RESUMEN

Polyphenolic compounds are common constituents of human and animal diets and undergo extensive metabolism by the gut microbiota before entering circulation. In order to compare the transformations of polyphenols from yerba mate, rosemary, and green tea extracts in the gastrointestinal tract, simulated gastrointestinal digestion coupled with colonic fermentation were used. For enhancing the comparative character of the investigation, colonic fermentation was performed with human, pig and rat intestinal microbiota. Chemical analysis was performed using a HPLC system coupled to a diode-array detector and mass spectrometer. Gastrointestinal digestion diminished the total amount of phenolics in the rosemary and green tea extracts by 27.5 and 59.2 %, respectively. These reductions occurred mainly at the expense of the major constituents of these extracts, namely rosmarinic acid (-45.7 %) and epigalocatechin gallate (-60.6 %). The yerba mate extract was practically not affected in terms of total phenolics, but several conversions and isomerizations occurred (e.g., 30 % of trans-3-O-caffeoylquinic acid was converted into the cis form). The polyphenolics of the yerba mate extract were also the least decomposed by the microbiota of all three species, especially in the case of the human one (-10.8 %). In contrast, the human microbiota transformed the polyphenolics of the rosemary and green extracts by 95.9 and 88.2 %, respectively. The yerba mate-extract had its contents in cis 3-O-caffeoylquinic acid diminished by 78 % by the human microbiota relative to the gastrointestinal digestion, but the content of 5-O-caffeoylquinic acid (also a chlorogenic acid), was increased by 22.2 %. The latter phenomenon did not occur with the rat and pig microbiota. The pronounced interspecies differences indicate the need for considerable caution when translating the results of experiments on the effects of polyphenolics performed in rats, or even pigs, to humans.

Asunto(s)

Colon , Depsidos , Digestión , Fermentación , Ilex paraguariensis , Extractos Vegetales , Polifenoles , Ácido Rosmarínico , Rosmarinus , Animales , Humanos , Extractos Vegetales/metabolismo , Rosmarinus/química , Ratas , Ilex paraguariensis/química , Porcinos , Depsidos/metabolismo , Depsidos/análisis , Polifenoles/metabolismo , Polifenoles/análisis , Colon/metabolismo , Colon/microbiología , Masculino , Cinamatos/metabolismo , Cinamatos/análisis , Microbioma Gastrointestinal , Té/química , Ácido Quínico/análogos & derivados , Ácido Quínico/metabolismo , Ácido Quínico/análisis , Catequina/análogos & derivados , Catequina/metabolismo , Catequina/análisis , Cromatografía Líquida de Alta Presión , Camellia sinensis/química

RESUMEN

Natural products are important sources of chemical diversity leading to unique scaffolds that can be exploited in the discovery of new drug candidates or chemical probes. In this context, chemical and biological investigation of ferns and lycophytes occurring in Brazil is an approach adopted by our research group aiming at discovering bioactive molecules acting on neurodegeneration targets. In the present study, rosmarinic acid (RA) isolated from Blechnum brasiliense showed an in vitro multifunctional profile characterized by antioxidant effects, and monoamine oxidases (MAO-A and MAO-B) and catechol-O-methyl transferase (COMT) inhibition. RA showed antioxidant effects against hydroxyl (HO(•)) and nitric oxide (NO) radicals (IC50 of 29.4 and 140 µM, respectively), and inhibition of lipid peroxidation (IC50 of 19.6 µM). In addition, RA inhibited MAO-A, MAO-B and COMT enzymes with IC50 values of 50.1, 184.6 and 26.7 µM, respectively. The MAO-A modulation showed a non-time-dependent profile, suggesting a reversible mechanism of inhibition. Structural insights on RA interactions with MAO-A and COMT were investigated by molecular docking. Finally, RA (up to 5 mM) demonstrated no cytotoxicity on polymorphonuclear rat cells. Taken together, our results suggest that RA may be exploited as a template for the development of new antioxidant molecules possessing additional MAO and COMT inhibition effects to be further investigated on in vitro and in vivo models of neurodegenerative diseases.

Asunto(s)

Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Cinamatos/farmacología , Depsidos/farmacología , Helechos/química , Animales , Antioxidantes/metabolismo , Antioxidantes/uso terapéutico , Sitios de Unión , Catecol O-Metiltransferasa/química , Catecol O-Metiltransferasa/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cinamatos/metabolismo , Cinamatos/uso terapéutico , Depsidos/metabolismo , Depsidos/uso terapéutico , Helechos/metabolismo , Humanos , Peróxido de Hidrógeno/toxicidad , Radical Hidroxilo/química , Radical Hidroxilo/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Simulación del Acoplamiento Molecular , Monoaminooxidasa/química , Monoaminooxidasa/metabolismo , Enfermedades Neurodegenerativas/tratamiento farmacológico , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Estructura Terciaria de Proteína , Ratas , Ácido Rosmarínico

RESUMEN

Snakebite envenoming is an important public health problem in many tropical and subtropical countries, and is considered a neglected tropical disease by the World Health Organization. Most severe cases are inflicted by species of the families Elapidae and Viperidae, and lead to a number of systemic and local effects in the victim. One of the main problems regarding viperidic accidents is prominent local tissue damage whose pathogenesis is complex and involves the combined actions of a variety of venom components. Phospholipases A2 (PLA2s) are the most abundant muscle-damaging components of these venoms. Herein, we report functional and structural studies of PrTX-I, a Lys49-PLA2 from Bothops pirajai snake venom, and the influence of rosmarinic acid (RA) upon this toxin's activities. RA is a known active component of some plant extracts and has been reported as presenting anti-myotoxic properties related to bothopic envenomation. The myotoxic activity of Lys49-PLA2s is well established in the literature and although no in vivo neurotoxicity has been observed among these toxins, in vitro neuromuscular blockade has been reported for some of these proteins. Our in vitro studies show that RA drastically reduces both the muscle damage and the neuromuscular blockade exerted by PrTX-I on mice neuromuscular preparations (by ∼80% and ∼90%, respectively). These results support the hypothesis that the two effects are closely related and lead us to suggest that they are consequences of the muscle membrane-destabilizing activity of the Lys49-PLA2. Although the C-terminal region of these proteins has been reported to comprise the myotoxic site, we demonstrate by X-ray crystallographic studies that RA interacts with PrTX-I in a different region. Consequently, a new mode of Lys49-PLA2 inhibition is proposed. Comparison of our results with others in the literature suggests possible new ways to inhibit bothropic snake venom myotoxins and improve serum therapy.

Asunto(s)

Bothrops , Cinamatos/metabolismo , Cinamatos/farmacología , Depsidos/metabolismo , Depsidos/farmacología , Lisina , Fosfolipasas A2/química , Fosfolipasas A2/metabolismo , Animales , Venenos de Crotálidos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Masculino , Ratones , Modelos Moleculares , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/fisiopatología , Fármacos Neuromusculares/antagonistas & inhibidores , Fármacos Neuromusculares/química , Fármacos Neuromusculares/metabolismo , Fármacos Neuromusculares/toxicidad , Inhibidores de Fosfolipasa A2 , Fosfolipasas A2/toxicidad , Unión Proteica , Conformación Proteica , Ácido Rosmarínico

RESUMEN

PrTX-I, a noncatalytic and myotoxic Lys49-phospholipase A(2) from Bothrops pirajai venom, was crystallized in the presence of the inhibitor rosmarinic acid (RA). This is the active compound in the methanolic extract of Cordia verbenacea, a plant that is largely used in Brazilian folk medicine. The crystals diffracted X-rays to 1.8 A resolution and the structure was solved by molecular-replacement techniques, showing electron density that corresponds to RA molecules at the entrance to the hydrophobic channel. The crystals belong to space group P2(1)2(1)2(1), indicating conformational changes in the structure after ligand binding: the crystals of all apo Lys49-phospholipase A(2) structures belong to space group P3(1)21, while the crystals of complexed structures belong to space groups P2(1) or P2(1)2(1)2(1).

Asunto(s)

Bothrops/metabolismo , Cinamatos/química , Venenos de Crotálidos/química , Depsidos/química , Fosfolipasas A2/química , Animales , Cinamatos/metabolismo , Venenos de Crotálidos/metabolismo , Cristalización , Cristalografía por Rayos X , Depsidos/metabolismo , Lisina/química , Modelos Moleculares , Fosfolipasas A2/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Ácido Rosmarínico