Effects Of PH And Coexisting Substances On The Interaction Between 6-Gingerol And Bovine Serum Albumin Studied By Multi-Spectroscopic Techniques

6-Gingerol,the main active ingredient in the gingerol component of ginger,has various biological activities,such as anti-inflammatory,antibacterial,antioxidant and anti-tumor.Serum albumin is the most abundant carrier protein in plasma,which has the function of transporting and maintaining the normal osmotic pressure of the blood.Serum albumin can bind with most exogenous and endogenous substances in the blood to form complexes,playing a role in transportation,distribution and metabolism.However,blood is a multi-component complex system,such as environmental p H,metal ions and nanoparticles,which directly influence the interaction between small molecule active substances and serum albumin,thereby affecting their generation,development and elimination in the organism.In this paper,bovine serum albumin（BSA）,which is highly homologous to human serum albumin,was used as a model protein to investigate the mechanism of interaction between 6-gingerol and BSA at different p H values（p H 3.0,4.5 and 7.4）,and to clarify the quenching constants,binding constant,number of binding sites and the type of binding force between them.Meanwhile,the effects of coexisting metal ions(Ca²⁺,Cu²⁺,Fe³⁺,Mg²⁺,Mn²⁺,Zn²⁺)and nanoparticles（liposomes,silica nanoparticles,corn starch nanoparticles）on the interaction between 6-gingerol and BSA were analyzed under simulated physiological p H conditions to elucidate the mechanism of 6-gingerol binding to BSA in a multivariate complex system.The results of the study will help to understand the transport and distribution of 6-gingerol in vivo and provide some theoretical basis for the subsequent application of 6-gingerol in clinical medicine.The main findings of this thesis are as follows:1.The effect of different p H values on the interaction between 6-gingerol and BSA were studied by UV-vis,fluorescence,circular dichroism,fourier transform infrared spectroscopy and molecular docking techniques.The results showed that 6-gingerol interacted with BSA at p H 3.0,4.5 and 7.4,and the mechanism of 6-gingerol causing fluorescence quenching of BSA was mainly the static quenching due to the formation of ground state complex,and the binding affinity between them was influenced by the conformation of BSA in different p H environments.At p H 3.0 and 4.5,the main driving forces in the binding of 6-gingerol to BSA were hydrophobic interactions,whereas at p H 7.4,hydrogen bonding and van der Waals forces were the main forces maintaining the stability of the 6-gingerol-BSA complex.The binding of 6-gingerol to BSA resulted in an increase in theα-helix content of BSA and a change in the secondary structure,and the effect of 6-gingerol on the secondary structure of BSA was greater at p H 7.4.Furthermore,the results of molecular docking revealed that 6-gingerol was inserted into the hydrophobic pocket of the active site I of the BSA IIA substructural domain,leading to a change in the conformation of BSA.2.The effect of coexisting metal ions on the interaction of 6-gingerol with BSA was studied under physiological p H 7.4 conditions using UV-vis,fluorescence and circular dichroism spectroscopy.The results indicated that there might be a competitive binding relationship between metal ions(Ca²⁺,Cu²⁺,Fe³⁺,Mg²⁺,Mn²⁺,and Zn²⁺)and6-gingerol,and subsequently affected the binding of BSA to 6-gingerol.The presence of metal ions reduced the quenching efficiency of 6-gingerol on the endogenous fluorescence of BSA,but did not change the fluorescence quenching mechanism and the action force type of 6-gingerol on BSA.The binding constants for the interaction of6-gingerol with BSA increased in the presence of Cu²⁺and Fe³⁺,while those decreased in the presence of Ca²⁺,Mg²⁺,Mn²⁺and Zn²⁺.In addition,the presence of metal ions altered the effect of 6-gingerol on the conformation of BSA and the microenvironment of amino acid residues,promoting the unfolding of BSA molecular structure in the6-gingerol-BSA system,resulting in varying degrees of reduction in theα-helix content.3.The effect of coexisting nanoparticles on the interaction of 6-gingerol with BSA was investigated using UV-vis,fluorescence and circular dichroism spectroscopy under simulated physiological p H environments.The results showed that a new ternary complex was formed between different types of nanoparticles such as liposomes,silica nanoparticles,corn starch nanoparticles and 6-gingerol and BSA.The presence of nanoparticles hindered the interaction between 6-gingerol and BSA,resulting in decreased the quenching constant of 6-gingerol with BSA,weakened the binding and slightly reduced the number of binding sites,but the fluorescence quenching mechanism of 6-gingerol against BSA was still a static quenching.In addition,the addition of nanoparticles attenuated the effect of 6-gingerol on the polarity of the microenvironment surrounding tryptophan（Trp）residues,leading to a reduction in theα-helix content of BSA in the 6-gingerol-BSA system and inducing the restoration of BSA secondary structure.