Investigation the interaction between procyanidin dimer and α-amylase: Spectroscopic analyses and molecular docking simulation

Accepted Manuscript Investigation the interaction between procyanidin dimer and αamylase: Spectroscopic analyses and molecular docking simulation

Taotao Dai, Jun Chen, Qian Li, Panying Li, Peng Hu, Chengmei Liu, Ti Li PII: DOI: Reference:

S0141-8130(17)35112-7 https://doi.org/10.1016/j.ijbiomac.2018.01.189 BIOMAC 9021

To appear in: Received date: Revised date: Accepted date:

21 December 2017 28 January 2018 29 January 2018

Please cite this article as: Taotao Dai, Jun Chen, Qian Li, Panying Li, Peng Hu, Chengmei Liu, Ti Li , Investigation the interaction between procyanidin dimer and α-amylase: Spectroscopic analyses and molecular docking simulation. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Biomac(2017), https://doi.org/10.1016/j.ijbiomac.2018.01.189

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ACCEPTED MANUSCRIPT Investigation the interaction between procyanidin dimer and α-amylase: Spectroscopic analyses and molecular docking simulation Taotao Daia, Jun Chena, Qian Lib, Panying Lia, Peng Hua, Chengmei Liua*, Ti Lia* State Key Laboratory of Food Science and Technology, Nanchang University, No.

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a

235 Nanjing East Road, Nanchang 330047, China

College of Bioengineering and Food, Hubei University of Technology, Wuhan

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b

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430068, China

Corresponding author:

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*Chengmei Liu

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E-mail address: [email protected]

Corresponding author:

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*Ti Li

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E-mail address: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT Procyanidins were reported to have an inhibitory effect on α-amylase, but the interaction mechanism between procyanidins and α-amylase was rarely reported. Spectroscopic and molecular docking techniques were utilized to explore the

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interaction between porcine pancreatic α-amylase (PPA) and B-type procyanidin

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dimer (PB2). PB2 decreased the intrinsic fluorescence and surface hydrophobicity of

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PPA, indicating that an interaction occurred and complex formed. The binding process of complex was spontaneous and the main interaction was hydrophobic force.

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Circular dichroism showed conformational changes of PPA with an increasing of

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α-helix and β-sheet structure. Molecular docking speculated that PB2 could form hydrophobic force with PPA by bind to the active sit (Asp 167, Asn 100, Arg 158, His

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201). This research can offer new insights into the mechanism of PB2 in inhibiting

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PPA catalysis and provide useful information on dietary recommendation of PB2 for the treatment of type 2 diabetes.

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Keywords: procyanidins; α-amylase; interaction.

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ACCEPTED MANUSCRIPT 1. Introduction Diabetes is one of the most significant public health issue in the world. According to the statistics from the International Diabetes Federation, Diabetes currently affects 425 million adults worldwide and is set to affect close to 700 million

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people by 2045 (IDF, 2017). Type 2 diabetes is the most prevalent among diabetes

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mellitus, and it has been linked to over consumption of foods rich in digestible

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carbohydrates, such as starch [1]. Generally, dietary carbohydrate digestion is mediated by digestive enzymes such as α-amylase which can rapidly convert

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digestible starch into maltose. Rapidly digested and absorbed dietary carbohydrates

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result in a rapid increase in the postprandial blood glucose level. For diabetic patients, the high blood glucose level after a meal face a challenge for managing

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meal-associated hyperglycemia. However, the dietary carbohydrate digestion and

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increase of the blood glucose concentration can be inhibited, once the carbohydrate digestive enzymes are inhibited.

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Currently, some anti-diabetic drugs available for inhibiting α-amylase, including

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acarbose, voglibose and miglitol, but these chemical drugs may cause undesirable side effects, such as adverse gastrointestinal symptoms and liver toxicity [2]. Thus, there is a considerable research interest in natural phytochemicals to inhibit the activity of α-amylase [3]. Studies have shown that polyphenolic compounds from plant-based foods or supplements have excellent inhibitory capacity to α-amylase [4]. Proanthocyanidins (PAs) are the most abundant phenolic compounds except lignin [5]. These phenolic compounds are oligomers and polymers of flavan-3-ol monomer units 3

ACCEPTED MANUSCRIPT (catechin/epicatechin, afzelechin/epiafzelechin, gallocatechin/epigallocatechin) and the linkage between the different units may be an A-type linkage (containing an ether bond in addition to the C–C bond) or a B-type linkage (C–C bond between monomers) [6, 7, 8]. The α-amylase inhibitory capacities of PAs from berry [9], longan pericarp

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[10], and sorghum [11], red rice [12] have been demonstrated. Numerous studies

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generally indicated that PAs with higher mean degree of polymerization (mDP) have

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better α-amylase inhibitory ability than that of lower mDP [13-15]. In addition, B-type oligomeric proanthocyanidins have also exhibited excellent inhibition of α-amylase

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[12, 16, 17]. However, these PAs extract are a mixture with different linking type and

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polymerization degree, the influence of PAs structure on to α-amylase inhibitory ability is not clear. The effect of other active compounds among PAs exact to

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α-amylase is also unclear. Hence, it is necessary to investigate how PAs bind to PPA

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and the interaction mechanism between PAs and PPA. In this research, the interaction between PPA (porcine pancreatic α-amylase) and

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PB2 compound (epicatechin unit and B-linked dimer) were investigated.

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Spectroscopic methods including fluorescence, circular dichroism, and ultraviolet in combination with molecular docking techniques were employed. This research can offer new insights into the mechanism of PB2 in inhibiting α-amylase catalysis and provide meaningful information on the dietary recommendation of PB2 for the treatment of type 2 diabetes.

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2. Materials and methods 2.1. Materials The porcine pancreatic α-amylase (PPA, Type VI-B, molecular weight: 51-54 KDa) and 1-anilinonaphthalene-8-sulfonic acid (ANS) were purchased from

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Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). B-type procyanidins dimer (PB2,

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CAS No: 29106-49-8, HPLC purity: 98.54%) was obtained from Aladdin Industrial

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Inc. (Shanghai, China). Phosphate buffer solution (PBS) was purchased from Solarbio

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(Beijing, China). PPA powder was dissolved in PBS (10 mmol/L, pH 6.9) to obtain stock solution (5 mg/mL)，and further diluted into 1 mg/mL. PB2 solution were also

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prepared in PBS (10 mmol/L, pH 6.9), and further diluted into various concentrations ranging from 0.05 to 1.73 mmol/L (0.05, 0.11, 0.21, 0.43, 0.86, and 1.73 mmol/L).

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The PPA and PB2 stock solutions were filtered through 0.45 μm filters (WondaDisc

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MCE, Shimadzu-gl) before analysis to remove any large particles. Ultrapure water

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was used throughout the study.

2.2. Fluorescence spectrum measurements

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The fluorescence spectrum at 298, 308 and 318 K were applied to estimate the interaction of PPA with PB2 using a fluorescence spectrofluorimeter (F-7000, Hitachi, Tokyo, Japan) equipped with 1.0 cm path-length quartz cell and thermostat bath. The formation of PPA-PB2 complexes was charactered by the fluorescence quenching method previously reported [18] with some modifications. The excitation wavelength was set at 295 nm with the width of the excitation and emission slit both adjusted at 2.5 nm and the emission spectra were collected from 300 to 450 nm. Briefly, 0.2 mL 5

ACCEPTED MANUSCRIPT of different concentrations (0.05-1.73 mmol/L) of PB2 solution were added into 3 mL 1 mg/mL PPA solution. The maximum fluorescence intensity was used to calculate the binding constant. All of the mixtures were held for 30 min to equilibrate before measurements. Fluorescence intensity was measured, and PB2 solution was used as

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blank to correct background of fluorescence. To exclude the influence of inner filter

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effects in UV-vis absorption, the following equation was used to correct fluorescence

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[19]:

Fc  Fm10( A1  A2 )/2 (1)

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where Fc and Fm stand for the corrected and measured fluorescence, respectively; A1

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and A2 represent the absorbance of PB2 at excitation and emission wavelengths, respectively.

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2.3. Surface hydrophobicity measurements

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The surface hydrophobicity was determined by the hydrophobicityfluorescence probe ANS using a fluorescence spectrophotometer (F-7000, Hitachi, Kyoto, Japan).

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The excitation slit was 5.0 nm and emission slit was 2.5 nm. The excitation

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wavelength was 390 nm. 16 μL 8 mmol/L ANS was added into 3.2 mL PPA-PB2 mixture solution (3.0 mL 5 mg/mL of PPA solution with 0.2 mL various concentrations of PB2). PB2 solution was used as blank to correct background of fluorescence.

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surface

hydrophobicity was

remarked

as

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relative

ANS-fluorescence intensity. 2.4. Circular dichroism measurements Conformational changes during PPA-PB2 interaction were investigated by 6

ACCEPTED MANUSCRIPT circular dichroism spectroscopy. The far-UV CD spectra of PPA were measured on CD spectrometer (Bio-Logic MOS 450, Claix, France) according to method previously reported [18]. All spectra of PPA in the presence of PB2 were recorded between 190 and 250 nm under constant nitrogen flush at room temperature after

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subtracting the background of PB2. The α-helix, β-sheet, β-turn, and random coil of

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PPA were analyzed from CD spectroscopic data by the online CONTIN method in

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DICHROWEB. The Website: (http://dichroweb.cryst.bbk.ac.uk/html/home.shtml).

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2.5. UV spectra determination

UV spectrum were recorded on a spectrophotometer (TU-1901, Persee, Beijing,

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China) according to method of Cai et al. [11] with some modifications. In brief, 0.2 mL of different concentrations (0.05-1.73 mmol/L) of PB2 solution were added into 3

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mL 1 mg/mL PPA solution. The absorption spectrum were recorded after 30 min, and

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collected from 240 to 330 nm. The appropriate blank responding to the PB2 solution

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were subtracted to correct background. 2.6. Molecular docking simulation

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Molecular docking simulation was performed as [20] with some modification, using Libdock algorithm in Discovery Studio 3.0 (Accelrys Inc., USA). The 3D structure of PPA (PDB: 1DHK) was obtained from the Protein Data Bank (http://www.rcsb.org/pdb). The 3D structure of PB2 was prepared by Chemoffice software, using the MMFF94 force field for energy minimization. After the PPA and PB2 were imported into Discovery Studio, the water molecules in PPA were removed, whereas all hydrogen atoms were added to the PPA file according the “Prepare 7

ACCEPTED MANUSCRIPT Protein” function at pH 6.9, temperature 25 oC. Then, the PPA structure was minimized by the algorithm of smart minimizer. The PB2 was optimized according the “Prepare Ligands” function at pH 6.9. Finally, the optimal structure of PPA and PB2 were given to the CHARMm force field. The docking site was defined from the

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recorded active site. Interaction between identified PB2 and protein structure of

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1DHK were observed in the software Discovery studio 2017 R2.

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2.7. Statistical analysis

The statistical analyses were performed using SPSS 16.0 (IBM Inc., USA). The

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mean values were compared by Tukey’s test at a 5% level of significance using

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one-way ANOVA.

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3. Results and discussion

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3.1. Fluorescence emission spectrum Fluorescence emission spectrum was applied to explore the interaction of PB2

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and PPA. PPA consists of 496 amino acid residues, forming a polypeptide chain with 17 tryptophan residues provided intrinsic fluorescence [11]. Thus, the excitation

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wavelength (295 nm) was selected to measure the fluorescence of PPA. The inner-filter effect refers to the absorption of excitation or emission the fluorophore [21]. The filter effect must be considered before using fluorescence data, firstly. From Fig 1A, inner filter effect can be excluded because of low absorption for maximum concentration of PB2 (0.11 mmol/L) at the excitation (295 nm) and emission (345 nm) wavelengths. The values were 0.039 (lower 0.1) and 0 at excitation and wavelengths

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ACCEPTED MANUSCRIPT emission, respectively. Thus, fluorescence was not necessary for calibration according to equation (1) [21]. The fluorescence spectra of PPA in the presence of PB2 at different concentrations are shown in Fig. 1B (298K), Fig. 1C (308K), Fig. 1D (318K), respectively. It can be observed that PPA exhibited a strong fluorescence emission

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band at 345 nm. The fluorescence intensity of PPA decreased significantly after

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addition of PB2, without any distinct shift at maximum band wavelength. This

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indicated that PB2 had a quenching effect on the intrinsic fluorescence of PPA which may be attributed to the bind of PB2 and PPA molecules to form complex and change

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the microenvironment of the tryptophan residues [22]. Many studies also found that

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the fluorescence intensity of enzyme decreased by increasing the phenolic content due to the interaction between digestive enzymes (α-amylase, α-glucosidase) and

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polyphenols (procyanidin [11], apigenin [23], kaempferol [24]) and forming

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complexes.

3.2. The fluorescence quenching mechanism and binding constant

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Generally, there are several potential factors of quenching, such as the inner-filter

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effect, dynamic quenching, and static quenching [21]. If the quenching is static, the quenching constant will decrease with the increasing temperature, as the stability of the protein-polyphenol complex will decrease with increasing temperature. On the contrary, for dynamic quenching, the quenching constant increases with the increase of temperature due to the collision effect [25]. The PPA fluorescence quenching data was analyzed using the Stern-Volmer equation (2) at three different temperatures (298, 308, and 318 K), which can confirm the type of fluorescence quenching mechanism 9

ACCEPTED MANUSCRIPT [26]. F0  1  K q 0 [Q]  1  K SV [Q] F

(2)

Where F0 and F are the fluorescence intensities without and with PB2, respectively; [Q] is the PB2 concentration; KSV is the Stern-Volmer quenching

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constant; Kq is the biomolecular quenching rate constant and τ0 is the average lifetime

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of the biomolecule without a quencher (τ0 =10-8 s) [27].

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From Fig. 1E, the Stern-Volmer plot showed good linearity at different temperatures, suggesting that the quenching mechanism is either a dynamic or static

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procedure [28]. The KSV (KSV= Kqτ0) values [1.60×103 (298 K), 1.48×103 (308 K), and

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1.18×103 M-1 (318 K)] were found to decrease gradually as the temperature rose, indicating the fluorescence quenching induced by the formation of PPA-PB2 complex

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(static type). The biomolecular quenching rate constant (Kq) was also higher than the

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maximum diffusion collision quenching constant value (2.0×1010 L·mol-1·s-1), which indicated that the quenching type was typical static quenching [29].

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For the static quenching, the binding constant (Ka) of the interaction between

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PPA and PB2 and the number of binding sites per protein (n) could be calculated according to a double logarithmic equation (3) [19]: log

F0  F  log K a  n log[Q] F

(3)

In Fig. 1F, the high linear correlation coefficient (R2>0.98) at different temperatures indicated the assumptions underlying the derivation of equation (3) were valid. The n values were all approximately equal to 1 suggesting that PPA had a single class of binding sites for PB2. All the obtained experimental results are summarized in 10

ACCEPTED MANUSCRIPT Table 1. The Ka values [0.37×102 (298 K), 1.59×102 (308 K), and 4.66×102 M-1 (318 K)] of PPA-PB2 complexes increased with increasing temperature. The calculated Ka values of PPA-PB2 complexes are lower than of PPA-sorghum procyanidins trimer complexes (Ka = 4.95×102 M-1) [11], suggested that there are a moderate affinity

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between PPA and PB2. Ho et al. [17] also reported that B-type procyanidins trimer

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have the better inhibitory ability to α-amylase than B-type procyanidins dimer. It can

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be speculate the B-type procyanidins with higher mDP have better inhibitory and binding ability to α-amylase. Numerous studies also had indicated that PAs polymers

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showed a stronger α-amylase inhibitory activity than that of PAs oligomers [14, 15].

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3.3. Thermodynamic parameters

The thermodynamic parameters were calculated to ascertain the main forces

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contributing to ligand-protein stability. The main interaction forces between small molecules and proteins may be hydrogen bonding, hydrophobic interactions, and

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electrostatic forces [30]. When ∆S > 0, ∆H > 0, hydrophobic forces dominate; when ∆S < 0, ∆H < 0, hydrogen bonds dominate; and when, ∆S > 0, ∆H < 0 electrostatic

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forces is involved in the interaction [31]. The entropy change (∆S), enthalpy change (∆H) and free energy change (ΔG) can be obtained by using Van’t Hoff equation [23]. ln K a  

H S  RT R

G  H  T S

(4) (5)

Here Ka is the binding constant obtained from equation (3) at the corresponding temperature, R is the gas constant (8.314 J·mol-1·K-1) and T is the absolute temperature (298, 308, and 318 K). The ∆S and ∆H values were obtained from the 11

ACCEPTED MANUSCRIPT intercept and slope of the linear Van’t Hoff plot based on lnKa versus 1/T. The free energy change (ΔG) were calculated from the equation (5). From Table 1, the negative ΔG indicated that the binding process of complex was spontaneous. The ∆S and ∆H values for the binding interaction between PB2 and

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PPA were 366 J·mol·K-1 and 99.94 kJ·mol-1, respectively. Both ΔS and ∆H values

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were more than 0, which indicated that the main force acting between PPA and PB2

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was hydrophobic interaction and the binding process was entropy driven. This result is consistent with other studies, which have reported that the driving forces for the

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binding of flavonoids (such as apigenin [23], pelargonidin [32], and anthocyanin [13])

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to globular proteins (α-glucosidase, and β-lactoglobulin) were mainly hydrophobic interactions.

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3.4. Protein surface hydrophobicity

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The surface hydrophobicity of PPA was charactered by relative ANS-binding fluorescence intensities of PPA with PB2. ANS was utilized to explore conformational

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changes in PPA and characterized surface exposure of hydrophobic sites [33]. In Fig.

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2, the surface hydrophobicity for PPA decreased progressively with increasing amount of PB2 from 0 to 108.03 μmol/L. The reduction in surface hydrophobicity of PPA-PB2 complexes speculated that the polarity increased and the surrounding solution polarity changed. The results are as reported by Jia et al. [33], who reported that the surface hydrophobicity of β-lactoglobulin-polyphenols (chlorogenic acid/EGCG) decreased and the forming of complexes might destroy the originally hydrophobic structure of protein and expose hydrophobic regions on PPA surface. 12

ACCEPTED MANUSCRIPT 3.5. Circular dichroism spectra Circular dichroism (CD) was utilized to monitor changes in the secondary structure (α-helix, β-sheet and β-turn) of PPA. The CD spectra of PPA in the absence and presence of PB2 are shown in Fig. 3A. The values of negative ellipticity increased

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with increasing PB2 concentration, indicating partial changes in the PPA secondary

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structure. The contents of secondary structures in PPA were calculated in Fig. 3A. The

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studies showed that free PPA contained around 12.7% α-helix, 7.0% β-sheet, 34.3% β-turn, and 46.0% random coil. After interaction with 108 μmol/L PB2, the contents

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of α-helix and β-turn increased to 34.7% and 12.7%, respectively, while the contents

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of β-sheet (27.7%) and random coil (24.9%) significantly decreased. Shu et al. [34] reported that erucic acid inserted into the hydrophobic surface of bovine serum

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albumin, which resulted in an increase of a-helix content. Xu et al. [35] also reported

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that ligand molecules inserted into the hydrophobic surface of glutelin which resulted in an increase in the amount of α-helix. Thus, it can be speculate that an increase in

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the amount of α-helix was caused by PB2 that inserted into the hydrophobic surface

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of PPA. The results correlated well with the analysis of surface hydrophobicity. 3.6. UV spectrum UV spectra of protein can be utilized to analyze the PPA structural changes and interaction [36]. Fig. 3B showed the ultraviolet spectra of PPA interacted with PB2 in concentrations ranging 0-108.03 μmol/L. The absorption peak at 275 nm of PPA due to π-π transition of peptide bond C=O group in tryptophan (Trp) and tyrosine (Tyr) aromatic [11]. The absorption of PPA successively increased with the increase of PB2 13

ACCEPTED MANUSCRIPT concentration (3.38 to 108.03 μmol/L), followed by a red shift. Similar results were shown in study of Cai et al. [11], who pointed that the increase of UV spectrum of PPA in the presence of sorghum procyanidins revealed the binding interaction between PPA and procyanidins. The results can also confirm the fluorescence

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quenching in the interaction of PPA with PB2.

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3.7. Computational docking simulation

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The computational docking (LibDock) simulation was applied to predict the

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precise binding sites of PB2 in RG and to confirm the experimental results described above. LibDock is a high throughput docking algorithm that positions catalyst

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generated ligand conformations in the protein active site, based on polar interaction sites (hotspots) [37]. Based on the LibDock protocol, the pose which have the highest

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Libdock score (110.327) corresponding to the most stable conformation, was selected

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for analysis about the interaction between PPA and PB2. Combined 3D docking mode (Fig. 4AB) and 2D schematic diagram (Fig. 4C), which can show clearly that PB2

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inserted into the cavity of PPA. From Fig. 4C, PB2 interacted with amino acid

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residues including Lys 200, Ile 235, Val 234, Ser 199, Ala 198, Asp 197, Glu 233, Arg 195, His 201, Asp 300, Leu 162, Tyr 151, Val 163, Ile 148, Gly 147, Gln 161and Gly 164, which were possible interaction sites between PB2 and PPA. Four hydrophobic interaction forces (one Pi-Pi T-shaped, there Pi-Alkyls) were found between PB2 and amino acid residues (His 201, Ile 235, Val 163, Ile 148). Due to the PB2 have many hydroxyl group, two hydrogen bonds also were found between PB2 and amino acid residue (Tyr 151, Ile 148). The distance of two hydrogen bonds (5.36 Å, 3.94 Å) and 14

ACCEPTED MANUSCRIPT hydrophobic forces (6.02 Å, 5.90 Å, 5.41 Å, 4.98 Å) were showed in Fig 4C. The main interaction force was hydrophobic interaction force which agreed with the result of thermodynamic parameter. From Fig. 4A, the PB2 located in the active site, which included Asp 167, Asn 100, Arg 158, His 201. Specially, His 201 interacted with PB2

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through the hydrophobic interaction forces and thus inhibited the activity of PPA.

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4. Conclusion

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This study demonstrated that bioactive flavonoids (procyanidin dimer) interacted

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with α-amylase using chemical methods in combination with molecular docking techniques. The fluorescence quenching of PPA was attributed to the formation of a

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stable PPA-PB2 complex. Hydrophobic interaction dominated the interaction process between PPA and PB2. The CD results showed that PPA-PB2 complex can induce

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changes in the secondary structure of PPA. The molecular docking simulations

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provided valuable information about the binding of the PB2 to the PPA surface. This research provides new insights into the inhibitory mechanism of PB2 on α-amylase.

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Acknowledgements

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The authors thank the National Nature Science Foundation of China (31460394, 31760441)

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Inhibitory activity, conformational change and mechanism, Food Res. Int. 100 (2017) 226-233. [27] J.R. Lakowicz, G. Weber, Quenching of protein fluorescence by oxygen. Detection of structural fluctuations in proteins on the nanosecond time scale, Biochemistry. 12(21) (1973) 4171-4179. [28] B.K. Paul, N. Ghosh, S. Mukherjee, Binding interaction of a prospective chemotherapeutic

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[35] X.F. Xu, W. Liu, J.Z. Zhong, L.P. Luo, C.M. Liu, S.J. Luo, L. Chen, Binding interaction between rice glutelin and amylose: Hydrophobic interaction and conformational changes, Int. J. Biol. Macromol. 81 (2015) 942-950. [36] D.G. Zhong, Y.P. Jiao, Y. Zhang, W. Zhang, N. Li, Q.H. Zuo, Q. Wang, W. Xue, Z.H. Liu, Effects

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simulation, J Biomol Struct Dy. (2016) 1-39.

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anti-cancerous secondary metabolites: ADMET based screening, molecular docking and dynamics

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Figure. 1 (A) Effect of the inner-filter effect on the fluorescence of PPA in a 1×1 cm cuvette. Absorbance values of the highest added concentrations are 0.11 mmol/L. Effect of PB2 on the intrinsic fluorescence spectrum of PPA at 298 K (B), 308 K (C), and 318 K (D). (E) The Stern-Volmer plots for the quenching of PPA by PB2 and the Kq of PPA-PB2 complex in 298 K,

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308K, and 318 K, respectively; (F) The plots for the static quenching of PPA by PB2 (298 K, 308

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K, and 318 K). CPPA=1 mg/mL; CPB2=0 to 0.108 mmol/L.

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CPPA=5 mg/mL; CPB2=0 to 108.03 μmol/L.

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Figure. 2 Changes in ANS-fluorescence intensity of PPA at different concentrations of PB2.

Figure. 3 (A) The profiles of PPA（5 mg/mL）secondary structure treated by PB2 with various

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concentrations, (0.00, 13.50, 27.01, 54.02, 108.03 μmol/L); (B) Ultraviolet spectra of PPA (1

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mg/mL) treated by PB2 with various concentrations from 0 to 108.03 μmol/L.

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Figure. 4 (A) 3D docking mode between PB2 and PPA simulated by Discovery Studio and the

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amino acid of active site (Asp 167, Asn 100, Arg 158, His 201); (B) The hydrophobic surface of protein receptor interact with PB2, the red colour and blue colour represent hydrophobicity and hydrophily, respectively; (C) 2D schematic interaction diagram between PB2 and PPA, the colour of amino acid residue is drawn by interaction.

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ACCEPTED MANUSCRIPT Table 1. Binding constants and thermodynamic parameters for the interaction between PPA and PB2 at different temperatures. ΔG (kJ· mol-1)

Regression equation

R2

K (×102 L·mol-1)

n

298

log［F0/F-1］=1.5678+0.5989 log［PB2］

0.947

0.37

0.60

-10.32

308

log［F0/F-1］=2.2014+0.7544 log［PB2］

0.990

1.59

0.75

-14.02

318

log［F0/F-1］=2.6682+0.8805 log［PB2］

0.966

4.66

0.88

-17.72

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ΔH (kJ· mol-1)

ΔS (kJ·mol·K-1)

99.94

0.37

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T/K

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ACCEPTED MANUSCRIPT Highlights：  Interaction between α-amylase (PPA) and procyanidin dimer (PB2) was studied.  The major PPA-PB2 interaction was hydrophobic interaction.  The interactions changed the conformation of PPA.

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 Spectroscopy and molecular docking explored the interaction mechanism.

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