EFFECT OF FLAVONOIDS AND PECTIN COMBINATIONS ON THE STRUCTURE OF PANCREATIC ALPHA-AMYLASE

Pancreatic alpha-amylase is continuously studied because its inhibition can provide key insights into reducing postprandial blood glucose levels in individuals with type 2 diabetes mellitus. Natural compounds in the daily diet have gained relevance for developing alternative management strategies for this disease. In this context, flavonoids have been studied as alpha-amylase inhibitors; however, their combined effect with other compounds present in vegetables, such as pectin, has not been fully studied. The mechanism underlying these effects and their activities on alpha-amylase remain fully unresolved. Therefore, this study evaluated the structural changes induced in porcine pancreatic alpha-amylase by flavonoids and pectin that could be associated with their enzymatic inhibition.

Porcine pancreatic alpha-amylase solution was prepared (1 mg mL^-1 in 50 mM HEPES buffer; pH 7.0). Intrinsic fluorescence was measured using a microplate reader at excitation and emission wavelengths of 290 nm and 320 nm, respectively. The assays were carried out in the absence and presence of the ligands, flavonoids, and pectin. Three different flavonoids—quercetin, hesperetin, and epicatechin—were tested across concentrations ranging from 10 to 400 µM. Each flavonoid was incubated with alpha-amylase to evaluate its effect on the protein intrinsic fluorescence. The same procedure was followed to assess the interaction between pectin and amylase, using pectin concentrations ranging from 0.01 to 0.5 mg mL^-1.

In the individual flavonoid-amylase assays, epicatechin did not exhibit a statistically significant quenching effect, whereas quercetin and hesperetin successfully quenched its intrinsic fluorescence. A static quenching mechanism was calculated for both compounds. Hesperetin seemed to be a stronger quencher with higher affinity than the other, maybe through the formation of more protein-ligand interactions, displaying values of k_q=7.8±0.16 x10¹¹ M^-1 s^-1, K_a= 5.57±0.20 x10³ M^-1; on one possible binding site (n=0.92±0.11). In contrast, a statistically significant fluorescence quenching was not observed for pectin. The quenching capacity observed for quercetin and hesperetin can be partially attributed to similar structural features, such as the C2-C3 double bond in the C-ring, which provides greater planarity to the flavonoid skeleton.

The best quenching effects for binary complexes were observed for pectin-quercetin and pectin-hesperetin. Nevertheless, pectin seemed to reduce the quenching effect of the lone flavonoid (higher K_a and n values, respectively). In conclusion, even though flavonoids such as hesperetin exhibited a high quencher capacity alone, their binary complex with pectin did not increase their quencher capacity. This lower effect may be attributed to more binding between flavonoid and pectin instead of flavonoid-protein binding. However, these complexes need further studies to analyze these possible flavonoid-pectin interactions.

The authors acknowledged UACJ for all the support.