Structure-affinity Relationship, Theory Model And Application Of Interaction Between Polyphenols And Serum Albumin

The interaction between bio-active compounds from Chinese medicine and proteins has attracted great interests among researchers,which can provide important information for their nutritional and medical effects on human health. The work in this thesis mainly concerns about the interaction between polyphenols and BSA.The flavonoids and resveratrol were selected to study the structure-affinity relationship,theory model and application about the interaction between small molecules and biomacromolecules.1.Analysis of interaction between puerarin and BSA by multispectroscopic methodThe interaction between puerarin and BSA is investigated by means of fluorescence spectroscopy,resonance light-scattering spectroscopy,infrared spectroscopy,and synchronous fluorescence spectra.The binding constants(K_a) between puerarin and BSA are 1.13×10⁴ L/mol(20℃),and 1.54×10⁴ L/mol (30℃),and the number of binding site(n) is 0.95±0.02.The experimental results shows that puerarin can be inserted into BSA and quenches the inner fluorescence by forming the puerarin-BSA complex.The addition of increasing puerarin to BSA solution leads to the gradual enhancement in RLS intensity, exhibiting the formation of the aggregate in solution.It is found that both static quenching and non-radiation energy transfer are the main reasons for the fluorescence quenching.The positive entropy change and enthalpy change indicate that the interaction of puerarin and BSA is driven mainly by hydrophobic interaction.The process of binding is a spontaneous process in which Gibbs free energy change is negative.2.Effect of hydroxyl groups in the ring B of flavonols on binding with BSAThe hydroxyl group in ring B of flavonols is a significant structural feature as free radical scavengers and antioxidants.In this paper,four flavonols (galangin,kaempferol,quercetin,and myricetin) with different hydroxyl groups in ring B are studied for their affinities for BSA by quenching the protein intrinsic fluorescence.From the spectra obtained,the quenching constants,the binding constants,and the number of binding sites are calculated.The hydroxyl groups in ring B of flavonols significantly affect the binding process;in general, the binding affinity increases with increasing hydroxyl groups in the ring B. The binding constants(K_a) are determined as:myricetin(4.54×10⁸ L/mol)＞quercetin(3.65×10⁷ L/mol)＞kaempferol(2.57×10⁶ L/mol)＞galangin(6.43×10⁵ L/mol).Addition of another hydroxyl group on the ring B can enhance the affinity for BSA by one order of magnitude.The hydrophobicity of these four flavonols is assessed by the partition coefficient values(K_ow) and the chromatographic retention factors(K') of these four flavonols are determined by HPLC.K' and lgK_ow are inversely proportional to lgK_a for flavonols with increasing hydroxyl groups on the ring B.These results showed that the hydrogen bond force plays an important role in binding flavonols to BSA. These results are also in agreement with the generally accepted structure -dependent free radical scavenger and antioxidant abilities of flavonols.3.Effect of glycosylation of flavonids on binding with serum albuminThe dietary sources of flavonoids are almost always glycosides,which in most cases are hydrolyzed to aglycones to produce effects.Four flavonoid aglycones(baicalein,quercetin,daidzein,and genistein) and their glycosides (baicalin,quercitrin,daidzin,and genistin) are studied for their affinities for BSA.The glycosylation of flavonoids affects the binding process;in general, the glycosylation decreases the binding affinity.Glycosidation could lower the affinity for BSA by one to three orders of magnitude depending on the conjugation site.For quercetin and quercitrin,the binding constants for BSA are 3.65×10⁷ and 6.47×10³ L/mol,respectively.For baicalein and baicalin,the binding constants are 4.54×10⁸ and 1.63×10⁶ L/mol,respectively.This result partly supports that the flavonoid aglycones are easier absorbed than flavonoid glycosides.The E_1/2 values of flavonoids are inversely proportional to the binding constants binding to BSA.Higher binding affinities with BSA are associated with higher anti-oxidant and free radical scavenger activities for this class of compounds.4.Investigate the mechanism of enhancement effect of EGCG on huperzine A inhibiting AChE activity by binding with BSA The mechanism of enhanced effect of(-)-epigallocatechin-3-gallate (EGCG) on huperzine A(Hup A) inhibiting acetylcholinesterase(AChE) was investigated by studying the binding process with BSA using multi-spectroscopic techniques.Hup A is a potent and reversible inhibitor of AChE and available currently in the market for Alzheimer's disease.EGCG is the main polyphenol in green tea and the most widely studied polyphenol for disease prevention.EGCG can enhance the inhibitory effect of Hup A on AChE and also can greatly prolong the inhibitory time.The fluorescence quenching and infrared spectra data indicate that there is a strong binding force between EGCG and BSA and Hup A hardly interacts with the main transport protein in blood.The binding constant(K_a) between EGCG and BSA was 2.96×10⁷ L/mol(37℃).In the presence of EGCG,Hup A can affect BSA.The synchronous fluorescence data shows that EGCG is close to Trp and Tyr residues and affects their microenvironment.After bound with EGCG,Hup A can affect BSA and Hup A remarkably affected the microenvironment of Tyr residues,but Hup A didn't affect the microenvironment of Trp residues. According to the above results,here showed the suggested mechanism of enhanced effect of EGCG on Hup A inhibiting AChE.At first,the EGCG-BSA complex forms.Hup A is suggested to bind to EGCG-BSA complex and locates in close proximity to the Tyr residues.The binding Hup A to EGCG also increases the affinity of EGCG for BSA through electron transfer.The enhanced transporting of Hup A in blood might be a cause of the enhanced effect of EGCG on Hup A inhibiting AChE.The binding constant(K_a) between GCG and BSA was 2.98×10⁵ L/mol(R=0.9930) and the number of binding site(n) was 1.22.The affinity of GCG to BSA is 100-times lower than that of EGCG for BSA.GCG and EGCG have the same structure;only the bond of C-2 is a epimer.5.A new model based on binomial distribution to analysis the interaction between drug and protein.A new model based on binomial distribution by means of fluorescence quenching was developed to gain insights into the interaction of small molecules with protein.Trans-resveratrol and BSA were used to test.The binding number maximum of Trans-resveratrol was determined to be 8.86 at 293.15 K,23.42 at 303.15 K and 33.94 at 313.15 K and the binding mechanism analyzed in detail.The apparent binding constants(Ka) between Trans-resveratrol and BSA were 5.02×10⁴(293.15 K),8.89×10⁴(303.15 K) and 1.60×10⁵ L/mol(313.15 K),and the binding distances(r) between Trans-resveratrol and BSA were 2.44,3.01,and 3.38 nm at 293.15,303.15,and 313.15 K,respectively.The negative entropy change and enthalpy change indicated that the interaction of Trans-resveratrol and BSA was driven mainly by van der Waals interactions and hydrogen bonds.The process of binding was a spontaneous process in which Gibbs free energy change was negative.The interaction of Trans-resveratrol and BSA was confirmed by synchronous fluorescence and FTIR spectra.