Título: Uma abordagem in vivo dos efeitos inibitórios in vitro dos ácidos fenólicos e falvonóides sobre a atividade da alfa-amilase pancreática.

 Orientadora: Profa. Dra. Rosane Marina Peralta 

 Data da defesa: 29/07/2022


Resumo: INTRODUCTION AND OBJECTIVES-Reduction in the activity of the α-amylases, which hydrolyze starch and glycogen has been proposed for diminishing the incidence of obesity and diabetes. Many plant extracts contain inhibitors of α-amylases and α-glucosidases and several molecules with this activity have been identified. In recent years, many polyphenols have been identified as inhibitors of pancreatic and salivary α-amylases and, in some cases, α–glucosidases. In this respect some of them have been extensively characterized. Their abilities to inhibit the α-amylases was tested using diverse substrates (starch, amylose, etc.) and different methods of measuring reaction rates. Binding to the enzymes was also extensively investigated using fluorescence and computational simulations. Taken together,the aforementioned evidence represents a highly respectable collection of data on the action of polyphenolic compounds on α- amylases and α-glucosidases under various in vitro conditions. In vivo confirmations are indispensable, considering translation of in vitro results to in vivo conditions are always uncertain for reasons that encompass, for example, doses, accessibility, conditions, substrate, and biotransformation. For this reason, due to the lack of in vivo experiments with polyphenols as putative inhibitors of starch absorption, this work aimed at investigating a series of 5 flavonoids (catechin, epicatechin, quercetin, rutin and naringenin) and 5 phenolic acids (gallic, syringic, vanillic, ferulic and sinapic acids), already known as inhibitors of α-amylases, for their possible inhibitory action on starch absorption. The method used was the starch tolerance test in mice and the results should provide a preliminary answer to the question if further in vivo or even clinical experiments with these compounds are of interest or not. The present work attempts at investigating if natural compounds that have been reported to be inhibitors of hydrolytic enzymes can also inhibit starch absorption in vivo.

 METHODS- The starch tolerance tests in mice were used for assaying starch absorption after administration of 100 mg/kg of five flavonoids (epicatechin, catechin, rutin, quercetin, naringenin) and five phenolic acids (gallic, syringic, vanillic, sinapic, ferulic), which are known to be in vitro inhibitors of α-amylases. Three types of solutions were administered: (a) solutions containing a single compound; (b) solutions containing a mixture of five different substances;

(c)               solution containing the positive control acarbose. Each experiment was conducted according to the following steps: 1) intragastric administration of one of type (a), (b) or (c) solutions; 2) intragastric administration of either starch or maltose; 3) sample collections for assaying blood glucose. These samples were collected from the tail vein at times 0, 30, 60, 90 and 120 minutes after starch administration. Glucose in these samples was assayed by means of a glucometer (AccuCheck®).

 MAIN RESULTS, DISCUSSION AND CONCLUSION- Among the flavonoids, only rutin inhibited starch absorption in mice, even though its effect was only 33% of the one exerted by the positive control acarbose. Among the five phenolic acids tested, only syringic acid produced a significant diminution in starch absorption, which was 47% of that one observed for acarbose. On the other hand, other three phenolic acids, namely gallic, sinapic and ferulic acid, promoted delay in the absorption of starch, which was characterized by a shift in the time peak of maximal blood glucose concentration, with no modification in the excess of glucose that effectively entered the circulation. Attempts at detecting eventual synergisms by administrating mixtures of flavonoids and phenolic acids were unsuccessful. These results indicate the need of careful translation of in vitro data on the inhibition of α-amylases and α-glucosidases to the in vivo conditions. In conclusion, no correlation between the reported strength by which polyphenols inhibit the starch hydrolyzing enzymes in vitro and their in vivo capacity in diminishing starch absorption. Instead of just speculating about the prospective utility of thean inhibitor as an anti-diabetic agent, the experimentation should always complement the in vitro characterization with in vivo experiments. This is presently the only way to acquire some certainty about the real