Study On The Interaction Of Synergistic Antioxidant Natural Polyphenols With Proteases

Natural active ingredients are widely used in agriculture,industry,food,pharmacy and other fields.As an important class of natural antioxidants,natural polyphenols have various biological activities such as anti-tumor,anti-viral,anti-bacterial,anti-inflammatory and anti-diabetic.A synergistic effect may be exhibited when two or more natural polyphenolic compounds are used in combination.Natural polyphenolic compounds have good application prospects as therapeutic drugs and food additives.They will inevitably bind with human protease after being taken into the human body,which will affect the biological activity of the active ingredient as well as the activity and stability of the enzyme.Studies of the binding of single and combined natural polyphenols with proteases can provide theoretical guidance for the use of natural polyphenols in functional foods and medicine.Based on the literature survey and the actual situation of our laboratory,a variety of spectral analysis,differential scanning calorimetry and molecular docking were used to study the interaction between natural polyphenols and human proteases.The activity and thermal stability of proteases,as well as the antioxidant activity,cytotoxicity and stability of active ingredients were also analyzed.This paper mainly includes the following three parts:Part Ⅰ: The single and combined binding of chlorogenic acid(CGA)and epigallocatechin gallate(EGCG)to lysozyme was investigated by fluorescence spectroscopy,UV–vis absorption spectroscopy,circular dichroism spectroscopy,dynamic light scattering and molecular docking.The thermodynamic parameters obtained by fitting the fluorescence data determined the static quenching mechanism of the binding of CGA and EGCG to lysozyme.During the spontaneous and exothermic binding of CGA and EGCG to lysozyme,electrostatic interaction was the main driving force for the binding of CGA to lysozyme,while hydrogen bonding and van der Waals forces played a major role for the binding of EGCG.CGA and EGCG competitively bound to the active site of lysozyme.The interaction of CGA and/or EGCG with lysozyme changed the secondary structure and the particle size of lysozyme,and inhibited the enzyme activity of lysozyme.Additionally,synergistic effects of CGA and EGCG in antioxidant activity and cytotoxicity were demonstrated.The maximum synergistic antioxidant effect and corresponding concentration of CGA and EGCG were obtained by response surface methodology.The effects of lysozyme on their activities such as antioxidant activity,cytotoxicity and stability were further studied.Part Ⅱ: The single and combined binding of caffeic acid(CA)and epicatechin gallate(ECG)to lysozyme was investigated by multispectroscopic methods(fluorescence spectroscopy,UV–vis absorption spectroscopy,Fourier transform infrared spectroscopy,circular dichroism spectroscopy,and dynamic light scattering),differential scanning calorimetry and molecular docking.The thermodynamic parameters of the binding of CA and ECG to the binary and ternary systems of lysozyme were obtained by the fitting of fluorescence data,and the static quenching mechanism of CA and ECG to lysozyme was determined.Hydrogen bonding,van der Waals forces and electrostatic interactions played a major role in the binding process.CA and ECG competitively bound with lysozyme in the ternary systems.The binding of CA and ECG to lysozyme affected the secondary structure and particle size of lysozyme,inhibited the activity of lysozyme and improved its thermal stability.Intracellular and extracellular antioxidant models and cytotoxicity experiments confirmed the synergistic antioxidant activity and cytotoxicity to He La cells.Finally,the presence of lysozyme enhanced the cellular antioxidant activity,cytotoxicity and stability of CA and ECG.Part Ⅲ: The single and combined binding of CGA/CA and gallic acid(GA)to trypsin was investigated by fluorescence spectroscopy,circular dichroism spectroscopy,dynamic light scattering,differential scanning calorimetry and molecular docking.The thermodynamic parameters of the CGA/CA/GA + trypsin binary system and the CGA/CA+ GA + trypsin ternary system were obtained by fitting the fluorescence data,and the static quenching mechanism of CGA,CA and GA on trypsin was determined.CGA/CA and GA competitively bound with trypsin in the ternary systems.The binding of CGA/CA/GA to trypsin was driven by enthalpy and changed the secondary structure of trypsin.In addition,the presence of CGA,CA and GA inhibited the enzymatic activity of trypsin and improved its thermostability.Antioxidant experiments showed that CGA/CA and GA had synergistic antioxidant activity,and the synergy of CA + GA was more obvious.The presence of trypsin enhanced the antioxidant activity of CGA and CA,but inhibited the antioxidant activity of GA.Finally,the results of stability experiment showed that the binary complex formed by CGA/CA/GA and trypsin had the strongest protective effect on polyphenols.