Introduction: Lauraceae alkaloids are a structurally diverse class of plant specialised secondary metabolites that play an important role in modern pharmacotherapy, being useful as well as model compounds for the development of synthetic analogues. However, alkaloids characterisation is challenging due to low concentrations, the complexity of plant extracts, and long processes for accurate structural determinations. Objective: The use of high-performance thin layer chromatography coupled with desorption electrospray ionisation multistage mass spectrometry (HPTLC DESI-MS n) as a fast tool to identify alkaloids present in Ocotea spixiana extract and evaluate the extract's acaricide activity. Methods: Ocotea spixiana twigs were extracted by conventional liquid-liquid partitioning. HPTLC analysis of the ethyl acetate extract was performed to separate isobaric alkaloids prior to DESI-MS n analysis, performed from MS 3 up to MS 7. The extract's acaricide activity against Rhipicephalus microplus was evaluated by in vitro (larval immersion test) and in silico tests. Results: HPTLC-DESI-MS n analysis was performed to identify a total of 13 aporphine and four benzylisoquinoline-type alkaloids reported for the first time in O. spixiana. In vitro evaluation of the extract and the alkaloid boldine showed significant activity against R. microplus larvae. It was established in silico that boldine had important intermolecular interactions with R. microplus acetylcholinesterase enzyme. Conclusion: The present study demonstrated that HPTLC-DESI-MS n is a useful analytical tool to identify isoquinoline alkaloids in plant extracts. The acaricide activity of the O. spixiana ethyl acetate extract can be correlated to the presence of alkaloids. K E Y W O R D S acaricide activity, alkaloids, HPTLC-DESI-MS n , Ocotea spixiana 1 | INTRODUCTION Ocotea is an expressive genus of the Lauraceae family, occurring mainly in tropical and subtropical areas. 1 Species of this genus are known for producing a variety of secondary metabolites, with neolignans, aporphine and benzylisoquinoline-type alkaloids as the most representative classes. 2,3 A wide range of biological activities from Ocotea species has been described in the literature, including

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The blood-brain barrier (BBB) is known to protect healthy brain cells from potentially dangerous chemical agents, but there are many evidences supporting the idea that this protective action is extended to tumor cells. Since the process of angiogenesis in brain tumors leads to BBB breakdown, biochemical characteristics of the BBB seem to be more relevant than physical barriers to protect tumor cells from chemotherapy. In fact, a number of resistance related factors were already demonstrated to be component of both BBB and tumor cells. The enzyme glutathione S-transferases (GST) detoxify electrophilic xenobiotics and endogenous secondary metabolites formed during oxidative stress. A role has been attributed to GST in the resistance of cancer cells to chemotherapeutic agents. This study characterized 8-methoxypsoralen (8-MOP) as a human GST P1-1 (hGST P1-1) inhibitor. To identify and characterize the potential inhibitory activity of 8-MOP, we studied the enzyme kinetics of the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with GSH catalyzed by hGST P1-1. We report here that 8-MOP competitively inhibited hGST P1-1 relative to CDNB, but there was an uncompetitive inhibition relative to GSH. Chromatographic analyses suggest that 8-MOP is not a substrate. Molecular docking simulations suggest that 8-MOP binds to the active site, but its position prevents the GSH conjugation. Thus, we conclude that 8-MOP is a promising prototype for new GST inhibitors pharmacologically useful in the treatment of neurodegenerative disorders and the resistance of cancer to chemotherapy.

A enzima UDP-N-acetilglicosamina pirofosforilase de Moniliophthora perniciosa (CCMB 0257), o fungo patogênico causador da doença vassoura-de-bruxa do Theobroma cacao, foi parcialmente purificada por precipitação com sulfato de amônio e cromatografia de gel filtração em Sephacryl S-200. O tampão de extração da enzima foi o fosfato de sódio, 0,050 mol L -1 , pH 7,0, contendo 1,0 mol L -1 de NaCl. A metodologia de superfície de resposta (MSR) foi usada para a obtenção do pH e temperatura ótima. Os resultados mostraram quatro diferentes isoenzimas (PyroMp I, PyroMp II, PyroMp III e PyroMp IV) que apresentaram pH ótimo na faixa de 6,9-8,4 e temperatura ótima variando entre 28 a 68 °C. A estrutura 3D de pirofosforilase de M. perniciosa foi obtida por modelagem comparativa. O modelo obtido mostrou uma boa qualidade, possuindo 78,6% de aminoácidos nas regiões energeticamente favoráveis. O modelo foi então submetido a simulações de dinâmica molecular (DM). O modelo apresentou uma boa qualidade geométrica após as simulações de DM (91,1% -gráfico de Ramachandran). A procura pelo sítio ativo da enzima mostrou que este é mantido extremamente conservado. Este modelo pode ser útil para desenvolvimento de inibidores contra a doença vassoura de bruxa.The enzyme UDP-N-acetylglucosamine pyrophosphorylase (PyroMp) from Moniliophthora perniciosa (CCMB 0257), a pathogenic fungal strain and the causative agent of the witches' broom disease in Theobroma cacao, was partially purified by precipitation with ammonium sulfate and gel filtration on Sephacryl S-200. The buffer for enzyme extraction was sodium phosphate, 0.050 mol L -1 , pH 7.0, containing 1.0 mol L -1 NaCl. Response surface methodology (RSM) was used to determine the optimum pH and temperature conditions. Four different isoenzymes (PyroMp I, PyroMp II, PyroMp III and PyroMp IV) were obtained with optimal pH ranging from 6.9-8.4 and optimum temperature ranging from 28 to 68 °C. The 3D structure of pyrophosphorylase of M. perniciosa was determined by comparative modeling. The model obtained showed a good quality, possessing 78.6% of amino acids in energetically allowed regions. The model was then submitted for DM simulation and showed a good geometric quality (91.1% Ramachandran plot). The active site of the enzyme was found to be extremely well conserved. This model will be useful for developing new inhibitors against witches' broom disease.Keywords: pyrophosphorylase, Moniliophthora perniciosa, kinetic characterization, heat stability, 3D structure, comparative modeling J. Braz. Chem. Soc. 1016 Purification, Characterization and Structural Determination of UDP-N-Acetylglucosamine Pyrophosphorylase IntroductionMoniliophthora perniciosa, the causative agent of the witches broom disease in Theobroma cacao, is responsible for major crop losses in South American and Caribbean cocoa plantations. 1,2 In 1989, witches broom disease of cocoa was identified in Bahia, the leading cocoa-growing region in Brazil. 2 In less than 10 years, production shrank from 383,000 tons to an estim...