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[alpha]-Glucosidase Inhibitory Mechanism of Phloridzin.

Byline: Jiangwei Liu, Yuzhen Chen, Benguo Liu and Guizhao Liang

Summary: Phloridzin is a chalcone with potential application in functional food and medicine. In this study, the [alpha]-glucosidase inhibitory mechanism of phloridzin was investigated by inhibitory test, fluorescence spectroscopy and molecular docking method. The IC50 value of phloridzin was determined at 0.68 mmol/L. With the increase of phloridzin concentration, the static fluorescence quenching of [alpha]-glucosidase could be observed. The quenching constant (Kq), binding constant (KA) and the number of binding site (n) of phloridzin to [alpha]-glucosidase were 6.1474x1012, 1.3243x105 and 1.076, respectively. The molecular docking result suggested that phloridzin could play the inhibitory role by binding the Lys510 and Glu374 of [alpha]-glucosidase with hydrogen bonds.

Keywords: Phloridzin, [alpha]-Glucosidase, Inhibitory mechanism, Fluorescence spectroscopy, Molecular docking.

Introduction

[alpha]-glucosidase in human is one of key digestive enzymes, which is responsible for the hydrolysis and absorbance of carbohydrates [1-2]. It is thought to be one of the main factors related to the postprandial hyperglycemia [3]. As a result, many artificial synthetic [alpha]-glucosidase inhibitors (acarbose, voglibose, etc.) were applied to control the blood sugar level [4-5]. But the significant side effects of these inhibitors, such as digestion disorders, hepatotoxicity and flatulence, were also observed [6-7]. As a result, many studies tend to screen the natural [alpha]-glucosidase inhibitors in food and medicinal plants [8-9].

Phloridzin widely exists in the fruit, leaf, stem and root of the apple tree. And it was also found in Lithocarpus ploystachyus Reld [10]. It is reported that phloridzin possesses many health-promoting functions, such as antioxidant, hypoglycaemic, anticardiovascular and antitumor activities [11-13]. It was referred that its hypoglycaemic activity contributed to its inhibitory capacities on the digestive enzymes. In view of this, the [alpha]-glucosidase inhibitory mechanism of phloridzin was investigated by inhibitory test, fluorescence spectroscopy and molecular docking method in this study.

Experimental

Chemicals

Phloridzin was obtained from Aladdin (Shanghai, China). [alpha]-Glucosidase (from Saccharomyces cerevisiae) and p-nitrophenyl-[alpha]-D-glucopyranoside (pNP-G) were purchased from Sigma Aldrich (St. Louis, MO). The ultrapure water from a Thermo GenPure UV/UF water system (Waltham, MA) was used. All other chemicals were of analytical grade.

[alpha]-Glucosidase inhibition assay

According to the previous study [14], the [alpha]-glucosidase inhibition assay was carried out with pNP-G as the substrate, which could be hydrolyzed to p-nitrophenol with the maximum absorbance at 405 nm by [alpha]-glucosidase. 1 mL of [alpha]-glucosidase (0.2 U/mL) and 0.6 mL of phloridzin at the different concentration (0,0.2,0.4,0.6,0.8,1.0 mM) were mixed and held at 37 AdegC for 10 min. Then, 1 mL of pNP-G was added to the above solution. The obtain mixture was incubated at 37 AdegC for 10 min. After addition of 1 mL ethanol to terminate the reaction, the obtained solution was diluted to 5 mL. And its absorbance at 405 nm was measured to determine the [alpha]-glucosidase inhibitory activity of phloridzin.

Fluorescence spectroscopy

The fluorescence analysis was performed according the previous report [15]. 4.6 mL of [alpha]-glucosidase and 0.4 mL of phloridzin at the different concentration (0,4,8,12,16,20 mM) were mixed and incubated at 37 AdegC for 10 min. The fluorescence spectrum of the obtained mixture was measured by using an Agilent Cary Eclipse fluorescence spectrophotometer (Santa Clara, CA). The excitation wavelength was 295 nm while the emission wavelength range was 305-450 nm. And both the slit widths for excitation and emission were set at 10 nm.

Molecular docking method

The molecular docking of phloridzin and [alpha]-glucosidase was carried out by using the Surflex-Dock program of SYBYL 8.1 software (Tripos Inc., USA) [16]. The crystal structure of [alpha]-glucosidase (PDB ID 4J5T) was from RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). The Protomol generation was accomplished base on the ligand method. The values for Bloat and Threshold were set at 0.5 and 0, respectively. The other parameters were defaulted. The output docking modes were evaluated by the scoring functions. And the docking mode with large Cscore and Total Score was used [17].

Statistical method

The data obtained in this study were expressed as the mean of three replicate determinations plus or minus the standard deviation (SD). The statistical comparisons were made by Student's t-test. The p values of < 0.05 were considered to be significant.

Results and Discussion

[alpha]-Glucosidase inhibitory activity

The inhibition of [alpha]-glucosidase was thought to be one of the available methods to control blood sugar level. It was reported that the polyphenols such as flavonoids and phenolic acids could effectively inhibit the enzyme activity of [alpha]-glucosidase [18-19]. In this study, the inhibition activity of phloridzin against [alpha]-glucosidase was measured with pNP-G as the substrate. As shown in Fig. 1, the linear relationship between phloridzin concentration and [alpha]-glucosidase activity could be observed. It was found that phloridzin was an effective [alpha]-glucosidase inhibitor with the IC50 value of 0.68 mmol/L.

Binding behavior

The fluorescence spectroscopy was widely applied to investigate the binding behavior of bioactive compounds with enzymes [20]. The intrinsic fluorescence of the enzyme coming from its amino acid residues (Trp, Tyr, Phe, etc.) could be influenced by the presence of the inhibitors. As shown in Fig. 2, when the excitation wavelength of [alpha]-glucosidase was set at 295 nm, its maximum emission intensity could be found at 330 nm. With the increase of the addition amount of phloridzin, the fluorescence intensity of [alpha]-glucosidase was observed as a gradual decrease, which meant that the fluorescence quenching happened. The fluorescence quenching includes static and dynamic types. The static fluorescence quenching attributes to the formation of the complex of enzymes and inhibitors.

The dynamic fluorescence quenching comes from the crash between enzymes and inhibitors [21]. For the dynamic fluorescence quenching, its quenching constant (Kq) could be calculated according to the Stern-Volmer equation:

F0 / F = 1 + Kq A*T0[Q]

Where F0 was the fluorescence peak intensity of [alpha]-glucosidase, F was the fluorescence peak intensity of [alpha]-glucosidase in the presence of phloridzin, Kq was the quenching constant, T0 was the average life span of tryptophan of the enzyme (T0 =10-8 s), and [Q] was the concentration of phloridzin.

For the dynamic fluorescence quenching, its binding constant (KA) and the number of binding site (n) could be obtained by using the following Lineweaver-Burk equation:

lg[(F0 - F ) / F ] = lg K A + n lg[Q]

As shown in Table-1, the quenching constant (Kq, 6.1474x1012) was far higher than the maximum constant for dynamic fluorescence quenching (2.0x1010), which suggested that the fluorescence quenching of [alpha]-glucosidase caused by phloridzin belonged to the static fluorescence quenching. According to Lineweaver-Burk analysis (Table-2), the binding constant (KA) and the number of binding site (n) were 1.3243x105 and 1.076, respectively. The result suggested that [alpha]-glucosidase should form the complex with phloridzin at the molar ratio of 1:1, which led to the decrease of the enzyme activity of [alpha]-glucosidase.

Table-1: The Stern-Volmer quenching constant (Kq) of phloridzin to [alpha]-glucosidase.

###Stern-Volmer equation###r2###Kq

###F0/F=6.1474x104[Q]+1.0000###0.9970###6.1474x1012

Table-2: The binding constant (KA) and the number of binding site (n) of phloridzin to [alpha]-glucosidase.

###r2###KA###n

###Lineweaver-Burk equation

lg(F0- F)/F = 1.076lg[Q]+5.122###0.9980###1.3243x105###1.076

Binding mode

Although the complex of enzymes and inhibitors have been characterized by a lot of instrumental methods such as UV, CD, NMR. But these techniques only provide the indirect information about the binding modes, which hinders the study about the inhibitory mechanism. Flavonoids usually play the inhibitory role by binding the enzymes and forming the stable complex [22]. Molecular docking is a common method to study this geometric and energy matching process, which can provide the binding mode and mechanism of the enzyme and its inhibitors [23-24]. In this study, it was carried out to speculate the binding mode between [alpha]-glucosidase and phloridzin.

As shown in Fig. 3, the amino acid residues of [alpha]-glucosidase around phloridzin were as following: Met1, Glu2, Glu3, Tyr4, Gln5, Lys6, Phe7, Thr8, Asn9, Glu10, Arg28, Tyr29, Val30, Phe373, Glu374, Leu375, Phe376, Leu412, Ala413, Ser414, Trp415, Phe416, Glu417, Met418, Lys509, Lys510, Ile511. And phloridzin could bind the Lys510 and Glu374 of [alpha]-glucosidase with hydrogen bonds. Those intermolecular hydrogen bonds could strengthen the binding between [alpha]-glucosidase and phloridzin, which effectively inhibited the activity of [alpha]-glucosidase.

Conclusions

The interaction of phloridzin and [alpha]-glucosidase could lead to the decrease of the activity of [alpha]-glucosidase. The IC50 value of phloridzin was determined at 0.68 mmol/L. And the static fluorescence quenching of [alpha]-glucosidase caused by phloridzin could be observed. The molecular docking result showed that phloridzin could bind Lys510 and Glu374 of [alpha]-glucosidase by hydrogen bonding, which led to the loss of [alpha]-glucosidase activity.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (No. 31771941 and 31771975), the Foundation of Henan Educational Committee (No. 15A550005), the Program for Science and Technology Innovation Talents in Universities of Henan Province (No. 14HASTIT019) and Henan Research Program of Foundation and Advanced Technology (No. 162300410177).

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Publication:Journal of the Chemical Society of Pakistan
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Date:Aug 31, 2018
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