Preliminary Study on the Inhibition and Mechanism of Citrus Flavonoids on Trypsin Activity
Citrus peel residue is the main byproduct of citrus processing, accounting for about 30-50% of the total fruit, and is often used to make feed or discarded directly. Leather residue is rich in functional components such as dietary fiber and flavonoids, and its development and utilization are beneficial for improving enterprise efficiency and protecting the environment.
Trypsin is an important digestive enzyme in both humans and animals, which can activate other digestive enzymes and play a crucial role in the metabolic process. Meanwhile, the occurrence of various diseases (pancreatitis, emphysema, cerebral edema, cancer, etc.) is related to excessive secretion of trypsin, and trypsin inhibitor drugs are often used for treatment in clinical practice.
Research has shown that some plant polyphenolic compounds exhibit certain inhibitory effects on trypsin, such as purple sweet potato anthocyanins showing a significant inhibitory effect on the catalytic activity of trypsin, causing static quenching of intrinsic fluorescence of trypsin, and the interaction between the two through hydrogen bonding and van der Waals forces; Quercetin, rutin, hesperetin, rutin, and quercetin all have varying degrees of trypsin inhibition; A flavonoid compound in Snow Chrysanthemum can inhibit the activity of trypsin and alleviate the damage of alcoholic acute pancreatitis in mice. As a common functional ingredient in health products, the effect of citrus flavonoid extract on trypsin activity is still unclear. In this study, Na benzoyl DL arginine p-nitroamide hydrochloride (BAPNA) was used as the substrate to investigate the effect of citrus flavonoids on trypsin activity and the types of inhibition. The mechanism of action between citrus flavonoids and trypsin was also studied by fluorescence spectroscopy, in order to provide reference for the development and utilization of citrus flavonoid special dietary foods and health foods.

The results of this experiment indicate that citrus flavonoids have an inhibitory effect on trypsin activity, and the higher the concentration of flavonoids, the stronger the inhibitory effect; At 37 ℃, the inhibition rate of 0.66mg/ml flavonoids on 0.5mg/ml trypsin was 29.9%. The inhibition of trypsin by citrus flavonoids is reversible and belongs to a mixed inhibition type of competitive inhibition and non competitive inhibition, with competitive inhibition dominating.

Fluorescence spectroscopy is commonly used to study the interactions between small molecules and proteins. This experiment found that citrus flavonoids (naringin) can weaken the fluorescence spectrum intensity of trypsin, causing a red shift in the fluorescence peak and exhibiting static quenching. Analysis of binding constants and binding sites revealed that naringin has one binding site with trypsin, and the main binding forces are hydrogen bonding and van der Waals forces. In the experiment, it was also found that the binding constant Ka between naringin and trypsin decreased with increasing temperature, with 1.10x106L/mol at 20 ℃ (293K) and 1.04x105L/mol at 35 ℃ (308K), indicating stronger binding at low temperatures. This is consistent with the inhibition rate of citrus flavonoids on trypsin at different temperatures: the inhibition rate of citrus flavonoids on trypsin was 50.7% at 0.66mh/ml at 27 ℃, higher than 29.9% at 37 ℃ and 18.0% at 47%.

This study reveals the inhibitory effect, types, and binding mechanisms of citrus flavonoids on trypsin, which can serve as a reference for the development of special dietary and health foods related to excessive secretion of trypsin, in order to slow down the occurrence of diseases and assist in treatment.