Anthelmintic, Antibacterial and Cytotoxicity Activity of Imidazole Alkaloids from Pilocarpus microphyllus Leaves.

Pilocarpus microphyllus Stapf ex Wardlew (Rutaceae), popularly known as jaborandi, is a plant native to the northern and northeastern macroregions of Brazil. Several alkaloids from this species have been isolated. There are few reports of antibacterial and anthelmintic activities for these compounds. In this work, we report the antibacterial and anthelmintic activity of five alkaloids found in P. microphyllus leaves, namely, pilosine, epiisopilosine, isopilosine, epiisopiloturine and macaubine. Of these, only anthelmintic activity of one of the compounds has been previously reported. Nuclear magnetic resonance, HPLC and mass spectrometry were combined and used to identify and confirm the structure of the five compounds. As regards the anthelmintic activity, the alkaloids were studied using in vitro assays to evaluate survival time and damaged teguments for Schistosoma mansoni adult worms. We found epiisopilosine to have anthelmintic activity at very low concentrations (3.125 µg mL-1 ); at this concentration, it prevented mating, oviposition, reducing motor activity and altered the tegument of these worms. In contrast, none of the alkaloids showed antibacterial activity. Additionally, alkaloids displayed no cytotoxic effect on vero cells. The potent anthelmintic activity of epiisopilosine indicates the potential of this natural compound as an antiparasitic agent. Copyright © 2017 John Wiley & Sons, Ltd.

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.

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.

Antinociceptive activity of the monoterpene α-phellandrene in rodents: possible mechanisms of action.

OBJECTIVES: The aim of this work was to investigate the antinociceptive property of α-phellandrene (α-PHE) in experimental nociception models and possible mechanisms involved. METHODS: Mass spectrometry was used to evaluate the purity and molecular mass of α-PHE. Macrophages from mice peritoneal cavity were used in an MTT test. Rodents were used in tests of chemical and mechanical nociception. In the study of the mechanisms, the animals were treated with pharmacological tools and then submitted to the glutamate test. KEY FINDINGS: α-PHE purity was 98.2% and molecular mass 136.1 Da. α-PHE did not show cytotoxicity. In the writhing and capsaicin tests, α-PHE promoted the antinociceptive effect in all evaluated doses (minimum dose 3.125 mg/kg). In the formalin test, α-PHE (50 mg/kg) was effective in inhibiting both phases. In the glutamate test, the monoterpene (12.5 mg/kg) decreased the nociceptive response. In carrageenan-induced hyperalgesia, α-PHE (50 mg/kg) decreased the hypernociception index. In the study of the mechanisms involved, pretreatment with naloxone reversed the α-PHE antinociceptive effect, the same occurred with glibenclamide, l-arginine, atropine and yohimbine. α-PHE did not show muscle relaxant activity or central depressant effects in open field and rota rod tests. CONCLUSIONS: α-PHE has an antinociceptive effect and it possibly involves the glutamatergic, opioid, nitrergic, cholinergic and adrenergic systems.