ABSTRACT Caryocar brasiliense Cambess., Caryocaraceae (pequi) is a typical Brazilian Cerrado tree. A previous study showed that the butanolic fraction of pequi leaves promotes endothelium-dependent relaxation mediated by nitric oxide and that it causes reversible hypotension in rats. In the present study, we investigated the cell signaling pathways associated with the butanolic fraction-induced nitric oxide release, and we characterized the chemical composition of its fraction. Vascular reactivity tests, a western blotting analysis, and a chemiluminescence assay were used to investigate the signaling pathways involved in the vasorelaxant effect of the butanolic fraction. Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry was used to characterize the butanolic fraction chemical composition. Vasorelaxation was mediated through the activation of the calmodulin and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways, leading to subsequent endothelial nitric oxide synthase phosphorylation and nitric oxide production, as evidenced by western blotting and chemiluminescence assays, respectively. The chemical characterization of the butanolic fraction revealed the presence of 72 oxygenated compounds, whose molecular formulae are compatible with phenolic compounds, suggesting a potential contribution of these compounds for the butanolic fraction vasorelaxant effect. These findings show that the calmodulin and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways are involved in the butanolic fraction-induced endothelial nitric oxide synthase activation and are promoted by polyphenol compounds present in the C. brasiliense leaves.

Eugenol is an aromatic compound found in several plant species. It presents important biological activities including cytotoxicity. In this paper, it is described the synthesis and the evaluation of the cytotoxic activity of eugenol derivatives bearing 1,2,3-triazole functionalities. Eugenol, extracted via hydrodistillation from dried flower buds of Eugenia caryophyllata (=Syzygium aromaticum), was submitted to alkylation reactions to afford two terminal alkynes in good yields. The key reaction involved in the preparation of eugenol derivatives corresponded to the Copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC), between alkynylated eugenol derivatives and different benzyl azides. The evaluation of the cytotoxicity of twenty seven synthesized derivatives against HL60 leukemia cell line revealed that at 100 µmol L-1, five of them, namely 4-((4-allyl-2-methoxyphenoxy)methyl)-1-(3-bromobenzyl)-1H-1,2,3-triazole (6n), 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-benzyl-1H-1,2,3-triazole (7a), 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(4-chlorobenzyl)-1H-1,2,3-triazole (7c), 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(4-iodobenzyl)-1H-1,2,3-triazole (7e) and 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(3-bromobenzyl)-1H-1,2,3-triazole (7m), were capable of significantly decreasing cell viability. These most active triazolic derivatives were also evaluated against B16F10 melanoma and Nalm6 leukemia cell lines. While only compound 7a was active against the former, compounds 6n, 7a, and 7m displayed activity against the latter. Derivative 7a was active against all cell lines. It is believed that eugenol derivatives bearing triazole functionalities may represent a scaffold to be explored toward the development of new agents against cancer.

Resumo A mastite bovina é um problema sanitário que afeta o gado leiteiro em todas as áreas produtoras do mundo, sendo de difícil controle e erradicação. A atividade antimicrobiana da oleorresina e do óleo essencial de Copaifera spp. foi verificada frente aos micro-organismos isolados de amostras de leite de vacas diagnosticadas com mastite subclínica grau III. A oleorresina de Copaifera ssp. apresentou boa atividade antimicrobiana (CIM≤100 µg/mL) frente às amostras de Staphylococcus coagulase positivo, Staphylococcus coagulase negativo, Streptococcus do grupo C, F, G e Corynebacterium spp. Já o óleo essencial de Copaifera spp. apresentou boa atividade antimicrobiana (CIM≤100 µg/mL) frente aos Staphylococcus coagulase negativo e Corynebacterium spp. Em relação à Escherichia coli, a atividade antimicrobiana da oleorresina e o óleo essencial foi inativa e fraca à inativa, respectivamente. Conclui-se que, frente às 55 bactérias isoladas, a olerresina apresentou melhor atividade antimicrobiana do que o óleo essencial de Copaifera spp. (p<0,0001). A análise por CG/EM identificou sesquiterpenos no óleo essencial e pelo método ESI FT-ICR MS, foi possível identificar ácidos diterpenos na oleorresina de Copaifera spp. Por fim, sugere-se que a oleorresina de Copaifera spp. é uma matéria-prima vegetal promissora no desenvolvimento de medicamento fitoterápico para o tratamento de mastite bovina.

Abstract The antimicrobial activities of the oleoresin (OR) and the essential oil (EO) of Copaifera spp. were checked against microorganisms isolated from milk samples of cows diagnosed with grade III subclinical mastitis. The OR had good antimicrobial activity (MIC ≤ 100 µg/mL) against samples of coagulase-positive Staphylococcus, coagulase-negative Staphylococcus, Streptococcus of groups C, F, and G, and Corynebacterium spp. Meanwhile, the EO had good antimicrobial activity (MIC≤100 µg/mL) against coagulase-negative Staphylococcus and Corynebacterium spp. The OR and the EO were inactive and weak to inactive, respectively, against Escherichia coli. Overall, the OR had better antimicrobial activity than the essential oil against the 55 bacterial isolates (p<0.0001). The GC/MS analysis identified sesquiterpenes in EO and by the ESI FT-ICR MS method, the identification of diterpenic acids in OR was possible. Therefore, this raw plant material is promising for the development of phytotherapeutic drugs against bovine mastitis.

Benzoato de metila e p-clorofenil acetato de metila reagem com benzilamina e pirrolidina levando às correspondentes amidas. Estas reações são mais rápidas na presença de 20 mol% de DBU, fornecendo os produtos com rendimentos levemente superiores. Quando um diéster derivado do ácido L-aspártico foi usado como substrato, a reação com benzilamina e pirrolidina foi quimiosseletiva para o éster metílico, levando às correspondentes amidas em bons rendimentos. Reação do monoéster metílico do ácido aspártico com estas aminas conduziu a amidas com um grupo ácido livre em C1. Anilina, menos básica e menos nucleofílica, não formou os produtos esperados tanto na ausência quanto na presença de DBU. Através do monitoramento da reação por ESI-MS, foi possível interceptar os intermediários-chave catiônicos formados nas reações entre o benzoato de metila e o p-clorofenil acetato de metila com a benzilamina, os quais foram caracterizados por ESI-MS/MS.

Methyl benzoate and methyl p-chlorophenyl acetate react with neat benzylamine and pyrrolidine to form the corresponding amides. These reactions are faster in the presence of 20 mol% of DBU providing slight better yields. When a diester derived from L-aspartic acid was used as substrate, the reaction with benzylamine and pyrrolidine in the presence of DBU was chemoselective and led to the corresponding amides in good yields. Reaction of aspartic acid monomethyl ester with these amines led to amides having a free carboxy group (at C1). Less nucleophilic and less basic aniline failed to form the expected products in both absence and presence of DBU. By monitoring the course of reaction by ESI-MS, key charged intermediates formed by the reactions of methyl benzoate and methyl p-chlorophenyl acetate with benzylamine were intercepted and further characterized by ESI-MS/MS.