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

Effects of EPI against adult <i>S</i>. <i>mansoni</i> BH strain (45 days post infection).

<p>Values are presented as mean± SEM. Number of animals/group = 10. Group treated with 100 mg /kg 3 animals died.</p><p>Asterisks indicate that difference compared to the control was significant with *** P<0.001, **P<0.01 and *P<0.05.</p><p>Effects of EPI against adult <i>S</i>. <i>mansoni</i> BH strain (45 days post infection).</p

Mass spectrum obtained from ESI+/Ion Trap.

<p>(A) free EPI with a pseudo molecular ion m/z 287.1 Da [M+H]⁺, (B) MS² with characteristic fragment at m/z 269.1 Da [M – H₂O + H]⁺, (C) MS³ with fragments at m/z 251.0 Da [M – 2H₂O + H⁺] and 168.06 Da with proposed chemical structure.</p

Neutral and ionic forms of EPI (A).

<p>Potentiometric pKa determination (B). Illustration of the pKa values of EPI predicted using the ChemAxon method (C). This graph shows the variation in the of distribution species vs pH values, and indicates that the ChemAxon predicted pKa values are respectively pKa1 = 6.40 and pKa2 = 13.90 (C). DSC (D) and DSC-TGA (E) data illustrating the thermal degradation that occurs around 220°C in a single step.</p

Effects of EPI against juvenile <i>S</i>. <i>mansoni</i> BH strain (treated 21 days post infection).

<p>Values are presented as mean ± SEM. Number of animals/group = 8.</p><p>Asterisks indicate that difference compared to the control was significant with ***P<0.001, **P<0.01 and *P<0.05.</p><p>Effects of EPI against juvenile <i>S</i>. <i>mansoni</i> BH strain (treated 21 days post infection).</p

Analytical HPLC used LiChrospher 60 RP column and eluted with potassium phosphate

<p>. (A) Standard EPI (20 µg/mL), (B) Standard pilocarpine (50 µg/mL), (C) “cultivated jaborandi leaves” solution, resulted from first extraction step, (D) “cultivated jaborandi acid” solution, obtained EPI under salt form, (E) Solution of “crude EPI” with some impurities as pilocarpine and other alkaloids, (F) last step of isolation showing EPI >98% purity.</p

EPI bond distances obtained through x-ray diffraction (Experimental) and DFT results (Calculated).

<p>Atom labels accordingly to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066702#pone-0066702-g004" target="_blank">Figure 4</a>.</p

Granuloma diameter (μm) in the liver of the mice from treated and untreated adult worm-infected groups 45 day post infection.

<p>Difference was significant ** <i>P</i><0.01.</p><p>Granuloma diameter (μm) in the liver of the mice from treated and untreated adult worm-infected groups 45 day post infection.</p

Histopathological study of organs sections of different groups of mice.

<p>Comparison between the histology of: liver (A,B,C), spleen (D,E,F), lung (G,H,I), kidney (J,K,L), and brain (M,N,O) obtained from Swiss mice (n = 4) treated with 0.0 (first column), 530 (second column) or 8000 (third column) mg/kg of EPI. The first and the second column refer to mice killed 7 days after assay and the third one represent organs from animals that died due to the administration of higher concentration of the drug. Light microscopy evaluation of these organs showed severe morphological changes in the spleen, lung, kidney and brain, but not in the parenchyma of liver in the animals treated with 8000 mg/kg of EPI. Focal alterations in the red pulp were observed in the spleen of animals treated with 530 mg/kg of the drug. Control group presented no morphological changes in the organs.</p