Latin American Applied Research 42:211-216 (2012)

STUDY OF THE INTERACTION OF GALANGIN, AND 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 , 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 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 -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. the quenching constant of maximum diffusion collision The binding distance between galangin, kaempferol, 10 -1 -1 was generally 2.0×10 L·mol ·s , and the KSV was in- quercetin and BSA can be calculated with the equation: creased with the temperature of the system increased. 6 6 6 E = 1− F / F0 = R0 /(R0 + r ), (3) Obviously, the quenching rate constants (KQ) of three -8 where F0 and F are the fluorescence intensities with and flavonols were far more than 1×10 s, and KSV was de- creased with the temperature increased. This means that without the existent of receptor, r the distance between the quenching is static quenching procedure by forming the donor and receptor, R0 the critical distance when conjugates of adduct. energy transfer efficiency was 50%. The value of R0 can be calculated with equation C. Calculation of binding constant 6 −25 2 4 R0 = 8.8×10 K N ΦJ, (4) In the process of static quenching, the relation of 2 quenching constant, fluorescence intensity and concen- where K is dipole orientation factor, N the refractive tration of the quencher can be described as follow (Ne- index of the medium in the system, Φ the fluorescence methy and Scheraga, 1962): quantum yield of donor, J the integral area of the over- lapped spectrums between the fluorescence spectrums log[](F − F) / F = log Kα + n log []Q (2) 0 of donor and the ultraviolet absorption spectrum. J can where Kα is the binding constant and n the number of be calculated with the equation: binding sites of the biomacromolecule. According to the 4 J = (∑ F(λ)ε (λ)λ Δλ)(∑ F(λ)Δλ), (5) plot with the left side of the equation as ordinate against logarithm of [Q] as abscissa, the value of Kα and n were where F(λ) and ε(λ) are the fluorescence intensity of the obtained and shown in Table 2. The high value of Kα donor and the molar absorptivity when the wavelength indicates there is a strong interaction force between is λ. In most conditions, K2, N and Φ is respectively 2/3, three flavonols and BSA. With the number of hydroxy 1.336 and 0.118. on the B-ring of flavonols increased, Kα and n in- Figure 3 shows the fluorescence spectra of BSA and creased. These results show the hydroxy on the B-ring the ultraviolet absorption spectra of galangin, kaempfe- of flavonols will strengthen the binding capacity of the rol and quercetin. With the equations mentioned, J, R0, small molecules. So it can be guessed that the hydroxy E, r were calculated and shown in Table 4. The binding on the B-ring of flavonols participates in the reaction distance of the galangin, kaempferol, quercetin and and becomes the important groups. Some literature re- BSA is respectively 3.38 nm, 3.64 nm and 3.75 nm. All ports the 3'-OH and 4'-OH on the B-ring of flavonols the distances are less than 7 nm, this indicates there is will enhance the antioxidant and eliminate free radical an energy transfer between three flavonols and BSA, ability, which can be attributed to their high binding ca- and the static fluorescence quenching of BSA is caused pacity (Liu et al., 2010; Xiao et al., 2008a). by non-radiation energy transfer. D. Binding mode of the Galangin, Kaempferol and F. Conformation investigation Quercetin with BSA To explore the structural change of BSA by addition The binding force between the drug molecules and pro- of three flavonols, we measured the synchronous fluo- tein may involve hydrophobic forces, Van Der Waals rescence spectra (Fig. 4) of BSA with various concen- interactions, electrostatic interactions and hydrogen tration of three flavonols. Synchronous fluorescence is a bonds, etc. According to the data of enthalpy change kind of simple and effective means to measure the fluo- rescence quenching and the possible shift of the maxi-

213 Z. D. FU, X. Q. CHEN, F. P. JIAO

of energy transfer compared with tyrosine. To tyrosine residues, there is a slight blue shift of the emission wa- velength, which shows the interaction changes the mi- croenvironment of tyrosine to an embedding state. While to tryptophan residues, there is a slight red shift of the maximum emission wavelength, which shows the interaction changes the microenvironment of tryptophan to an exposed state.

IV. CONCLUSION In this paper, the interaction between three flavonols (galangin, kaempferol and quercetin) and BSA was stu- died by fluorescence spectroscopy. The experimental results indicated that the probable quenching mechan- ism of fluorescence of BSA by three flavonols is a static quenching procedure and the binding reaction is sponta- neous. The binding force is largely mediated by hydro- phobic forces. The results obtained from synchronous fluorescence spectra show that the structure of BSA mo- lecules is changed dramatically in the presence of three flavonols. The fluorescence intensity of BSA exhibits remarkable decrease along with appreciable blue-shift of its maximum emission wavelength upon addition of the three compounds, respectively.

ACKNOWLEDGEMENTS We wish to acknowledge the support given to this work by the China National Natural Science Foundation (project No. 20805058) and China Postdoctoral Science Foundation (Project No. 20080431023).

REFERENCES Chenetal, Z.Y., “Antioxidant activity of natural flavono- ids is governed by number and location of their aro- matic hydroxyl groups,” Chem. Phys Lipids, 79, 157–163 (1996). Fig. 3: The overlaps of the absorption spectra of galangin (a), Fuentes, M., N.J. Scenna, P.A. Aguirre and M.C. Mus- kaempferol (b), quercetin (c) UV and fluorescence spectra of -6 -1 -1 sati, “Anaerobic digestion of carbohydrate and pro- BSA. BSA, 1.0 × 10 mol· L ; Flavonols, 1.0 × 10-6 mol· L tein-based wastewaters in fluidized bed bioreactors,” mum emission wavelength λmax, relative to the altera- Lat. Am. Appl. Res., 37, 235-242 (2007). tion of the polarity around the chromophore microenvi- Jiao, F.P., X.Q. Chen, Z.D. Fu, Y.H. Hu and Y.H. ronment. Δλ, representing the value of difference be- Wang, “Synthesis and structural characterization of tween excitation and emission wavelengths, is an impor- L-(-)-malic acid pillared layered double hydrox- tant operating parameter. When the Δλ is 15 nm, the ides,” Lat. Am. Appl. Res., 39, 127-130 (2009). synchronous fluorescence spectrometry shows the spec- Keli, S.O., M.G.L. Hertog, E. Feskens and D. Krom- tral characters of tyrosine residues, and when the Δλ is hout, “Dietary flavonoids antioxidant vitamins and 60 nm, tryptophan residues are shown. When Δλ is set incidence of stroke: the Zutphen study,” Arch. In- at 15 or 60 nm the shift of the λmax and the fluores- tern. Med., 156, 637–642 (1996). cence quenching of BSA imply the alteration of polarity Knekt, P.J., R. Jarvinen, A. Reunanen and J. Maatela, microenvironment around Tyr or Trp residues and the “ intake and coronary mortality in Finland: state of drug binding to BSA. a cohort study, ” Br. Med. J., 312, 478–81 (1996). The obviously fluorescence quenchings in two situa- tions show three flavonols, tyrosine and tryptophan re- Lien, E., S. Ren, H. Bui and R. Wang, “Quantitative sidues can bind simultaneously (Fig. 4). However, structure-activity relationship analysis of phenolic when the Δλ is 60 nm, the fluorescence decline is much antioxidants,” Free Radic. Biol. Med., 26, 285–294 larger than 15 nm. It can be concluded that three flavo- (1999). nols are closer with tryptophan or have higher efficiency

214 Latin American Applied Research 42:211-216 (2012)

Fig.4: The synchronous fluorescence spectrometry of galangin (1, 2), kaempferol (3, 4), quercetin (5, 6) and BSA. Δλ=15nm (A), Δλ=60nm (B), [BSA]=1.0×10–6 mol·L–1; [galangin]=1×10–5 mol·L–1; a–f: 0,100,200,300,400,500 µL of galangin. [kaempfe- rol]=1×10–5 mol·L–1; a–f: 0,100,200,300,400,500 µL of kaempferol.

Liu, X., X.Q. Chen, J.B. Xiao, J.Y. Zhao, F, Jiao and X, Ross, P.D. and S. Subramanian, “Thermodynamics of Jiang, “Effect of nydrogenation on Ring C of Flavo- protein association reaction: forces contribution to nols on their Affinity for Bovine Serum Albumin,” stability,” Biochemistry, 20, 3096-3102 (1981). J. Solution Chem., 39, 533-542 (2010) Soares, S., N. Mateus and V. Freitas, “Interaction of Malonga, H.J., F. Neault and H.A. Tajmir-Riahi, Different Polyphenols with Bovine Serum Albumin “DNase I – DNA interaction alters DNA and protein (BSA) and Human Salivary r-Amylase (HSA) by conformations,” DNA Cell Biol., 25, 393-398 Fluorescence Quenching,” J. AgricFood Chem., 55, (2006). 6726–6735 (2007). Nemethy, G., H.A. Scheraga, “Structure of water and Tian, J.N., J.Q. Liu, X. Tian, Z. Hu and X.G. Cheng, hydrophobic in proteins,” J. Phys. Chem., 66, 1773– “Study of the interaction of kaempferol with bovine 1789 (1962). serum albumin,” J. Mol. Struct., 691, 197 – 202 Riihimaki, L.H., M.J. Vainio, J.M.S. Heikura, K.H. (2004). Valkonen, V.T. Virtanen and P.M. Vuorela, “Bind- Xiang, G.H., C.L. Tong and H.Z. Lin, “Nitroaniline ing of phenolic compounds and their derivatives to Isomers Interaction with Bovine Serum Albumin bovine and reindeer ß-lactoglobulin,” J. Agric. Food and Toxicological Implications,” J Fluoresc., 18, Chem., 56, 7721–7729 (2008). 671–678 (2008).

215 Z. D. FU, X. Q. CHEN, F. P. JIAO

Xiao, J., X. Chen, L. Zhang , S.G. Talbot, G.C. Li and M. Xu, “Investigation of the mechanism of enhanced effect of EGCG on huperzine A’s inhibition of ace- tylcholinesterase activity in rats by a multispectros- copic method,” J. Agric. Food Chem., 56, 910–915 (2008a). Xiao, J.B., X.Q. Chen, X.Y. Jiang, M. Hilczer and M. Tachiya, “Probing the interaction of trans-resveratrol with bovine serum albumin: a fluorescence quench- ing study with Tachiya model,” J. Fluoresc., 18, 671–678 (2008b). Xiao, J.B., M. Suzuki, X.Y. Jiang, X.Q. Chen and K. Yamamoto, M. Xu, “Influence of B-ring hydroxyla- tion on interactions of flavonols with bovine serum albumin,” J. Agric. Food Chem., 56, 2350–2356 (2008c). Yan, Z.Y., X.F. Shao and L.Yan, “Interaction between gatifloxacin and bovine serum albumin,” J. Chin. Pharm. Sci., 40, 33 – 37 (2005).

Received: November 11, 2009. Accepted: August 22, 2011. Recommended by Subject Editor Ana Lea Cukierman.

216