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

Abstract

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.

Anthelmintic, Antibacterial and Cytotoxicity Activity of Imidazole Alkaloids fromPilocarpus microphyllusLeaves

“…EPI–[(3S,4R)‐3‐[(S)‐hydroxy(phenyl)methyl]‐4‐[(1‐methylimidazol‐4‐yl)methyl]oxolan‐2‐one] had 15.623 min as RT, and the m/z was 287 Da [M + H] + ; these results are in accordance with the work of Veras et al . () (data not shown). EPIIS–[3S‐[3alpha(S*),4beta]‐dihydro‐3‐(beta‐hydroxybenzyl)‐4‐[(1‐methyl‐1H‐imidazol‐5 yl)methyl]furan‐2(3H)‐one, ISO–[3S‐[3alpha(S*),4beta]‐dihydro‐3‐(alpha‐hydroxybenzyl)‐4‐[(1‐methyl‐1H‐imidazol‐5‐yl)methyl]furan‐2(3H)‐one and PILO–[3R‐[3alpha(R*),4alpha]]‐dihydro‐3‐(alpha‐hydroxybenzyl)‐4‐[(1‐methyl‐1H‐imidazol‐5‐yl)methyl]furan‐2(3H)‐one; for ISO and PILO, the identification was possible by HPLC through different RT, 17.816 and 19.245 min, respectively.…”

“…The antibacterial activity of many naturally occurring alkaloids has been reported in the literature (Navarro and Delgado, ; An et al ., ), and some have been proposed for use as anthelmintic agents and to combat neglected diseases (Veras et al ., ; Moraes, ; de Moraes, ). Table summarizes previously known and now‐reported data on the five alkaloids studied in this work.…”

In vitro effects of Pilocarpus microphyllus extracts and pilocarpine hydrochloride on Rhipicephalus (Boophilus) microplus

“…microphyllus, from which pilocarpine is extracted, is native and widely spread in Piauí, Maranhão and Pará states of Brazil (PINHEIRO, 1997). Currently, the annual industrial production of Jaborandi leaves is 10,000 kg, of which approximately 1% is pilocarpine (VÉRAS et al, 2013) demonstrating the production potential of this medicinal species and its pilocarpine alkaloid. It is extremely important to emphasize that Brazil is the only supplier of this active pharmaceutical ingredient (API) for the international pharmaceutical industry (BRANDÃO et al, 2008) and studies with P. microphyllus alkaloids and its industrial applications are of fundamental importance for a large number of economically precarious communities whose main source of income is the species harvest in underdeveloped regions of northeastern Brazil (LIMA et.…”

“…The use of certain processes to optimize extraction of the P. microphyllus EE may result in the possibly more efficient recovery of extracts with higher concentrations of pilocarpine alkaloid. The acidification of P. microphyllus leaves and the further extraction with polar solvents such as water and ethanol favor the extraction of total alkaloids that can be subsequently submitted to fractionation and prepurification processes (VÉRAS et al, 2013). Due to the high cost of obtaining isolated pilocarpine hydrochloride, obtaining extracts with a higher content of that alkaloid is an alternative that could possibly be used as a low-cost acaricide.…”

Natural Products with Antischistosomal Activity

“…Despite the high concentration (>500 μM), epiisopiloturine showed selective antischistosomal activity and exhibited no cytotoxicity to mammalian cells [49]. Recently, an industrial scale process for extraction, purification and isolation of epiisopiloturine has been described [87].…”