Evaluation of non-clinical toxicity of extract and vouacapans from fruits of Pterodon pubescens Benth.

Pterodon pubescens Benth. is widely used in folk medicine for the treatment of inflammatory conditions, with the activity attributed to the compounds with a vouacapan moiety, however, few studies report the toxicological evaluation of the extract and safety issues related to the species. Herein the non-clinical toxicity, in in vivo and in vitro tests, of dichloromethane crude extract of Pterodon pubescens fruits (PPE) and vouacapan diterpene furan isomer´s mixture (1:1) 6α-hydroxy-7ß-acetoxy-vouacapan-17ß-oate methyl ester and 6α-acetoxy-7ß-hydroxy-vouacapan-17ß-oate methyl ester isomers (VDFI mixture) is reported. Toxicological evaluation of 110-day repeated dose oral toxicity study, as hematological, biochemical, and histopathological parameters demonstrated that animals (male and female Wistar rats) treated with PPE presented no signs of toxicity, nevertheless daily high dose administration (500 mg/Kg) altered the metabolic homeostasis of animals that manifested microgoticular hepatic steatosis. Biochemical and histopathological results of animals (female Swiss mice) treated daily with VDFI mixture, at the highest dose (300 mg/Kg), indicated liver toxicity in one animal causing acute hepatotoxicity. Alkaline Comet assay demonstrated that PPE and VDFI mixture increased the percentage of DNA fragmentation without interfering with the tail moment parameter, but only VDFI mixture (30 µg/mL) presented statistical difference. In the micronucleus induction test, PPE and VDFI mixture did not demonstrate mutagenic potential. Our data provide evidence for the safety use of PPE and VDFI mixture in lower doses enabling further clinical studies and the development of herbal medicine.

Preparation and characterization of microcapsules of Pterodon pubescens Benth. by using natural polymers

An oleaginous fraction obtained from an alcohol extract of the fruit of Pterodon pubescens Benth. (FHPp) was microencapsulated in polymeric systems. These systems were developed using a complex coacervation method and consisted of alginate/medium-molecular-weight chitosan (F1-MC), alginate/chitosan with greater than 75% deacetylation (F2-MC), and alginate/low-molecular-weight chitosan (F3-MC). These developed systems have the potential to both mask the taste of the extract, and to protect its constituents against possible chemical degradation. The influence of the formulation parameters and process were determined by chemical profiling and measurement of the microencapsulation efficiency of the oleaginous fraction, and by assessment of microcapsule morphology. The obtained formulations were slightly yellow, odorless, and had a pleasant taste. The average diameters of the microcapsules were 0.4679 µm (F2-MC), 0.5885 µm (F3-MC), and 0.9033 µm (F1-MC). The best formulation was F3-MC, with FHPp microencapsulation efficiency of 61.01 ± 2.00% and an in vitro release profile of 75.88 ± 0.45%; the content of vouacapans 3-4 was 99.49 ± 2.80%. The best model to describe the release kinetics for F1-MC and F3-MC was that proposed by Higuchi; however, F2-MC release displayed first-order kinetics; the release mechanism was of the supercase II type for all formulations.

Uma fração oleaginosa obtida do extrato etanólico de frutos de Pterodon pubescens Benth (FHPp) foi microencapsulada em um sistema polimérico. Estes sistemas foram desenvolvidos utilizando o método de coacervação complexa, constituído de alginato/quitosana massa molecular média (F1-MC), alginato/quitosana com desacetilação superior a 75% (F2-MC) e alginato/quitosana de massa molecular baixa (F3-MC). Estes sistemas desenvolvidos têm o potencial tanto de mascarar o sabor do extrato, quanto de protegê-lo de possível degradação química. A influência dos parâmetros de formulação e processo foram determinadas por caracterização química, determinação da eficiência de microencapsulação da fração oleaginosa e por avaliação morfológica da microcápsula. As formulações mostraram-se ligeiramente amareladas, inodoras e com sabor agradável. Os diâmetros médios das microcápsulas foram de 0,4679 µm (F2-MC), 0,5885 µm (F3-MC) e 0,9033 µm (F1-MC). A melhor formulação foi F3-MC, considerando-se que apresentou eficiência de encapsulação de 61,01 ± 2,00%, e perfil de liberação in vitro de 75,88 ± 0,45%; o conteúdo dos vouacapanos 3-4 foi 99,49 ± 2,80%. O melhor modelo para descrever a cinética de liberação foi o modelo proposto por Higuchi para F1-MC e F3-MC, entretanto, para F2-MC foi o modelo de primeira ordem, e o mecanismo de liberação foi do tipo super caso II para todas as formulações.

Avaliação dos mecanismos envolvidos na inibição da proliferação e na indução da apoptose de linhagem leucêmica pelo óleo de sementes de Pterodon pubescens e suas frações/subfrações / Assessment of the mechanisms involved in inhibiting proliferation and inducing apoptosis of lineage leukemia by oil seed Pterodon pubescens and its fractions/subfractions

Alterações no ciclo celular e desenvolvimento de tumor encontram-se intimamente ligados. Expressão inadequada e/ou mutações de quinases dependentes de ciclinas, ciclinas e inibidores de quinases dependentes de ciclinas têm sido descritas em vários tipos de cânceres. Portanto, a busca por novas molédulas que têm como alvo eventos em tal via bioquímica tem aumentado ao longo dos anos. Como as plantas são importantes fontes de novas potenciais drogas quimioterápicas, o estudo da fitoquímica, aliado às pesquisas farmacológicas, permite desvendar novas substâncias com potencial anti câncer. Embora efeitos antitumorais tenham sido demonstrados para extratos de Pterodon pubescens, os mecanismos que induzem estes efeitos ainda não estão esclarecidos. Portanto, o presente trabalho teve como objetivos subfracionar a fração hexano do extrato bruto de frutos de Pterodon pubescens, avaliar alguns mecanismos pelos quais a subfração mais ativa exerce seu efeito antiproliferativo sobre linhagem leucêmica (K562) e tentar identificar compostos envolvidos em tais efeitos. O extrato bruto (OPp) foi obtido por maceração em etanol e posteriormente foi feita uma partição líquido-liquido obtendo-se a fração Hexano (HEX). O subfracionamento por coluna de sílica da HEX resultou em oito subfrações, sendo que a subfração 5 (SF5) a que apresentou menor complexidade por cromatografia gasosa e maior efeito biológico. A citotoxicidade foi avaliada pelo método de redução do sal tetrazol (MTT) e a proliferação celular pela síntese de DNA e curva de crescimento. As fases do ciclo celular e a apoptose foram analisados por citometria de fluxo e a expressão do RNA mensageiro por RT-PCR. A expressão de proteína foi avaliada por Western blotting e a identificação de compostos por CG-EM e 13C-RMN. A SF5 exerceu efeito citotóxico maior do que o extrato bruto, fração HEX e as outras subfrações. Na síntese de DNA e curva de crescimento, a SF5 exerceu efeito inibitório tempo e concentração...

Alterations in the cell cycle and tumor development are closely connected. Inapropriated expression and/or mutations of cyclin dependet kinases, cyclins and cyclin kinase inhibitors have been described in many kinds of cancer. So, the search of new molecules that have such biochemical pathway as target has increased over the years. Since plants are important sources of new potential chemotherapic drugs, phytochemical studies combined to pharmacological investigations permit the discover of new substances with potential anti-cancer properties. Although anti tumor effects have been demonstrated for Pterodon pubescens extracts, its mechanisms are still obscure. Therefore, the aims of present work were to subfractionate the HEX fraction of Pterodon pubescens, evaluate some mechanisms by which the most active subfraction exerts its antiproliferative effect on leukemic cell line (K562) and try to elucidate compounds involved in such effects. The crude extract (PpO) was obtained by maceration in ethanol with further liquid-liquid partition yielding the Hexan fraction (HEX). The HEX subfractioning by silica column yielded 8 subfractions, being the subfraction 5 (SF5) that with minor complexity by gas chromatography and greater biological effect. The citotoxicity was evaluated by tetrazolium salt reduction (MTT) and cellular proliferation by DNA synthesis and growth curve. The cell cycle phases and apoptosis were analyzed by flow cytometry and messenger RNA expression by RT-PCR. The protein expression was evaluated by Western blotting and the identification of compounds by GC-MS and 13C-NMR. SF5 induced greater citotoxic effects when compared to crude extract, HEX fraction or the others subfractions. On DNA synthesis and growth curve, SF5 induced time and concentration dependent inhibiory effect, also greater than HEX fraction. The cell cycle analysis showed an increase of cells on G1 phase with consequently reduction of S and G2/M phases. SF5 inhibited the MAP...