Estudo da atividade antinitrosante, antioxidante e inibição enzimática por extratos hidroetanólicos de Baccharis crispa Spreng.(Asteraceae) por eletroforese capilar de zona

Abstract

Capillary electrophoresis stands out as one of the most promising analytical techniques due to its automated and reliable character, as well as being a flexible, accurate, highly efficient separation technique requiring only small amounts of sample for analysis. Thus, capillary electrophoresis has also been recognized as a suitable technique to study enzyme and radical reactions, including the enzyme inhibition activity of natural product extracts and phytochemicals. In this work, we applied three established methods using capillary electrophoresis to analyze the ability of Baccharis crispa extracts by maceration with 70% ethanol and 96% ethanol for 5 days, which can inhibit the enzymes acetylcholinesterase and α-glycosidase, and which can also perform antinitrosant and antioxidant activity. In the antinitrosant test, the ability of the extracts to capture nitrosating species was evaluated by observing their presence in the electropherograms. For this analysis, nitrite, nitrate, and thiocyanate were used as a mixture of standards. The evaluation of the antinitrosative activity consists in the capture of nitrite by the sample, reducing its initial concentration. The antioxidant test was performed using the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) and quercetin as antioxidant standard. In this analysis, the area of the DPPH peak was observed, which represents the amount captured by the sample and the internal standard. For the analysis of the inhibition of the two enzymes, the reaction of the extracts with the enzymes occurred inside the capillary through multiple injections. The amount of end product of the substrates of each enzyme, allowed the calculation of the inhibition capacity of the extracts. In this study, we demonstrate the usefulness of capillary electrophoresis as a tool for the evaluation of pharmacological properties of medicinal plants, using Baccharis crispa, and report that it is possible to use different modes of capillary electrophoresis as a tool to analyze reactions and inhibitory activity. In addition, capillary electrophoresis has advantages such as reduced amount of solvents, samples and standards used, reduced analysis time and automation. In the development of the antinitrosive activity, it was not possible to use the macerated extracts due to the interference of the methanol used in the dilution of the residues and therefore it was opted to use the infusion as extraction method. In this analysis there was 27.19% nitrite capture by the infusion. The samples extracted with ethanol at 70% and 96% in concentration of 1 mg L, obtained antioxidant capacity of 50.30% (equivalent to 34.84mg of quercetin per gram of extract) and 42.22% (equivalent to 28.29mg of quercetin per gram of extract) respectively when incubated and 58.40% (equivalent to 29.7.0mg of quercetin per gram of extract) and 38.98% (equivalent to 18.71mg of quercetin per gram of extract) respectively with incubation. In enzyme reaction analyses, the enzyme acetylcholinesterase was inhibited by 7.80% by the 70% maceration extract and 6.89% with the 96% maceration extract. The enzyme αglycosidase was inhibited by 0.51% by the 70% maceration extract, but the 96% maceration extract was not able to show inhibitory activity for this enzyme. In this study we can see the versatility of different methods employing capillary electrophoresis to analyze three types of activities, along with the advantages that this technique can provide.