Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 151 (2015) 576–590

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Molecular interactions of flavonoids to pepsin: Insights from spectroscopic and molecular docking studies Hua-jin Zeng a, Ran Yang b,⇑, Huili Liang a, Ling-bo Qu b,c a

School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, PR China College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, PR China c School of Chemistry and Chemical Engineering, Henan University of Technology, Zhengzhou 450001, PR China b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 The inhibitory effects of 10 flavonoids

The complex was formed by non-covalent reactions between flavonoids (baicalein here) and pepsin, which resulted in the significant decrease in the fluorescence intensity of pepsin. The molecular docking study shows that flavonoids are located in the hydrophobic cavity of pepsin. Since the binding of flavonoids affected the microenvironment of the pepsin activity site, flavonoids caused the inhibition of pepsin activity.

on pepsin were measured in vitro.  Binding mechanisms were investigated by spectroscopic and docking methods.  Pepsin fluorescence was quenched via static quenching with r less than 7 nm.  The interaction of Pepsin with flavonoids occurred in the hydrophobic cavity.  The common residues lining the flavonoids in the catalytic site were investigated.

a r t i c l e

i n f o

Article history: Received 16 March 2015 Received in revised form 17 June 2015 Accepted 18 June 2015 Available online 20 June 2015 Keywords: Flavonoid Pepsin Interaction Fluorescence spectroscopy Molecular docking

a b s t r a c t In the work described on this paper, the inhibitory effect of 10 flavonoids on pepsin and the interactions between them were investigated by a combination of spectroscopic and molecular docking methods. The results indicated that all flavonoids could bind with pepsin to form flavonoid–pepsin complexes. The binding parameters obtained from the data at different temperatures revealed that flavonoids could spontaneously interact with pepsin mainly through electrostatic forces and hydrophobic interactions with one binding site. According to synchronous and three-dimensional fluorescence spectra and molecular docking results, all flavonoids bound directly into the enzyme cavity site and the binding influenced the microenvironment and conformation of the pepsin activity site which resulted in the reduced enzyme activity. The present study provides direct evidence at a molecular level to understand the mechanism of digestion caused by flavonoids. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction

⇑ Corresponding author. E-mail address: [email protected] (R. Yang). http://dx.doi.org/10.1016/j.saa.2015.06.059 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.

Pepsin, an enzyme expressed as a prototype of zymogen and pepsinogen and was released by the chief cells in the stomach to degrade food proteins into peptides, was the first animal enzyme

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to be discovered by Theodor Schwann in 1836 [1]. As an important digestive protease in the stomach, pepsin is responsible for the most of the digestive activities [2]. When the food enters the stomach, pepsin will not only digest the protein of the food, but also interact with ingredients of the food at the same time, and then its activity may be affected by these compounds. And what is worse, some adverse effects, such as hiccup singultation, nausea and vomiting, will be caused in this process. Therefore, in order to evaluate the toxicity and binding mechanism of these small molecules that enter the stomach through food and drug, recently several reports were investigated on the interactions between pepsin and some molecules [3–7]. Flavonoids are the important phytonutrient components that occur in edible plants, vegetables, fruits and plant-originated foodstuffs [8]. Therefore, flavonoids may be a class of natural compounds that people take daily through the consumption of plant food. Generally, the trace mount of flavonoids in food do not cause obviously indigestive symptoms. However, due to exhibiting broad pharmaceutical activities, several flavonoids were extracted from plants as the main component of drugs in clinic, such as Lpriflavone Tablets and Silybin Capsules. According to published results and the data on file with the manufacture of these drugs, some adverse effects on digestion would occur in some patients even if they took the normal dosage [9]. The reason for this might be due to indigestion caused by flavonoids. Therefore, in order to improve the safety of drug usage in clinical, it is very significant to investigate the inhibitory effect of flavonoids on pepsin and learn about the knowledge that whether the drug could interact with the pepsin, what the mechanism of this action was in this process. Recently, several public and scientific interests have been focused on the interactions of flavonoids with some proteins, such as lysozyme [10], human serum albumin [11], bovine serum

HO

2. Experimental 2.1. Reagents The pepsin was obtained from Sigma–Aldrich Chemical Co. (USA) and was used without further purification. Flavonoids were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (China) and dissolved in methanol to form a 1.0  103 mol L1 solution, which was used to determine the binding sites of flavonoids on pepsin. 0.2 mol L1 of Citric acid–sodium citrate buffer solutions containing 0.1 mol L1 NaCI were prepared to adjust the acidity of the system pH 2.0, which is the most common pH for pepsin digests. Water was purified with a Milli-Q purification system (USA). All the chemicals were of analytical-reagent grade and used without further purification.

OH

HO

O

albumin [12] and tyrosinase [13]. However, to the best of our knowledge, little concern was placed on the inhibition of flavonoids on the activity of pepsin and the bindings of them to pepsin. In order to reveal the reason of indigestion caused by flavonoid, in the present study the inhibitory effect of 10 flavonoids (including baicalein, apigenin, luteolin, keampferol, quercetin, morin, liquiritigenin, naringenin, daidzein and genistein, structures shown in Fig. 1) on pepsin was investigated in vitro. Moreover, to further reveal the mechanism of digestion caused by these flavonoids, the interactions between 10 flavonoids and pepsin were studied by multiple spectroscopic techniques and molecular modeling in this study. This study provides basic data for clarifying the binding mechanism of flavonoids with pepsin and is help for understanding the symptoms of indigestion after oral administration of some flavonoid-contained drugs.

O

OH

HO

O

OH

HO OH

O

OH

O

Baicalein

OH

Apigenin

O

Luteolin OH

HO

HO OH

O

OH

O

OH

OH

O

OH

Quercetin O

OH

HO

O

OH

OH

O

Naringenin HO

O

O

OH O

O

Morin

O

Daidzein

OH OH

O

Liquiritigenin HO

HO

OH

OH

Keamferol HO

O

HO

OH OH

O

Genistein Fig. 1. The molecular structures of the tested flavonoids.

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2.2. Pepsin activity measurement

100 90 80 Inhibitive rate (%)

The enzyme activity was detected by the method with some modifications [14]. 2.5  106 mol L1 of pepsin in buffer solution (pH 2.0) was mixed with various concentrations of flavonoid at 37 °C for 10 min, and then 1.0 mL of 5% bovine hemoglobin solution was added in. After 10 min, 5.0 mL of 5% trichloroacetic acid was added to terminate the reaction. The mixture was stood for 10 min, and then centrifuged at 4000 rpm for 15 min. After addition of 300 lL of Fehling’s solution and 3.0 mL of NaOH to the supernatant, the mixture was incubated at 37 °C for 15 min and then the value of OD660 was measured using a spectrophotometer. The activity of pepsin can be calculated by the following equation:

70 60 50 40 30 20 10 0

Inhibitive rate ð%Þ ¼ ðOD660 blank  OD660 sample Þ=OD660 blank  100

0

2

4

6 -5

8

10

-1

Concentration (×10 mol L )

2.3. Fluorescence measurements All fluorescence spectra were recorded on a Hitachi F-2500 fluorescence spectrophotometer with a 1.0 cm quartz cell. The experimental temperature was maintained by recycling water throughout the quartz cell. The excitation wavelength was 280 nm and the excitation and emission slit widths were set at 5 nm. The scan speed was 1200 nm min1 and photo multiplier tube (PMT) voltage was 600 V. The UV–vis spectrum was recorded on a Shimadzu UV-2450 spectrophotometer (Shimadzu, Japan) equipped with a 1.0 cm quartz cell. Synchronous fluorescence spectra of pepsin in the absence and presence of flavonoids were measured (Dk = 15 nm, kex = 250– 320 nm and Dk = 60 nm, kex = 250–320 nm, respectively). The excitation and emission slit widths were set at 5 nm. The scan speed was 1200 nm min1 and PMT voltage was 600 V. The three dimensional fluorescence spectra were performed under the following conditions: the emission wavelength range was selected from 270 to 500 nm, the initial excitation wavelength was set to 200 nm, and the scanning number was 15 with the increment of 5 nm. 2.4. Molecular docking Docking calculations were performed by using AutoDock 4.0. The structure of flavonoids was generated by Chemdraw Ultra 8.0 and the energy minimized conformation of flavonoids was obtained by Gaussian 03 software. Docking calculations were carried out on a pepsin model (PDB code 5PEP, http://www.rcsb.org/ pdb/home/home.do). With the aid of AutoDock, the ligand root of flavonoids was detected and rotatable bonds were defined. Essential hydrogen atoms and Kollman united atom type charges were added into the pepsin protein model. Docking simulations were performed using the local search method to search for the optimum binding site of small molecules to the protein. To recognize the binding sites in pepsin, blind docking was carried out and grid maps of 90 Å  100 Å  90 Å grid points and 0.375 Å spacing were generated. The AutoDocking parameters were used as following: GA population size: 100; maximum number of energy evaluations: 250,000. The conformation with the lowest binding free energy was used for further analysis. 3. Results and discussion 3.1. Effect of flavonoids on pepsin activity In order to reveal whether flavonoids can affect on the activity of pepsin after they enter the organism through food and drug, the effect of flavonoids on the pepsin activity in vitro was investigated.

baicalein morin

apigenin liquritigenin

luteolin naringenin

keampferol daidzein

quercetin genistein

Fig. 2. The effect of flavonoids on pepsin activity in vitro.

As shown in Fig. 2, with the increasing of flavonoid concentration, the inhibitive rate was increased significantly and the 50% inhibitive rate (IC50) was calculated and summarized in Table 1. These results indicate that flavonoids can inhibit the activity of pepsin. 3.2. Fluorescence quenching In the past few years, fluorescence method has been widely applied to study the interaction of drugs and proteins and can provide abundant information about the binding parameters [15,16]. In order to investigate the binding of the 10 flavonoids to pepsin, the fluorescence emission spectra were recorded in the range of 300–500 nm upon excitation at 280 nm. Under these conditions, no fluorescence spectra of flavonoid itself were observed. Fig. 3 illustrated the fluorescence emission spectra of pepsin in the presence of various concentrations of flavonoids at 293 K. As shown in Fig. 3, it was found that the addition of flavonoids not only led to a significant reduction in the fluorescence signal, but a light wavelength shift of the maximum (kmax) occurred in some flavonoid– pepsin systems. Blue wavelength shifts of kmax were observed in keampferol–pepsin (from 340 to 335 nm), quercetin–pepsin (from 340 to 333 nm) and morin–pepsin (from 340 to 334 nm) systems, and red wavelength shifts were discovered in baicalein–pepsin (from 340 to 355 nm), liquiritigenin–pepsin (from 340 to 345 nm) and naringenin–pepsin (from 340 to 347 nm) systems. The shift in the position of emission maximum corresponds to the changes of the polarity around the chromophore molecule, suggesting that flavonoids have formed non-covalent compounds with pepsin, which could quench the fluorescence of pepsin. To confirm quenching process of flavonoids to the fluorescence of pepsin, the Stern–Volmer equation shown in Eq. (1) was applied [10]:

F 0 =F ¼ 1 þ K q s0 ½Q  ¼ 1 þ K sv ½Q

ð1Þ

where F0 and F are the fluorescence intensity in the absence and presence of quencher, respectively, [Q] is the concentration of the quencher, s0 is the fluorescence lifetime without the quencher and its value is 108 s [17], Kq is the quenching rate constant of bio-molecule and Ksv is the Stern–Volmer dynamic quenching constant. According to Eq. (1), the quenching constant of flavonoids to pepsin were calculated and listed in Table 2. It is well known that the maximum scatter collision quenching constant of various quencher with the biopolymer is 2.0  1010 L mol1 S1 [18]. Obviously, the rate constant of pepsin quenching procedure

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H.-j. Zeng et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 151 (2015) 576–590 Table 1 The effect of the tested flavonoids on pepsin activity in vitro (n = 3). Flavonoid

Equation

r

IC50 (mol L1)

Baicalein Apigenin Luteolin Keampferol Quercetin Morin Liquiritigenin Naringenin Daidzein Genistein

y = 11.653x  20.971 y = 7.2454x + 11.541 y = 4.3146x + 24.756 y = 5.3807x + 29.884 y = 4.3931x + 36.407 y = 6.4776x + 8.4415 y = 6.4051x + 9.8018 y = 5.3542x + 10.792 y = 0.0731x3  1.1127x2 + 5.6188x + 29.599 y = 0.1171x3  1.6166x2 + 7.9139x + 17.488

0.9770 0.9835 0.9701 0.9856 0.9946 0.9968 0.9830 0.9861 0.9974 0.9986

6.09  105 5.31  105 5.85  105 3.74  105 3.09  105 6.42  105 6.28  105 7.32  105 10.59  105 9.81  105

900

Baicalein

900

Apigenin

800 800

700 Fluorescence intensity

Fluorescence intensity

700

600 500 400 300 200

600 500 400 300 200

100

100

0 290

310

330

350

370

390

410

430

0

450

290

310

Wavelength(nm)

350

370

900

Luteolin

800

800

700

700 Fluorescence intensity

Fluorescence intensity

900

600 500 400 300

390

Keampferol

600 500 400 300

200

200

100

100 0

0 290

310

330

350

370

390

410

430

280

450

300

320

340

360

380

400

Wavelength (nm)

Wavelength (nm)

900

1000

Quercetin

800

900

700

800 Fluorescence intensity

Fluorescence intensity

330

Wavelength (nm)

600 500 400 300 200

Morin

700 600 500 400 300 200

100

100

0 290

310

330

350

370

390

Wavelength (nm)

410

430

0 290

310

330

350

370

390

410

430

Wavelength (nm)

Fig. 3. Effect of flavonoids on pepsin fluorescence. Conditions: peaks from up to down Cbaicalein = (0, 3.0, 6.0, 9.0, 12.0, 15.0, 20.0, 25.0, 30.0, 35.0)  106 mol L1, Capigenin = (0, 3.0, 6.0, 9.0, 12.0, 15.0, 18.0, 21.0, 25.0, 30.0)  106 mol L1, Cluteolin = (0, 2.0, 4.0, 6.0, 9.0, 12.0,15.0, 18.0, 20.0, 25.0)  106 mol L1, Ckeampferol = (0, 5.6, 11.1, 16.7, 19.4, 22.2, 27.7, 33.3, 38.8, 44.4)  106 mol L1, Cquercetin = (0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0)  106 mol L1, Cmorin = (0, 5.0, 10.0, 15.0, 20.0, 25.0,30.0, 35.0, 40.0, 45.0)  106 mol L1, Cliquiritigenin = (0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0)  106 mol L1, Cnaringenin = (0, 3.0, 5.0, 8.0, 11.0, 14.0,17.0, 20.0, 25.0, 30.0)  106 mol L1, Cdaidzein = (0, 10.0, 15.0, 20.0, 25.0, 30.0,35.0, 40.0, 45.0, 50.0)  106 mol L1, Cgenistein = (0, 3.7, 7.4, 11.1, 12.9, 14.8, 18.5, 22.2, 25.9, 29.6)  106 mol L1; Cpepsin = 2.5  105 mol L1, T = 293 K.

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800

900

700

800 700

Fluorescence intensity

fluorescence intensity

Naringenin

Liquiritigenin

600 500 400 300 200

600 500 400 300 200 100

100

0

0 290

310

330

350

370

390

410

290

430

310

900

Daidzein

370

390

Genistein

800

700

700 Fluorescence intensity

Fluorescence intensity

350

900

800

600 500 400 300 200

600 500 400 300 200

100

100

0 290

330

Wavelength (nm)

Wavelength(nm)

310

330

350

370

390

0 290

310

Wavelength (nm)

330 350 Wavelength (nm)

370

390

Fig. 3 (continued)

Table 2 Stern–Volmer constants for the interaction of pepsin with flavonoids at different temperatures (n = 3). Flavonoid Baicalein Apigenin Luteolin Keampferol Quercetin Morin Liquiritigenin Naringenin Daidzein Genistein a b

T (K) 293 310 293 310 293 310 293 310 293 310 293 310 293 310 293 310 293 310 293 310

Equations F0/F = 0.2307[Q] + 0.9206 F0/F = 0.2258[Q] + 0.9186 F0/F = 0.5825[Q] + 0.8758 F0/F = 0.5203[Q] + 0.9086 F0/F = 0.5408[Q] + 0.9421 F0/F = 0.5308[Q] + 0.919 F0/F = 0.7264[Q] + 0.9415 F0/F = 0.6451[Q] + 0.9807 F0/F = 0.7187[Q] + 0.6927 F0/F = 0.5759[Q] + 0.7892 F0/F = 0.4096[Q] + 0.98371 F0/F = 0.3023[Q] + 0.9426 F0/F = 0.396[Q] + 0.8901 F0/F = 0.3721[Q] + 0.8967 F0/F = 0.3441[Q] + 0.7936 F0/F = 0.3240[Q] + 0.7767 F0/F = 0.3061[Q] + 0.8322 F0/F = 0.3282[Q] + 0.8055 F0/F = 0.3212[Q] + 0.9289 F0/F = 0.3453[Q] + 0.9314

Ksv (L mol1) 4

2.31  10 2.26  104 5.83  104 5.20  104 5.41  104 5.31  104 7.26  104 6.45  104 7.19  104 5.76  104 4.10  104 3.02  104 3.96  104 3.72  104 3.44  104 3.24  104 3.06  104 3.28  104 3.21  104 3.45  104

Kq (L mol1) 12

2.31  10 2.26  1012 5.83  1013 5.20  1013 5.41  1012 5.31  1012 7.26  1012 6.45  1012 7.19  1012 5.76  1012 4.10  1012 3.02  1012 3.96  1012 3.72  1012 3.44  1012 3.24  1012 3.06  1012 3.28  1012 3.21  1012 3.45  1012

Ra

SDb

0.9668 0.9696 0.9886 0.9956 0.9912 0.9840 0.9653 0.9386 0.9774 0.9664 0.9904 0.9941 0.9895 0.9835 0.9693 0.9797 0.9816 0.9778 0.9890 0.9894

0.14 0.15 0.09 0.05 0.07 0.10 0.14 0.23 0.03 0.02 0.07 0.06 0.09 0.12 0.21 0.26 0.10 0.12 0.05 0.05

The correlation coefficient. The standard deviation.

initiated by flavonoids were all greater than the Kq of the scatter procedure, which demonstrated that the above quenching were the static quenching resulted from the formation of non-covalent compounds between pepsin and flavonoids.

equilibrium between free and bound molecules could be represented by the following equation [19]:

3.3. Binding constant and binding sites

where [Qd] and [Qp] are the concentrations of drug molecule and protein, respectively. Ka is the binding constant. By plot of log (F0  F)/F versus log (1/{[Qd]  (F0  F) [Qp]/F0}), the number binding sites n and binding constant Ka of the interaction between

For the static quenching, when small molecules bind independently to a set of equivalent sites on a macromolecule, the

log ½ðF 0  FÞ=F ¼ n log K a  n log ð1=f½Q d   ðF 0  FÞ½Q p =F 0 gÞ

ð2Þ

H.-j. Zeng et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 151 (2015) 576–590

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Fig. 4. Overlapping of fluorescence spectra of pepsin (Cpepsin = 2.5  105 mol L1) with absorption spectra of flavonoids (Cflavonoid = 1.0  104 mol L1).

flavonoids and pepsin can be calculated and the results are summarized in Table 3. From Table 3, it can be found that the values of n at the experimental temperatures were approximately equal to one, indicating that the existence of just a single binding site in pepsin for each flavonoid during their interaction. The value of Ka is of the order of 104 L mol1, indicating that a strong interaction exists between each flavonoid and pepsin. Moreover, Ka (apigenin–pepsin) > Ka (naringenin–pepsin), Ka (daidzein-pepsin) > Ka (liquiritigenin–pepsin), Ka (genistein–pepsin) > Ka (naringenin–pepsin) and Ka (apigenin–pepsin) > Ka (baicalein–pepsin) at the same temperature indicate that the flavonoids with a C2,3 double bond, unsubstituted hydroxyl groups at C5, C7, C40 and a ketone group at position C4 can bind with pepsin more tightly. However, it can be seen from Ka (keampferol-pepsin) > Ka (luteolin–pepsin), Ka (luteolin–pepsin) > Ka (quercetin–pepsin) and Ka (luteolin–pepsin) > Ka (morin–pepsin) that the effect of hydroxyl group at C3 on this process was a matter of controversy [20,21]. These results were consistent with the antioxidant activity of flavonoids [22].

3.4. The nature of the binding forces The binding forces between small molecule and bio-molecule often include van der Waals forces, electrostatic forces, hydrophobic interactions and hydrogen bonds within the binding site [15]. According to the data of enthalpy change (DH°) and entropy change (DS°), the model of interaction between drug and biomolecule can be concluded: (1) DH° > 0 and DS° > 0, hydrophobic forces; (2) DH° < 0 and DS° < 0, van der Waals interactions and hydrogen bonds; (3) DH° < 0 and DS° > 0, electrostatic interactions [23]. To obtain those thermodynamic parameters between flavonoids and pepsin, the Eqs. (3–5) were used: 

lnðk2 =k1 Þ ¼ DH =Rð1=T 1  1=T 2 Þ 

DG ¼ RT ln k 



ð3Þ ð4Þ



DS ¼ ðDG  DH Þ=T

ð5Þ

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Fig. 4 (continued)

Table 3 The binding constant Ka and relative thermodynamic parameters of the flavonoid–pepsin systems (n = 3). Flavonoid

T (K)

Ka (L mol1)

n

Ra

SDb

DH° (kJ mol1)

DG° (kJ mol1)

DS° (J mol1 K1)

Baicalein

293 310 293 310 293 310 293 310 293 310 293 310 293 310 293 310 293 310 293 310

5.81  104 5.48  104 6.91  104 6.54  104 10.45  104 9.48  104 13.60  104 13.19  104 8.45  104 7.78  104 4. 18  104 4.16  104 3.97  104 3.77  104 5.58  104 5.09  104 4.18  104 4.16  104 5.71  104 5.06  104

0.99 0.97 1.02 1.21 0.82 1.09 1.22 1.21 0.91 0.90 1.18 1.41 1.27 1.33 0.93 1.02 1.56 1.43 1.06 1.14

0.9736 0.9713 0.9822 0.9311 0.9561 0.9680 0.9927 0.9959 0.9945 0.9811 0.9923 0.9894 0.9982 0.9863 0.9869 0.9499 0.9983 0.9969 0.9685 0.9669

0.15 0.19 0.09 0.18 0.10 0.10 0.06 0.11 0.05 0.08 0.05 0.07 0.03 0.07 0.08 0.08 0.02 0.03 0.09 0.10

1.19

33.58 34.86 31.41 32.66 29.64 30.68 27.14 28.58 28.58 29.54 27.14 28.58 26.72 28.12 32.03 33.29 25.67 27.29 26.40 28.70

108.69

Apigenin Luteolin Keampferol Quercetin Morin Liquiritigenin Naringenin Daidzein Genistein a b

1.90 2.69 2.41 0.31 2.41 2.61 0.55 2.28 4.09

73.40 90.45 85.31 105.73 85.31 82.27 105.63 95.00 104.06

The correlation coefficient. The standard deviation.

The thermodynamic parameters for the interaction of flavonoids with pepsin are shown in Table 3. The negative sign for DG° means that the interactions between flavonoids and pepsin are spontaneous processes. The electrostatic interactions are the main driving force in the binding of pepsin with baicalein, apigenin, luteolin, keampferol, quercetin, morin, liquiritigenin, and naringenin and hydrophobic interactions are playing major roles in daidzein and genistein binding to pepsin. However, as shown in Table 3, the value of DH° in quercetin–pepsin and naringenin– pepsin systems are close to zero, indicating that hydrophobic

interaction also play a very important role in the formation of these two complexes. Meanwhile, the different amounts of hydroxyls exist in molecular structure of flavonoids, thus the hydrogen bonding formation might also participate in the binding processes. 3.5. Energy transfer between flavonoid and pepsin According to Förster’s non-radiative energy transfer theory [24], the distance between donor and acceptor can be calculated by the following equation:

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where E is the energy transfer efficiency, R0 is the critical distance between the donor and acceptor when their transfer efficiency is 50%, K2 is the spatial orientation factor of the dipole, N is the refractive index of medium, U is the quantum yield of the donor, and J is the overlap integral of the fluorescence emission spectrum of the donor with the absorption spectrum of the acceptor (shown in Fig. 4), which can be calculated by the equation:

Table 4 The distance parameters of pepsin with flavonoids. J (cm3 moL1 L)

Flavonoid Baicalein Apigenin Luteolin Keampferol Quercetin Morin Liquiritigenin Naringenin Daidzein Genistein

15

9.89  10 1.72  1014 2.25  1014 2.47  1014 1.30  1014 1.59  1014 1.09  1014 3.99  1015 3.21  1015 5.44  1015

R0 (nm)

E

r (nm)

2.90 3.18 3.33 3.38 3.04 3.14 2.95 2.49 2.41 2.63

0.41 0.39 0.37 0.26 0.16 0.27 0.21 0.40 0.18 0.29

3.08 3.43 3.64 4.02 4.00 3.71 3.68 2.67 3.10 3.05

J ¼ RðFðkÞ  eðkÞ  k4  DkÞ=RðFðkÞ  DkÞ

E ¼ 1  F=F 0 ¼ R60 =ðR60 þ r6 Þ

ð6Þ

R60 ¼ 8:8  1025 K 2  U  N4  J

ð7Þ

300

where F(k) is the fluorescence intensity of the fluorescence donor at wavelength k, and e(k) is the molar absorption coefficient of the acceptor at wavelength k. In the present case, K2 = 2/3, N = 1.366, and U = 0.118 [25]. According to Eqs. (5–7), E, r, R0, and J of the flavonoid-pepsin interaction could be calculated and listed in Table 4. The values of R0 and r for flavonoids were all less than 7 nm, which implied that the non-radiative energy transfer from pepsin to flavonoids had occurred with high probability.

Baicalein (Δλ=15 nm)

800

250

Baicalein (Δλ=60 nm)

700 600

Fluorescence intensity

Fluorescence intensity

ð8Þ

200 150 100

500 400 300 200

50 100

0

0

260

270

280

290

300

310

250

260

Wavelength (nm)

280

290

300

310

Wavelength (nm)

Apigenin (Δλ=15nm)

500

270

Apigenin (Δλ=60 nm)

800 700 Fluorescence intensity

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Fig. 5. Synchronous fluorescence spectra of interaction between pepsin and flavonoids at room temperature. Conditions as same as Fig. 3.

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Fig. 5 (continued)

Moreover, the acquisition of 0.5R0 < r < 1.5R0 mean that the calculated results were well-predicted by Förster’s theory. The value of E was high, which indicated that the energy could be transferred from pepsin to flavonoids. As a consequence, the intrinsic fluorescence emission of pepsin would be quenched by the binding of flavonoids.

3.6. Conformational investigations Synchronous fluorescence spectra were developed in 1971 and were obtained by simultaneously scanning excitation and emission monochromators while maintaining a constant wavelength interval between them. When the D-value (Dk) between excitation and emission wavelength were set at 15 or 60 nm, the synchronous fluorescence gives the characteristic information of tyrosine (Tyr) or tryptophan (Trp) residues, respectively [26]. By investigating

the synchronous fluorescence spectra of Tyr and Trp residues, the conformational changes of pepsin could be explored. The synchronous fluorescence spectra of pepsin upon addition of flavonoids at Dk = 15 and 60 nm were shown in Fig. 5. As shown in Fig. 5, a weak shift can be observed when Dk = 15 nm and Dk = 60 nm, and at the same time, the fluorescence intensity of pepsin decreased regularly along with the addition of flavonoid. This implied that the binding of flavonoid to pepsin was mainly located close to Tyr and Trp residues. Considering the minor shift in the emission peaks, it can be concluded that the microenvironment surrounding Tyr and Trp residues undergoes a slight change in the presence of flavonoid. In addition, the minor shifts in the synchronous fluorescence spectra express a slight change in the conformation of pepsin upon binding with flavonoids. Three-dimensional fluorescence spectroscopy is another powerful method for providing conformational and structural information of protein [27]. The three-dimensional fluorescence spectra of

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Liquiritigenin (Δλ=15nm)

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Fig. 6. The three-dimensional CGenistein = 25.0  106 mol L1, Cpepsin = 2.5  105 mol L1.

fluorescence contour spectra of pepsin and flavonoid–pepsin systems. Conditions: Cbaicalein = Capigenin = Cnaringenin = Cluteolin = 20.0  106 mol L1, Cquercetin = 40.0  106 mol L1, Ckeampferol = Cmorin = Cliquiritigenin = Cdaidzein = 45.0  106 mol L1,

pepsin and flavonoid–pepsin systems were shown in Fig. 6. Peak 1 is the Rayleigh scattering peak (kem = kex), peak 2 (kem/kex = 280/340 nm) mainly reveals the spectral feature of Tyr and Trp residues. Because when pepsin is excited at 280 nm, it

primarily display the intrinsic fluorescence of Trp and Tyr residues, and the phenylalanine residue fluorescence can be negligible. In the absence of flavonoid, the relative fluorescence intensity of peak 2 and the stoke shift (kem – kex) of pepsin were 810 and 60 nm,

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Fig. 6 (continued)

respectively; after the addition of flavonoids, the relative fluorescence intensity of peak 2 and the stoke shifts have changed obviously in all flavonoid–pepsin systems, which indicated that the interactions between flavonoids and pepsin induced microenvironment and conformation changed in pepsin and this result was consistent with that of synchronous fluorescence spectra.

3.7. Molecular modeling investigation To identify the precise binding sites on pepsin, a molecular modeling investigation was performed to simulate the binding mode between pepsin and flavonoids. From the docking calculation, the lowest energy-ranked results of flavonoid–pepsin

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conformations were summarized in Table 5. It can be seen from Table 5 that the observed free energy change of binding for flavonoid–pepsin systems was not extremely close to the experimental data from Table 3. The reason for this result may be the differences between the X-ray structure of the pepsin in crystal and that of the aqueous system in this study. Generally, the total binding free energy can be divided into electrostatic energy and nonelectrostatic energy, such as hydrophobic,

polar and hydrogen. As shown in Fig. 7, all flavonoids were located in the hydrophobic cavity of pepsin and were surrounded by the hydrophobic and hydrophilic amino acid residues (shown in Table 6). Therefore, it can be concluded that the interactions between flavonoids and pepsin were mainly electrostatic forces and hydrophobic interactions in nature. These results were consistent with the results obtained from the thermodynamic parameter analysis.

Table 5 The lowest energy-ranked results of flavonoid–pepsin binding conformations. Flavonoid

Binding energy (kcal mol1)

Inhib constant (lM)

Ligand efficiency

Internal energy

Baicalein Apigenin Luteolin Keampferol Quercetin Morin Liquiritigenin Naringenin Daidzein Genistein

6.53 (27.33 kJ mol1) 6.57 (27.50 kJ mol1) 6.39 (26.75 kJ mol1) 6.57 (27.50 kJ mol1) 6.66 (27.88 kJ mol1) 6.13 (25.66 kJ mol1) 5.86 (24.53 kJ mol1) 5.83 (24.40 kJ mol1) 5.9 (24.70 kJ mol1) 5.98 (25.03 kJ mol1)

16.36 15.40 20.68 15.37 13.18 32.34 50.26 53.31 47.17 41.51

0.33 0.33 0.3 0.31 0.3 0.28 0.31 0.29 0.31 0.3

6.7 6.93 7.5 7.72 8.1 7.09 6.41 6.24 6.45 6.81

Fig. 7. Docked pose corresponding to the minimum energy conformation for flavonoid binding to pepsin cavity. Detailed illustration of the amino acid residues lining the binding site in the pepsin cavity. Green molecule displays flavonoids; broken lines display hydrogen bonds. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 7 (continued)

Table 6 The amino acid residues lining the binding site in pepsin cavity and hydrogen bonds between flavonoid and pepsin. Flavonoids

Baicalein Apigenin Luteolin Keampferol Quercetin Morin Liquritigenin Naringenin Daidzein Genistein

Amino acid residues lining the binding site and hydrogen bonds Hydrophobic amino acid

Hydrophilic amino acid

Hydrogen bond

Gly76, Ile213, Gly217, Ser219, Leu220, Met290, Val292, Ile301 Gly34, Asn37, Ile73, Tyr75, Gly76, Ile128, Ser129, Ala130, Tyr189, Gly217, Ile30, Gly34, Tyr75, Gly76, Phe117, Tyr189, Gly217, Leu220 Ile30, Gly34, Gly76, Tyr75, Phe111, Phe117, Ile120, Tyr189, Gly217, Leu220 Ile30, Gly34, Tyr75, Gly76, Phe111, Phe117, Ile120, Tyr189, Gly217, Leu220 Tyr75, Gly76, Ile120, Tyr189, Ile213, Gly217, Met290, Val292, Leu299, Ile301 Ile30, Gly34, Tyr75, Gly76, Gly78, Phe111, Leu112, Phe117, Ile120, Tyr189, Gly217 Ile30, Tyr75, Phe111, Phe117, Ile120, Gly217, Ser219, Leu220 Gly34, Ile73, Tyr75, Gly76, Ile128, Tyr189, Gly217 Gly34, Tyr75, Gly76, Ile128, Tyr189, Gly217

Thr77, Asp215, Thr218, Thr222, Glu288 Asp32, Ser35, Ser36, Thr74, Asp215, Thr218 Glu13, Asp32, Ser35, Thr77, Asp215, Thr218, Ser219 Glu13, Asp32, Ser35, Thr77, Asp215, Thr218, Ser219 Glu13, Asp32, Thr74, Thr77, Asp215, Thr218, Ser219 Asp32, Ser35, Thr77, Asp215, Thr218 Asp32, Thr77

Gly217 (1.9977 Å), Ser219 (1.9447 Å), Glu288 (1.6709 Å)

Glu13, Asp32, Ser35, Thr77, Thr218, Glu288 Asp32, Ser35, Thr74, Thr77 Ser35, Ser36, Asp32, Thr77, Asp215, Thr218

Asp32 (1.8544 Å), Glu288 (1.7079 Å)

It was reported that the catalytic site of pepsin was formed by two aspartate residues (Asp32 and Asp215), one of which had to be protonated, and the other deprotonated for the protein to be active [28]. As shown in Table 6, both two residues appeared in baicalein–, apigenin–, luteolin–, keampferol–, quercetin– and genistein–pepsin systems, however, only Asp32 residue appeared in morin–, liquiritigenin–, naringenin– and daidzein–pepsin systems. These results can be explained why the binding constant of the former six systems was larger than that of the latter four systems in the binding processes. In addition, due to the presence of several ionic and polar groups near the probe molecule, there was also considerable number of hydrogen bonds in flavonoid–pepsin systems. Therefore,

Ile128 (1.9095 Å), Asp215 (2.0511 Å) Glu13 (2.1360 Å), Asp32 (1.6854 Å), Gly34 (1.9878 Å), Ser219 (2.1041 Å), Glu13 (1.9260 Å), Asp32 (2.1851 Å), Gly34 (1.8082 Å), Asp215 (1.9501 Å), Glu13 (2.1371 Å), Asp32 (2.3384 Å), Gly34 (1.9804 Å), Ser219 (2.0050 Å), Asp32 (1.7936 Å), Thr77 (2.1695 Å), Asp215 (1.7527 Å) Tyr189 (2.2666 Å)

Gly76 (2.1137 Å), Thr77 (1.8501 Å), Ile128 (1.8483 Å) Asp32 (2.1439 Å), Gly34 (2.8586 Å), Thr77 (1.8065 Å), Asp215 (3.0830 Å), Ile218 (1.9646 Å)

hydrogen bonding was also an important force in the binding process. As shown in Table 6, in some flavonoid–pepsin systems one of the hydrogen bonds was formed with the Asp32 and/or Asp215 residues, which might be associated with the activity of pepsin.

4. Conclusion In this study, the interactions between 10 flavonoids and pepsin were investigated by a combination of experimental and computational methods. The results indicated that all flavonoid could effectively quench the fluorescence of pepsin via static quenching and could bind spontaneously with pepsin mainly through electrostatic

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forces and hydrophobic interactions, as well as hydrogen bonding. Synchronous and three-dimensional fluorescence spectra and molecular docking results indicated that conformation and microenvironment of pepsin were obviously changed in the presence of flavonoids. Because the binding of flavonoid affected the microenvironment of the pepsin activity site, the activities of pepsin were inhibited by flavonoids. All these experimental results and theoretical data in this study would be help for understanding the digestion caused by flavonoids. Acknowledgements We grateful acknowledge the financial support of the National Natural Science Foundation of China (U1304823). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2015.06.059. References [1] O.C. Aszmann, J. Reconstr. Microsurg. 16 (2000) 291–296. [2] A. Gole, C. Dash, M. Rao, M. Sastry, Chem. Commun. 4 (2000) 297–298. [3] Y.B. Huang, J. Yan, B.Z. Liu, Z. Yu, X.Y. Gao, Y.C. Tang, Y.Q. Zi, Spectrochim. Acta A 75 (2010) 1024–1029. [4] V. Boeris, Y. Micheletto, M. Lionzo, N.P. Silveira, G. Pico, Carbohydr. Polym. 84 (2011) 459–464.

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