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Sep 6, 2017 - The IC50 values for apigenin and baicalein were 146.8 ± 7.1 and 446.4 ±. 23.9 μM, respectively, against α-amylase. The combination o...
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Dietary Flavonoids and Acarbose Synergistically Inhibit α‑Glucosidase and Lower Postprandial Blood Glucose Bo-wei Zhang,† Xia Li,† Wen-long Sun,† Yan Xing,† Zhi-long Xiu,† Chun-lin Zhuang,*,‡ and Yue-sheng Dong*,† †

School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, Liaoning, China School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China

‡

S Supporting Information *

ABSTRACT: The inhibition of porcine pancreatic α-amylase and mammalian α-glucosidase by 16 individual flavonoids was determined. The IC50 values for baicalein, (+)-catechin, quercetin, and luteolin were 74.1 ± 5.6, 175.1 ± 9.1, 281.2 ± 19.2, and 339.4 ± 16.3 μM, respectively, against α-glucosidase. The IC50 values for apigenin and baicalein were 146.8 ± 7.1 and 446.4 ± 23.9 μM, respectively, against α-amylase. The combination of baicalein, quercetin, or luteolin with acarbose showed synergistic inhibition, and the combination of (+)-catechin with acarbose showed antagonistic inhibition of α-glucosidase. The combination of baicalein or apigenin with acarbose showed additive inhibition of α-amylase at lower concentrations and antagonistic inhibition at a higher concentration. Kinetic studies of α-glucosidase activity revealed that baicalein alone, acarbose alone, and the combination showed noncompetitive, competitive, and mixed-type inhibition, respectively. Molecular modeling revealed that baicalein had higher affinity to the noncompetitive binding site of maltase, glucoamylase, and isomaltase subunits of αglucosidase, with glide scores of −7.64, −6.98, and −6.88, respectively. (+)-Catechin had higher affinity to the active sites of maltase and glucoamylase and to the noncompetitive site of isomaltase. After sucrose loading, baicalein dose-dependently reduced the postprandial blood glucose (PBG) level in mice. The combination of 80 mg/kg baicalein and 1 mg/kg acarbose synergistically lowered the level of PBG, and the hypoglycemic effect was comparable to 8 mg/kg acarbose. The results indicated that baicalein could be used as a supplemental drug or dietary supplement in dietary therapy for diabetes mellitus. KEYWORDS: baicalein, acarbose, synergistic inhibition, postprandial blood glucose, α-glucosidase

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INTRODUCTION Diabetes is one of the most serious global health emergencies of the 21st century. The international diabetes federation estimated that approximately 415 million people suffered from diabetes and that there were 318 million people with impaired glucose tolerance in 2015. Diabetes is associated with metabolic disorders that occur when the body cannot produce enough insulin or cannot use insulin, and it is diagnosed by observing the level of fasting blood glucose (FBG) or postprandial blood glucose (PBG). Consistently high levels of blood glucose in diabetes patients can lead to serious complications that affect the heart, blood vessels, kidneys, nerves, and eyes.1 One therapeutic approach to treat diabetes is to delay the glucose absorption by inhibiting digestive enzymes such as α-amylase and αglucosidases.2 Dietary carbohydrates are hydrolyzed into disaccharides and polysaccharides by α-amylase, which is secreted by the salivary glands and pancreas.3 The polysaccharides and disaccharides are then processed into monosaccharides by α-glucosidases.4 αGlucosidases are divided into maltase-glucoamylase and sucraseisomaltase, each of which is composed of two active subunits located on the C- and N-terminus of their original proteins, respectively.5 All four subunits of the α-glucosidases can hydrolyze maltose. However, only the C-terminal subunit of sucrase-isomaltase can hydrolyze sucrose.6,7 It has been reported that inhibiting the digestion of dietary carbohydrates can stop the progression of diabetes mellitus.8 Acarbose, an α-glucosidase © 2017 American Chemical Society

inhibitor, can suppress the digestion of carbohydrates and consequently delay the absorption of glucose and reduce the postprandial hyperglycemia.9 However, the mechanism of the hypoglycemic effect of acarbose is limited to the inhibition of digestive enzymes, and acarbose was found to be less effective than metformin and sulfonylureas in long-term treatment of diabetes.10,11 Growing evidence suggested that the combination of antidiabetic natural products with acarbose can enhance the efficacy of acarbose and/or extend the simple mechanism of its hypoglycemic activity. The combination of berry extracts with acarbose showed synergistic inhibition of α-amylase and αglucosidases.12 The combination of black tea extracts and acarbose exhibited a synergistic inhibition against intestinal αglucosidase and a hypoglycemic effect.13 Cyanidin derivatives in combination with acarbose synergistically alleviate postprandial hyperglycemia in rats.14 Our previous research suggested that the coadministration of Oroxylum indicum seed extracts with acarbose could extend the mechanism of the hypoglycemic effect of acarbose and enhance the efficacy of acarbose by up to 5fold in an 8-week treatment of diabetes in mice.15 In addition, individual inhibitions of α-amylase and α-glucosidases by natural Received: Revised: Accepted: Published: 8319

June 1, 2017 September 4, 2017 September 6, 2017 September 6, 2017 DOI: 10.1021/acs.jafc.7b02531 J. Agric. Food Chem. 2017, 65, 8319−8330

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Journal of Agricultural and Food Chemistry plant extracts16 or phenolic compounds such as kaempferol,17 caffeic acid,18 and chlorogenic acid5 have been reported. These data suggested that dietary constituents administered either individually or in combination can be alternative agents to prevent and treat diabetes. Flavonoids are widely distributed in plants and are present in considerable quantities in human diets, including in vegetables, fruits, tea, and red wine, and they are regarded as the major effective constituent.19,20 Several clinical and basic research studies have shown that flavonoids have positive effects for the treatment of multiple diseases, including diabetes,20 obesity,21 cardiovascular diseases,22 and cancer.23 As a class of αglucosidase inhibitors,24,25 the flavonoids also display PBGcontrolling effects.26 A previous study and our previous research have shown that flavonoids or flavonoid-containing natural products exhibited synergistic PBG-lowering and α-glucosidase inhibitory effects with acarbose.14,27 However, flavonoids have a rich structural diversity that includes at least six subgroups (including flavones, flavonols, flavanones, isoflavones, flavan-3ols, and anthocyanins).28 The synergistic PBG-lowering effect of the combination of flavonoids other than cyanidin derivatives and acarbose is still unclear, and the mechanism of the synergistic effect of flavonoids and acarbose needs to be revealed. In the present study, the inhibition of α-glucosidases and αamylase by dietary flavonoids and the combination with acarbose was screened. The individual and combined PBG-lowering effects of the selected compounds were evaluated in vivo. The mechanism of the synergism was analyzed and evaluated through in vitro and molecular docking studies.

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or 9 mM maltose was added to the reaction mixture as the substrate. The mixture was then incubated for 30 min at 37 °C. After the incubation, the reaction was terminated by incubating the reaction mixture at 100 °C for 10 min. The amount of the liberated glucose in the supernatant was determined with a commercial assay kit based on the glucose oxidase method. Except for a change in the concentration of sucrose, experiments to determine the type of inhibition by baicalein on mammalian α-glucosidase activity were carried out in the same manner. The type of inhibition was determined by Lineweaver−Burk plots and further confirmed by a nonlinear regression analysis with the Levenberg−Marquardt algorithm (Sigma Plot; Systat Software Inc., San Jose, CA, USA).5 The nonlinear regression method, which could avoid additional errors introduced by linear regression, was also used to calculate the kinetic parameters. Evaluation of Synergy in the α-Glycosidase Inhibition Assay. The inhibitors were added individually or in combination at different concentrations in the α-glycosidase assay. The combination index (CI), which is a quantification of the degree of inhibitor interactions based on the median-effect principle developed by Chou and Talalay, were determined using the CompuSyn software.31 The equation for the median-effect principle is as follows:

⎛f ⎞ log⎜⎜ a ⎟⎟ = m log D − m log Dm ⎝ fu ⎠

(1)

where fa is the fraction affected by dose D, f u is the unaffected fraction ( f u = 1 − fa), m is the coefficient signifying the shape of the dose−effect curve, D is the dose of the inhibitor, and Dm is the median-effect dose (IC50 in this article). The equation for the CI is expressed as follows: CI =

MATERIALS AND METHODS

Chemicals. Acarbose was purchased from Bayer Healthcare Co., Ltd. (Berlin, Germany). All flavonoid standards were purchased from Must Biotechnology Co., Ltd. (Chengdu, China). Starch, maltose, and sucrose were purchased from Solarbio Co., Ltd. (Beijing, China). Mammalian α-glucosidase was prepared by our lab. Porcine pancreatic α-amylase was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The glucose test kit (ACCU-CHEK) used for the in vivo assays was purchased from Roche Diagnostics GmbH (Germany). The glucose assay kit used for the in vitro assays was purchased from Nanjing Jiancheng Bioengineering Institute. (Jiangsu, China). α-Amylase Assay. α-Amylase (EC 3.2.1.1) activity was determined following the method previously reported with slight modifications.29 Briefly, baicalein or acarbose and 0.1 U/mL porcine pancreatic αamylase were preincubated in phosphate-buffered saline (pH 7.0) for 15 min at 37 °C. Fifty microliters (50 μL) of 0.4% (w/v) starch as the substrate was added to the reaction mixture for a total volume of 200 μL, and the reaction mixture was incubated at 37 °C for 10 min. After the incubation, the reaction was stopped by the addition of 1 mL of dinitrosalicylic (DNS) reagent (1% 3,5-dinitrosalicylic acid, 0.2% phenol, 0.05% Na2SO3, and 1% NaOH in aqueous solution) to the reaction mixture. The mixtures were then heated for 10 min at 100 °C. After the mixture was cooled to room temperature, the absorbance was read at 540 nm with a spectrophotometer. Acarbose was used as a positive control. α-Glucosidase Assay. The α-glucosidase solution was prepared as previously reported with minor modifications.30 The rats were sacrificed after a 16-h starvation period. The small intestinal tissues were excised and homogenized in 100 mM phosphate buffer saline with 14 mM NaCl (1:10 dilution; w/v). After a centrifugation for 15 min at 12000g, the supernatant was collected and used as the enzyme solution. The α-glucosidase activity (maltase, EC 3.2.1.20; sucrase, EC 3.2.1.48) was determined following the method of a previous report.29 Briefly, 100 μL of the enzyme solution containing 0.002 U/mL of αglucosidase activity and 100 μL of the sample (baicalein or acarbose) were preincubated for 15 min at 37 °C. Then, 100 μL of 27 mM sucrose

(D)1 (D)2 + (Dx )1 (Dx )2

(2)

where (D)1 and (D)2 are the doses of inhibitors that produce a certain level of inhibition in the combination system, and (Dx)1 and (Dx)2 are the doses of inhibitors added alone that lead to the same level of inhibition. The combined inhibition was divided into synergistic (CI < 0.9), additive (CI = 0.9−1.1), or antagonistic (CI > 1.1) inhibition, according to the CI values. Molecular Docking. Molecular docking was performed to predict the binding site and efficacy between the α-glucosidase proteins and flavonoids using the Schrodinger Maestro 10.2 software package. NTerminal sucrase-isomaltase (PDB: 3LPP), N-terminal maltaseglucoamylase (PDB: 2QMJ), and C-terminal maltase-glucoamylase (PDB: 3TOP) were examined in this study. All proteins were subjected to cavity analysis and ranked based on their expanded van der Waals volume for binding evaluation using Sitemap following the software instructions.32 For each protein structure, the four best ranked cavities were selected with SiteScores > 0.9 and volumes > 60. The protein preparation was performed following a previous report.33 The docking grid (32 × 32 × 32 Å) was generated based on the chosen binding sites. The original ligand within the PDB structure was then redocked into the corresponding protein structure without any constraints. The reliability of the docking procedure was evaluated by comparing the CαRMSD between the positions of heavy atoms of the ligand in the calculated crystal structure. This procedure redocked acarbose, miglitol, salacinol, and kotalanol with low CαRMSD values (Figure S1) and was used in the subsequent in silico evaluation. The 5 best conformations were generated based on the empirical glide score (kcal/mol). The interactions between the subunits of the α-glucosidase and flavonoids were predicted. Blind docking was also carried out using Autodock 4.2 to cross-check the docking results for active site binding,34 with the following settings: maximum number of 25,000,000 energy evaluation, number of generations = 27,000, mutation rate = 0.02, maximum number of iterations = 300, and number of docking runs = 100. The grid size was 120 × 120 × 120 Å with a spacing of 0.375 Å centered on the active site for each protein. 8320

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Figure 1. Structures of the flavonoids and the structure−activity relationships of flavonoids on the inhibition of mammalian α-glucosidase. Animals. Kunming mice (18−22 g, 6 weeks old) and SD rats (180− 220 g) were purchased from Dalian Medical University SPF experimental animal center (Dalian, China). The animals were provided a standard rodent diet and free access to water and were maintained at a temperature of 20−22 °C. The animals and protocols used in this study were approved by the Animal Care Committee of Dalian Medical University. Oral Sugar Tolerance Test. The oral sugar tolerance test was performed according to a previous report.35 Kunming mice that had been fasted for 16 h were randomly divided into several groups. A baicalein-loaded self-microemulsifying drug delivery system was prepared according to previous reports.36 The final concentrations of ethyl oleate, PEG 400, and TWEEN-80 used in the oral sugar tolerance test were 2%, 2%, and 4% (w/w), respectively. The vehicle (control group); baicalein at 40, 80, and 240 mg/kg; or acarbose at 0.3, 1, and 8 mg/kg was orally administered to the mice. Sucrose or glucose was orally administered at 2 g/kg body weight simultaneously with the drugs. Blood samples were collected from the tail vein at 0, 30, 60, and 120 min, and the levels of blood glucose were measured with a glucose test kit. Statistical Analysis. The statistical analysis was performed using SPSS 17.0. All the results were expressed as the mean ± standard deviation. Comparisons between groups were analyzed using one-way ANOVA followed by Fisher LSD test. A p value less than 0.05 was

judged as statistically significant. The concentration of the inhibitors that caused 50% inhibition (IC50) of the enzyme activity was calculated by nonlinear regression.

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RESULTS AND DISCUSSION

Individual Inhibition of Mammalian α-Glucosidase and Porcine Pancreatic α-Amylase by Flavonoids. Sixteen dietary flavonoids, which have various hydroxylation and glycosylation patterns across the A, B, or C rings and belong to 5 subgroups of flavonoids, including flavones, flavonols, flavanones, isoflavones, and flavan-3-ols (Figure 1), were selected and tested for their inhibition of mammalian α-glucosidase and porcine pancreatic α-amylase. For the maltose-hydrolyzing activity, the IC50 value of acarbose was 0.4 ± 0.1 μM, which was similar to that reported previously.37 Baicalein and (+)-catechin showed the strongest inhibition among the tested flavonoids with IC50 values of 74.1 ± 5.6 μM and 175.1 ± 9.1 μM (Table 1, Figure S2), respectively. The IC50 value for baicalein was similar to that in a previous report.38 Quercetin and luteolin showed weaker inhibition than baicalein, with IC50 values of 281.2 ± 19.2 μM and 339.4 ± 16.3 8321

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Journal of Agricultural and Food Chemistry Table 1. Individual Inhibition of Flavonoids against Mammalian α-Glucosidase and Porcine Pancreatic α-Amylase maltase flavonoid

α-amylase

sucrase

IC50 (μM)

inhibna (%)

IC50 (μM)

74.1 ± 5.6 >400 339.4 ± 16.3 >400 >400 >400

81.3 ± 4.1 4.2 ± 2.6 53.9 ± 3.8 24.6 ± 2.9 44.1 ± 3.4 4.8 ± 2.0

14.6 ± 2.7 >400 >400 >400 >400 >400

281.2 ± 19.2 >400 >400

58.7 ± 4.8 41.2 ± 5.1 5.1 ± 1.2

>400 >400 >400 >400 >400 >400

inhibna (%)

IC50 (μM)

inhibna (%)

80.0 ± 6.2 5.5 ± 2.1 16.0 ± 0.9 8.9 ± 3.7 25.2 ± 1.4 26.6 ± 4.2

446.4 ± 23.9 >400 >400 146.8 ± 7.1 >400 >400

47.2 ± 3.8 24.5 ± 3.0 54.3 ± 4.4 79.3 ± 2.9 −7.4 ± 1.0 −4.9 ± 2.2

>400 >400 >400

16.0 ± 2.5 −3.9 ± 3.1 16.5 ± 2.2

>400 >400 >400

45.7 ± 4.1 −5.4 ± 2.6 41.8 ± 2.3

27.6 ± 3.1 7.4 ± 2.2 7.2 ± 2.4

>400 >400 >400

0.5 ± 0.2 3.1 ± 1.1 16.0 ± 1.9

>400 >400 >400

4.1 ± 0.7 3.2 ± 1.1 20.6 ± 2.8

28.1 ± 3.6 −6.8 ± 2.4 2.4 ± 1.2

>400 >400 >400

15.3 ± 2.4 14.7 ± 3.0 −14.1 ± 2.3

394.2 ± 17.1 >400 >400

52.1 ± 4.6 21.4 ± 2.6 32.4 ± 2.0

71.7 ± 4.7 96.5 ± 7.4

>400 1.2 ± 0.3

flavone baicalein chrysin luteolin apigenin scutellarin baicalin flavonol quercetin rutin kaempferol flavanone dihydromyricetin hesperetin naringenin isoflavone puerarin daidzein genistein flavan-3-ol (+)-catechin acarbose a

175.1 ± 9.1 0.4 ± 0.1

23.9 ± 2.5 90.1 ± 5.6

>400 5.3 ± 3.1

−11.1 ± 1.9 86.2 ± 6.1

Inhibition by 400 μM flavonoids. The values shown are the averages of triplicate assays.

μM, respectively. The IC50 values for the other flavonoids were greater than 400 μM (Table 1). Comparison of IC50 values showed that the inhibition of maltase by baicalein was 1/200 of that by acarbose. For the sucrose-hydrolyzing activity, the IC50 value for acarbose was 1.2 ± 0.3 μM. Baicalein showed the strongest inhibition among the tested flavonoids. The IC50 value for baicalein was 14.6 ± 2.7 μM (Table 1), which was 1/12 of that of acarbose. The relative inhibition intensity of baicalein to acarbose showed that baicalein exhibited a stronger inhibition against sucrase than maltase. The inhibitory activities of all the other tested flavonoids were weak at 400 μM (Table 1). For the α-amylase activity, the IC50 value for acarbose was 5.3 ± 3.1 μM (Table 1), which is consistent with the previously reported value of 6.46 μM.35 Apigenin showed the strongest inhibition (Table 1). Luteolin, baicalein, quercetin, and kaempferol showed weaker inhibition than apigenin. The IC50 values for apigenin and baicalein were 146.8 ± 7.1 μM and 446.4 ± 23.9 μM. Comparison of IC50 values suggested that the inhibitory activity of apigenin and baicalein were 1/28 and 1/84 of that of acarbose, respectively. The inhibition of individual flavonoids against α-glucosidase and α-amylase revealed a structure−activity relationship. Generally, the tested flavonoids showed relatively stronger individual inhibition of the maltose-hydrolyzing activity than sucrose-hydrolyzing activity. For the maltose-hydrolyzing activity, the fact that baicalein showed stronger inhibition than chrysin suggested that the 6-hydroxyl group is important for exerting inhibition (Figure 1, Table 1), which is consistent with a previous report.38 The importance of the 3′- and 4′-hydroxyl groups can be shown by comparing the inhibition of luteolin and apigenin, scutellarin and baicalin, and quercetin and kaempferol, respectively. The importance of the 6-hydroxyl group was greater than that of the 4′-hydroxyl group as baicalein showed a stronger inhibition than apigenin. The introduction of 7-glucuronic acid

group decreased the inhibition as baicalein showed a stronger inhibitory activity than baicalin. Genistein showed very weak inhibition compared with apigenin, suggesting that the linkage of the B-ring at the 2-position is important for the inhibitory activity. (+)-Catechin showed stronger inhibition than quercetin, suggesting that the lack of a 4-carbonyl group in the C-ring can enhance the inhibition. For α-amylase activity, comparisons of the inhibition by apigenin vs chrysin and scutellarin vs baicalin showed that the 4′hydroxyl groups were important (Table 1). The fact that baicalein showed stronger inhibition than chrysin suggested that the 6-hydroxyl group is important for the inhibitory activity. The inhibition by apigenin was greater than that of naringenin, suggesting that the nearly planar structure of the C-ring is important for the inhibitory activity. Apigenin exhibited stronger inhibition than genistein, suggesting that linkage of the B-ring at the 2-position can enhance the inhibitory activity. Combined Inhibition of Mammalian α-Glucosidase and Porcine Pancreatic α-Amylase by Flavonoids and Acarbose. The combined inhibition of flavonoids with acarbose against α-glucosidase and α-amylase was determined. CI values, a quantitative measurement for the evaluation of combined effect, were used to assess whether combinations provided more (synergistic, CI < 0.9), equivalent (additive, CI = 0.9−1.1), or less (antagonistic, CI > 1.1) inhibition against α-glucosidase and α-amylase than that of flavonoids and acarbose alone.31 For the maltose-hydrolyzing activity, the combination of baicalein and acarbose strongly enhanced the inhibition compared with individual compounds. For example, the combination of baicalein at 17.5 μM and acarbose at 0.194 μM caused 62.4% inhibition of the activity of α-glucosidase (Figure 2A), which is higher than the inhibition caused by baicalein alone at 35.0 μM or acarbose alone at 0.387 μM (twice the concentration of each compound). The calculated CI values were less than 0.41 at all dosages, suggesting a strongly 8322

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Figure 2. 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. CI < 0.9, CI = 0.9−1.1, and CI > 1.1 indicate synergism, additive effect, and antagonism, respectively. The values shown are the means of triplicate assays ± standard error.

synergistic interaction between baicalein and acarbose. The CI values for the combinations of either quercetin or luteolin with acarbose were between 0.51 and 0.83, which suggests moderate synergism (Figure 2B and 2C). For the combination of (+)-catechin and acarbose, the CI values were less than 0.9 at low concentrations but were higher than 1.1 at the highest concentration, which suggests antagonism (Figure 2D). No obvious enhancement was found when acarbose was combined with the flavonoids that displayed weak or no individual inhibition (data not shown), suggesting that inhibitory activity of each compound in the combination was necessary for exerting synergistic inhibition. For the sucrose-hydrolyzing activity, the combination of baicalein and acarbose strongly enhanced the inhibition compared with the individual compounds (Figure 3). The CI values were between 0.61 and 0.70 at lower concentrations (3.19, 6.39, and 10.7 μM for baicalein), suggesting synergism; and the CI value was 0.39 at a higher concentration (14.8 μM for baicalein), suggesting strong synergism. These data indicated that the synergistic inhibition on sucrase became stronger at higher concentration of baicalein and acarbose. For the α-amylase activity, the combinations of flavonoids with acarbose did not show any obvious enhancement of the inhibition, compared with that observed for acarbose or the various flavonoids added individually. For example, the combination of baicalein at 148 μM and acarbose at 1.86 μM caused 35.1% inhibition of the activity of α-amylase, which is

Figure 3. 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. CI < 0.9, CI = 0.9−1.1, and CI > 1.1 indicate synergism, additive effect, and antagonism, respectively. The values shown are the means of triplicate assays ± standard error.

lower than the inhibition caused by baicalein alone at 296 μM and close to the inhibition caused by acarbose at 3.72 μM (twice the concentration of each compound). Most calculated CI values were higher than 0.9, suggesting additive inhibition or antagonistic inhibition. The only exception was the combination of apigenin at 46.3 μM and acarbose at 0.93 μM, with a CI value of 0.71, suggesting weak synergistic inhibition. (Figure 4). 8323

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Figure 4. 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. CI < 0.9, CI = 0.9−1.1, and CI > 1.1 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−Menten plots of acarbose alone (B), baicalein alone (C), and 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.

synergistic inhibition of α-glucosidase by the combination of baicalein, luteolin, and quercetin with acarbose. Boath et al. have reported a synergistic effect of acarbose and black currant extracts.40 However, no synergistic inhibitory interaction of flavones or flavonol compounds with acarbose has been reported. Interestingly, (+)-catechin showed antagonistic but not synergistic effect at higher concentrations in our present work. Gao et

Because the combined effect cannot be evaluated by a simple arithmetic sum of the inhibition produced by individual inhibitors, it is difficult to differentiate the synergistic, antagonistic, and additive effects, until Chou and Talalay introduced CI value to distinguish the three types of inhibition. This method have been widely used for the evaluation of drug combination. 39 Using this method, we determined the 8324

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substrate. Thus, baicalein is expected to bind to the noncompetitive binding site and enhance the affinity of enzyme for acarbose, and finally reduce the affinity of enzyme for substrate. This speculation was supported by a previous study, in which black tea extract was found to be a noncompetitive inhibitor against α-glucosidases and it exhibited synergism with acarbose. Furthermore, black tea extract also reduced the affinity of enzyme for substrate.13 However, further study is still needed to clarify the exact change in the α-glucosidases caused by flavonoid binding. On the other hand, Adisakwattana et al. also reported that cyanidin-3-rutinoside, another noncompetitive inhibitor of αglucosidase, showed synergistic inhibition with acarbose.14 Taking these data into consideration, binding at the noncompetitive site of α-glucosidase might be the basic requirement for an inhibitor that could exert synergistic inhibition with acarbose. Molecular Docking. To identify the binding sites for the flavonoids in the intestinal glucosidases, we performed molecular docking for baicalein, (+)-catechin, quercetin, luteolin, and acarbose with α-glucosidases using Schrodinger software. As no crystal structure of rat α-glucosidase is currently available, crystal structures of three available human α-glucosidase subunits, 2QMJ (N-terminal of maltase-glucoamylase), 3TOP (Cterminal of maltase-glucoamylase), and 3LPP (N-terminal of sucrase-isomaltase), were used. The sequence homology of the three subunits to their corresponding human counterparts is 92%, 90%, and 86%, respectively, within 10 Å around the predicted binding sites (Figures S3−S5) of baicalein. Four binding sites in each protein were chosen according to the site score values that were calculated based on their expanded van der Waals volume for binding evaluation and ranked using the sitemap panel (Figure 6), which was similar to those previously reported.32 For each protein, the active site was included in the chosen sites. For N-terminal maltase-glucoamylase (2QMJ), the glide score for acarbose in the active site (site 2) was −7.34 kcal/mol (Table 3). Baicalein was predicted to bind to site 1, with a better glide score of −7.64 kcal/mol compared to the interaction with the active site (site 2, −5.50 kcal/mol). Three major interactions were observed to contribute to the maintenance of the binding: (a) hydrogen bonds between Phe535 and Ser521 and the hydroxyl group in the A-ring (Figure 7); (b) a hydrogen bond between Lys776 and the carbonyl group in the C-ring; and (c) hydrophobic interactions in a large pocket (Val779, Leu286, Ala780, Pro287, and Ile523). (+)-Catechin was predicted to bind to the active site (site 2), with a glide score of −6.55 kcal/mol. Like acarbose, (+)-catechin bound to the −1 and +1 sugarbinding site residues Asp327, Asp443, and Asp542 through hydrogen bonds with the hydroxyl groups in the B- and C-rings. Luteolin and quercetin were predicted to bind to site 1, with glide scores of −8.15 and −7.05 kcal/mol, respectively. The Hbonding interactions of hydroxyl and carbonyl groups with Val779, Asp777, Lys776, Phe535, and Ser521 for luteolin and Asp777, Lys776, Phe535, and Ser521 for quercetin were predicted to be the predominant interactions (Figures S6A, S6B). For C-terminal maltase-glucoamylase (3TOP), the glide score for acarbose in the active site (site 3) was −8.00 kcal/mol (Table 3). Baicalein was predicted to bind to site 1, with a glide score (−6.98 kcal/mol) better than that for its interaction with active site (site 3, −5.70 kcal/mol). The hydroxyl group in the A-ring formed hydrogen bonds with Pro1327 and Glu1284 (Figure 8).

al. have reported that the combination of epigallocatechin gallate and acarbose showed synergistic inhibition at lower concentrations and antagonistic inhibition at higher concentrations, and that the CI values increased with the concentrations of the inhibitors.41 Although these authors used yeast α-glucosidase in their studies, their results were comparable to our observation. When combined with acarbose, baicalein showed a lower CI value against maltose-hydrolysis than those of luteolin and quercetin, indicating stronger synergistic inhibition. This result suggests that either the introduction of 6-hydroxyl group or the removal of 3′-, 4′-hydroxyl groups is important for exerting synergism (Figure 1). The fact that quercetin showed stronger synergistic inhibition with acarbose than that of (+)-catechin suggested that the introduction of a 4-carbonyl group in the Cring could enhance the synergism. Type of Inhibition by Baicalein and Acarbose on Mammalian α-Glucosidase. The types of inhibition by baicalein and its combination with acarbose against mammalian α-glucosidase were determined by both Lineweaver−Burk and Michaelis−Menten plots. The Lineweaver−Burk plots of acarbose generated straight lines which had intersections on the Y-axis, suggesting competitive inhibition (Figure 5A). This data was consistent with the previous report.42 The straight lines generated by baicalein alone and the combination had intersections on the X-axis and in the second quadrant, suggesting noncompetitive and mixed-type inhibitions, respectively. The types of inhibition were further confirmed by the Michaelis−Menten plots using the best fit of nonlinear regression analysis with the Levenberg−Marquardt algorithm. Compared with the control, the addition of acarbose significantly increased the Michaelis constant (Km), but the maximum reaction velocity (Vmax) was not significantly changed; the addition of baicalein significantly reduced the Vmax, but the Km was not significantly changed (Figure 5B, Figure 5C, and Table 2). When a combination of baicalein and acarbose was added in a Table 2. Kinetic Parameters for Mammalian Sucrase control baicalein acarbose acarbose + baicalein (1:9.56)

Km (mM)

Vmax (μM/min)

15.2 ± 1.9 14.1 ± 2.2 29.5 ± 2.3# 43.6 ± 4.1#,†

387 ± 40.8 275 ± 34.9#a 388 ± 31.7 247 ± 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. a

fixed ratio, the Km value was significantly higher than those of both acarbose alone and the control, and the Vmax was significantly lower than that of the control (Figure 5D and Table 2). The values of Km, Vmax, and their patterns confirmed that baicalein alone, acarbose alone, and the combination exhibited noncompetitive, competitive, and mixed-type inhibition, respectively. Besides, the significantly higher Km value of the combination than that of acarbose alone also indicated that the combination caused greater reduction of the affinity of enzyme for substrate. This result indicated that, when used in combination, the inhibitors had stronger binding capacity at the competitive binding site, i.e., active site or substrate-binding site. The fact that baicalein alone did not change the Km value suggested that baicalein, a noncompetitive inhibitor, cannot bind to the competitive binding site and thereby reduce the affinity for 8325

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Figure 6. Selected binding sites of maltase (2QMJ), glucoamylase (3TOP), and isomaltase (3LPP).

Table 3. Binding Glide Scores of Acarbose, Baicalein, Luteolin, Quercetin, and (+)-Catechin on α-Glucosidase Proteins docking glide score (kcal/mol) 2QMJ (maltase)

3TOP (glucoamylase)

3LPP (isomaltase)

a

binding site

site score

baicalein

luteolin

quercetin

(+)-catechin

acarbose

1a 2 (active site) 3 4 1 2 3 (active site) 4 1 2 3 (active site) 4

1.06 0.96 0.95 0.95 1.12 1.05 1.03 0.96 1.11 1.08 1.00 0.92

−7.64 −5.50 −6.16 −4.54 −5.08 −6.98 −5.70 −6.11 −5.91 −6.88 −4.68 −6.49

−8.15 −5.41 −7.10 −6.48 −7.40 −6.73 −7.52 −5.39 −5.93 −6.47 −4.95 −6.24

−7.05 −5.39 −7.03 −6.59 −7.15 −6.93 −7.19 −5.88 −5.05 −5.97 −4.98 −5.92

−6.01 −6.55 −6.04 −6.12 −6.86 −6.96 −7.70 −7.19 −5.93 −8.79 −5.81 −5.93

−6.73 −7.34 −5.37 −5.13 −5.71 −5.98 −8.00 −5.12 ncb −4.97 −7.13 −5.54

The binding site underlined was predicted to have the strongest affinity with baicalein. bNo binding conformation was produced.

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.

acarbose, (+)-catechin bound to the −1 and +1 sugar-binding site residues His1584, Asp1279, Asp1526, Arg1510, and Asp1157 through hydrogen bonds. Luteolin and quercetin were predicted to bind to active site (site 3) with glide scores of

The carbonyl group in the C-ring formed hydrogen bonds with Leu1291. The B-ring formed hydrophobic interactions with Pro1405 and Leu1401. (+)-Catechin was predicted to bind to the active site (site 3) with a glide score of −7.70 kcal/mol. Like 8326

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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.

−7.52 and −7.19 kcal/mol, respectively. The H-bonding interactions of hydroxyl and carbonyl groups with His1584, Asp1526, Asp1279, and Asp1157 for both luteolin and quercetin were predicted to be the predominant interactions (Figures S6C, S6D). For N-terminal sucrase-isomaltase (3LPP), the glide score for acarbose in the active site (site 3) was −7.13 kcal/mol (Table 3). Baicalein, luteolin, quercetin, and (+)-catechin were predicted to bind to site 2, with better glide scores of −6.88, −6.47, −5.97, and −8.79 kcal/mol, respectively, compared with their interactions with the active site (site 3, −4.68, −4.98, −4.98, and −5.81 kcal/ mol, respectively). Asp806 formed hydrogen bonds with the hydroxyl group in the A-ring of baicalein (Figure 9). The residues (His600, Ile552, and Gly562) formed hydrogen bonds with the hydroxyl group of (+)-catechin. The B-rings of both baicalein and (+)-catechin formed hydrophobic interactions in a large pocket (Phe551, Ile552, Ala565, and Ala566).

For all three subunits of the α-glucosidases, the noncompetitive site was predicted to have a much higher binding affinity to baicalein. Quercetin and luteolin were predicted to have higher affinities for the noncompetitive site of maltase (2QMJ) and isomaltase (3LPP) and the active site of glucoamylase (3TOP). In contrast, (+)-catechin was predicted to have a higher affinity for the active sites of maltase (2QMJ) and glucoamylase (3TOP) but a higher affinity for the noncompetitive site for isomaltase (3LPP). These results are consistent with the previous reports that (+)-catechin is a noncompetitive inhibitor of isomaltase (3LPP) but could bind to different binding sites of maltase and glucoamylase.5 These observations, together with the facts that baicalein displayed stronger synergism than quercetin and luteolin and (+)-catechin displayed antagonism at higher concentrations in the enzyme assays, suggested that noncompetitive binding in the αglucosidases would enhance the synergistic inhibition, whereas competitive binding at the active sites of α-glucosidases at higher 8327

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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).

concentrations might lead to antagonistic inhibition between flavonoids and acarbose. In addition, H-bonds were formed between the carbonyl group of baicalein and the residues in the noncompetitive sites when binding to maltase (2QMJ) and glucoamylase (3TOP) but not isomaltase (3LPP), which suggested that the carbonyl group in the flavonoids may be important in producing the noncompetitive inhibition against maltase (2QMJ) and glucoamylase (3TOP). Hypoglycemic Effect of Baicalein and Coadministration of Acarbose. The hypoglycemic effect of baicalein and coadministration of acarbose was evaluated through sucrose tolerance test in mice, and the time courses for the levels of blood glucose were measured before and 30, 60, 90, and 120 min after sucrose loading (2 g/kg) (Figure 10). The PBG level of the control group increased and peaked at 30 min and then decreased. The baicalein- and acarbose-administered groups exhibited dose-dependent decrease in the level of PBG compared with the control group. Baicalein at 80 and 200 mg/kg and acarbose at 1 and 8 mg/kg decreased the levels of PBG significantly 30 min after sucrose loading (Figure 10A and Figure 10B). The combination of baicalein at 80 mg/kg and acarbose at 1 mg/kg significantly reduced the levels of PBG 30 and 60 min after sucrose loading compared with the control group. This reduction was greater than that observed in the groups that were administered the individual agents, 80 mg/kg baicalein or 1 mg/ kg acarbose (Figure 10C). PBG levels after the coadministration of baicalein (80 mg/kg) and acarbose (1 mg/kg) were not significantly different from those receiving 8 mg/kg acarbose alone. Our data suggested that the coadministration of baicalein with acarbose generated a synergistic hypoglycemic effect in vivo, which was consistent with the synergistic effects of combination

in vitro. The effective dose of acarbose could be reduced by 87.5%. The glucose tolerance test was also performed to exclude the influences other than carbohydrate digestion. Glucose was administered orally to the fasted mice. No significant difference in PBG levels was observed among the baicalein (80 mg/kg), acarbose (8 mg/kg), and control groups (Figure 10D). This result suggested that the PBG lowering effect of baicalein resulted from the inhibition of carbohydrate digestion. We reported that baicalein could enhance the efficacy of acarbose both in vitro and in vivo for the first time. It is known that the hypoglycemic effect of acarbose is limited to the inhibition of carbohydrate digestion. Long-term treatment with baicalein, on the other hand, has also been reported to have an antidiabetic effect through the promotion of islet β-cell function,43 the activation of AMPKα/IRS/Akt pathway,44 the suppression of inflammatory responses,45 and the reduction of oxidative stress.46 Thus, baicalein and acarbose can inhibit the αglucosidase in the gut lumen synergistically to prevent hyperglycemia; and the absorbed baicalein is expected to increase the insulin resistance, improve the complications, and restore the pancreatic function. Therefore, baicalein might extend the simple mechanism of acarbose alone, and enhance the efficacy of acarbose. Baicalein alone also showed PBG lowering effect in vivo. Thus, our present work is expected to expand the usage of baicalein in diabetes treatment as a dietary supplement. Flavonoids are widely distributed in plants and are found in considerable quantities in human diets, such as fruit, vegetables, nuts, seeds, fruits, tea, and red wine. Baicalein was found in abundance in several natural products such as Oroxylum indicum and Scutellaria baicalensis.45 The dose of baicalein in our in vivo 8328

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monotherapy in patients with Type 2 diabetes: a 24-week, double-blind, randomized trial. Diabetic Med. 2008, 25, 435−441. (3) Jones, K.; Sim, L.; Mohan, S.; Kumarasamy, J.; Liu, H.; Avery, S.; Naim, H. Y.; Quezada-Calvillo, R.; Nichols, B. L.; Pinto, B. M.; Rose, D. R. Mapping the intestinal alpha-glucogenic enzyme specificities of starch digesting maltase-glucoamylase and sucrase-isomaltase. Bioorg. Med. Chem. 2011, 19, 3929−3934. (4) Sim, L.; Willemsma, C.; Mohan, S.; Naim, H. Y.; Pinto, B. M.; Rose, D. R. Structural basis for substrate selectivity in human maltaseglucoamylase and sucrase-isomaltase N-terminal domains. J. Biol. Chem. 2010, 285, 17763−17770. (5) Simsek, M.; Quezada-Calvillo, R.; Ferruzzi, M. G.; Nichols, B. L.; Hamaker, B. R. Dietary phenolic compounds selectively inhibit the individual subunits of maltase-glucoamylase and sucrase-isomaltase with the potential of modulating glucose release. J. Agric. Food Chem. 2015, 63, 3873−3879. (6) Cezard, J. P.; Conklin, K. A.; Das, B. C.; Gray, G. M. Incomplete intracellular forms of intestinal surface membrane sucrase-isomaltase. J. Biol. Chem. 1979, 254, 8969−8975. (7) Kolínská, J.; Kraml, J. Separation and characterization of sucroseisomaltase and of glucoamylase of rat intestine. BBA-Enzymology 1972, 284, 235−247. (8) Rademacher, T. W.; Parekh, R. B.; Dwek, R. A. Glycobiology. Annu. Rev. Biochem. 1988, 57, 785−838. (9) Mohan, S.; Eskandari, R.; Pinto, B. M. Naturally occurring sulfonium-ion glucosidase inhibitors and their derivatives: a promising class of potential antidiabetic agents. Acc. Chem. Res. 2014, 47, 211−225. (10) Derosa, G.; Maffioli, P. Efficacy and safety profile evaluation of acarbose alone and in association with other antidiabetic drugs: a systematic review. Clin. Ther. 2012, 34, 1221−36. (11) Van de Laar, F. A.; Lucassen, P. L.; Akkermans, R. P.; Van de Lisdonk, E. H.; Rutten, G. E.; Van Weel, C. Alpha-glucosidase inhibitors for type 2 diabetes mellitus. Cochrane Database Syst. Rev. 2005, DOI: 10.1002/14651858.CD003639.pub2. (12) Grussu, D.; Stewart, D.; McDougall, G. J. Berry polyphenols inhibit alpha-amylase in vitro: identifying active components in rowanberry and raspberry. J. Agric. Food Chem. 2011, 59, 2324−2331. (13) Satoh, T.; Igarashi, M.; Yamada, S.; Takahashi, N.; Watanabe, K. Inhibitory effect of black tea and its combination with acarbose on small intestinal alpha-glucosidase activity. J. Ethnopharmacol. 2015, 161, 147− 155. (14) Adisakwattana, S.; Yibchok-Anun, S.; Charoenlertkul, P.; Wongsasiripat, N. Cyanidin-3-rutinoside alleviates postprandial hyperglycemia and its synergism with acarbose by inhibition of intestinal alpha-glucosidase. J. Clin. Biochem. Nutr. 2011, 49, 36−41. (15) Sun, W.; Sang, Y.; Zhang, B.; Yu, X.; Xu, Q.; Xiu, Z.; Dong, Y. Synergistic effects of acarbose and an Oroxylum indicum seed extract in streptozotocin and high-fat-diet induced prediabetic mice. Biomed. Pharmacother. 2017, 87, 160−170. (16) Chai, T. T.; Kwek, M. T.; Ong, H. C.; Wong, F. C. Water fraction of edible medicinal fern Stenochlaena palustris is a potent alphaglucosidase inhibitor with concurrent antioxidant activity. Food Chem. 2015, 186, 26−31. (17) Peng, X.; Zhang, G.; Liao, Y.; Gong, D. Inhibitory kinetics and mechanism of kaempferol on alpha-glucosidase. Food Chem. 2016, 190, 207−215. (18) Xu, D.; Wang, Q.; Zhang, W.; Hu, B.; Zhou, L.; Zeng, X.; Sun, Y. Inhibitory activities of caffeoylquinic acid derivatives from Ilex kudingcha C.J. Tseng on alpha-glucosidase from Saccharomyces cerevisiae. J. Agric. Food Chem. 2015, 63, 3694−3703. (19) Courts, F. L.; Williamson, G. The Occurrence, Fate and Biological Activities of C-glycosyl Flavonoids in the Human Diet. Crit. Rev. Food Sci. Nutr. 2015, 55, 1352−1367. (20) Kawser Hossain, M.; Abdal Dayem, A.; Han, J.; Yin, Y.; Kim, K.; Kumar Saha, S.; Yang, G. M.; Choi, H. Y.; Cho, S. G. Molecular Mechanisms of the Anti-Obesity and Anti-Diabetic Properties of Flavonoids. Int. J. Mol. Sci. 2016, 17, 569.

study was 80 mg/kg. Therefore, the dose for human administration was estimated to be 8.8 mg/kg using body surface area model,47,48 which corresponds to approximately 3 times the average daily human intake of flavonoids.49 This is a relatively safe dose, on the basis that treatments with baicalein at 800 mg twice a day for 10 days50 or a single dose of 100−2800 mg51 were reported to be safe and well tolerated by volunteers. Besides, all the adverse events reported, including blurred vision and plasma fibrinogen decrease, were rated as “mild” and resolved without further treatment. Therefore, baicalein alone or the combination could be regarded as a low-toxicity agent. In conclusion, the combination of baicalein, quercetin, or luteolin with acarbose synergistically inhibited α-glucosidase in vitro, and baicalein lowered PBG levels in vivo. The mechanism of the synergistic inhibition is the noncompetitive inhibition of the α-glucosidases by the flavonoids and acarbose. These results indicated the potential of baicalein for reducing the effective dose and extending the simple mechanism of the hypoglycemic effect of acarbose. Also, baicalein may be used as a supplemental drug or dietary supplement for diabetes mellitus.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b02531. CαRMSD, inhibition of α-amylase, amino acid sequences, and binding positions and interaction patterns of luteolin and quercetin with α-glucosidases (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*School of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong Road, Dalian 116024, China. E-mail: [email protected]. Tel/fax: +86-411-84706344. *School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China. E-mail: zclnathan@163. com. Tel: 86-21-81871258-804. ORCID

Chun-lin Zhuang: 0000-0002-0569-5708 Yue-sheng Dong: 0000-0001-5010-6426 Funding

This work was supported by the National Natural Science Foundation of China (81172966 and 81502978), Shanghai “ChenGuang” project (16CG42), and Open Fund of Key Laboratory of Biotechnology and Bioresources Utilization (Dalian Minzu University), State Ethnic Affairs Commission & Ministry of Education, China. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED PBG, postprandial blood glucose; FBG, fasting blood glucose; OISE, Oroxylum indicum seed extracts; DNS, dinitrosalicylic; CI, combination index

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REFERENCES

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DOI: 10.1021/acs.jafc.7b02531 J. Agric. Food Chem. 2017, 65, 8319−8330