Epiisopilosine alkaloid has activity against Schistosoma mansoni in mice without acute toxicity.

Schistosomiasis is a disease caused by parasites of the genus Schistosoma, currently affecting more than 200 million people. Among the various species of this parasite that infect humans, S. mansoni is the most common. Pharmacological treatment is limited to the use of a single drug, praziquantel (PZQ), despite reports of parasite resistance and low efficacy. It is therefore necessary to investigate new potential schistosomicidal compounds. In this study, we tested the efficacy of epiisopilosine (EPIIS) in a murine model of schistosomiasis. A single dose of EPIIS (100 or 400 mg/kg) administered orally to mice infected with adult S. mansoni resulted in reduced worm burden and egg production. The treatment with the lower dose of EPIIS (100 mg/kg) significantly reduced total worm burden by 60.61% (P < 0.001), as well as decreasing hepatosplenomegaly and egg excretion. Scanning electron microscopy revealed morphological changes in the worm tegument after treatment. Despite good activity of EPIIS in adult S. mansoni, oral treatment with single dose of EPIIS 100 mg/kg had only moderate effects in mice infected with juvenile S. mansoni. In addition, we performed cytotoxicity and toxicological studies with EPIIS and found no in vitro cytotoxicity (in HaCaT, and NIH-3T3 cells) at a concentration of 512 µg/mL. We also performed in silico analysis of toxicological properties and showed that EPIIS had low predicted toxicity. To confirm this, we investigated systemic acute toxicity in vivo by orally administering a 2000 mg/kg dose to Swiss mice. Treated mice showed no significant changes in hematological, biochemical, or histological parameters compared to non-treated animals. Epiisopilosine showed potential as a schistosomicidal drug: it did not cause acute toxicity and it displayed an acceptable safety profile in the animal model.

Effect of neem (Azadirachta indica A. Juss) leaf extract on resistant Staphylococcus aureus biofilm formation and Schistosoma mansoni worms.

ETHNOPHARMACOLOGICAL RELEVANCE: There are ethnopharmacological reports supporting the use of neem (Azadirachta indica A. Juss) leaf against bacterial and worm infections. However there is a lack of studies about its effect on bacterial biofilm formation and Schistosoma mansoni worms. This study reports the in vitro effects of neem leaf ethanolic extract (Neem EE) on Methicillin-resistant Staphylococcus aureus (MRSA) biofilm and planktonic aggregation formation, and against S. mansoni worms. MATERIALS AND METHODS: Quantification of the Azadirachtin (AZA), thought to be one of their main compounds related to biological effects, was performed. The effect of sub-inhibitory concentrations of Neem EE on biofilm formation and planktonic aggregates of S. aureus was tested using the crystal violet dye method and atomic force microscopy (AFM) analysis, respectively. Changes in S. mansoni motor activity and death of worms were analyzed in vitro after exposition to the extract. Treated schistosomes were also examined using confocal laser scanning microscopy. RESULTS: It was observed the presence of AZA in the extract (0.14 ± 0.02 mg/L). Testing Neem EE sub-inhibitory concentrations, a significant biofilm adherence inhibition from 62.5 µg/mL for a sensitive S. aureus and 125 µg/mL for two MRSA strains was observed. AFM images revealed that as the Neem EE concentration increases (from 250 to 1000 µg/mL) decreased ability of a chosen MRSA strain to form large aggregates. In relation of anti-schistosoma assay, the extract caused 100% mortality of female worms at a concentration of 50 µg/mL at 72 h of incubation, while 300 µg/mL at 24h of incubation was required to achieve 100% mortality of male worms. The extract also caused significant motor activity reduction in S. mansoni. For instance, at 96 h of incubation with 100 µg/mL, 80% of the worms presented significant motor activity reduction. By the confocal microscopy analysis, the dorsal surface of the tegument of worms exposed to 300 µg/mL (male) and 100 µg/mL (female) of the extract showed severe morphological changes after 24h of treatment. CONCLUSIONS: Neem leaf ethanolic extract presented inhibitory effect on MRSA biofilm and planktonic aggregation formation, and anthelmintic activity against S. mansoni worms.

Anthelmintic activity in vivo of epiisopiloturine against juvenile and adult worms of Schistosoma mansoni.

Schistosomiasis is a serious disease currently estimated to affect more that 207 million people worldwide. Due to the intensive use of praziquantel, there is increasing concern about the development of drug-resistant strains. Therefore, it is necessary to search for and investigate new potential schistosomicidal compounds. This work reports the in vivo effect of the alkaloid epiisopiloturine (EPI) against adults and juvenile worms of Schistosoma mansoni. EPI was first purified its thermal behavior and theoretical solubility parameters charaterised. In the experiment, mice were treated with EPI over the 21 days post-infection with the doses of 40 and 200 mg/kg, and 45 days post-infection with single doses of 40, 100 and 300 mg/kg. The treatment with EPI at 40 mg/kg was more effective in adult worms when compared with doses of 100 and 300 mg/kg. The treatment with 40 mg/kg in adult worms reduced parasite burden significantly, lead to reduction in hepatosplenomegaly, reduced the egg burden in faeces, and decreased granuloma diameter. Scanning electron microscopy revealed morphological changes to the parasite tegument after treatment, including the loss of important features. Additionally, the in vivo treatment against juvenile with 40 mg/kg showed a reduction of the total worm burden of 50.2%. Histopathological studies were performed on liver, spleen, lung, kidney and brain and EPI was shown to have a DL50 of 8000 mg/kg. Therefore EPI shows potential to be used in schistosomiasis treatment. This is the first time that schistosomicidal in vivo activity of EPI has been reported.

Nanopharmaceutical approach of epiisopiloturine alkaloid carried in liposome system: preparation and in vitro schistosomicidal activity.

Schistosomiasis is a neglected tropical disease caused by blood flukes of the genus Schistosoma. This disease control has been widely made by praziquantel-reference drug, but resistance to this drug has already been found. There has been the finding of an imidazole alkaloid in jaborandi leaves-epiisopiloturine, which has known activity against adult, young and egg forms of Schistosoma mansoni. This alkaloid is an apolar molecule with difficult solubility; therefore, the liposomal structure of epiisopiloturine was proposed. Liposomes are carrying structures of drugs that may enhance solubility of compounds such as epiisopiloturine. In this work, we report in vitro epiisopiloturine-loaded liposomes effect formed by different concentrations of lipids 9:1 (weight ratio) dipalmitoylphosphatidylcholine:cholesterol and 8:2 (weight ratio) dipalmitoylphosphatidylcholine:cholesterol. Results have showed that epiisopiloturine extraction and isolation have been successful through high-performance liquid chromatography-HPLC and its purity confirmed through mass spectrometry has showed 287 Da molecular mass. Formulations from 9:1 DPPC:cholesterol and 8:2 DPPC:cholesterol with loaded EPI (300 microg/ml) have killed parasites at 100% after incubation 96 h and 120 h, respectively. Confocal microscopy employed to observe morphological alterations in the tegument of adult form of Schistosoma mansoni. Details from interaction, between epiisopiloturine and liposome, have been achieved by semi-empirical AM1 calculations, which have showed that epiisopiloturine inside is more stable than the outside form, at least 10 kcal. This is first time that schistosomicidal activity has been reported for epiisopiloturine-loaded into liposome.

Industrial Scale Isolation, Structural and Spectroscopic Characterization of Epiisopiloturine from Pilocarpus microphyllus Stapf Leaves: A Promising Alkaloid against Schistosomiasis.

This paper presents an industrial scale process for extraction, purification, and isolation of epiisopiloturine (EPI) (2(3H)-Furanone,dihydro-3-(hydroxyphenylmethyl)-4-[(1-methyl-1H-imidazol-4-yl)methyl]-, [3S-[3a(R*),4b]]), which is an alkaloid from jaborandi leaves (Pilocarpus microphyllus Stapf). Additionally for the first time a set of structural and spectroscopic techniques were used to characterize this alkaloid. EPI has shown schistomicidal activity against adults and young forms, as well as the reduction of the egg laying adult worms and low toxicity to mammalian cells (in vitro). At first, the extraction of EPI was done with toluene and methylene chloride to obtain a solution that was alkalinized with ammonium carbonate. The remaining solution was treated in sequence by acidification, filtration and alkalinization. These industrial procedures are necessary in order to remove impurities and subsequent application of the high performance liquid chromatography (HPLC). The HPLC was employed also to remove other alkaloids, to obtain EPI purity higher than 98%. The viability of the method was confirmed through HPLC and electrospray mass spectrometry, that yielded a pseudo molecular ion of m/z equal to 287.1 Da. EPI structure was characterized by single crystal X-ray diffraction (XRD), (1)H and (13)C nuclear magnetic resonance (NMR) in deuterated methanol/chloroform solution, vibrational spectroscopy and mass coupled thermal analyses. EPI molecule presents a parallel alignment of the benzene and the methyl imidazol ring separated by an interplanar spacing of 3.758 Å indicating a π-π bond interaction. The imidazole alkaloid melts at 225°C and decomposes above 230°C under air. EPI structure was used in theoretical Density Functional Theory calculations, considering the single crystal XRD data in order to simulate the NMR, infrared and Raman spectra of the molecule, and performs the signals attribution.

Anti-inflammatory and antinociceptive activity of epiisopiloturine, an imidazole alkaloid isolated from Pilocarpus microphyllus.

The aim of this study was to investigate the antinociceptive and anti-inflammatory activities of epiisopiloturine (1), an imidazole alkaloid found in the leaves of Pilocarpus microphyllus. The anti-inflammatory activity of 1 was evaluated using several agents that induce paw edema and peritonitis in Swiss mice. Paw tissue and peritoneal fluid samples were obtained to determine myeloperoxidase (MPO) activity or tumor necrosis factor (TNF)-α and interleukin (IL)-1ß levels. The antinociceptive activity was evaluated by acetic acid-induced writhing, the hot plate test, and pain induction using formalin. Compared to vehicle treatment, pretreatment with 1 (0.1, 0.3, and 1 mg/kg, ip) of mice significantly reduced carrageenan-induced paw edema (p < 0.05). Furthermore, compound 1 at a dose of 1 mg/kg effectively inhibited edema induced by dextran sulfate, serotonin, and bradykinin, but had no effect on histamine-induced edema. The administration of 1 (1 mg/kg) following carrageenan-induced peritonitis reduced total and differential peritoneal leukocyte counts and also carrageenan-induced paw MPO activity and TNF-α and IL-1ß levels in the peritoneal cavity. Pretreatment with 1 also reduced acetic acid-induced writhing and inhibited the first and second phases of the formalin test, but did not alter response latency in the hot plate test. Pretreatment with naloxone reversed the antinociceptive effect of 1.

Prevalence of human immunodeficiency virus drug resistance mutations and subtypes in drug-naive, infected individuals in the army health service of Rio de Janeiro, Brazil.

The prevalence of mutations that confer resistance to antiretroviral drugs was examined in 56 drug-naive, human immunodeficiency virus type 1 (HIV-1)-infected individuals from the Army Health Service in Rio de Janeiro, Brazil. No primary protease inhibitor mutations were found, but secondary mutations were observed in 51.2% of the samples. Fourteen percent of the viruses had reverse transcriptase inhibitor-associated mutations. Comparative analysis of protease secondary mutations from four different time periods in drug-naive patients in the city of Rio de Janeiro has indicated constant rates for particular mutations. Changes in CD4 cell counts and HIV viral load over time in subtype B- and non-B-infected drug-naive patients were not significantly different.

Epidemiological aspects of the brazilian spotted fever: serological survey of dogs and horses in an endemic area in the state of Sao Paulo, Brazil

Com o objetivo de obter informacoes sobre a febre maculosa brasileira, um estudo em animais domesticos foi conduzido no municipio de Pedreira, Sao Paulo, Brasil, onde 17 casos humanos foram notificados. Amostras de soro obtidas de animais foram testadas pelo teste de imunofluorescencia indireta para deteccao de anticorpos para rickettsia do grupo da febre maculosa. Soro reatividade foi observada em 12 (36,4 por cento) dos 33 caes e sete (77,8 por cento) dos nove equinos procedentes da area endemica. Para comparacao, amostras de sangue de caes e de equinos procedentes de area nao endemica foram testadas e quatro (12,9 por cento) dos 31 caes e tres dos 10 equinos foram positivos...