Inhibition of Pancreatic Α-Amylase by Resveratrol Derivatives: Biological Activity and Molecular Modelling Evidence for Cooperativity Between Viniferin Enantiomers

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Article Inhibition of Pancreatic α-amylase by Resveratrol Derivatives: Biological Activity and Molecular Modelling Evidence for Cooperativity between Viniferin Enantiomers

Luce M. Mattio 1 , Mauro Marengo 1, Chiara Parravicini 2, Ivano Eberini 2, Sabrina Dallavalle 1, Francesco Bonomi 1, Stefania Iametti 1 and Andrea Pinto 1,*

1 Department of Food, Environmental and Nutritional Sciences (DeFENS), University of Milan, Via Celoria 2, 20133 Milano, Italy 2 Department of Pharmacological and Biomolecular Sciences (DiSFeB) & cDSRC, University of Milan, Via Balzaretti 9, 20133 Milano, Italy * Correspondence: [email protected]; Tel.: +39-02-50316814

Received: 6 August 2019; Accepted: 2 September 2019; Published: 5 September 2019

Abstract: To improve the current understanding of the role of stilbenoids in the management of diabetes, the inhibition of the pancreatic α-amylase by resveratrol derivatives was investigated. To approach in a systematic way, the mechanistic and structural aspects of the interaction, potential bioactive agents were prepared as single molecules, that were used for the biological evaluation of the determinants of inhibitory binding. Some dimeric stilbenoids—in particular, viniferin isomers— were found to be better than the reference drug acarbose in inhibiting the pancreatic α-amylase. Racemic mixtures of viniferins were more effective inhibitors than the respective isolated pure enantiomers at an equivalent total concentration, and displayed cooperative effects not observed with the individual enantiomers. The molecular docking analysis provided a thermodynamics-based rationale for the measured inhibitory ability and for the observed synergistic effects. Indeed, the binding of additional ligands on the surface of the alpha-amylase was found to decrease the dissociation constant of inhibitors bound to the active site of the enzyme, thus providing a mechanistic rationale for the observed inhibitory synergies.

Keywords: resveratrol; stilbenoids; pancreatic alpha-amylase; enzyme inhibition; molecular docking; food bioactives synergism

1. Introduction Diabetes (type 1 or 2) is nowadays the most common serious metabolic disorder and one of the most challenging health problems [1,2]. Diabetes is characterized by persistent hyperglycaemia related to alterations in the glucose and lipid metabolism, including eﬀects on key regulatory enzymes, and to associated oxidative stress. The management of blood glucose level may be accomplished by the use of oral hypoglycaemic drugs, such as biguanides, insulin secretagogues, and inhibitors of enzymes involved in the release of simple sugars from polysaccharides and in their uptake [3]. Pancreatic α-amylase is an endo-hydrolase acting on 1,4-glucosidic linkages in linear regions of suitable length in starch, glycogen, and in various oligosaccharides. The activity of the enzyme releases simpler sugars that are then converted into glucose for intestinal absorption by other enzymes, including the brush-border α-glucosidase. Therefore, the inhibition of α-amylase activity results in decreased bioavailability of oligosaccharides and absorbable sugars and, consequently, in a decrease of the postprandial hyperglycaemia. Alpha-amylase inhibitors (e.g., acarbose), competitively and reversibly

Molecules 2019, 24, 3225; doi:10.3390/molecules24183225 www.mdpi.com/journal/molecules Molecules 2019, 24, 3225 2 of 14 inhibit the enzyme, but are reportedly associated with gastrointestinal side effects such as flatulence, abdominal pain, and diarrhoea [4,5]. Naturally occurring bioactives are currently considered precious elements of inspiration and an important source of structures for the development of novel nutraceuticals and pharmaceuticals [6]. The antioxidant activity and the related health benefits of dietary plant polyphenols are well documented, but there is an increasing evidence that polyphenols may have other beneficial effects in the management of diabetes, independent of their antioxidant capacities. In particular, they have been involved in direct modulation of the activity of key enzymes involved in the carbohydrate and lipid metabolism [7,8]. In this broad context, one particular group of the wood-derived natural products—namely, the stilbenoids family—has become increasingly popular, due to the wide spectrum of biological activities and to their intriguing molecular structures. Hydroxylated stilbenes—oligomers of resveratrol (3,5,40-trihydroxystilbene) ranging from dimers to octamers—form one of the most interesting and therapeutically relevant groups of plant-derived polyphenols. Due to the wide variety of biological activities (anti-bacterial, anti-HIV, anti-inflammatory, and anti-proliferative) shown by stilbenoids, resveratrol-based medicinal chemistry has become rapidly evolving in the past decade, covering a broad range of therapeutic applications [9]. Numerous studies have demonstrated that resveratrol can have antidiabetic effects through diverse mechanisms that may address multiple molecular targets, resulting in a significant therapeutic action in the whole organism [10]. The complex physiological action of resveratrol as an antidiabetic agent has been attributed to its capacity to modulate different pathways and to its action on a diversity of molecular targets. A far-from-exhaustive list of protein targets for resveratrol includes phosphodiesterases, adenylyl cyclases, kinases, sirtuins, transcription factors, cytokines, etc. [11]. The vast range of activities and differing potencies attributed to resveratrol may be more easily accounted for by assuming that some of its possible oxidation products, including some of the naturally-occurring dimeric stilbenoids, were involved in eliciting specific effects. Indeed, the modes of action and the activities of resveratrol “in vivo” may be partially, or even solely, due to the activities of the oligomeric derivatives generated from the monomeric precursor in a biological environment. The products of stilbenoid oligomerization have a high structural complexity and a three-dimensional architecture that is not present in the resveratrol itself, thus leading to a higher probability for selective binding to a specific macromolecular target (be that of catalytic or regulatory proteins, receptors, or transporters). To tackle in the most proper way the mechanistic and structural issues related to the specificity of these interactions, each potential bioactive agent needs to be prepared as a single molecule, and the individual species should be submitted to a biological evaluation separately. This “single molecule” approach used in this study has multiple advantages: (1) It allows to pinpoint the molecular determinants of the activity of individual chemical species; (2) it prevents the effects of possible competition among species present in variable concentration in the “natural” extracts most often used in this type of studies; (3) it makes it possible to highlight possible synergies among the individual bioactive species under investigation, as well as of therapeutically relevant synergies with synthetic compounds of pharmacological relevance. In this report, in order to improve the current understanding of the determinants of the interaction of stilbenoids with one of key proteins in the glucose metabolism, we investigated the capacity of individual resveratrol derivatives to inhibit the pancreatic alpha-amylase, with the aim of providing structural insights into molecular features relevant to the biological activity of each chemical species. The issue of possible competition or synergy among the most active compounds was also tested by using appropriate combinations of pure enantiomers. Computational approaches based on molecular docking were used to elucidate the mode of binding of each of the active compounds to the target protein and to evaluate the thermodynamic stability of their various binding modes. Molecules 2019, 24, 3225 3 of 14

2. Results

2.1. PreparationMolecules of 2019 Individual, 24, x FOR PEER Resveratrol REVIEW Derivatives 3 of 15

To better2.1. Preparation understand of Individual the molecularResveratrol Derivatives determinants of the interaction of stilbenoids with the Molecules 2019, 24, x FOR PEER REVIEW 3 of 15 alpha-amylaseTo and better to understand clarify which the molecular physical determinants properties of canthe interaction be exploited of stilbenoids in the with development the alpha- of more potent congeners,2.1.amylase Preparation and we to aimed ofclarify Individual towhich perform Resveratrol physical preliminary propertiesDerivatives ca n structure-activity be exploited in the development relationship of more (SAR) potent studies. Our first objectivecongeners, wasTo better to we haveunderstand aimed access to perform the to molecular some preliminary representative determinants structur ofe-activity the monomers interaction relationship of (compounds stilbenoids (SAR) studies. with1– the4 Our, Figurealpha- first 1). amylaseobjective and was to to clarify have accesswhich tophysical some representative properties can monomers be exploited (compounds in the development 1–4, Figure of more1). potent OH congeners,OH we aimed to performOMe preliminary structure-activityOH relationship (SAR) studies. Our first MeO objective was to have access to some representativeHO monomers (compounds 1–4, FigureOH 1). OH MeO HO OH OH OMe OH OMe OH OH OH MeO resveratrol (1) pterostilbene (2) HO piceatannol (3) 3,5-dimethoxy-piceatannolOH (4) OH MeO HO OH OH OH OMe OH OH OH 3,5-dimethoxy-piceatannol (4) resveratrol (1) pterostilbeneHO (2) piceatannol (3) MeO H OMe HO HO OMe OH OH OH OH OH OHOH H MeO HO H MeO HO OH OMe OH HO HOHO OMe OH OH O OH OH O OH HO O OH H (±)-trans-δ-viniferin (5)(±)-trans-ε-viniferin (6) (±)-pallidol (7) MeO(±)-pterostilbene trans-dehydrodimer (8) HO OH OH OH HO O HO O OH O Figure 1.FigureStructures 1. Structures of the of representativethe representativestilbenoid stilbenoid monomers monomers (1–4) ( and1–4 )dimers and dimers(5–8). (5–8). (±)-trans-δ-viniferin (5)(±)-trans-ε-viniferin (6) (±)-pallidol (7) (±)-pterostilbene trans-dehydrodimer (8) ResveratrolResveratrol (1) and (1 pterostilbene) and pterostilbene (2 ()2 are) are commerciallycommercially available. available. Piceatannol Piceatannol (3) was obtained (3) was by obtained Figure 1. Structures of the representative stilbenoid monomers (1–4) and dimers (5–8). by the partialthe partial modification modification of of a a reported reported procedur proceduree [12]. [ 12Pterostilbene]. Pterostilbene was oxidized was with oxidized IBX in withN, N- IBX in N, dimethylformamide (DMF), and the intermediate O-quinone was reduced with methanolic NaBH4 N-dimethylformamideResveratrol (DMF),(1) and pterostilbene and the intermediate (2) are commerciallyO-quinone available. was Piceatannol reduced (3) with was obtained methanolic by NaBH4 theto obtain partial compound modification 4, whichof a reported was demethylated procedure [12]. in a Pterostilbenemodest yield was(20%) oxidized (Figure with 2). Alternatively, IBX in N, N- to obtain compoundcompound 3 was4, which obtained was from demethylated peracetylated resveratrol in a modest 9, which yield was (20%)selectively (Figure deacetylated2). Alternatively, by dimethylformamide (DMF), and the intermediate O-quinone was reduced with methanolic NaBH4 compoundtothe3 obtain wasCandida obtainedcompound antarctica from4 lipase, which peracetylated to wasgive demethylated compound resveratrol 10 .in The a modest treatment9, which yield of (20%)10 was with (Figure selectively IBX and 2). removalAlternatively, deacetylated of the by the Candida antarcticacompoundtwo acyl groupslipase 3 was by toobtained the give hydrazine compoundfrom peracetylated monohydrate10. The resveratrol [13] treatment finally 9 gave, which ofpiceatannol 10waswith selectively 3 IBX. and deacetylated removal by of the two acyl groupsthe by Candida the hydrazine antarctica lipase monohydrate to give compound [13] finally10. The treatment gave piceatannol of 10 with IBX3. and removal of the two acyl groups by the hydrazine monohydrate [13] finally gave piceatannol 3.

Figure 2. Synthesis of monomers 3–4 and dimers 5–8. Reagents and conditions: a) 1-IBX, DMF, r.t., 80

min; 2-NaBH4 MeOH 0 °C, 62%; b) BBr3, DCM, −35 °C to r.t., N2, 22 h, 20%; c) Ac2O, TEA, DCM, r.t., overnight, 92%; d) CAL-B (Candida antarctica lipase), toluene/n-BuOH, 40 °C, 6 h, 81%; e) IBX, DMF, Figure 2. SynthesisFigure 2. Synthesis of monomers of monomers3–4 3and–4 and dimers dimers 55–8.. Reagents Reagents and and conditions: conditions: a) 1-IBX, a) DMF, 1-IBX, r.t., DMF,80 r.t., 80 r.t., 80 min, 42%; f) NH2NH2, MeOH, r.t., 30 min, 81%; g) 1-HRP, 30 min, 40 °C, acetone: Citrate buffer min; 2-NaBH4 MeOH 0 °C, 62%; b) BBr3, DCM, −35 °C to r.t., N2, 22 h, 20%; c) Ac2O, TEA, DCM, r.t., min; 2-NaBH 4 MeOH 0 ◦C, 62%; b) BBr3, DCM, 35 ◦C to r.t., N2, 22 h, 20%; c) Ac2O, TEA, DCM, r.t., overnight, 92%; d) CAL-B (Candida antarctica lipase),− toluene/n-BuOH, 40 °C, 6 h, 81%; e) IBX, DMF, overnight, 92%; d) CAL-B (Candida antarctica lipase), toluene/n-BuOH, 40 ◦C, 6 h, 81%; e) IBX, DMF, r.t., r.t., 80 min, 42%; f) NH2NH2, MeOH, r.t., 30 min, 81%; g) 1-HRP, 30 min, 40 °C, acetone: Citrate buffer 80 min, 42%; f) NH2NH2, MeOH, r.t., 30 min, 81%; g) 1-HRP, 30 min, 40 ◦C, acetone: Citrate buffer pH 5 (1:1); 2-H2O2, 15 min, 40 ◦C, overall yield 61%; h) 1-HRP, 30 min, 40 ◦C, acetone: Phosphate buffer pH 8 (1:1); 2-H2O2, 90 min, 40 ◦C, overall yield 21%; i) 1 - HRP, 30 min, 40 ◦C, acetone: Citrate buffer pH 5 (1:1); 2-H2O2, 90 min, 40 ◦C, overall yield 49%; j) FeCl3 6H2O, MeOH : H2O 1:1, 15%. Molecules 2019, 24, 3225 4 of 14

Successively, we focused on dimeric compounds, whose three-dimensional architecture should improve the selectivity of their binding to an enzymatic target with respect to the planar monomers. However, low natural abundance and difficult extraction procedures prompted us to develop a synthetic strategy for dimers as well, based on a biomimetic approach. Resveratrol dimers are naturally obtained by an oxidative radical coupling. The regioisomeric dimerization modes constitute the foundation for the biosynthesis of a diverse collection of high-order resveratrol oligomers. Thus, a biomimetic synthetic strategy was followed [14], which allowed to obtain dimers (5–8, see Figure1) in quantities suitable for their systematic biological evaluation. Trans-δ-viniferin (5), pallidol (7), and pterostilbene-trans-dihydrodimer (8) were obtained from resveratrol (1) and pterostilbene (2) by treatment with horseradish peroxidase (HRP) and H2O2 [15,16] (Figure2). Trans-ε-viniferin (6) was obtained through a metal-catalyzed oxidative coupling by subjecting resveratrol to one-electron oxidation by ferric ions (FeCl 6H O) [17]. 3· 2 Regardless of whether the resveratrol oxidation was HRP-or metal-catalyzed [14], the first key step in dimerization is the formation of a phenoxy radical, which delocalizes due to the high conjugation of the stilbenoid nucleus and may be stabilized in position 8 or in position 3 and 10. Subsequent radical coupling (8-8’ coupling; 8-10’coupling; 8-3’ coupling) leads to the formation of very reactive p-quinone intermediates, which readily undergo intramolecular Friedel-Craft cyclization, giving respectively pallidol (7), trans-ε-viniferin (6) and trans-δ-viniferin (5). As a final step, the enantiomers of both the trans-δ-viniferin (5) and trans-ε-viniferin (6) were separated by the preparative chiral HPLC (Figure3).

Moleculescomparison 2019, 24 with, x FOR control PEER REVIEW runs (all including 15% MeOH, as MeOH was the solvent used for preparing5 of 15 stock solutions of compounds 1–8). None of the compounds tested here—at the concentrations used in this study—showed a signiﬁcant inhibitory activity towards the puriﬁed α-glucosidase.

Figure 3. Cont.

Figure 3. Chromatogram of the separation of two enantiomers of (±)-trans-δ-viniferin ((5), top) and (±)-trans-ε-viniferin ((6), bottom).

2.2. Alpha-amylase Inhibitory Activity The monomeric and dimeric stilbenoids described above were tested for their ability to inhibit the enzyme α-amylase, using acarbose as a reference compound. The method used to evaluate the enzyme activity relies on a two-step procedure. In a first step, α-amylase acts on a modified p- nitrophenyl-α-D-maltoheptaoside, releasing products that may be hydrolyzed by an excess α- glucosidase present in the assay mixture, therefore allowing spectrophotometric quantitation of the released p-nitrophenolate anion. The data obtained with various inhibitors are expressed in comparison with control runs (all including 15% MeOH, as MeOH was the solvent used for preparing stock solutions of compounds 1–8). None of the compounds tested here—at the concentrations used in this study—showed a significant inhibitory activity towards the purified α-glucosidase.

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Figure 3. Chromatogram of the separation of two enantiomers of ( )-trans-δ-viniferin ((5), top) and Figure 3. Chromatogram of the separation of two enantiomers of (±)-± trans-δ-viniferin ((5), top) and ( )-transε-ε-viniferin ((6), bottom). (±)-±trans- -viniferin ((6), bottom). TheMolecules results 2019, of 24, a x preliminaryFOR PEER REVIEW screening are reported in Figure4. Among the tested compounds6 of 15 (all 2.2. Alpha-amylase Inhibitory Activity at a 10 mM ﬁnal concentration), the four monomers (1–4) and pallidol (7) showed a modest inhibitory The results of a preliminary screening are reported in Figure 4. Among the tested compounds activityThe (21–45%)monomeric compared and dimeric to acarbose stilbenoids (86% inhibition,described atabove a 1 mM were ﬁnal tested concentration). for their ability Conversely, to inhibit the (all at a 10 mM final concentration), the four monomers (1–4) and pallidol (7) showed a modest the enzyme α-amylase, using acarbose as a referenceδ compound. The εmethod used to evaluate the inhibitoryinhibitory capacity activity of the (21–45%) dimeric compared species transto acarbose- -viniferin (86% (inhibition,5) and trans at -a -viniferin1 mM final ( 6concentration).) was comparable enzyme(~90%Conversely, inhibitionactivity relies the at 10inhibitory on mM) a two-step to capacity that observed procedure.of the dimeric with In species aa 1first mM transstep, acarbose.-δ -viniferinα-amylase It is(5 ) interestingactsand ontrans a- εmodified-viniferin to note thatp- nitrophenyl-pterostilbene(6) wasα -D-maltoheptaoside,comparable (2) and its dimer(∼90% (inhibition8) decreasedreleasing at 10products the mM) enzyme to that activity observedmay ofbeonly withhydrolyzed 21%a 1 andmM by 28%,acarbose. an respectively, excess It is α- glucosidaseindicatinginteresting thatpresent the to note freein the that hydroxyl assay pterostilbene mixture, groups (2 in)therefore and other its similardimer allowing (8 species) decreased spectrophotometric play the a relevantenzyme activity role quantitation in of the only interaction 21% of the releasedwith theand p-nitrophenolate enzyme. 28%, respectively, indicatinganion. The that thedata free obta hydroxylined groupswith various in other similarinhibitors species are play expressed a relevant in comparisonrole in with the interaction control runs with (all the including enzyme. 15% MeOH, as MeOH was the solvent used for preparing stock solutions of compounds 1–8). None of the compounds tested here—at the concentrations used in this study—showed a significant inhibitory activity towards the purified α-glucosidase.

Figure 4. Inhibition of the pancreatic α-amylase by the various compounds presented in Figure1, each Figure 4. Inhibition of the pancreatic α-amylase by the various compounds presented in Figure 1, at a 10 mM final concentration. The effect of a 1 mM acarbose is shown for comparison. each at a 10 mM final concentration. The effect of a 1 mM acarbose is shown for comparison. Overall, the results in Figure4 suggest that the racemic trans-δ-viniferin (5) and trans-ε-viniferin Overall, the results in Figure 4 suggest that the racemic trans-δ-viniferin (5) and trans-ε-viniferin (6) were(6) were the most the most efficacious efficacious inhibitors inhibitors of of the the pancreatic pancreatic alpha-amylase.alpha-amylase. This This observation observation prompted prompted us to evaluateus to evaluate whether whether the the inhibitory inhibitory capacity capacity ofof compoundscompounds (5 (5) )and and (6 ()6 was) was correlated correlated with with their their geometricgeometric features. features. The racemicThe racemic forms forms and theand pure the enantiomerspure enantiomers of each of compoundeach compound were testedwere tested as for as their forinhibitory their capacityinhibitory at a 1 capacity mM final at concentration,a 1 mM final concentration, always in always comparison in comparison with a 1with mM a 1 acarbose. mM acarbose. The The data in Figuredata5 show in Figure that the5 show enantiomers that the enantiomers with the ( Rwith, R) the absolute (R, R) configurationabsolute configuration have a greaterhave a greater inhibitory inhibitory efficacy than those of the (S, S) series, confirming that the inhibitory activity could be linked to the spatial arrangement of substituents on the 2,3-dihydrobenzofuran ring. Moreover, quite surprisingly, the data in Figure 5 also show that the racemic forms of both the dimeric species have a greater inhibitory efficacy than their respective pure enantiomers, at equivalent total concentrations. To further elucidate this finding, inhibition studies were carried out over a range of inhibitor concentrations (zero to 0.1 mM, Figure 6) much lower than that used in previous experiments. These experiments were carried out using either purified enantiomers or their equimolar mixtures, meant to simulate the simultaneous presence of the racemic form of each viniferin while avoiding possible effects of minor impurities that could have been present in the original racemic mixtures.

Molecules 2019, 24, 3225 6 of 14 eﬃcacy than those of the (S, S) series, conﬁrming that the inhibitory activity could be linked to the spatialMolecules arrangement 2019, 24, x FOR of substituents PEER REVIEW on the 2,3-dihydrobenzofuran ring. 7 of 15

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Figure 5. Residual enzyme activity in the presence of viniferin isomers, of their purified enantiomeric Figure 5. Residual enzyme activity in the presence of viniferin isomers, of their purified enantiomeric forms, and of their racemic forms. Final concentration of each inhibitor species (including acarbose and forms, and of their racemic forms. Final concentration of each inhibitor species (including acarbose resveratrol)and resveratrol) was 1 mM. was 1 mM. Moreover, quite surprisingly, the data in Figure5 also show that the racemic forms of both the dimeric species have a greater inhibitory efficacy than their respective pure enantiomers, at equivalent total concentrations. To further elucidate this finding, inhibition studies were carried out over a range of inhibitor concentrations (zero to 0.1 mM, Figure6) much lower than that used in previous experiments. These experimentsFigure 5. Residual were enzyme carried activity out using in the presence either purified of viniferin enantiomers isomers, of their or purified their equimolar enantiomeric mixtures, meant to simulateforms, and theof their simultaneous racemic forms. presence Final concentration of the racemic of each inhi formbitor of species each (including viniferin acarbose while avoiding and resveratrol) was 1 mM. possible effects of minor impurities that could have been present in the original racemic mixtures.

Figure 6. Concentration dependence of the α-amylase inhibition by purified enantiomeric forms of trans-δ-viniferin (5) and trans-ε-viniferin (6), and by equimolar mixtures of their purified enantiomers, simulating the presence of a racemic form of each species.

The comparative analysis presented in Figure 6 confirm the higher inhibitory efficacy of trans-δ- viniferin (5) with respect to trans-ε-viniferin (6), as well as the different ability of the enantiomers with respect to inhibitory binding to the enzyme. In both cases, the “simulated” racemic forms of either molecule have a greater efficacy in inhibiting the enzyme than their respective pure Figure 6. Concentration dependence of the α-amylase inhibition by puriﬁed enantiomeric forms of enantiomers,Figure 6. Concentrationat an equivalent dependence total concentration. of the α-amylase inhibition by purified enantiomeric forms of trans-transδ-viniferin-δ-viniferin (5) and (5) andtrans trans-ε-viniferin-ε-viniferin (6 (),6), and and byby equimolar mixtures mixtures of oftheir their purified puriﬁed enantiomers, enantiomers, simulating simulating the presencethe presence of aof racemic a racemic form form of of each each species.species.

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The increased efficacy of the enantiomeric mixture is particularly evident in the case of trans-δ- The comparativeviniferin (5), where analysis the mode presented of binding in of Figurethe two 6enantiomers confirm may the involve higher multiple inhibitory and non- e fficacy of trans-δ-viniferinequivalent (5) withsites on respect the target to transprotein.-ε -viniferinIndeed, as sh (6own), as in well Figure as 7, the the dianalysisfferent of ability inhibitory of thebinding enantiomers of trans-δ-viniferin (5) through the Hill equation indicated facilitated binding of the (S, S) enantiomer with respectat a to high inhibitory concentration. binding The markedly to the enzyme. sigmoidal Inbinding both observed cases, thefor the “simulated” (S, S) enantiomer racemic was forms of either moleculecharacterized have a by greater a Hill cooperativity efficacy in inhibitingcoefficient (n theH) close enzyme to fourthan and a theirrelatively respective high average pure value enantiomers, app μ H H app at an equivalentof Ki total(58 M). concentration. In contrast, values of n were close to unity for the (R, R) enantiomer (n = 1.5; Ki μ The increased= 43 M) and e ffiforcacy the high-affinity of the enantiomeric binding observed mixture for an equimolar is particularly mixture of the evident (R, R) and in (S, the S) case of enantiomers (nH = 1.2; Kiapp = 12 μM). trans δ - -viniferinAside (5), from where confirming the modethe presence of bindingof multiple of binding the twosites for enantiomers the enantiomeric may forms involve of trans- multiple and non-equivalentδ-viniferin ( sites5), with on athe distinct target affinity protein. and efficacy, Indeed, the as cooperative shown in effects Figure discussed7, the analysisabove point of to inhibitory binding ofthetrans facilitated-δ-viniferin binding (when5) through other binding the Hillsites on equation the target indicated protein (not facilitatednecessarily close binding to regions of the (S, S) enantiomerrelated at a high to inhibitory concentration. effects) are The occupied. markedly This sigmoidaloffers a possible binding explanation observed of the for high the inhibitory (S, S) enantiomer efficacy of enantiomeric mixtures (or of racemic forms)H with respect to pure enantiomers. We was characterizedhypothesize by that a Hill sequential cooperativity binding of coea “lowfficient affinity” (n enantiomer) close to to four the enzyme and a relativelyhaving the “high high average app H H value of Ki affinity”(58 µenantiomerM). In contrast, on the active values site ofmay n resultwere in closea strong to decrease unity for in the the dissociation(R, R) enantiomer constant of (n = 1.5; app Ki = 43 theµM) high-affinity and for the enantiomer. high-affi Innity other binding words, our observed hypothesis for implies an equimolar that the simultaneous mixtureof binding the ( R, R) and of different Henantiomers app“locks” one of them into the alpha-amylase active site, decreasing its (S, S) enantiomers (n = 1.2; Ki = 12 µM). apparent dissociation constant Kiapp and consequently increasing its inhibitory ability.

Figure 7. HillFigure plot 7. Hill for plot the for inhibition the inhibition of α of-amylase α-amylase by by didifferentfferent enantiomeric enantiomeric forms forms of trans- ofδ-viniferintrans-δ-viniferin (5). Lines are(5). theLines best are the fit best to the fit to experimental the experimental data, data, drawn drawn by by using using a three-parameters a three-parameters Hill equation Hill equation H (y=a*x^nHH/([Ki]^nH + x^nH). (y=a*xˆn /([Ki]ˆn + xˆn ). 2.3. Molecular Docking Studies Aside from confirming the presence of multiple binding sites for the enantiomeric forms of trans-δ-viniferinSupporting (5), with evidence a distinct for the affi datanity interpretation and efficacy, offered the above cooperative came through effects molecular discussed docking above point studies. The various panels of Figure 8 show the top-scoring binding pose for individual enantiomeric to the facilitatedspecies of binding compounds when (5) otherand (6) bindingin the amylase sites onactive the site, target whereas protein their (notdetailed necessarily molecular close to regions relatedrecognition to inhibitory mechanism eisff reportedects) are in occupied.the schematic Thisviews opresentedffers a possiblein Figure 9. explanation In short, binding of the high inhibitory efreeffi cacyenergy of values enantiomeric obtained from mixtures molecular (or ofdocking racemic confirm forms) that withthe amylase respect active to pure site may enantiomers. We hypothesizeaccommodate that sequential all these bindingspecies, although of a “low with affi dinity”fferent enantiomer affinities, as to reported the enzyme in Supporting having the “high Information Table S1. The affinities of the investigated compounds for the enzyme, as assessed affinity” enantiomerthrough the docking on the activescores, are site cons mayistent result with inthe a efficacy strong of decrease individual in species the dissociation as inhibitors, constantas of the high-affimadenity evident enantiomer. by the enzymatic In other assays words, reported our hypothesisand commented implies in the previous that the subsection. simultaneous binding of different enantiomers “locks” one of them into the alpha-amylase active site, decreasing its apparent app dissociation constant Ki and consequently increasing its inhibitory ability.

2.3. Molecular Docking Studies Supporting evidence for the data interpretation offered above came through molecular docking studies. The various panels of Figure8 show the top-scoring binding pose for individual enantiomeric species of compounds (5) and (6) in the amylase active site, whereas their detailed molecular recognition mechanism is reported in the schematic views presented in Figure9. In short, binding free energy values obtained from molecular docking confirm that the amylase active site may accommodate all these species, although with different affinities, as reported in Supporting Information Table S1. The affinities of the investigated compounds for the enzyme, as assessed through the docking scores, are Molecules 2019, 24, 3225 8 of 14 consistent with the efficacy of individual species as inhibitors, as made evident by the enzymatic assays reported andMolecules commented 2019, 24, x FOR in PEER the REVIEW previous subsection. 9 of 15

FigureMolecules 8. Top-scoringFigure 2019 8., 24Top-scoring, x FOR binding PEER binding REVIEW poses poses between between the the pig pig pancreatic pancreaticα α-amylase-amylase and and the the enantiomeric enantiomeric10 of 15 forms of the transforms-viniferin of the speciestrans-viniferin in the species protein in the active protein site: active A, ( site:S, S )-A,trans (S, S)--δtrans-viniferin;-δ-viniferin; B, (B,R ,(R,)- Rtrans)-trans--δ-viniferin; δ C, (S, S)-trans-viniferin;-ε-viniferin; C, (S, S)- D,trans (R-ε, -viniferin;R)-trans -D,ε-viniferin. (R, R)-trans-ε-viniferin.

Docking studies (Figure 8; figure 9, and Table S1 in the Supporting Information) highlight the relevance of hydroxyl groups in the interaction with the enzyme, explaining the virtual absence of inhibitory effects for some of the derivatives under scrutiny, even at concentration orders of magnitude higher than those where viniferins proved effective. In this regard, not all phenols were created equal, since each of the species with a high inhibitory capacity has a different set of protein residues as specific interactors, as made evident by the schematics presented in Figure 9. Quite expectedly, the set of protein interactors and the nature of the interactions was different for the two viniferin isomers, in consideration of their vastly different overall geometry. Perhaps more excitingly, enantiomers of each species were found to differ in their mode of docking to the protein. The presence of differences in the spatial arrangement of substituents at their stereogenic centers resulted in a completely different set of protein interactors for other- structurally similar-portions of the molecule. In more general terms, Figures 8 and 9 suggest that each of the most efficient inhibitors has a specific binding mode. Taking into account the specificity of the binding (as indicated by computational data) and the efficacy of individual stilbenoids derivatives (see Figure 1), it seems that the most active species described in this study (with Kiapp in the 10–20 micromolar range) do not just behave as “hydrophobic plugs” with respect to limiting access of the substrate to the active site of the enzyme.

Figure 9. Ligand interaction diagram for the molecular docking top-scoring complexes between the pig pancreatic α-amylase and various enantiomeric forms of the trans-viniferin isomers in the protein Figure 9. Ligand interaction diagram for the molecular docking top-scoring complexes between the active site. Interactions are recapitulated in the ﬁgure legend. pig pancreatic α-amylase and various enantiomeric forms of the trans-viniferin isomers in the protein active site. Interactions are recapitulated in the figure legend.

On the other hand, computational data point out the ability of the α-amylase active site to accommodate compounds with sensibly different geometric features. Active site binding in the arrangements proposed here takes advantage of a pocket in the protein interior—in close proximity of the active site, which is supposedly close to the surface of the protein and meant to harbour large and hydrophilic species—able to accommodate with relative ease the bulky substituents on the 2,3- dihydrobenzofuran core of stilbenoid dimers. Molecular modelling was also applied to explore the possibility of gaining mechanistic insights into the “racemic effect” amply discussed in a previous subsection when commenting inhibition data, and took into account the minimum energy mode of simultaneous binding of different enantiomers of trans-δ-viniferin (5) to α-amylase. Our modelling approach started from the top-scoring complexes of the trans-δ-viniferin enantiomers presented in Figures 8 and 9, in order to explain the synergistic inhibition effect of this racemic mixture on the pancreatic α-amylase. In the proposed model (Figure 10), (S, S)-trans-δ-viniferin is the most affine compound for the enzyme active site. The possibility for its enantiomeric form, (R, R)-trans-δ-viniferin, to bind in a more external protein site and to stabilize the enzyme complex with (S, S)-trans-δ-viniferin has been demonstrated to be thermodynamically feasible. As a result, simultaneous binding of different

Molecules 2019, 24, 3225 9 of 14

Docking studies (Figure8; Figure9, and Table S1 in the Supporting Information) highlight the relevance of hydroxyl groups in the interaction with the enzyme, explaining the virtual absence of inhibitory effects for some of the derivatives under scrutiny, even at concentration orders of magnitude higher than those where viniferins proved effective. In this regard, not all phenols were created equal, since each of the species with a high inhibitory capacity has a different set of protein residues as specific interactors, as made evident by the schematics presented in Figure9. Quite expectedly, the set of protein interactors and the nature of the interactions was different for the two viniferin isomers, in consideration of their vastly different overall geometry. Perhaps more excitingly, enantiomers of each species were found to differ in their mode of docking to the protein. The presence of differences in the spatial arrangement of substituents at their stereogenic centers resulted in a completely different set of protein interactors for other-structurally similar-portions of the molecule. In more general terms, Figures8 and9 suggest that each of the most e fficient inhibitors has a specific binding mode. Taking into account the specificity of the binding (as indicated by computational data) and the efficacy of individual stilbenoids derivatives (see Figure1), it seems that the most active app species described in this study (with Ki in the 10–20 micromolar range) do not just behave as “hydrophobic plugs” with respect to limiting access of the substrate to the active site of the enzyme. On the other hand, computational data point out the ability of the α-amylase active site to accommodate compounds with sensibly different geometric features. Active site binding in the arrangements proposed here takes advantage of a pocket in the protein interior—in close proximity of the active site, which is supposedly close to the surface of the protein and meant to harbour large and hydrophilic species—able to accommodate with relative ease the bulky substituents on the 2,3-dihydrobenzofuran core of stilbenoid dimers. Molecular modelling was also applied to explore the possibility of gaining mechanistic insights into the “racemic effect” amply discussed in a previous subsection when commenting inhibition data, and took into account the minimum energy mode of simultaneous binding of different enantiomers of trans-δ-viniferin (5) to α-amylase. Our modelling approach started from the top-scoring complexes of the trans-δ-viniferin enantiomers presented in Figures8 and9, in order to explain the synergistic inhibition effect of this racemic mixture on the pancreatic α-amylase. In the proposed model (Figure 10), (S, S)-trans-δ-viniferin is the most affine compound for the enzyme active site. The possibility for its enantiomeric form, (R, R)-trans-δ-viniferin, to bind in a more external protein site and to stabilize the enzyme complex with (S, S)-trans-δ-viniferin has been demonstrated to be thermodynamically feasible. As a result, simultaneous binding of different enantiomeric forms stabilizes the resulting complex, and thus promotes an overall increase in the inhibitory capacity. Molecular docking calculations, followed by MM, showed that the involved change in the binding free energy computed through the MM-GBSA approach (namely, 66.54 kcal/mol for (S, − S)-trans-δ-viniferin in the binding site with externally bound (R, R)-trans-δ-viniferin versus 44.58 − kcal/mol for (S, S)-trans-δ-viniferin in the active site by itself; corresponding to a ∆∆G of 21.96 kcal/mol) were fully compatible with the changes in the inhibitory capacity observed in the experiments reported in the various panels of Figure6 and analyzed in terms of coperativity through the Hill plot presented in Figure7. Molecules 2019, 24, x FOR PEER REVIEW 11 of 15

Molecules 2019, 24, 3225 10 of 14 enantiomeric forms stabilizes the resulting complex, and thus promotes an overall increase in the inhibitory capacity.

Figure 10. TopTop scoring scoring pose pose of of (R (R, R, R)-)-transtrans-δ--viniferinδ-viniferin to tothe the pig pig pancre pancreaticatic alpha alpha amylase amylase with with (S, S (S)-, Strans)-trans-δ-viniferin-δ-viniferin already already bound bound to toits itsactive active site. site.

3. DiscussionMolecular docking calculations, followed by MM, showed that the involved change in the bindingThe free overall energy evidence computed gathered through in the this MM-GBSA study highlights approach that: (namely, 1) Some −66.54 dimeric kcal/mol stilbenoids for (S, S)- species—mosttrans-δ-viniferin notably, in the the binding naturally site occurring with externally isomers of boundtrans-viniferins—may (R, R)-trans-δ-viniferin be way more versus effi cacious−44.58 thankcal/mol acarbose for ( inS, inhibitingS)-trans-δ-viniferin the pancreatic in theα-amylase; active site 2) theby itself; geometric corresponding features of individualto a ΔΔG viniferinof 21.96 isomerskcal/mol) a ffwereect their fully inhibitory compatible ability; with 3) the racemic change mixturess in the of eitherinhibitory the transcapacity-δ- or observedtrans-ε-viniferin in the areexperiments more effective reported inhibitors in the various than the panels respective of Figure isolated 6 and pure analyzed enantiomers; in terms 4) of the coperativity molecular dockingthrough analysisthe Hill plot provides presented thermodynamics-based in Figure 7. evidence for the observed inhibitory ability (or lack of it) of the various stilbenoids considered in this study, as well as a mechanistic rationale for the synergistic e3.ff Discussionect observed in racemic mixtures of the trans-viniferin isomers. PointThe overall 1 and 2 inevidence the above gathered list are ofin particular this study interest, highlights also because that: intestinal1) Some concentrationsdimeric stilbenoids of the mostspecies—most efficacious notably, stilbenoid the dimers naturally may beoccurring higher than isomers expected of fortrans a given-viniferins—may food on a composition-only be way more basis,efficacious dueto than their acarbose poor absorption in inhibiting at the mucosalthe pancreatic level. In α this-amylase; frame, it2) is the worth geometric also reminding features that of theindividual dimeric viniferin stilbenoid isomers species affect may formtheir throughinhibitory a simple ability; metabolic 3) racemic transformation mixtures of either of their the precursors, trans-δ- or thattrans are-ε-viniferin usually are much more more effective abundant inhibitors than oligomers than the respective in common isolated foods andpure beverages. enantiomers; Notably, 4) the viniferinsmolecular havedocking been analysis reported provides to represent thermodynami up to 20% ofcs-based the total evidence stilbenoids for inthe wine observed [18]. In inhibitory addition, proceduresability (or lack are availableof it) of forthe thevarious selective stilbenoids recovery considered of viniferins in fromthis food-processingstudy, as well as byproducts a mechanistic [19]. However,rationale for viniferins the synergistic recovered effect from observed byproducts in racemic are a mixture mixtures of isomers,of the trans with-viniferin a relative isomers. abundance of the individualPoint 1 and species 2 in the being above dependent list are of on particular the source. interest, also because intestinal concentrations of the mostPoint 3efficacious in the list abovestilbenoid represents dimers the mostmay novel—andbe higher mostthan unexpected—outcomeexpected for a given of ourfood studies. on a Ascomposition-only a matter of fact, basis, synergistic due to e fftheirects poor appear absorption to be at work at the in mucosal many of level. the reported In this protein-phenolicsframe, it is worth inhibitoryalso reminding interactions that [the20, 21dimeric]. However, stilbenoid neither sp theecies nature may of theform involved through chemical a simple species metabolic nor the moleculartransformation basis of their these precursors, effect appears that to are have usua beenlly investigatedmuch more abundant in sufficient than detail, oligomers in particular in common when chemicallyfoods and beverages. similar components Notably, couldviniferins be involved. have been To reported our best knowledge,to represent thisup isto the20% first of reportthe total in whichstilbenoids chemically in wine pure [18]. species In addition, were used procedures to carry are out available detailed analysis for the selective of the geometric recovery and of structuralviniferins determinantsfrom food-processing of these synergisticbyproducts e ff[19].ects. However, From a practical viniferins standpoint, recovered the from observation byproducts that are the a mixture racemic mixturesof isomers, of with dimeric a relative stilbenoids abundance are more of the effective individual than thespecies isolated being enantiomers dependent oonffers the circumstantial source. supportPoint for 3 thein “foodthe list better above than represents pills” intervention the most novel—and strategies, andmost could unexpected—outcome offer useful guidelines of our for thestudies. structural As a matter design of of fact, molecules synergistic with improvedeffects appear efficacy to be as at well work as forin many their usageof the andreported dosage, protein- either alonephenolics or in inhibitory combination. interactions [20,21]. However, neither the nature of the involved chemical species nor the molecular basis of these effect appears to have been investigated in sufficient detail,

Molecules 2019, 24, 3225 11 of 14

In this frame, it is worth noting that preliminary experiments indicate that dimeric stilbenoids do not compete with acarbose in the inhibition of the pancreatic α-amylase. On the contrary, the evidence gathered so far points to a possible synergy among various classes of the inhibitory species, even when apparently unlikely because of their remarkable structural differences. The possible occurrence of a similar synergism in other enzymes relevant to the human health seems a research territory still unchartered and much worth exploring. Finally, point 4 in the list above underlines the relevance of molecular docking studies in this type of investigation, in particular when used in combination with the biological activity data. In the case reported here, this combination of approaches offers a clue for explaining the synergistic effects mentioned above, and could provide useful hints as for their preventive or therapeutic use.

4. Materials and Methods

4.1. Chemistry Unless otherwise specified, chemicals were from Sigma-Aldrich (Milan, Italy), as were the Candida antarctica lipase and the horseradish peroxidase used for some of the synthetic approaches reported here. All reagents and solvents were of reagent grade or were purified by standard methods before use. Melting points were determined on a model B-540 Büchi apparatus and are uncorrected. Optical rotation determinations were carried out using a Jasco P-1010 spectropolarimeter (Jasco Europe, Cremella, Italy), coupled with a Haake N3-B thermostat. NMR data were acquired using a Varian Mercury-300 MHz spectrometer (Varian, Palo Alto, CA, USA). Chemical shifts (δ values) and coupling constants (J values) are given in ppm and Hz, respectively. Chiral HPLC analyses were performed using a Kromasil 5-AmyCoat column (4.6 mm i.d. 250 mm, Nouryon Separation Products, Bohus, × Sweden), fitted to a Jasco PU-980 pump and a Jasco UV-975 detector. Runs were carried out in 60/40 (v/v) hexane/iPrOH + 0.1% TFA at a flow rate of 1 mL min-1, monitoring the eluate at 280 nm. Preparative chiral HPLC was performed with a Kromasil 5-AmyCoat column (21.2 i.d. 250 mm), fitted to a 1525 × Extended Flow Binary HPLC pump and a Waters 2489 UV/Vis detector (both from Waters, Milan, Italy). The solvent was 60/40 (v/v) hexane/iPrOH + 0.1% TFA, at a flow rate of 15 mL min-1, monitoring the eluate at 280 nm. Isolation and purification of the compounds were performed by a flash column chromatography on silica gel 60 (230–400 mesh). The analytical thin-layer chromatography (TLC) was conducted on Supelco TLC plates (silica gel 60 F254, aluminum foil). Structural characterizations of the various stilbenoid derivatives mentioned above are detailed in the Supplementary materials and are in accordance with literature reports [22–29].

4.2. Enzymatic Inhibition Porcine pancreatic α-amylase [EC 3.2.1.1] was from Sigma-Aldrich (Milan, Italy). Activity was monitored by using the K-AMYLSD kit from Megazyme (Bray, Ireland), with modifications. An aliquot of α-amylase (0.03 mL, 0.1 µM in 50 mM phosphate buffer, pH 6.8) was added to 0.120 mL of 50 mM phosphate buffer, pH 6.8, in a microtiter plate well. The investigated compounds where then added as methanolic solutions (0.03 mL) of appropriate concentration. The reaction was started by adding 0.02 mL of the substrate solution provided with the kit, resulting in a final concentration of 0.4 µM substrate. The kit-provided substrate solution also contained an excess of thermostable bacterial α-glucosidase, that is required for the release of the p-nitrophenolate anion from the p-nitrophenyl glucosides produced by the activity of α-amylase on the chemically modified maltoheptaoside. At appropriate times, the reaction was stopped by adding 0.1 mL of 0.2 M Na2CO3, and the absorbance was read in a microplate reader set at 405 nm. Blanks were prepared in the absence of the pancreatic α-amylase. Controls were otherwise complete reaction mixtures, containing 15% methanol (v/v), but no bioactive species. Acarbose (from a stock solution in methanol) was used as the reference inhibitor. A minimum of three independent replications was carried out for each assay. Molecules 2019, 24, 3225 12 of 14

4.3. Computational Procedures All the computational procedures were carried out with a Schrödinger Maestro BioLuminate v. 2019-01 (http://schrodinger.com) with the OPLS3e force field, as previously described [30]. The crystallographic structure of the pig pancreatic alpha amylase was downloaded from RCSB PDB (id: 1HX0) and was checked, fixed, and relaxed through the Protein Preparation Wizard. The trans-viniferin species and enantiomeric form database was sketched and prepared with the LigPrep, carefully checking the stereocenters. Molecular docking was carried out through the Glide Ligand Docking with a standard precision (SP) according to a well-established protocol [31]. The active site was identified through the Site Map tool and confirmed according to the annotations of the UniProt pig pancreatic alpha amylase entry (AMYP_PIG). The prime MM-GBSA was run in order to estimate the binding energy of the (S, S)-trans-δ-viniferin to the active site of the pig pancreatic alpha amylase in the presence and absence of (R, R)-trans-δ-viniferin.

4.4. Statistical Analysis The analysis of variance on the enzymatic inhibition data was performed adopting the least significant difference (LSD). Data were processed by using the Statgraphics XV, version 15.1.02 (StatPoint, Warrenton, VA, USA). The appropriate non-linear regression routines in SigmaPlot (rev. 10, Jandel Scientific, San Rafael, CA, USA) were used for graphical and numerical analysis of the inhibition data.

Supplementary Materials: The following are available online, Characterization of compounds 3–10; Table S1: Docking score of top scoring complexes. Author Contributions: L.M.M. and M.M. contributed equally to this manuscript. L.M.M. performed the synthesis and purification of the tested compounds, M.M. carried out the enzymatic inhibition measurements, and C.P. carried out the molecular docking. I.E., S.D., and S.I. analyzed the data; L.M.M., M.M., and C.P. drafted the manuscript; A.P. and F.B. designed the research and had primary responsibility for the final content of the report. All authors have read and approved the final manuscript. Acknowledgments: This work was financially supported by “Piano di Sostegno alla Ricerca 2015/2017-Linea 2_Azione A” of the University of Milan (DeFENS, to A.P.; DiSFeB, to I.E.), as well as by grants from MIUR “Progetto Eccellenza” and MIUR “Finanziamento annuale individuale delle attività di base di ricerca”. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds 3–10 are available from the authors.

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