Natural Prenylchalconaringenins and Prenylnaringenins as

Jan 29, 2017 - (32) The isomaltase crystal structure was downloaded from the Protein Data Bank (PDB) (3A4A). The protein was ... Animal procedures wer...
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Natural Prenylchalconaringenins and Prenylnaringenins as Antidiabetic Agents: α‑Glucosidase and α‑Amylase Inhibition and in Vivo Antihyperglycemic and Antihyperlipidemic Effects Hua Sun,*,† Dong Wang,† Xiaotong Song,† Yazhou Zhang,† Weina Ding,† Xiaolin Peng,† Xiaoting Zhang,† Yashan Li,† Ying Ma,‡ Runling Wang,‡ and Peng Yu*,† †

China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, Tianjin University of Science and Technology, Tianjin 300457, China ‡ School of Pharmacy, Tianjin Medical University, Tianjin 300070, China S Supporting Information *

ABSTRACT: Inhibition of α-glucosidase and α-amylase decreases postprandial blood glucose levels and delays glucose absorption, making it a treatment strategy for type 2 diabetes. This study examined in vivo and in vitro antidiabetic activities of natural prenylchalconaringenins 1 and 2 and prenylnaringenins 3 and 4, found in hops and beer. 3′-Geranylchalconaringenin (2) competitively and irreversibly inhibited α-glucosidase (IC50 = 1.08 μM) with activity 50-fold higher than that of acarbose (IC50 = 51.30 μM) and showed moderate inhibitory activity against α-amylase (IC50 = 20.46 μM). Docking analysis substantiated these findings. In addition, compound 2 suppressed the increase in postprandial blood glucose levels and serum levels of total cholesterol and triglycerides in streptozotocin-induced diabetic mice. Taken together, these results suggest that 2 has dual inhibitory activity against α-glucosidase and α-amylase and alleviates diabetic hyperglycemia and hyperlipidemia, making it a potential functional food ingredient and drug candidate for management of type 2 diabetes. KEYWORDS: prenylchalconaringenin, prenylnaringenin, diabetes, α-glucosidase, α-amylase, docking

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knowledge, antidiabetic activity of prenylated flavonoids 1−4 has not been reported. Type 2 diabetes, one of the most prevalent metabolic diseases in the world, is characterized by hyperglycemia and hyperlipidemia. Inhibition of α-glucosidase and α-amylase, which digest dietary starch into glucose, is studied as a method for controlling blood sugar levels.23 α-Amylase catalyzes digestion of carbohydrates by hydrolyzing α-(1, 4)-glycosidic linkages and producing maltose and glucose from starch. αGlucosidase catalyzes the final step of carbohydrate digestion and releases glucose from oligosaccharides. α-Glucosidase and α-amylase dual inhibitors slow the release of glucose from starch and oligosaccharides, delaying glucose absorption and decreasing postprandial blood glucose levels.24 The current focus of our research is identifying natural bioactive prenylated flavonoids and their derivatives to facilitate the application of these compounds as functional foods and drug candidates.25,26 Our previous studies have identified several series of potent α-glucosidase inhibitors.26−28 In this study, we evaluate the in vitro α-glucosidase and α-amylase inhibitory activities of compounds 1−4 and the in vivo antidiabetic activity [in streptozotocin (STZ)-induced diabetes mice] of the most potent flavonoid 2. To the best of our knowledge, this is the first study of in vitro and in vivo antidiabetic activity of prenylated flavonoids 1−4.

INTRODUCTION The hop cones, flowers of widely cultivated common hop (Humulus lupulus L. Cannabaceae), are used by the brewing industry to give beer characteristic aroma and flavor.1 In recent years, beneficial effects of this plant have received an increased amount of attention. Hops contain flavonoids, terpenoids, phloroglucinol, and catechins2 and are widely used to treat menopausal vasomotor symptoms,3,4 cancer,5 joint pain, and loss of appetite.4 Xanthohumol, the principal flavonoid of H. lupulus, displays anticancer, antimicrobial, antioxidant, antiinflammatory, and estrogenic activities.6,7 Metabolism and in vivo pharmacokinetics of xanthohumol have been investigated.8,9 3′-Prenylated chalconaringenins desmethylxanthohumol (1) and 3′-geranylchalconaringenin (2) and 8-prenylated naringenins 8-prenylnaringenin (3) and 8-geranylnaringenin (4) (Figure 1) are desmethylated isomerization products of xanthohumol present in small quantities in beer and hops. The medicinal value of these compounds is not well understood. Desmethylxanthohumol (1) was isolated from hops in 198810 and found to display anticancer11 and antioxidant activity.12,13 3′-Geranylchalconaringenin (2) and 8-prenylnaringenin (3) were isolated from hops in 19971 and shown to exhibit cytotoxic, antioxidant,14 and endocrine activities.15 8-Prenylnaringenin (3),16 a metabolite of isoxanthohumol, is the most potent phytoestrogen discovered to date and shows anticancer activity.17−19 8-Geranylnaringenin (4) possesses weak phytoestrogenic15 and anticancer20 potencies. Recently, xanthohumol was found to inhibit α-glucosidase21 and improve dysfunctional glucose and lipid metabolism in vitro.22 To the best of our © 2017 American Chemical Society

Received: Revised: Accepted: Published: 1574

December 4, 2016 January 28, 2017 January 28, 2017 January 29, 2017 DOI: 10.1021/acs.jafc.6b05445 J. Agric. Food Chem. 2017, 65, 1574−1581

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Figure 1. Structures of natural flavones 1−6 and acarbose.

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target pH (7.0 ± 2.0), choosing stereoisomers, and geometry minimization with the OPLS2005 force field.31 Molecular docking was performed using the flexible docking protocol in Discovery Studio 3.5.32 The isomaltase crystal structure was downloaded from the Protein Data Bank (PDB) (3A4A). The protein was prepared by removing water and adding hydrogen atoms (“Clean Protein” protocol). In the first step of flexible docking, sidechain rotamers of protein residues were sampled using the ChiFlex algorithm.33 Asp 69, His 112, Arg 213, Asp 215, Glu 276, His 351, Asp 352, and Arg 442, key residues in the catalytic active site, were selected as being flexible. Low-energy conformations of ligands were selected with the “BEST” algorithm.34 The binding site was defined using all atoms within 10 Å of the cocrystallized ligand. After protein side chains had been minimized, docking was performed using the CDOCKER protocol of Discovery Studio, including a final simulated annealing and energy minimization of ligand poses. Materials and Protocols in Vivo. Animals and Diets. Male Kunming mice (18−22 g) were procured from The Institute for Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC) [Beijing, China, License SCXK (Jun) 2012-0004]. Animal procedures were approved by the Tianjin Medical University Institutional Animal Care and performed in strict compliance with local and national ethical guidelines. The mice were kept in polypropylene cages (five in each cage) and housed under laboratory conditions (18−23 °C, 55−60% humidity, 12 h light/dark cycle). The mice were fed a standard pellet diet for 1 week after arrival and randomly divided into two groups: mice fed a standard pellet diet (normal mice) and mice fed a high-fat, high-fructose diet to induce type 2 diabetes. All mice had free access to food and water. Induction of Experimental Type 2 Diabetic Mice and Treatment.35 After eating the high-fat, high-fructose food for 3 weeks, the mice were subjected to a 12 h fast. Type 2 diabetes was induced by an intraperitoneal injection of STZ [100 mg/kg of body weight, dissolved in 0.05 M citrate buffer (pH 4.5) (Sigma)] and 2 weeks of the high-fat, high-fructose diet before the initiation of treatment. Fasting blood from the tail vein was used to determine blood glucose concentrations. The mice were classified as type 2 diabetes if blood glucose levels were >11.0 mM. Type 2 diabetic mice were fed the high-fat, high-fructose diet throughout the study. Blood glucose and body weight were checked every week. Type 2 diabetic mice were divided into four groups with 10 mice per group. Every day, 10 mL of saline/kg, 100 mg of acarbose in saline/kg, 50 mg of compound 2 in saline/kg, and 100 mg of compound 2 in saline/kg were orally administered to groups 1 (diabetic control), 2, 3, and 4, respectively. Determination of Blood Glucose Levels. Blood glucose levels were determined as previously described.36 After receiving treatment for 14 days and fasting overnight, all mice were orally administered maltose (2 g/kg) 60 min after treatment. Blood samples were taken from the tail vein after 0, 30, 60, and 120 min, and blood glucose was measured using a glucometer (Sinocare, Changsha, China). Areas under the curve (AUCs) were calculated with the trapezoidal rule. Determination of Blood Lipid Levels. Blood lipid levels were determined as previously described.35 At the end of the experimental period, blood samples were collected and the serum samples obtained by centrifugation (2000g for 20 min) were stored at −20 °C for further analysis. Serum total cholesterol (TC) and triglycerides (TGs) were

MATERIALS AND METHODS

Materials and in Vitro Protocols. Materials. Desmethylxanthohumol, 3′-geranylchalconaringenin, 8-prenylnaringenin, and 8-geranylnaringenin were isolated from hops and identified (see the Supporting Information for spectral data), as previously described.29 Baker’s yeast α-glucosidase, hog pancreas α-amylase, almond β-glucosidase, pnitrophenyl α-D-glucopyranoside (pNαGP), 2-chloro-4-nitrophenyl αD -maltotrioside (G3-CNP), p-nitrophenyl β- D -glucopyranoside (pNβGP), acarbose, and bis(8-anilinonaphthalene-1-sulfonate) (bisANS) were purchased from Sigma-Aldrich (St. Louis, MO). Dialysis bags (MD 34-3.5-5) were obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Enzyme Inhibition Assays. Commercially available α-glucosidase, α-amylase, and β-glucosidase were selected as the target proteins, with pNαGP, G3-CNP, and pNβGP as their substrates, respectively. Compounds 1−6 and acarbose were dissolved in dimethyl sulfoxide. α-Glucosidase, β-glucosidase, pNαGP, and pNβGP were dissolved in potassium phosphate buffer (0.05 M, pH 6.8). α-Amylase and G3CNP were dissolved in potassium phosphate buffer (0.1 M, pH 6.0). Enzymatic reaction mixtures were composed of enzymes (20 μL), substrates (30 μL), test compounds (10 μL), and potassium phosphate buffer (140 μL) and incubated at 37 °C for 30 min. Enzymatic activity was detected by spectrophotometry at 405 nm. α-Glucosidase, αamylase, and β-glucosidase were used at final concentrations of 4, 25, and 2 milliunits in potassium phosphate buffer with pNαGP (0.075 mM), G3-CNP (0.5 mM), and pNβGP (0.75 mM) as substrates, respectively. Results are the average of three independent experiments performed in duplicate. Kinetics of Enzyme Inhibition. The kinetics of α-glucosidase inhibition was determined using the inhibition assays from the previous section, in the presence of different concentrations of compound 2 (0, 1, 2, and 3 μM) and substrate (0.15, 0.3, 0.6, and 1.2 mM). Kinetics was analyzed using the Lineweaver−Burk plots of the substrate concentration and velocity. Dialysis Experiment. α-Glucosidase alone (400 μL, 0.01 unit/mL) or α-glucosidase (400 μL, 0.01 unit/mL) treated with 2 (20 μM) was dialyzed in buffer with dialysis bags (Solarbio) at 4 °C for 24 h. After dialysis, the only α-glucosidase group was treated with 2 for 30 min. The residual α-glucosidase activity of the contents in the dialysis bags was analyzed as described above.21 Intrinsic Fluorescence Measurements. α-Glucosidase (1 unit) was pretreated with 2 (0, 1, 5, 20, 100, 300, and 500 μM) for 30 min at 25, 31, and 37 °C. Intrinsic fluorescence spectra (330−400 nm) were recorded using an Infinite M200PRO fluorescence spectrophotometer (Tecan Inc., Grödig, Austria) with an excitation wavelength of 295 nm.30 Hydrophobicity Analysis of α-Glucosidase Using Bis-ANS. αGlucosidase (1 unit) was incubated with different concentrations of 2 (0, 0.01, 0.1, 1, 3, 5, 20, and 50 μM) at 37 °C for 30 min. A fluorescence probe, Bis-ANS (30 μL, 100 μM), was added and the mixture incubated at 37 °C for an additional 15 min. Fluorescence (400−700 nm) was measured (λex = 400 nm) using a Synergy H1 microplate spectrofluorometer (BioTek, Winooski, VT). Docking Study. Compounds 1, 2, and 5 and acarbose were prepared using the “Ligand Preparation” module of Discovery Studio. The preparation consisted of generating possible ionization states at a 1575

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(IC50 = 51.30 μM) and displayed moderate inhibitory activity against α-amylase (IC50 = 20.46 μM). α-Glucosidase Inhibitory Mechanisms of Compound 2. Compound 2 Inhibits α-Glucosidase Competitively. To determine the mechanisms of α-glucosidase inhibition by compound 2, kinetic analysis was performed using Lineweaver−Burk plots. Compound 2 (0, 1, 2, and 3 μM) was added to α-glucosidase dissolved in potassium phosphate buffer (20 μL, 0.04 unit/mL) and incubated at 37 °C for 30 min. pNαGP at different concentrations (0.15, 0.3, 0.6, and 1.2 mM) was added to initiate the reaction. A series of Lineweaver−Burk plots were obtained (Figure 2A). The results showed that the values of 1/Km increased with increasing concentrations of 2, whereas the intercept on the y-axis remained constant, indicating that 2 is a competitive inhibitor of α-glucosidase. Compound 2 Inhibits α-Glucosidase Irreversibly. Inhibition of α-glucosidase by different concentrations of 2 is illustrated in Figure 2B. Plotting velocity versus enzyme concentration in the presence of different concentrations of 2 resulted in a family of parallel straight lines. The slopes of the lines were unaffected with increasing inhibitor concentrations, indicating that 2 was an irreversible inhibitor. Compound 2 Quenches the Intrinsic Fluorescence of αGlucosidase. The interaction of compound 2 with αglucosidase was examined using fluorescence quenching experiments. Stern−Volmer plots for the quenching of αglucosidase by 2 at different temperatures (25, 31, and 37 °C) are presented in Figure 3A and Table 2. The results show that the KSV values (Stern−Volmer quenching constants) for the interaction between 2 and α-glucosidase decreased with an increase in temperature. Consequently, fluorescence quenching was mainly controlled by a static mechanism resulting from the formation of the compound 2−α-glucosidase complex.30 As shown in Figure 3B, at 25 °C, α-glucosidase displayed an intrinsic fluorescence emission peak belonging to tryptophan residues21 at 334 nm after being excited at 295 nm, whereas 2 did not fluoresce from 320 to 400 nm. The fluorescence intensity of compound 2-treated (1−500 μM) α-glucosidase at 334 nm was gradually quenched with increasing amounts of 2, in a concentration-dependent manner. These results indicated that compound 2 directly interacted with α-glucosidase and quenched its intrinsic fluorescence. Compound 2 Reduces the Hydrophobicity of α-Glucosidase. To further study the binding and interaction between 2 and α-glucosidase, the surface hydrophobicity of α-glucosidase was measured using an extrinsic fluorescence probe bis-ANS.37

analyzed using commercial diagnostic kits (Jiancheng, Nanjing, China). Statistical Analysis. All data are expressed as means ± the standard deviation (SD) or means ± the standard error of the mean (SEM). Results were analyzed by one-way analysis of variance (ANOVA) or two-tailed independent Student’s t tests. Significant differences were determined by homogeneity of variance and least significant difference (LSD) multiple comparisons using SPSS version 21.0.

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RESULTS AND DISCUSSION In Vitro α-Glucosidase, α-Amylase, and β-Glucosidase Inhibition. α-Glucosidase and α-amylase inhibitory activities of prenyl and geranyl flavonoids 1−4, nonprenylated scaffolds naringenin (5) and chalconaringenin (6), and acarbose (Figure 1) were investigated. To explore specificity toward αglucosidase-related enzymes, inhibitory activity against βglucosidase was evaluated. As shown in Table 1, all flavonoids Table 1. α-Glucosidase and α-Amylase Inhibition IC50 (μM)a no.

name

αglucosidase

αamylase

βglucosidase

1 2 3 4 5 6

3′-prenylchalconaringenin 3′-geranylchalconaringenin 8-prenylnaringenin 8-geranylnaringenin chalconaringenin naringenin acarboseb

22.42 1.08 45.92 3.77 20.02 44.65 51.30

85.92 20.46 >100 15.38 >100 >100 2.21

>50 >50 >50 >50 >50 >50 >50

a

Results are the average of three independent experiments, each performed in duplicate. Standard deviations were less than ±10%. b Reference compound.

exhibited moderate to good inhibition of α-glucosidase. Geranyl flavonoids 2 and 4 showed potencies higher than those of prenyl flavonoids 1 and 3 and their nonprenylated precursors 5 and 6, indicating that the geranyl group is essential for α-glucosidase inhibition. In addition, compounds 2 and 4 were more potent upon being used for α-amylase inhibition. No apparent inhibitory activity against β-glucosidase was observed for any of the compounds, with IC50 values of >50 μM. Overall, geranyl flavonoids 2 and 4 displayed dualinhibitory activity against α-glucosidase and α-amylase. 3′Geranylchalconaringenin (2) showed α-glucosidase inhibitory activity (IC50 = 1.08 μM) 50-fold higher than that of acarbose

Figure 2. Inhibitory kinetics of compound 2 on α-glucosidase. (A) Lineweaver−Burk plots of 2 [(▲) 3, (▼) 2, (■) 1, and (●) 0 μM]. (B) Relationship of the α-glucosidase activity and enzyme concentration (4, 6, and 8 milliunits/mL) at different concentrations of 2 [(●) 0, (▲) 4, and (▼) 5 μM]. 1576

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Figure 3. Variation of the intrinsic fluorescence spectra of α-glucosidase. (A) Stern−Volmer plots for fluorescence quenching at three different temperatures. (B) The enzyme (1 unit/mL) was incubated with compound 2 (1−500 μM) for 30 min at 25 °C.

crystal structure of isomaltase (PDB entry 3A4A) from S. cerevisiae that was reported to be a more suitable template because its sequence is 71% identical and 84% similar with that of S. cerevisiae α-glucosidase. Besides, the main interactions of acarbose with S. cerevisiae α-glucosidase and S. cerevisiae isomaltase are quite similar.38−40 Flavones 1, 2, and 5 were docked in the binding pocket of isomaltase and compared to acarbose (Figure 5). Chalcone scaffolds of the compounds are oriented toward the core of the binding pocket, allowing the chalcone substituents to interact closely with important residues in the active site. Five hydrogen bonds are established between the phenolic hydrogens and oxygens of chalconaringenin (5) and the side chains of Lys 156, Ser 157, Ser 241, Asp 242, and Gln 353. Phenyl rings A and B of 5 form π-interactions with Lys 156, Tyr 158, Arg 315, and Gln 353 (Figure 5B). Introduction of a hydrophilic prenyl group on C-3′ of ring A (compound 1) led to the formation of H-bonds with Phe 178, Asp 242, Gln 353, and Glu 277 and πinteractions with Phe 303 and Arg 442 (Figure 5D), resulting in an altered docking pose and an altered orientation of 1 in the active pocket, compared to those of 5. Calculated docking scores of 1 (47 kcal/mol) and 5 (49 kcal/mol) were similar, consistent with their similar potency (Table 1). Introduction of a longer geranyl side chain on C-3′ (compound 2) allows for interactions (Figure 5F) deeper in the binding pocket, which stabilize 2 in the active site. In addition, four H-bonds and three π-interactions were formed between hydroxyl and benzyl groups of 2 and key residues of the active pocket. These interactions improve the fit of 2 in the active pocket, resulting in a binding conformation similar to that of acarbose (Figure 5G). The calculated docking score of 2 (58 kcal/mol) was significantly higher than scores of 1 and 5, consistent with the experimental observation (Table 1) that 2 is a more efficient enzyme inhibitor. Compound 2 Reduces Blood Glucose Levels in Vivo. To understand the basis of suppression for compound 2 with a carbohydrate, we furthermore investigated the influence of compound 2 on blood glucose levels with an in vivo maltose loading test. α-Glucosidase and α-amylase hydrolyze maltose, releasing glucose. Therefore, blood glucose levels after oral administration of maltose indirectly reflect the activities of αglucosidase and/or α-amylase. Postprandial blood glucose levels at 15 and 30 min of mice treated with compound 2 (50 and 100 mg/kg) were lower than those of control and acarbose-treated (100 mg/kg) diabetic mice (Figure 6A). AUCs for glucose in treated diabetic mice were reduced by 16.4% (46.1 mmol h−1 L−1) for acarbose (100 mg/kg), 22.1%

Table 2. Quenching Constants (KSV) and Associated Correlation Coefficients for the Interaction between Compound 2 and α-Glucosidase at Different Temperatures

a

T (°C)

KSV (×104 L/mol)

Ra

25 31 37

1.69 1.54 1.29

0.9990 0.9829 0.9874

R is the correlation coefficient for the KSV values.

Excited at 400 nm, bis-ANS did not exhibit a fluorescence signal from 430 to 700 nm (Figure 4). However, a significantly

Figure 4. Fluorescence spectra of the α-glucosidase−bis-ANS complex in the absence and presence of different concentrations of 2 (0.01−50 μM).

increased fluorescence intensity was observed when bis-ANS was incubated with α-glucosidase, whose maximal absorption peak was at 496 nm. When the enzyme−bis-ANS complex was treated with 2, the fluorescence of the complex decreased remarkably in a concentration-dependent manner. These results suggested that 2 might compete at the binding sites of the enzyme−bis-ANS complex and/or reduce the surface hydrophobicity of α-glucosidase. Docking Analysis. Molecular docking was performed on flavones 1, 2, and 5 to identify binding modes that could explain the effects of the H, prenyl, and geranyl substituents on activity. As the crystal structure of α-glucosidase from Saccharomyces cerevisiae (the enzyme used in our biological assay) is not available, a docking study was conducted using the 1577

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Figure 5. Binding modes of 1, 2, 5, and acarbose in the enzyme active site. Docking of (A) 5, (C) 2, and (E) 1 and corresponding two-dimensional interaction diagrams (B, D, and F, respectively). Blue and green dotted lines indicate hydrogen bonds, whereas π-interactions are represented by orange lines. Residues involved in hydrogen bonds, charge, and polar interactions (pink circles) or van der Waals interactions (green circles) are shown. (G) Compound 2 (yellow) and acarbose (gray) superimposed in the active pocket.

(43.0 mmol h−1 L−1) for compound 2 (50 mg/kg), and 26.2% (40.7 mmol h−1 L−1) for compound 2 (100 mg/kg) compared to that of the STZ control group (55.1 mmol h−1 L−1) (Figure 6B). These results suggest that administration of 2 significantly suppressed postprandial hyperglycemia in diabetic mice. Compound 2 Reduces Serum Levels of Total Cholesterol and Triglycerides. As shown in Table 3, a significant increase in

serum levels of TC and TGs was observed in STZ-induced diabetic mice compared to the control (P < 0.01). In diabetic mice treated with 2 (50 and 100 mg/kg) and acarbose (100 mg/kg), TC levels were decreased by 35.4, 37.4, and 28.2%, respectively, whereas TG levels were decreased by 33.0, 46.0, and 20.4%, respectively, compared to those of STZ control mice. 1578

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Figure 6. Blood glucose levels after administration of 2 in diabetic mice. (A) Blood glucose concentrations of mice after an oral load of maltose. (B) Effects of 2 on the AUC of the oral load of maltose in diabetic and normal mice. Data are represented as means ± SEM (n = 10). *P < 0.05 vs model; **P < 0.01 vs model; ***P < 0.001 vs model; #P < 0.001 vs normal.

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Table 3. TC and TG Levels of Mice Groupsa group normal STZ control 2 2 acarbose

dosage (mg/kg)

50 100 100

TC (mmol/L) 1.32 1.95 1.26 1.22 1.40

± ± ± ± ±

0.38 1.04 0.68 0.48b 0.67

* Supporting Information

TG (mmol/L) 5.33 14.88 9.97 8.04 11.85

± ± ± ± ±

ASSOCIATED CONTENT

S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b05445. Nuclear magnetic resonance and mass spectral signals of compounds 1−4 (PDF)

0.39 3.37 2.20c 2.14d 2.91

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Data are expressed as means ± SD (n = 10). bP < 0.05 vs STZ control. cP < 0.01 vs STZ control. dP < 0.001 vs STZ control.

a

AUTHOR INFORMATION

Corresponding Authors

*Telephone: +86-22-60912562. Fax: +86-22-60602298. E-mail: [email protected]. *E-mail: [email protected].

Several mechanisms have been proposed for antihyperlipidemic effects of hops extract in vivo models: inhibition of absorption of fat from the intestine, suppressed activity of enzymes involved in fatty acid synthesis, and reduced level of SREBP-1c mRNA expression.41 Xanthohumol, a hops analogue of compound 2, decreased the rate of de novo lipogenesis by inhibiting SREBP maturation in the mouse liver42 and reducing the level of hepatic mRNA expression of genes involved in fatty acid synthesis and gluconeogenesis.43 α-Glucosidase and αamylase inhibition could be the antihyperlipidemic mechanism underlying compound 2-induced reduction of serum TC and TGs levels. In conclusion, this study examined the antidiabetic activity of natural prenylchalconaringenins 1 and 2 and prenylnaringenins 3 and 4 from hops. 3′-Geranylchalconaringenin (2) was identified as a potent α-glucosidase and moderate α-amylase dual inhibitor in vitro and shown to inhibit α-glucosidase in a competitive and irreversible manner. Fluorescence quenching of α-glucosidase was shown to be a static process resulting from the formation of the compound 2−α-glucosidase complex. The results of fluorescence quenching and surface hydrophobicity analysis of α-glucosidase indicated that direct binding of 2 to αglucosidase induced conformational changes in the protein. In addition, hydrogen bonding, π, van der Waals, and electrostatic interactions indicated in the molecular docking study, together with a good shape match to the active pocket, could explain the high affinity of 2 for α-glucosidase. Compound 2 at doses of 50 and 100 mg/kg significantly ameliorated the increase in postprandial blood glucose, serum TC, and serum TG levels in STZ-induced diabetic mice. Taken together, the results of this study indicate that 2 exhibits antidiabetic activity in vitro and in vivo and possesses developmental potential as a functional food and antidiabetic drug candidate. The metabolism and pharmacokinetics of compound 2 are the subjects of ongoing studies in our laboratories.

ORCID

Hua Sun: 0000-0001-9920-6778 Funding

This study was supported by the National Natural Science Foundation of China (21502138) and the Scientific Research Foundation (20110115) and Laboratory Innovation Foundation of Undergraduate (1504A301) of the Tianjin University of Science and Technology. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to the Research Centre of Modern Analytical Technology of the Tianjin University of Science and Technology for nuclear magnetic resonance measurements and matrix-assisted laser desorption ionization time-of-flight analysis.

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REFERENCES

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DOI: 10.1021/acs.jafc.6b05445 J. Agric. Food Chem. 2017, 65, 1574−1581