Interaction mechanism between α-glucosidase and A-type trimer procyanidin revealed by integrated spectroscopic analysis techniques

International Journal of Biological Macromolecules 143 (2020) 173–180

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

Interaction mechanism between α-glucosidase and A-type trimer procyanidin revealed by integrated spectroscopic analysis techniques Li Zhao a,1, Luming Wen a,1, Qun Lu a,b,c, Rui Liu a,b,c,⁎ a b c

College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural University), Ministry of Education, Wuhan 430070, China Wuhan Engineering Research Center of Bee Products on Quality and Safety Control, Wuhan 430070, China

a r t i c l e

i n f o

Article history: Received 13 October 2019 Received in revised form 2 December 2019 Accepted 3 December 2019 Available online 06 December 2019 Keywords: Procyanidins α-Glucosidase Inhibition effect Interaction mechanism

a b s t r a c t α-Glucosidase is an important enzyme in human intestine, and inhibition of its activity can lower blood sugar levels to effectively prevent hyperglycaemia induced tissue damage. Here, we investigated the inhibitory activities of procyanidins with different structures on α-glucosidase and the underlying mechanism. The results showed that the IC50 of catechin and compounds 2–7 on α-glucosidase was lower than that of acarbose. Atype procyanidins might have better inhibitory activity than B-type procyanidins. In addition, there was no positive correlation between the polymerization degree of A-type procyanidin oligomer and its inhibitory effect on α-glucosidase. Compound 7 (A-type trimer) with the best inhibitory effect reversibly inhibited the activity of αglucosidase in a mixed-type manner. Fluorescence data confirmed that the intrinsic fluorescence of αglucosidase was quenched by compound 7 through static-dynamic quenching. The calculated thermodynamic parameters indicated that their binding was spontaneous and driven by hydrophobic interaction, which was also confirmed by the UV spectrum experiment. Besides, circular dichroism analysis displayed that their binding resulted in conformational changes of α-glucosidase characterized by a decrease in α-helix and an increase in βsheet. The results demonstrate the ability of procyanidins to intervene in the progression of type 2 diabetes by inhibiting α-glucosidase. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Diabetes is a metabolic disease characterized by the rise of blood sugar, and may cause complications such as cardiovascular and cerebrovascular diseases and sensory neuropathy [1]. The progression of diabetes can be controlled by supplementing insulin, improving insulin resistance and controlling blood sugar [2]. In recent years, with better understanding of the pathogenesis of diabetes and the development of drug treatment, many hypoglycemic drugs have been applied to clinical treatment of diabetes, such as α-glucosidase inhibitor, which is widely used in the treatment of type 2 diabetes. The hypoglycemic mechanism is that when the food enters the small intestine, α-glucosidase inhibitor can inhibit the hydrolysis of the disaccharide or oligosaccharide on the small intestinal mucosa to reduce the absorption of carbohydrates, resulting in a lower blood sugar level after dinner [3]. Acarbose and voglibose are used as the main α-glucosidase inhibitors; however,

⁎ Corresponding author at: College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China. E-mail address: [email protected] (R. Liu). 1 These authors contributed to the work equally.

https://doi.org/10.1016/j.ijbiomac.2019.12.021 0141-8130/© 2019 Elsevier B.V. All rights reserved.

their application is largely limited by some intestinal side effects [4]. Therefore, researchers have attempted to find natural α-glucosidase inhibitors in plants as alternatives to overcome the drawbacks of existing inhibitors. As a result, some studies have reported that some compounds from plants, such as alkaloids, polyphenols and flavonoids, have inhibitory activity for α-glucosidase [5]. Procyanidins are also known as polymeric flavan-3-ols, and are formed by polymerization of the monomers catechin and epicatechin. They can be divided into two types (A and B) according to the connection bond between monomers [6]. B-type procyanidins are mainly found in plants such as cereals, nuts, berries, and beans, while A-type procyanidins are only found in a few plants such as litchi and peanut skin [7]. Procyanidins possess many good physiological functions such as prevention of diabetes and cardiovascular and cerebrovascular diseases, as well as anti-inflammatory and anti-tumor effects [8–11]. In recent years, researchers have begun to notice the hypoglycemic function of procyanidins. Kang et al. extracted two phenolic substances from blueberries, namely BAE-LMW and BAE-PAC, with IC50 values of 0.242 mg/mL and 0.915 mg/mL for α-glucosidase, respectively [12]. Kong et al. found that water-soluble extract of grape seed has a higher content of GSAE, whose IC50 value is 25.25 ± 0.53 g/mL [13]. Lavelli et al. showed that the seven phenolic substances extracted from grape skin also have good inhibitory effects on α-glucosidase [14]. These

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studies were based on the crude extracts of procyanidins, all of which exhibited certain inhibitory effects on α-glucosidase. Due to the difficulty in separating and preparing procyanidins with different structures, there has been little research on the effects of well-defined procyanidins on α-glucosidase. As a result, the mechanism underlying the inhibitory effects of procyanidins on α-glucosidase remains largely unknown. The interactions between polyphenols and proteins are generally divided into covalent bonds, hydrophobic interactions, hydrogen bonds, and electrostatic interactions [15]. The interactions between small molecules and proteins can be characterized by using UV–visible absorption spectroscopy, fluorescence spectroscopy, circular dichroism, infrared spectroscopy, and molecular docking [16–18]. Under the laboratory conditions, Yu et al. discovered that SPC tetramers led to a static fluorescence quenching of the catalytic region of glucosyltransferases-I, changing the microenvironment of its aromatic amino acid residues and secondary structure [19]. In this study, the inhibitory activity experiments and inhibition kinetics experiments were carried out with well-defined procyanidins to reveal the inhibition types of procyanidins on α-glucosidase. Besides, the interaction between procyanidins and α-glucosidase and the resulting changes in the secondary structure of α-glucosidase were elucidated by using spectral methods. Our findings provide a theoretical basis for using procyanidins to regulate the blood glucose level of patients with type 2 diabetes.

calculated by the following formulas [22]. 1 Km 1 1 ¼  þ V Vmax ½S Vmax Slope ¼

Km Km ½I þ Ki  Vmax Vmax

Intercept ¼

1 1 ½I  þ Kis  Vmax Vmax

2.4. Fluorescence spectrum measurements Fluorescence spectrum measurements were performed on a RF5301PC fluorescence spectrophotometer (Shimadzu, Japan) equipped with a 1.0 cm quartz cell at a constant temperature water bath. Excitation slit and emission slit were set as 5 nm. The excitation wavelength of 285 nm was chosen and the fluorescence emission spectra were recorded in the range of 300–450 nm. The experiment was conducted in a constant temperature water bath at two temperatures (290 K and 310 K). 1 mL procyanidin solution at different concentrations was added to 1 mL of α-glucosidase sample, and the mixture was incubated in a quartz cuvette for 1 min before the measurements. Every spectrum was scanned for three times.

2. Materials and methods 2.5. Measurements of UV absorption spectra 2.1. Materials α-Glucosidase from Saccharomyces cerevisiae was purchased from Sigma-Aldrich Co. (St Louis, MO, USA). 4-nitrophenyl-α-Dglucopyranoside (pNPG), catechin, and epicatechin were purchased from Shang Hai Yuanye Biotechnology Company (Shang Hai, China). Compounds 1–7 were provided by our group, and were dissolved in a phosphate buffer solution of pH 6.80 (0.1 M PBS). Na2HPO4, NaH2PO4, Na2CO3, and other reagents were of analytical grade.

UV absorption spectra of the reaction systems were measured on a UV-1800 UV/vis spectrophotometer (Shimadzu, Japan) with a 1.0 cm quartz cuvette. Absorption spectra of α-glucosidase (0.5 U/mL) with different concentrations of procyanidins (same volume PBS as blank) were recorded in the range of 195–300 nm. The spectra of αglucosidase, procyanidin, and α-glucosidase-procyanidin mixture were measured, respectively. 2.6. Measurements of circular dichroism (CD)

2.2. Comparison of the inhibitory effects of different procyanidins and their structural units on α-glucosidase Measurement of the inhibitory activities of procyanidins and their structural units on α-glucosidase was performed with the method of Liu et al. and Kang et al. with some modifications [20,21]. 40 μL of samples (the blank group was added with the same volume of 0.1 M sodium phosphate buffer at pH 6.8) and 20 μL of α-glucosidase solution (0.4 U/mL) were pre-incubated in a 96-well plate for 10 min at 37 °C. Then, 40 μL pNPG (0.5 mM) was added into each microplate well. After incubation for 20 min at 37 °C, 50 μL Na2CO3 solution (0.1 M) was used to terminate the process. The absorbance values were determined at 405 nm by microplate reader (Multiskan GO, Thermo Fisher, USA). In this experiment, acarbose was used as the positive control group, and the inhibition rate was calculated according to the formula:
Inhibitory Rate ð%Þ ¼ Acontrol −Asample =Acontrol  100%

Far-UV CD spectra of α-glucosidase (1 U/mL) in the absence or presence of procyanidins were measured with Jasco-810 spectrophotometer (JASCO, Japan) at 25 °C and under continuous nitrogen inflation. Experiment related parameters included: test temperature, room temperature; wavelength, 195–240 nm; instrument sensitivity, 2 mdeg/cm; path length of quartz cell, 1 mm; scan rate, 100 nm/min; and scan time, 0.5 s. Circular dichroism (CD) of α-glucosidase with varying concentrations of procyanidins (same volume PBS as blank) was recorded in the range of 195–240 nm. 2.7. Statistical analysis All the data were determined three times and expressed as means ± standard deviations (n = 3). One-way analysis of variance (ANOVA) was used to analyze differences among treatments (SPSS 20.0 for Windows). The mean values were compared using Duncan's multiple range test. P b 0.05 was considered to be statistically significant. 3. Results and discussion

2.3. Kinetics of the inhibition of α-glucosidase by procyanidins The types of inhibition of α-glucosidase by procyanidins were assayed by varying the concentration of α-glucosidase (0.125, 0.25, 0.5, 0.75, 1 and 1.25 U/mL) and pNPG (0.25, 0.5, 1, 2 and 4 mM), respectively. At 37 °C, the enzyme inhibition reaction was recorded by measuring the absorbance of the microplate reader at 405 nm for 5 min, to obtain the corresponding enzymatic reaction rate. Ki and Kis can be

3.1. Inhibitory effects of different kinds of procyanidins and their structural units on α-glucosidase Fig. 1 shows the inhibitory effects of procyanidins with different structures and their structural units on α-glucosidase. All different samples showed certain inhibitory effects on α-glucosidase in a concentration-dependent manner. The IC50 values of different samples on α-glucosidase are presented in Table 1. Catechin and compounds

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Fig. 1. Inhibitory effect of different samples on α-glucosidase. (A) Inhibition rates of catechin on α-glucosidase. (B) Inhibition rates of epicatechin on α-glucosidase. (C–I) Inhibition rates of compounds 1–7 on α-glucosidase, respectively. The lower case letters indicate significant differences in inhibitory rate on α-glucosidase between different inhibitor concentrations (P b 0.05).

2–7 showed lower IC50 (IC50 = 25.28 ± 0.67 μg/mL–370.29 ± 2.21 μg/mL) on α-glucosidase than the positive control (acarbose, IC50 = 376.28 ± 10.49 μg/mL), suggesting that they have better inhibitory effects on α-glucosidase and can be potentially used as α-glucosidase inhibitors [23]. Compounds 1–4 are all procyanidin dimers, and the IC50 values of Btype procyanidins (compounds 1 and 2) were higher than those of Atype procyanidins (compounds 3 and 4). Hence, A-type procyanidins Table 1 IC50 values of α-glucosidase inhibition by procyanidins and their structural units. Sample name

Sample type

IC50 (μg/mL)

Compound 7 Compound 3 Compound 4 Compound 5 Compound 6 Compound 2 Catechin Acarbose Compound 1 Epicatechin

A-type trimer A-type dimer A-type dimer A-type trimer A-type trimer B-type dimer monomer Positive control B-type dimer Monomer

25.28 ± 0.67a 73.71 ± 0.21b 99.02 ± 1.15c 128.49 ± 4.17d 158.84 ± 4.91e 223.12 ± 8.93f 370.29 ± 2.21g 376.28 ± 10.49g 626.15 ± 6.92h 1046.50 ± 21.84i

Notes: Results are significantly different with different lower-case letters using Duncan's multiple range test (P b 0.05). Acarbose was used here as a positive control for comparison with samples.

may have better inhibitory activity on α-glucosidase than B-type procyanidins. In addition, comparison of the IC50 values of the dimers and trimers of A-type procyanidins showed that there was no positive correlation between the polymerization degree of A-type procyanidins and their inhibitory effects on α-glucosidase. Also, both catechin and epicatechin are the structural units of procyanidins, but the IC50 value of catechin was only one-third that of epicatechin due to the difference in their spatial conformation. In order to explore the type and mechanism of the inhibition of procyanidins on α-glucosidase in the subsequent experiments, compound 7 with better inhibitory effect was selected. The structure of compound 7 is shown in Fig. 2. 3.2. Inhibition kinetics of procyanidins on α-glucosidase Inhibitors can alter enzyme activity by interacting with the enzymes. Previous studies have shown that acarbose and oligosaccharides mainly combine reversibly with α-glucosidase to competitively inhibit the activity of α-glucosidase, slowing down the decomposition of sucrose into glucose and fructose and thus reducing the absorption of intestinal glucose, so as to relieve postprandial hyperglycemia. Compound 7 (Atype trimer), which showed the best inhibitory effect on αglucosidase, was selected to investigate the inhibition kinetics. Enzyme inhibitors usually have two modes of inhibition on enzymes: reversible inhibition and irreversible inhibition. At constant substrate

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concentration of compound 7, the longitudinal intercept gradually increases, indicating that compound 7 is a mixed reversible inhibitor [26]. The slope and intercept of different straight lines obtained by double reciprocal curves were plotted with different concentrations of compound 7 (Fig. 3C and D), and the inhibition constant (Ki, Kis) was calculated and shown in Table 2. A lower inhibition constant would lead to better binding ability of the inhibitor to the enzyme, resulting in a stronger inhibitory activity [27]. The Ki of compound 7 was lower than Kis, indicating that its affinity with free enzymes is greater than that with enzyme-substrate complex. Thus, it can be concluded that compound 7 is a mixed inhibitor dominated by competitive inhibition [21,26]. 3.3. Inhibitory mechanism of compound 7 on α-glucosidase

Fig. 2. The structure of compound 7.

concentration, the reaction rates of different concentrations of αglucosidase and compound 7 were determined, and the results are shown in Fig. 3A. The slope of the sample group was lower than that of the control group. Increase in the concentration of compound 7 (inhibitor) resulted in a decrease in the slope, indicating that compound 7 is a reversible inhibitor of α-glucosidase [24]. It can be concluded that compound 7 does not reduce the amount of active enzyme, but only causes a decrease in the activity of the enzyme [25]. At constant enzyme concentration, the reaction rates of α-glucosidase and compound 7 were determined under different concentrations of procyanidin and substrate. Taking the reciprocal of the substrate concentration as the abscissa and the reciprocal of the enzyme reaction rate as the ordinate, a Lineweaver-Burk plot curve was shown in Fig. 3B. The four straight lines intersect at a point in the second quadrant, and with increasing

3.3.1. Fluorescence quenching mechanism of α-glucosidase by compound 7 Fluorescence quenching is a common method for studying protein/ ligand interactions because the quencher may cause conformational changes in the protein. It has the advantages of spectral simplification, reduction of the spectral bandwidth, and avoidance of some disturbing effects [28,29]. Fluorescence spectra are sensitive to the microenvironment of amino acid chromophores. Tyrosine and phenylalanine are the fluorescent chromophores in the amino acids of α-glucosidase [30]. Table 3 shows the fluorescence intensity of α-glucosidase under the conditions of λem = 300–450 nm and different λex. When the excitation wavelength was 285 nm, α-glucosidase showed the highest fluorescence intensity. At the emission wavelength of 300–450 nm, compound 7 hardly produced fluorescence. α-glucosidase had the maximum fluorescence intensity at 334 nm, and with increasing concentration of compound 7, the fluorescence intensity of α-Glucosidase decreased in a regular way (Fig. 4A and B). It is possible that compound 7 interacts with α-glucosidase, causing the quenching of endogenous fluorescence in α-glucosidase to varying degrees [31]. The addition of

Fig. 3. (A) Inhibition kinetics of compound 7 on α-glucosidase. Concentrations of compound 7 = 0, 5, 10, and 15 μg/mL for curve a → d, respectively. (B) Lineweaver-Burk plots for the inhibition of compound 7 at different concentrations on α-glucosidase. Concentration of α-glucosidase = 0.5 U/mL, and concentrations of compound 7 = 0, 5, 10, and 15 μg/mL for curves a → d, respectively. (C) and (D) represent the secondary plot of the slop and the intercept of the straight line versus concentration of compound 7, respectively.

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Table 2 Lineweaver-Burk curve equations and inhibition constants for inhibition of α-glucosidase by compound 7. Grouping

Concentration (μg/mL)

Lineweaver-Burk equation

R2

Kis

Ki

Control group Compound 7

0.0 5.0 10.0 15.0

Y = 12.150x + 18.994 Y = 18.679x + 19.269 Y = 20.830x + 21.999 Y = 27.008x + 23.140

0.9984 0.9984 0.9978 0.9986

/ 57.97

/ 15.39

compound 7 caused a significant blue shift in the maximum fluorescence absorption wavelength of α-glucosidase (from 334 nm to 314 nm), resulting in a gradual decrease of the microenvironment polarity around the amino acid residues in α-glucosidase [32,33]. To reveal the possible quenching mechanism of compound 7 on αglucosidase, the fluorescence quenching data were analyzed by the

Table 3 Fluorescence intensity of α-glucosidase under λem = 300–450 nm and different λex. λex (nm)

Fluorescence intensity (AU) 1

2

3

Mean

275 280 285 290 295 300

169.70 193.70 196.30 170.80 128.80 67.91

167.40 193.40 195.60 169.70 128.70 67.83

169.90 194.70 196.10 171.10 128.90 68.45

169.00 ± 1.39c 193.93 ± 0.68b 196.00 ± 0.36a 170.53 ± 0.74c 128.80 ± 0.10d 68.06 ± 0.34e

Notes: Results are significantly different with different lower-case letters using Duncan’s multiple range test (P b 0.05).

following modified form of the Stern-Volmer equation [34]: F0 ¼ ð1 þ KDÞ½Q ð1 þ KSÞ½Q  ¼ 1 þ Kapp ½Q ; F where F0 and F are the fluorescence intensities of α-glucosidase before and after the addition of compound 7, respectively, Kapp is the SternVolmer apparent quenching constant (Kapp = (KD + KS) + KDKS[Q]2), KD and KS are the dynamic and static quenching constants, respectively, and [Q] is the concentration of compound 7. The Stern-Volmer plot was based on fluorescence data measured at 290 and 310 K (Fig. 4C). The apparent quenching constant Kapp and the correlation coefficient R2 were calculated (Table 4). There are generally two types of fluorescence quenching: static quenching and dynamic quenching. Static quenching is caused by a complex formed from the interaction between the quencher and ground-state fluorescent molecules. Dynamic quenching is caused by material collision or energy transfer between the quencher and the excited fluorescent molecules [35]. In many cases, the fluorophore can be quenched both by dynamic and by static with the same quencher. When this is the case, the Stern-

Fig. 4. Effect of compound 7 on the intrinsic fluorescence spectra of α-glucosidase at 290 K (A) and 310 K (B). a–e: Concentrations of compound 7 = 0, 0.4, 0.6, 0.8, and 1.0 mg/mL, and the line in the bottom (f) shows the fluorescence spectrum of compound 7 when α-glucosidase concentration = 0. (C) The Stern-Volmer plots for the quenching of α-glucosidase by compound 7. (D) The plots for the static quenching of α-glucosidase by compound 7 (290 K and 310 K), Cα-glucosidase = 1 U/mL; Ccompound 7 = 0.4, 0.6, 0.8, and 1 mg/mL.

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Table 4 Quenching constant Ksv, binding constant KA and relative thermodynamic parameters of the compound 7-α-glucosidase interaction at different temperatures. T

Kapp (×103 L/mol)

Ra

KA (×106L/mol)

n

Rb

ΔH (KJ mol−1)

ΔG (KJ mol−1)

ΔS (KJ mol−1 K−1)

290 310

6.2430 ± 0.012 7.3463 ± 0.019

0.9969 0.9974

9.352 ± 0.047 8.696 ± 0.307

2.052 ± 0.002 2.017 ± 0.008

0.9980 0.9977

2.7179 ± 1.532 2.7179 ± 1.532

−33.2711 ± 2.930 −35.7531 ± 3.026

0.1241 ± 0.005 0.1241 ± 0.005

Ra is the correlation coefficient for the Stern-Volmer plots. Rb is the correlation coefficient for the KA values.

Volmer plot exhibits an upward curvature, concave toward the yaxis at high [Q] [34]. In the case, the K app values [6.2430 × 10 3 (290 K), and 7.3463 × 103 M−1 (310 K)] were found to increase gradually with increasing temperature, which is a characteristic of hydrophobic interactions [36]. Then, the binding constant and number of binding sites between compound 7 and α-glucosidase were analyzed by the following equation [37]: ð F0−FÞ lg ¼ lg KA þ nlg½Q ; F where KA is the apparent binding constant, which can be determined by the intercept of the curves; n is the number of binding sites, which can be determined by the slope of the curves; and [Q] is the concentration of compound 7. The relationship between lg [(F0-F)/(F)] and lg [Q] is shown in Fig. 4D. The binding constant KA and the binding site n were calculated from the slope and intercept of the linear equation (Table 4). The KA value [9.352 × 106 (290 K), and 8.696 × 106 M−1 (310 K)] of the αglucosidase-compound 7 complex decreased with increasing temperature, suggesting a decrease in the stability of the complex [38]. The binding site n of compound 7 to α-glucosidase was about 2, indicating that the molar ratio of compound 7 to α-glucosidase is 1:2 in the formed complex. 3.3.2. Driving force analysis by thermodynamics To elucidate the binding between compound 7 and α-glucosidase, the thermodynamic parameters, including Gibbs free energy change

Fig. 5. UV absorption spectra of compound 7, α-glucosidase and the compound 7-α-glucosidase system. (a, e): the absorbance spectrum of α-glucosidase only (cα-glucosidase = 0.5 U/mL) and compound 7 only (c compound 7 = 100 μg/mL); (b → d): absorbance spectrum of the compound 7-α-glucosidase system when the concentration of compound 7 is 100, 60, and 40 μg/mL, respectively, and the concentration of α-glucosidase is 0.5 U/mL; (f): the calculated curve from (a) + (e).

(ΔG), enthalpy change (ΔH) and entropy change (ΔS), were calculated by van't Hoff equation [36]:

ln KA ¼ ΔH=RT þ ΔS=R

ΔG ¼ ΔH–TΔS Here KA is the binding constant, R is the gas constant (8.314 J·mol−1·K−1), and T is the absolute temperature (290 K and 310 K). Thermodynamic parameters for the interaction between compound 7 and α-glucosidase are presented in Table 4. ΔG b 0 indicates that the binding of compound 7 to α-glucosidase is spontaneous. Besides, the values of ΔH and ΔS were calculated to be 2.7179 ± 1.532 kJ mol−1 and 0.1241 ± 0.005 KJ mol−1 k−1, respectively. According to the theory of Ross and Subramanian, if ΔS N 0 and ΔH N 0, hydrophobic interactions are the predominant driving force in the interaction process of compound 7 and α-glucosidase [39]. However, hydrogen binding should not be ignored since the experiment was conducted in aqueous solution and there were many hydroxyl groups on the molecules of compound 7 and α-glucosidase. 3.3.3. UV absorption spectrum measurements Changes in the microenvironment around amino acid residues in the protein molecule can lead to changes in the UV absorption wavelength of the protein. Hence, UV spectra can be used to explore the structural changes of protein and formation of protein-ligand complex [40,41]. Fig. 5 shows the UV absorption spectra of α-glucosidase in the absence or presence of compound 7. Clearly, there are two main absorption peaks in the UV spectra of α-glucosidase. The strong absorption peak is at about 210 nm, which reflects the absorption of the enzyme skeleton, and there is a weak absorption peak near 280 nm due to the presence of aromatic amino acids (Trp, Tyr and Phe) [42]. Obviously, with the addition of different concentrations of compound 7, the peak of α-

Fig. 6. Effect of compound 7 on the CD spectrum of α-glucosidase. Concentration of αglucosidase = 1 U/mL; a → d: Concentration of compound 7 = 0, 10, 20, 30 μg/mL.

L. Zhao et al. / International Journal of Biological Macromolecules 143 (2020) 173–180 Table 5 Effect of different concentrations of compound 7 (μg/mL) on the secondary structure of αglucosidase. Compound 7 (ug/mL)

α-Glucosidase secondary structure (%) α-Helix (%)

β-Sheet (%)

β-Turn (%)

Random coil (%)

Total (%)

0 10 20 30

15.9 1.3 0.3 0

39 74.2 74.7 76.9

10.2 0 0 0

34.9 24.5 25.0 23.1

100 100 100 100

glucosidase at 207 nm showed different degrees of red shift and decrease in color. After the addition of the lowest concentration of compound 7, the peak red shift was 0.7 nm, and with the increasing concentration of compound 7, the amplitude of the red shift also increased. These results show that the addition of compound 7 changed the structure of α-glucosidase. It is possible that the interaction between α-glucosidase and compound 7 causes loosening and stretching of protein skeleton in the α-glucosidase molecule and an increase in the hydrophobicity of the enzyme microenvironment [33], which is consistent with the fluorescence spectrum measurement result that the binding of compound 7 to α-glucosidase was mainly hydrophobic. 3.3.4. Secondary structure analysis by CD CD spectroscopy is a powerful and sensitive technique for monitoring the secondary structure changes of proteins [43,44]. Here, CD measurement was performed to investigate whether compound 7 can induce changes in the secondary structure of α-glucosidase [45], and the results are shown in Fig. 6 and Table 5. With the addition of compound 7, the two negative peaks gradually disappeared, and the peak intensity near 195 nm also decreased, indicating a sharp decrease in α-helix. On the contrary, a significant negative peak appeared at 215 nm, indicating a significant increase in the number of β-sheets [46]. In addition, some other secondary structure changes can be found in Table 5. The α-helix and the β-turn completely disappeared with a reduction of the irregular curl. The hydrogen bond maintains the stability of the secondary structure of proteins, and changes in the secondary structure indicate that the position where hydrogen bond is formed has been changed accordingly. Our results show that compound 7 interacts with α-glucosidase and changes its secondary structure, resulting in changes in the microenvironment of the amino acid residues, which may reduce the enzyme activity and further influence the absorption of glucose by human small intestine. 4. Conclusion Procyanidins of different configurations have certain inhibitory effects on α-glucosidase, and the IC50 values of catechin and compounds 2–7 on α-glucosidase (IC50 = 25.28 ± 0.67 μg/mL −370.29 ± 2.21 μg/mL) are lower than those of the positive control (acarbose, IC50 = 376.28 ± 10.49 μg/mL). Compound 7 (A-type trimer) has the best inhibitory effect, and the inhibition type is reversible mixed inhibition. The interaction mechanism between α-glucosidase and compound 7 was investigated by spectroscopic methods. Fluorescence quenching results indicate that compound 7 spontaneously interacts with α-glucosidase through a static-dynamic quenching process, and the interaction is hydrophobic. UV-absorption spectroscopy suggests that the binding of compound 7 to α-glucosidase would result in a red shift at 210 nm due to the stretching of the peptide chain in the α-glucosidase molecule and changes in the microenvironment of the amino acid residues. CD analysis shows that compound 7 reduces the percentages of α-helix, β-turn and random curl and increases β-sheet in α-glucosidase. These findings may provide a theoretical basis for investigating the binding mechanism between procyanidins and α-glucosidase, and implications

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for developing new agents for hyperglycaemia damage. And it should be noted that this study was performed in an in vitro chemical system. However, the matrix of real food systems is complex, which contains not only carbohydrates but also certain amounts of proteins with strong binding ability to polyphenols. The inhibitory effect of procyanidins on digestive enzymes (such as α-glucosidase and α-amylase) may be lower than expected due to the presence of proteins in the food system, which thereby affects the regulatory effect of procyanidins on blood sugar in vivo. This phenomenon has attracted the attention of researchers [14]. Therefore, it is necessary to protect these procyanidins from being combined with proteins in the food system and deliver the procyanidins to human body through in vivo experimental evaluations and certain measures such as embedding (maltodextrin embedding and microencapsulation) [47,48], which may help to achieve better regulatory effect of procyanidins on blood sugar levels.

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