Characterization of the variation in the imidazole alkaloid profile of Pilocarpus microphyllus in different seasons and parts of the plant by electrospray ionization mass spectrometry fingerprinting and identification of novel alkaloids by tandem mass spectrometry

Abstract

Pilocarpus microphyllus (Rutaceae), popularly known as jaborandi, is the only commercial source of an imidazole alkaloid named pilocarpine. In the present study, the variation in the profile of imidazole alkaloids in different seasons and in different parts of the P. microphyllus plant during the summer was analyzed by electrospray ionization mass spectrometry in the positive ion mode [ESI(+)-MS]. The fingerprints of these extracts repeatedly presented similar ions which were mass-selected and studied by tandem mass spectrometry (ESI-MS/MS and ESI-MS/MS/MS) and high-resolution mass spectrometry, resulting in the characterization of eight imidazole alkaloids. The data from the ESI(+)-MS fingerprints were analyzed by principal component analysis (PCA), showing that pilocarpine was present mainly in the summer, whereas in the autumn mainly pilosine and winter anhydropilosine were found. Three alkaloids, reported for the first time in extracts of P. microphyllus, were found. Analysis of the distribution of alkaloids in different parts of the plant during the summer showed that, although pilocarpine was present throughout the plant, 13-nor-8(11)-dihydropilocarpine was found mainly in the stem, pilosine and anhydropilosine were present mainly in the intermediary leaves, and the three new alkaloids were mainly found in the leaflets and petioles. Based on the dissociation patterns of these alkaloids, we observed that there were three structurally related groups of alkaloids differing in their distribution in the plant tissues and responding differently to seasonal variations. These results also indicate that these three groups of alkaloids could belong to intermediate, parallel or competitive pathways for pilocarpine formation biosynthesis.

Production of imidazole alkaloids in cell cultures of jaborandi as affected by the medium pH

“…Therefore, lineage P would be interesting for commercial exploration using bioreactors, since it is friable and produces and excretes exclusively pilocarpine. In contrast, although lineage PP produces pilosine and pilocarpine, it is an interesting model to study the biosynthesis of imidazole alkaloids in jaborandi since the biosynthesis routes of pilocarpine and pilosine seem to be related (Abreu et al 2007b;Sawaya et al 2008).…”

“…The high production on a specific day and the release of alkaloids to the medium might be an attempt of the cells to buffer the pH of the medium. In aqueous solution, pilocarpine may epimerize to isopilocarpine which, in turn, hydrolyzes to isopilocarpic acid (Abreu et al 2007b). Unfortunately, diastereoisomers cannot be distinguished from their original structures by the ESI-MS method used here (Abreu et al 2007a;Sawaya et al 2008).…”

Screening species of Pilocarpus (Rutaceae) as sources of pilocarpine and other imidazole alkaloids

“…This method reduces the possibility of degradation, isomerisation and artifacts which haunt classical phytochemical techniques. Initial studies with direct insertion ESI-MS fingerprinting allowed the characterization of previously undetected alkaloids from this species (Abreu et al 2007). The purpose of the present study was to compare the alkaloid profile of eight samples of leaves (from seven different species) of Pilocarpus grown in two regions of Brazil and to determine their pilocarpine content.…”

Fingerprinting of propolis by easy ambient sonic-spray ionization mass spectrometry

“…We performed the first systematic study applying ESI-MS fingerprinting to propolis [19], analyzing over 40 propolis samples, mainly from Brazil. Since then, ESI-MS fingerprinting has been applied to a wide variety of food and beverage samples [20][21][22] as well as natural products [23].…”