RESUMO

Malaria, an infectious disease that kills more than 438,000 people per year worldwide, is a major public health problem. The emergence of strains resistant to conventional therapeutic agents necessitates the discovery of new drugs. We previously demonstrated that various substances, including terpenes, have antimalarial activity in vitro and in vivo. Nerolidol is a sesquiterpene present as an essential oil in several plants that is used in scented products and has been approved by the US Food and Drug Administration as a food-flavouring agent. In this study, the antimalarial activity of nerolidol was investigated in a mouse model of malaria. Mice were infected with Plasmodium berghei ANKA and were treated with 1000 mg/kg/dose nerolidol in two doses delivered by the oral or inhalation route. In mice treated with nerolidol, parasitaemia was inhibited by >99% (oral) and >80% (inhalation) until 14 days after infection (P <0.0001). On Day 30 post-infection, the survival rate of orally treated mice was 90% compared with 16% in controls (P <0.0001). In contrast, inhalation-treated mice showed a survival rate of 50% vs. 42% in controls (P > 0.05). The toxicity of nerolidol administered by either route was not significant, whilst genotoxicity was observed only at the highest dose tested. These results indicate that combined use of nerolidol and other drugs targeting different points of the same isoprenoid pathway may be an effective treatment for malaria.

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RESUMO

Previous studies have shown that fosmidomycin, risedronate, and nerolidol exert antimalarial activity in vitro. We included squalestatin, an inhibitor of the isoprenoid metabolism in Erwinia uredovora, and found that combinations of compounds which act on different targets of the plasmodial isoprenoid pathway possess important supra-additivity effects.

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RESUMO

Nerolidol is a naturally occurring sesquiterpene found in the essential oils of many types of flowers and plants. It is frequently used in cosmetics, as a food flavoring agent, and in cleaning products. In addition, nerolidol is used as a skin penetration enhancer for transdermal delivery of therapeutic drugs. However, nerolidol is hemolytic at low concentrations. A simple and fast GC-MS method was developed for preliminary quantification and assessment of biological interferences of nerolidol in mouse plasma after oral dosing. Calibration curves were linear in the concentration range of 0.010-5 µg/mL nerolidol in mouse plasma with correlation coefficients (r) greater than 0.99. Limits of detection and quantification were 0.0017 and 0.0035 µg/mL, respectively. The optimized method was successfully applied to the quantification of nerolidol in mouse plasma.

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O preparo de suspensão bacteriana é procedimento importante para avaliação da inflamação e podeser obtida por diferentes métodos que precisam ou não do passo de armazenamento. O objetivo destetrabalho foi investigar a influência do armazenamento de suspensão de Staphylococcus aureus na viabilidade bacteriana e sua influência na inflamação in vivo. A suspensão bacteriana de S. aureus ATCC 6538 foi preparada visualmente seguindo o grau 4 da Escala de McFarland. A viabilidade bacteriana nessa solução foi determinada pela contagem de UFC, a qual foi utilizada para induzir artrite séptica e peritonite. O armazenamento por 24 h reduziu as UFC de S. aureus. Essa redução da viabilidadebacteriana resultou em diminuição da hiperalgesia mecânica, edema e recrutamento leucocitário na artriteséptica, e recrutamento leucocitário e produção de citocinas na peritonite bacteriana. Estes resultadosdemonstraram que o armazenamento de suspensão bacteriana afetou sua viabilidade, resultando emdiminuição da resposta inflamatória in vivo, sugerindo a importância de padronizar procedimentos parao preparo de suspensão bacteriana. Uma abordagem concebível seria determinar o número de UFC em um específico grau da Escala de McFarland, o qual permitirá o preparo e o uso de uma suspensão bacteriana no mesmo dia para os testes in vivo.

The preparation of bacterial suspension is an important procedure used in laboratories for inflammatoryevaluation protocols and can be obtained by different methods that need or not need step storage. The aimof this work was investigate the influence of storage of Staphylococcus aureus suspension in bacterialviability and its influence in bacteria-induced inflammation in vivo. The bacterial suspension of S. aureus ATCC 6538 was prepared accordingly to 4th degree of McFarland’s Scale by visual comparison. This suspension was used to determine by CFU counting the bacterial viability and for administration to the animals to induce septic arthritis and peritonitis. Twenty four hours of storage reduced the S. aureus CFU. As a consequence of reduced bacterial viability, was detected reduced mechanical hyperalgesia, edema and leukocyte recruitment in septic arthritis and leukocyte recruitment and cytokine production bacterial peritonitis. These results demonstrate that storage of bacterial suspension affected bacterialviability, which resulted in diminished inflammatory response in vivo, raising the importance of standardprocedures for bacterial suspension preparation. A conceivable approach would be to determine the number of CFU at a specific McFarland’s scale degree, which will allow the preparation and use a bacterial suspension in the same day for in vivo testing.

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A Dietilnitrosamina (DEN), uma substância reconhecidamente hepatotóxica e carcinogênica, foiutilizada na indução da necrose hepática centrolobular em ratos isogênicos Lewis divididos em 5 grupos de 5 animais. O objetivo deste estudo foi avaliar o efeito quimiopreventivo da epigalocatequina-3-galato (EGCG), de Camellia sinensis (chá verde) no tratamento da hepatotoxicidade celular induzida pela DEN. Foi mensurada a concentração sérica da alanina aminotransferase (ALT) e aspartato aminotransferase(AST) dos diferentes grupos experimentais. No ensaio bioquímico para AST e ALT, houve diferença significativa entre os valores médios do grupo controle (163±70,32) comparado ao grupo DEN (1631±1039,44), sugerindo que a DEN influencia na função hepática. Entretanto, não houve diferença significativa entre o grupo DEN e o tratado com epigalocatequina. A lactato desidrogenase (LDH) éconsiderada um marcador bioquímico comum para avaliação da progressão tumoral, e em relação ao LDH, as amostras não apresentaram diferenças significativas entre o grupo DEN (1385,5±43,13) e DEN + EGCG 150mg ou DEN + EGCG 200mg 1537,5±1010,45). Neste trabalho foi demonstrado que a epigalocatequina nas concentrações de 150 e 200 mg/Kg não induziram alterações hepáticas e também não foi possível verificar nenhuma quimioproteção pela EGCG em animais inicialmente tratados comDEN durante 24 horas. Sendo assim, novos experimentos com diferentes concentrações de EGCG sãonecessários para comprovar seu possível efeito quimioprotetor.

Diethylnitrosamine (DEN), a known hepatotoxic and carcinogenic substance, was used in the induction of centrilobular hepatic necrosis in isogenic Lewis rats divided into 5 groups with 5 animals. The aim of this study was to evaluate the chemopreventive effect of epigallocatechin-3-gallate (EGCG)from Camellia sinensis (green tea) in the treatment of cellular hepatotoxicity induced by DEN. It was measured the serum concentration of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of the different experimental groups. In the biochemistry assay for AST and ALT, there was significant difference between median values of control group (163±70.32) compared to DEN group(1631±1039.44), suggesting that DEN influences on hepatic function. However, there was no significant difference between DEN group to that treated with epigallocatechin. Lactate dehydrogenase (LDH) is considered a common biochemical marker for evaluation of tumor progression, and regarding LDH,the samples presented no significant differences between the DEN group (1385.5±43.13) and DEN + EGCG 150mg or DEN + EGCG 200mg (1537.5± 1010.45). In this work it was demonstrated that epigallocatechin concentrations of 150 and 200 mg/kg did not induce liver alterations and though was not verified any chemoprotective effect by EGCG in animals initially treated with DEN for 24 hours. Moreover, new experiments with different concentrations of EGCG are needed to verify its possiblechemoprotector effect.

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In the present study, nasal mucus from patients with leprosy were analyzed by PCR using specific primers for Lsr2 gene of Mycobacterium leprae. The presence of Lsr2 gene in the nasal mucus was detected in 25.80% of patients with paucibacillari leprosy, and 23.07% of contacts. Despite the absence of clinical features in the contact individuals, it was possible to detect the presence of Lsr2 gene in the nasal mucus of these individuals. Therefore, PCR detection of M. leprae targeting Lsr2 gene using nasal mucus samples could contribute to early diagnosis of leprosy.

RESUMO

O chá verde (Camellia sinensis) e seus principais componentes, as catequinas, apresentam várias atividades biológicas, dentre elas a ação antioxidante e quimioprotetora contra agentes hepatotóxicos, como a dietilnitrosamina (DEN), um conhecido agente citotóxico. Este trabalho tem como objetivo avaliar e compreender os mecanismos da ação do extrato do chá verde (ECV), como agente antioxidante e quimioprotetor. Foram realizados testes in vivo com ratos Wistar machos utilizando o ECV a 2% (2mg/100mL) via oral, como única fonte de água, durante 35 dias. Para verificar a capacidade antioxidante e quimioprotetora do ECV utilizou-se a DEN (200mg/kg) como agente lesivo. Os resultados indicam que o chá verde, nas doses próximas as que são ingeridas pela população, não é capaz de inibir significativamente a lesão causada pela alta dose da DEN, mas os resultados indicaram uma tendência à quimioproteção, sugerindo que o chá verde pode agir como substância preventiva mesmo em baixas doses, frente a um agente hepatotóxico classicamente conhecido.

Green tea (Camellia sinensis) and its main components, the catechins present several biological activities such as: antioxidant and chemoprotective action against hepatotoxic agent as diethylnitrosamine (DEN), a known cytotoxic substance. This work has the objective to evaluate and understand the green tea extract (GTE) mechanisms of action as a chemoprotector agent. Therefore, tests in vivo with Wistarrats using GTE 2% (2mg/100mL) was taken orally as the only source of water for 35 days. To verify the antioxidant and chemoprotective capacity of GTE, DEN (200mg/kg) was used as a lesion agent. The results indicate that green tea if the dosage close to the ones taken by the population is not able to expressively inhibit the lesion caused for the high dose of DEN, but the results indicated a tendency to chemoprotection, suggesting to green tea may act as a prevention substance even in low dosage in presence of a classically known hepatotoxic agent.

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The increasing resistance of malarial parasites to almost all available drugs calls for the identification of new compounds and the detection of novel targets. Here, we establish the antimalarial activities of risedronate, one of the most potent bisphosphonates clinically used to treat bone resorption diseases, against blood stages of Plasmodium falciparum (50% inhibitory concentration [IC50] of 20.3±1.0 µM). We also suggest a mechanism of action for risedronate against the intraerythrocytic stage of P. falciparum and show that protein prenylation seems to be modulated directly by this drug. Risedronate inhibits the transfer of the farnesyl pyrophosphate group to parasite proteins, an effect not observed for the transfer of geranylgeranyl pyrophosphate. Our in vivo experiments further demonstrate that risedronate leads to an 88.9% inhibition of the rodent parasite Plasmodium berghei in mice on the seventh day of treatment; however, risedronate treatment did not result in a general increase of survival rates.

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RESUMO

O melaço de cana-de-açúcar é uma fonte de carbono alternativa de menor custo que a glicose tradicionalmente utilizada em processos fermentativos. No presente estudo, um biopolímero solúvel de Agrobacterium radiobacter k84 (ARB) foi obtido por fermentação utilizando melaço de cana de açúcar como fonte de carbono em um processo com rendimento de 10,0 g.L-1. O ARB é composto de minerais (40%), carboidratos (35%) e proteínas (15%). O teste do efeito citotóxico do ARB in vitro nas concentrações de 2,5 mg/mL, 5,0 mg/mL e 10,0 mg/mL em células LLC MK2 (Rim de Macaco Rhesus) revelou uma concentração citotóxica 50% (CC50) de 9,32 mg/mL. Em estudo in vivo de toxicidade oral durante 30 dias, camundongos Swiss receberam por gavagem soluções de ARB nas concentrações de 5 mg/mL, 15 mg/mL, 50 mg/mL e 150 mg/mL (aproximadamente 25 mg/kg/dia, 75 mg/kg/dia, 250 mg/ kg/dia e 750 mg/kg/dia). Os resultados não apresentaram sinais hematológicos ou histopatológicos de efeitos adversos, levando a definir a dose sem efeito adverso observado (NOAEL) como 150 mg/mL (aproximadamente 750 mg/kg/dia).

Sugar cane molasses is a cheaper carbon source alternative than glucose traditionally used in fermentation processes. In the present study a biopolymer soluble from Agrobacterium radiobacter k84 (ARB) was obtained by fermentation using sugar cane molasses as a carbon source in a process with yield of 10.0 g.L-1. The ARB is composed by minerals (40%), carbohydrate (35%) and protein (15%). In vitro test of the cytotoxic effect of ARB at concentrations 2.5 mg/mL, 5.0 mg/mL and 10.0 mg/mL in LLC MK2 (Rhesus Monkey Kidney) cells revealed a 50% cytotoxic concentration (CC50) of 9.32 mg/mL. In a 30-day in vivo oral toxicity study, Swiss mice were administered ARB by gavage at 5 mg/mL, 15 mg/mL, 50 mg/mL and 150 mg/mL (approximately 25 mg/kg/day, 75 mg/kg/day, 250 mg/kg/day and 750 mg/kg/day). The results did not present any hematological or histopathological signs of adverse effects, leading us to define the no observed adverse effect level (NOAEL) as 150 mg/mL (approximately 750 mg/kg/day).

RESUMO

O melaço de cana-de-açúcar é uma fonte de carbono alternativa de menor custo que a glicose tradicionalmente utilizada em processos fermentativos. No presente estudo, um biopolímero solúvel de Agrobacterium radiobacter k84 (ARB) foi obtido por fermentação utilizando melaço de cana de açúcar como fonte de carbono em um processo com rendimento de 10,0 g.L-1. O ARB é composto de minerais (40%), carboidratos (35%) e proteínas (15%). O teste do efeito citotóxico do ARB in vitro nas concentrações de 2,5 mg/mL, 5,0 mg/mL e 10,0 mg/mL em células LLC MK2 (Rim de Macaco Rhesus) revelou uma concentração citotóxica 50% (CC50) de 9,32 mg/mL. Em estudo in vivo de toxicidade oral durante 30 dias, camundongos Swiss receberam por gavagem soluções de ARB nas concentrações de 5 mg/mL, 15 mg/mL, 50 mg/mL e 150 mg/mL (aproximadamente 25 mg/kg/dia, 75 mg/kg/dia, 250 mg/ kg/dia e 750 mg/kg/dia). Os resultados não apresentaram sinais hematológicos ou histopatológicos de efeitos adversos, levando a definir a dose sem efeito adverso observado (NOAEL) como 150 mg/mL (aproximadamente 750 mg/kg/dia).

Sugar cane molasses is a cheaper carbon source alternative than glucose traditionally used in fermentation processes. In the present study a biopolymer soluble from Agrobacterium radiobacter k84 (ARB) was obtained by fermentation using sugar cane molasses as a carbon source in a process with yield of 10.0 g.L-1. The ARB is composed by minerals (40%), carbohydrate (35%) and protein (15%). In vitro test of the cytotoxic effect of ARB at concentrations 2.5 mg/mL, 5.0 mg/mL and 10.0 mg/mL in LLC MK2 (Rhesus Monkey Kidney) cells revealed a 50% cytotoxic concentration (CC50) of 9.32 mg/mL. In a 30-day in vivo oral toxicity study, Swiss mice were administered ARB by gavage at 5 mg/mL, 15 mg/mL, 50 mg/mL and 150 mg/mL (approximately 25 mg/kg/day, 75 mg/kg/day, 250 mg/kg/day and 750 mg/kg/day). The results did not present any hematological or histopathological signs of adverse effects, leading us to define the no observed adverse effect level (NOAEL) as 150 mg/mL (approximately 750 mg/kg/day).