SULFATION PATHWAYS Potential benefits of a sulfated

httpsdoiorg101530JME-18-0031httpjmeendocrinology-journalsorg copy 2018 Society for Endocrinology

Printed in Great BritainPublished by Bioscientifica Ltd

A sulfated resveratrol derivativeM Correia-da-Silva V Rocha et al

Journal of Molecular Endocrinology

M27ndashM3961 2

THEMATIC RESEARCH

SULFATION PATHWAYS

Potential benefits of a sulfated resveratrol derivative for topical application

Marta Correia-da-Silva12 Veroacutenica Rocha3 Claacuteudia Marques3 Claacuteudia M Deus45 Adriana Marques-Carvalho4 Paulo J Oliveira45 Andreia Palmeira12 Madalena Pinto12 Emiacutelia Sousa12 Joseacute Manuel Sousa Lobo3 and Isabel Filipa Almeida3

1Laboratory of Organic and Pharmaceutical Chemistry Department of Chemical Sciences Faculty of Pharmacy University of Porto Porto Portugal2CIIMAR ndash Interdisciplinary Centre of Marine and Environmental Research Matosinhos Portugal3UCIBIO REQUIMTE Laboratory of Pharmaceutical Technology Department of Drug Sciences Faculty of Pharmacy University of Porto Porto Portugal4CNC ndash Center for Neuroscience and Cell Biology University of Coimbra UC Biotech Building Biocant Park Cantanhede Portugal5IIIUC ndash Institute for Interdisciplinary Research University of Coimbra Coimbra Portugal

Correspondence should be addressed to E Sousa esousaffuppt

(M Correia-da-Silva and V Rocha contributed equally to this work)

This paper is part of a thematic section on Sulfation Pathways The guest editors for this section were Jonathan Wolf Mueller and Paul Foster

Abstract

Resveratrol (RSV) is a polyphenolic compound with antioxidant anti-inflammatory and

anti-aging properties partly associated with sirtuin 1 (SIRT1)-activation in the skin However

poor water solubility may limit RSV efficacy This work aimed to clarify the interest of

a new synthetic water-soluble RSV derivative (resveratrol glucoside sulfate RSV-GS) for

topical application Resveratrol glucoside sulfate was synthesized using microwave-assisted

sulfation Cytotoxicity assays were performed with the keratinocyte HaCaT cell line using

MTT reduction neutral red uptake Alamar Blueresazurin reduction trypan blue exclusion

and measurement of ATP concentration Western blotting was used to evaluate SIRT1

protein content Regarding SIRT1 binding an in silico docking study was performed using

AutoDock Vina Our results showed that the synthetic derivative RSV-GS was 1000 times

more soluble in water than RSV and its non-sulfated glucoside No relevant decrease in

HaCaT cell viability was observed for concentrations up to 5 mM for RSV-GS and up to

500 μM for resveratrol glucoside while a significant decrease in HaCaT viability occurred

from 100 μM for RSV RSV-GS and RSV showed a similar behavior regarding protective effect

against oxidative stress-induced cytotoxicity SIRT1 protein content increased after treatment

with 500 μM of RSV-GS and 100 μM of RSV Moreover in silico studies predicted that RSV-GS

binds more stably to SIRT1 with a lower binding free energy than RSV Although these

results support the possible use of RSV-GS in topical formulations in vivo safety and efficacy

studies are needed before considering the use of RSV-GS in commercial products

Introduction

Resveratrol (RSV) 354prime-trihydroxystilbene is a polyphenolic compound found in the seeds and skin of grapes red wine peanuts and berries (Kasiotis et al 2013)

Resveratrol acts as a toxin that protects the plant against injurious microorganisms UV radiation mechanical injury and heavy metal pollution For these reasons RSV

Journal of Molecular Endocrinology (2018) 61 M27ndashM39

Key Words

f resveratrol

f cytotoxicity

f sirtuin modulation

f topical antioxidants

-18-0031

Downloaded from Bioscientificacom at 03212022 042208AMvia free access

M28M Correia-da-Silva V Rocha et al

A sulfated resveratrol derivative 61 2Journal of Molecular Endocrinology

is a phytoalexin (Amri et al 2012 Tang et al 2014) The most abundant form of RSV in nature is resveratrol 3-O-β-D-glucoside (RSV-G) (Regev-Shoshani et al 2003) also called piceid or polydatin

Several studies demonstrated that RSV has interesting properties regarding skin care (Baxter 2008 Ndiaye et al 2011 Park et al 2014) The biological effects of RSV are supported by mechanistic data that indicates the involvement of increased expression and activation of the protein deacetylase sirtuin 1 (SIRT1) (Baur amp Sinclair 2006 Price et al 2012) Sirtuin 1 is expressed in skin keratinocytes (Blander et al 2009) Resveratrol acting as a SIRT1 activator protects the skin against oxidative stress events and consequently prevents skin cell damage (Cao et al 2009)

Resveratrol occurs in cis-(Z) and trans-(E) isomeric forms in nature trans-resveratrol is the most common and biologically active isomeric form as well as the most photo- and thermal-stable isomer (Baxter 2008 Carlotti et al 2012) Fast trans-cis isomerization (Detoni et al 2012 Sanna et al 2012) and poor water solubility (Lepak et al 2015) limits RSV efficacy and constrains the design of topical formulations Besides classical formulation development (Amri et al 2012 Summerlin et al 2015) chemical conjugations such as sulfation (Hoshino et al 2010) glycosylation (Regev-Shoshani et al 2003) oligomerization (Matsuura et al 2015) and other derivations (Regev-Shoshani et al 2003 Medina et al 2010 Mattarei et al 2013) are being employed to overcome these limitations

Synthetic RSV derivatives with improved water solubility may be obtained with similar or better activities toward SIRT1 activation In this work the validation of a synthetic water-soluble RSV derivative (resveratrol glucoside sulfate RSV-GS) for skin care was evaluated and compared with the parent compounds RSV and RSV-G by studying the cytotoxic and protective effects (lysosomal integrity cell membrane damage and metabolic activity) and the alterations of SIRT1 protein content using a human keratinocyte cell line and performing in silico affinity studies for SIRT1

Materials and methods

Reagents

Dulbeccorsquos Modified Eaglesrsquos Medium (DMEM) with 25 mM d-glucose and 1 mM pyruvate inactivated fetal bovine serum (FBS) penicillin-streptomycin solution Dulbeccorsquos phosphate buffered saline (DPBS) without

calcium chloride and magnesium chloride 025 trypsin-ethylenediaminetetraacetic acid (EDTA) solution were supplied by Gibco by Life Technologies Dimethyl sulfoxide (DMSO) 04 trypan blue solution neutral red (NR) solution and resazurin sodium salt were purchased from Sigma-Aldrich AlamarBlue was provided by Thermo Scientific 3-[45-Dimethyl-2-yl]-25-diphenyl tetrazolium bromide (MTT) was acquired from Life Technologies Sirtuin-1 and actin antibodies were obtained from Abcam and Millipore respectively In all experiments purified water was used and obtained using a Direct-Q Water Purification System (Merck Millipore) with a reverse osmosis process trans-Resveratrol was purchased from Fragon (Satildeo Paulo Brazil) trans-resveratrol 3-szlig-D-glucopyranoside (572691) and triethylamine sulfur trioxide adduct (S 5139) were purchased from Sigma-Aldrich UVVis spectra were traced with a synergy HT microplate reader from BioTek with GEN5 software Melting point was obtained in a Koumlfler microscope and was not corrected Infrared (IR) spectra were recorded on a Nicolet iS10 from Thermo Scientific with Smart OMNI-Transmission accessory (Software 188 OMNIC 83) in KBr Nuclear magnetic resonance (NMR) spectra were taken in DMSO-d6 at room temperature on Bruker AMC AVANCE 300 instrument (Bruker Biosciences Corporation Billerica MA USA) instrument High-resolution mass spectrometry (HRMS) results were obtained in CACTI services Vigo Spain

Synthesis of resveratrol glucoside sulfate (RSV-GS)

A mixture of trans-resveratrol 3-szlig-D-glucopyranoside (05 g 128 mmol) and triethylamine sulfur trioxide adduct (4 equivOH) in dimethylacetamide (10 mL) was stirring and heated for 30 min at 100degC under MW irradiation (200 W) After cooling the mixture was poured into acetone (200 mL) under basic conditions (triethylamine) and left at 4degC for 24 h The formed crude oil was washed with ether dissolved in a minimum volume of water and applied on a DOWEX cation exchange resin (Na+ form) After evaporation until dryness a light brown solid was obtained (85 yield) NMR and HRMS were according to previously reported data (Correia-da-Silva et al 2011)

Water solubility determination

The solubility was tested at the maximum solubility class of the European Pharmacopeia (very soluble gt1000 mgmL) by dissolving a given amount in ultra-pure water (UPW) (RSV and RSV-G 2 mg in 200 mL RSV-GS 50 mg in

M2961 2M Correia-da-Silva V Rocha et al

A sulfated resveratrol derivativeJournal of Molecular Endocrinology

50 microL) and stirring at 25degC for 1 h After visual inspection RSV-GS was the only soluble compound Suspensions of RSV and RSV-G were filtered through a 022 microm membrane filter with a syringe After filtration 200 μL of each solution was transferred to a microplate well and a 110 dilution was made The UVVis Spectra were traced at 310 and 340 nm for RSV and RSV-G respectively and the concentration was determined according to the standard calibration curve obtained for each compound Three standard calibration curves were obtained with 4 different concentrations for each compound (RSV 0010 0005 00010 and 00005 mgmL RSV-G 0050 0025 0005 and 00025 mgmL) The solubility assay was repeated in three independent days

Cell culture conditions

The immortalized human keratinocyte cell line (HaCaT) was obtained from Cell Lines Service (CLS Eppelheim Germany) (Boukamp et al 1988) The method of authentication involved characterization of DNA Profile (STR) Cells were maintained at 37degC in a humidified atmosphere containing 5 CO2 in Dulbeccorsquos modified Eaglersquos medium (D31966) supplemented with 10 FBS 2 mM glutamine and 1 penicillinstreptomycin (growth medium) HaCaT cell line was bought from the vendor at passage number 31 and experiments were performed after that passage

Cytotoxicity testingCytotoxicity was evaluated using different assays namely MTT reduction NR uptake Alamar Blue reduction and trypan blue exclusion HaCaT cells were incubated with different concentrations of each compound (RSV and RSV-G 25 50 100 350 and 500 μM RSV-GS 100 350 500 1000 and 5000 μM) Cytotoxicity assessment of RSV in this cell line was already published by our group (Rocha et al 2017) but the results were reproduced here for comparison purposes For the trypan blue assay the concentration range used was slightly different and is described below DMSO (10) was used as positive control and cells treated with the solvent as negative control Cell viability was calculated with regard to negative control which was set to 100 viability IC50 was calculated by linear regression All absorbance readings were performed on a multi-mode microplate reader (Synergy HT BioTek Instruments) A minimum of three independent experiments were performed for each assay

MTT reduction assayHaCaT cells (1 times 104 cellswell 96-well microplates) were incubated with different concentrations of RSV RSV-G or RSV-GS After 24-h incubation cells were washed with DPBS and 05 mgmL MTT solution (serum free DMEM) was added After 2-h incubation DMSO was added to dissolve the formazan crystals and absorbance was measured at 570 nm

Neutral red uptake assayHaCaT cells (2 times 104 cellswell 96-well microplates) were incubated with different concentrations of RSV RSV-G or RSV-GS After 24-h incubation NR solution (DMEM) was added (50 microgmL) to cells previously washed with DPBS Cells were washed with DPBS after 3 h of incubation and a solution of 50 ethanol1 acetic acid was added to extract the NR dye captured within the cells The plates were placed for 10 min in a microplate shaker at room temperature and protected from light Absorbance was measured at 540 nm

Trypan blue exclusion assayHaCaT cells (1 times 105 cellswell 12-well microplates) were incubated with different concentrations of RSV RSV-G or RSV-GS (RSV and RSV-G 25 200 and 500 μM RSV-GS 100 500 and 5000 μM) After 24 h of incubation cells were trypsinized and counted using trypan blue vital dye (11)

Measurement of cell protection against oxidative stressProtective effects of RSV and derivatives against H2O2- and menadione-induced cytotoxicity were evaluated using resazurin assay and ATP levels determination HaCaT cells were seeded as described in each protocol described below and incubated with different concentrations of each compound (RSV and RSV-G 25 50 100 350 and 500 μM RSV-GS 100 350 500 1000 and 5000 μM) during 24 h Vehicle controls received an equivalent amount of DMSO only which never exceeded 01 vv Afterward 9 mM H2O2 or 25 microM menadione were added during 3 h A minimum of four independent experiments were performed for each assay

Sulforhodamine B (SRB) assayThe SRB assay was used to detect the loss of cell mass resulting from H2O2- or menadione-induced toxicity Cells were seeded at a concentration of 20000 cellscm2

in 96-well plates with a final volume of 100 microL per well Cells were treated with 78 156 312 625 125 and 250 μM menadione and 19 39 78 156 and 312 mM H2O2 during 3 h At specific time points the incubation medium was removed and cells were fixed in 1 acetic acid in ice-cold methanol for at least 1 day Cells were then incubated with 005 (wv) SRB reagent dissolved in 1 acetic acid for 1 h at 37degC Unbound dye was removed with 1 acetic acid Dye bound to cell proteins was extracted with 10 mM Tris-base solution pH 10 After SRB labeling absorbance was measured in a BioTek Cytation 3 spectrophotometer at 510 nm and the amount of dye released is proportional to the number of cell mass in each well (Vichai amp Kirtikara 2006)

Alamar Blueresazurin assayHaCaT cells (2 times 104 cellswell 96-well microplates) were incubated with different concentrations of RSV RSV-G or RSV-GS After 24-h incubation 10 AlamarBlue was added to each well and incubated for 4 h Finally the absorbance at 570 and 600 nm was determined

The resazurin assay was used to measure the cellular viability based on metabolic activity of living cells through the fluorescence intensity Cells were seeded at a concentration of 20000 cellscm2 in 96-well plates with a final volume of 100 microL per well Resazurin stock solution (1 mgmL) was prepared in PBS 1times and stored at minus20degC Cells were treated as described in protective testing section At specific time points cell media from each well was removed carefully and cells were washed with PBS 1times After removing the PBS 1times 100 μL of resazurin solution (1100 dilution in growth medium from a stock solution) was added to each well and incubated for 4 h at 37degC with a 5 CO2 atmosphere Resorufin fluorescence was measured in a BioTek Cytation 3 spectrophotometer using excitation wavelength of 540 nm and emission of 590 nm (Silva et al 2016) The spectral characteristics of DMEM and resazurin and menadione (25 microM) were determined in 96-well plate by BioTek Cytation 3 spectrophotometer using wavelength between 350 and 700 nm (visible spectrum)

ATP levels determinationIntracellular ATP levels were measured by using CellTiter-Glo Luminescent Cell Viability Assay (Promega) following manufacturerrsquos instructions Cells were seeded at a concentration of 20000 cellscm2 in white opaque-bottom 96-well plates with a final volume of 100 microL per well Cells were treated as described in protective testing

section At specific time points 50 microL of CellTiter-Glo Reagent (CellTiter-Glo Buffer + CellTiter-Glo Substrate) was added to the cells Contents were mixed for 2 min on an orbital shaker to induce cell lysis and after 10 min of incubation at 22degC the luminescence signal was monitored in a BioTek Cytation 3 spectrophotometer (BioTek Instruments) ATP standard curve was also generated following manufacturerrsquos instructions The luminescence signal obtained was proportional to the amount of ATP present in solution

Protein semi-quantification by western blottingHaCaT cells were treated with two concentrations of each compound (RSV and RSV-G 25 and 100 μM RSV-GS 100 and 500 μM) during 24 h Vehicle controls only received an equivalent amount of DMSO which never exceeded 01 vv To obtain total cellular extracts all cells were harvested by trypsinization and washed with PBS 1times In order to collect the cells one centrifugation step was performed for 5 min at 1000 g (4degC) The cellular pellet was resuspended in cell lysis buffer 1times (Bio-Rad 9803) supplemented with 100 microM phenylmethylsulfonyl fluoride (PMSF) Protein content was determined by the Bradford method using bovine serum albumin as a standard (Bradford 1976) An equivalent amount of protein (20 microg) for each sample was separated by electrophoresis on 10 SDSndashpolyacrylamide gel (SDSndashPAGE) and transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane After blocking with 5 milk in TBST (50 mM TrisndashHCl pH 8 154 mM NaCl and 01 Tween 20) for 2 h at room temperature PVDF membranes were incubated overnight at 4degC with primaries antibodies against SIRT1 (11000 ab110304 Abcam) and actin (15000 MAB1501 Millipore) Membranes were incubated with the respective alkaline phosphatase conjugated secondary antibody anti-mouse IgG (12500) HRP-linked antibody for 1 h at room temperature Membranes were incubated with the ECL detection substrate (from Bio-Rad) and imaged with the Biospectrum Multispectral imaging system (UVP LLC Upland CA Cambridge UK) Densities of each band were calculated with ImageJ 145S program

Docking study

The 3D structures of RSV and RSV-GS were drawn using HyperChem 75 (Froimowitz 1993) being minimized by the semi-empirical PolakndashRibiere conjugate gradient method (RMS lt 01 kcalAring mol) (Zhang et al 2006) Docking simulations between SIRT1 with bound peptide

substrate (pdb code 5BTR) (Cao et al 2015) and the small molecules were undertaken in AutoDock Vina (Scripps Research Institute USA) (Seeliger amp de Groot 2010 Trott amp Olson 2010) AutoDock Vina considered the target conformation as a rigid unit while the ligands were allowed to be flexible and adaptable to the target Vina searched for the lowest binding affinity conformations and returned 9 different conformations for each ligand AutoDock Vina was run using an exhaustiveness of 8 and a grid box with the dimensions of 170 190 and 170 Aring engulfing the crystallographic RSV-binding site To validate the docking approach for the protein structure used the root mean square distance (rmds) between co-crystallized modulator RSV and docked RSV was evaluated Conformations and interactions were visualized using PyMOL version 13 (Lill amp Danielson 2011)

Molecular dynamics simulation

Previous to the molecular dynamics (MD) simulations the SIRT1peptideRSV-GS and SIRT1peptideRSV complexes obtained from docking were placed inside a 6 Aring margin spherical droplet containing 17234 water molecules and their energy minimized using the MMFF94x force field (Cheng et al 2000) until RMS gradient lt01 kcalAringmol The MD simulations (Hospital et al 2015 Mortier et al 2015) were performed using MOE-dynamic implemented in MOE 201409 (Chemical Computing Groups Montreal Canada) (Vilar et al 2008) MD simulations were done by choosing MMFF94x force field and NVT (N total atom V volume T temperature) ensemble and Noseacute-Poincareacute-Andersen (NPA) algorithm (Sturgeon amp Laird 2000) with 0002 ps time step and sampling every 05 ps The system was heated from 0 to 300 K in 100 ps (heat stage) followed by a 2000 ps of a production stage at 300 K the system was then cooled back to 0 K in 100 ps (cooling stage) (Karplus amp McCammon 2002) The system behavior was monitored by the analysis of root mean square deviation (RMSD) and potential energy over the course of the simulation

Statistical analysis

Statistical evaluation of cytotoxicity results was carried out using one-way analysis of variance (ANOVA) followed by the Dunnett post hoc test Statistical analyses were performed using the SPSS software (v 230 IBM) Statistical analysis of data involving studies of cell protection by the different molecules and Western blotting was performed by using GraphPad Prism 601 (GraphPad Software) and presented as mean plusmn standard deviation (sd) for the

number of experiments indicated in the legends of the figures Data normality was tested with KolmogorovndashSmirnov and ShapirondashWilk test Statistical analysis of two groups was performed using the non-parametric MannndashWhitney test or two-way ANOVA followed by Sidak post hoc test Values with P lt 005 were considered as statistically significant ()

Results

Synthesis and solubility of RSV-GS

The synthesis of RSV-GS was accomplished by sulfation of RSV-G with triethylamine sulfur trioxide adduct (4 equivOH) in dimethylacetamide Applying MW irradiation the reaction time decreased to only 30 min and a significant increase of the yield was achieved (85) when compared to the previously described procedure in which conventional heating was used (Correia-da-Silva et al 2011) Also the use of SPE-WCX in the purification process contributed to improve the isolated yield of RSV-GS Solubility of RSV-GS in water was found to be gt1000 mgmL (Fig 1) and accordingly to the European Pharmacopoeia RSV-GS can be considered lsquovery solublersquo This synthetic derivative is more than 1000 times more soluble in water than RSV or RSV-G (lt1 mgmL Fig 1) Water solubility of RSV and RSV-G was also evaluated in the same conditions for comparison purposes and the obtained values (Fig 1) were similar to those previously reported (Park et al 2012)

Cytotoxicity testing

To investigate the potential benefits of the water-soluble RSV-GS different cytotoxicity assays in HaCaT cells were performed in the presence of RSV-GS as also as in the presence of its parent non-sulfated compounds RSV-G and RSV The results showed that RSV promoted a concentration-dependent decrease in cell viability In general a significant decrease in HaCaT viability occurred at 100 μM In the NR reduction assay (Fig 2) exposure to 25 μM of RSV caused a significant decrease

trans-RSV RSV-G RSV-GS

Chemicalstructure

Watersolubilityat 25degC

0029 plusmn 0005mgmL

0079 plusmn 0012mgmL gt1000 mgmL

Figure 1Structure and solubility of resveratrol and its derivatives

in cell viability comparing to the negative control (Fig 2B) but the cell viability was around 80 which can be considered non-cytotoxic (Technical Committee ISOTC 194 2009) A previous reported study performed in a primary keratinocyte line showed that RSV did not induce significant cytotoxicity up to 50 μM (Wu et al 2014) which is consistent with the majority of our results IC50 values were around 400 μM for all the assays except for MTT (17447 plusmn 1118 μM) which was the most sensitive assay (Table 1) This assay was also more sensitive when performed in HaCaT cells (Mukherjee et al 2012) Of notice the cell density used to perform the MTT test in the present work was lower than the one used for the other cytotoxicity tests in agreement with optimization studies that defined the optimal experimental conditions Thus it is not possible to exclude the hypothesis that the lower IC50 values obtained for the MTT assay might be because cells were exposed to higher levels of the compound These remarks further reinforce the importance of using different in vitro cytotoxicity tests which rely on different mechanisms of cell death The cytotoxicity assays used herein assess the lysosomal integrity (NR uptake) cell membrane damage (trypan blue) and metabolic activity (MTT and AlamarBlueresazurin reduction) Considering the RSV-G the concentration range studied was limited by the maximum concentration of the solvent (DMSO) that could be used without affecting cell viability (1) Even that it was possible to observe that RSV-G did not affect cell viability at the tested concentrations (25ndash500 μM) Interestingly the sulfated derivative RSV-GS only induced a significant reduction of HaCaT viability (70) in the MTT reduction assay when a very high concentration was used (5000 μM) (Fig 2A) A minimum tenfold increase in IC50 value can be expected for RSV-GS in comparison with RSV These results are in accordance with previous data reported for sulfated small molecules in which no cytotoxicity was observed even at the millimolar range namely in HCT-116 and SW-480 human colon cancer cell lines human genital ME180 HeLa and primary human foreskin fibroblast cells mouse lymphocytic leukemia P-388 cancer cell lines human MT-4 MCF-7 and KB cells (Correia-da-Silva et al 2014)

Figure 2Cytotoxic effect of RSV and derivatives in HaCaT cells Cell viability of HaCaT cell line exposed to RSV and derivatives determined by the MTT reduction assay (A) neutral red uptake assay (B) Alamar Blue reduction assay (C) and trypan blue exclusion assay (D) Control 1 = cells treated with RSV and RSV-G solvent (DMSO) (A) 100 plusmn 1368 (B) 100 plusmn 1752 (C)

100 plusmn 2913 and Control 2 = cells treated with cell medium (A) 100 plusmn 1616 (B) 100 plusmn 1426 (C) 100 plusmn 1157 (D) 100 plusmn 044 Each data point represents the mean plusmn sd of at least three independent experiments P le 005 vs control (no additions)

Protective effects on models of oxidative stress-induced cytotoxicity

Protective effects of RSV through several mechanisms of action have been demonstrated extensively in several disease models (Fukui et al 2010 Albuquerque et al 2015) To evaluate whether RSV-GS prevented H2O2- and menadione-induced cytotoxicity metabolic activity and ATP levels were evaluated Hydrogen peroxide and menadione are frequently used as cell stressors since they induce reactive oxygen species (ROS) formation oxidative DNA damage mitochondrial dysfunction and trigger apoptotic pathway often resulting in an all-or-nothing result (Criddle et al 2006 Loor et al 2010 Reis et al 2015 Xiang et al 2016) Firstly dosendashresponse curves of H2O2 (9 mM) and menadione (25 microM) were obtained to choose cytotoxic concentrations for HaCaT cells (Supplementary Fig 1 see section on supplementary data given at the end of this article) Because the range of concentrations used did not allow for an accurate IC50 determination and considering the large drop of ATP concentrations (often higher than 80) after stressor incubation we opted to use concentrations of stressors around the IC20 based on metabolic viability results In fact the fact that ATP depletion may be an early consequence of different toxicants has been previously demonstrated (Swiss et al 2013) Resveratrol per se decreased metabolic activity (Fig 3A and B) and ATP levels (Fig 4A and B) starting at 350 microM Also RSV did not prevent the decrease in metabolic activity resulting from H2O2 (Fig 3A) and menadione (Fig 3B) treatment for all concentrations tested while RSV increased ATP levels after H2O2 treatment at 25 and 50 microM (Fig 4A) RSV-G per se had no effect in the metabolic activity (Fig 3C and D) and ATP levels (Fig 4C and D However RSV-G protected against the decrease on HaCaT metabolic activity after H2O2 treatment at 25 50 and 100 microM (Fig 3C) and protected from menadione-induced decrease in metabolic activity at 25 microM (Fig 3D) In contrast ATP depletion was not prevented after both treatments (Fig 4C and D) Regarding RSV-GS per se 100

350 and 500 microM decreased metabolic activity (Fig 3E and F) while no alterations in ATP levels were observed with exception of increased ATP levels after pre-treatment with 5000 microM RSV-GS (Fig 4E and F) Moreover RSV-GS did not protect from H2O2 and menadione-induced decrease metabolic activity (Fig 3E and F) and ATP depletion (Fig 4E and F) The results obtained using resazurin in the presence of menadione as a stressor must be analyzed carefully By obtaining the absorption spectra of resazurin in the presence of menadione a slight increase in the spectrum of the former was observed Therefore it was not possible to conclude whether this type of interaction also occurs inside cells during the experimental protocol (Supplementary Fig 2)

Western blotting docking and MD studies

Sirtuins are attractive therapeutic targets for metabolic and aging-related diseases (Lavu et al 2008 Shih amp Donmez 2013) Sirtuins employ a conserved catalytic core domain to catalyze deacetylation by transferring the acetyl group from the acetyl-lysine of proteins to NAD+ (Sauve 2010) Sirtuins activating compounds such as RSV-related polyphenols can promote survival of human cells However their mode of action is still not completely understood (Chung et al 2010 Jayasena et al 2013)

Initially SIRT1 protein content was semi-quantified using Western blotting after treatment with RSV and derivatives The obtained results (Fig 5) showed that RSV and RSV-GS increased SIRT1 protein content in HaCaT cells at 100 and 500 microM respectively while no alterations were observed after RSV-G treatment Moreover in order to predict the molecular mechanism of RSV-GS an in silico docking study was performed between the small molecules RSV-GS and the known activator RSV and the target SIRT1 As AutoDockVina was able to return a RSV pose below a preselected RMSD value from the known conformation (bellow 2 Aring) it was assumed that it could predict docking poses accurately The RMSD value found

Table 1 degIC50 values (mean plusmn sd) and the values of the lowest concentration that promote a statistically significant decrease of

HaCaT viability in comparison with control for different cytotoxicity assays

Cytotoxicity assays

MTT reduction AlamarBlue reduction Neutral Red uptake Trypan Blue exclusion

IC50 plusmn sd Conc IC50 plusmn sd Conc IC50 plusmn sd Conc IC50 plusmn sd Conc

RSV (microM) 17447 plusmn 1118 100 38727 plusmn 688 350 38319 plusmn 5381 25 41262 plusmn 1479 200RSV-GS (microM) gt5000 5000 gt5000 ndash gt5000 ndash gt5000 5000RSV-G (microM) gt500 ndash gt500 ndash gt500 ndash gt500 ndash

(IC50) concentration that promotes a reduction of 50 in cell viabilityConc Concentration at which a statistically significant (P le 005) decrease in cell viability in comparison with control occurs

for crystallographic RSV vs docked RSV was 03 Aring which was considered a good threshold value for validating a structure for use in molecular docking (Fig 6C) (Hevener et al 2009) Both RSV and RSV-GS bind on top of the substrate peptide (Fig 5A) they bind in close proximity and directly contact each other through π-π stacking interactions and polar contacts It has been described that peptide leads to the formation of the activator-binding site by influencing these regions (Gertz et al 2012) The RSV-GSpeptideSIRT1 and RSVpeptideSIRT1 complexes obtained on the docking studies presented a free energy of minus91 and minus82 kcalmol respectively Therefore RSV-GS binds more stably to SIRT1 (lower binding free energy) than the parent compound RSV The 6 sulfate groups of RSV-GS increased the number of polar interactions with SIRT1-binding pocket (interactions with residues Pro-211 Thr-219 Gln-222 Ile-223 Asn-226 Glu-230 Asp-298 and Phe-414) (Fig 5C) Both compounds RSV and RSV-GS adopted a similar binding pose in SIRT1-binding pocket with the phenol groups establishing interactions with the sub-pocket formed by residues Pro-211 and Asn-226 (Dai et al 2015) However RSV-GS adopted a pose that also reached the surfaces formed by residues Asp-298 and Glu-230 (Fig 5B) Similar to RSV (Gertz et al 2012) RSV-GS shall close SIRT1 active site opening thereby trapping the bound peptide and increasing the interaction interface

by contacting the substrate fluorophore directly This interaction may lead to a substrate binding mode more suitable for the subsequent deacetylation step (Cao et al 2015)

To gain detailed insight in the energetic and geometric behavior of the SIRT1peptideRSV-GS complex in aqueous solution a 2 ns MD simulation was performed based on the aforementioned docking complex structure considering the effects of the target flexibility and the explicit water solvation Conformations of SIRT1peptideRSV-GS complex along simulation time (Fig 7A B C and D) RMSD and potential energy plots of complex conformations with respect to time (Fig 7E) were obtained

During the simulation period the RSV-GS and peptide positions on the binding pocket was kept constant with slight variations in the conformation as expected Water molecules established hydrogen interactions with polar groups on both RSV-GS and peptide (Fig 7A B C and D) By calculating the RMSD values of the RSV-GS backbone heavy atoms relative to the coordinates of the initial (energy-minimized) structure the overall changes in RSV-GS atomic coordinates were monitored during MD simulations in water (Fig 6E) After an initial fast-rise region the RMSD values changed little reaching a more constant value after approximately 1 ns of simulation

Figure 3Effects of RSV and derivatives in metabolic activity of HaCaT cells after oxidative stress Cells were incubated with 25 50 100 350 and 350 microM RSV or RSV-G or with 100 350 500 1000 and 5000 microM RSV-GS for 24 h followed by incubation with hydrogen peroxide (H2O2) and menadione for 3 h Metabolic viability of HaCaT cells incubated with RSV (A and B) RV-G (C and D) and RSV-GS (E and F) were obtained by resazurin assay as described in lsquoMaterials and methodsrsquo section Data are expressed as mean plusmn sd of 4 independent experiments For white bars stands for P lt 005 vs control stands for P lt 001 vs control and stands for P lt 0001 vs control For black bars $stands for P lt 005 vs control $$stands for P lt 001 vs control and $$$$stands for P lt 00001 vs control For the comparison between the two groups stands for P lt 005 vs control group and stands for P lt 0001 vs control group

(27 Aring) indicating the validity of the complex in solution and at human body temperature (300 K) The system was then cooled for 100 ps to get stable energies (minus12 times 103 kcalmol) (Fig 7D and E) For RSV the RMSD reached an equilibrium at 1 ns (12 Aring) and the system arrived to a minimum energy of minus6151 kcalmol at the end of the MD

simulation (Supplementary data and Fig 3) Therefore SIRT1peptideRSV-GS was more stable in water than SIRT1peptideRSV and the RSV-GS conformation in the cavity of SIRT1 suffered less significant variations than RSV (lower RMSD) during the period of the simulation

Discussion

The physicochemical properties of RSV have restricted its use in topical products One particular challenge is the poor water solubility as confirmed in this work (0029 plusmn 0005 mgmL) that prevents the use of high concentrations of pure RSV into topical formulations In fact the leading topical RSV products often contain less than 1 of pure RSV (Farris et al 2013) The synthetic persulfated resveratrol glucoside described in this work was found to be around 1000-fold more soluble in water than RSV Topical formulations comprise a wide array of physical systems being oil in water creams one of the most used for which water is the external phase and the prominent ingredient in their composition This favors the inclusion of water-soluble compounds in meaningful concentrations regarding biological activity and a hydrophilic RSV derivative represents a clear advantage over the parent molecule The improvement in RSV penetration has been studied with several approaches including formulation design and the use of pro-molecules (Zhang et al 2007 Hung et al 2008) Enhancement of RSV bioavailability in the epidermis upon application of a water-soluble pro-molecule has been reported (Zhang et al 2007)

Toxicological and protective testing in a human keratinocyte cell line is a logical approach for compounds intended for skin care application Remarkable differences were observed between the cytotoxic effects of RSV-GS and RSV Resveratrol decreased the cell viability more than 50 at 500 microM while RSV-GS did not decrease the cell viability for more than 30 even at 5000 microM However the non-sulfated RSV-G was also much less toxic than RSV

Figure 4Effects of RSV and derivatives in ATP levels of HaCaT cells in oxidative stress models Cells were incubated with 25 50 100 350 and 350 microM RSV or RSV-G or with 100 350 500 1000 and 5000 microM RSV-GS for 24 h followed by incubation with hydrogen peroxide (H2O2) and menadione for 3 h Intracellular ATP levels of HaCaT cells incubated with RSV (A and B) RSV-G (C and D) and RSV-GS (E and F) were obtained by CellTiter-Glo Luminescent Cell Viability Assay respectively as described in materials and methods section Data are expressed as mean plusmn sd of 4 independent experiments For white bars stands for P lt 00001 vs control For black bars $$$$stands for P lt 00001 vs control For the comparison between the two groups stands for P lt 00001 vs control group

Figure 5RSV and RSV-GS increased SIRT1 protein content Cells were incubated with 25 and 100 microM RSV or RSV-G and with 100 and 500 microM RSV-GS for 24 h Basal sirtuin 1 content after RSV or derivatives treatment was semi-quantified by Western blotting analysis (A and B) Blots are representative from four different cell preparations Data are expressed as mean plusmn sd of 4 different experiments P lt 005 denotes a significant difference from control

(in contrast to RSV no decrease in cell viability was observed in the presence of 500 microM of RSV-G) It is possible to argue that while glycosylation had guaranteed the non-toxicity of RSV sulfation guaranteed both water solubility and non-toxicity Besides water solubility this adds another advantage to RSV-GS the possibility of use RSV-GS in higher concentrations on topical products than RSV

Some similarities between the protective effects of RSV-GS and RSV against well-characterized oxidative stress inducers were observed Interestingly enough RSV-GS and RSV (the positive control) did not prevent H2O2- and menadione-induced decrease metabolic activity however RSV at 25 and 50 microM protected from ATP depletion induced by H2O2 while RSV-GS did not Despite some authors have shown in cells the protective effects of RSV against H2O2-induced toxicity (Ovesna et al 2006) some authors also described the opposite effect (Fabre et al 2011) anticipating that RSV and RSV-GS effects are cell type and dose dependent and that studies involving different variable stressors and incubation times make different studies hard to compare Furthermore it was also described that RSV at 50 microM increased the susceptibility to stressors (Fabre et al 2011) hence the use of resveratrol as a positive control does not always represent a universal assumption This is in accordance with the results obtained where RSV

but not RSV-GS increased menadione-induced toxicity Nevertheless it is known that RSV induces cell cycle arrest and that cells in S-phase are significantly more susceptible to H2O2 than in other phases of the cell cycle (Leroy et al 2001 Frankenberg-Schwager et al 2008) which can explain the obtained results Moreover the increase in ATP levels observed after 5 mM RSV-GS might be a metabolic adaptive response which precedes programmed cell death (Los et al 2002)

Nevertheless we also demonstrated here that RSV and RSV-GS increased SIRT1 protein content which is in agreement with others reports (Price et al 2012 Deus et al 2017) Sirtuin-activating compounds such as RSV-related polyphenols can promote survival of human cells although their mechanism is not completely understood (Chung et al 2010 Jayasena et al 2013) To clarify whether the studied molecules directly interact with SIRT1 in silico studies in this work have predicted that RSV-GS establishes polar interactions with eight SIRT1 residues

Figure 6(A) Surface representation for SIRT1 (pdb code 5BTR) bound to crystallographic substrate peptide (dark gray sticks) and RSV (black sticks) and docked RSV (white sticks) and RSV-GS (light gray) (B) Detailed view of RSV-GS (light gray) on the binding site (C) Detailed view of crystallographic RSV (black sticks) and docked RSV (white sticks) on the binding site Crystallographic peptide substrate is not shown for easier interpretation Polar interactions are represented as broken lines Relevant amino acids are labeled andor represented in capped sticks

Figure 7Snapshots of the geometries of SIRT1peptideRSV-GS complexes obtained from the MD runs in water at (A) 100 (B) 700 (C) 1400 and (D) 2200 ps SIRT1 is represented as a surface peptide and RSV-GS are shown in dark gray and light gray sticks respectively for simplification only the oxygen (small sphere) of the water molecules are represented hydrogen interactions established between peptide or RSV-GS and water molecules are depicted with broken lines (E) RMSD graph of backbone atoms of RSV-GS in the complex as a function of time and potential energy plot during MD simulation

whereas RSV establishes only two polar interactions At this point the extent of hydrolysis by sulfatases in the skin is uncertain However even if biotransformation of RSV-GS partially occurs the effectiveness is not expected to be compromised since binding to SIRT1 was predicted to be more efficient and high concentrations of RSV-GS will be possible to incorporate on topical products

Supplementary dataThis is linked to the online version of the paper at httpsdoiorg101530JME-18-0031

Declaration of interestThe authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported

FundingThis work was supported through national funds provided by Foundation for Science and Technology from the Minister of Science Technology and Higher Education (FCTMCTES ndash PIDDAC) and European Regional Development Fund (ERDF) through the COMPETE ndash Programa Operacional Factores de Competitividade (POFC) (POCI-01-0145-FEDER- 016659007440 POCI-01-0145-FEDER-016790 and POCI-01-0145-FEDER-016793) PPCDT ndash Promover a Produccedilatildeo Cientiacutefica e Desenvolvimento Tecnoloacutegico e a Constituiccedilatildeo de Redes Temaacuteticas (Project 3599) RIDT ndash Reforccedilar a Investigaccedilatildeo o Desenvolvimento Tecnoloacutegico e a Inovaccedilatildeo (Project 9471) under the projects PTDCDTP-FTO24332014 PTDCMAR-BIO46942014 and PTDCAAG-TEC07392014 in the framework of the programme PT2020

Author contribution statementM C S synthesized the compound and determined its physicochemical properties and E S and M P analyzed the data V R performed the cytotoxicity analysis with HaCaT cells and I A and C M analyzed the data A P performed the in silico studies C M D A M C and P J O performed measurements of protective effects of RSV and derivatives against stressors and Western blotting analysis M C S V R and A P wrote the manuscript while all authors gave significant contributions in discussion and revision E S and I A conceived the study design

ReferencesAlbuquerque RV Malcher NS Amado LL Coleman MD dos Santos DC

Borges RS Valente SAS Valente VC amp Monteiro MC 2015 In vitro protective effect and antioxidant mechanism of resveratrol induced by dapsone hydroxylamine in human cells PLos ONE 10 e0134768 (httpsdoiorg101371journalpone0134768)

Amri A Chaumeil JC Sfar S amp Charrueau C 2012 Administration of resveratrol what formulation solutions to bioavailability limitations Journal of Controlled Release 158 182ndash193 (httpsdoiorg101016jjconrel201109083)

Baur JA amp Sinclair DA 2006 Therapeutic potential of resveratrol the in vivo evidence Nature Reviews Drug Discovery 5 493ndash506 (httpsdoiorg101038nrd2060)

Baxter RA 2008 Anti-aging properties of resveratrol review and report of a potent new antioxidant skin care formulation Journal of Cosmetic Dermatology 7 2ndash7 (httpsdoiorg101111j1473-2165200800354x)

Blander G Bhimavarapu A Mammone T Maes D Elliston K Reich C Matsui MS Guarente L amp Loureiro JJ 2009 SIRT1 promotes differentiation of normal human keratinocytes Journal of Investigative Dermatology 129 41ndash49 (httpsdoiorg101038jid2008179)

Boukamp P Petrussevska RT Breitkreutz D Hornung J Markham A amp Fusenig NE 1988 Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line Journal of Cell Biology 106 761ndash771 (httpsdoiorg101083jcb1063761)

Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Analytical Biochemistry 72 248ndash254 (httpsdoiorg1010160003-2697(76)90527-3)

Cao C Lu S Kivlin R Wallin B Card E Bagdasarian A Tamakloe T Wang WJ Song X Chu WM et al 2009 SIRT1 confers protection against UVB- and H2O2-induced cell death via modulation of p53 and JNK in cultured skin keratinocytes Journal of Cellular and Molecular Medicine 13 3632ndash3643 (httpsdoiorg101111j1582-4934200800453x)

Cao D Wang M Qiu X Liu D Jiang H Yang N amp Xu R-M 2015 Structural basis for allosteric substrate-dependent stimulation of SIRT1 activity by resveratrol Genes and Development 29 1316ndash1325 (httpsdoiorg101101gad265462115)

Carlotti ME Sapino S Ugazio E Gallarate M amp Morel S 2012 Resveratrol in solid lipid nanoparticles Journal of Dispersion Science and Technology 33 465ndash471 (httpsdoiorg101080019326912010548274)

Cheng A Best SA Merz KM Jr amp Reynolds CH 2000 GBSA water model for the Merck molecular force field (MMFF) Journal of Molecular Graphics and Modelling 18 273ndash282 (httpsdoiorg101016S1093-3263(00)00038-3)

Chung S Yao H Caito S Hwang JW Arunachalam G amp Rahman I 2010 Regulation of SIRT1 in cellular functions role of polyphenols Archives of Biochemistry and Biophysics 501 79ndash90 (httpsdoiorg101016jabb201005003)

Correia-da-Silva M Sousa E Duarte B Marques F Cunha-Ribeiro LM amp Pinto MM 2011 Dual anticoagulantantiplatelet persulfated small molecules European Journal of Medicinal Chemistry 46 2347ndash2358 (httpsdoiorg101016jejmech201103016)

Correia-da-Silva M Sousa E amp Pinto MM 2014 Emerging sulfated flavonoids and other polyphenols as drugs nature as an inspiration Medicinal Research Reviews 34 223ndash279 (httpsdoiorg101002med21282)

Criddle DN Gillies S Baumgartner-Wilson HK Jaffar M Chinje EC Passmore S Chvanov M Barrow S Gerasimenko OV Tepikin AV et al 2006 Menadione-induced reactive oxygen species generation via redox cycling promotes apoptosis of murine pancreatic acinar cells Journal of Biological Chemistry 281 40485ndash40492 (httpsdoiorg101074jbcM607704200)

Dai H Case AW Riera TV Considine T Lee JE Hamuro Y Zhao H Jiang Y Sweitzer SM Pietrak B et al 2015 Crystallographic structure of a small molecule SIRT1 activator-enzyme complex Nature Communications 6 7645 (httpsdoiorg101038ncomms8645)

Detoni CB Souto GD da Silva AL Pohlmann AR amp Guterres SS 2012 Photostability and skin penetration of different E-resveratrol-loaded supramolecular structures Photochemistry and Photobiology 88 913ndash921 (httpsdoiorg101111j1751-1097201201147x)

Deus CM Serafim TL Magalhaes-Novais S Vilaca A Moreira AC Sardao VA Cardoso SM amp Oliveira PJ 2017 Sirtuin 1-dependent resveratrol cytotoxicity and pro-differentiation activity on breast cancer cells Archives of Toxicology 91 1261ndash1278 (httpsdoiorg101007s00204-016-1784-x)

Fabre KM Saito K DeGraff W Sowers AL Thetford A Cook JA Krishna MC amp Mitchell JB 2011 The effects of resveratrol and selected metabolites on the radiation and antioxidant response Cancer Biology and Therapy 12 915ndash923 (httpsdoiorg104161cbt121017714)

Farris P Krutmann J Li YH McDaniel D amp Krol Y 2013 Resveratrol a unique antioxidant offering a multi-mechanistic approach for treating aging skin Journal of Drugs in Dermatology 12 1389ndash1394

Frankenberg-Schwager M Becker M Garg I Pralle E Wolf H amp Frankenberg D 2008 The role of nonhomologous DNA end joining conservative homologous recombination and single-strand annealing in the cell cycle-dependent repair of DNA double-strand breaks induced by H(2)O(2) in mammalian cells Radiation Research 170 784ndash793 (httpsdoiorg101667RR13751)

Froimowitz M 1993 HyperChem a software package for computational chemistry and molecular modeling Biotechniques 14 1010ndash1013

Fukui M Choi HJ amp Zhu BT 2010 Mechanism for the protective effect of resveratrol against oxidative stress-induced neuronal death Free Radical Biology and Medicine 49 800ndash813 (httpsdoiorg101016jfreeradbiomed201006002)

Gertz M Nguyen GT Fischer F Suenkel B Schlicker C Franzel B Tomaschewski J Aladini F Becker C Wolters D et al 2012 A molecular mechanism for direct sirtuin activation by resveratrol PLoS ONE 7 e49761 (httpsdoiorg101371journalpone0049761)

Hevener KE Zhao W Ball DM Babaoglu K Qi J White SW amp Lee RE 2009 Validation of molecular docking programs for virtual screening against dihydropteroate synthase Journal of Chemical Information and Modeling 49 444ndash460 (httpsdoiorg101021ci800293n)

Hoshino J Park E-J Kondratyuk TP Marler L Pezzuto JM van Breemen RB Mo S Li Y amp Cushman M 2010 Selective synthesis and biological evaluation of sulfate-conjugated resveratrol metabolites Journal of Medicinal Chemistry 53 5033ndash5043 (httpsdoiorg101021jm100274c)

Hospital A Goni JR Orozco M amp Gelpi JL 2015 Molecular dynamics simulations advances and applications Advances and Applications in Bioinformatics and Chemistry 8 37ndash47 (httpsdoiorg102147AABCS70333)

Hung CF Lin YK Huang ZR amp Fang JY 2008 Delivery of resveratrol a red wine polyphenol from solutions and hydrogels via the skin Biological and Pharmaceutical Bulletin 31 955ndash962 (httpsdoiorg101248bpb31955)

Jayasena T Poljak A Smythe G Braidy N Munch G amp Sachdev P 2013 The role of polyphenols in the modulation of sirtuins and other pathways involved in Alzheimerrsquos disease Ageing Research Reviews 12 867ndash883 (httpsdoiorg101016jarr201306003)

Karplus M amp McCammon JA 2002 Molecular dynamics simulations of biomolecules Nature Structural Biology 9 646ndash652 (httpsdoiorg101038nsb0902-646)

Kasiotis KM Pratsinis H Kletsas D amp Haroutounian SA 2013 Resveratrol and related stilbenes their anti-aging and anti-angiogenic properties Food and Chemical Toxicology 61 112ndash120 (httpsdoiorg101016jfct201303038)

Lavu S Boss O Elliott PJ amp Lambert PD 2008 Sirtuinsndashnovel therapeutic targets to treat age-associated diseases Nature Reviews Drug Discovery 7 841ndash853 (httpsdoiorg101038nrd2665)

Lepak A Gutmann A Kulmer ST amp Nidetzky B 2015 Creating a water-soluble resveratrol-based antioxidant by site-selective enzymatic glucosylation Chembiochem 16 1870ndash1874 (httpsdoiorg101002cbic201500284)

Leroy C Mann C amp Marsolier MC 2001 Silent repair accounts for cell cycle specificity in the signaling of oxidative DNA lesions Yeast 18 S37ndashS37 (httpsdoiorg101093emboj20112896)

Lill MA amp Danielson ML 2011 Computer-aided drug design platform using PyMOL Journal of Computer-Aided Molecular Design 25 13ndash19 (httpsdoiorg101007s10822-010-9395-8)

Loor G Kondapalli J Schriewer JM Chandel NS Vanden Hoek TL amp Schumacker PT 2010 Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis Free Radical Biology and Medicine 49 1925ndash1936 (httpsdoiorg101016jfreeradbiomed201009021)

Los M Mozoluk M Ferrari D Stepczynska A Stroh C Renz A Herceg Z Wang ZQ amp Schulze-Osthoff K 2002 Activation and caspase-mediated inhibition of PARP a molecular switch between fibroblast necrosis and apoptosis in death receptor signaling Molecular Biology of the Cell 13 978ndash988 (httpsdoiorg101091mbc01-05-0272)

Matsuura BS Keylor MH Li B Lin Y Allison S Pratt DA amp Stephenson CRJ 2015 A scalable biomimetic synthesis of resveratrol dimers and systematic evaluation of their antioxidant activities Angewandte Chemie International Edition 54 3754ndash3757 (httpsdoiorg101002anie201409773)

Mattarei A Azzolini M Carraro M Sassi N Zoratti M Paradisi C amp Biasutto L 2013 Acetal derivatives as prodrugs of resveratrol Molecular Pharmaceutics 10 2781ndash2792 (httpsdoiorg101021mp400226p)

Medina I Alcaacutentara D Gonzaacutelez MJ Torres P Lucas R Roque J Plou FJ amp Morales JC 2010 Antioxidant activity of resveratrol in several fish lipid matrices effect of acylation and glucosylation Journal of Agricultural and Food Chemistry 58 9778ndash9786 (httpsdoiorg101021jf101472n)

Mortier J Rakers C Bermudez M Murgueitio MS Riniker S amp Wolber G 2015 The impact of molecular dynamics on drug design applications for the characterization of ligand-macromolecule complexes Drug Discovery Today 20 686ndash702 (httpsdoiorg101016jdrudis201501003)

Mukherjee SG OrsquoClaonadh N Casey A amp Chambers G 2012 Comparative in vitro cytotoxicity study of silver nanoparticle on two mammalian cell lines Toxicology in Vitro 26 238ndash251 (httpsdoiorg 101016jtiv201112004)

Ndiaye M Philippe C Mukhtar H amp Ahmad N 2011 The grape antioxidant resveratrol for skin disorders promise prospects and challenges Archives of Biochemistry and Biophysics 508 164ndash170 (httpsdoiorg101016jabb201012030)

Ovesna Z Kozics K Bader Y Saiko P Handler N Erker T amp Szekere T 2006 Antioxidant activity of resveratrol piceatannol and 33prime44prime55prime-hexahydroxy-trans-stilbene in three leukemia cell lines Oncology Reports 16 617ndash624 (httpsdoiorg103892or163617)

Park H Kim J Choi KH Hwang S Yang SJ Baek NI amp Cha J 2012 Enzymatic synthesis of piceid glucosides using maltosyltransferase from caldicellulosiruptor bescii DSM 6725 Journal of Agricultural and Food Chemistry 60 8183ndash8189 (httpsdoiorg101021jf302127a)

Park J Park JH Suh HJ Lee IC Koh J amp Boo YC 2014 Effects of resveratrol oxyresveratrol and their acetylated derivatives on cellular melanogenesis Archives of Dermatological Research 306 475ndash487 (httpsdoiorg101007s00403-014-1440-3)

Price NL Gomes AP Ling AJY Duarte FV Martin-Montalvo A North BJ Agarwal B Ye L Ramadori G Teodoro JS et al 2012 SIRT1 is required for AMPK activation and the beneficial effects of resveratrol on mitochondrial function Cell Metabolism 15 675ndash690 (httpsdoiorg101016jcmet201204003)

Regev-Shoshani G Shoseyov O Bilkis I amp Kerem Z 2003 Glycosylation of resveratrol protects it from enzymic oxidation Biochemical Journal 374 157ndash163 (httpsdoiorg101042bj20030141)

Reis AC Alessandri AL Athayde RM Perez DA Vago JP Avila TV Ferreira TPT de Arantes ACS Coutinho DD Rachid MA et al 2015 Induction of eosinophil apoptosis by hydrogen peroxide promotes the resolution of allergic inflammation Cell Death and Disease 6 e1632 (httpsdoiorg101038cddis2014580)

Rocha V Marques C Figueiredo JL Gaio AR Costa PC Sousa Lobo JM amp Almeida IF 2017 In vitro cytotoxicity evaluation of resveratrol-loaded nanoparticles focus on the challenges of in vitro

methodologies Food and Chemical Toxicology 103 214ndash222 (httpsdoiorg101016jfct201703017)

Sanna V Roggio AM Siliani S Piccinini M Marceddu S Mariani A amp Sechi M 2012 Development of novel cationic chitosan-and anionic alginate-coated poly(DL-lactide-co-glycolide) nanoparticles for controlled release and light protection of resveratrol International Journal of Nanomedicine 7 5501ndash5516 (httpsdoiorg102147IJNS36684)

Sauve AA 2010 Sirtuin chemical mechanisms Biochimica et Biophysica Acta 1804 1591ndash1603 (httpsdoiorg101016jbbapap201001021)

Seeliger D amp de Groot BL 2010 Ligand docking and binding site analysis with PyMOL and AutodockVina Journal of Computer-Aided Molecular Design 24 417ndash422 (httpsdoiorg101007s10822-010-9352-6)

Shih J amp Donmez G 2013 Mitochondrial sirtuins as therapeutic targets for age-related disorders Genes and Cancer 4 91ndash96 (httpsdoiorg1011771947601912474931)

Silva FS Starostina IG Ivanova VV Rizvanov AA Oliveira PJ amp Pereira SP 2016 Determination of metabolic viability and cell mass using a tandem resazurinsulforhodamine B assay Current Protocols in Toxicology 68 2241ndash22415 (httpsdoiorg101002cptx1)

Technical Committee ISOTC 194 2009 ISO 10993-52009 biological evaluation of medical devices ndash part 5 tests for in vitro cytotoxicity

Sturgeon JB amp Laird BB 2000 Symplectic algorithm for constant-pressure molecular dynamics using a nose-poincare thermostat Journal of Chemical Physics 112 3474ndash3482 (httpsdoiorg1010631480502)

Summerlin N Soo E Thakur S Qu Z Jambhrunkar S amp Popat A 2015 Resveratrol nanoformulations challenges and opportunities International Journal of Pharmaceutics 479 282ndash290 (httpsdoiorg101016jijpharm201501003)

Swiss R Niles A Cali JJ Nadanaciva S amp Will Y 2013 Validation of a HTS-amenable assay to detect drug-induced mitochondrial toxicity

in the absence and presence of cell death Toxicology In Vitro 27(6)1789ndash1797 (httpsdoi 101016jtiv201305007)

Tang PC Ng YF Ho S Gyda M amp Chan SW 2014 Resveratrol and cardiovascular health ndash promising therapeutic or hopeless illusion Pharmacological Research 90 88ndash115 (httpsdoiorg101016jphrs201408001)

Trott O amp Olson AJ 2010 AutoDock Vina improving the speed and accuracy of docking with a new scoring function efficient optimization and multithreading Journal of Computational Chemistry 31 455ndash461 (httpsdoiorg101002jcc21334)

Vichai V amp Kirtikara K 2006 Sulforhodamine B colorimetric assay for cytotoxicity screening Nature Protocols 1 1112ndash1116 (httpsdoiorg101038nprot2006179)

Vilar S Cozza G amp Moro S 2008 Medicinal chemistry and the molecular operating environment (MOE) application of QSAR and molecular docking to drug discovery Current Topics in Medicinal Chemistry 8 1555ndash1572 (httpsdoiorg102174156802608786786624)

Wu Z Uchi H Morino-Koga S Shi W amp Furue M 2014 Resveratrol inhibition of human keratinocyte proliferation via SIRT1ARNTERK dependent downregulation of aquaporin 3 Journal of Dermatological Science 75 16ndash23 (httpsdoiorg101016jjdermsci201403004)

Xiang JM Wan CY Guo R amp Guo DZ 2016 Is hydrogen peroxide a suitable apoptosis inducer for all cell types Biomed Research International 2016 6 (httpsdoiorg10115520167343965)

Zhang L Zhou W amp Li D-H 2006 A descent modified PolakndashRibiegraverendashPolyak conjugate gradient method and its global convergence IMA Journal of Numerical Analysis 26 629ndash640 (httpsdoiorg101093imanumdrl016)

Zhang G Flach CR amp Mendelsohn R 2007 Tracking the dephosphorylation of resveratrol triphosphate in skin by confocal Raman microscopy Journal of Controlled Release 123 141ndash147 (httpsdoiorg101016jjconrel200708001)

Received in final form 14 March 2018Accepted 27 March 2018

Abstract

Introduction

Reagents





MTT reduction assay

Neutral red uptake assay

Trypan blue exclusion assay

Measurement of cell protection against oxidative stress

Sulforhodamine B (SRB) assay

Alamar Blueresazurin assay

ATP levels determination

Protein semi-quantification by western blotting

Docking study



Results





Discussion

Supplementary data

Declaration of interest

Funding

Author contribution statement

References

is a phytoalexin (Amri et al 2012 Tang et al 2014) The most abundant form of RSV in nature is resveratrol 3-O-β-D-glucoside (RSV-G) (Regev-Shoshani et al 2003) also called piceid or polydatin

Several studies demonstrated that RSV has interesting properties regarding skin care (Baxter 2008 Ndiaye et al 2011 Park et al 2014) The biological effects of RSV are supported by mechanistic data that indicates the involvement of increased expression and activation of the protein deacetylase sirtuin 1 (SIRT1) (Baur amp Sinclair 2006 Price et al 2012) Sirtuin 1 is expressed in skin keratinocytes (Blander et al 2009) Resveratrol acting as a SIRT1 activator protects the skin against oxidative stress events and consequently prevents skin cell damage (Cao et al 2009)

Resveratrol occurs in cis-(Z) and trans-(E) isomeric forms in nature trans-resveratrol is the most common and biologically active isomeric form as well as the most photo- and thermal-stable isomer (Baxter 2008 Carlotti et al 2012) Fast trans-cis isomerization (Detoni et al 2012 Sanna et al 2012) and poor water solubility (Lepak et al 2015) limits RSV efficacy and constrains the design of topical formulations Besides classical formulation development (Amri et al 2012 Summerlin et al 2015) chemical conjugations such as sulfation (Hoshino et al 2010) glycosylation (Regev-Shoshani et al 2003) oligomerization (Matsuura et al 2015) and other derivations (Regev-Shoshani et al 2003 Medina et al 2010 Mattarei et al 2013) are being employed to overcome these limitations

Synthetic RSV derivatives with improved water solubility may be obtained with similar or better activities toward SIRT1 activation In this work the validation of a synthetic water-soluble RSV derivative (resveratrol glucoside sulfate RSV-GS) for skin care was evaluated and compared with the parent compounds RSV and RSV-G by studying the cytotoxic and protective effects (lysosomal integrity cell membrane damage and metabolic activity) and the alterations of SIRT1 protein content using a human keratinocyte cell line and performing in silico affinity studies for SIRT1

Reagents

Dulbeccorsquos Modified Eaglesrsquos Medium (DMEM) with 25 mM d-glucose and 1 mM pyruvate inactivated fetal bovine serum (FBS) penicillin-streptomycin solution Dulbeccorsquos phosphate buffered saline (DPBS) without

calcium chloride and magnesium chloride 025 trypsin-ethylenediaminetetraacetic acid (EDTA) solution were supplied by Gibco by Life Technologies Dimethyl sulfoxide (DMSO) 04 trypan blue solution neutral red (NR) solution and resazurin sodium salt were purchased from Sigma-Aldrich AlamarBlue was provided by Thermo Scientific 3-[45-Dimethyl-2-yl]-25-diphenyl tetrazolium bromide (MTT) was acquired from Life Technologies Sirtuin-1 and actin antibodies were obtained from Abcam and Millipore respectively In all experiments purified water was used and obtained using a Direct-Q Water Purification System (Merck Millipore) with a reverse osmosis process trans-Resveratrol was purchased from Fragon (Satildeo Paulo Brazil) trans-resveratrol 3-szlig-D-glucopyranoside (572691) and triethylamine sulfur trioxide adduct (S 5139) were purchased from Sigma-Aldrich UVVis spectra were traced with a synergy HT microplate reader from BioTek with GEN5 software Melting point was obtained in a Koumlfler microscope and was not corrected Infrared (IR) spectra were recorded on a Nicolet iS10 from Thermo Scientific with Smart OMNI-Transmission accessory (Software 188 OMNIC 83) in KBr Nuclear magnetic resonance (NMR) spectra were taken in DMSO-d6 at room temperature on Bruker AMC AVANCE 300 instrument (Bruker Biosciences Corporation Billerica MA USA) instrument High-resolution mass spectrometry (HRMS) results were obtained in CACTI services Vigo Spain

A mixture of trans-resveratrol 3-szlig-D-glucopyranoside (05 g 128 mmol) and triethylamine sulfur trioxide adduct (4 equivOH) in dimethylacetamide (10 mL) was stirring and heated for 30 min at 100degC under MW irradiation (200 W) After cooling the mixture was poured into acetone (200 mL) under basic conditions (triethylamine) and left at 4degC for 24 h The formed crude oil was washed with ether dissolved in a minimum volume of water and applied on a DOWEX cation exchange resin (Na+ form) After evaporation until dryness a light brown solid was obtained (85 yield) NMR and HRMS were according to previously reported data (Correia-da-Silva et al 2011)

The solubility was tested at the maximum solubility class of the European Pharmacopeia (very soluble gt1000 mgmL) by dissolving a given amount in ultra-pure water (UPW) (RSV and RSV-G 2 mg in 200 mL RSV-GS 50 mg in

Docking study

Results

Chemicalstructure

Cytotoxicity assays

Discussion

Abstract

Introduction

Reagents





MTT reduction assay








Docking study



Results





Discussion

Supplementary data


Funding


References

Docking study

Results

Chemicalstructure

Cytotoxicity assays

Discussion

Abstract

Introduction

Reagents





MTT reduction assay








Docking study



Results





Discussion

Supplementary data


Funding


References

Docking study

Results

Chemicalstructure

Cytotoxicity assays

Discussion

Abstract

Introduction

Reagents





MTT reduction assay








Docking study



Results





Discussion

Supplementary data


Funding


References

Results

Chemicalstructure

Cytotoxicity assays

Discussion

Abstract

Introduction

Reagents





MTT reduction assay








Docking study



Results





Discussion

Supplementary data


Funding


References

Cytotoxicity assays

Discussion

Abstract

Introduction

Reagents





MTT reduction assay








Docking study



Results





Discussion

Supplementary data


Funding


References

Cytotoxicity assays

Discussion

Abstract

Introduction

Reagents





MTT reduction assay








Docking study



Results





Discussion

Supplementary data


Funding


References

Discussion

Abstract

Introduction

Reagents





MTT reduction assay








Docking study



Results





Discussion

Supplementary data


Funding


References

Discussion

Abstract

Introduction

Reagents





MTT reduction assay








Docking study



Results





Discussion

Supplementary data


Funding


References

Abstract

Introduction

Reagents





MTT reduction assay








Docking study



Results





Discussion

Supplementary data


Funding


References

Abstract

Introduction

Reagents





MTT reduction assay








Docking study



Results





Discussion

Supplementary data


Funding


References

Abstract

Introduction

Reagents





MTT reduction assay








Docking study



Results





Discussion

Supplementary data


Funding


References

Abstract

Introduction

Reagents





MTT reduction assay








Docking study



Results





Discussion

Supplementary data


Funding


References

Documents

SULFATION PATHWAYS Potential benefits of a sulfated