| Flavonoids are a group of natural active compounds that are abound in nature and possess various favorable biological effects in remedy of many diseases.Studies on the interactions between bioactive small molecules (especially such as flavonoids drugs) and biomacromolecules (such as serum albumin) are of important reference value for pharmacokinetics, biochemistry, pharmaceutics and clinic medicine, which can provide an important physical parameters and pharmaceuticals standard. Bovine serum albumin (BSA) was selected as the model protein in this paper. The interactions between eight flavonoids drugs (QU, MY, DQU, DMY, ECG, EGCG, EGC,GC) and BSA were investigated by the fluorescence spectroscopy and UV-visible absorption spectroscopy.This paper were performed on four sections as follows:(1)The interaction of dihydromyricetin (DMY) and BSA under simulated physiological conditions was investigated by means of fluorescence emission spectroscopy, UV-visible absorption spectroscopy and synchronous fluorescence spectroscopy, and the effect of metal ions on the combination was studied.The experimental results showed that DMY quenched the intrinsic fluorescence of BSA by static quenching mechanism.The quenching constant Ksv, binding constant Ka and binding sites n at 15℃,25℃and 37℃was calculated respectively; The effect of DMY on the BSA secondary structure was analysed, and the thermodynamic parameters indicated that the interaction was driven mainly by hydrophobic and electrostatic forces;The binding distance between BSA and DMY was determined as 3.43 nm based on Forster theory. The presence of Co2+, Cu2+,Ni2+and K+ increased the binding constants of DMY-BSA system, especially Cu2+, could enhance the affinity to BSA by 2 orders of magnitude.(2) Two flavonols (quercetin and myricetin) and their corresponding hydrides (dihydroquercetin and dihydromyricetin) were studied for their ability to bind with BSA by fluorescence spectroscopy under simulated physiological conditions.The effect of hydrogenation on ring C and the numbers of OH on ring B on the affinity for BSA was discussed. Experimental results showed that the other three flavonols could quench BSA fluorescence significantly, except for dihydroquercetin.The binding constants were 1.84×108(myricetin),3.82×107(quercetin), and 1.36×104 L.mol-1 (dihydromyricetin) respectively, which indicated that hydrogenation on ring C could lower the affinity to BSA by 4 orders of magnitude, while increasing one OH on ring B could enhance the affinity to BSA by 1 order of magnitude.(3) Selecting epicatechin gallate (ECG) as the research object, the effect of three different temperatures (17℃,27℃and 37℃)and four different metal ions (Co2+, Zn2+, Cu2+ and Ni2+) on binding with BSA was discussed by means of fluorescence spectroscopy under simulated physiological pH condition.The results showed that the presence of metal ions did not change the type of quenching, but enhance the quenching effect of ECG on the BSA fluorescence, and the main forces in the binding process were changed from the hydrophobic into electrostatic force; Ni2+, Cu2+ increased the binding constants of ECG-BSA system, while Co2+, Zn2+ decreased the binding constants.(4) To explore structure-affinity relationship, four catechins drugs (ECG, EGCQ EGC,and GC) were selected to investigate the binding laws with BSA by means of fluorescence spectroscopy. The results showed that the binding constant of EGCG was bigger than that of ECG, followed by EGC.EGC and GC are pairs of enantiomers, but the combination effects of GC with BSA was lower significantly. The reason may be that BSA is a chiral selector, and it is selective to chiral compounds. |
