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Orientador : Raquel Marques Braga Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica Doutorado

INTRODUCTION: Species of the genera Psychotria and Palicourea are sources of indole alkaloids, however, the distribution of alkaloids within the plants is not known. Analysing the spatial distribution using desorption electrospray ionisation mass spectrometry imaging (DESI-MSI) has become attractive due to its simplicity and high selectivity compared to traditional histochemical techniques.OBJECTIVES: To apply DESI-MSI to visualise the alkaloid distribution on the leaf surface of Psychotria prunifolia and Palicourea coriacea and to compare the distributions with HPLC-MS and histochemical analyses.METHODOLOGY: Based upon previous structure elucidation studies, four alkaloids targeted in this study were identified using high resolution mass spectrometry by direct infusion of plant extracts, and their distributions were imaged by DESI-MSI via tissue imprints on a porous Teflon surface. Relative quantitation of the four alkaloids was obtained by HPLC-MS/MS analysis performed using multiple-reaction monitoring (MRM) mode on a triple quadrupole mass spectrometer.RESULTS: Alkaloids showed distinct distributions on the leaf surfaces. Prunifoleine was mainly present in the midrib, while 10-hydroxyisodeppeaninol was concentrated close to the petiole; a uniform distribution of 10-hydroxyantirhine was observed in the whole leaf of Psychotria prunifolia. The imprinted image from the Palicourea coriacea leaf also showed a homogeneous distribution of calycanthine throughout the leaf surface.CONCLUSION: Different distributions were found for three alkaloids in Psychotria prunifolia, and the distributions found by MSI were in complete accordance with HPLC-MS analysis and histochemical results. The DESI-MSI technique was therefore demonstrated to provide reliable information about the spatial distribution of metabolites in plants. Copyright © 2017 John Wiley & Sons, Ltd.

Aspidosperma macrocarpon belongs to the family Apocynaceae and is endemic to Americas and mainly found from Mexico to Argentina. It is known in Brazil as “guatambu” or “peroba”. Crude extracts and their fractions from leaves were assayed against human cancer cells lines: glioma (U251), melanoma (UACC-62), mammary (MCF-7), ovarian expressing the multidrug resistance phenotype (NCI-ADR/RES), lung (NCI-H460), prostate (PC-3), kidney (786-0), ovarian (OVCAR-3), colon (HT-29) and leukemia (K-562). The crude extract (EAM), hexane (HA) and chloroform (CA) fractions were the most active fractions against K-562 with GI50 values low than 1 µg/mL. Also, CA was moderate active against OVCAR-3 and NCI-ADR/RES cells lines. This phytochemical study allowed to identify the known kopsanone, kopsinine, ursolic acid, rutin, 5-O-caffeoylquinic acid, and 3,5-O-dicaffeoylquinic acid. The kopsanone was also evaluated against human cancer cell lines and showed activity to the U251 and K-562 cell lines, with GI50 values of 20.6 µg/mL and 8.7 µg/mL, respectively.

Indole alkaloids (IAs) are known for their pharmacological activities, including for neurodegenerative diseases (NDs) treatment. In fact, IAs have been calling the attention due to their multifunctionality, interacting with different targets mainly related to NDs. In this study, two β-carbolinic IA (βCIA), namely kopsanone (1) and N(4)-oxide-kopsanone (2), were evaluated for their monoamine oxidase (MAO) inhibitory potential. Both βCIAs were obtained from Aspidosperma macrocarpon. For isolation, the leaves’ methanol extract was submitted to acid-base extraction. The alkaloid fraction (0.47 g) was chromatographed in silica open column using mixtures of CHCl3:MeOH, affording 1 (85 mg) and 2 (14.7 mg). The in vitro assays were carried out using kynuramine as non-selective substrate of human MAO-A and MAO-B supersomes (BD Gentest). Compound 1 was identified based on data comparison to previous studies. Compound 2 analysis, afforded a mass spectrum with a characteristic [M-16]+ fragment and downfield shift in somesignals in NMR analysis, indicating aN-oxide derivative of 1. It was observed that 2 impaired MAO-A activity with an IC50 value of 4.8 μM, while 1 exhibited ten times higher potency. Molecular docking (GOLD, version 5.2, CCDC) was performed aiming at understanding the binding of 1 in MAO-A. Compound 1 was able to bind the MAO-A active site in the same pocket occupied by harmine. Its indole moiety faces to the FAD co-factor, making π-π stacking interactions with Y407. Van der Waals contacts are observed between 1 and Y69, Q215, I335, L337, and F352, contributing to stabilize the protein-ligand complex. The GoldScore retrieved for the docking of 1 in MAO-A was 51.6 while the value obtained for the re-docking of harmine (IC50 = 0.002 μM) was 79.1, suggesting that the in silico scores are in agreement with the experimental data. These results corroborate to the fact that IAs can be used as scaffolds aiming the inhibition of ND related enzymes.

The phytochemical investigation of Sabicea brasiliensis roots led to the isolation of 5-O-caffeoylquinic acid, 3,5- and 4,5-O-dicaffeoylquinics acids, scopoletin, ursolic acid, a mixture of β-sitosterol, stigmasterol and campesterol, daucosterol and saccharose. The structures of the isolated compounds were assigned on the basis of one- and two-dimensional NMR spectroscopic methods and by comparison with literature data. The anti-inflammatory and antioxidant activities of the crude methanolic extract and its fractions were analyzed.

The continuity of the phytochemical study of crude extracts of P. prunifolia's roots and branches led to the isolation of five indole-β-carboline alkaloids. Among them, the 10-hydroxy-iso-deppeaninol and N-oxide-10-hydroxy-antirhine derivatives are described here for the first time. The structures were achieved through 1D and 2D NMR, IR and HRMS analyses. The branches and roots crude extracts and the alkaloids 14-oxoprunifoleine and strictosamide showed selective activity against L. amazonensis, with IC50 values of 16.0 and 40.7 µg per mL, respectively. A continuidade do estudo fitoquímico de P. prunifolia com a análise dos extratos etanólicos obtidos a partir de suas raízes e galhos levou ao isolamento de cinco alcaloides indol-β-carbolínicos dos quais, dois derivados, o 10-hidróxi-iso-deppeaninol e o N-óxido-10-hidróxi-antirhina são descritos pela primeira vez. As estruturas foram determinadas por análise de técnicas espectroscópicas de IV, EMAR e RMN (¹H e 13C, 1D e 2D). A avaliação da atividade frente à Leishmania amazonensis e Trypanosoma cruzi, mostrou que os extratos brutos e os alcaloides 14-oxoprunifoleína e estrictosamida inibiram as formas promastigotas de L. amazonensis, com valores de CI50 de 16,0 e 40,7 µg per mL, respectivamente.