Characterization of Maltase and Sucrase Inhibitory Constituents from Rhodiola Crenulata

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Article Characterization of Maltase and Sucrase Inhibitory Constituents from Rhodiola crenulata

1, 2, 2, Wen-Tai Li †, Yu-Hsuan Chuang † and Jung-Feng Hsieh * 1 National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei 11221, Taiwan; [email protected] 2 Department of Food Science, Fu Jen Catholic University, Taipei 242, Taiwan; [email protected] * Correspondence: [email protected]; Tel.: +886-2-2905-2516 These authors contributed equally to the work. † Received: 29 September 2019; Accepted: 31 October 2019; Published: 2 November 2019

Abstract: The inhibitory properties of epicatechin-(4β,8)-epicatechingallate (B2-3’-O-gallate), epicatechin gallate (ECG), and epicatechin (EC) isolated from Rhodiola crenulata toward maltase and sucrase were investigated. The half-maximal inhibitory concentration (IC50) values for maltase were as follows: B2-3’-O-gallate (1.73 1.37 µM), ECG (3.64 2.99 µM), and EC (6.25 1.84 µM). ± ± ± Inhibition kinetic assays revealed the inhibition constants (Ki) of the mixed-competitive inhibitors of maltase, as follows: B2-3’-O-gallate (1.99 0.02 µM), ECG (3.14 0.04 µM), and EC (7.02 0.26 µM). ± ± ± These compounds also showed a strong inhibitory activity toward sucrase, and the IC50 values of B2-3’-O-gallate, ECG, and EC were 6.91 3.41, 18.27 3.99, and 18.91 3.66 µM, respectively. ± ± ± Inhibition kinetic assays revealed the inhibition constants (Ki) of the mixed-competitive inhibitors of sucrase as follows: B2-3’-O-gallate (6.05 0.04 µM), ECG (8.58 0.08 µM), and EC (13.72 ± ± ± 0.15 µM). Overall, these results suggest that B2-3’-O-gallate, ECG, and EC are potent maltase and sucrase inhibitors.

Keywords: maltase; sucrase; inhibitor; inhibition kinetics assay; Rhodiola crenulata

1. Introduction Over the past three decades, the number of people with diabetes mellitus has quadrupled, with the result being that diabetes mellitus is now the ninth leading cause of death worldwide. Approximately 1 in 11 adults have diabetes mellitus, and roughly 90% of those cases are type 2. The most pronounced symptom of diabetes mellitus is an abnormal postprandial increase in blood glucose levels [1,2]. It is widely believed that control over postprandial hyperglycemia is crucial to the effective treatment of diabetes mellitus [3]. It is well known that dietary carbohydrates are broken down into monosaccharides by hydrolytic enzymes, α-glucosidases, which are absorbed into the intestinal brush border membrane. The final step of carbohydrate digestion can be catalyzed by α-glucosidases. Furthermore, a number of physiologically important enzymes are involved in the process of digesting dietary carbohydrates [4,5]. Thus, one approach to controlling this digestion process involves delaying the absorption of glucose by inhibiting α-glucosidase activity [6,7]. α-Glucosidases are divided into four hydrolase types, i.e., maltase (Enzyme Commission (EC) 3.2.0.20), sucrase (EC 3.2.1.48), glucoamylase (EC 3.2.1.3), and isomaltase (EC 3.2.1.10). These four enzymes form two complexes with different substrate specificities, maltase–glucoamylase and sucrase–isomaltase complexes [8]. Among them, maltase is the major enzyme responsible for the digestion and absorption of dietary starch, whereas sucrase can hydrolyze sucrose [9]. Maltase is a membrane-bound enzyme that binds to microvilli on intestinal enterocytes. It can hydrolyze the α-1,4 linkages of maltose residues to release a single glucose molecule [10,11]. In addition, sucrase

Foods 2019, 8, 540; doi:10.3390/foods8110540 www.mdpi.com/journal/foods Foods 2019, 8, 540 2 of 13 is localized in the brush border membranes of the small intestine. This enzyme can hydrolyze the α-1,2 linkages of sucrose to release glucose and fructose [12]. By acting against these enzymes in the gut, maltase and sucrase inhibitors reduce postprandial glucose levels by restraining the liberation of glucose from oligosaccharides and disaccharides [4]. Nijpels et al. [13] reported that diabetes could be managed by reducing the impact of glycemia by inhibiting maltase and sucrose activity through the regular consumption of the antihyperglycemic drug acarbose. In one intervention study, the daily consumption of acarbose for a period of three years was shown to reduce the risk of developing type 2 diabetes by 6%, compared to a control group that was not consuming acarbose. In a study by Kwon et al. [14], water extracts of Rhodiola crenulata (R. crenulata) were shown to significantly reduce the inhibitory activity of α-glucosidase. The genus Rhodiola L. (Crassulaceae) comprises approximately 90 species found throughout the world, and more than 70 species are found in the western and northern regions of Asia. Among these, R. crenulata, is an important member of the Crassulaceae family. For centuries, the roots of R. crenulata have been used as a health supplement. It is possible that these extracts could be used as a supplement for postprandial hyperglycemia associated with diabetes mellitus. In our previously study, we isolated and characterized α-glucosidase inhibitory constituents, including epicatechin-(4β,8)-epicatechingallate (B2-3’-O-gallate), epicatechin gallate (ECG), and epicatechin (EC) from R. crenulata [15]. These results clearly demonstrated the strong inhibitory effects of B2-3’-O-gallate, ECG, and EC against α-glucosidase activity. The above potent maltase and sucrase inhibitors were considered for use in treating diabetes mellitus. Although the inhibition of α-glucosidases by B2-3’-O-gallate, ECG, and EC has previously been reported, the inhibitory properties of B2-3’-O-gallate, ECG, and EC toward maltase and sucrase have not been examined. Thus, this paper describes the inhibition kinetics of B2-3’-O-gallate, ECG, and EC toward maltase and sucrase. Our primary objective in this study was to elucidate the inhibitory effects of 2-3’-O-gallate, ECG, and EC on maltase and sucrase.

2. Materials and Methods

2.1. Preparation of B2-3’-O-gallate, ECG, and EC from R. crenulata Dried roots of R. crenulata were obtained from a traditional Chinese medicine pharmacy in Chiayi in south Taiwan. The authenticity was confirmed by Dr. Hsiang-Wen Tseng (Industrial Technology Research Institute, Taiwan) using DNA sequencing technology and an internal transcribed spacer sequence database. Isolates of B2-3’-O-gallate, ECG, and EC were prepared from a crude extract of R. crenulata in accordance with the methods reported by Chu et al. [15]. Briefly, a total of 90 g R. crenulata roots was milled and extracted using distilled water (900 mL). After centrifugation at 12,000 g for × 15 min, the supernatant was collected and freeze-dried to yield 16 g of water extract. The extract that displayed IC value for maltase was 5.23 0.18 µg/mL, whereas the extract that displayed IC value 50 ± 50 for sucrase was 21.42 0.45 µg/mL. The water extract underwent column chromatography on a vacuum ± manifold using solid-phase extraction (SPE) cartridges. Each sample (50 mg) was chromatographed on a SPE cartridge. The samples were then eluted stepwise using 0%, 10%, 20%, 30%, and 40% methanol in distilled water. A total of 5 fractions, one for each methanol elution (20 mL), were collected. The samples were concentrated using a rotary evaporator and then freeze-dried. The total yield of the fractions was 92.4%, whereas the yields of the individual fractions were as follows: 0% elution (67.6%), 10% elution (16.5%), 20% elution (5.1%), 30% (2.3%), and 40% (0.9%). The 30% fraction was redissolved in distilled water and subjected to column chromatography on a HPLC high-performance liquid chromatography (HPLC) system comprising a pump (PU-980, JASCO, Tokyo, Japan) and a detector (UV-970, JASCO) with a C18 packed column (4.6 mm 250 mm, 5 µm Spherical, Dikma × Technologies Inc.). A gradient elution from H2O/acetonitrile (86:14) to H2O/acetonitrile (72:28) was used to isolate B2-3’-O-gallate, ECG, and EC. Elution began with a solvent flow rate of 1 mL/min for the collection of B2-3’-O-gallate, ECG, and EC. The total yields were as follows: B2-3’-O-gallate (8.7 mg), ECG (6.1 mg), and EC (7.6 mg). Foods 2019, 8, 540 3 of 13

2.2. Maltase Activity Assay Maltase activity was characterized using the methods outlined by Adisakwattana et al. [16] with slight modiﬁcations. Maltose and maltase derived from Saccharomyces cerevisiae (S. cerevisiae) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). A reaction mixture was formulated from 30 µL of maltose (86.3 mM) with 20 µL of maltase (0.33 units/mL) and 50 µL of phosphate buﬀer (100 mM, pH 7.0). The mixture was incubated at 37 ◦C for 10 min and then at 100 ◦C for a further 10 min to stop the reaction. The glucose concentrations released by the reaction mixture were determined via the glucose oxidase method (glucose assay kit, Sigma Chemical Co., St. Louis, MO, USA) using a VersaMax microplate reader (Molecular Devices Corporation, Sunnyvale, CA, USA) at an absorbance wavelength of 540 nm.

2.3. Maltase Inhibitory Activity of B2-3’-O-gallate, ECG, EC, and Quercetin In this study, we focused on B2-3’-O-gallate, ECG, EC, and quercetin, with quercetin (Sigma Chemical Co., St. Louis, MO, USA) as positive controls. All of the samples were assessed in terms of maltase inhibitory activity under a range of concentrations (0–30 µM). Maltase inhibitory activity was evaluated by dissolving the samples directly in phosphate buﬀer solution (100 mM, pH 7.0). Note that each sample was assayed in triplicate. Inhibitory activity was estimated in terms of IC50 values based on percent inhibition, as follows:

percent inhibition (%) = [(A A )] 100%/A (1) without sample − with sample × without sample 2.4. Sucrase Activity Assay Estimates of sucrase activity were obtained using the method outlined by Akkarachiyasit et al. [17] with slight modiﬁcations. Sucrose and sucrase derived from Leuconostoc mesenteroides were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The reaction mixture comprised 40 µL of sucrose (480 mM) with 10 µL of sucrase (0.33 units/mL) in 50 µL of phosphate buﬀer solution (100 mM, pH 7.0). The mixtures were incubated at 37 ◦C for 60 min and then at 100 ◦C for a further 10 min to stop the reaction. The glucose concentrations released from the reaction mixtures were estimated via the glucose oxidase method (glucose assay kit, Sigma Chemical Co., St. Louis, MO, USA) using a VersaMax microplate reader (Molecular Devices Corporation, Sunnyvale, CA, USA) at an absorbance wavelength of 540 nm.

2.5. Sucrase Inhibitory Activity of B2-3’-O-gallate, ECG, EC, and Quercetin In this study, we focused on B2-3’-O-gallate, ECG, EC, and quercetin, with quercetin (Sigma Chemical Co., St. Louis, MO, USA) as positive controls. All samples were assessed in terms of sucrase inhibitory activity under a range of concentrations (0–50 µM). Inhibitory activity was assessed by dissolving samples directly in phosphate buﬀer solution (100 mM, pH 7.0). Inhibitory activity was estimated in terms of IC50 values based on percent inhibition, as follows:

percent inhibition (%) = [(A A )] 100%/A (2) without sample − with sample × without sample 2.6. Lineweaver–Burk Plots and Dixon Plots Lineweaver–Burk plot analysis was used to identify the mode of inhibition of B2-3’-O-gallate, ECG, EC, and quercetin on maltase and sucrase. Kinetics assays were conducted using substrates of various concentrations with and without inhibitors. Maltose was used as a substrate for maltase, where the initial velocity was expressed as the rate of absorbance at 540 nm per min. Sucrose was used as a substrate for sucrase, where the initial velocity was expressed as the rate of absorbance at 540 nm per 18 min. Dixon plot analysis was used to determine the competitive inhibition constant (Ki) and uncompetitive inhibition constant (Ki’). Ki was used as the equilibrium constant for the Foods 2019, 8, x FOR PEER REVIEW 4 of 13 Foods 2019, 8, 540 4 of 13 of the maltase inhibitors were derived from Dixon plots in accordance with the methods described by Cornish-Bowden [18]. inhibition of binding to maltase and sucrase, whereas Ki’ was used as the equilibrium constant for the inhibition of binding to maltase–maltose and sucrase–sucrose complexes. The Ki and Ki’ values of 2.7. Statistical Analysis the maltase inhibitors were derived from Dixon plots in accordance with the methods described by Cornish-BowdenData are expressed [18]. as the mean ± standard deviation. All data analysis was performed using Statistical Analysis System (SAS software release 9.4 for Windows, version 13.2, SAS Institute, Inc., 2.7.Cary, Statistical NC, USA). Analysis Statistically significant differences between treatment groups were determined usingData one-way are expressed ANOVA as followed the mean by Duncan’sstandard multiple deviation. range All test. data Three analysis replicates was performed of each sample using ± Statisticalwere assayed, Analysis and the System level (SAS of significance software release was set 9.4 at forp < Windows,0.05. version 13.2, SAS Institute, Inc., Cary, NC, USA). Statistically significant differences between treatment groups were determined using 3. Results and Discussion one-way ANOVA followed by Duncan’s multiple range test. Three replicates of each sample were assayed, and the level of significance was set at p < 0.05. 3.1. Maltase and Sucrase Inhibitory Activity of B2-3’-O-gallate, ECG, EC, and Quercetin 3. ResultsPotent and maltase Discussion and sucrase inhibitors have been considered for use for treating diabetes mellitus [19]. Thus, we investigated the inhibition of maltase and sucrase by B2-3’-O-gallate, ECG, and EC 3.1. Maltase and Sucrase Inhibitory Activity of B2-3’-O-gallate, ECG, EC, and Quercetin derived from R. crenulata. Figure 1 compares the inhibitory activity of B2-3’-O-gallate, ECG, and EC withPotent that of maltase quercetin, and which sucrase is a inhibitors leading candid have beenate for considered the treatment for useof diabetes for treating mellitus diabetes [20]. mellitusQuercetin [19 was]. Thus, used wein this investigated study as a the standard inhibition inhibitor, of maltase which and reduces sucrase the by hydrolysis B2-3’-O-gallate, of maltose ECG, by andinhibiting EC derived the activity from R. of crenulata maltase.. FigureWang1 et compares al. [21] reported the inhibitory that quercetin activity of showed B2-3’- O high-gallate, inhibitory ECG, andactivity, EC with with that an ofIC quercetin,50 value of which4.8 ± 0.4 is amM leading against candidate rat maltase. for the We treatment assessed of the diabetes inhibitory mellitus effects [20 of]. QuercetinB2-3’-O-gallate, was used ECG, in EC, this and study quercetin as a standard on maltase inhibitor, at various which concentrations. reduces the hydrolysis The addition of maltoseof B2-3’- byO-gallate, inhibiting ECG, the EC, activity and ofquercetin maltase. at Wang a concentration et al. [21] reported of 10 μM that was quercetin shown showedto significantly high inhibitory enhance activity,maltase withinhibition, an IC asvalue follows: of 4.8 B2-3’-0.4O mM-gallate against (90.7%), rat maltase. ECG (87.7%), We assessed EC (66.3%), the inhibitory and quercetin effects 50 ± of(56.3%). B2-3’-O -gallate, ECG, EC, and quercetin on maltase at various concentrations. The addition of B2-3’-O-gallate, ECG, EC, and quercetin at a concentration of 10 µM was shown to significantly enhance maltase inhibition, as follows: B2-3’-O-gallate (90.7%), ECG (87.7%), EC (66.3%), and quercetin (56.3%).

Figure 1. Inhibitory eﬀects of epicatechin-(4β,8)-epicatechingallate (B2-3’-O-gallate), epicatechin gallate (ECG),Figure epicatechin1. Inhibitory (EC), effects and quercetin of epicatechin-(4 on maltase.β,8)-epicatechingallate Each value is represented (B2-3’- asO the-gallate), mean epicatechinstandard ± deviationgallate (ECG), of triplicate epicatechin measurements. (EC), and quercetin on maltase. Each value is represented as the mean ± standard deviation of triplicate measurements. Table1 lists the structures of B2-3’- O-gallate, ECG, EC, and quercetin. The IC50 values for maltase were as follows: B2-3’-O-gallate (1.73 1.37 µM), ECG (3.64 2.99 µM), EC (6.25 1.84 µM), and ± ± ±

Foods 2019, 8, 540 5 of 13

quercetin (8.33 3.91 µM). The strength of the IC values was in the following order: B2-3’-O-gallate ± 50 < ECG < EC < quercetin. We also compared the inhibitory activities of B2-3’-O-gallate, ECG, EC, and quercetin toward sucrase. As shown in Figure2, the addition of B2-3’- O-gallate, ECG, EC, and µ quercetin atFoods a concentration 2019, 8, x FOR PEER of 20REVIEWM was shown to signiﬁcantly enhance sucrase inhibition, as6 follows: of 13 Foods 2019, 8, x FOR PEER REVIEW 6 of 13 B2-3’-O-gallateFoods (84.6%),2019, 8, x FOR ECG PEER (56.1%), REVIEW EC (51.4%), and quercetin (51.7%). 6 of 13 Table 1. Structure, molecular weight, molecular formula, and IC50 values of maltase and sucrase Foods Table2019, 8 , 1.x FORStructure, PEER REVIEW molecular weight, molecular formula, and IC50 values of maltase and sucrase6 of 13 Tableinhibitors. 1. Structure, molecular weight, molecular formula, and IC50 values of maltase and sucrase Table 1. Structure,inhibitors. molecular weight, molecular formula, and IC50 values of maltase and inhibitors. Table 1. Structure, molecular weight, molecular formula, and IC50 values of maltase anda sucrase sucrase inhibitors. Molecular Molecular IC50 (μM) Inhibitor Structure Molecular Molecular IC50 (μM) a inhibitors.Inhibitor Structure MolecularWeight MolecularFormula MaltaseIC50 (μM)Sucrase a Inhibitor Structure MolecularWeight Formula Maltase IC (SucraseµM) a Weight FormulaMolecular Maltase 50 Sucrasea Inhibitor Structure Molecular Molecular IC50 (μM) Inhibitor Structure Weight Formula Epicatechin- Weight Formula MaltaseMaltase Sucrase Sucrase Epicatechin- Epicatechin-(4β,8)- (4β,8)- epicatechingallat(4β,8)- 731.16 C37H30O16 1.73 ± 1.37 6.91 ± 3.41 epicatechingallatEpicatechin- 731.16 C37H30O16 1.73 ± 1.37 6.91 ± 3.41 epicatechingallate (B2-3’-O- 731.16 C37H30O16 1.73 ± 1.37 6.91 ± 3.41 Epicatechin-(4β,8)-epicatechingallatee (B2-3’(4β,8)--O- e (B2-3’gallate)-O- 731.16 C H O 1.73 1.37 6.91 3.41 (B2-3’-O-gallate)epicatechingallatgallate) 731.16 C37H3030O1616 1.73 ± 1.37± 6.91 ± 3.41± e gallate)(B2-3’-O-

gallate)

Epicatechin Epicatechin 442.37 C22H18O10 3.64 ± 2.99 18.27 ± 3.99 gallateEpicatechin (ECG) 442.37 C22H18O10 3.64 ± 2.99 18.27 ± 3.99 Epicatechin gallategallate (ECG) (ECG) 442.37442.37 C C2222HH1818OO1010 3.643.64 ± 2.992.99 18.27 18.27± 3.99 3.99 gallate (ECG) ± ± Foods 2019, 8, x FOR PEEREpicatechin REVIEW 5 of 13 442.37 C22H18O10 3.64 ± 2.99 18.27 ± 3.99 gallate (ECG)

Table 1 lists the structures of B2-3’-O-gallate, ECG, EC, and quercetin. The IC50 values for maltase

were as follows:Epicatechin EpicatechinB2-3’- (EC) O-gallate (EC) (1.73 ± 1.37 μM), 290.27290.27ECG (3.64C C 1515±HH 142.9914OO6 6 μM), 6.256.25 ±EC 1.841.84 (6.25 18.91 ± 18.91 ±1.84 3.66 μ3.66M), and Epicatechin (EC) 290.27 C15H14O6 6.25 ± 1.84± 18.91 ± 3.66± Epicatechin (EC) 290.27 C15H14O6 6.25 ± 1.84 18.91 ± 3.66 quercetin (8.33 ± 3.91 μM). The strength of the IC50 values was in the following order: B2-3’-O-gallate

Epicatechin (EC) 290.27 C15H14O6 6.25 ± 1.84 18.91 ± 3.66 < ECG < EC < quercetin. We also compared the inhibitory activities of B2-3’-O-gallate, ECG, EC, and quercetin towardQuercetin sucrase. As shown in Figure 2,302.24 the addition C15H10 ofO7 B2-3’-8.33O-gallate,3.91 18.98ECG,2.53 EC, and Quercetin 302.24 C15H10O7 8.33 ± 3.91± 18.98 ± 2.53± Quercetin 302.24 C15H10O7 8.33 ± 3.91 18.98 ± 2.53 quercetin at a concentrationQuercetin of 20 μM was shown302.24 to significantlyC15H10O7 enhance 8.33 ± 3.91 sucrase 18.98 ± 2.53inhibition, as

a follows: B2-3’-O-gallateQuercetin (84.6%),IC 50 values ECG are expressed(56.1%), asEC the (51.4%),302.24 mean standard andC15 Hquercetin10 deviation.O7 8.33 (51.7%). ± 3.91 18.98 ± 2.53 a IC50 values are expressed as the mean± ± standard deviation. a IC50 values are expressed as the mean ± standard deviation. a IC50 values are expressed as the mean ± standard deviation. 3.2. Mode of Inhibition ofa B2-3’-O-gallate, ECG, EC, and Quercetin Toward Maltase 3.2. Mode of Inhibition of B2-3’-O-gallate,IC50 values are expressed ECG, EC, as andthe meanQuercetin ± standard Toward deviation. Maltase 3.2. Mode of Inhibition of B2-3’-O-gallate, ECG, EC, and Quercetin Toward Maltase We also evaluated the inhibition kinetics of B2-3’-O-gallate, ECG, EC, and quercetin toward We also evaluated the inhibition kinetics of B2-3’-O-gallate, ECG, EC, and quercetin toward maltase.3.2. ModeWe alsoIn of thisInhibition evaluated study, of the B2-3’-O-gallate,the Michaelis inhibition constant kineticsECG, EC,(K m)of and B2-3’-of Quercetin maltaseO-gallate, was Toward 1.01ECG, MaltasemM, EC, whereas and quercetin in the study toward by maltase. In this study, the Michaelis constant (Km) of maltase was 1.01 mM, whereas in the study by maltase.Pyner et Inal. this[22], study, the K mthe value Michaelis of maltase constant was (7.5Km) mM. of maltase The Lineweaver–Burk was 1.01 mM, whereas plots of inB2-3’- the Ostudy-gallate by PynerWe et al.also [22], evaluated the Km valuethe inhibition of maltase kinetics was 7.5 of mM. B2-3’- ThOe -gallate,Lineweaver–Burk ECG, EC, plotsand ofquercetin B2-3’-O -gallatetoward Pyner(Figure et 3A), al. [22], ECG the (Figure Km value 3B), ECof maltase (Figure was3C), 7.5 and mM. quercetin The Lineweaver–Burk (Figure 3D) did notplots intersect of B2-3’- theO-gallate x- or y- (Figuremaltase. 3A), In this ECG study, (Figure the 3B), Michaelis EC (Figure constant 3C), (andKm) quercetin of maltase (Figure was 1.01 3D mM,) did whereasnot intersect in the the study x- or byy- (Figureaxis, which 3A), isECG indicative (Figure of 3B), mixed-type EC (Figure inhibition 3C), and. quercetinA number (Figure of mixed-type 3D) did notmaltase intersect inhibitors the x- haveor y- axis,Pyner which et al. [22],is indicative the Km valueof mixed-type of maltase inhibition was 7.5 mM.. A number The Lineweaver–Burk of mixed-type maltase plots of inhibitorsB2-3’-O-gallate have axis,previously which beenis indicative reported. of Inmixed-type a previous inhibition study, kinetic. A number analysis of revealedmixed-type that maltase maltase inhibitors was inhibited have previously(Figure 3A), been ECG reported. (Figure 3B), In a EC pr evious(Figure study, 3C), and kinetic quercetin analysis (Figure revealed 3D) didthat not maltase intersect was the inhibited x- or y- previouslyby cinnamic been acid reported. derivatives, In a includingprevious study, caffeic kinetic acid, ferulicanalysis acid, revealed and isoferulic that maltase acid, was in inhibiteda mixed- byaxis, cinnamic which is acid indicative derivatives, of mixed-type including inhibition caffeic ac. Aid, number ferulic ofacid, mixed-type and isoferulic maltase acid, inhibitors in a mixed- have byinhibition cinnamic manner acid derivatives,[16]. Our previous including results caffeic indicated acid, ferulicthat B2-3’- acid,O -gallateand isoferulic (Ki = 0.30 acid, ± 0.03 in aμ M,mixed- Ki’ = inhibitionpreviously manner been reported. [16]. Our In previous a previous results study, indicated kinetic thatanalysis B2-3’- revealedO-gallate that (Ki maltase= 0.30 ± 0.03was μinhibitedM, Ki’ = inhibition1.42 ± 0.01 manner μM), ECG [16]. (K Ouri = 0.21 previous ± 0.04 results μM, K i’indicated = 2.34 ± that0.06 B2-3’-μM), Oand-gallate quercetin (Ki = (0.30Ki = ±1.44 0.03 ± μ 0.04M, Kμi’M, = 1.42by cinnamic ± 0.01 μM), acid ECG derivatives, (Ki = 0.21 including± 0.04 μM, caffeic Ki’ = 2.34acid, ± ferulic0.06 μM), acid, and and quercetin isoferulic (K i acid,= 1.44 in ± a0.04 mixed- μM, 1.42Ki’ =± 9.330.01 μ±M), 0.10 ECG μM) (K iwere = 0.21 mixed-competitive ± 0.04 μM, Ki’ = 2.34 inhibitors ± 0.06 μ M),of α and-glucosidase quercetin [15].(Ki = Furthermore,1.44 ± 0.04 μM, a Kinhibitioni’ = 9.33 manner± 0.10 μ[16].M) Ourwere previous mixed-competitive results indicated inhibitors that B2-3’- of αO-glucosidase-gallate (Ki =[15]. 0.30 Furthermore,± 0.03 μM, Ki’ a= KLineweaver–Burki’ = 9.33 ± 0.10 μplotM) indicatedwere mixed-competitive that epigallocatechin inhibitors gallate of wasα-glucosidase a competitive [15]. inhibitorFurthermore, against a Lineweaver–Burk1.42 ± 0.01 μM), ECG plot ( Kindicatedi = 0.21 ± 0.04that μepigallocatechinM, Ki’ = 2.34 ± 0.06 gallate μM), was and a quercetincompetitive (Ki =inhibitor 1.44 ± 0.04 against μM, Lineweaver–Burkmaltose substrate forplot maltase, indicated and that the Kepigallocatechini calculated from gallate a Dixon was plot a competitivewas 5.93 ± 0.26 inhibitor μM [25]. against maltoseKi’ = 9.33 substrate ± 0.10 forμM) maltase, were mixed-competitiveand the Ki calculated inhibitors from a Dixon of α -glucosidaseplot was 5.93 [15].± 0.26 Furthermore, μM [25]. a maltoseThe substrate Dixon plots for maltase,in Figure and 4 revealed the Ki calculated that all of from the acompounds Dixon plot werewas 5.93 mixed-type ± 0.26 μM inhibitors [25]. of Lineweaver–BurkThe Dixon plots plot in indicatedFigure 4 revealedthat epigallocatechin that all of the gallate compounds was a werecompetitive mixed-type inhibitor inhibitors against of maltaseThe withDixon the plots following in Figure Ki values:4 revealed B2-3’- thatO-gallate all of the (1.99 compounds ± 0.02 μM; were Figure mixed-type 4A), ECG inhibitors (3.14 ± 0.04 of maltasemaltose withsubstrate the following for maltase, Ki andvalues: the KB2-3’-i calculatedO-gallate from (1.99 a Dixon± 0.02 plotμM; was Figure 5.93 4A), ± 0.26 ECG μM (3.14 [25]. ± 0.04 maltaseμM; Figure with 4B), the ECfollowing (7.02 ± K 0.26i values: μM; B2-3’-FigureO -gallate4C), and (1.99 quercetin ± 0.02 μ(7.81M; Figure± 0.10 4A),μM; ECGFigure (3.14 4D). ± 0.04The μM; TheFigure Dixon 4B), plots EC (7.02 in Figure ± 0.26 4 μrevealedM; Figure that 4C), all ofand the qu compoundsercetin (7.81 were ± 0.10 mixed-type μM; Figure inhibitors 4D). The of μstrengthM; Figure of the4B), K iEC values (7.02 was ± 0.26 as follows: μM; Figure B2-3’- 4C),O-gallate and qu< ECGercetin < EC (7.81 < quercetin. ± 0.10 μ M;The Figure Ki’ values 4D). were The strengthmaltase withof the the Ki followingvalues was K asi values: follows: B2-3’- B2-3’-OO-gallate-gallate (1.99 < ECG ± 0.02 < EC μ M;< quercetin. Figure 4A), The ECG Ki’ values (3.14 ± were 0.04 strengthas follows: of B2-3’-the KiO values-gallate was (3.73 as ±follows: 0.08 μM), B2-3’- ECGO-gallate (6.38 ± 0.04 < ECG μM), < EC EC < (18.88 quercetin. ± 0.22 The μM), Ki and’ values quercetin were asμM; follows: Figure B2-3’- 4B), OEC-gallate (7.02 (3.73± 0.26 ± 0.08μM; μ FigureM), ECG 4C), (6.38 and ± 0.04quercetin μM), EC (7.81 (18.88 ± 0.10 ± 0.22 μM; μM), Figure and quercetin4D). The as(14.97 follows: ± 0.75 B2-3’- μM).O-gallate Note that (3.73 all ± 0.08of the μM), K iECG values (6.38 were ± 0.04 lower μM), than EC (18.88 the K ±i’ 0.22 values. μM), As and mentioned quercetin (14.97strength ± 0.75of the μ KM).i values Note wasthat as all follows: of the B2-3’-Ki valuesO-gallate were < lowerECG < than EC < thequercetin. Ki’ values. The KiAs’ values mentioned were (14.97previously, ± 0.75 K iμ isM). the Note equilibrium that all constantof the K fori values the inhibition were lower of binding than the to maltase,Ki’ values. whereas As mentioned Ki’ is the previously,as follows: B2-3’- Ki is theO-gallate equilibrium (3.73 ± constant0.08 μM), for ECG the (6.38 inhibition ± 0.04 μofM), binding EC (18.88 to maltase, ± 0.22 μ whereasM), and quercetinKi’ is the previously,equilibrium K constanti is the equilibrium for the inhibition constant of for binding the inhibition to the maltase–maltose of binding to maltase, complex. whereas In a Kpreviousi’ is the equilibrium(14.97 ± 0.75 constant μM). Note for thatthe inhibitionall of the ofK i bindingvalues wereto the lower maltase–maltose than the Ki’ complex. values. AsIn amentioned previous equilibriumreport by Cortés constant et al.for [26], the inhibitionKi values ofare binding smaller to than the maltase–maltoseKi’ values in cases complex. of reversible In a previous mixed- reportpreviously, by Cortés Ki is the et equilibriumal. [26], Ki valuesconstant are for smaller the inhibition than K ofi’ valuesbinding in to cases maltase, of reversiblewhereas K i’mixed- is the reportcompetitive by Cortés inhibition. et al. This [26], is Kani indicationvalues are that smaller inhibitor–enzyme than Ki’ values binding in cases affinity of reversibleexceeds inhibitor– mixed- competitiveequilibrium inhibition.constant for This the is inhibitionan indication of thatbinding inhibitor–enzyme to the maltase–maltose binding affinity complex. exceeds In a inhibitor– previous Figure 2.competitiveenzyme–substrateInhibitory inhibition. eﬀects binding of This epicatechin-(4 affinity,is an indication resultingβ,8)-epicatechingallate, that in inhibitor–enzyme the mixed-competitive epicatechin binding inhibition affinity gallate, exceedsof maltase. epicatechin, inhibitor– These enzyme–substratereport by Cortés etbinding al. [26], affinity, Ki values resulting are smaller in the mixed-competitivethan Ki’ values in inhibition cases of reversibleof maltase. mixed- These andFigure quercetinenzyme–substratefindingscompetitive 2. Inhibitory onsuggest inhibition. sucrase. effects that binding B2-3’-of This Each epicatechin-(4 affinity, isO an-gallate, value indication resulting isECG,β represented,8)-epicatechingallate, that EC,in inhibitor–enzymeth ande mixed-competitive quercetin as the mean maepicatechin bindingy bind inhibitionstandard affinity to gallate,either exceedsof deviationmaltase.maltase epicatechin, inhibitor– orThese ofthe findings suggest that B2-3’-O-gallate, ECG, EC, and quercetin may bind± to either maltase or the and quercetinfindings enzyme–substrate onsuggest sucrase. that binding EachB2-3’- valueaffinity,O-gallate, is resultingrepresente ECG, EC, ind th andase mixed-competitivethe quercetin mean ±ma standardy bind inhibition todeviation either of maltasemaltase. of triplicate orThese the triplicate measurements. measurements. findings suggest that B2-3’-O-gallate, ECG, EC, and quercetin may bind to either maltase or the

As shown in Table 1, the IC50 values for sucrase were as follows: B2-3’-O-gallate (6.91 ± 3.41 μM), ECG (18.27 ± 3.99 μM), EC (18.91 ± 3.66 μM), and quercetin (18.98 ± 2.53 μM). The strength of the IC50 values was in the following order: B2-3’-O-gallate < ECG < EC < quercetin. We can see that the inhibitory effects of quercetin on sucrase were far less pronounced than those of B2-3’-O-gallate, ECG, and EC (p < 0.05). There has already been considerable research into the use of these polyphenols as maltase and sucrase inhibitors as an alternative to pharmaceutical treatments such as acarbose for the management of type 2 diabetes. Pyner et al. [22] reported that the IC50 values of polyphenol-rich green tea extract, acarbose, and epigallocatechin gallate for rat maltase were 0.035 ± 0.005 mg/mL, 0.42 ± 0.02 μM, and 14.0 ± 2.0 μM, respectively. Furthermore, the IC50 values of polyphenol-rich green tea extract, acarbose, and epigallocatechin gallate for rat sucrase were 1.8 ± 0.3 mg/mL, 12.3 ± 0.6 μM, and 950 ± 86 μM, respectively. In addition, quercetin showed inhibitory activities with IC50 values of 3.5 ± 0.3 mM against rat sucrose [21]. Furthermore, mogroside IV (IC50 = 12 mM), siamenoside I (IC50 = 10 mM), and mogroside III (IC50 = 1.6 mM) isolated from Siraitia grosvenori also have maltase inhibitory activity [23]. Kim et al. [24] isolated a bromophenol, bis (2,3-dibromo-4,5- dihydroxybenzyl)ether, from the red alga Polyopes lancifolia. That compound had an IC50 value of 1.00 ± 0.03 mM against sucrose in rats. According to the above results, B2-3’-O-gallate, ECG, and EC have the potential to be maltase and sucrase inhibitors.

Foods 2019, 8, 540 6 of 13

As shown in Table1, the IC values for sucrase were as follows: B2-3’-O-gallate (6.91 3.41 µM), 50 ± ECG (18.27 3.99 µM), EC (18.91 3.66 µM), and quercetin (18.98 2.53 µM). The strength of the IC ± ± ± 50 values was in the following order: B2-3’-O-gallate < ECG < EC < quercetin. We can see that the inhibitory eﬀects of quercetin on sucrase were far less pronounced than those of B2-3’-O-gallate, ECG, and EC (p < 0.05). There has already been considerable research into the use of these polyphenols as maltase and sucrase inhibitors as an alternative to pharmaceutical treatments such as acarbose for the management of type 2 diabetes. Pyner et al. [22] reported that the IC50 values of polyphenol-rich green tea extract, acarbose, and epigallocatechin gallate for rat maltase were 0.035 0.005 mg/mL, 0.42 0.02 µM, and ± ± 14.0 2.0 µM, respectively. Furthermore, the IC values of polyphenol-rich green tea extract, acarbose, ± 50 and epigallocatechin gallate for rat sucrase were 1.8 0.3 mg/mL, 12.3 0.6 µM, and 950 86 µM, ± ± ± respectively. In addition, quercetin showed inhibitory activities with IC values of 3.5 0.3 mM 50 ± against rat sucrose [21]. Furthermore, mogroside IV (IC50 = 12 mM), siamenoside I (IC50 = 10 mM), and mogroside III (IC50 = 1.6 mM) isolated from Siraitia grosvenori also have maltase inhibitory activity [23]. Kim et al. [24] isolated a bromophenol, bis (2,3-dibromo-4,5-dihydroxybenzyl)ether, from the red alga Polyopes lancifolia. That compound had an IC value of 1.00 0.03 mM against sucrose in rats. 50 ± According to the above results, B2-3’-O-gallate, ECG, and EC have the potential to be maltase and sucrase inhibitors.

3.2. Mode of Inhibition of B2-3’-O-gallate, ECG, EC, and Quercetin Toward Maltase We also evaluated the inhibition kinetics of B2-3’-O-gallate, ECG, EC, and quercetin toward maltase. In this study, the Michaelis constant (Km) of maltase was 1.01 mM, whereas in the study by Pyner et al. [22], the Km value of maltase was 7.5 mM. The Lineweaver–Burk plots of B2-3’-O-gallate (Figure3A), ECG (Figure3B), EC (Figure3C), and quercetin (Figure3D) did not intersect the x- or y-axis, which is indicative of mixed-type inhibition. A number of mixed-type maltase inhibitors have previously been reported. In a previous study, kinetic analysis revealed that maltase was inhibited by cinnamic acid derivatives, including caﬀeic acid, ferulic acid, and isoferulic acid, in a mixed-inhibition manner [16]. Our previous results indicated that B2-3’-O-gallate (Ki = 0.30 0.03 µM, Ki’ = 1.42 0.01 ± ± µM), ECG (Ki = 0.21 0.04 µM, Ki’ = 2.34 0.06 µM), and quercetin (Ki = 1.44 0.04 µM, Ki’ = 9.33 ± ± ± ± 0.10 µM) were mixed-competitive inhibitors of α-glucosidase [15]. Furthermore, a Lineweaver–Burk plot indicated that epigallocatechin gallate was a competitive inhibitor against maltose substrate for maltase, and the Ki calculated from a Dixon plot was 5.93 0.26 µM[25]. ± Foods 2019, 8, x FOR PEER REVIEW 7 of 13

Foodsmaltase–maltose2019, 8, 540 complex. Nonetheless, the binding sites and underlying mechanisms of inhibition7 of 13 have yet to be elucidated.

Figure 3. Lineweaver–Burk plots for the inhibition of maltase by maltase inhibitors, with maltose as Figure 3. Lineweaver–Burk plots for the inhibition of maltase by maltase inhibitors, with maltose as the substrate. (A) B2-3’-O-gallate. (B) ECG. (C) EC. (D) Quercetin. the substrate. (A) B2-3’-O-gallate. (B) ECG. (C) EC. (D) Quercetin. The Dixon plots in Figure4 revealed that all of the compounds were mixed-type inhibitors of 3.3. Mode of Inhibition of B2-3’-O-gallate, ECG, EC, and Quercetin Toward Sucrase maltase with the following Ki values: B2-3’-O-gallate (1.99 0.02 µM; Figure4A), ECG (3.14 0.04 µM; ± ± FigureIn4 B),our EC analysis (7.02 of0.26 theµ M;inhibition Figure4 C),kinetics and quercetin of B2-3’- (7.81O-gallate,0.10 ECG,µM; EC, Figure and4D). quercetin The strength toward of ± ± thesucrose,Ki values the Michaelis was as follows: constant B2-3’- (Km)O-gallate was 100.4< ECG mM.< InEC a previous< quercetin. report The byKi Pyner’ values et wereal. [22], as follows:the Km B2-3’-valueO of-gallate sucrase (3.73 activity0.08 wasµM), 9.5 ECGmM. (6.38The Lineweaver–Burk0.04 µM), EC (18.88 plots0.22 of B2-3’-µM),O and-gallate quercetin (Figure (14.97 5A), ECG0.75 ± ± ± ± µ(FigureM). Note 5B), that EC all (Figure of the K5C),i values and werequercetin lower (Figure than the 5D)Ki’ did values. not intersect As mentioned the x- previously,or y-axis, whichKi is the is equilibriumindicative of constant mixed-type for theinhibition. inhibition In ofprevious binding st toudies, maltase, a number whereas of Kmixed-i’ is thetype equilibrium sucrase inhibitors constant forhave the been inhibition reported. of binding Cyaniding-3-rutinoside to the maltase–maltose (a natural complex. anthocyanin) In a previous was reportfound byin Cortlitchié sand et al. sweet [26], Kcherry.i values Kinetics are smaller analysis than revealedKi’ values that in sucrase cases of was reversible inhibited mixed-competitive by cyanidin-3-rutinoside inhibition. in Thisa mixed- is an indicationtype manner that [27]. inhibitor–enzyme In addition, ferulic binding acid affinity and exceeds isoferulic inhibitor–enzyme–substrate acid inhibited sucrase in binding a mixed-type affinity, resultingmanner [16]. in the Furtherm mixed-competitiveore, a Lineweaver–Burk inhibition of maltase.plot indicated These findingsthat valienamine suggest that was B2-3’- a competitiveO-gallate, ECG,inhibitor EC, against and quercetin the sucrose may bindsubstrate to either of sucrase, maltase and or the the maltase–maltose Ki calculated from complex. a Dixon Nonetheless, plot was 0.77 the bindingμM [12]. sites and underlying mechanisms of inhibition have yet to be elucidated.

Foods 2019, 8, x FOR PEER REVIEW 8 of 13 Foods 2019, 8, 540 8 of 13

Figure 4. Dixon plots for the inhibition of maltase by maltase inhibitors, with maltose as the substrate. (FigureA) B2-3’- 4. ODixon-gallate. plots (B for) ECG. the inhibition (C) EC. (D of) Quercetin.maltase by maltase inhibitors, with maltose as the substrate. (A) B2-3’-O-gallate. (B) ECG. (C) EC. (D) Quercetin. 3.3. Mode of Inhibition of B2-3’-O-gallate, ECG, EC, and Quercetin Toward Sucrase InThe our Dixon analysis plots of in the Figure inhibition 6 indicated kinetics that of B2-3’-these Ocompounds-gallate, ECG, were EC, mixe andd-type quercetin inhibitors toward of sucrose,sucrase with the Michaelisthe following constant Ki values: (Km) B2-3’- wasO 100.4-gallate mM. (6.05 In a± previous0.04 μM; Figure report 6A), by Pyner ECG (8.58± et al. [0.0822], μ theM; KFigurem value 6B), of EC sucrase (13.72 activity ± 0.15 μ wasM; Figure 9.5 mM. 6C), The and Lineweaver–Burk quercetin (14.05 plots± 0.03 of μ B2-3’-M; FigureO-gallate 6D). The (Figure strength5A), ECGof the (Figure Ki values5B), was EC as (Figure follows:5C), B2-3’- and quercetinO-gallate (Figure< ECG <5 D)ECdid < quercetin. not intersect The theKi’ valuesx- or werey-axis, as which follows: is indicativeB2-3’-O-gallate of mixed-type (31.84 ± 0.34 inhibition. μM), ECG In (15.74 previous ± 0.32 studies, μM), EC a number (21.36 ± of 0.30 mixed-type μM), and sucrase quercetin inhibitors (18.54 ± have0.07 μ beenM). Note reported. that the Cyaniding-3-rutinoside Ki values of B2-3’-O-gallate, (a natural ECG, anthocyanin) EC, and quercetin was found were in lower litchi than and the sweet Ki’ cherry.values, Kineticsindicating analysis that revealedthese compounds that sucrase were was inhibitedmixed-competitive by cyanidin-3-rutinoside inhibitors of insucrase. a mixed-type These mannerfindings [ 27suggest]. In addition, that B2-3’- ferulicO-gallate, acid and ECG, isoferulic EC, and acid inhibitedquercetin sucrasemay bind in a to mixed-type either sucrase manner or [ 16the]. Furthermore,sucrase–sucrose a Lineweaver–Burk complex. Nonetheless, plot indicated the binding that valienaminesites and underlying was a competitive mechanisms inhibitor of inhibition against thehave sucrose yet to substratebe elucidated. of sucrase, and the Ki calculated from a Dixon plot was 0.77 µM[12].

3.4. Inhibition Scheme of B2-3’-O-gallate, ECG, EC, and Quercetin for Maltase and Sucrase Figure 7 presents the inhibition schemes of the maltase and sucrase inhibitors (B2-3’-O-gallate, ECG, EC, and quercetin). Figure 7A shows that maltase hydrolyzes carbohydrates by acting on 1,4-α linkages. Maltase inhibitors counter enzymes in the gut to restrain the liberation of glucose from oligosaccharides and disaccharides, resulting in a reduction in postprandial glucose levels [4]. In the maltase inhibitory activity assay, maltase could hydrolyze maltose to glucose, while maltase inhibitors prevent this maltase activity. Ki and Ki’ are the dissociation constants of the maltase– inhibitor complex and the maltase–maltose–inhibitor complex, respectively. According to our results, B2-3’-O-gallate (Ki = 1.99 ± 0.02 μM, Ki’ = 3.73 ± 0.08 μM), ECG (Ki = 3.14 ± 0.04 μM, Ki’ = 6.38 ± 0.04

Foods 2019, 8, x FOR PEER REVIEW 9 of 13

μM), EC (Ki = 7.02 ± 0.26 μM, Ki’ = 18.88 ± 0.22 μM), and quercetin (Ki = 7.81 ± 0.10 μM, Ki’ = 14.97 ± 0.75 μM) are mixed-competitive inhibitors of maltase. Note that mixed-competitive inhibitors bind at sites that are distinct from maltose active sites; however, they bind to either maltase or the maltase– maltose complex. Moreover, a similar inhibition scheme was also observed in the results of B2-3’-O- gallate, ECG, EC, and quercetin against sucrase (Figure 7B). In the sucrase inhibitory activity assay, sucrase could hydrolyze sucrose to glucose, while sucrase inhibitors prevent this sucrase activity [28]. Ki and Ki’ are the dissociation constants of the sucrase–inhibitor complex and the sucrase–sucrose– inhibitor complex, respectively. According to our results, B2-3’-O-gallate (Ki = 6.05 ± 0.04 μM, Ki’ = 18.54 ± 0.07 μM), ECG (Ki = 8.58 ± 0.08 μM, Ki’ = 15.74 ± 0.32 μM), EC (Ki = 13.72 ± 0.15 μM, Ki’ = 21.36 ± 0.30 μM), and quercetin (Ki = 14.05 ± 0.03 μM, Ki’ = 31.84 ± 0.34 μM) are mixed-competitive inhibitors of sucrase. Mixed-competitive inhibitors bind at sites that are distinct from sucrose active sites; however, they bind to either sucrase or the sucrase–sucrose complex. Note that B2-3’-O-gallate, ECG, Foodsand 2019EC ,all8, 540had significant inhibitory effects on maltase as well as sucrase. 9 of 13

Figure 5. Lineweaver–Burk plots for the inhibition of sucrase by sucrase inhibitors, with sucrose as the substrate.Figure 5. Lineweaver–Burk (A) B2-3’-O-gallate. plots (B) ECG.for the (C inhibition) EC. (D) Quercetin.of sucrase by sucrase inhibitors, with sucrose as the substrate. (A) B2-3’-O-gallate. (B) ECG. (C) EC. (D) Quercetin. The Dixon plots in Figure6 indicated that these compounds were mixed-type inhibitors of sucrase with the following Ki values: B2-3’-O-gallate (6.05 0.04 µM; Figure6A), ECG (8.58 0.08 µM; ± ± Figure6B), EC (13.72 0.15 µM; Figure6C), and quercetin (14.05 0.03 µM; Figure6D). The strength ± ± of the Ki values was as follows: B2-3’-O-gallate < ECG < EC < quercetin. The Ki’ values were as follows: B2-3’-O-gallate (31.84 0.34 µM), ECG (15.74 0.32 µM), EC (21.36 0.30 µM), and quercetin ± ± ± (18.54 0.07 µM). Note that the Ki values of B2-3’-O-gallate, ECG, EC, and quercetin were lower ± than the Ki’ values, indicating that these compounds were mixed-competitive inhibitors of sucrase. These ﬁndings suggest that B2-3’-O-gallate, ECG, EC, and quercetin may bind to either sucrase or the sucrase–sucrose complex. Nonetheless, the binding sites and underlying mechanisms of inhibition have yet to be elucidated. Foods 2019, 8, 540 10 of 13 Foods 2019, 8, x FOR PEER REVIEW 10 of 13

Figure 6. Dixon plots for the inhibition of sucrase by sucrase inhibitors, with sucrose as the substrate. Figure 6. Dixon plots for the inhibition of sucrase by sucrase inhibitors, with sucrose as the substrate. (A) B2-3’-O-gallate. (B) ECG. (C) EC. (D) Quercetin. (A) B2-3’-O-gallate. (B) ECG. (C) EC. (D) Quercetin. 3.4. Inhibition Scheme of B2-3’-O-gallate, ECG, EC, and Quercetin for Maltase and Sucrase Figure7 presents the inhibition schemes of the maltase and sucrase inhibitors (B2-3’- O-gallate, ECG, EC, and quercetin). Figure7A shows that maltase hydrolyzes carbohydrates by acting on 1,4- α linkages. Maltase inhibitors counter enzymes in the gut to restrain the liberation of glucose from oligosaccharides and disaccharides, resulting in a reduction in postprandial glucose levels [4]. In the maltase inhibitory activity assay, maltase could hydrolyze maltose to glucose, while maltase inhibitors prevent this maltase activity. Ki and Ki’ are the dissociation constants of the maltase–inhibitor complex and the maltase–maltose–inhibitor complex, respectively. According to our results, B2-3’-O-gallate (Ki = 1.99 0.02 µM, Ki’ = 3.73 0.08 µM), ECG (Ki = 3.14 0.04 µM, Ki’ = 6.38 0.04 µM), EC (Ki = ± ± ± ± 7.02 0.26 µM, Ki’ = 18.88 0.22 µM), and quercetin (Ki = 7.81 0.10 µM, Ki’ = 14.97 0.75 µM) are ± ± ± ± mixed-competitive inhibitors of maltase. Note that mixed-competitive inhibitors bind at sites that are distinct from maltose active sites; however, they bind to either maltase or the maltase–maltose complex. Moreover, a similar inhibition scheme was also observed in the results of B2-3’-O-gallate, ECG, EC, and quercetin against sucrase (Figure7B). In the sucrase inhibitory activity assay, sucrase could hydrolyze sucrose to glucose, while sucrase inhibitors prevent this sucrase activity [28]. Ki and Ki’ are the dissociation constants of the sucrase–inhibitor complex and the sucrase–sucrose–inhibitor complex, respectively. According to our results, B2-3’-O-gallate (Ki = 6.05 0.04 µM, Ki’ = 18.54 0.07 µM), ± ± ECG (Ki = 8.58 0.08 µM, Ki’ = 15.74 0.32 µM), EC (Ki = 13.72 0.15 µM, Ki’ = 21.36 0.30 µM), and ± ± ± ± quercetin (Ki = 14.05 0.03 µM, Ki’ = 31.84 0.34 µM) are mixed-competitive inhibitors of sucrase. ± ± Mixed-competitive inhibitors bind at sites that are distinct from sucrose active sites; however, they

Foods 2019, 8, 540 11 of 13 bind to either sucrase or the sucrase–sucrose complex. Note that B2-3’-O-gallate, ECG, and EC all had signiﬁcantFoods 2019, 8, inhibitoryx FOR PEER eREVIEWﬀects on maltase as well as sucrase. 11 of 13

Figure 7. Inhibition schemes for B2-3’-O-gallate, ECG, EC, and quercetin toward maltase (A) and Figure 7. Inhibition schemes for B2-3’-O-gallate, ECG, EC, and quercetin toward maltase (A) and sucrase (B). E: enzyme, S: substrate, I: inhibitor, P: product. sucrase (B). E: enzyme, S: substrate, I: inhibitor, P: product. 4. Conclusions 4. Conclusions B2-3’-O-gallate, ECG, and EC isolated from R. crenulata displayed maltase and sucrase inhibitory B2-3’-O-gallate, ECG, and EC isolated from R. crenulata displayed maltase and sucrase inhibitory properties in a mixed-competitive mode. The IC50 values of these compounds for both maltase andproperties sucrose in werea mixed-competitive in the following mode. order: The EC >ICECG50 values> B2-3’- of theseO-gallate. compounds Among for these both compounds,maltase and sucrose were in the following order: EC > ECG > B2-3’-O-gallate. Among these compounds, B2-3’-O- B2-3’-O-gallate had the highest maltase and sucrose inhibitory activities. We observed that the IC50 valuesgallate ofhad B2-3’- theO highest-gallate, maltase ECG, and and EC sucrose were lowerinhibitory than thoseactivities. of quercetin, We observed which that means the thatIC50 theyvalues have of greaterB2-3’-O potential-gallate, ECG, for the and prevention EC were oflower hyperglycemia than those of associated quercetin, with which maltase means or th sucrase.at they Ourhave ﬁndings greater potential for the prevention of hyperglycemia associated with maltase or sucrase. Our findings clearly identify B2-3’-O-gallate, ECG, and EC as likely candidates for the treatment of diabetes mellitus, warranting further in vivo studies.

Author Contributions: Formal Analysis, Data Curation, Preparation of the Research Work, Y.H.C.; Writing, Data Analysis, W.T.L.; Investigation of the Study, and contribution to Review and Editing, J.F.H.

Foods 2019, 8, 540 12 of 13 clearly identify B2-3’-O-gallate, ECG, and EC as likely candidates for the treatment of diabetes mellitus, warranting further in vivo studies.

Author Contributions: Formal Analysis, Data Curation, Preparation of the Research Work, Y.-H.C.; Writing, Data Analysis, W.-T.L.; Investigation of the Study, and contribution to Review and Editing, J.-F.H. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

1. Sancho, R.A.S.; Pastore, G.M. Evaluation of the effects of anthocyanins in type 2 diabetes. Food Res. Int. 2012, 46, 378–386. [CrossRef] 2. Zheng, Y.; Ley, S.H.; Hu, F.B. Global aetiology and epidemiology of type 2 diabetes mellitus and its complications. Nat. Rev. Endocrinol. 2018, 14, 88–98. [CrossRef][PubMed] 3. Zanoveli, J.M.; Morais, H.D.; Dias, I.C.; Schreiber, A.K.; Souza, C.P.; Cunha, J.M. Depression associated with diabetes: From pathophysiology to treatment. Curr. Diabetes Rev. 2016, 12, 165–178. [CrossRef][PubMed] 4. Girish, T.K.; Pratape, V.M.; Prasada Rao, U.J.S. Nutrient distribution, phenolic acid composition, antioxidant and α-glucosidase inhibitory potentials of black gram (Vigna mungo L.) and its milled by-products. Food Res. Int. 2012, 46, 370–377. [CrossRef] 5. Rose, D.R.; Chaudet, M.M.; Jones, K. Structural studies of the intestinal α-glucosidases, maltase-glucoamylase and sucrase-isomaltase. J. Pediatr. Gastroenterol. Nutr. 2018, 66, S11–S13. [CrossRef] 6. Peytam, F.; Adib, M.; Shourgeshty, R.; Mohammadi-Khanaposhtani, M.; Jahani, M.; Imanparast, S.; Faramarzi, M.A.; Mahdavi, M.; Moghadamnia, A.A.; Rastegar, H.; et al. Design and synthesis of new imidazo[1,2-b]pyrazole derivatives, in vitro α-glucosidase inhibition, kinetic and docking studies. Mol. Divers. 2019.[CrossRef] 7. Silva, C.P.D.; Soares-Freitas, R.A.M.; Sampaio, G.R.; Santos, M.C.B.; do Nascimento, T.P.; Cameron, L.C.; Ferreira, M.S.L.; Arêas, J.A.G. Identification and action of phenolic compounds of Jatobá-do-cerrado (Hymenaea stignocarpa Mart.) on α-amylase and α-glucosidase activities and flour effect on glycemic response and nutritional quality of breads. Food Res. Int. 2019, 116, 1076–1083. [CrossRef] 8. Simsek, M.; Quezada-Calvillo, R.; Nichols, B.L.; Hamaker, B.R. Phenolic compounds increase the transcription of mouse intestinal maltase-glucoamylase and sucrase-isomaltase. Food Funct. 2017, 8, 1915–1924. [CrossRef] 9. Toda, M.; Kawabata, J.; Kasai, T. α-Glucosidase inhibitors from clove (Syzgium aromaticum). Biosci. Biotechnol. Biochem. 2000, 64, 294–298. [CrossRef] 10. Kawakami, K.; Li, P.; Uraji, M.; Hatanaka, T.; Ito, H. Inhibitory effects of pomegranate extracts on recombinant human maltase-glucoamylase. J. Food Sci. 2014, 79, H1848–H1853. [CrossRef] 11. Lee, B.H.; Hamaker, B.R. Maltase has most versatile α-hydrolytic activity among the mucosal α-glucosidases of the small intestine. J. Pediatr. Gastroenterol. Nutr. 2018, 66, S7–S10. [CrossRef][PubMed] 12. Zheng, Y.G.; Shentu, X.P.; Shen, Y.C. Inhibition of porcine small intestinal sucrose by valienamine. J. Enzyme Inhib. Med. Chem. 2005, 20, 49–53. [CrossRef][PubMed] 13. Nijpels, G.; Boorsma, W.; Dekker, J.M.; Kostense, P.J.; Bouter, L.M.; Heine, R.J. A study of the effects of acarbose on glucose metabolism in patients predisposed to developing diabetes: The Dutch acarbose intervention study in persons with impaired glucose tolerance (DAISI). Diabetes Metab. Res. Rev. 2008, 24, 611–616. [CrossRef][PubMed] 14. Kwon, Y.I.; Jang, H.D.; Shetty, K. Evaluation of Rhodiola crenulata and Rhodiola rosea for management of type II diabetes and hypertension. Asia Pac. J. Clin. Nutr. 2006, 15, 425–432. [PubMed] 15. Chu, Y.H.; Wu, S.H.; Hsieh, J.F. Isolation and characterization of α-glucosidase inhibitory constituents from Rhodiola crenulata. Food Res. Int. 2014, 57, 8–14. [CrossRef] 16. Adisakwattana, S.; Chantarasinlapin, P.; Thammarat, H.; Yibchok-Anun, S. A series of cinnamic acid derivatives and their inhibitory activity on intestinal α-glucosidase. J. Enzyme Inhib. Med. Chem. 2009, 24, 1194–2000. [CrossRef][PubMed] 17. Akkarachiyasit, S.; Charoenlertkul, P.; Yibchok-anun, S.; Adisakwattana, S. Inhibitory activities of cyanidin and its glycosides and synergistic effect with acarbose against intestinal α-glucosidase and pancreatic α-amylase. Int. J. Mol. Sci. 2010, 11, 3387–3396. [CrossRef] Foods 2019, 8, 540 13 of 13

18. Cornish-Bowden, A. A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors. Biochem. J. 1974, 137, 143–144. [CrossRef] 19. 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. [CrossRef] 20. Kim, J.H.; Kang, M.J.; Choi, H.N.; Jeong, S.M.; Lee, Y.M.; Kim, J.I. Quercetin attenulates fasting and postprandial hyperglycemia in animal models of diavetes mellitus. Nutr. Res. Pract. 2011, 5, 107–111. [CrossRef] 21. Wang, H.; Du, Y.J.; Song, H.C. α-Glucosidase and α-amylase inhibitory activities of guava leaves. Food Chem. 2010, 123, 6–13. [CrossRef] 22. Pyner, A.; Nyambe-Silavwe, H.; Williamson, G. Inhibition of human and rat sucrase and maltase activities to assess antiglycemic potential: Optimization of the assay using acarbose and polyphenols. J. Agric. Food Chem. 2017, 65, 8643–8651. [CrossRef][PubMed] 23. Suzuki, Y.A.; Murata, Y.; Inui, H.; Sugiura, M.; Nakano, Y. Triterpene glycosides of Siraitia grosvenori inhibit rat intestinal maltase and suppress the rise in blood glucose level after a single oral administration of maltose in rats. J. Agric. Food Chem. 2005, 53, 2941–2946. [CrossRef][PubMed] 24. Kim, K.Y.; Nguyen, T.H.; Kurihara, H.; Kim, S.M. α-Glucosidase inhibitory activity of bromophenol puriﬁed from the red alga Polyopes lancifolia. J. Food Sci. 2010, 75, H145–H150. [CrossRef] 25. Nguyen, T.T.H.; Jung, S.H.; Lee, S.; Ryu, H.J.; Kang, H.K.; Moon, Y.H.; Kim, Y.M.; Kimura, A.; Kim, D. Inhibitory eﬀects of epigallocatechin gallate and its glucoside on the human intestinal maltase inhibition. Biotechnol. Bioprocess Eng. 2012, 17, 966–971. [CrossRef] 26. Cortés, A.; Cascante, M.; Cárdenas, M.L.; Cornish-Bowden, A. Relationships between inhibition constants, inhibitor concentrations for 50% inhibition and types of inhibition: New ways of analysing data. Biochem. J. 2001, 357, 263–268. [CrossRef] 27. Adisakwattana, S.; Yibchok-anun, S.; Charoenlertkul, P.; Wongsasiripat, N. Cyanidin-3-rutinoside alleviates postprandial hyperglycemia and its synergism with acarbose by inhibition of intestinal α-glucosidase. J. Clin. Biochem. Nutr. 2011, 49, 36–41. [CrossRef] 28. Abdelhady, M.I.; Shaheen, U.; Bader, A.; Youns, M.A. A new sucrase enzyme inhibitor from Azadirachta indica. Pharmacogn. Mag. 2016, 12, S293–S296. [CrossRef]

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