Inhibition of chrysin on xanthine oxidase activity and its inhibition mechanism

International Journal of Biological Macromolecules 81 (2015) 274–282

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Inhibition of chrysin on xanthine oxidase activity and its inhibition mechanism Suyun Lin a , Guowen Zhang a,∗ , Yijing Liao b , Junhui Pan a a b

State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China College of Pharmacy, Nanchang University, Nanchang 330047, China

a r t i c l e

i n f o

Article history: Received 3 February 2015 Received in revised form 6 August 2015 Accepted 7 August 2015 Available online 11 August 2015 Keywords: Xanthine oxidase Chrysin Competitive inhibitor Inhibition mechanism Multispectroscopic methods Molecular simulation

a b s t r a c t Chrysin, a bioactive flavonoid, was investigated for its potential to inhibit the activity of xanthine oxidase (XO), a key enzyme catalyzing xanthine to uric acid and finally causing gout. The kinetic analysis showed that chrysin possessed a strong inhibition on XO ability in a reversible competitive manner with IC50 value of (1.26 ± 0.04) × 10−6 mol L−1 . The results of fluorescence titrations indicated that chrysin bound to XO with high affinity, and the interaction was predominately driven by hydrogen bonds and van der Waals forces. Analysis of circular dichroism demonstrated that chrysin induced the conformational change of XO with increases in ␣-helix and ␤-sheet and reductions in ␤-turn and random coil structures. Molecular simulation revealed that chrysin interacted with the amino acid residues Leu648, Phe649, Glu802, Leu873, Ser876, Glu879, Arg880, Phe1009, Thr1010, Val1011 and Phe1013 located within the active cavity of XO. The mechanism of chrysin on XO activity may be the insertion of chrysin into the active site occupying the catalytic center of XO to avoid the entrance of xanthine and causing conformational changes in XO. Furthermore, the interaction assays indicated that chrysin and its structural analog apigenin exhibited an additive effect on inhibition of XO. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Xanthine oxidase (XO) is a homodimer with each monomer containing one molybdenum molybdopterin (Mo-pt), one flavin adenine dinucleotide (FAD) and two distinct [2Fe–2S] centers [1]. It is a critical enzyme in purine catabolism, and a widely distributed protein in mammalian tissues. XO has been extensively studied from a biochemical perspective for many years [2]. Physiologically, XO is responsible for the final two steps of purine catabolism [3]: catalyzing hypoxanthine to xanthine and then to uric acid. The oxidation occurs in the Mo-pt center accompanying with generating reactive oxygen species (ROS), superoxide and hydrogen peroxide [4]. An elevated level of blood uric acid (hyperuricemia) is the underlying cause of gout, and the excessive deposition of uric acid in joints can induce painful inflammation. Moreover, hyperuricemia is related to some other diseases, such as obesity and high blood pressure [5]. The concomitant subversive free radicals are considered to be associated with inducing DNA damage [6], ischemia-reperfusion injury, heart attacks, stroke and renal hypoxia [7,8]. Thus, in order to inhibit the oxidation of xanthine and then to reduce the

∗ Corresponding author. E-mail address: [email protected] (G. Zhang). http://dx.doi.org/10.1016/j.ijbiomac.2015.08.017 0141-8130/© 2015 Elsevier B.V. All rights reserved.

production of uric acid, the therapeutic of inhibitors against XO has been proposed in the prevention of hyperuricemia and gout. Allopurinol, an effective XO inhibitor, is still the main drug for treating hyperuricemia, but it exhibits some side effects such as vasculitis, hypersensitivity syndrome, reactive oxygen species (ROS) induced diseases, Stevens–Johnson syndrome and renal toxicity [9]. Hence, searching for new XO inhibitors with safe and high potential is of great importance, not only for treating gout but also combating various complications. A number of XO inhibitors have been reported including purine analog, biphenyl derivative, aryl pyrazole derivative and flavonoid [10]. As a kind of natural inhibitors in fruits, vegetables, tea and other natural products with a variety of biological activities and low toxicity, flavonoids have attracted much attention in recent years. Early studies have identified several flavonoids with good inhibitory ability on XO. For example, Takahama et al. have reported that quercetin, a natural flavonoid inhibited XO activity by 50% in a competitive manner [11]. Luteolin, a kind of flavonoid which exists widely in fruits and vegetables, has been found to inhibit XO activity with an inhibition constant (Ki ) of (1.9 ± 0.7) × 10−6 mol L−1 [4]. Studies in vitro have suggested that apigenin as another kind of common flavonoid also possessed a stronger inhibition activity against XO with respect to xanthine compared with allopurinol, and their Ki values were (0.61 ± 0.31) × 10−6 mol L−1 and

S. Lin et al. / International Journal of Biological Macromolecules 81 (2015) 274–282

(0.34 ± 0.22) × 10−6 mol L−1 , respectively [1]. These results highlighted the potential of flavonoids in the treatment of gout. Thus, as a flavonoid with analogous structure to apigenin, chrysin has also been investigated for its potential inhibition on XO. Chrysin is a main active ingredient of food and natural plants, such as honey, passiflora and propolis [12] and Oroxylum indicum [13] which is one herbal medicine usually used in China and other Southeastern Asian countries. It has been shown to have multiple biological activities including anti-bactericidal, anti-inflammatory, antioxidant, anti-hypertension and anti-cancer effects [12]. Besides, >Costantino et al. have reported the XO inhibitory ability of chrysin with IC50 values of (1.41 − 1.82) × 10−6 mol L−1 [14], however, the study was limited to the enzymatic activity assay, unfortunately, no report is available on the inhibitory mechanism of chrysin against XO. Therefore, clarifying the inhibition mechanism of chrysin on XO activity may provide new insights into the applications of chrysin as a XO inhibitor. As flavonoid compounds, chrysin (5,7-dihydroxy flavone) and apigenin (4 ,5,7-trihydroxy flavone) possess similar structure and inhibitory ability on XO activity alone, their possible synergistic effect on the inhibition of XO activity attracted our attention. Theoretically, the possible favorable outcomes for synergism include: increasing the efficacy of the therapeutic effect, decreasing the dosage but increasing or maintaining the same efficacy to avoid toxicity, and providing efficient synergism against target [15]. Cai et al. have investigated the synergistic effect of the three phenolic acids in fermented oats, their results indicated that each binary combination of p-coumaric, ferulic and caffeic acid has a synergistic effect [16]. Earlier studies have also shown that hesperidin and diazepam have the synergy in terms of sedative and sleeping, the use of hesperidin may thus decrease the effective therapeutic doses of diazepam [17]. The present study was thus aimed to determine the inhibition mechanism and inhibition kinetics of chrysin on XO and the effect of apigenin on inhibition of XO activity by chrysin in vitro through UV–vis absorption measurements, fluorescence titrations, circular dichroism (CD) analysis and molecular docking studies. This study has the potential to provide guidance for the development of more potent XO inhibitors. 2. Materials and methods 2.1. Materials XO (Cat. No. X-4875, 35.7 units mL−1 ) from bovine milk and xanthine (Cat. No. X-0626) were both purchased from Sigma–Aldrich Co. (St. Louis, USA) and then prepared as stock solutions with 0.05 mol L−1 Tris–HCl buffer (pH 7.4). The stock solutions of XO (5.0 × 10−6 mol L−1 ) and xanthine (5.0 × 10−4 mol L−1 ) were both freshly prepared just before the experiments. Chrysin and apigenin (analytical grade) from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) were dissolved in absolute ethyl alcohol to prepare stock solution (5.0 × 10−3 mol L−1 ) respectively, and the final alcoholic concentrations in the experiments were less than 1% with no effect on the enzymatic structure and activity (assured by experiments). All stock solutions were stored at 0–4 ◦ C, all other reagents were of analytical reagent grade, and freshly ultrapure water (18.2 M cm) was used throughout the study by using Millipore Simplicity Water Purification System (Millipore, Molsheim, France). 2.2. Enzyme activity assay Assay of XO activity was carried out by ultraviolet–visible (UV–vis) spectroscopy as reported earlier [18] with minor

275

modifications by using a UV-2450 spectrophotometer (Shimadzu, Japan) with a 1.0 cm quartz cell. Briefly, a series of assay solutions prepared in Tris–HCl buffer (0.05 mol L−1 , pH 7.4) consisting of a constant concentration of XO (7.5 × 10−8 mol L−1 ) and various amounts of inhibitor solutions were incubated for 3 h at 37 ◦ C. Then the assay was initiated by adding the substrate xanthine (final concentration was 5.0 × 10–5 mol L−1 ) to the 2.0 mL reaction mixture. The absorbance of the mixtures was measured at 290 nm every 10 s at room temperature. Averages of three replicates are presented. The enzymatic activity assay without inhibitor was defined as 100%. Thus, the relative enzymatic activity (%) = (slope of reaction kinetics equation obtained by reaction with inhibitor)/(slope of reaction kinetics equation obtained by reaction without inhibitor) × 100% [19]. 2.3. Kinetic analysis for inhibitory type The same method as XO activity assay was used to determine the inhibitory type of chrysin against XO. The inhibition type was analyzed from the Lineweaver–Burk plot. For competitive inhibition, the Lineweaver–Burk equation can be written as: 1

v

=

Km Vmax



1+

[I] Ki

 1

[S]

+

1 Vmax

(1)

Secondary plot can be plotted from: app

Km

=

Km [I] + Km Ki

(2)

where Ki and Km denote the inhibition constant and Michaelis–Menten constant, respectively, their values can be calculated from above equations.  is the enzyme reaction rate in the absence and presence of chrysin. [I] and [S] are the concentraapp tions of inhibitor and substrate, respectively. Km represents the app apparent Michaelis–Menten constant. The secondary plot of Km vs. [I] is linearly fitted, assuming a single inhibition site or a single class of inhibition site [20]. 2.4. Fluorescence titrations Fluorescence titrations were conducted by a Hitachi spectrofluorimeter Model F-7000 (Hitachi, Japan) equipped with a thermostat bath and a 150 W xenon lamp to characterize the binding properties of chrysin with XO. The fixed concentration of XO (5.0 × 10−7 mol L−1 ) was titrated with varying concentration of chrysin from 0 to 3.0 × 10−5 mol L−1 in a total of 3.0 mL. After equilibrating for 5 min, the fluorescence spectra of the solutions were measured at different temperatures (298, 301 and 310 K) in the wavelength range of 300–500 nm upon excitation 280 nm. The widths of both the excitation and emission slits were set at 5 nm. The background fluorescence of the buffer (0.05 mol L−1 Tris–HCl, pH 7.4) was subtracted from the chrysin–XO complexes. In order to eliminate the possibility of re-absorption and inner filter effects in UV absorption, all the fluorescence data were corrected for absorption of excitation light and emitted light by the following relationship [21]: Fc = Fm e(A1 +A2 )/2

(3)

where Fc and Fm represent the corrected and measured fluorescence. A1 and A2 are the absorbance of chrysin at excitation and emission wavelengths, respectively. The competitive assays were operated by using allopurinol as a site marker. This assay was performed by adding various amount of chrysin to the allopurinol–XO mixture containing XO and allopurinol at the same concentration of 5.0 × 10−7 mol L−1 . The synchronous fluorescence spectra were measured with the

276

S. Lin et al. / International Journal of Biological Macromolecules 81 (2015) 274–282

excitation and emission wavelength interval () stabilized at 15 and 60 nm over the range of 270–370 nm. 2.5. Determination of energy transfer between XO and chrysin According to Förster’s non-radiative energy transfer theory [22], the energy transfer efficiency E, is not only related to the distance (r) between the donor and acceptor but also influenced by the critical energy transfer distance R0 . In this study, the concentrations of chrysin and XO were both fixed at 5.0 × 10−7 mol L−1 , the UV–vis absorption spectrum of chrysin and the emission spectrum of XO were recorded ranging from 300 to 500 nm.

Obtained results were expressed as means ± standard deviation (n = 3). One-way analysis of variance (ANOVA) was performed by using Origin 8.0 followed by multiple tests, in order to determine the significant difference at p < 0.05. 3. Results and discussion 3.1. Effect of chrysin on XO activity Fig. 1A shows the inhibition effects of three inhibitors (chrysin, apigenin and allopurinol, structure shown in the inset of Fig. 1A) on

2.6. Circular dichroism (CD) studies The CD spectra of XO in the absence and presence of chrysin were recorded in a wavelength range of 200–250 nm by a BioLogic MOS 450 CD spectrometer (Bio-Logic, Claix, France) with a 1.0 mm path length quartz cuvette at a speed of 120 nm min−1 under constant nitrogen flush. The concentration of XO was kept at 1.0 × 10−6 mol L−1 , and the molar ratios of chrysin to XO varied as 0:1, 1:1 and 2:1, respectively. All observed CD spectra were corrected for the buffer signal, and results were expressed as ellipticity in millidegrees. The contents of different secondary structures (␣-helix, ␤-sheet, ␤-turn and random-coil) of XO were calculated with CD spectroscopic data by the online SELCON3 program (http:// dichroweb.cryst.bbk.ac.uk/html/home.shtml) [23]. 2.7. Interaction analysis of chrysin with apigenin on XO activity The interaction analysis of chrysin with apigenin on XO activity was carried out according to a previous report [16] with slight modifications. Va and Vb represent the relative remaining enzymatic activity of XO in the presence of chrysin and apigenin, respectively. Vab denotes the relative enzymatic activity in the presence of chrysin–apigenin mixture. The values of Vab and Vc (the calculated value of Va × Vb ) were used to evaluate the interaction between chrysin and apigenin. When the values of Vab − Vc below −0.1 were considered as synergistic interaction (SY) which means they synergistically enhance inhibitory ability against XO with respect to xanthine, while its values between −0.1 and +0.1 were regarded as additive interaction (AD). 2.8. Molecular modeling Molecular modeling of the drug–XO complex was operated on docking program AutoDock4.2. It docked flexible ligand into a rigid protein conformationally and exhibited the detail of binding. The crystal structure of XO (PDB ID: 3ETR) was downloaded from the Protein Data Bank (http://www.rcsb.org/pdb), then all water molecules were removed and polar hydrogens and assigned Gasteiger charges were added. The 3D structure of ligand was depicted in Chem3D Ultra 8.0. A dimension grid box ˚ was defined to enclose the active site with (110 A˚ × 100 A˚ × 110 A) ˚ Finally, docking with the default a certain grid spacing (0.729 A). parameters except for changing the docking runs to100 times. In the outcomes of docking, the best-scoring docked model (the lowest docking energy) of the ligand was selected to represent its most favorable binding mode predicted by this program. The outputs were further manifested by using the PyMol [19]. 2.9. Statistical analysis The effect of chrysin on XO was conducted three times with three different sample preparations. All data were analyzed using SAS statistical package (version 8.0, SAS Institute, Cary, NC, USA).

Fig. 1. (A) Inhibitory effects of chrysin, apigenin and allopurinol on XO activity (pH 7.4, T = 298 K). c(XO) = 7.5 × 10−8 mol L−1 and c(xanthine) = 5.0 × 10−5 mol L−1 . Their structures were shown in the inset. (B) Plots of  vs. [XO]. c(xanthine) = 5.0 × 10−5 mol L−1 , and c(chrysin) = 0, 1.0, 2.0, 5.0 and 10.0 × 10−6 mol L−1 for curves a → e, respectively. (C) Lineweaver–Burk plots. c(XO) = 7.5 × 10−8 mol L−1 , and c(chrysin) = 0, 1.0, 2.0 and 3.0 × 10−6 mol L−1 for curve a → d, respectively; the inset was the secondary plot of slope vs. [chrysin]. Values are expressed as the mean ± SD (n = 3).

S. Lin et al. / International Journal of Biological Macromolecules 81 (2015) 274–282

277

XO activity. All the inhibitors effectively inhibited XO activity in a dose-dependent manner (p < 0.05). The IC50 (loss of 50% enzymatic activity) values of allopurinol (positive control) and apigenin were (2.93 ± 0.02) × 10−6 and (3.57 ± 0.03) × 10−6 mol L−1 , respectively, while the IC50 value of chrysin was (1.26 ± 0.04) × 10−6 mol L−1 which was nearly similar with that of previous report [14]. Among them, chrysin exhibited the highest inhibitory effect compared to that of the other two inhibitors (p < 0.05) at concentrations over 0.25 × 10–6 mol L−1 . Structurally, both chrysin and apigenin possess two-benzene rings, which has been proved to be very important in the inhibition on the activity of XO [24], whereas apigenin possesses an extra C-4 hydroxy which might be the main reason of causing different inhibition from chrysin. Apigenin exhibited a lower inhibitory ability than chrysin, suggesting that the addition of a 4 hydroxy has negative effect on IC50 value, which could be explained by the destabilization of polar hydroxy of benzene ring B stretching into the hydrophobic region of active cavity of XO and resulting in lowering binding affinity [1,25]. Comparatively, the non-polar benzene ring B of chrysin was more stable in the hydrophobic cavity which was propitious to steady the binding of chrysin to XO finally resulted in a lower IC50 . 3.2. The competitive inhibition by chrysin The inhibitory type of chrysin against XO was analyzed by Lineweaver–Burk double reciprocal plots. As shown in Fig. 1B, the straight lines all passed through the origin but the slopes decreased with increasing concentrations of the inhibitor, indicating that chrysin inhibited XO activity reversibly [26]. The vertical axis intercept (1/Vmax ) of the double-reciprocal Lineweaver–Burk plots remained and horizontal axis intercept (−1/Km ) increased with rising concentrations of chrysin (Fig. 1C), which suggested that chrysin was a competitive inhibitor. The secondary plot (inset of Fig. 1C) was linearly fitted with Ki value of (5.70 ± 0.14) × 10−7 mol L−1 . It can be inferred that chrysin bound in a single class of inhibition sites on XO, to the same redox center as xanthine, which was suggested to be the molybdenum center [18]. 3.3. Quenching of XO fluorescence upon chrysin binding As the results of inhibition kinetics determined through above assays, the fluorescence experiments were performed to further investigate the binding mechanism of chrysin to XO. Fluorescence quenching means the abatement of the fluorescence intensity of a sample due to some processes, including many kinds of molecular interactions like excited state reactions, molecular rearrangements, energy transfer, ground state complex formation and collisional quenching [27]. When the excitation wavelength was set at 280 nm, the fluorescence emission at about 340 nm was mainly attributed to its intrinsic fluorescent amino acid residues (tryptophan, tyrosine and phenylalanine), fluorescence quenching was described by the Stern–Volmer equation [28,29]: F0 = 1 + KSV [Q] = 1 + Kq 0 [Q] F

(4)

where F0 and F represent the fluorescence intensities of XO in the absence and presence of the quencher, respectively, KSV is the Stern–Volmer quenching constant, Kq is the quenching rate constant of the biomolecule (Kq = KSV / 0 ). [Q] is the concentration of the quencher,  0 is the average lifetime of biomolecule in the absence of quencher, and its value is 10−8 s [28]. As shown in Fig. 2A, the fluorescence intensity of XO with maxima emission at 340 nm quenched gradually without obvious shift of the emission peak with increasing amounts of chrysin to XO solution, which is a direct evidence for the interaction between chrysin and XO. As shown in Fig. 2B and Table 1, with the increase of

Fig. 2. (A) Fluorescence spectra of XO in the presence of chrysin at various concentrations (pH 7.4, T = 298 K, ex = 280 nm). c(XO) = 5.0 × 10−7 mol L−1 , and c(chrysin) = 0, 3.0, 6.0, 9.0, 12.0, 15.0, 18.0, 21.0, 24.0, 27.0 and 30.0 × 10−6 mol L−1 for curves a → k, respectively; curve x shows the emission spectrum of chrysin only, c(chrysin) = 5.0 × 10−7 mol L−1 . (B) The Stern–Volmer plots for the fluorescence quenching of XO by chrysin at 298 K, 304 K and 310 K. Values are expressed as the mean ± SD (n = 3).

temperature, the KSV values decreased significantly (p < 0.05) and the values of Kq (2.24 × 1013 , 1.42 × 1013 and 0.89 × 1013 L mol−1 s−1 at 298, 304 and 310 K, respectively) were much higher than the maximum scatter collision quenching constant (2.0 × 1010 L mol−1 s−1 ) of various quenchers with biopolymers [30]. These results indicated that the fluorescence quenching mechanism of XO by chrysin was static quenching rather than dynamic quenching [31,32]. 3.4. Binding constant and binding site It is assumed that small molecules bind independently into a set of equivalent sites on a macromolecule. The binding constant Ka and the number of binding sites n can be calculated from the flowing equation [33]: log

F0 − F 1 = n log Ka − n log (F −F)[P ] F [Qt ] − 0 F t

(5)

0

where [Qt ] and [Pt ] denote the total concentrations of chrysin and XO, respectively. The values of Ka and n for the chrysin–XO complexation at the three temperatures were calculated (Table 1) from the intercept and slope of the regression curve of log(F0 − F)/F versus log 1/([Qt ] − (F0 − F)[Pt ]/F0 ). The high Ka values (in the order of 105 L mol−1 ) and the high linear correlation coefficient R suggested a strong affinity for the chrysin–XO interaction. The values

278

S. Lin et al. / International Journal of Biological Macromolecules 81 (2015) 274–282

Table 1 The quenching constants KSV , binding constant Ka and relative thermodynamic parameters for the interaction of chrysin with XO at different temperatures.a T (K)

KSV (×105 L mol−1 )

Rb

Ka (×105 L mol−1 )

Rc

n

H (kJ mol−1 )

G (kJ mol−1 )

S (J mol−1 K−1 )

298 304 310

2.24 ± 0.05 1.42 ± 0.03 0.89 ± 0.04

0.9914 0.9932 0.9858

7.98 ± 0.02 3.35 ± 0.04 1.01 ± 0.01

0.9983 0.9882 0.9897

1.04 1.15 1.13

−130.07 ± 0.12

−31.64 ± 0.02 −29.66 ± 0.02 −27.68 ± 0.02

−330.06 ± 0.04

a b c

Values are the means of triplicate assays. R is the correlation coefficient for the KSV values. R is the correlation coefficient for the Ka values.

Fig. 3. The overlap of fluorescence spectrum of XO (black) and ultraviolet spectrum of chrysin (blue) at 298 K. c(XO) = c(chrysin) = 5.0 × 10−7 mol L−1 . (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

of n were found to be close to one at different temperatures, implying that there was a single class of binding sites on XO for chrysin [34]. The competitive assays showed that the Ka value of the chrysin–XO complex decreased significantly from 7.98 × 105 to 3.00 × 104 L mol−1 in the absence and presence of allopurinol (5.0 × 10−7 mol L−1 ). Due to the high rate of decrease of Ka in the competitive assays as other competitive probes [35], it could be deduced that chrysin may possessed a matched ability to compete with allopurinol for the same binding site which was proved to be the active cavity of XO. Allopurinol with the same concentration had a little effect on the Ka of apigenin–XO complex (from 1.10 × 104 to 1.07 × 104 L mol−1 ), implying that apigenin could not compete the same binding site with allopurinol. These results further supported the result of a lower IC50 of chrysin than apigenin. 3.5. Thermodynamic analysis Determination of the thermodynamic parameters (enthalpy change H and entropy change S) can clarify the acting force of the chrysin–XO complexation. The values of H and S were determined from the van’t Hoff equation: log Ka = −

S H + 2.303RT 2.303R

G = H − T S

(6) (7)

Here Ka and R are the binding constant and the gas constant (8.314 J mol−1 K−1 ) respectively and the temperatures are 298, 304 and 310 K. Generally, hydrogen bonds, van der Waals forces, electrostatic forces and hydrophobic interactions are regarded as the main forces acting during non-covalent interactions between small molecules and macromolecules [36]. As shown in Table 1, the negative signal for G meant that the interaction of chrysin with XO was favorable and spontaneous. The negative values of H (−130.07 ± 0.05 kJ mol−1 ) and S (−330.06 ± 0.16 J mol−1 K−1 ) suggested that the binding was an exothermic process, and the

Fig. 4. Synchronous fluorescence spectra of XO in the absence and presence of chrysin c(XO) = 5.0 × 10−7 mol L−1 , and c(chrysin) = 0, 3.0, 6.0, 9.0, 12.0, 15.0, 18.0, 21.0, 24.0, 27.0 and 30.0 × 10−6 mol L−1 for curves a → k, respectively. (A)  = 15 nm, (B)  = 60 nm.

complexation was predominately driven by hydrogen bonds and van der Waals forces [19]. 3.6. Energy transfer between XO and chrysin According to Foster’s non-radiative energy transfer theory, the distance r between XO (donor) and bound-drug chrysin (acceptor) could be determined as previously described. The overlap of the UV–vis absorption spectrum of chrysin with the fluorescence emission spectrum of XO is shown in Fig. 3. For ligand–protein interaction, 2 = 2/3, N = 1.336 and ˚ = 0.207 [21]. The values of J (the overlap integral), R0 , E and r were calculated to be 3.577 × 10−14 cm3 L mol−1 , 3.33 nm, 0.208 and 4.16 nm,

0.02AD 0.12 0.14 −0.02AD 0.19 0.17 −0.04AD

c

Values are the means of triplicate assays. SY = synergistic interaction. AD = additive interaction. a

b

0.21 −0.05AD 0.30 0.25

0.25

0.01AD 0.19 0.20 −0.04AD 0.30 0.26 −0.09AD 0.30 −0.06AD 0.47 0.41

0.39

−0.01AD 0.25 0.24 −0.06AD 0.39 0.33 −0.06AD 0.43 −0.08ADc 0.60 0.52

0.49

−0.03AD 0.30 0.27 −0.08AD 0.47 0.39 0.72

Chrysin (5.0 × 10−7 mol L−1 ) Chrysin (1.0 × 10−6 mol L−1 ) Chrysin (1.3 × 10−6 mol L−1 ) Chrysin (2.0 × 10−6 mol L−1 )

0.60

0.60 0.53 −0.12SYb

−0.07AD

Vab − Vc Expected (Vc ) Observed (Vab ) Vab − Vc Observed (Vab ) Vab − Vc Expected (Vc )

The synchronous fluorescence spectroscopy can provide information about the molecular environment in the vicinity of the chromophore groups (tyrosine and tryptophan) of XO [38]. As shown in Fig. 4A and B, both of the synchronous fluorescence intensities of tyrosine ( = 15 nm) and tryptophan ( = 60 nm) residues decreased remarkably with the addition of chrysin to XO solution, the maximum emission wavelengths showed unchanged and a visible red-shift (from 284 to 288 nm), respectively, suggesting that the microenvironment of tyrosine residue had no obvious change, while the polarity increased and the hydrophobicity decreased around tryptophan residue [39]. Further analysis of conformational changes of XO was conducted by CD measurements, a sensitive technique to detect the secondary structural change in protein. As shown in Fig. 5, the CD spectrum of XO exhibited two negative double humped peaks in the ultraviolet region at 208 and 222 nm, which were the characteristic peaks caused by the transition of ␲ → ␲ and n → ␲* of ␣-helix structure [40]. With the molar ratios of chrysin to XO increased from 0:1 to 1:1 and 2:1, the CD signal of XO was obviously enhanced. The calculated contents of ␣-helix and ␤-sheet from the CD spectroscopic data increased from 36.8% to 44.5%, 50.2%, and from 11.8% to 11.9%, 12.4%, while the ␤-turn and random coil structures decreased from 26.3% to 22.5%, 20.1%, and from 25.1% to 21.7%, 17.3%, respectively (Table 2). The results indicated that the interaction between chrysin and polypeptide chains of XO altered the hydrogen bond networks and the secondary structures of XO molecules. The increase of ␣-helix assumed that the more compact structure of XO might make it harder for substrate to locate into the active cavity and led to a lower enzymatic activity [18].

Table 3 Interaction effects of apigenin on the inhibition activity of chrysin against XO at different concentrations.a

3.7. Conformational changes

Interaction

respectively. The r value was lower than 8 nm, and 0.5R0 < r < 1.5R0 , suggesting that there was high probability of the energy transfer from XO to chrysin [19]. Furthermore, the larger r value in comparison to R0 further supported the presence of static quenching mechanism in the binding of chrysin to XO [37].

Observed (Vab )

25.1 21.1 17.3

Vab − Vc

26.3 22.5 20.1

Expected (Vc )

11.8 11.9 12.4

Observed (Vab )

36.8 44.5 50.2

Value

0:1 1:1 2:1

Value

Random coil (%)

Interaction

␤-Turn (%)

Value

␤-Sheet (%)

Apigenin (2.0 × 10–6 mol L−1 )

␣-Helix (%)

279

Apigenin (1.0 × 10−6 mol L−1 )

Molar ratio [chrysin]:[XO]

Apigenin (3.0 × 10−6 mol L−1 )

Table 2 The contents of secondary structures of free XO and chrysin–XO systems (CD spectra) at pH 7.4.

Expected (Vc )

Interaction

Fig. 5. The CD spectra of XO in the presence of increasing amounts of chrysin. c(XO) = 2.0 × 10−6 mol L−1 , the molar ratios of chrysin to XO were 0:1 (a), 1:1 (b) and 2:1 (c), respectively.

Value

Apigenin (10.0 × 10−6 mol L−1 )

Interaction

S. Lin et al. / International Journal of Biological Macromolecules 81 (2015) 274–282

280

S. Lin et al. / International Journal of Biological Macromolecules 81 (2015) 274–282

Fig. 6. (A) Cluster analyses of the AutoDock docking runs of chrysin with XO, the inset was the optimal binding free energies of chrysin and XO according to docking result (marked in red). (B) The results of molecule simulation, and the red box indicates the putative binding site of ligands; (a) the molecular of XO displaying the respective binding of chrysin (yellow carbons), apigenin (light pink carbons) and allopurinol (pink carbons) at the active pocket which marked in green; (b) predicted binding mode of chrysin docked into the active pocket of XO by molecule simulation. Chrysin (yellow carbons) and the hydrophobic residues (gray sticks) surrounding the chrysin inhibitor were displayed as stick structures and labeled with their residue numbers on a ribbon model background. Mo atom (indigo) was represented too. The dashed lines indicate hydrogen-bonding interactions; (c) a close view of the superposition of the docked structures of chrysin (yellow carbons) and allopurinol (pink carbons, blue nitrogen) in the active site of XO. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.8. Interaction of chrysin and apigenin on XO inhibitory effect The inhibitory effect of the combination of chrysin and apigenin on XO activity was determined, and four concentrations of chrysin (apigenin) were chosen based on their corresponding inhibitory effects (the relative enzymatic activities were 85%, 70%, 55% and 35%, respectively, Fig. 1). As shown in Table 3, the combination of chrysin and apigenin showed mainly additive interaction on inhibition of XO activity. Mostly, the differences of Vab and the corresponding Vc were between −0.1 and 0.1, which indicated that those two flavonoids exhibited only the additive effect on the inhibition of XO activity. Interestingly, when chrysin

(5.0 × 10−7 mol L−1 ) and apigenin (1.0 × 10−6 mol L−1 ) were added alone, Va and Vb were 0.85, therefore, Vc was calculated to be 0.72. However, the observed Vab was 0.60 when apigenin and chrysin were added together, the difference (Vab − Vc = −0.12) was below −0.1, suggesting that they had synergistic effect at this low concentration. These results indicated that the inhibition on XO exerted by chrysin could not be significantly altered by apigenin. Both chrysin and apigenin inhibited the activity of XO in a competitive manner by binding in the active cavity [25]. The additive interaction might be explained coupling with the result of molecule simulation. As displayed in Fig. 6Ba, chrysin and apigenin bound into the same active cavity (marked in green) of XO but with different

S. Lin et al. / International Journal of Biological Macromolecules 81 (2015) 274–282

dispositions, or rather, they had respective non-interfering regional location, both ultimately hampered the locating of substrate then represented separate inhibition toward XO. Comparatively, the location of chrysin was very close to the location of allopurinol that it might make allopurinol much harder to locate it, this result was consistent with the result of competitive assays. It was assumed that their different dispositions related to their structures might result in the different IC50 values. 3.9. Computational docking analysis The simulation docking was carried out to illustrate visually the interaction between chrysin and XO. According to the cluster analysis of the AutoDock docking runs of chrysin with XO (Fig. 6A), the optimal binding free energies (marked in red) is calculated and listed in the inset (Gbinding = −2.87 kcal mol−1 ). The binding energy was slightly higher than G (−7.56 kcal mol−1 ) obtained from above thermodynamic analysis at 298 K, which could be due to the lack of desolvation energy, because the Autodock was performed under simulation of vacuum environment [41]. The visual bindings of chrysin, apigenin and allopurinol into the active cavity (marked in green) of XO are depicted in Fig. 6Ba. The active cavity was a long and narrow channel leading to the active site (Mo-pt cofactor) of XO. Once the cavity was bound by an inhibitor, the channel and surrounding space were mostly plugged, which might result in blocking of the landing of xanthine (substrate) and ultimately preventing its oxidation. As shown in Fig. 6Bb, chrysin was located in the pocket surrounded by several amino acid residues (Leu648, Phe649, Glu802, Leu873, Ser876, Glu879, Arg880, Phe1009, Thr1010, Val1011, and Phe1013). These relative residues were all among the Mo-pt domain. Glu802 and Arg880 were proved to play key roles in the hydroxylation of substrate xanthine [42], suggesting that chrysin had the same binding region as the substrate. Theoretically, the two-benzene rings of chrysin were thought to represent aromatic interactions (␲–␲ effects) with the benzene rings of Val1011 and Phe1013, which might be favorable for tight binding [43]. The ring B of chrysin was inserted into the hydrophobic cavity within residues Leu648, Phe649, Glu802, Leu873 and Phe1013, these hydrophobic interactions might also be propitious for stabilizing the cavity. C-5 hydroxyl and C-7 hydroxyl groups of the A ring and the ether group of C ring formed hydrogen bonds with active-site residues Thr1010, Glu879 and Ser876, respectively, indicating hydrogen bond was another main role in the binding of chrysin to XO, which confirmed the result in Section 3.5. The conjecture that C-5 and C-7 hydroxyl groups of flavonoid assisted in representing the inhibitory activity against XO has been supported by previous studies [1,24]. The docking result of superposition of chrysin and allopurinol in the active site of XO was shown in Fig. 6Bc. Allopurinol located in the same hydrophobic cavity as chrysin by interacting with relative amino acid residues. In contrast, chrysin interacted more extensively with surrounding environment in the active cavity via C-5 and C-7 hydroxyl groups, the ether group and the benzene ring B. Therefore, with all these interactions, the lower IC50 value of chrysin than allopurinol was observed (Fig. 1A). The docking superposition implied their rivalry of binding, which was consistent with the results of competitive fluorescence experiments. 4. Conclusions The result indicated that chrysin could inhibit efficiently the activity of XO and act as a potential natural substitute of allopurinol. Chrysin bound spontaneously into the active cavity of XO to form the chrysin–XO complex with one high-affinity binding site and the binding was mainly driven by hydrogen bonds and van der Waals forces. Chrysin statically quenched the intrinsic

281

fluorescence of XO. The increased compactness (increase in ␣-helix content) of XO induced by chrysin was considered to reduce the landing of substrate [18], and caused the less hydrophobic environment of XO which was in turn favorable for the binding of hydroxyl group of chrysin. The molecule docking revealed that the related amino acid residues in active cavity were Leu648, Phe649, Glu802, Leu873, Ser876, Glu879, Arg880, Phe1009, Thr1010, Val1011 and Phe1013. Additionally, there was an additive effect between chrysin and its structural analog apigenin. However, it might be great helpful in treating gout or even other diseases if some of the synergistic compounds can be found by using this method. Then it will be possible to increase the efficacy of treating gout with less dosage of drugs. Further in vivo studies are required to evaluate the effectiveness of chrysin in decreasing uric acid concentration in the blood. This study has provided important insights into the inhibitory mechanism of chrysin on XO, which will be helpful for clinical applications of chrysin as a XO inhibitor and a food functional ingredient. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Nos. 21167013, 31460422 and 31060210), the Natural Science Foundation of Jiangxi Province (20142BAB204001 and 20143ACB20006), the Joint Specialized Research Fund for the Doctoral Program of Higher Education (20123601110005), the Program of Jiangxi Provincial Department of Science and Technology (20141BBG70092), the Research Program of State Key Laboratory of Food Science and Technology of Nanchang University (SKLF-ZZB-201305 and SKLF-ZZA-201302), and the Jiangxi Provincial Postgraduate Innovation Fund (YC2014S057). References [1] C.M. Lin, C.S. Chen, C.T. Chen, Y.C. Liang, J.K. Lin, Molecular modeling of flavonoids that inhibits xanthine oxidase, Biochem. Biophys. Res. Commun. 294 (2002) 167–172. [2] C. Enroth, B.T. Eger, K. Okamoto, T. Nishino, T. Nishino, E.F. Pai, Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: structure-based mechanism of conversion, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 10723–10728. [3] R. Hille, Structure and function of xanthine oxidoreductase, Eur. J. Inorg. Chem. (2006) 1913–1926. [4] J.M. Pauff, R. Hille, Inhibition studies of bovine xanthine oxidase by luteolin, silibinin, quercetin, and curcumin, J. Nat. Prod. 72 (2009) 725–731. [5] M. Mazzali, J. Hughes, Y.G. Kim, J.A. Jefferson, D.H. Kang, K.L. Gordon, H.Y. Lan, S. Kivlighn, R.J. Johnson, Elevated uric acid increases blood pressure in the rat by a novel crystal-independent mechanism, Hypertension 38 (2001) 1101–1106. [6] H. Xiong, Y. Chen, X. Zhang, H. Gu, S. Wang, An electrochemical biosensor for the rapid detection of DNA damage induced by xanthine oxidase-catalyzed Fenton reaction, Sens. Actuators B 181 (2013) 85–91. [7] M.E. Inkster, M.A. Cotter, N.E. Cameron, Treatment with the xanthine oxidase inhibitor, allopurinol, improves nerve and vascular function in diabetic rats, Eur. J. Pharmacol. 561 (2007) 63–71. [8] U.Z. Malik, N.J. Hundley, G. Romero, R. Radi, B.A. Freeman, M.M. Tarpey, E.E. Kelley, Febuxostat inhibition of endothelial-bound XO: implications for targeting vascular ROS production, Free Radic. Biol. Med. 51 (2011) 179–184. [9] P. Pacher, A. Nivorozhkin, C. Szabó, Therapeutic effects of xanthine oxidase inhibitors: renaissance half a century after the discovery of allopurinol, Pharmacol. Rev. 58 (2006) 87–114. [10] C.H. Wang, S. Li, The research progress of xanthine oxidase inhibitors, Foreign Med. Sci. (Sect. Pharm.) 33 (2006) 351–354. [11] U. Takahama, Y. Koga, S. Hirota, R. Yamauchi, Inhibition of xanthine oxidase activity by an oxathiolanone derivative of quercetin, Food. Chem. 126 (2011) 1808–1811. [12] P.C. Lv, T.T. Cai, Y. Qian, J. Sun, H.L. Zhu, Synthesis, biological evaluation of chrysin derivatives as potential immunosuppressive agents, Eur. J. Med. Chem. 46 (2011) 393–398. [13] A.L. Sun, Q.H. Sun, R.M. Liu, Isolation and purification of baicalein and chrysin from the extracts of oroxylum indicum by high speed counter-current chromatography, Chin. J. Anal. Chem. 34 (2006) S243–S246. [14] L. Costantino, G. Rastelli, A. Albasini, A rational approach to the design of flavones as xanthine oxidase inhibitors, Eur. J. Med. Chem. 31 (1996) 693–699.

282

S. Lin et al. / International Journal of Biological Macromolecules 81 (2015) 274–282

[15] T.C. Chou, Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies, Pharmacol. Rev. 58 (2006) 621–681. [16] S. Cai, O. Wang, M. Wang, J. He, Y. Wang, D. Zhang, et al., In vitro inhibitory effect on pancreatic lipase activity of subfractions from ethanol extracts of fermented oats (Avena sativa L.) and synergistic effect of three phenolic acids, J. Agric. Food Chem. 60 (2012) 7245–7251. [17] S.P. Fernández, C. Wasowski, A.C. Paladini, M. Marder, Synergistic interaction between hesperidin, a natural flavonoid, and diazepam, Eur. J. Pharmacol. 512 (2005) 189–198. [18] J.K. Yan, G.W. Zhang, J.H. Pan, Y.J. Wang, ␣-Glucosidase inhibition by luteolin: kinetics, interaction and molecular docking, Int. J. Biol. Macromol. 64 (2014) 213–223. [19] Y.J. Wang, G.W. Zhang, J.K. Yan, D. Gong, Inhibitory effect of morin on tyrosinase: insights from spectroscopic and molecular docking studies, Food Chem. 163 (2014) 226–233. [20] Y.X. Si, Z.J. Wang, D. Park, H.Y. Chung, S.F. Wang, L. Yan, et al., Effect of hesperetin on tyrosinase: inhibition kinetics integrated computational simulation study, Int. J. Biol. Macromol. 50 (2012) 257–262. [21] S. Bi, L. Yan, Y. Wang, B. Pang, T. Wang, Spectroscopic study on the interaction of eugenol with salmon sperm DNA in vitro, J. Lumin. 132 (2012) 2355–2360. [22] T. Föster, O. Sinanoglu, Modern Quantum Chemistry, Academic Press, New York, 1996, pp. 93–94. ˇ Podlipnik, N.P. Ulrih, Interactions of different [23] M. Skrt, E. Benedik, C. polyphenols with bovine serum albumin using fluorescence quenching and molecular docking, Food. Chem. 135 (2012) 2418–2424. [24] T. Hayashi, K. Sawa, M. Kawasaki, M. Arisawa, M. Shimizu, N. Morita, Inhibition of cow’s milk xanthine oxidase by flavonoids, J. Nat. Prod. 51 (1988) 345–348. [25] D.E. Van Hoorn, R.J. Nijveldt, P.A. Van Leeuwen, Z. Hofman, L. M’Rabet, D. De Bont, K. Van Norren, Accurate prediction of xanthine oxidase inhibition based on the structure of flavonoids, Eur. J. Pharmacol. 451 (2002) 111–118. [26] Y.X. Si, S. Ji, W. Wang, N.Y. Fang, Q.X. Jin, Y.D. Park, et al., Effects of boldine on tyrosinase: inhibition kinetics and computational simulation, Process Biochem. 48 (2013) 152–161. [27] J.H. Kim, R. Hille, Reductive half-reaction of xanthine oxidase with xanthine. Observation of a spectral intermediate attributable to the molybdenum center in the reaction of enzyme with xanthine, J. Biol. Chem. 268 (1993) 44–51. [28] W. Peng, F. Ding, Y.T. Jiang, Y. Sun, Y.K. Peng, Evaluation of the biointeraction of colorant flavazin with human serum albumin: insights from multiple spectroscopic studies, in silico docking and molecular dynamics simulation, Food Funct. 5 (2014) 1203–1217. [29] Z. Chen, D. Wu, Monodisperse BSA-conjugated zinc oxide nanoparticles based fluorescence sensors for Cu2+ ions, Sens. Actuators B 192 (2014) 83–91.

[30] G.W. Zhang, Y.D. Ma, L. Wang, Y.P. Zhang, J. Zhou, Multispectroscopic studies on the interaction of maltol, a food additive, with bovine serum albumin, Food Chem. 133 (2012) 264–270. [31] Y. Mu, L. Li, S.Q. Hu, Molecular inhibitory mechanism of tricin on tyrosinase, Spectrochim. Acta Part A 107 (2013) 235–240. [32] Y. He, P. Yin, H. Gong, J. Peng, S. Liu, X. Fan, S. Yan, Characterization of the interaction between mercaptoethylamine capped CdTe quantum dots with human serum albumin and its analytical application, Sens. Actuators B 157 (2011) 8–13. [33] Y.Q. Wang, H.M. Zhang, G.C. Zhang, S.X. Liu, Q.H. Zhou, H.Z. Fei, Z.T. Liu, Studies of the interaction between paraquat and bovine hemoglobin, Int. J. Biol. Macromol. 41 (2007) 243–250. [34] N. Shahabadi, M. Maghsudi, Z. Kiani, M. Pourfoulad, Multispectroscopic studies on the interaction of 2-tert-butylhydroquinone (TBHQ), a food additive, with bovine serum albumin, Food Chem. 124 (2011) 1063–1068. [35] Y. Teng, R. Liu, C. Li, Q. Xia, P. Zhang, The interaction between 4-aminoantipyrine and bovine serum albumin: multiple spectroscopic and molecular docking investigations, J. Hazard. Mater. 190 (2011) 574–581. [36] P.D. Ross, S. Subramanian, Thermodynamics of protein association reactions: forces contributing to stability, Biochemistry 20 (1981) 3096–3102. [37] S.S. Kalanur, J. Seetharamappa, V.K.A. Kalalbandi, Characterization of interaction and the effect of carbamazepine on the structure of human serum albumin, J. Pharm. Biomed. Anal. 53 (2010) 660–666. [38] Y. Teng, R. Liu, S. Yan, X. Pan, P. Zhang, M. Wang, Spectroscopic investigation on the toxicological interactions of 4-aminoantipyrine with bovine hemoglobin, J. Fluoresc. 20 (2010) 381–387. [39] J.Q. Lu, F. Jin, T.Q. Sun, X.W. Zhou, Multi-spectroscopic study on interaction of bovine serum albumin with lomefloxacin–copper (II) complex, Int. J. Biol. Macromol. 40 (2007) 299–304. [40] N. Shahabadi, M. Maghsudi, S. Rouhani, Study on the interaction of food colourant quinoline yellow with bovine serum albumin by spectroscopic techniques, Food Chem. 135 (2012) 1836–1841. [41] W.J. Hu, L. Yan, D. Park, H.O. Jeong, H.Y. Chung, J.M. Yang, G.Y. Qian, Kinetic, structural and molecular docking studies on the inhibition of tyrosinase induced by arabinose, Int. J. Biol. Macromol. 50 (2012) 694–700. [42] T. Nishino, K. Okamoto, B.T. Eger, E.F. Pai, T. Nishino, Mammalian xanthine oxidoreductase-mechanism of transition from xanthine dehydrogenase to xanthine oxidase, FEBS J. 275 (2008) 3278–3289. [43] K. Okamoto, B.T. Eger, T. Nishino, S. Kondo, E.F. Pai, T. Nishino, An extremely potent inhibitor of xanthine oxidoreductase crystal structure of the enzyme-inhibitor complex and mechanism of inhibition, J. Biol. Chem. 278 (2003) 1848–1855.