Study On The Inhibitory Effect And Mechanism Of Polyphenol Oxidase By Hesperetin And Diosmetin

Polyphenol oxidase（PPO）is widely present in vegetables,fruits and many other organisms.PPO catalyzes the oxidation of phenols to melanin.In the presence of O₂,PPO o-hydroxylates monophenol,which then oxidizes o-diphenol to dopa quinone,and finally forms melanin.This process is usually accompanied by the deepening of the system color.In the food industry,browning caused by PPO can reduce the nutritional value of food,and the color of browning can also affect the consumer’s desire to consume,making the food lose its commercial value and causing huge economic losses.At present,the common PPO inhibitors on the market,such as kojic acid and hydroquinone,have poor stability and are prone to side effects after long-term use.Flavonoids have a wide range of physiological activities,and they are cheaper and safer to use.Therefore,it has become an important direction for natural plant active ingredients to control enzymatic browning of fruits and vegetables and treat skin diseases such as pigmentation,to find natural PPO inhibitors with high efficiency,few toxic and side effects,and to explore their inhibition mechanisms.It can provide certain experimental basis and theoretical guidance for the development of new PPO inhibitors and their practical application in the food and pharmaceutical fields.In this study,the inhibitory activities of hesperetin and diosmetin against PPO were determined and systematically investigated their molecular mechanism using a variety of spectroscopic techniques combined with computer simulation.The combined effect index（CI）value was used to evaluate the combined inhibitory effect of hesperetin,diosmetin and kojic acid against PPO,and preliminatively explored their combined inhibitory mechanism.In addition,the effects of Na Cl concentrations and p H values on hesperetin inhibiting PPO,binding with PPO,and changing PPO’s conformation were evaluated,and the main results were as follows:1.Both hesperetin and diosmetin effectively inhibited PPO activity,whose IC₅₀values were 776.0±15.5μM and 302.9±1.82μM,indicating that the inhibitory ability of diosmetin was stronger than that of hesperetin.Hesperetin and diosmetin reversibly inhibited PPO activity,were mixed PPO inhibitors,their inhibition constants K_ivalues were 569.4±6.24μM and 327.9±2.46μM,their apparent coefficientαwere greater than 1,suggesting that they were more likely to bind to free PPO.2.MCR-ALS analysis confirmed that hesperetin/diosmetin bound with PPO through the non covalent interaction and formed PPO-hesperetin/diosmetin complex.Both of hesperetin and diosmetin quenched the endogenous fluorescence of PPO in a static manner.At the measured temperatures,the binding constants were all 10⁴orders of magnitude,indicating that both hesperetin and diosmetin had moderate binding strength with PPO.The n values at the experimental temperatures were all close to 1,indicating that there was only one or one class of hesperetin/diosmetin’s binding sites in PPO.The main driving force of hesperetin/diosmetin binding with PPO was hydrophobic interaction.Synchronous fluorescence results showed that hesperetin enhanced the polarity of the microenvironment around tryptophan residues in PPO,but had no effect on that of tyrosine residues.Diosmetin had little effect on the polarity of the microenvironment around tyrosine and tryptophan residues.Circular dichroism（CD）analysis showed that hesperetin/diosmetin induced the increase ofα-helix content,the decrease ofβ-folding and random curling contents,and the tightening of enzyme structure.Molecular docking results showed that hesperetin/diosmetin entered the hydrophobic cavity of enzyme,bound to the binuclear copper active center of the enzyme,and interacted with amino acids around the active center of PPO,induced the conformational change of PPO,prevented substrate from entering PPO’s active center,thus reducing the catalytic activity of PPO.The molecular dynamics simulation results showed that hesperetin/diosmetin reduced the stability and hydrophobicity of the enzyme,and enhanced the compactness of the enzyme structure.3.Combination index（CI）analysis showed that hesperetin combined with kojic acid had a synergistic inhibitory effect on PPO in the experimental concentration range,while diosmetin combined with kojic acid showed antagonistic effect at low concentration and synergistic effect at high concentration,and the combination of hesperetin and diosmetin showed antagonistic effect.The presence of hesperetin was beneficial to the interaction between kojic acid and PPO,and the binding constant of kojic acid and PPO increased slightly,K_avalue increased from（2.0±0.2）×10³L mol^-1to（2.9±0.1）×10³L mol^-1.In the presence of hesperetin（kojic acid）,kojic acid could still interact with PPO-hesperetin complex（hesperetin could still interact with PPO-kojic acid complex）through hydrogen bonds,hydrophobic forces,and van der Waals forces.Hesperetin（kojic acid）directly bound to the active center of PPO,occupied the binding site of the substrate on PPO,while kojic acid（hesperetin）bound near the hydrophobic cavity of PPO,hindered the entry of the substrate into the active center of PPO.Hesperetin and kojic acid inhibited the catalysis of PPO on substrates in different ways,and their combined use showed a synergistic inhibition on PPO.4.The effect of Na Cl concentrations and p H values on hesperetin inhibiting PPO activity,binding with PPO and changing PPO’s conformation were investigated.The changes in Na Cl concentrations and p H values did not change the type of fluorescence quenching of PPO by hesperetin,and the main driving force for their binding was still the hydrophobic interaction.The presence of Na Cl promoted the interaction between hesperetin and PPO in the reaction system,and increased the binding constant K_avalue of hesperetin and PPO.When the p H values increased（p H5.0→6.0→7.0→8.0）,the K_avalue decreased,and the binding affinity of hesperetin to PPO weakened.With the increase of Na Cl concentrations,theα-helix content of PPO decreased,theβ-fold and random coil contents increased,and the structure of the enzyme became more loose,which might facilitate hesperetin to enter the active center of PPO and bind with PPO.Changes in p H values also affected the molecular conformation of PPO.At p H 7.0,the contents ofα-helix andβ-fold in PPO were the highest（35.10%and 26.55%,respectively）,and the content of random coil was the lowest（26.75%）.With the increase of p H（p H 8.0）or decrease of p H（p H 5.0 and p H 6.0）,the contents ofα-helix andβ-fold in PPO decreased,and the content of random coil increased,indicating that either acidic or alkaline environment would loosen the structure of the enzyme.Regardless of the changes of Na Cl concentration and p H value in the reaction system,the addition of hesperetin increased theα-helix content of PPO,decreased the random coil content,and made the structure of PPO more compact.Molecular dynamics simulations showed that the addition of Na Cl made the PPO system more unstable,but the presence of hesperetin enhanced the stability of the system and weakened the effect of Na Cl on the stability of PPO-hesperetin complex.