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

<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

Principal coordinate analysis of 11 morphological quantitative variables and 111 ISSR binary molecular markers from three groups of <i>Pilocarpus microphyllus</i> (S01, S02, S03), representing two cultivated lines.

<p>Green line (black: S01). Traditional line (red: S02; blue: S03)</p

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

Primers used for generating polymorphic ISSR markers in <i>Pilocarpus microphyllus</i> genotypes, showing the respective levels of polymorphic loci.

<p>Primers used for generating polymorphic ISSR markers in <i>Pilocarpus microphyllus</i> genotypes, showing the respective levels of polymorphic loci.</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

Genetic variation of the 15 individuals from the three groups of <i>Pilocarpus microphyllus</i> (S01, S02, S03) analyzed with GenAlEx 6.5 software.

<p>Genetic variation of the 15 individuals from the three groups of <i>Pilocarpus microphyllus</i> (S01, S02, S03) analyzed with GenAlEx 6.5 software.</p

Pairwise genetic distances (as PhiPT values) between the three groups (S01, S02, S03) of cultivated <i>Pilocarpus microphyllus</i>, based on ISSR marker data analyzed with GenAlEx 6.5 software.

<p>Pairwise genetic distances (as PhiPT values) between the three groups (S01, S02, S03) of cultivated <i>Pilocarpus microphyllus</i>, based on ISSR marker data analyzed with GenAlEx 6.5 software.</p

Chromatograms and mass spectra of alkaloid standards from <i>Pilocarpus microphyllus</i> analyzed by LC/MS.

<p>Pilocarpine, showing the retention time at 15.05 min and molecular weight for mass spectrometry, electron spray [M + H]⁺ at 209 Da. (B) Epiisopiloturine, showing the retention time at 17.05 min and molecular weight for mass spectrometry, electron spray [M + H] ⁺ at 287 Da.</p