Mestrado em Ciências Farmacêuticas

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Avaliação da atividade anti-Acanthamoeba castellanii de frações alcaloídicas de diferentes espécies da família Amaryllidaceae e da licorina
(Universidade Federal do Espírito Santo, 2017-04-04) Rosa, Maressa Dietrich; Borges, Warley de Souza; Bueloni, Cinthia Furst Leroy Gomes; Rott, Marilise Brittes; Kitagawa, Rodrigo Rezende
Free-living amoebae (FLA) are protozoa of wide environmental dispersion. Acanthamoeba is classified as one of the genres of FLA and can behave as an elective parasite in humans, causing two diseases: Amebic Granulomatous Encephalitis (AGE), a cronic disease of the central nervous system, difficult to treat; and Amebic Keratitis (AK), a very painful ocular cornea disease that can lead to Blindness and loss of the eyeball in more critical cases. The treatment for CA is very difficult, long, toxic and does not present complete effectiveness against existing cases. The great challenge of the treatment is the high toxicity and the resistance to the medicines. The use of plants as research sources for new amebicidal bioactive compounds has shown a good alternative; since these have high amoebicidal power and low toxicity. The objective of this study was to investigate the in vitro antiAcanthamoeba castellanii activity of 22 alkaloid enriched fractions of 12 species of Amaryllidaceae and the alkaloid lycorine on Acanthamoeba castellanii trophozoites using 3- (4,5-dimethyl) 5-diphenyltetrazolium (MTT). Chlorhexidine 0.02% was used as a positive control. The species Hippeastrum canastrense, Hippeastrum dinizcruziae, Crinum giganteum Hippeastum puniceum and lycorine showed a concentration-dependent effect, with H. canastrense and H. diniz-cruziae at the concentration of 2000 μg.mL-1, with a high amebicide (100% Inhibition) greater than chlorhexidine (86% inhibition). The cytotoxicity of these fractions and the lycopene were also evaluated on MDCK mammalian cells by the use of the MTT dye and showed a concentration-dependent effect, and H. diniz-cruziae at concentrations of 250, 500 and 1000 μg.mL-1 showed low cytotoxicity (5 to 7% inhibition) less than chlorhexidine (97% inhibition). The IC50 value calculated for H. canastrense, H. dinizcruziae, Crinum giganteum, H. puniceum and lycorine was from 285.61 to 962.75 μg.mL-1 and the selectivity index (SI) was 1, 27 to 7.0. The H. diniz-cruziae species presented promising results, with high amebicidal power (100% inhibition), low cytotoxicity (5 to 7% inhibition), lower IC50 value of 285.61 μg.mL-1 and higher IS value (7,0), being a promising target for the discovery of new drugs with antiAcanthamoeba castellanii activity.
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.
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 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.
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.
Avaliação socioeconômica, demográfica, parasitológica e hematológica de comunidades quilombolas do norte do Espírito Santo, Brasil
(Universidade Federal do Espírito Santo, 2017-03-31) Brauer, Alline Mikaele Nunes Wildemberg; Gradella, Débora Barreto Teresa; Souza, Marco Antônio Andrade de; Pinto, Hudson Alves; Gazzinelli, Sandro Eugênio Pereira
Brazil is considered the country, extra Africa, that presents the largest contingent of black population. However, it is still possible to observe especially in the quilombola communities a scenario marked by socioeconomic marginalization and precarious living and health conditions. The present work is a cross-sectional, descriptive and quantitative study with the objective of performing a socioeconomic, demographic, parasitological and hematological evaluation of the inhabitants of quilombola communities located in the northern region of Espírito Santo, Brazil. The socioeconomic and demographic evaluation was performed by family unit, through the application of a questionnaire based on the National Survey by Household Sample. For the investigation of intestinal parasites the methods of spontaneous sedimentation (HPJ) and Kato Katz were used. In the hematological investigation, the complete blood count, the hemoglobinopathies (osmotic resistance test, alkaline electrophoresis in cellulose acetate and high performance liquid chromatography) were performed. The results were evaluated using descriptive and inferential statistics tools, using Pearson's chi-square test and Poisson regression analysis, adopting the 95% confidence interval (p <0.05). It was observed that of the 76 household heads interviewed, 90.79% (n = 69) had channeled water in the residence and 72.37% (n = 55) reported that the origin of the water used by them is well or spring . In addition, 98.68% (n = 75) reported having a bathroom at home, of which 90.67% (n = 68) reported having a rudimentary septic tank and 63.16% (n = 48) claimed that the waste generated was buried or burned. Regarding the general health of the population, 78.94% (n = 60) reported having access to public health services, 64.47% (n = 49) reported having ever had parasitic diseases and 63, 15% (n = 48) claimed to know how they are transmitted. On the other hand, 92.10% (n = 70) reported that they did not know if they had sickle cell disease or sickle cell trait and 94.73% (n = 72) do not know how they are acquired. The parasitological tests showed positive results in 48% (n = 72) of the total samples analyzed (150), and 25% (n = 18) of these samples had two or more parasites. The most frequent parasite was Ascaris lumbricoides (19.4%, n = 14) and among the commensals Entamoeba coli (55.6%, n = 40), followed by Endolimax nana (16.7%, n = 12). Considering the hematological evaluation, of the 192 analyzed samples, a frequency of 13.54% (n = 26) of individuals with anemia was observed and 80.77% (n = 21) were of mild severity. There was predominance of normocytic anemia (92.31%, n = 24) and normocytic anemia (73.07%, n = 19). Regarding the hemoglobinopathies, 9.37% (n = 18) of the individuals presented variant hemoglobins confirmed by high-performance liquid chromatography. The presence of heterozygosis for Hb AS was observed in 6.77% (n = 13) and for Hb AC in 2.60% (n = 5). The results suggest the existence of precarious socioeconomic conditions in the quilombola communities in the Northern region of Espírito Santo and indicate the need to implement public health measures aimed at reducing, preventing and treating intestinal parasitic diseases. The considerable prevalence of variant hemoglobins in the region and the lack of knowledge about this subject demonstrate the importance of enabling health professionals to attend hemoglobinopathies, especially in genetic counseling.
Efeito de antimicrobianos sobre biofilmes de stenotrophomonas maltophilia em diferentes estágios de formação
(Universidade Federal do Espírito Santo, 2018-12-06) Carvalhais, Bárbara Ellen Santos; Santos, Kênia Valéria dos; Schuenck, Ricardo Pinto; Spano, Liliana Cruz
Stenotrophomas maltophilia is a non-fermenting carbohydrate Gram-negative bacillus, which although of environmental origin, has been reported in bacteremias related to catheter use, urinary tract infections, endocarditis, and respiratory tract infections, especially in immunosuppressed patients. In addition to the intrinsic resistance to several antimicrobials, another characteristic of S. maltophilia that makes it an important opportunistic pathogen is its ability to form biofilms. Thus, our objective was to evaluate the effect of antimicrobials indicated for the treatment of S. maltophilia infections on biofilm in different stages of formation (adhesion, exponential growth and maturation). We evaluated 19 clinical samples and one reference sample (ATCC 13637) of S. maltophilia identified by biochemical methods and confirmed by amplification of the 23S region of ribosomal RNA. Samples were evaluated for similarity by ERIC-PCR. After this initial characterization, the samples were submitted to the following tests: (i) biofilm formation capacity; (ii) biofilm formation kinetics; (iii) antimicrobial susceptibility profile to chloramphenicol (CHL), levofloxacin (LVX), ceftazidime (CAZ), sulfamethoxazole-trimethoprim (SXT) and gentamicin (GEN) by the broth microdilution method and (iv) determination of the minimum inhibitory concentration of antimicrobial agents against biofilm (MIC-b) at different stages of formation. The samples showed genetic heterogeneity in the technique used. All were moderate producers of biofilm, showing adhesion until the 4th hour of incubation, exponential growth between the 4th and 8th hour and mature biofilm between 8 and 24 hours of incubation. All clinical samples in their planktonic forms were sensitive to LVX and SXT and most were sensitive to CHL. Higher resistance frequencies were for CAZ and GEN. We observed that bacterial susceptibility decreases as the biofilm becomes mature. The antimicrobial with the best biofilm activity regardless of the formation time was GEN followed by LVX. SXT which is the first choice antimicrobial for the treatment of S. maltophilia infections was the least effective against biofilms along with CAZ. The determination of the susceptibility profile of the samples in the biofilm shows that GEN was the best antimicrobial activity at all formation times. CAZ was the least effective against 24 hour biofilm and SXT was the least effective in 4 and 6 hour biofilm. Considering the MIC-b/MIC ratio, we can organize antimicrobials in the following decreasing order of activity against mature (24h) biofilms: GEN, LVX, CHL, SXT and CAZ. Based on these findings, we conclude that GEN may be a good option against infections related to S. maltophilia biofilms in regions where this microorganism is susceptible to aminoglycosides. LVX remains a good option against S. maltophilia biofilms. On the other hand, SXT that is currently considered the gold standard for infection by S. maltophilia should not be used in infections where biofilms may be present.
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.
Efeito do tratamento padrão com atropina e pralidoxima na prevenção das alterações comportamentais e bioquímicas após intoxicação aguda pelo organofosforado clorpirifós
(Universidade Federal do Espírito Santo, 2016-03-31) Siqueira, Alciene Almeida; Sampaio, Karla Nívea; Harres, Vanessa Beijamini; Pires, Rita Gomes Wanderley; Siqueira, Ionara Rodrigues
Organophosphorus insecticides (OP), used in agriculture in many countries worldwide, exert their toxicity by inhibiting the activity of both central and peripheral cholinesterases, leading to the accumulation of acetylcholine at cholinergic synapses. Clinical studies have shown that chronic exposure to OP at low doses can induce affective disorders such as depression. Our research group verified that acute poisoning by one of those agents, chlorpyrifos (CPF) 20mg/kg, induced a depressivelike behaviour in adults male Wistar rats on the Forced Swimming Test (FST). Standard treatment for OP poisoning involves the use of atropine (ATR), a muscarinic antagonist, to treat the symptoms of cholinergic syndrome and a reativator of cholinesterases, such as pralidoxime (2-PAM), to restore the peripheral enzymatic activity. Thus, our aim was to assess if standard treatment would attenuate or reverse behavioral and biochemical changes in the FST, plasma, hippocampus, striatum and prefrontal cortex of rats 24 hours and 30 days after acute exposure to CPF. Animals were submitted to pre-exposure in the FST and immediately after they received intraperitoneal injections of CPF or saline (SAL). One hour later, animals were evaluated with regards to acute toxicity and then they were treated with SAL, ATR 10 mg/kg, 2-PAM 40mg/kg or ATR+2-PAM. Acute toxicity evaluation was carried out hour to hour for up to 4 hours after CPF or SAL injections. Twenty-four hours post-poisoning, half of the animals were decapitated in order to collect blood for determination of plasma cholinesterase (ChE) activity and the brain were dissected for determination of acetylcholinesterase (AChE) activity related to this time. The other half of animals was submitted to the sessions test (24 hours) and re-test (30 days after poisoning) in the FST, in which was measured the immobility time (in seconds) for 5 minutes. After the re-test session, the animals were decapitated and samples were collect for determination of cholinesterase activity 30 days after acute poisoning. CPF induced signs of acute toxicity and decreased plasma ChE activity .CPF increased immobility time in the test session (a depressivelike effect), but not in the re-test (30 days). ATR attenuated the depressive-like effect of CPF. 2-PAM reactivated AChE in the prefrontal cortex but not in the hippocampus and striatum 24 hours after poisoning. Thirty days later, behavioral and biochemical changes were absent. These results show that: (1) Behavioral and biochemical changes induced by CPF were transient and partially reversed by treatment with ATR and 2-PAM; (2) The possible accumulation of ACh by hippocampal AChE inhibition seems to be related to increased immobility time on the FST. This study suggests that even acute poisoning within a short period of time may impair mental health of subjects exposed to OP. It also suggests that the standard treatment for OP poisoning seems to be insufficient to reverse all of the changes caused by the OP exposure.
Efeitos da exposição crônica ao organofosforado clorpirifós sobre a função cardiorespiratória
(Universidade Federal do Espírito Santo, 2018-08-31) Batista, Thatiany Jardim; Sampaio, Karla Nívea; Silva, Valdo José Dias da; Bissoli, Nazare Souza; Santos, Leonardo dos
Previous studies of our group showed that acute exposure to high doses of organophosphorus compound (OP), chlorpyrifos (CPF), impaired cardiorespiratory reflexes in rats. In addition, some studies indicate that prolonged exposure to OP compounds impairs cardiovascular function, producing cardiac hypertrophy and altering the mechanical properties of the aorta. If prolonged CPF exposure also affects tonic and reflex cardiorespiratory function remained to be explored. Wistar rats were injected with CPF intraperitoneally (i.p.) for 4 weeks (three times a week) or 12 weeks (once a week) at the following doses: 7 mg/kg (CPF 7; 4 weeks, n=14; 12 weeks, n=14) and 10 mg/kg (CPF 10; 4 weeks, n=9; 12 weeks, n=12). The control group received saline (NaCl, 0.9%) for the same period (SAL, 4 weeks, n=9, 12 weeks, n=13). At the end of the treatment, the femoral artery and vein were catheterized under anesthesia with tribromoethanol (250 mg/kg, i.p.), to allow pressure recordings and drugs administration, respectively. After 24 hours of recovery from surgery, pulsatile arterial pressure (PAP) was recorded, from which diastolic blood pressure (DBP), systolic blood pressure (SBP), mean arterial pressure (MAP) and heart rate (HR) were derived. Respiratory rate (fR), tidal volume (VT) and minute volume (VE) were obtained by whole-body plethysmography. The chemorreflex responses were evaluated by intravenous injections of KCN (10, 20, 40 and 80 μg) while the spontaneous baroreflex was evaluated from the PAS and pulse interval (PI) recordings using the sequence method. These recordings were also used to evaluate heart rate variability (HRV) in time and frequency domain (spectral analysis). Finally, the activities of plasma acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were also quantified. The results showed that the enzymatic activity of the CPF intoxicated animals was significantly lower than the control. In addition, CPF 10 group treated for 4 weeks presented a lower weight gain. No differences were observed in the baseline values of DBP, SBP, MAP, HR and fR, as well as in the HRV time-domain indices, either after 4 or 12 weeks of treatment. In the spectral analysis an increase was only observed for the very low frequency band (VLF). Animals treated with CPF presented an impairment of the baroreflex gain, for ascending ramps, and of the baroreflex effectiveness index (BEI). The chemoreflex bradycardic response was significantly reduced in the CPF treated rats for both treatment periods when compared to the control groups. No difference among groups was observed for the chemoreflex pressor response nor the tachypneic response for the two treatment periods. On the other hand, CPF 10 group had an increase in VT, for both treatment periods, and an increase in the VE, only after 1 month treatment. Similar to what was previously demonstrated after acute exposure to CPF, low doses chronic exposure also impairs cardiorespiratory function in rats. These results may have important clinical implications for workers, such as farmers, who are frequently exposed to these 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.
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.
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.
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.

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