Latin American Applied Research 42:211-216 (2012) STUDY OF THE INTERACTION OF GALANGIN, KAEMPFEROL AND QUERCETIN WITH BSA Z. D. FU†‡, X. Q. CHEN† and F. P. JIAO† †College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, P.R. China. ‡ Changsha Research Institute of Mining and Metallurgy, Changsha, Hunan 410012, P.R China [email protected] Abstract−− The interactions between Bovine Se- ing the fluorescence parameters, information concerning rum Albumin (BSA) and three flavonols, galangin, the structural changes in biomacromolecule can be ob- kaempferol and quercetin were studied by means of tained. There have been several studies on fluorescence fluorescence spectroscopy. The fluorescence intensity quenching of proteins induced by flavonoids and other of BSA exhibits remarkable decrease along with ap- polyphenols (Riihimaki et al., 2008; Xiao et al., 2008c). preciable blue-shift of its maximum emission wave- This paper studied the interaction between BSA and length upon addition of the three compounds, re- three flavonols, galangin, kaempferol and quenching by spectively. The respective binding constant Kα and fluorescence spectroscopy, and compared the difference number of binding sites of each compound were cal- of those reactions. culated, and the quenching mechanism was pro- II. MATERIALS AND METHODS posed. Based on the values of thermodynamic para- A. Apparatus meters, the binding of each compound proceeds The fluorescence spectra were recorded on a JASCO spontaneously with BSA. The binding distance be- FP-6500 spectrofluorometer equipped with a thermos- tween each and BSA was obtained by Foerster's di- tated compartment using 1.0cm quartz cuvette. The pH pole-dipole non-radiation energy transfer mechan- measurements were carried out on a PHS-3C Exact Dig- ism. ital pH meter, which was calibrated with standard pH Keywords−− Galangin, Kaempferol, Quercetin, buffers. flavonols, Bovine Serum Albumin, Fluorescence B. Reagents Spectroscopy. BSA (V, Sigma) diluted into 1.0×10-5 mol·L-1 as the re- I. INTRODUCTION serving solution; galangin, kaempferol, quercetin (Shan- The interaction between bio-macromolecules and drugs hai u-sea biotech co., ltd. purity>99%) were dissolved has attracted great interest among researchers since sev- into the mixture of methanol and water, whose volume eral decades (Xiang et al., 2008; Soares et al., 2007). ratio is 1: 1, then diluted into 1.0×10-4 mol L-1 as the re- Among bio-macromolecules, serum albumin is the most serving solution. Tris-HCl buffer (0.20 mol/L, pH 7.4) abundant protein in the circulatory system of man or an- containing 0.10 mol/L NaCl was selected to keep the pH imal, which carries plenty of drugs to all places of the value and maintain the ionic strength of the solution. All body (Malonga et al., 2006). The drug–protein interac- the reagents used in this experiment were analytical tion may result in the formation of a stable protein–drug grade, and the water was newly doubly-distilled and complex, which has important effect on the distribution, deionized. free concentration and the metabolism of drug in the C. Fluorescence spectrum analysis blood stream (Xiao et al., 2008a). Therefore, studies on 1.0 mL BSA solution and the volume of flavonol solu- the binding of drug with protein will facilitate interpre- tion indicated in Fig. 1 were added into 10 mL volume- tation of the metabolism and transporting process of tric flasks respectively. After diluting to 10 mL with drug, and will help to explain the relationship between deionized water and mixing thoroughly, each flask was structures and functions of protein. kept in a constant temperature water bath at 290 K, 300 The interaction between molecules including hydro- K and 310 K for 1h. Fixed λex at 280 nm, each solution gen bonding, ionic and van der Waals interactions (Jiao was scanned to determine the fluorescence intensity in et al., 2009). Protein–drug interactions play an impor- the range of 290nm to 450 nm. Then absorption spec- tant role in a variety of biological processes (Fuentes et trum of each compound solution (1.0×10-6 mol·L-1) was al., 2007). scanned in the range of 250 nm to 400 nm. Flavonoids have been suggested to have several po- tential health benefits due to their antioxidant activities, III. RESULTS AND DISCUSSION which are attributed to the presence of phenolic hydrox- A. Characteristics of fluorescence spectra yl moieties on the structure (Keli et al., 1996; Knekt et Fluorescence quenching spectra of BSA in the presence al., 1996). of various concentrations of galangin, kaempferol and Fluorescence spectroscopy is an appropriate method quercetin are shown in Fig.1. With the increase of the to determine the interaction between small molecules concentration of galangin, kaempferol and quercetin, the and biomacromolecules (Xiao et al., 2008b). By analyz- fluorescence of BSA were quenched regularly, and the 211 Z. D. FU, X. Q. CHEN, F. P. JIAO concentration of the quencher. Dynamic quenching was assumed as the type of the fluorescence quenching between three flavonols and BSA, so the process will accord with the equation above. Figure 2 shows the Stern-Volmer curves in dif- ferent temperature plotted with F0/F as ordinate against [Q] as abscissa. The figure shows the curves were li- near, and with the temperature increased, all of the slopes decreased. According to the Stern-Volmer equa- tion, Kq and KSV were calculated and shown in Table 1. The results show that the KSV decreases with increas- ing temperature, indicating that the probable quenching mechanism of fluorescence of BSA by three flavonols is a static quenching procedure, resulting in forming fla- vonols-BSA complexs and the stability of the complex decreases with increasing temperature. Table 1: The quenching constants between BSA and flavones -1 Flavonols Temperature/ºC KSV/L mol Kq R Galangin 17 7.557×105 7.557×1013 0.995 37 5.933×105 5.933×1013 0.992 Kaempferol 17 7.254×105 6.854×1013 0.996 37 5.524×105 5.124×1013 0.998 Quercetin 17 6.147×105 5.869×1013 0.991 37 4.788×105 5.214×1013 0.996 Fig. 1: Effects of galangin (A), kaempferol (B) and Quercetin (C) on fluorescence spectra of BSA at 37 ºC.c (BSA) = 1.0× 10-6 mol·L-1, c (Flavonols ) = 1× 10-5 mol·L-1, a–k: 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0µl. maximum emission wavelengths of three flavonols have a slight blue shift. The results show that galangin, kaempferol and quercetin have a reaction with BSA, making a change of the microenvironment of at least one of the two indole rings in the BSA (Tian et al., 2004). Complexes would be generated from three flavo- nols and BSA, which have little or no fluorescence. B. Type of the fluorescence quenching The system of quenching can be divided into static quenching and dynamic quenching. The dynamic quenching is an interaction process between quencher and excited state molecule of the fluorescent substance, which follows the Stern-Volmer equation (Yan et al., 2005). The Eq. (1) represents the fluorescence intensity of the fluorescent substance. (1) F0 / F = 1+ Kqτ 0 []Q = 1+ KSV []Q , where F0 and F are the fluorescence intensities before and after the addition of the quencher, Kq the rate con- stant of bimolecular quenching, τ0 the average lifetime of the electronically excited state of BSA in the absence Fig. 2: Stern-Volmer curves of BSA quenched by galangin (a), kaempferol (b) and Quercetin (c). of quencher, KSV the kinetic quenching constant, [Q] the 212 Latin American Applied Research 42:211-216 (2012) Table 2: The binding constants of BSA with flavones (ΔH) and entropy change (ΔS), the model of interaction Flavonols Kα n Correlation coefficient between drug and biomolecule can be concluded (Ross Galangin 5.939×106 1.038 0.998 and Subramanian, 1981): (1) ΔH>0, ΔS>0, hydrophobic Kaempferol 6.733×106 1.121 0.996 forces; (2) ΔH<0, ΔS>0, electrostatic interactions. When 6 Quercetin 8.382×10 1.256 0.993 the change of temperature is not much, the ΔH can be Table 3: The thermodynamic parameters of the interactions regard as a constant. The thermodynamic parameters between BSA and flavones were calculated by the first law of thermodynamics and Flavonols ΔH (kJ·mol-1) ΔS (J·K-1) ΔG (kJ·mol-1) the binding constant of each compound at different tem- Galangin -3.552 118.3 -40. 2 perature is shown in Table 3. From the results shown in Kaempferol -3.552 120.1 -40.5 Table 3, it can be found ΔG of every compound is less Quercetin -3.552 123.5 -41.8 than zero, this shows the reaction is carried out by itself and hydrophobic forces is the major force. Table 4: The distance parameter of BSA with flavones E. Binding distance Flavonols J (cm3·Lmol-1) R (nm) E r (nm) 0 According to Foerster's dipole-dipole non-radiation ener- Galangin 1.95×10-14 2.78 0.36 3.38 gy transfer mechanism, in the similar concentration of Kaempferol 1.86×10-14 2.64 0.29 3.64 donor and acceptor condition, if the fluorescence spec- Quercetin 1.91×1014 2.73 0.31 3.75 trum of donor and UV of receptor have enough overlap, According to the literatures (Lien et al., 1999; Che- and the distance between them is less than 7nm, it is like- netal, 1996), for dynamic quenching, the lifetime of bio- ly to occur Non-energy radiation between donor and reci- logical macromolecules was generally about 1×10-8 s, pient, resulting in donor fluorescence quenching.