Anthelmintic, Antibacterial and Cytotoxicity Activity of Imidazole Alkaloids fromPilocarpus microphyllusLeaves

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.info:eu-repo/semantics/publishedVersio

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), 1H and 13C 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

Gastric Antiulcerogenic and Hypokinetic Activities of Terminalia fagifolia Mart. & Zucc. (Combretaceae)

The acute toxicity, the antioxidant activity, and the pharmacological activity on the gastrointestinal tract of rodents of the ethanolic extract (TFEE) from the bark of Terminalia fagifolia Mart. & Zucc. (Combretaceae) and of its aqueous (TFAqF), hydroalcoholic (TFHAF), and hexanic (TFHEXF) partition fractions have been evaluated. TFEE presented low acute toxicity, antioxidant, and antiulcerogenic activity against ethanol-induced ulcers, which was partially blocked by pretreatment with L-NAME and indomethacin. It reduced the total acidity and raised the pH of gastric secretion. Additionally, TFEE delayed gastric emptying and slightly inhibited the small intestinal transit and also presented a weakly antidiarrheal activity. The antiulcerogenic and antioxidant activity were also detected in TFAqF and TFHAF but not in TFHEXF. The antisecretory and gastroprotective activity of TFEE partially involve the nitric oxide and prostaglandin participation. Nevertheless, TFEE, TFAqF, and TFHAF drastically reduced the mucus layer adhered to the gastric wall of rats treated with ethanol or indomethacin. Complementary studies are required in order to clarify the paradox of the presence of a gastroprotector activity in this plant that, at the same time, reduces the mucus layer adhered to the gastric wall

Characterization and Biological Activities of Ocellatin Peptides from the Skin Secretion of the Frog Leptodactylus pustulatus

Eight new peptides were isolated from the skin secretion of the frog Leptodactylus pustulatus and their amino acid sequences determined by de novo sequencing and by cDNA cloning. Structural similarities between them and other antimicrobial peptides from the skin secretion of Leptodactylus genus frogs were found. Ocellatins-PT1 to -PT5 (25 amino acid residues) are amidated at the C-terminus, while ocellatins-PT6 to -PT8 (32 amino acid residues) have free carboxylates. Antimicrobial activity, hemolytic tests, and cytotoxicity against a murine fibroblast cell line were investigated. All peptides, except for ocellatin-PT2, have antimicrobial activity against at least one Gram negative strain. Ocellatin-PT8 inhibited the growth of Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Salmonella choleraesuis strains with MICs in the 60−240 μM range. No significant effect was observed in human erythrocytes and in a murine fibroblast cell line after exposure to the peptides at MICs. A comparison between sequences obtained by both direct HPLC-MS de novo sequencing and cDNA cloning demonstrates the secretion of mature peptides derived from a pre-pro-peptide structure

Structural and spectroscopic characterization of epiisopiloturine-metal complexes, and anthelmintic activity <i>vs</i>. <i>S. mansoni</i>

<p>Epiisopiloturine (EPI), extracted from leaves of <i>Pilocarpus microphyllus</i>, a plant originally from the Amazon and Savanna regions in Brazil, was described as a potential drug against Schistosomiasis, a neglected severe disease. Herein, EPI was complexed with copper(II) or zinc(II) salts and the isolated species, [Cu(epi)₄](ClO₄)₂ (<b>1</b>) and [Zn(epi)₂Cl₂] (<b>2</b>), were structurally and spectroscopically characterized. By using X-ray diffraction, the crystal structures of both metal complexes were determined, indicating a square pyramidal geometry for copper for <b>1</b> and a tetrahedral environment around zinc for <b>2</b>. EPR spectra of <b>1</b> show a typical tetragonal environment around the central metal ion with some tetrahedral distortion, both in the solid state and in frozen acetonitrile solution, in accordance with crystallographic data. For <b>2</b>, NMR spectra have bands consistent with a tetrahedral species in solid state or in DMSO-d₆ solution. These spectroscopic characterization data were further supported by Density Functional Theory calculations, showing that these metal complexes are also stable in solution. Those metal complexes were tested against adult worms of <i>Schistosoma mansoni</i>, in comparison to the free alkaloid as anthelmintic agent. Coordination with copper(II) improved the alkaloid schistosomicidal properties, while binding to zinc(II) decreased its activity.</p

Seasonal change in main alkaloids of jaborandi (<i>Pilocarpus microphyllus</i> Stapf ex Wardleworth), an economically important species from the Brazilian flora

<div><p><i>Pilocarpus microphyllus</i> Stapf ex Wardleworth (jaborandi, Rutaceae) is one of the most important Brazilian medicinal species owing to its content of pilocarpine (PIL), an alkaloid used for treating glaucoma and xerostomia. This species contains another alkaloid, epiisopiloturine (EPI), which has demonstrated effectiveness against schistosomiasis. The aim of this work was to assess seasonal changes of PIL and EPI in three populations of cultivated <i>P</i>. <i>microphyllus</i> from northeastern Brazil over one year, including the dry and rainy seasons. Alkaloid profiles were correlated to phenotypic and genetic patterns in the morphological and molecular characterizations. PIL was the primary alkaloid and its levels differed among populations in all months except September. The S01 population (green line) showed an especially high PIL content compared to populations S02 and S03 (traditional line), which had similar alkaloid contents. PIL content gradually decreased in the three populations in the rainy season.EPI content was significantly different between the green line (S01) and the traditional line (S02 and S03).S01 had a significantly lower EPI content in all months, demonstrating that it was not the best source for EPI extraction. Inter simple sequence repeat (ISSR) markers and morphological analyses clearly separated S01 from S02 and S03, in agreement with the alkaloid results. This study shows the first correlation between the chemical, morphological, and molecular markers of <i>P</i>. <i>microphyllus</i> and highlights the potential benefits of a multidisciplinary research approach aimed at supporting both industry and conservation of natural resources.</p></div

Pilocarpine (%, w/w) contents in S01, S02, and S03 over one year, measured with HPLC.

<p>Pilocarpine (%, w/w) contents in S01, S02, and S03 over one year, measured with HPLC.</p

<i>Pilocarpus microphyllus</i>Stapf ex Wardleworth(jaborandi).

<p>(A) <i>P</i>. <i>microphyllus</i>, highlighting the terminal leaflets (Photo: Anidro do Brasil Extrações S.A). (B) <i>P</i>. <i>microphyllus</i> harvested with pruning shears (Photo: Anidro do Brasil Extrações S.A.).</p

Rainfall and pilocarpine content (%) in the dry and rainy seasons.

<p>(A) Rainfall distribution (mm) during the dry and rainy seasons [0.00 mm; 109.69 mm (<i>p</i> = 0.0003), respectively] in a <i>P</i>. <i>microphyllus</i> plantation, Piauí state, northeastern Brazil. (B) Percentage of PIL (w/w) by the dry and rainy seasons [0.727; 0.683 (<i>p</i> = 0.5637), respectively] from S01. (C) Percentage of PIL (w/w) by the dry and rainy seasons [0.560; 0.350 (<i>p</i> = 0.0149), respectively] from S02. (D) Percentage of PIL (w/w) by the dry and rainy seasons [0.687; 0.415 (<i>p</i> = 0.0030), respectively] from S03.</p

Analysis of molecular variance (AMOVA) of the three groups of cultivated <i>Pilocarpus microphyllus</i>, analyzed by GenAlEx 6.5 software.

<p>Analysis of molecular variance (AMOVA) of the three groups of cultivated <i>Pilocarpus microphyllus</i>, analyzed by GenAlEx 6.5 software.</p