Kaempferol as Selective Human MAO-A Inhibitor: Analytical Detection

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Article

Kaempferol as Selective Human MAO-A Inhibitor: Analytical Detection in Calabrian Red Wines, Biological and Molecular Modelling Studies Maria Concetta Gidaro, Christian Astorino, Anél Petzer, Simone Carradori, Francesca Alcaro, Giosuè Costa, Anna Artese, Giancarlo Rafele, Francesco Maria Russo, Jacobus Petrus Petzer, and Stefano Alcaro J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b06043 • Publication Date (Web): 28 Jan 2016 Downloaded from http://pubs.acs.org on February 3, 2016

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Journal of Agricultural and Food Chemistry

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Kaempferol as Selective Human MAO-A Inhibitor: Analytical Detection

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in Calabrian Red Wines, Biological and Molecular Modelling Studies

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Maria Concetta Gidaro†, Christian Astorino‡, Anél Petzer§, Simone Carradori⊥, Francesca Alcaro†,

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Giosuè Costa†, Anna Artese†, Giancarlo Rafele║, Francesco M. Russo‡, Jacobus P. Petzer§, Stefano

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Alcaro*,†

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†

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Universitario “S. Venuta”, Viale Europa, Loc. Germaneto, 88100 Catanzaro, Italy.

Dipartimento di Scienze della Salute, Università “Magna Græcia” di Catanzaro, Campus

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‡

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§

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Africa.

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⊥Department

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Chieti, Italy.

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║

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Corresponding Author

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*

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Campus “S. Venuta”, Viale Europa, 88100 Catanzaro, Italy. E-mail: [email protected] Phone: +39 0961

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3694197

Dipartimento ARPACal di Crotone, via E. Fermi s.n.c. 88900 Crotone, Italy. Centre of Excellence for Pharmaceutical Sciences, North-west University, Potchefstroom 2520, South

of Pharmacy, “G. D’Annunzio” University of Chieti-Pescara, Via dei Vestini 31, 66100

Slow Wine, Via di Mendicità Istruita, 12042, Bra (Cuneo), Italy

Prof. Stefano Alcaro, Dipartimento di Scienze della Salute, Università “Magna Græcia” di Catanzaro,

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Title running header: Kaempferol MAO-A Inhibitor in Calabrian Red Wines

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Abstract

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The purpose of this work was to determine the kaempferol content in three red wines of Calabria, a

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southern Italian region with a great number of certified food products. Considering that wine cultivar,

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climate and soil influence the qualitative and quantitative composition in flavonoids of Vitis vinifera L.

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berries, the three analyzed samples were taken from the 2013 vintage. Moreover, the “Gaglioppo”

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samples with assigned Controlled Origin Denomination (DOC), were also investigated in the

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production of years 2008, 2010 and 2011. In addition to the analysis of kaempferol, which is present in

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higher concentration than in other Italian wines, in vitro assays were performed in order to evaluate, for

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the first time, the inhibition of the human monoamine oxidases (hMAO-A and hMAO-B). Molecular

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recognition studies were also carried out to provide insight into the binding mode of kaempferol and

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selectivity of inhibition of the hMAO-A isoform.

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Keywords

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Flavonoids, red wine, monoamine oxidase, inhibition, computational chemistry.


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Introduction

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There are over three thousand articles in PubMed1 reporting the isolation and/or biological properties of

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kaempferol. A mini-review of Calderón-Montaño et al.2 provides a comprehensive summary of the

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information about the distribution in the plant kingdom, the biological activities and pharmacokinetic

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properties of this flavonoid. Monoamine oxidases (MAOs) consist of two isoforms: MAO-A and

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MAO-B. The function of MAOs is to catalyze the α-carbon 2-electron oxidation of amine substrates in

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the peripheral tissues and brain. Both MAO-A and MAO-B contain covalently linked 8-α-S-thioether

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FAD cofactors and represent significant therapeutic targets: MAO-B inhibitors have been proposed as

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coadjuvants in the treatment of Parkinson’s disease,3,

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extensively used as antidepressant agents.5

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In the literature, kaempferol has been shown to act as a selective inhibitor of the MAO-A isoform,

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however experimental studies were not carried out using human enzymes. For example, Ryu et al.

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showed that kaempferol inhibits rat brain mitochondrial MAO-A with an IC50 value of 10 µM, while no

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inhibition against the MAO-B isoform was observed.6 Sloley et al. confirmed this result with

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kaempferol isolated from leaves of Ginkgo biloba.7 Recently, some flavonoids structurally similar to

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kaempferol were also isolated from Propolis, thus proving that natural products represent a resource for

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the discovery of new MAO inhibitors.8 In the field of nutraceutics, computational techniques and in

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particular Virtual Screening play an important role in the discovery of new hit compounds. This

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reduces the cost and time of the research.9 In silico studies also provide additional insight into the

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possible mechanism of action and binding mode of active compounds against MAOs, as previously

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reported by some of us in other manuscripts.10-12

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The Phenol Explorer database13 reports the content of kaempferol in several foods. In red wine,

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relatively high concentrations of kaempferol were reported for the South African Shiraz (0.36 mg/100

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whereas MAO-A inhibitors have been



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mL) and Cabernet Sauvignon (0.35 mg/100 mL).14 Regarding Italian red wines, a number of different

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varieties were previously analyzed for kaempferol content. These include “Chianti” and “Nero

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D’Avola” from North and South Italy, respectively. Collectively, Italian red wines possess low

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concentrations of kaempferol in the range of 0.02-0.04 mg/100 mL.15,

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quantitative compositions in flavonoids of Vitis vinifera L. berries are affected by several factors, such

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as grape variety, place of growing, wine cultivar, climate and soil.17

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The purpose of this study was to determine the kaempferol content of three Calabrian red wines named

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“Magliocco”, with assigned Protected Denomination Origin (PDO), “Gaglioppo”, with assigned

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Controlled Origin Denomination (DOC) and “Nerello”, a variety with Protected Geographical

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Indication (PGI) in Calabria, an Italian district. In addition to the analysis of kaempferol in these red

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wines, in vitro assays were also performed in order to evaluate, for the first time, the inhibition of the

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human monoamine oxidases (hMAO-A and hMAO-B) by kaempferol. Molecular recognition studies

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were carried out to provide insight into binding modes and inhibition selectivity against MAOs.

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Quercetin, a flavonoid which differs from kaempferol by one hydroxyl group in the 3' position, is also a

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well-known MAO-A inhibitor and was thus included in the biological and computational experiments

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of this study.18

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Materials and methods

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Chemicals

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Commercial standards of kaempferol, quercetin, harmine and safinamide were purchased from Sigma-

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Aldrich (Milan, Italy). All chromatographic solvents were HPLC grade and were purchased from Carlo

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Erba (Milan, Italy): acetonitrile, ethanol (EtOH) 96%, ammonia solution (NH4OH) 28% and

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hydrochloric acid (HCl) 37%. Ultrapure Water (18 MΏ) was produced by a Milli-Q (MerckMillipore,

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Darmstadt, Germany) water purification system.

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Wine samples 4 ACS Paragon Plus Environment

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In fact, qualitative and

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The red wine samples were taken from the 2013 vintage and were kindly provided by the Calabrian

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farms: the PDO “Magliocco” by Terre di Cosenza DOP Azienda Agricola G. Calabrese; the DOC

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“Gaglioppo” by A’vita Azienda Vinicola De Franco and the PGI “Nerello” by Azienda Vinicola

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Tramontana since 1890, respectively produced in the north, center and south of Calabria. The DOC

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“Gaglioppo” was also investigated in the vintages of 2008, 2010 and 2011.

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Preparation of red wine extracts

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Optimization of extraction and hydrolysis procedure of flavonol conjugates in wines have been

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described by Hertog et al.19-21 In the present study same changes have been done as follows. At first,

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each wine sample (50 mL) was distilled, at atmospheric pressure, in order to obtain 2.0 mL of a residue

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which was cooled and dissolved with 10 mL of EtOH and 3.2 mL of HCl 4 N. In the next step, the

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resulting suspension was filtered and the aliquot was heated in a water bath at 50 °C for 20 min, then

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cooled and neutralized with concentrated ammonia. Finally, an additional filtration was carried out and

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the final volume of the solution was adjusted to 10 mL with EtOH. The clear or slightly coloured

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solution was used for the High-Performance Liquid Chromatography (HPLC) analysis.

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HPLC analysis

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The chromatographic system consisted of an Agilent 1100 Series equipped with a thermostated column

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compartment and a diode array Detector, analytical λ 365 nm reference λ 450 nm. Reversed-phase

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separation was carried out at 30 °C using a ZORBAX Eclipse XDB-C18 column (150 × 4.6 mm i.d.; 5

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µm) with the injection volume set to 20 µL. Considering the experimental procedure adopted by

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McDonald et al,21 after the optimization study, we selected as mobile phase a mixture of (A) water and

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(B) acetonitrile. Then we applied a flow rate of 0.8 mL/min in a gradient mode as follows: (i) 0.0–2.0

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min (A and B, 95:5, v/v); (ii) 30.0–32.0 min (A and B, 00:100, v/v) and (iii) 32.0–35.0 min (A and B,

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95:5, v/v). Kaempferol was identified by comparing the UV spectrum (Figure 1S) and retention time

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with those obtained from pure standard solutions (Figure 2S). Flavanol quantification was based on 5 ACS Paragon Plus Environment


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peak area and the corresponding concentration was expressed as mg/100 mL. All determinations were

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carried out in quadruplicate. A validation of the above mentioned variations was carried out working

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directly with known amounts of standard kaempferol solutions and also by adding them to wine

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samples during the extraction phase or the hydrolysis steps.

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Analytical quality control

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Stock solutions of kaempferol (1000 mg/L) in acetonitrile were prepared and stored in a refrigerator at

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+4 °C. Four different standard solutions were prepared by dilution of the stock solution to the final

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concentrations of 0.5-5.0 mg/L. Calibration curves were constructed by plotting analyte peak area

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versus analyte concentration. Quintupled injections were made for each concentration level and a

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weighted linear regression was applied (R2= 0.9997). The recovery for kaempferol was at least 90% of

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the analyte added.

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Biochemistry

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The measurement IC50 values for the inhibition of MAO

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To determine IC50 values for the inhibition of human MAO-A and MAO-B by kaempferol, quercetin

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and the reference inhibitors, harmine and safinamide, the commercially available recombinant human

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enzymes expressed in insect cells were used (Sigma-Aldrich).22 Briefly, the enzyme reactions were

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conducted in 96-well microtiter plates in a potassium phosphate buffer (100 mM, pH 7.4, made

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isotonic with KCl). The reaction volume was 200 µL and kynuramine was added at a concentration of

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50 µM. The test inhibitors were dissolved in DMSO and added at concentrations of 0.003–100 µM,

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yielding a final DMSO concentration of 4%. After the addition of MAO-A (0.0075 mg protein/mL) or

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MAO-B (0.015 mg protein/mL) the microtiter plates were incubated for 20 min at 37 ºC. Sodium

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hydroxide (80 µL of 2 N) was added to terminate the reactions and the microtiter plates were analyzed

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by fluorescence spectrophotometry (λex = 310; λem = 400 nm).23 For each experiment, a linear

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calibration curve constructed with 4-hydroxyquinoline (0.047–1.56 µM) was included. To determine 6 ACS Paragon Plus Environment

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the IC50 values, the inhibition data were fitted to the one site competition model incorporated into the

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Prism 5 software package (GraphPad). IC50 values were reported as the mean ± standard deviation (SD)

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of triplicate measurements.22

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Dialysis of MAO-A

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Dialysis experiments were conducted according to the published protocol.22,

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concentration of 0.03 mg protein/mL was pre-incubated (for 15 min at 37 °C) in the absence and

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presence of the test inhibitors, kaempferol and quercetin, and the reference irreversible inhibitor,

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pargyline (IC50 = 13 µM).25 The inhibitor concentrations in these samples were 4 × IC50 and the final

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volume 0.8 mL. The samples were subsequently dialysed for 24 h in potassium phosphate buffer (100

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mM, pH 7.4) containing 5% sucrose as outer buffer (80 mL). The outer buffer was replaced with fresh

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buffer at 3 h and 7 h after the start of dialysis. Slide-A-Lyzer dialysis cassettes (Thermo Scientific) with

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a molecular weight cut-off of 10000 and a sample volume capacity of 0.5–3 mL were used for these

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studies. The samples were diluted 2-fold to yield a final inhibitor concentration of 2 × IC50 and the

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residual MAO-A activity was measured as described for the measurement of IC50 values. For this

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purpose, kynuramine was added at a concentration of 50 µM. Samples containing MAO-A and

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kaempferol or quercetin were not dialysed and instead incubated at 4 °C for the same time period (24

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h). The residual MAO-A activities were reported as the mean ± standard deviation (SD) of triplicate

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measurements.

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Lineweaver-Burk plots

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Lineweaver-Burk plots for the inhibition of MAO-A were constructed by using kynuramine at eight

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different concentrations for each plot (15–250 µM).22 For each test inhibitor, six Lineweaver-Burk

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plots were constructed. The inhibitor concentrations selected for these six plots were: 0 × IC50, ¼ ×

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IC50, ½ × IC50, ¾ × IC50, 1 × IC50 and 1¼ × IC50. The concentration of MAO-A in the enzyme reactions

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was 0.015 mg protein/mL, and MAO-A activities were measured as described for the measurement of 7 ACS Paragon Plus Environment

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MAO-A at a


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IC50 values. Ki values were estimated from plots of the slopes of the Lineweaver-Burk plots versus

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inhibitor concentration, where the x-axis intercept equals –Ki. Ki values were also estimated by global

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fitting of the inhibition data to the Michaelis-Menten equation using the Prism 5 software package.

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Molecular modelling studies

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Protein Preparation

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The X-ray complexes of hMAO-A/harmine and hMAO-B/safinamide (our reference inhibitors) were

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downloaded from the Protein Data Bank (PDB)26 with the respective accession codes 2Z5X27 and

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2V5Z.28 For each PDB file, the “Protein Preparation Wizard”29 function in Maestro 9.7 of the

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Scrodinger Suite 201430 was used in order to add hydrogens, assign partial charges, build side chains

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and loops with missing atoms. The final step in the protein preparation process was to refine the

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structure with an energy restrained minimization, using OPLS-2005 as force field, until a final root

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mean square deviation (RMSD) of 0.30 Å with respect to the X-ray structure. The co-crystallized

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ligands were used to generate the docking grid box and were then removed prior to grid generation in

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the next step.

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Ligand Preparation

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The kaempferol and quercetin structures were imported as 3D SDF file from the PubChem site,31 into

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the Maestro GUI 9.7 of the Scrodinger Suite 2014.30 Protonated and tautomeric forms at pH 7.4 for

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each molecule were calculated using the “LigPrep” module.32 The lowest energy conformations,

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obtained by using the OPLS-2005 force field, were used as starting points for the docking simulations.

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The positive controls harmine and safinamide, removed from the PDB crystal structures, were

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submitted to the same protocol.

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QM-Polarized Ligand Docking

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The Quantum Mechanics/Molecular Mechanics (QM/MM) docking experiments were performed by

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the Schrödinger QM-Polarized Ligand Docking Protocol (QPLD).33 The QPLD protocol is aimed at 8 ACS Paragon Plus Environment

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improving the partial charges on the ligand atoms in a Glide docking run by replacing them with

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charges derived from QM calculations on the ligand in the field of the receptor. This protocol is

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summarized as follows: Rigid Receptor Docking (RRD) was performed using Glide 5.9 and the top ten

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poses of each ligand were used to calculate the polarizable ligand charges, induced by the protein, by

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means of QSite 5.9 at the B3LYP/6-31G* level. Finally, the ligands with QM/MM modified charges

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were re-docked and ten poses of each ligand were saved. Finally, the Glide energy value was used as

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scoring function in order to rank the poses.

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Prime MM-GBSA free energy

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For each ligand, the ten docked poses generated by the QPLD, in complex with the X-ray crystal

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structures (2Z5X and 2V5Z), were energy minimized using Prime 3.5.34 Binding free energies (dG

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Bind) of each complex were calculated using the Prime/MM-GBSA method with OPLS-2005 as force

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field and the default setting. The complexes with the best dG bind values were further submitted to

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Molecular Dynamics simulations (MDs).

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Molecular Dynamics simulations

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Desmond 3.7 system, with OPLS-2005 force field, was used to perform the MDs of 10 ns with the NPT

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ensemble, Temperature (T) of 10 K and pressure (P) of 1 atm.35 For each ligand: the complex was

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solvated with TIP3P water model in a cubic box; the prepared systems were neutralized using the

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appropriate number of Na+ counterions and the salt concentration was set to 0.15 M. The “Simulation

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Interactions Diagram” (SID) utility of Desmond 3.7 was used to display the ligand-target interactions

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during the MDs. Protein interactions with the ligands were monitored throughout the simulation and

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were categorized by type (Figures 3S-4S). Protein-ligand interactions (or 'contacts') were categorized

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into four types: Hydrogen Bonds, Hydrophobic, Ionic and Water Bridges. Each interaction type

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contains more specific subtypes, which can be explored through the SID panel. The stacked bar charts

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were normalized over the course of the trajectory: for example, a value of 0.7 suggests that for the 70% 9 ACS Paragon Plus Environment


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of the simulation time the specific interaction is maintained. Values over 1.0 are possible as some

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protein residue may make multiple contacts of same subtype with the ligand. Finally, the Pymol

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1.7.0.036 molecular graphics system was used to visualize the molecules in the last frames of the MDs

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and to generate all figures.

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Results and Discussion

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The effects of temperature on grape berry composition have been studied extensively and a recent

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publication by Song et al. confirmed that sunlight and UV exposure significantly increase

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anthocyanins, total pigment, total phenolics and tannin content.37 Moreover, Spayd et al. reported an

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increased concentration of the 3-glycosides of kaempferol with exposure to solar radiation.38 Based on

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this evidence we carried out a preliminary study on three samples of monocultivar Calabrian red wine

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from the 2013 vintage in order to determine the kaempferol content (in units of mg/100 mL) (Figure 1).

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The PDO “Magliocco”, the DOC “Gaglioppo” and the PGI “Nerello” were respectively selected from

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the north, center and south of Calabria district in order to check whether the kaempferol concentration

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differs within the same region and with respect to other Italian red wines.

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Free kaempferol was not found in the analyzed samples, but it was only detected after hydrolysis of its

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conjugated forms. In fact, conjugated flavonoids are generally present in higher concentrations than

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their free counterparts.39 The great number of anthocyanins present in the red grape extract usually

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cause interference in the chromatographic separation and identification of flavonoids.40 Therefore, the

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assignment of the kaempferol peak (Figure 1S) was confirmed by comparison of the retention time of

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its corresponding reference standard (Figure 2S), analyzed under the same chromatographic conditions.

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The analytical results, reported in Table 1, showed the highest concentration of kaempferol in the DOC

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“Gaglioppo” grape variety, equal to 0.12 mg/100 mL. The northern PDO “Maglioppo” displayed the

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lowest kaempferol concentration (0.08 mg/100 mL). However, compared to the national average of

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0.02-0.04 mg/100 mL, these Calabrian red wines may be considered as having high kaempferol 10 ACS Paragon Plus Environment

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content. Finally, we extended the study to the different vintage of “Gaglioppo”, namely 2008, 2010 and

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2011, available by A’vita Azienda Vinicola De Franco. Unfortunately, it was not possible to correlate

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the vintage effect on the phenolic profile, because suppliers did not record some particular relevant

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weather indicators (temperatures, sunlight exposure and vine water status). However, the results

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reported in Table 2 showed, in all cases, that kaempferol content was higher than in other Italian red

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wines.

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To investigate the MAO inhibitory properties of kaempferol, quercetin and the reference inhibitors,

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harmine and safinamide, the recombinant human enzymes were used. To measure MAO activity,

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kynuramine served as a non-specific MAO substrate (Figure 1). Kynuramine is metabolized by MAO-

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A and MAO-B to yield 4-hydroxyquinoline, which can conveniently be quantified by fluorescence

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spectrophotometry. By measuring MAO activities in the presence of different inhibitor concentrations,

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sigmoidal dose-response curves were constructed from which IC50 values were determined. The results,

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given in Table 3, showed that kaempferol acts as a potent human MAO-A inhibitor with an IC50 value

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of 0.525 µM. This is in accordance to literature reports of the MAO inhibitory properties of this

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compound (6, 7). Quercetin (IC50 = 3.98 µM) also was a selective MAO-A inhibitor, but with lower

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potency compared to kaempferol. Both compounds, however, do not inhibit human MAO-B at a

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maximal tested concentration of 100 µM. As expected, the reference inhibitors harmine and safinamide

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are potent hMAO inhibitors with the anticipated selectivities for MAO-A and MAO-B, respectively. To

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examine the reversibility of inhibition of kaempferol and quercetin, these inhibitors (at concentration

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equal to 4 × IC50) were pre-incubated with MAO-A, and subsequently dialysed. For reversible

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inhibition, dialysis was expected to remove the inhibitor and restore enzyme activity to 100% of the

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MAO activity recorded in the absence of inhibitor (negative control). As shown in Figure 2, dialysis

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indeed restored MAO-A activity to 201% (kaempferol) and 129% (quercetin) of the negative control.

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However, in mixtures (not dialysed) of kaempferol and quercetin with MAO-A, inhibition persisted 11 ACS Paragon Plus Environment


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with MAO-A activity at 50% and 31%, respectively. As expected, dialysis did not restore MAO-A

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activity following inhibition by the irreversible inhibitor, pargyline, with activity at only 3.2% (Figure

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1). These data demonstrated that kaempferol and quercetin are reversible inhibitors of human MAO-A.

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Interestingly, kaempferol appeared to prevent time-dependent loss of MAO-A activity since the

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residual activity of MAO-A after 24 h of dialysis in the presence of kaempferol (201%) is significantly

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higher than experiments conducted in the absence of kaempferol (100%). In this study 52% of MAO-A

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activity is lost during 24h dialysis, and is thus almost completely prevented by the presence of

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kaempferol. The molecular basis of this protective effect is not clearly understood and requires further

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investigation. Quercetin displayed a similar protective effect although with lower potency. It should be

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mentioned that D-amphetamine, a classic competitive inhibitor (Ki = 20 µM, human placental MAO-A)

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may also protect human MAO-A from loss of activity.41 This compound has been included in the

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protocol for the purification of MAO-A from human placental tissues. In this instance D-amphetamine

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(3 mM) is added during ion exchange chromatography. Slightly lower MAO-A yield has been obtained

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when D-amphetamine is omitted.

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Based on the finding that kaempferol and quercetin are reversible and selective MAO-A inhibitors and

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Lineweaver-Burk plots were constructed to unravel the inhibition mechanism of MAO-A by these

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inhibitors. For each inhibitor a set of six plots were constructed by employing eight different

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concentrations (15-250 µM) of the substrate (kynuramine) for each plot. The inhibitor concentrations

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selected for the six plots were: 0 × IC50, ¼ × IC50, ½ × IC50, ¾ × IC50, 1 × IC50 and 1¼ × IC50. These

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Lineweaver-Burk plots were shown in Figure 3 and were indicative of a competitive inhibition of

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MAO-A by both kaempferol and quercetin. This is evident from the fact that the plots of each set

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intersected on the y-axis. From the Lineweaver-Burk plots, Ki values for the inhibition of MAO-A by

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kaempferol and quercetin were estimated at 0.455 and 6.31 µM, respectively. Global fitting of the


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inhibition data to the Michaelis-Menten equation yielded Ki values of 0.362 ± 0.021 µM (R2 = 0.99)

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and 4.24 ± 0.305 µM (R2 = 0.99) for kaempferol and quercetin, respectively.

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To investigate the molecular mechanism of hMAO-A inhibition by kaempferol and quercetin, 3D

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structures of the ligands were obtained from the PubChem31 database in the SDF format and loaded

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into the Maestro 9.7 graphical user.30 The X-ray crystal structures of the MAO enzymes used for the

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molecular modelling studies were selected by considering that the co-crystallized inhibitors are used as

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positive controls in our in vitro assays. Successively, hMAO-A in complex with harmine and hMAO-B

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in complex with safinamide were obtained from the Protein Data Bank (PDB)26 (accession codes 2Z5X

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and 2V5Z, respectively).27, 28 The molecular recognition studies were performed by means of the QM-

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Polarized Ligand Docking (QPLD),33 an approach recently used to investigate the binding mode of

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several compounds.42 For the flexible structures of kaempferol and quercetin, 10 binding orientations in

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each enzyme were predicted and submitted to the Prime/MMGBSA method in order to calculate free

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binding energies (dG Bind), expressed in Kcal/mol.34 The positive control inhibitors, harmine and

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safinamide, were used to generate the docking grid box before being removed from the X-ray crystal

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structures. These control inhibitors were redocked using the same protocol as that carried out for

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kaempferol and quercetin. The theoretical results were reported in Table 3 and showed that the two

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flavonoids were better accommodated in the binding pocket of hMAO-A compared to hMAO-B.

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Regarding the hMAO-A isoform, the dG Bind values were respectively equal to -49.52 Kcal/mol for

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kaempferol and -48.35 Kcal/mol for quercetin, thus confirming the biological data of the in vitro assays

294

(Table 3).

295

The complexes were further submitted to 10 ns of Molecular Dynamic (MD) simulations in order to

296

analyze the ligand-target interactions and to investigate the contributions of the amino acids of the

297

catalytic site in their molecular recognition. In Figure 4 we collected the graphical representations of

298

the MD final frames for kaempferol and quercetin in complex with hMAO-A and hMAO-B, 13 ACS Paragon Plus Environment


Page 14 of 29

299

respectively. The “Simulation Interaction Diagram” (SID) utility of the Desmond 3.7 package35 was

300

used to visualize protein interactions with the ligands monitored throughout the simulation. These

301

interactions were categorized by type and summarized, as shown in the plots included in the Supporting

302

Information (Figures 3S-4S). These results confirmed that the specificity of the reversible inhibitors

303

was mainly caused by the different size and shape of the substrate/inhibitor cavity, restricted by ILE335

304

and PHE208 in hMAOA, which correspond to TYR326 and ILE199 in hMAO-B.27 In fact, kaempferol

305

binding mode to the hMAO-A active site is stabilized by the hydrophobic interactions with these key

306

residues for a longer time than in hMAO-B. In hMAO-A kaempferol maintained for 80% of the total

307

simulation time hydrophobic interactions with ILE335 and for 90% of the simulation time with

308

PHE208 (Figure 3S). On the contrary, in hMAO-B, kaempferol maintained these hydrophobic contacts

309

for only 30% of the simulation time with ILE199 and for 80% with TYR326. In addition, a crucial

310

difference in MAO inhibition between the two polyphenols can be attributed to the greater number of

311

protein interactions for kaempferol, and particularly to its H-bond contacts with ALA111, ASN181 and

312

VAL210, maintained for a long time period during the simulation (Figures 4 and 3S). In conclusion,

313

natural products, considered as an important resource of bioactive compounds such as flavonoids, have

314

been often investigated in order to identify the chemical components involved in several beneficial

315

effects for health. In the nutraceutical field, computational techniques have been successfully used both

316

for the prediction of ligand-target binding affinity and to better understand the molecular basis of the

317

biological response.

318

Abbreviations used

319

dG Bind: Free Binding Energy

320

DOC: Controlled Origin Denomination

321

FAD: Flavin Adenine Dinucleotide

322

hMAO: Human Monoamine Oxidase 14 ACS Paragon Plus Environment

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323

MAOs: Monoamine Oxidases

324

MD: Molecular Dynamic

325

PDO: Protected Denomination Origin

326

PGI: Protected Geographical Indication

327

QM/MM: Quantum Mechanics/Molecular Mechanics

328

QPLD: QM-Polarized Ligand Docking

329

RMSD: Root Mean Square Deviation

330

RRD: Rigid Receptor Docking

331

SD: Standard Deviation

332

SID: Simulation Interactions Diagram

333

Acknowledgments

334

This work was supported by Interregional Research Center for Food Safety and Health at the Magna

335

Græcia University of Catanzaro (MIUR PON a3_00359). The authors thank Dr. Simon Cross,

336

Molecular Discovery Ltd, for the corrections in the language style of manuscript.

337 338

Supporting Information description

339

In Figure 1S HPLC chromatograms of the three Calabrian red wine samples, from the 2013 vintage,

340

were displayed with the kaempferol peak identified by comparison of the retention time of its

341

corresponding reference standard shown in Figure 2S. In Figures 3S and 4S we reported SID plots

342

showing the protein-ligand interactions, established during the MD simulation, between the selected

343

flavonoids and amino acid residues of hMAO-A and hMAO-B, respectively.

344



Page 16 of 29

345

References

346

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monoamine oxidase B as reliable and efficient enzyme source for inhibitor screening. Bioorg Med

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selective and reversible hMAO-B inhibitors. Eur J Med Chem. 2013, 66, 221-227.

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25. Strydom, B.; Bergh, J.J.; Petzer, JP. The inhibition of monoamine oxidase by 8-(2phenoxyethoxy)caffeine analogues. Arzneimittelforschung. 2012, 62, 513-518. 26. http://www.rcsb.org/


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27. Son, S.Y.; Ma, J.; Kondou, Y.; Yoshimura, M.; Yamashita, E.; Tsukihara, T. Structure of human

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28. Binda, C.; Wang, J.; Pisani, L.; Caccia, C.; Carotti, A.; Salvati, P.; Edmondson, D.E; Mattevi, A.

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Structures of human monoamine oxidase B complexes with selective noncovalent inhibitors:

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safinamide and coumarin analogs. J Med Chem. 2007, 50, 5848-5852.

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29. Schrödinger Suite 2014-1 Protein Preparation Wizard; Epik version 2.7, Schrödinger, LLC, New

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31. https://pubchem.ncbi.nlm.nih.gov/

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version 5.9, Schrödinger, LLC, New York, NY, 2014.

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34. Prime version 3.5, Schrödinger, LLC, New York, NY, 2014.

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Maestro-Desmond Interoperability Tools, version 3.7, Schrödinger, New York, NY, 2014.

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36. The PyMOL Molecular Graphics System, Version 1.7.0.0 Schrödinger, LLC

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37. Song, J.; Smart, R.; Wang, H.; Dambergs, B.; Sparrow, A.; Qian, M.C. Effect of grape bunch

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sunlight exposure and UV radiation on phenolics and volatile composition of Vitis vinifera L. cv.

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38. Spayd, S.E.; Tarara, J.M.; Mee, D.L.; Ferguson, J.C. Separation of sunlight and temperature effects on the composition of Vitis vinifera cv. Merlot Berries. Am J Enol Vitic. 2002, 53, 3.



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39. Tsanova-Savova, S.; Ribarova, F. Free and conjugated Myricetin, Quercetin, and Kaempferol in Bulgarian red wines. J Food Comp Anal. 2002, 15, 639-645.

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40. Castillo-Muñoz, N.; Gómez-Alonso, S.; García-Romero, E.; Hermosín-Gutiérrez, I. Flavonol

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profiles of Vitis vinifera red grapes and their single-cultivar wines. J Agric Food Chem. 2007, 55,

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41. Weyler, W.; Salach, JI. Purification and properties of mitochondrial monoamine oxidase type A from human placenta. J Biol Chem. 1985, 260, 13199-207.

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42. Reddy, K.K.; Singh, S.K. Insight into the binding mode between N-methyl pyrimidones and

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prototype foamy virus integrase-DNA complex by QM-polarized ligand docking and molecular

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dynamics simulations. Curr Top Med Chem. 2015, 15, 43-49.


Page 21 of 29


447 448

Figures captions

449

Figure 1. 2D chemical structures of the flavonoids kaempferol and quercetin; pargyline and harmine,

450

hMAO-A inhibitors; safinamide, hMAO-B inhibitor, and kynuramine, a non-specific MAO substrate.

451

Figure 2. Dialysis restores hMAO-A activity after inhibition by kaempferol (top) and quercetin

452

(bottom) but not after inhibition by the irreversible inhibitor, pargyline. Inhibition by kaempferol and

453

quercetin, however, persists in samples not dialysed.

454

Figure 3. Lineweaver-Burk plots for the inhibition of hMAO-A by kaempferol (A) and quercetin (B).

455

The insets are replots of the slopes of the Lineweaver-Burk plots versus inhibitor concentration.

456

Figure 4. MD final frames and H-bond contacts of: kaempferol in the hMAO-A (a) and hMAO-B

457

binding sites (c) and quercetin in the hMAO-A (b) and hMAO-B binding sites (d). Kaempferol and

458

quercetin are rendered, respectively, as dark green and light green carbon sticks. The binding pocket

459

amino acids and FAD are labeled as grey carbon sticks. The enzyme is shown as a cyan cartoon and all

460

non-carbon atoms are colored according to atom types.



Page 22 of 29

461 Table 1. Range and median value in mg/100 mL of the kaempferol concentration, with relative standard deviation (SD), for the three analyzed Calabrian red wines from the 2013 vintage.

Red wine sample Magliocco Gaglioppo Nerello

Range 0.083-0.091 0.094-0.128 0.106-0.124

Median value 0.087 0.111 0.115

462


SD 0.004 0.017 0.009

Page 23 of 29


Table 2. Range and median value in mg/100 mL of the kaempferol concentration, with relative standard deviation (SD), for the available vintage of the DOC “Gaglioppo” red wine.

Vintage 2008 2010 2011

Range 0.081-0.089 0.089-0.107 0.101-0.109

Median value 0.085 0.098 0.105

463


SD 0.004 0.009 0.004


Page 24 of 29

Table 3. Experimental IC50 values in µM and theoretical dG Bind values in Kcal/mol of 1) kaempferol and quercetin for both MAO isoforms; 2) harmine, the co-crystallized inhibitor of hMAO-A (2Z5X X-ray structure) and 3) safinamide, the co-crystallized inhibitor of hMAO-B ( 2V5Z X-ray structure).

hMAO-A Compound

hMAO-B

kaempferol quercetin harmine

0.525 ± 0.035 3.98 ± 0.265 0.0029 ± 0.00042

dG Bind -49.52 -48.35 -46.07

safinamide

/

/

IC50

IC50 >100 >100 / 0.0479 ± 0.00472

464


dG Bind -42.66 -46.98 / -73.70

Page 25 of 29


OH

OH HO

HO

O

O

OH

N

OH

OH OH O

OH O

N H harmine

NH2

H2N

N

O

pargyline

quercetin

kaempferol

O F

NH2

O HN

safinamide

Figure 1.


O kynuramine


A: B: C: D:

200

Rate (%)

150

No inhibitor Kaempferol Pargyline Kaempferol -

dialysed dialysed dialysed not dialysed

100

50

0

A

B

A: B: C: D:

125 100

Rate (%)

C

D

No inhibitor Quercetin Pargyline Quercetin -

dialysed dialysed dialysed not dialysed

75 50 25 0

A

B

C

D

Figure 2..


Page 26 of 29

Page 27 of 29


A Slope

1200

100

1/V (%)

75

800 400

−0.6 −0.3

0.0 0.3 [I], µ M

0.6

50 25 0 −0.02

0.00

B

1/V (%)

75

0.06

1200

Slope

100

0.02 0.04 1/[S]

800 400

−6 −4 −2 0 2 [I], µ M

4

6

50 25 0 −0.02

0.00

0.02 0.04 1/[S]

0.06

Figure 3.



Figure 4.


Page 28 of 29

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For Table of Contents Only


Kaempferol as Selective Human MAO-A Inhibitor: Analytical Detection

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