Cell Suspension as a Tool to Study the Biosynthesis of Pilocarpine in Jaborandi

Abstract

Jaborandi (Pilocarpus microphyllus) is a species that naturally occurs in the North and Northeast of Brazil, whose leaves produce pilocarpine (an imidazole alkaloid that has been used to treat glaucoma and xerostomy), the biosynthesis of which is still uncertain. The aim of this work was to establish cell lineages and select them according to an alkaloid profile similar to the one from Jaborandi leaves. The induction of callus was done in different culture media and growth regulators. Calluses from primary cultures or those subcultured several times were used as explants for the obtainment of six cell lineages. Alkaloids content analyses and growth curves showed that lines obtained from primary cultures produced more alkaloids and a better development. Cell lines from 12 subcultures presented a decrease in pilocarpine and pilosine production. After 24 subcultures, the production of alkaloids remained constant. ESI-MS analysis showed that cell culture extracts have the same alkaloid composition as extracts made from leaves. The results indicate that cell suspensions can be used as a model to study the biosynthesis of the imidazole alkaloid in P. microphyllus.

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

“…Previously, we have observed a strong influence of medium pH on the production of pilocarpine and the related alkaloid pilosine in calluses obtained from jaborandi leaf petioles (Abreu et al 2005) and, later, we validated cell suspensions as a model to study the biosynthesis of imidazole alkaloids in jaborandi (Abreu et al 2007a). In this work, two jaborandi cell lineages were studied regarding their ability to produce and release pilocarpine and pilosine in the medium, as well as the effects of pH on the partition of these alkaloids between cell and medium.…”

“…Calluses obtained from jaborandi leaf petioles were used to establish cell suspension cultures (Abreu et al 2007a). Two cell lineages (P and PP) were selected for this work due to their rapid growth rate and visual aspects, but mainly because they differed in the production and release of pilocarpine and pilosine in the culture medium, as shown by preliminary analyses (data not shown).…”

“…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). Together with the other alkaloids detected in the cell and in the media, the pH might be controlled by alkaloid production, release and/or modification of the acidic form.…”

“…Although information on the content of pilocarpine in jaborandi is available (Pinheiro 1997;Andrade-Neto et al 1996), very little is known about its biosynthesis (Avancini et al 2003;Abreu et al 2005Abreu et al , 2007aSandhu et al 2006;Sawaya et al 2008).…”

Metabolic Alterations in Different Developmental Stages ofPilocarpus microphyllus

“…For this reason, deeper insight into the biosynthesis and metabolic regulation of imidazole alkaloids could potentially allow manipulation or metabolic engineering favouring pilocarpine synthesis over the biosynthesis of other alkaloids. We previously reported on pilocarpine regulation under stress conditions [6], molecular diversity in jaborandi [7], characterization of the imidazole alkaloid profile in different seasons and identification of novel alkaloids [8], selection of cell suspensions lines for biosynthesis studies [9], and cell lines producing only pilocarpine [10]. A preliminary study using jaborandi from different developmental stages showed that mature plants produce similar amounts of pilosine and pilocarpine, but during juvenile stages only pilocarpine was found.…”