patients with DM2, who are users or not of insulin therapy, have increased oxidative stress and inflammation, which may contribute to the various complications of the disease.
Estudo químico e biológico de Hippeastrum puniceum (Lam.) Kuntze (Amaryllidaceae)
(Universidade Federal do Espírito Santo, 2017-02-23) Soprani, Letícia Carlesso; Borges, Warley de Souza; Jamal, Claudia Masrouah; 1º membro da banca
Hippeastrum puniceum is a specie that belongs to Amaryllidaceae This family presents a very specific group of alkaloids known as Amaryllidaceae alkaloids, (group of isoquinoline alkaloids), showing several biological activities such as antioxidant activity, inhibition of the enzyme acetylcholinesterase, antiviral action, antifungal, antiparasitic, and others. The purpose of this work was to study the chemical composition of the bulbs of the species Hippeastrum puniceum (Lam.) Kuntz., and evaluate the possible biological activity. The acidbase extraction was performed for the methanolic extract of H. puniceum bulbs, obtaining 03 other fractions: Hp01 (Hexan), Hp02 (Ethyl Acetate) e Hp03 (Ethyl Acetate:Methanol 3:1). Then were used classics techniques of fractionation and purification. Tests were performed to evaluate the antioxidant activity against the synthetic radical DPPH and ABTS, as well as the determination of in vitro thrombolytic activity that evaluated the capacity of the extracts promote clot lysis. Analysis of the extracts by GC-MS revealed the presence of 11 alkaloids: 9-O-demethyllicoramine, licoramine, galantamine, assoanine, kirkine, 8- demethylmaritidine, pancratinine, 11-hydroxyvittatine, pseudolicorine, 2αhydroxyhomolycorine and licorine. They were isolated by standard techniques of isolation and purification of the alkaloids 9-O-demethyllycoramine, 9- demethyl-2α-hydroxyhomolycorine, licorine and tazettine. The fractions HpEb, Hp02 and Hp03 showed antioxidant capacity. The results obtained demonstrate the progress in the phytochemical study of Hippeastrum puniceum. However it is necessary to search for other methodologies to evaluate the biological potential of this species.
Avaliação da qualidade e da compatibilidade fármaco-excipientes das formulações farmacêuticas sólidas do mercado contendo meloxicam
(Universidade Federal do Espírito Santo, 2017-03-15) Silveira, Lucas Melo da; Oliveira, Marcelo Antônio de; Jamal, Claudia Masrouah; França, Hildegardo Seibert
Meloxicam (MLX) is a non-steroidal anti-inflammatory, cyclooxygenase (COX) inhibitor, used to relieve inflammation and pain. MLX have a preferential affinity for COX-2, which is associated with a lower incidence of gastrointestinal side effects. The drug belongs to Class II of the Biopharmaceutical Classification System (BCS) where dissolution is the bioavailability limiting step. In view of this classification, it is a fundamental to carry out further studies regarding the compatibility between drug and excipients, mechanisms and kinetics of degradation reactions, since any changes directly influence the quality of the product. The aim of the present work is to evaluate the solid pharmaceutical formulations containing MLX found on the market defining more suitable excipients to improve the stability of pharmaceutical formulations. Thermal analysis techniques were used to characterize and evaluate the compatibility between the drug and the excipients present in the market formulations. Method by high performance liquid chromatography was developed and validated for quantification of MLX and possible degradation products. Alternative method by UV spectrophotometry was validated for quantification of MLX in the formulations. In the study of compatibility between drug-excipient was found incompatibility with magnesium stearate, red iron oxide, povidone, sodium starch glycolate and mannitol. In the study of intrinsic stability, the drug was subjected to conditions of forced degradation where it was unstable in alkaline medium. In the evaluation of the solid state kinetics, the MLX degraded according to AvramiErofeyev A4 model, presenting a shelf life of about 6 years for the raw material under inert condition. In the evaluation of the quality control of the products of the market, it was observed that the Lab10 and Lab11 were disapproved in the dissolution test. As for the evaluation of the dissolution profile only two products have been shown to be pharmaceutical equivalents
Estudo químico em alcaloides de Hippeastrum reginae (L.) Herb. (Amaryllidaceae)
(Universidade Federal do Espírito Santo, 2017-03-20) Oliveira, Marcos Vinícius Lacerda de; Borges, Warley de Souza; Cunha Neto, Álvaro; Vieira, Paulo Cezar
Objectives: The main objective of the present work was to carry out a chemical study on alkaloids of the species Hippeastrum reginae, promoting the isolation and characterization of these compounds. Methods: The plant was separated in bulbs and leaves for the extraction with methanol. After this step, the acid-base extraction and then the partition with solvents of different polarities were made to obtain the fractions enriched in alkaloids. The enriched fractions were initially analyzed by gas chromatography coupled to mass spectrometry (GC/MS) and then fractionated by chromatographic techniques such as Sephadex exclusion column, column chromatography and preparative thin layer chromatography. For the isolation of some alkaloids, high performance liquid chromatography (HPLC) and for the identification of the nuclear magnetic resonance (NMR) technique were used. Results: The CG/MS analyze of the extracts evidenced the presence of 11 alkaloids, of which four had no fragmentation pattern compatible with the alkaloid library, and may be unpublished in the literature. Eighteen alkaloids of Amaryllidaceae were isolated, most of them belonging to the homolycorine class. Of these compounds, six new alkaloids of Amaryllidaceae were identified, four of which were fully elucidated (2--methoxy-7-hydroxyhomolycorine, 2--7-dihydroxyhomolycorine, 2-- methoxyhomolycorine and 2-α-hydroxyalbomaculine), while Two (7-hydroxyhomolycorine and N-oxide albomaculine) analyzes were performed for 1H-NMR and high resolution masses. Conclusion: The H. reginae plant had a high alkaloid content and 18 compounds were isolated. Albomaculine and 2--7- dimethoxyhomolycorine alkaloids were the most abundant in GC / MS analysis. Six alkaloids not found in the literature were identified in this work and show a great potential for the discovery of new active compounds.
Estudo químico de alcaloides nas raízes de Worsleya procera (Lem.) Traub (Amaryllidaceae)
(Universidade Federal do Espírito Santo, 2017-07-31) Salarini, Karla Loriatto Gonring; Borges, Warley de Souza; Gonçalves, Rita de Cássia Ribeiro; Kuster, Ricardo Machado
Comprising more than 1600 species worldwide, the Amaryllidaceae family is distinguished by the production of isoquinolinic alkaloids which are assigned biological activities such as: antitumor, anti-inflammatory, analgesic, antimicrobial and reversible acetylcholinesterase inhibitory action. In Brazil, 141 species of Amaryllidaceae have been described, of which 62 are endemic and some are threatened of extinction, as is the case of Worsleya procera (Lem.) Traub, the only representative of the genus Worsleya. This species occurs exclusively in the State of Rio de Janeiro (RJ) and no published phytochemical studies have been found for this species. In order to carry out the phytochemical study on alkaloids of the species, samples of the roots of Worsleya procera (Lem.) Traub were collected in the Permanent Protection Area of Petrópolis (RJ) and after maceration and acid-base extraction, extracts enriched in alkaloids were obtained. Preliminary analysis by gas chromatography coupled to a mass spectrometer (CG-MS) identified 12 compounds already known, as well as other 18 non-identified by the University of Barcelona database. Using chromatographic, spectroscopic and spectrometric techniques, it was possible to isolate and identify the alkaloids lycorine, tazettine, 3-epimacronine, homolycorine, albomaculine, ismine and trisphaeridine. The roots of Worsleya procera are a rich source of alkaloids, especially of lycorine and tazettine. The identification of these compounds may contribute to chemotaxonomy of this species as well as in research involving biological activities due to the amount of isolated alkaloids.
Avaliação in vitro e in vivo dos efeitos da intoxicação aguda com o inseticida organofosforado, clorpirifós, e da eficácia do tratamento farmacológico empregado na intoxicação sobre a modulação cardiorrespiratória tônica e reflexa
(Universidade Federal do Espírito Santo, 2017-08-08) Felippe, Igor Simões Assunção; Harres, Vanessa Beijamini; Sampaio, Karla Nívea; Paton, Julian Francis Richmond; Silva, Valdo José Dias da
In Brazil, the usage of agrochemicals has grown significantly in recent years, making the country one of the world leaders in consumption. Organophosphates (OFs) have been associated with cardiovascular diseases and intoxication induced by these compounds has a high mortality rate. Previous studies from our laboratory have shown that acute exposure to an OP compound, chlorpyrifos (CPF), in rats impairs the cardiovascular responses of the baroreflex and chemoreflex. However, it was not possible to address in this study the effects induced by the exposure to this compound on the respiratory function, despite the important modulation that the cardiovascular reflexes, particularly the chemoreflex, exert on respiratory parameters. Considering that the cardiorespiratory function plays a major role in the survival of intoxicated individuals, it seems of pivotal importance the development of detailed studies, using in vitro and in vivo approaches, to investigate the effects of CPF exposure on the tonic and reflex cardiorespiratory modulation. Additionally, the present controversy surrounding the standard treatment adopted in OP intoxication and the absence of studies focusing on antidote treatment and restoration of OPinduced cardiovascular damage, further reinforces the importance of studies in the field. In the present study, in vitro and in vivo protocols were used to evaluate the effects of acute intoxication with CPF on tonic and reflex cardiorespiratory activity and to evaluate whether the pharmacological treatment with the antidotes atropine (ATR) and pralidoxime (2-PAM) was capable of reversing the damage on the chemoreflex function previously observed. Two groups (CPF and control) were submitted to the in vitro protocol involving the working heart-brain stem preparation (WHBP). In this preparation, basal activities of the phrenic (PNA), recurrent laryngeal (RLN) and thoracic sympathetic (tSNA) nerves, in addition to heart rate and perfusion pressure were recorded. Associated with these recordings, in situ activation of the chemoreflex and baroreflex was also performed, followed by procedures of bilateral vagotomy and blockade with atenolol and hexamethonium. In the in vivo model, animals were grouped according to different combinations of treatments with CPF, ATR and 2-PAM. Twenty-four hours after treatment, activation and records of the chemoreflex function were performed. The presence of intoxication signs, ataxia, tremor and sialorrhea, was evaluated along three hours after receiving injections of CPF or saline; and after the treatment with ATR and/or 2-PAM. For both in vitro and in vivo models, the activities of plasma butyrylcholinesterase (BuChE) and cerebral acetylcholinesterase (AChE) in the brainstem were quantified. Additionally, in the in vivo protocol, punchs of brain stem sections containing the nucleus ambiguous, rostral ventrolateral medulla (RVLM), the pre-Bötzinger complex (preBöTC) and the nucleus of the solitary tract (NTS) were collected for individualized quantification of cerebral AChE activity. Our data showed that acute exposure to CPF led to impairment of the chemoreflex responses either in the in vitro or in vivo models. Additionally in the in vitro study, an impairment of baroreflex function was also observed, indicated by the reduced reflex-induced inhibition of the sympathetic activity in poisoned animals. Concerning the respiratory function in the WHBP, a reduction in the amplitude of PNA and of the RLN post-inspiratory discharge was also observed in intoxicated animals when compared to the control group. CPF poisoned animals also presented a higher tachycardia following vagal section when compared to control group. Blockade with hexamethonium induced an increase in perfusion pressure of CPF intoxicated animals. In the in vivo model, treatment with the antidotes, ATR and 2-PAM, induced differentiated effects on chemoreflex responses in intoxicated animals. Treatment with 2-PAM restored the impairment of the chemoreflex-induced hypertension observed in CPF intoxicated animals. On the other hand, treatment with atropine attenuated the impairment of the bradycardic response of the chemoreflex observed in poisoned animals. Both treatments seemed to exert a protective effect on the tachypneic response of the chemoreflex. All CPF intoxicated animals showed a marked inhibition of the plasma butyrylcholinesterase and acetylcholinesterase activities either in the brainstem or in the isolated punchs. CPF animals also exhibited signs of acute intoxication, which were attenuated or abolished by the antidote treatments. Our data indicate that CPF intoxication induces an impairment of the chemo and baroreflex responses and that the antidote treatment seems to partially restore the damage observed in the cardiorespiratory function.
Epidemiologia molecular e caracterização da resistência de amostras de Acinetobacter baumannii e Pseudomonas aeruginosa resistentes aos carbapenêmicos provenientes de hospitais da grande Vitória-ES
(Universidade Federal do Espírito Santo, 2017-08-30) Vallorini, Thalita Pereira Cabral; Schuenck, Ricardo Pinto; 1º membro da banca
The emergence and spread of antimicrobial resistance among non-fermenting Gram-negative bacilli, such as Acinetobacter baumannii and Pseudomonas aeruginosa, is a worldwide problem. Carbapenems are beta-lactam antimicrobials indicated for the treatment of serious infections caused by these agents, however, the emergence of multiresistant pathogens seriously threatens the use of this class of drug in the hospital environment. The present work aimed to characterize clinical samples of P. aeruginosa and A. baumannii resistant to cabapenems, as well as the presence of beta-lactam resistance genes, epidemiological profile and susceptibility to antimicrobials used in clinical routine. The antimicrobial susceptibility was performed by disc diffusion and antimicrobial gradient tests. PCR was used to the detection of beta-lactamases-encoding genes and the genetic polymorphism was analyzed by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) techniques. The species included in this study were: A. baumannii (n = 26) and P. aeruginosa (n = 15). The most of the isolates presented a multiresistance profile to the tested antimicrobials, highlighting the resistance to colistin in nine isolates of A. baumannii. Among the A. baumannii isolates, the prevalent carbapenemase gene (92.3%) was blaOXA-23 and in P. aeruginosa isolates the blaVIM gene was prevalent (33.3%). The PFGE analysis showed the prevalence of two pulsotypes among the A. baumannii: abA (34.6%) and abB (23%), while the P. aeruginosa isolates presented distinct pulsotypes, demonstrating the polyclonal origin of isolates. MLST was performed in five isolates of P. aeruginosa presenting different pulsotypes and STs 357, 2321, 1121, 244 and 227 were found, with two clonal complexes of world importance: CC235 and CC244. In addition, ST357, ST2321 and ST1121 were described for the first time in Brazil.
Epidemiologia, caracterização microbiológica e avaliação dos custos com o tratamento de infecções do pé diabético
(Universidade Federal do Espírito Santo, 2018-03-08) Batista, Julianne Soares Jardim Lacerda; Schuenck, Ricardo Pinto; Spano, Liliana Cruz; Gonçalves, Rita de Cássia Ribeiro
Diabetic foot infection (DFI) is one of the most serious and costly complications of diabetic patients. This study aimed to analyze the microbiological and epidemiological aspects of DFI and to determine the costs of treating these patients. We collected clinical and epidemiological data from the medical records. To analyze the costs, we collected data referring to the real cost invested in the treatment of each patient. The antimicrobial susceptibility was determined using agar diffusion or E-test. Genetic polymorphisms were analyzed by the PFGE technique. The study included 46 patients with DFI; the majority were men (58.7%) who were elderly (mean age 63.9 ± 10.8 years) with associated cardiovascular diseases (60.8%). The amputation rate was 56.5 %. The average treatment cost of DFI was US$ 9,936.84 (ranging from US$ 14.19 to US$ 58,230.71). Forty-seven isolates were collected, and a predominance of Gram-negative microorganisms (70.8 %) was observed. PFGE revealed a large clonal diversity. Time and costs of hospitalization and antimicrobial treatment were higher in cases of multidrug-resistant and polymicrobial infections. We observed that DFIs are serious complications with long times of hospitalization and costly antimicrobial treatment. Bacterial resistance and polymicrobial nature of wounds can increase the costs of such morbidity.
Estudo do efeito de naftopiranonas de espécies de paepalanthus sp sobre Helicobacter pylori, macrófagos ativados e linhagem celular de adenocarcinoma gástrico
(Universidade Federal do Espírito Santo, 2018-03-19) Ardisson, Juliana Santa; Rodrigues, Ricardo Pereira; Kitagawa, Rodrigo Rezende; Fronza, Marcio; Schuenck, Ricardo Pinto
The naphthopyranones paepalantine and 5-methoxy-3,4-dehydroxanthomegnin were isolated from Paepalanthus sp and showed antioxidant, anti-inflammatory, antitumor and antimicrobial potential, including anti-H. pylori. H. pylori infection is one of the main causes of gastric cancer. The infection causes an excessive inflammatory response through the neutrophils and macrophages infiltration, increasing the release of reactive species and inducing the production of pro-inflammatory mediators. In the gastric environment, H. pylori expresses the urease enzyme, increasing the pH of the medium through ammonia production. In this context, our aim was to evaluate the activity of naphthopyranones in H. pylori, immunomodulation in macrophages and cytotoxic action in gastric adenocarcinoma cell line and to confirm the potential of interaction of these substances in the urease and iNOS binding sites through molecular docking studies. The determination of the minimum inhibitory concentration (MIC) of the naphthopyranones for H. pylori was performed by broth microdilution technique for further analysis of the synergistic activity with metronidazole and for morphological analysis by scanning electron microscopy (SEM). The evaluation of the urease inhibition by the naphthopyranones was performed in vitro and in silico. The immunomodulatory activity of naphthopyranones was evaluated detecting the nitric oxide and cytokines (TNF-α, IL-1β, and IL-6) released by macrophages, and the interaction potential of the naphthopyranones within iNOS binding site was studied in silico. The reduction of the pathogenicity of H. pylori in macrophages was investigated after treatment with sub-inhibitory concentrations of the naphthopyranones. Finally, cytotoxic activity in gastric adenocarcinoma cells (AGS) was evaluated by the MTT assay. 5-methoxy-3,4-dehydroxanthomegnin is shown to inhibit H. pylori (metronidazole resistant strain) synergistically with metronidazole, reducing its MIC to 8 μg/mL. The results of the scanning electron microscopy, that evaluated the H. pylori morphology, evidenced that the naphthopyrananones alter the H. pylori morphology, indicating a possible role on the Penicilin-binding protein (PBPs) due to the bacterial cell wall modifications. It was also verified that H. pylori grow under naphthopyranones sub-inhibitory concentrations causes reduction of the activation of macrophages by inhibition of NO production in 60.21 ± 1.8% for paepalantine and 47.50 ± 0.6% for 5- methoxy-3,4-dehydroxanthomegnin. Regarding urease inhibition, no significant inhibitory activity of the samples and no favorable interactions with the major aminoacid residues in the active site of this enzyme was observed. The samples demonstrated immunomodulatory potential by inhibiting the proinflammatory cytokines produced by LPS stimulated macrophages (TNF-ɑ inhibition of 36.07 ± 2.63% and 60.19 ± 0.12% and IL-6 inhibition of 36.91 ± 0.57% and 92.35 ± 0.15% of paepalantine and 5-methoxy-3,4-dehydroxanthomegnin respectively, both at 25 μg/mL) and NO (99.5 ± 5.31% for paepalantine and 76.9 ± 4.11% for 5-methoxy-3,4- dehydroxanthomegnin at a concentration of 12.5 μg/mL) and also demonstrated interaction potential in the active site of the iNOS enzyme. In addition, naphthopyranones demonstrated satisfactory cytotoxic activity in AGS, with IC50 of 24.56 ± 2.3 μg/mL for paepalantine and 17.45 ± 1.6 μg/mL for 5-methoxy-3,4- dehydroxanthomegnin. In general, the naphthopyranones demonstrate potential for future use in the treatment and prevention of H. pylori infection as well as the diseases related to this infection, especially gastric cancer.

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