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Dietary Flavonoids and Acarbose Synergistically Inhibit alpha-Glucosidase and Lower Postprandial Blood Glucose Bowei Zhang, Xia Li, Wenlong Sun, Yan Xing, Zhilong Xiu, Chunlin Zhuang, and Yuesheng Dong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02531 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 7, 2017
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Journal of Agricultural and Food Chemistry
Dietary Flavonoids and Acarbose Synergistically Inhibit α-Glucosidase and Lower Postprandial Blood Glucose
1 2 3 4 5 6
Bo-wei Zhanga, Xia Lia, Wen-long Suna, Yan Xinga, Zhi-long Xiua, Chun-lin Zhuang*b, Yue-sheng
7
Dong*a
8
a
School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, Liaoning, China
9 10
b
School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China
11 12 13
Corresponding author: :
14
Yue-sheng Dong, Ph. D.
15
School of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong Road,
16
Dalian 116024. China; E-mail:
[email protected]; Tel./Fax: +86-411-84706344
Associate Professor
17 18
Chun-lin Zhuang, Ph. D. Associate Professor
19
School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433,
20
China; E-mail:
[email protected]; Tel: 86-21-81871258-804
21 22
Running title:
23
Synergistic inhibition of α-glucosidase by baicalein and acarbose. 1
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Abstract
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The inhibition of porcine pancreatic α-amylase and mammalian α-glucosidase by sixteen
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individual flavonoids was determined. The IC50 values for baicalein, (+)-catechin, quercetin, and
27
luteolin were 74.1 ± 5.6, 175.1 ± 9.1, 281.2 ± 19.2, and 339.4 ± 16.3 µM, respectively, against
28
α-glucosidase. The IC50 values for apigenin and baicalein were 146.8 ± 7.1 and 446.4 ± 23.9 µM,
29
respectively, against α-amylase. The combination of baicalein, quercetin or luteolin with acarbose
30
showed synergistic inhibition, and the combination of (+)-catechin with acarbose showed
31
antagonistic inhibition of α-glucosidase. The combination of baicalein or apigenin with acarbose
32
showed additive inhibition of α-amylase at lower concentrations and antagonistic inhibition at a
33
higher concentration. Kinetic studies of α-glucosidase activity revealed that baicalein alone, acarbose
34
alone, and the combination showed non-competitive, competitive, and mixed-type inhibition,
35
respectively. Molecular modeling revealed that baicalein had higher affinity to the non-competitive
36
binding site of maltase, glucoamylase, and isomaltase subunits of α-glucosidase, with glide scores of
37
-7.64, -6.98, and -6.88, respectively. (+)-Catechin had higher affinity to the active sites of maltase
38
and glucoamylase and to the non-competitive site of isomaltase. After sucrose loading, baicalein
39
dose-dependently reduced the postprandial blood glucose (PBG) level in mice. The combination of
40
80 mg/kg baicalein and 1 mg/kg acarbose synergistically lowered the level of PBG, and the
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hypoglycemic effect was comparable to 8 mg/kg acarbose. The results indicated that baicalein could
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be used as a supplemental drug or dietary supplement in dietary therapy for diabetes mellitus.
43 44
Key words:baicalein, acarbose, synergistic inhibition, postprandial blood glucose, α-glucosidase
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Journal of Agricultural and Food Chemistry
Introduction
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Diabetes is one of the most serious global health emergencies of the 21st century. The international
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diabetes federation estimated that approximately 415 million people suffered from diabetes and that
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there were 318 million people with impaired glucose tolerance in 2015. Diabetes is associated with
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metabolic disorders that occur when the body cannot produce enough insulin or cannot use insulin,
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and it is diagnosed by observing the level of fasting blood glucose (FBG) or PBG. Consistently high
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levels of blood glucose in diabetes patients can lead to serious complications that affect the heart,
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blood vessels, kidneys, nerves, and eyes.1 One therapeutic approach to treat diabetes is to delay the
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glucose absorption by inhibiting digestive enzymes such as α-amylase and α-glucosidases.2
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Dietary carbohydrates are hydrolyzed into disaccharides and polysaccharides by α-amylase, which
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is secreted by the salivary glands and pancreas.3 The polysaccharides and disaccharides are then
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processed
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maltase-glucoamylase and sucrase-isomaltase, each of which is composed of two active subunits
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located on the C- and N-terminus of their original proteins, respectively.5 All four subunits of the
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α-glucosidases can hydrolyze maltose. However, only the C-terminal subunit of sucrase-isomaltase
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can hydrolyze sucrose.6, 7 It has been reported that inhibiting the digestion of dietary carbohydrates
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can stop the progression of diabetes mellitus.8 Acarbose, an α-glucosidase inhibitor, can suppress the
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digestion of carbohydrates and consequently delay the absorption of glucose and reduce the
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postprandial hyperglycemia.9 However, the mechanism of the hypoglycemic effect of acarbose is
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limited to the inhibition of digestive enzymes, and acarbose was found to be less effective than
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metformin and sulfonylureas in long-term treatment of diabetes.10, 11
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into
monosaccharides
by
α-glucosidases.4
α-Glucosidases
are
divided
into
Growing evidence suggested that the combination of anti-diabetic natural products with acarbose 3
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can enhance the efficacy of acarbose and/or extend the simple mechanism of its hypoglycemic
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activity. The combination of berry extracts with acarbose showed synergistic inhibition of α-amylase
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and α-glucosidases.12 The combination of black tea extracts and acarbose exhibited a synergistic
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inhibition against intestinal α-glucosidase and a hypoglycemic effect.13 Cyanidin derivatives in
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combination with acarbose synergistically alleviate postprandial hyperglycemia in rats.14 Our
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previous research suggested that the co-administration of Oroxylum indicum seed extracts with
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acarbose could extend the mechanism of the hypoglycemic effect of acarbose and enhance the
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efficacy of acarbose by up to 5-fold in a 8-week treatment of diabetes in mice.15 In addition,
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individual inhibitions of α-amylase and α-glucosidases by natural plant extracts16 or phenolic
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compounds such as kaempferol,17 caffeic acid,18 and chlorogenic acid5 have been reported. These
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data suggested that, either administered individually or in combination, dietary constituents can be
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alternative agents to prevent and treat diabetes.
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Flavonoids are widely distributed in plants and are present in considerable quantities in human
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diets, including in vegetables, fruits, tea, and red wine, and are regarded as the major effective
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constituent.19, 20 Several clinical and basic research studies have shown that flavonoids have positive
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effects for the treatment of multiple diseases, including diabetes,20 obesity,21 cardiovascular
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diseases,22 and cancer.23 As a class of α-glucosidase inhibitors,24,
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PBG-controlling effects.26 A previous study and our previous research have shown that flavonoids or
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flavonoid-containing natural products exhibited synergistic PBG-lowering and α-glucosidase
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inhibitory effects with acarbose.14,
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includes at least six subgroups (including flavones, flavonols, flavanones, isoflavones, flavans-3-ol
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and anthocyanins).28 The synergistic PBG-lowering effect of the combination of flavonoids other
27
25
the flavonoids also display
However, flavonoids have a rich structural diversity that
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than cyanidin derivatives and acarbose is still unclear, and the mechanism of the synergistic effect of
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flavonoids and acarbose needs to be revealed.
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In the present study, the inhibition of α-glucosidases and α-amylase by dietary flavonoids and the
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combination with acarbose was screened. The individual and combined PBG-lowering effects of the
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selected compounds were evaluated in vivo. The mechanism of the synergism was analyzed and
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evaluated through in vitro and molecular docking studies.
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Materials and methods
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Chemicals
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Acarbose was purchased from Bayer Healthcare Co., Ltd (Berlin, Germany). All flavonoid
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standards were purchased from Must Biotechnology Co., Ltd (Chengdu, China). Starch, maltose and
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sucrose were purchased from Solarbio Co., Ltd (Beijing, China). Mammalian α-glucosidase was
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prepared by our lab. Porcine pancreatic α-amylase was purchased from Sigma Chemical Co (St.
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Louis, MO, USA). The glucose test kit (ACCU-CHEK) used for the in vivo assays was purchased
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from Roche Diagnostics GmbH (Germany). The glucose assay kit used for the in vitro assays was
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purchased from Nanjing Jiancheng Bioengineering Institute. (Jiangsu, China).
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α-Amylase assay
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α-Amylase (EC 3.2.1.1) activity was determined following the method previously reported with
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slight modifications.29 Briefly, baicalein or acarbose and 0.1 U/mL porcine pancreatic α-amylase
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were pre-incubated in phosphate-buffered saline (pH 7.0) for 15 min at 37°C. Fifty microliters (50
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µL) of 0.4% (w/v) starch as the substrate was added to the reaction mixture for a total volume of 200
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µL, and the reaction mixture was incubated at 37°C for 10 min. After the incubation, the reaction was
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stopped by the addition of 1 mL dinitrosalicylic (DNS) reagent (1% 3, 5-dinitrosalicylic acid, 0.2% 5
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phenol, 0.05% Na2SO3, and 1% NaOH in aqueous solution) to the reaction mixture. The mixtures
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were then heated for 10 min at 100°C. After the mixture was cooled to room temperature, the
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absorbance was read at 540 nm with a spectrophotometer. Acarbose was used as a positive control.
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α-Glucosidase assay
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The α-glucosidase solution was prepared as previously reported with minor modifications.30 The
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rats were sacrificed after a 16-h starvation period. The small intestinal tissues were excised and
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homogenized in 100 mM phosphate buffer saline with 14 mM NaCl (1:10 dilution; w/v). After a
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centrifugation for 15 min at 12000 g, the supernatant was collected and used as the enzyme solution.
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The α-glucosidase activity (maltase: EC 3.2.1.20, sucrase: EC 3.2.1.48) was determined following
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the method of a previous report.29 Briefly, 100 µL of the enzyme solution containing 0.002 U/mL of
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α-glucosidase activity and 100 µL of the sample (baicalein or acarbose) were pre-incubated for 15
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min at 37°C. Then, 100 µL of 27 mM sucrose or 9 mM maltose was added to the reaction mixture as
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the substrate. The mixture was then incubated for 30 min at 37°C. After the incubation, the reaction
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was terminated by incubating the reaction mixture at 100°C for 10 min. The amount of the liberated
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glucose in the supernatant was determined with a commercial assay kit based on the glucose oxidase
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method. Except for a change in the concentration of sucrose, experiments to determine the type of
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inhibition by baicalein on mammalian α-glucosidase activity were carried out in the same manner.
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The type of inhibition was determined by Lineweaver-Burk plots and further confirmed by a
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nonlinear regression analysis with the Levenberg-Marquardt algorithm (Sigma Plot; Systat Software
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Inc., San Jose, CA, USA).5 The nonlinear regression method, which could avoid additional errors
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introduced by linear regression, was also used to calculate the kinetic parameters.
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Evaluation of synergy in the α-glycosidase inhibition assay 6
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The inhibitors were added individually or in combination at different concentrations in the
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α-glycosidase assay. The combination index (CI), which is a quantification of the degree of inhibitor
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interactions based on the median-effect principle developed by Chou and Talalay, were determined
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using the Compusyn software.31
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The equation for the median-effect principle is as follows:
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(1)
140
where fa is the fraction affected by dose D, fu is the unaffected fraction (fu = 1 - fa), m is the
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coefficient signifying the shape of the dose-effect curve, D is the dose of the inhibitor, and Dm is the
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median-effect dose (IC50 in this article).
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The equation for the CI is expressed as follows: (2)
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where (D)1 and (D)2 are the doses of inhibitors that produce a certain level of inhibition in the
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combination system, and (Dx)1 and (Dx)2 are the doses of inhibitors added alone that lead to the
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same level of inhibition.
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The combined inhibition was divided into synergistic (CI1.1) inhibition, according to the CI values.
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Molecular docking
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Molecular docking was performed to predict the binding site and efficacy between the
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α-glucosidase proteins and flavonoids using the Schrodinger Maestro 10.2 software package.
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N-terminal sucrase-isomaltase (PDB: 3LPP), N-terminal maltase-glucoamylase (PDB: 2QMJ) and
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C-terminal maltase-glucoamylase (PDB: 3TOP) were examined in this study. All proteins were 7
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subjected to cavity analysis and ranked based on their expanded van der Waals volume for binding
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evaluation using Sitemap following the software instructions.32 For each protein structure, the four
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best ranked cavities were selected with SiteScores > 0.9 and volumes > 60. The protein preparation
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was performed following a previous report.33 The docking grid (32 × 32 × 32 Å) was generated
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based on the chosen binding sites. The original ligand within the PDB structure was then re-docked
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into the corresponding protein structure without any constraints. The reliability of the docking
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procedure was evaluated by comparing the CαRMSD between the positions of heavy atoms of the
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ligand in the calculated crystal structure. This procedure re-docked acarbose, miglitol, salacinol, and
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kotalanol with low CαRMSD values (Figure S1) and was used in the subsequent in silico evaluation.
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The 5 best conformations were generated based on the empirical glide score (kcal/mol). The
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interactions between the subunits of the α-glucosidase and flavonoids were predicted. Blind docking
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was also carried out using Autodock 4.2 to cross-check the docking results for active site binding,34
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with the following settings: maximum number of 25,000,000 energy evaluation, number of
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generations = 27,000, mutation rate = 0.02, maximum number of iterations = 300, and number of
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docking runs = 100. The grid size was 120 × 120 × 120 Å with a spacing of 0.375 Å centered on the
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active site for each protein.
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Animals
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Kunming mice (18~22 g, 6 weeks old) and SD rats (180~220 g) were purchased from Dalian
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Medical University SPF experimental animal center (Dalian, China). The animals were provided a
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standard rodent diet and free access to water and were maintained at a temperature of 20~22°C. The
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animals and protocols used in this study were approved by the Animal Care Committee of Dalian
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Medical University. 8
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Oral sugar tolerance test
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The oral sugar tolerance test was performed according to a previous report.35 Kunming mice that
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had been fasted for 16 h were randomly divided into several groups. A baicalein-loaded
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self-microemulsifying drug delivery system was prepared according to previous reports.36 The final
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concentrations of ethyl oleate, PEG 400 and tween-80 used in the oral sugar tolerance test were 2%,
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2% and 4% (w/w), respectively. The vehicle (control group); baicalein at 40, 80, and 240 mg/kg; or
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acarbose at 0.3, 1 and 8 mg/kg was orally administered to the mice. Sucrose or glucose was orally
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administered at 2 g/kg body weight simultaneously with the drugs. Blood samples were collected
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from the tail vein at 0, 30, 60 and 120 min, and the levels of blood glucose were measured with a
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glucose test kit.
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Statistical Analysis
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The statistical analysis was performed using SPSS 17.0. All the results were expressed as the mean
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± standard deviation. Comparisons between groups were analysed using one-way ANOVA followed
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by Fisher LSD test. A p value less than 0.05 was judged as statistically significant. The concentration
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of the inhibitors that caused 50% inhibition (IC50) of the enzyme activity was calculated by nonlinear
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regression.
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Results and Discussion
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Individual inhibition of mammalian α-glucosidase and porcine pancreatic α-amylase by
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flavonoids
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Sixteen dietary flavonoids, which have various hydroxylation and glycosylation patterns across the
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A, B, or C rings and belong to 5 subgroups of flavonoids, including flavones, flavonols, flavanones,
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isoflavones, and flavans-3-ol (Figure 1), were selected and tested for their inhibition of mammalian 9
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α-glucosidase and porcine pancreatic α-amylase.
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For the maltose-hydrolyzing activity, the IC50 value of acarbose was 0.4 ± 0.1 µM, which was
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similar to that reported previously.37 Baicalein and (+)-catechin showed the strongest inhibition
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among the tested flavonoids with IC50 values of 74.1 ± 5.6 µM and 175.1 ± 9.1 µM (Table 1, Figure
203
S2), respectively. The IC50 value for baicalein was similar to that in a previous report.38 Quercetin
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and luteolin showed weaker inhibition than baicalein, with IC50 values of 281.2 ± 19.2 µM and 339.4
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± 16.3 µM, respectively. The IC50 values for the other flavonoids were greater than 400 µM (Table 1).
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Comparison of IC50 values showed that the inhibition of maltase by baicalein was 1/200 of that by
207
acarbose.
208
For the sucrose-hydrolyzing activity, the IC50 value for acarbose was 1.2 ± 0.3 µM. Baicalein
209
showed the strongest inhibition among the tested flavonoids. The IC50 value for baicalein was 14.6 ±
210
2.7 µM (Table 1), which was 1/12 of that of acarbose. The relative inhibition intensity of baicalein to
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acarbose showed that baicalein exhibited a stronger inhibition against sucrase than maltase. The
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inhibitory activities of all the other tested flavonoids were weak at 400 µM (Table 1).
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For the α-amylase activity, the IC50 value for acarbose was 5.3 ± 3.1 µM (Table 1), which is
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consistent with the previously reported value of 6.46 µM.35 Apigenin showed the strongest inhibition
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(Table 1). Luteolin, baicalein, quercetin and kaempferol showed weaker inhibition than apigenin.
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The IC50 values for apigenin and baicalein were 146.8 ± 7.1 µM and 446.4 ± 23.9 µM. Comparison
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of IC50 values suggested that the inhibitory activity of apigenin and baicalein were 1/28 and 1/84 of
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that of acarbose, respectively.
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The inhibition of individual flavonoids against α-glucosidase and α-amylase revealed a
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structure-activity relationship. Generally, the tested flavonoids showed relatively stronger individual 10
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inhibition of the maltose-hydrolyzing activity than sucrose-hydrolyzing activity. For the
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maltose-hydrolyzing activity, the fact that baicalein showed stronger inhibition than chrysin
223
suggested that the 6-hydroxyl group is important for exerting inhibition (Figure 1, Table 1), which is
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consistent with a previous report.38 The importance of the 3'- and 4'-hydroxyl groups can be shown
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by comparing the inhibition of luteolin and apigenin, scutellarin and baicalin, quercetin and
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kaempferol, respectively. The importance of the 6-hydroxyl group was greater than that of the
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4'-hydroxyl group as baicalein showed a stronger inhibition than apigenin. The introduction of
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7-glucuronic acid group decreased the inhibition as baicalein showed a stronger inhibitory activity
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than baicalin. Genistein showed very weak inhibition compared with apigenin, suggesting that the
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linkage of the B-ring at the 2-position is important for the inhibitory activity. (+)-Catechin showed
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stronger inhibition than quercetin, suggesting that the lack of a 4-carbonyl group in the C-ring can
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enhance the inhibition.
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For α-amylase activity, comparisons of the inhibition by apigenin vs chrysin and scutellarin vs
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baicalin, showed that the 4'-hydroxyl group were important (Table 1). The fact that baicalein showed
235
stronger inhibition than chrysin suggested that the 6-hydroxyl group is important for the inhibitory
236
activity. The inhibition by apigenin was greater than that of naringenin, suggesting that the nearly
237
planar structure of the C-ring is important for the inhibitory activity. Apigenin exhibited stronger
238
inhibition than genistein, suggesting that linkage of the B-ring at the 2-position can enhance the
239
inhibitory activity.
240
Combined inhibition of mammalian α-glucosidase and porcine pancreatic α-amylase by
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flavonoids and acarbose
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The combined inhibition of flavonoids with acarbose against α-glucosidase and α-amylase were 11
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determined. CI values, a quantitative measurement for the evaluation of combined effect, were used
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to assess whether combinations provided more (synergistic, CI1.1) inhibitions against α-glucosidase and α-amylase than
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those of flavonoids and acarbose alone.31
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For the maltose-hydrolyzing activity, the combination of baicalein and acarbose strongly enhanced
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the inhibition compared with individual compounds. For example, the combination of baicalein at
249
17.5 µM and acarbose at 0.194 µM caused 62.4% inhibition of the activity of α-glucosidase (Figure
250
2A), which is higher than the inhibition caused by baicalein alone at 35.0 µM or acarbose alone at
251
0.387 µM (twice the concentration of each compound). The calculated CI values were less than 0.41
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at all dosages, suggesting a strongly synergistic interaction between baicalein and acarbose. The CI
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values for the combinations of either quercetin or luteolin with acarbose were between 0.51 and 0.83,
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which suggests moderate synergism (Figure 2B and 2C). For the combination of (+)-catechin and
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acarbose, the CI values were less than 0.9 at low concentrations but were higher than 1.1 at the
256
highest concentration, which suggests antagonism (Figure 2D). No obvious enhancement was found
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when acarbose was combined with the flavonoids that displayed weak or no individual inhibition
258
(data not shown), suggesting that inhibitory activity of each compounds in the combination was
259
necessary for exerting synergistic inhibition.
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For the sucrose-hydrolyzing activity, the combination of baicalein and acarbose strongly enhanced
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the inhibition compared with the individual compounds (Figure 3). The CI values were between 0.61
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and 0.70 at lower concentrations (3.19, 6.39, and 10.7 µM for baicalein), suggesting synergism. And
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the CI value was 0.39 at a higher concentration (14.8 µM for baicalein), suggesting strong synergism.
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These data indicated that the synergistic inhibition on sucrase became stronger at higher 12
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concentration of baicalein and acarbose.
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For the α-amylase activity, the combinations of flavonoids with acarbose did not show any
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obvious enhancement of the inhibition, compared with that observed for acarbose or the various
268
flavonoids added individually. For example, the combination of baicalein at 148 µM and acarbose at
269
1.86 µM caused 35.1% inhibition of the activity of α-amylase, which is lower than the inhibition
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caused by baicalein alone at 296 µM and close to the inhibition caused by acarbose at 3.72 µM
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(twice the concentration of each compound). Most calculated CI values were higher than 0.9,
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suggesting additive inhibition or antagonistic inhibition. The only exception was the combination of
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apigenin at 46.3 µM and acarbose at 0.93 µM, with a CI value of 0.71, suggesting weak synergistic
274
inhibition. (Figure 4).
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Because the combined effect cannot be evaluated by a simple arithmetic sum of the inhibition
276
produced by individual inhibitors, it is difficult to differentiate the synergistic, antagonistic and
277
additive effects, until Chou and Talalay introduced CI value to distinguish the three types of
278
inhibition. This method have been widely used for the evaluation of drug combination.39 Using this
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method, we determined the synergistic inhibition of α-glucosidase by the combination of baicalein,
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luteolin, and quercetin with acarbose. Boath et al has reported a synergistic effect of acarbose and
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black currant extracts.40 However, no synergistic inhibitory interaction of flavones or flavonol
282
compounds with acarbose has been reported. Interestingly, (+)-catechin showed antagonistic but not
283
synergistic effect at higher concentrations in our present work. Gao et al has reported that the
284
combination of epigallocatechin gallate and acarbose showed synergistic inhibition at lower
285
concentrations and antagonistic inhibition at higher concentrations, and that the CI values increased
286
with the concentrations of the inhibitors.41 Although these authors used yeast α-glucosidase in their 13
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studies, their results were comparable to our observation.
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When combined with acarbose, baicalein showed a lower CI value against maltose-hydrolysis than
289
those of luteolin and quercetin, indicating stronger synergistic inhibition. This result suggests that
290
either the introduction of 6-hydroxyl group or the removal of 3'-, 4'-hydroxyl groups is important for
291
exerting synergism (Figure 1). The fact that quercetin showed stronger synergistic inhibition with
292
acarbose than that of (+)-catechin suggested that the introduction of a 4-carbonyl group in the C-ring
293
could enhance the synergism.
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Type of inhibition by baicalein and acarbose on mammalian α-glucosidase
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The types of inhibition by baicalein and its combination with acarbose against mammalian
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α-glucosidase were determined by both Lineweaver-Burk and Michaelis-Menton plots. The
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Lineweaver-Burk plots of acarbose generated straight lines which had intersections on the Y-axis,
298
suggesting competitive inhibition (Figure 5A). This data was consistent with the previous report.42
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The straight lines generated by baicalein alone and the combination had intersections on the X-axis
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and in the second quadrant, suggesting non-competitive and mixed-type inhibitions, respectively.
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The types of inhibition were further confirmed by the Michaelis-Menton plots using the best fit of
302
nonlinear regression analysis with the Levenberg-Marquardt algorithm. Compared with the control,
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the addition of acarbose significantly increased the Michaelis constant (Km), but the maximum
304
reaction velocity (Vmax) was not significantly changed; the addition of baicalein significantly reduced
305
the Vmax, but the Km was not significantly changed (Figure 5B, Figure 5C, and Table 2). When a
306
combination of baicalein and acarbose was added in a fixed ratio, the Km value was significantly
307
higher than those of both acarbose alone and the control, and the Vmax was significantly lower than
308
that of the control (Figure 5D and Table 2). The values of Km, Vmax, and their patterns confirmed 14
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that baicalein alone, acarbose alone, and the combination exhibited non-competitive, competitive,
310
and mixed-type inhibition, respectively. Besides, the significantly higher Km value of the
311
combination than that of acarbose alone also indicated that the combination caused greater reduction
312
of the affinity of enzyme for substrate. This result indicated that, when used in combination, the
313
inhibitors had stronger binding capacity at the competitive binding site, i.e. active site or
314
substrate-binding site. The fact that baicalein alone did not change the Km value suggested that
315
baicalein, a non-competitive inhibitor, cannot bind to the competitive binding site and thereby reduce
316
the affinity for substrate. Thus, baicalein is expected to bind to the non-competitive binding site and
317
enhance the affinity of enzyme for acarbose, and finally reduce the affinity of enzyme for substrate.
318
This speculation was supported by a previous study, in which black tea extract was found to be a
319
non-competitive inhibitor against α-glucosidases and it exhibited synergism with acarbose.
320
Furthermore, black tea extract also reduced the affinity of enzyme for substrate.13 However, further
321
study is still needed to clarify the exact change in the α-glucosidases caused by flavonoid binding.
322
On the other hand, Adisakwattana et al also reported that cyanidin-3-rutinoside, another
323
non-competitive inhibitor of α-glucosidase, showed synergistic inhibition with acarbose.14 Taking
324
these data into consideration, binding at the non-competitive site of α-glucosidase might be the basic
325
requirement for an inhibitor that could exert synergistic inhibition with acarbose.
326
Molecular docking
327
To identify the binding sites for the flavonoids in the intestinal glucosidases, we performed
328
molecular docking for baicalein, (+)-catechin, quercetin, luteolin, and acarbose with α-glucosidases
329
using Schrodinger software. As no crystal structure of rat α-glucosidase is currently available, crystal
330
structures
of
three
available
human
α-glucosidase
subunits,
15
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2QMJ
(N-terminal
of
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331
maltase-glucoamylase), 3TOP (C-terminal of maltase-glucoamylase) and 3LPP (N-terminal of
332
sucrase-isomaltase), were used. The sequence homology of the three subunits to their corresponding
333
human counterpart is 92%, 90%, and 86%, respectively, within 10 Å around the predicted binding
334
sites (Figure S3-S5) of baicalein. Four binding sites in each protein were chosen according to the
335
site score values that were calculated based on their expanded van der Waals volume for binding
336
evaluation and ranked using the sitemap panel (Figure 6), which was similar to those previously
337
reported.32 For each protein, the active site was included in the chosen sites.
338
For N-terminal maltase-glucoamylase (2QMJ), the glide score for acarbose in the active site (site 2)
339
was -7.34 kcal/mol. Baicalein was predicted to bind to site 1, with a better glide score of -7.64
340
kcal/mol compared to the interaction with the active site (site 2, -5.50 kcal/mol). Three major
341
interactions were observed to contribute to the maintenance of the binding: (a) hydrogen bonds
342
between Phe535 and Ser521 and the hydroxyl group in the A-ring (Figure 7); (b) a hydrogen bond
343
between Lys776 and the carbonyl group in the C-ring; and (c) hydrophobic interactions in a large
344
pocket (Val779, Leu286, Ala780, Pro287 and Ile523). (+)-Catechin was predicted to bind to the
345
active site (site 2), with a glide score of -6.55 kcal/mol. Like acarbose, (+)-catechin bound to the -1
346
and +1 sugar-binding site residues Asp327, Asp443 and Asp542 through hydrogen bonds with the
347
hydroxyl groups in the B- and C-rings. Luteolin and quercetin were predicted to bind to site 1, with
348
glide scores of -8.15 and -7.05 kcal/mol, respectively. The H-bonding interactions of hydroxyl and
349
carbonyl groups with Val779, Asp777, Lys776, Phe535, and Ser521 for luteolin and Asp777, Lys776,
350
Phe535, and Ser521 for quercetin were predicted to be the predominant interactions (Figure S6A,
351
S6B).
352
For C-terminal maltase-glucoamylase (3TOP), the glide score for acarbose in the active site (site 3) 16
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was -8.00 kcal/mol (Table 4). Baicalein was predicted to bind to site 1, with a glide score (-6.98
354
kcal/mol) better than that for its interaction with active site (site 3, -5.70 kcal/mol). The hydroxyl
355
group in the A-ring formed hydrogen bonds with Pro1327 and Glu1284 (Figure 8). The carbonyl
356
group in the C-ring formed hydrogen bonds with Leu1291. The B-ring formed hydrophobic
357
interactions with Pro1405 and Leu1401. (+)-Catechin was predicted to bind to the active site (site 3)
358
with a glide score of -7.70 kcal/mol. Like acarbose, (+)-catechin bound to the -1 and +1
359
sugar-binding site residues His1584, Asp1279, Asp1526, Arg1510, and Asp1157 through hydrogen
360
bonds. Luteolin and quercetin was predicted to bind to active site (site 3) with glide scores of -7.52
361
and -7.19 kcal/mol, respectively. The H-bonding interactions of hydroxyl and carbonyl groups with
362
His1584, Asp1526, Asp1279, and Asp1157 for both luteolin and quercetin were predicted to be the
363
predominant interactions (Figure S6C, S6D).
364
For N-terminal sucrase-isomaltase (3LPP), the glide score for acarbose in the active site (site 3)
365
was -7.13 kcal/mol (Table 4). Baicalein, luteolin, quercetin and (+)-catechin were predicted to bind
366
to site 2, with better glide scores of -6.88, -6.47, -5.97, and -8.79 kcal/mol, respectively, compared
367
with their interactions with the active site (site 3, -4.68, -4.98, -4.98, and -5.81 kcal/mol,
368
respectively). Asp806 formed hydrogen bonds with the hydroxyl group in the A-ring of baicalein
369
(Figure 9). The residues (His600, Ile552, and Gly562) formed hydrogen bonds with the hydroxyl
370
group of (+)-catechin. The B-rings of both baicalein and (+)-catechin formed hydrophobic
371
interactions in a large pocket (Phe551, Ile552, Ala565, and Ala566).
372
For all three subunits of the α-glucosidases, the non-competitive site was predicted to have a much
373
higher binding affinity to baicalein. Quercetin and luteolin were predicted to have higher affinities
374
for the non-competitive site of maltase (2QMJ) and isomaltase (3LPP) and the active site of 17
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375
glucoamylase (3TOP). In contrast, (+)-catechin was predicted to have a higher affinity for the active
376
sites of maltase (2QMJ) and glucoamylase (3TOP) but a higher affinity for the non-competitive site
377
for isomaltase (3LPP). These results are consistent with the previous reports that (+)-catechin is a
378
non-competitive inhibitor of isomaltase (3LPP) but could bind to different binding sites of maltase
379
and glucoamylase.5 These observations, together with the facts that baicalein displayed stronger
380
synergism than quercetin and luteolin and (+)-catechin displayed antagonism at higher
381
concentrations in the enzyme assays, suggested that non-competitive binding in the α-glucosidases
382
would enhance the synergistic inhibition, whereas competitive binding at the active sites of
383
α-glucosidases at higher concentrations might lead to antagonistic inhibition between flavonoids and
384
acarbose. In addition, H-bonds were formed between the carbonyl group of baicalein and the
385
residues in the non-competitive sites when binding to maltase (2QMJ) and glucoamylase (3TOP) but
386
not isomaltase (3LPP), which suggested that the carbonyl group in the flavonoids may be important
387
in producing the non-competitive inhibition against maltase (2QMJ) and glucoamylase (3TOP).
388
Hypoglycemic effect of baicalein and co-administration of acarbose
389
The hypoglycemic effect of baicalein and co-administration of acarbose was evaluated through
390
sucrose tolerance test in mice, and the time courses for the levels of blood glucose were measured
391
before and 30, 60, 90, and 120 min after sucrose loading (2 g/kg) (Figure 10). The PBG level of the
392
control group increased and peaked at 30 minutes and then decreased. The baicalein- and
393
acarbose-administered groups exhibited dose-dependent decrease in the level of PBG compared with
394
the control group. Baicalein at 80 and 200 mg/kg and acarbose at 1 and 8 mg/kg decreased the levels
395
of PBG significantly 30 min after sucrose loading (Figure 10A and Figure 10B). The combination
396
of baicalein at 80 mg/kg and acarbose at 1 mg/kg significantly reduced the levels of PBG 30 and 60 18
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min after sucrose loading compared with the control group. This reduction was greater than that
398
observed in the groups that were administered the individual agents, 80 mg/kg baicalein or 1 mg/kg
399
acarbose (Figure 10C). PBG levels after the co-administration of baicalein (80 mg/kg) and acarbose
400
(1 mg/kg) were not significantly different from those receiving 8 mg/kg acarbose alone. Our data
401
suggested that the co-administration of baicalein with acarbose generated a synergistic hypoglycemic
402
effect in vivo, which was consistent with the synergistic effects of combination in vitro. The effective
403
dose of acarbose could be reduced by 87.5%.
404
The glucose tolerance test was also performed to exclude the influences other than carbohydrate
405
digestion. Glucose was administered orally to the fasted mice. No significant difference in PBG
406
levels was observed among the baicalein (80 mg/kg), acarbose (8 mg/kg), and control groups
407
(Figure 10D). This result suggested that the PBG lowering effect of baicalein resulted from the
408
inhibition of carbohydrate digestion.
409
We reported that baicalein could enhance the efficacy of acarbose both in vitro and in vivo for the
410
first time. It is known that the hypoglycemic effect of acarbose is limited to the inhibition of
411
carbohydrate digestion. Long-term treatment with baicalein, on the other hand, has also been
412
reported to have an anti-diabetic effect through the promotion of islet β-cell function,43 the activation
413
of AMPKα/IRS/Akt pathway,44 the suppression of inflammatory responses,45 and the reduction of
414
oxidative stress.46 Thus, baicalein and acarbose can inhibit the α-glucosidase in the gut lumen
415
synergistically to prevent hyperglycemia. And the absorbed baicalein is expected to increase the
416
insulin resistance, improve the complications and restore the pancreatic function. Therefore,
417
baicalein might extend the simple mechanism of acarbose alone, and enhance the efficacy of
418
acarbose. Baicalein alone also showed PBG lowering effect in vivo. Thus, our present work is 19
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419
expected to expand the usage of baicalein in diabetes treatment as a dietary supplement.
420
Flavonoids are widely distributed in plants and are found in considerable quantities in human diets,
421
such as fruit, vegetables, nuts, seeds, fruits, tea, and red wine. Baicalein was found in abundance in
422
several natural products such as Oroxylum indicum and Scutellaria baicalensis.45 The dose of
423
baicalein in our in vivo study was 80 mg/kg. Therefore, the dose for human administration was
424
estimated to be 8.8 mg/kg using body surface area model,47, 48 which corresponds to approximately 3
425
times the average daily human intake of flavonoids.49 This is a relatively safe dose, on the basis that
426
treatments with baicalein at 800 mg twice a day for 10 days50 or a single dose of 100-2800 mg51 were
427
reported to be safe and well tolerated by volunteers. Besides, all the adverse events reported,
428
including blurred vision and plasma fibrinogen decreased, were rated as “mild” and resolved without
429
further treatment. Therefore, baicalein alone or the combination could be regarded as a low-toxicity
430
agent.
431
In conclusion, the combination of baicalein, quercetin, or luteolin with acarbose synergistically
432
inhibited α-glucosidase in vitro, and baicalein lowered PBG levels in vivo. The mechanism of the
433
synergistic inhibition is the non-competitive inhibition of the α-glucosidases by the flavonoids and
434
acarbose. These results indicated the potential of baicalein for reducing the effective dose and
435
extending the simple mechanism of the hypoglycemic effect of acarbose. Also, baicalein may be
436
used as a supplemental drug or dietary supplement for diabetes mellitus.
437
Abbreviations Used
438
Postprandial blood glucose (PBG)
439
Fasting blood glucose (FBG)
440
Oroxylum indicum seed extracts (OISE) 20
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441
Dinitrosalicylic (DNS)
442
Combination index (CI)
443
Funding
444
This work was supported by the National Natural Science Foundation of China (81172966 and
445
81502978), Shanghai “ChenGuang” project (16CG42), and Open Fund of Key Laboratory of
446
Biotechnology and Bioresources Utilization (Dalian Minzu University), State Ethnic Affairs
447
Commission & Ministry of Education, China.
448
Supporting information description
449
The re-docked acarbose, miglitol, salacinol and kotalanol and the CαRMSD values (Figure S1).
450
The Individual inhibition of α-amylase by acarbose (A) and baicalein (B) (Figure S2). The amino
451
acid sequence of human maltase (2QMJ) and the sequence homology compared with rat maltase
452
(Figure S3). The amino acid sequence of human glucoamylase (3TOP) and the sequence homology
453
compared with rat maltase (Figure S4). The amino acid sequence of human isomaltase (3LPP) and
454
the sequence homology compared with rat maltase (Figure S5). The binding positions and
455
interaction patterns of luteolin and quercetin with α-glucosidases (Figure S6).
21
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Tables Table 1. Individual inhibition of flavonoids against mammalian α-glucosidase and porcine pancreatic α-amylase Maltase
Sucrase
α-Amylase
Flavonoid IC50 (µM)
Inhibitiona (%)
IC50 (µM)
Inhibitiona (%)
IC50 (µM)
Inhibitiona (%)
Baicalein
74.1 ± 5.6
81.3 ± 4.1
14.6 ± 2.7
80.0 ± 6.2
446.4 ± 23.9
47.2 ± 3.8
Chrysin
> 400
4.2 ± 2.6
> 400
5.5 ± 2.1
> 400
24.5 ± 3.0
Luteolin
339.4 ± 16.3
53.9 ± 3.8
> 400
16.0 ± 0.9
> 400
54.3 ± 4.4
Apigenin
> 400
24.6 ± 2.9
> 400
8.9 ± 3.7
146.8 ± 7.1
79.3 ± 2.9
Scutellarin
> 400
44.1 ± 3.4
> 400
25.2 ± 1.4
> 400
-7.4 ± 1.0
Baicalin
> 400
4.8 ± 2.0
> 400
26.6 ± 4.2
> 400
-4.9 ± 2.2
Quercetin
281.2 ± 19.2
58.7 ± 4.8
> 400
16.0 ± 2.5
> 400
45.7 ± 4.1
Rutin
> 400
41.2 ± 5.1
> 400
-3.9 ± 3.1
> 400
-5.4 ± 2.6
Kaempferol
> 400
5.1 ± 1.2
> 400
16.5 ± 2.2
> 400
41.8 ± 2.3
Dihydromyricetin
> 400
27.6 ± 3.1
> 400
0.5 ± 0.2
> 400
4.1 ± 0.7
Hesperetin
> 400
7.4 ± 2.2
> 400
3.1 ± 1.1
> 400
3.2 ± 1.1
Naringenin
> 400
7.2 ± 2.4
> 400
16.0 ± 1.9
> 400
20.6 ± 2.8
Puerarin
> 400
28.1 ± 3.6
> 400
15.3 ± 2.4
394.2 ± 17.1
52.1 ± 4.6
Daidzein
> 400
-6.8 ± 2.4
> 400
14.7 ± 3.0
> 400
21.4 ± 2.6
Genistein
> 400
2.4 ± 1.2
> 400
-14.1 ± 2.3
> 400
32.4 ± 2.0
175.1 ± 9.1
71.7 ± 4.7
> 400
23.9 ± 2.5
> 400
-11.1 ± 1.9
0.4 ± 0.1
96.5 ± 7.4
1.2 ± 0.3
90.1 ± 5.6
5.3 ± 3.1
86.2 ± 6.1
Flavone
Flavonol
Flavanone
Isoflavone
Flavans-3-ol (+)-Catechin Acarbose a
Inhibition by 400 µM flavonoids. The values shown are the averages of triplicate assays. 25
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Table 2. Kinetic parameters for mammalian sucrase Km (mM)
Vmax (µM/min)
Control
15.2 ± 1.9
387 ± 40.8
Baicalein
14.1 ± 2.2
275 ± 34.9#
Acarbose
29.5 ± 2.3#
388 ± 31.7
Acarbose + Baicalein (1:9.56)
43.6 ± 4.1#†
247 ± 20.4#
The values shown are the averages of triplicate assays. #p < 0.01 compared with the control group, and †p < 0.01 compared with the acarbose alone group.
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Table 3. The binding glide scores of acarbose, baicalein, luteolin, quercetin, and (+)-catechin on α-glucosidase proteins Docking glide score (kcal/mol) Binding Site a
Site Score
baicalein
luteolin
quercetin
(+)-catechin
acarbose
2QMJ (maltase)
1 2 (active site) 3 4
1.06 0.96 0.95 0.95
-7.64 -5.50 -6.16 -4.54
-8.15 -5.41 -7.10 -6.48
-7.05 -5.39 -7.03 -6.59
-6.01 -6.55 -6.04 -6.12
-6.73 -7.34 -5.37 -5.13
3TOP (glucoamylase)
1 2 3 (active site) 4
1.12 1.05 1.03 0.96
-5.08 -6.98 -5.70 -6.11
-7.40 -6.73 -7.52 -5.39
-7.15 -6.93 -7.19 -5.88
-6.86 -6.96 -7.70 -7.19
-5.71 -5.98 -8.00 -5.12
1 2 3 (active site)
1.11 1.08 1.00
-5.91 -6.88 -4.68
-5.93 -6.47 -4.95
-5.05 -5.97 -4.98
-5.93 -8.79 -5.81
N.C.b -4.97 -7.13
4
0.92
-6.49
-6.24
-5.92
-5.93
-5.54
3LPP (isomaltase)
a
The binding site underlined was predicted to have the strongest affinity with baicalein.
b
No binding conformation was produced.
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Figure legends Figure 1. The structures of the flavonoids and the structure-activity relationships of flavonoids on the inhibition of mammalian α-glucosidase. Figure 2. The inhibitory effect of the combination of acarbose and flavonoids against mammalian α-glucosidase. The inhibitory effect of the combination of acarbose with baicalein (A), quercetin (B), luteolin (C) or (+)-catechin (D), using maltose as substrate. CI values above data points were calculated by CompuSyn software. CI1.1 indicate synergism, additive effect, and antagonism, respectively. The values shown are the means of triplicate assays ± standard error. Figure 3. The inhibitory effect of the combination of acarbose and baicalein against mammalian α-glucosidase, using sucrose as substrate. CI values above data points were calculated by CompuSyn software. CI1.1 indicate synergism, additive effect, and antagonism, respectively. The values shown are the means of triplicate assays ± standard error. Figure 4. The inhibitory effect of the combination of acarbose with flavonoids against porcine pancreatic α-amylase and mammalian α-glucosidase. The inhibition of the combination of acarbose with apigenin (A) or baicalein (B) against porcine pancreatic α-amylase. CI values above data points were calculated by CompuSyn software. CI1.1 indicate synergism, additive effect, and antagonism, respectively. The values shown are the averages of triplicate assays ± standard error. Figure 5. Lineweaver-Burk plots of the combination (A) and Michaelis-Menton plots of acarbose alone (B), baicalein alone (C), the combination of baicalein and acarbose (D), against mammalian α-glucosidase, using sucrose as substrate. The values shown are the means of triplicate assays ± standard error. 28
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Figure 6. The selected binding sites of maltase (2QMJ), glucoamylase (3TOP), and isomaltase (3LPP). Figure 7. Binding sites for acarbose, baicalein, and (+)-catechin in the N-terminal maltase-glucoamylase (2QMJ) protein. The atoms of the protein are colored as follows: carbon – green, oxygen – red, nitrogen – blue. The carbon atoms of acarbose are colored light blue; the carbon atoms of baicalein are colored orange; the carbon atoms of (+)-catechin are colored purple. The hydrogen bonds are shown by yellow dashed lines. Figure
8.
Binding sites of acarbose, baicalein, and (+)-catechin
in the C-terminal
maltase-glucoamylase (3TOP) protein. The atoms of the protein are colored as follows: carbon – green, oxygen – red, nitrogen – blue. The carbon atoms of acarbose are colored light blue; the carbon atoms of baicalein are colored orange; the carbon atoms of (+)-catechin are colored purple. The hydrogen bonds are shown by yellow dashed lines. Figure
9. Binding sites of acarbose, baicalein, and (+)-catechin in the
N-terminal
sucrase-iasomaltase (3LPP) protein. The atoms of the protein are colored as follows: carbon – green, oxygen – red, nitrogen – blue. The carbon atoms of acarbose are colored light blue; the carbon atoms of baicalein are colored orange; the carbon atoms of (+)-catechin are colored purple. The hydrogen bonds are shown by yellow dashed lines. Figure 10. PBG-lowering effect of baicalein or acarbose alone or their combination. Baicalein and/or acarbose and sucrose (A, B and C) or glucose (D) were orally administered to the mice. The values represent the means ± SEM (n=10; *p < 0.05, #p < 0.01 compared with control group.).
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Figures
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Figure 2.
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Figure 3.
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Figure 4.
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Figure 5.
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Figure 6.
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Figure 7.
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Figure 8.
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Figure 9.
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Figure 10.
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Graphic abstract for table of contents
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