Neuroprotective and Anti-inflammatory Effects of Pterostilbene Metabolites in Human Neuroblastoma SH-SY5Y and RAW 264.7 Macrophage Cells

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ "This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of Agricultural and Food Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.jafc.9b07147." 1 Neuroprotective and anti-inflammatory effects of pterostilbene 1 metabolites in human neuroblastoma SH-SY5Y and RAW 264.7 2 macrophage cells 3 4 Pablo Peñalver,†,* Sonia Zodio,† Ricardo Lucas,‡ María Violante de-Paz‡ and Juan Carlos 5 Morales†,* 6 7 Affiliations 8 †Department of Biochemistry and Molecular Pharmacology, Instituto de Parasitología y 9 Biomedicina López Neyra, CSIC, PTS Granada, Avda. del Conocimiento, 17, 18016 Armilla, 10 Granada, Spain. 11 ‡Department of Organic and Pharmaceutical Chemistry, School of Pharmacy, University of 12 Seville, c/ Prof. García González, 2, 41012, Seville, Spain. 13 14 15 16 17 18 19 20 21 22 23 24 25 KEYWORDS 26 Polyphenols, neuroprotective effect, inflammation, metabolites. 27 2 ABSTRACT 28 Oxidative stress is known to be a key factor in many neurodegenerative diseases. Inflammation 29 also plays a relevant role in a myriad of pathologies such as diabetes and atherosclerosis. 30 Polyphenols coming from dietary sources, such as pterostilbene, may be beneficial in this type of 31 diseases. However, most of them are rapidly metabolized and excreted yielding very low phenolic 32 bioavailability what makes difficult to find out which are the mechanisms responsible for the 33 observed bioactivity. Herein, we evaluate the effects of pterostilbene and its metabolites against 34 H2O2-induced cell damage in human neuroblastoma SH-SY5Y cells and against LPS-challenged 35 RAW 264.7 macrophages. Among the metabolites tested, 3-methyl-4´-glucuronate-resveratrol 36 (also called 4’-glucuronate pinostilbene, PIN-GlcAc, 11) prevented neuronal death via attenuation 37 of ROS levels and increased REDOX activity in neurons. This compound is also able to 38 ameliorate LPS-mediated inflammation on macrophages via inhibition of IL-6 and NO 39 production. Thus, polyphenol from dietary sources could be part of potential functional foods 40 designed to ameliorate the onset and progression of certain neurodegenerative diseases via 41 oxidative stress reduction. 42 43 44 45 46 47 48 49 50 51 52 53 54 55 3 INTRODUCTION 56 Oxidative stress is known to be a relevant factor in the progress of neurodegenerative diseases 57 such as Alzheimer’s disease, Parkinson’s disease or ischemia.1 Stress is caused by a disparity 58 between the reactive oxygen species (ROS) and reactive nitrogen species (RNS) generated, and 59 the endogenous antioxidants present in cells.2 ROS are usually detected in apoptotic cell death as 60 well as in neurodegenerative processes in the brain.3 61 Inflammation is the defense mechanism of the human body to injurious stimuli, such as tissue 62 harm, trauma, or infection. It is mediated by activated immune cells such as monocytes and 63 macrophages.4 Chronic inflammation is related to a wide variety of diseases such as asthma, 64 psoriasis, obesity, atherosclerosis or cancer.5 Thus, clinical strategies to fight chronic 65 inflammation and related diseases like cancer include blocking theinflammatory responses and 66 the inhibition of proinflammatory mediator production. 67 Inflammation is mediated by inflammatory cytokines such as interleukins IL-6 and IL-1β, tumor 68 necrosis factor (TNF)-α, interferon (IFN)-γ and by inflammatory mediators, such as nitric oxide 69 (NO) and prostaglandin E2 (PGE2).6 The main pro-inflammatory stimuli comprise cytokines, UV 70 irradiation, mitogens and bacterial lipopolysaccharide (LPS) which modulate their effects by 71 inducing the activation of transcription factor NFκB.7-8 72 Dietary polyphenols have been reported to display anti-apoptotic, anti-inflammatory and 73 antioxidant properties (preventing proteins oxidation, lipid peroxidation, and ROS generation, 74 having thus a potential neuroprotective effect). In fact, these effects have been observed on in 75 vitro and in vivo models of toxicity and neurological disorders.9-10 Moreover, polyphenols from 76 red wine, nuts berries and other fruits, such as resveratrol (RES), pterostilbene (PTER) and 77 ellagitannins (ETs), have proven to exert neuroprotective effects on various animal models of 78 neurodegeneration.11-14 Nonetheless, dietary source polyphenols show poor bioavailability due to 79 an extensive metabolism by colonic microbiota and through phase II metabolism in the liver,15 80 preventing them from reaching systemic tissues.9, 16 81 4 Therefore, the compounds really responsible for the in vivo neuroprotective and antioxidant 82 effects of polyphenols are not entirely known. While dietary polyphenols show poor 83 bioavailability, their metabolites can actually reach the brain,17-22 and the human bloodstream.23- 84 24 So, phenolic metabolites should be taken into account as potential candidates to exert the 85 biological activity attributed to the polyphenolic dietary precursors. In fact, protocatechuic Acid, 86 a major metabolite of anthocyanins found in green tea, wine and berries, has been reported to 87 prevent PC12 neuronal cells death caused by amyloid-β toxicity.25 Similarly, Gonzalez-Sarrías et 88 al. showed neuroprotective activity of physiologically relevant polyphenol-derived metabolites 89 such as gallic acid, ellagic acid (EA), 3,4-dihydroxyphenylpropionic acid and 3,4- 90 dihydroxyphenylacetic acid in a H2O2-challenged SH-SY5Y cells model.26 91 92 Pterostilbene (PTER) is a naturally occurring stilbenoid, analogue of RES, mainly found in 93 blueberries. PTER has shown improved oral bioavailability and potency in comparison with 94 RES.27-28 The antioxidant, anti-inflammatory, and anticancer activity of PTER has been deeply 95 studied in the literature.29-33 The metabolic fate of PTER in mice was investigated by Shon et al.34 96 and seven metabolites were detected in urine using liquid chromatography/atmospheric pressure 97 chemical ionization and electrospray ionization tandem mass spectrometry. Two of those 98 metabolites come from direct chemical modification of PTER, its 4’-glucuronate (PTER-GlcAc, 99 7) and its 4’-sulphate (PTER-S, 8) derivatives (see Table 1). The other five metabolites come 100 from modification of two intermediates formed in vivo but not detected, the mono-demethylated 101 pterostilbene (also called pinostilbene, PIN, 9) and the mono-3’-hydroxylated pterostilbene (3’- 102 OH-PTER). These metabolites are PIN-4’-glucuronate (PIN-GlcAc, 11) and PIN-4’-sulphate 103 (PIN-S, 12) from PIN (Table 1), and 4’-sulphate-3’-OH-PTER, 4’-glucuronate-3’-OH-PTER and 104 4’-glucuronate-3’-sulphate-PTER from the intermediate 3’-OH-PTER (not shown in Table 1). 105 PIN is also found in the amazonian climber Gnetum venosum and in the bark of Pinus sibirica.35- 106 36 Most importantly, PIN is a major metabolite of PTER in the colon of mice fed with PTER and 107 another stilbenoid, analogue of RES. PIN has shown to play an important role in the anti-colon 108 cancer effects elicited when PTER was orally administered to mice.37 109 5 PTER metabolites are probably produced in a similar way to those of RES which is mainly 110 metabolized in the liver into the corresponding mono-glucuronides or mono-sulfates by phase II 111 enzymes and, to a lower extent, into piceatannol by phase I enzymes, and into dihydroresveratrol 112 by human microbiota.38 113 In this paper, the anti-inflammatory and antioxidant activity of several PTER metabolites 114 (compounds 7-12, Table 1), at physiologically relevant concentrations, were determined. RES 115 and its metabolites (compounds 2-5, Table 1) were also prepared and evaluated for direct 116 comparison with the PTER metabolites. An LPS-induced inflammation in RAW 264.7 117 macrophages and an H2O2-induced neurotoxicity in human neuroblastoma SH-SY5Y cells 118 models were chosen for these purposes. 119 120 MATERIALS AND METHODS 121 General information 122 All chemicals were obtained from chemical suppliers and used without further purification, 123 unless otherwise noted. RES (1) and PTER (6) were obtained from Sigma Aldrich. All reactions 124 were monitored by TLC on precoated Silica-Gel 60 plates F254, and detected by heating with 125 Mostain (500 ml of 10% H2SO4, 25g of (NH4)6Mo7O24•4H2O, 1g Ce(SO4)2•4H2O). Products were 126 purified by flash chromatography with silica gel60 (200-400 mesh). NMR spectra were recorded 127 on 300, 400 or 500 MHz NMR equipment, at room temperature for solutions in CDCl3, D2O or 128 CD3OD. Chemical shifts are referred to the solvent signal and are expressed in ppm. 2D NMR 129 experiments (COSY, TOCSY, ROESY, and HMQC) were carried out when necessary to assign 130 the corresponding signals of the new compounds. Sephadex LH 20, Reverse phase column and 131 then ion-exchanged with Dowex 50W were used in the purification of several glucuronic and 132 sulfate metabolites. High resolution mass spectra (HRMS) were obtained on an ESI/quadrupole 133 mass spectrometer (WATERS, ACQUITY H CLASS). If necessary, the purity was determined 134 by high performance liquid chromatography (HPLC). Purity of all final compounds was 95% or 135 higher. 136 6 Synthesis 137 4´-O-(2,3,4-tri-O-pivaloyl-  -D-glucuronic acid methyl ester)-3,5-dimethoxy-Resveratrol (14). 138 To a solution of trichloroacetimidate 1339 (354 mg, 0.58 mmol) and Pterostilbene 6 (100 mg, 0.39 139 mmol) in anhydrous CH2Cl2 (5 mL) at 0ºC, BF3∙OEt2 (0.145 mmol, 18 L) was added. After 30 140 min the reaction was warmed at room temperature for another 30 min. TLC showed the formation 141 of a major product and consumption of the glycosyl donor. The reaction was then quenched with 142 NEt3 and concentrated in vacuo. The resulting residue was purified by flash column 143 chromatography (hexane: ethyl acetate from 6:1 to 2:1) to afford 14 (162 mg, 60%) as a yellow 144 glassy solid; δH (300 MHz, CDCl3) 7.35 (d, J = 8.7 Hz, 2 H, Harom), 6.97-6.81 (m, 4 H, 2 Harom, 145 2x=CH), 6.56 (d, J = 2.1 Hz, 2 H, Harom), 6.31 (t, J = 2.1 Hz, 1 H, Harom), 5.42-5.25 (m, 3 H, 146 H-3, H-4, H-2), 5.06 (d, J = 7.5 Hz, 1 H, H-1), 4.16 (d, J = 9.6 Hz, 1 H, H-5), 3.74 (s, 6 H, OCH3), 147 3.66 (3 H, COOCH3), 1.08, 1.07 (2 s, 27 H, C(CH3)3); δC (75 MHz, CDCl3) 177.0, 176.5, 176.4, 148 166.9 (C=O), 160.9, 156.5, 139.3, 132.6, 128.2, 127.9, 127.8, 117.1, 104.5, 99.9 (=C, Carom), 149 99.5 (C-1), 73.0 (C-5), 71.4 (C-3), 70.5 (C-2), 69.1 (C-4), 55.4, 52.3 (CH3O), 38.8 (C(CH3)3), 150 27.2, 27.1 (C(CH3)3); (HRMS (ES+) Calcd. for C38H50O12Na (M+) 721.3200, found: 721.3186. 151 Potassium 4´-O-(  -D- glucopyranosyluronic acid)-3,5-dimethoxy-resveratrol (7). A suspension 152 of 14 (90 mg, 0.128 mmol) and K2CO3 (110 mg, 0.79 mmol) was prepared in a mixture of 153 methanol:water:dioxane (1:1:1, 9 mL), and the mixture was stirred at room temperature for 24 h. 154 Then, KOH (100 mg, 1.78 mmol) was added and the reaction was stirred for 48 h and and later 155 neutralized with IR-120 H+ resin. The solvents were then removed and the residue was purified 156 by RP-C18 column eluting with water:methanol (1:6) to obtain 40 mg (70 % yield). δH (300 MHz, 157 CD3OD) 7.48 (d, J = 8.7 Hz, 2 H, Harom), 7.14-7.08 (m, 3 H, 2 Harom, 1x=CH), 6.97 (d, J = 158 16.4 Hz, 2 H, =CH), 6.69 (d, J = 2.1 Hz, 2 H, Harom), 6,38 (t, J = 2.1 Hz, 1 H, Harom), 4.97-4.95 159 (m, 1 H, H-1), 3.78 (m, 7 H, H-5, OCH3), 3.55-3.53 (m, 3 H, H-3, H-4, H-2); δC (75 MHz, CD3OD) 160 174.8 (C=O), 161.0, 157.5, 139.7, 131.7, 128.1, 127.3, 126.9, 116.7, 103.9, 100.1 (=C, Carom), 161 99.3 (C-1), 76.4 (C-5), 75.2 (C-3), 73.3 (C-2), 72.2 (C-4), 54.4 (CH3O); (HRMS (ES+) Calcd. for 162 C22H25O9Na (M+) 433.1499, found: 433.1487. 163 7 Sodium Resveratrol-3,5-dimethoxy-4’-sulfate (8). Pterostilbene (3,5-dimethoxy resveratrol, 6, 164 100 mg, 0.39 mmol) was dissolved in dry DMF in a 2-5 mL microwave reaction vial containing 165 magnetic stirrer bar and fitted with a septum, which was then pierced with a needle. Sulfur 166 trioxide–trimethylamine complex (2 eq., 0.78 mmol, 108 mg) was then added (this complex has 167 been previously washed with H2O, MeOH, and CH2Cl2 and dried under high vacuum). Microwave 168 based sulfation reaction was performed using a Biotage Initiator synthesizer in sealed reaction 169 vessels.40-42 The closed vial was then evacuated under high vacuum for 30 min and the mixture 170 was subjected to microwave radiation for 40 min at 120 ºC (50-60W average power). MeOH (1 171 mL) and CH2Cl2 (1 mL) were added, and the solution was layered on the top of a Sephadex LH- 172 20 chromatography column which was eluted with MeOH to obtain the corresponding 173 triethylammonium salt that was then purified through RP C18 chromatography with MeOH-H2O 174 (80:20). Finally, the product was eluted through an ion exchange colum with dowex Na+ to afford 175 the corresponding sodium salt 8 (120 mg, 86% yield). δH (300 MHz, CD3OD) 7.39-7.36 (m, 3 H, 176 Harom), 7.07-6.85 (m, 5 H, 2 Harom, 2x=CH), 6.35 (s, 1 H, Harom),3.78 (s, 6 H, OCH3); δC (75 177 MHz, CD3OD) 161.0, 160.9, 139.8, 128.9, 128.6, 127.6, 125.3, 115.1 (=C, Carom), 55.4 (CH3O); 178 (HRMS (ES+) Calcd. for C16H15O6S (M+) 335.0595, found:.335.0612 179 (E)-4-[3-Methoxy-5-(tert-butyldimethylsilyloxy) styryl]phenol or 3-Methoxy-5-(tert- 180 butyldimethylsilyloxy)-resveratrol (15). To a solution of 3-methoxy-resveratrol (650 mg, 2.68 181 mmol) in DMF (anhydrous, 10 mL) cooled in an ice-water bath under argon, TBDMSCl (202 mg, 182 1.34 mmol, 0.5 equiv) and imidazole (225 mg, 3.35 mmol, 1.0 equiv) were added sequentially. 183 The reaction mixture was stirred for 30 min at 0º C and another 30 min at room temperature. The 184 pale yellow reaction mixture was diluted with EtOAc (50 mL), cast into a separatory funnel, and 185 washed with water (2x25 mL), brine (25 mL), and the organic phase was dried (Na2SO4). 186 Filtration and concentration in vacuo afforded the crude that was purified by flash column 187 chromatography (hexane:ethyl acetate:methanol from 10:1 to 3:1) to afford 15 ( 200 mg, 21%). 188 δH (300 MHz, CDCl3) 7.45 (d, J = 8.7 Hz, 2 H, Harom), 6.96-6.82 (m, 4 H, 2 Harom, 2x=CH), 189 6.61 (d, J = 2.0Hz, 2 H, Harom), 6,31 (t, J = 2.0 Hz, 1 H, Harom), 3.85 (s, 3 H, OCH3), 1.03 (s, 9 190 8 H, C(CH3)3), 0.26 (s, 6 H, Si(CH3)2); δC (75 MHz, CDCl3) 159.3, 157.0, 156.7, 139.8, 130.0, 191 128.7, 127.8, 126.3, 114.2, 111.0, 106.6, 106.4 (=C, Carom), 55.4 (CH3O), 25.7 (C(CH3)3), 18.2 192 (C(CH3)3), -4.35 (Si(CH3)2). (HRMS (ES+) Calcd. for C21H29O3Si (M+1) 357.1886, found: 193 357.1878. 194 4´-O-(2,3,4-tri-O-pivaloyl-  -D-glucuronic acid methyl ester)-3-methoxy-5-(tert- 195 butyldimethylsilyloxy) Resveratrol (16). To a solution of trichloroacetimidate 13 (382 mg, 0.63 196 mmol) and compound 15 (150 mg, 0.42 mmol) in anhydrous CH2Cl2 (5 mL) at 0ºC, BF3 ∙OEt2 197 (0.157 mmol, 20 L) was added. After 30 min the reaction was warmed at room temperature for 198 another 30 min. TLC showed the formation of a major product and consumption of the glycosyl 199 donor. The reaction was then quenched with NEt3 and concentrated in vacuo. The resulting 200 residue was purified by flash column chromatography (hexane: ethyl acetate from 8:1 to 6:1) to 201 afford 16 (200 mg, 60%) as a yellow glassy solid; δH (300 MHz, CDCl3) 7.22 (d, J = 8.7 Hz, 2 H, 202 Harom), 6.79-6.59 (m, 4 H, 2 Harom, 2x=CH), 6.53-6.47 (m, 2 H, Harom), 6,17 (t, J = 2.1 Hz, 1 203 H, Harom), 5.29-5.11 (m, 3 H, H-3, H-4, H-2), 4.92 (d, J = 7.8 Hz, 1 H, H-1), 4.03 (d, J = 9.9 Hz, 204 1 H, H-5), 3.62 (s, 3 H, OCH3), 3.55 (3 H, COOCH3), 0.96, 0.95, 0.94 (3 s, 27 H, C(CH3)3), 0.78 205 (s, 9 H, SiC(CH3)3), 0.00 (s, 6 H, Si(CH3)2); δC (75 MHz, CDCl3) 177.0, 176.5, 176.4, 166.8 206 (C=O), 159.5, 157.9, 156.8, 139.9, 129.7, 129.1, 127.8, 125.9, 114.1, 113.3, 108.1, 107.9 (=C, 207 Carom), 99.6 (C-1), 72.9 (C-5), 71.5 (C-3), 70.5 (C-2), 69.1 (C-4), 55.3, 52.9 (CH3O), 38.8, 38.7, 208 30.9 (C(CH3)3, Si C(CH3)3), 27.2, 27.1, 25.6 (C(CH3)3, SiC(CH3)3,), -4.42 (Si (CH3)2); (HRMS 209 (ES+) Calcd. for C43H62O12NaSi (M+) 821.3908, found 821.3896. 210 Potassium 4´-O-(  -D- glucopyranosyluronic acid)-3-methoxy-resveratrol (11). A suspension 211 of 16 (150 mg, 0.187 mmol) and KF (2eq., 22 mg, 0.374 mmol) was prepared in methanol (4 mL) 212 and stirred for 2 h. Then, K2CO3 (180 mg, 1.30 mmol) was added and the mixture was stirred at 213 room temperature for 24 h. Later, KOH (100 mg, 1.78 mmol) was added and reaction was stirred 214 for 48 h, and finally, it was neutralized with IR-120 H+ resin. The solvents were removed and the 215 residue was purified by RP-C18 column eluting with water:methanol (1:6). Fractions containing 216 the major product were concentrated to afford compound 11 (65 mg, 84%). δH (300 MHz, 217 15 In the case of the pretreatment experiments (adding the corresponding compounds previously to 374 H2O2 addition), we observed mild neuroprotective effects for every compound at 1 and 10 μM 375 (Figure 2A and 2B, right), and at 100 μM only for the three glucuronated derivatives screened (5, 376 7 and 11). The rest of compounds didn’t show neuroprotection at the highest concentration tested, 377 possibly due to their intrinsic toxicity to neuronal cells (see Figure 1). 378 In fact, González-Sarrias et al. also found no effect for RES and its metabolites under similar 379 conditions of co-treatment experiments on SH-SY5Y neuronal cells, and only a slight effect under 380 the pre-treatment experiments.26 On the other hand, they found that other phenolic natural 381 products such as gallic acid, ellagic acid, 3,4-dihydroxyphenylpropionic acid and 3,4- 382 dihydroxyphenylacetic acid, at 10 μM concentration, significantly attenuated the H2O2-induced 383 cytotoxicity in comparison with H2O2 treatment alone. 384 385 Effects on Intracellular ROS Accumulation and on REDOX Activity Induced by H2O2 386 Addition. The effects on intracellular ROS levels and REDOX activity in neuronal SH-SY5Y 387 cells upon addition of RES and PTER metabolites are summarized in Figures 3 and 4, 388 respectively. The protective effect of the metabolites on H2O2-induced oxidative stress was 389 determined at 2h incubation time since H2O2 diminishes cell viability up to 50% at 24h for some 390 compounds (see Figure 2). The intracellular ROS accumulation and REDOX activity in the cells 391 with each metabolite alone (without H2O2), was measured 6 h after treatments. 392 In absence of H2O2, the intracellular ROS levels in SH-SY5Y cells was slightly lower than in 393 control cells after treatment with RES and its metabolites (Figure 3a, left). This is particularly 394 true for RES (1). In the presence of PTER and its metabolites, ROS levels were very close to the 395 control basal levels (except for compound 8, see Figure 3b, left). 396 In the case of metabolite and H2O2 co-treatments (Figures 3a and 3b, center), most compounds 397 were able to decrease the ROS levels in comparison with the H2O2 control (H2O2 triggers ROS 398 production up to a 40%). Some compounds were even capable of diminishing ROS production to 399 16 levels below the basal levels before H2O2 addition (see compounds 1, 3 and 8, Figures 3a and 3b, 400 center). Some PTER metabolites (7, 10-12) were active alleviating ROS production at 10 and 100 401 M, but not at 1M. 402 In the case of pre-incubation with RES, PTER or their metabolites before H2O2 addition (Figures 403 3a and 3b, right), again, most of the compounds were able to decrease the ROS levels compared 404 to the H2O2 control. Remarkably, all PTER metabolites (except compound 7 at 1 M) were 405 capable of restoring ROS production under the basal levels before H2O2 addition. This is also true 406 for RES (1) at 10 and 100 M, but not at 1M. 407 Regarding REDOX activity, RES, PTER and all their metabolites, except 6 and 8 at 100 M 408 (concentration at which both compounds proved to be very toxic to SH-SY5Y cells, see Figure 409 1), exhibited an enhanced basal REDOX activity compared to control cells (Figures 4a and 4b, 410 left). The same trend is observed both, in the co-treatment and the pre-treatment experiments 411 with H2O2, being most derivatives able to activate the intracellular REDOX activity above the 412 initial levels before H2O2 addition (H2O2 treatment led to a decrease of REDOX activity of up to 413 a 30%, see Figure 4b, center). 414 415 Anti-inflammatory effects of RES, PTER and their Metabolites on LPS-challenged RAW 416 264.7 macrophages. 417 All RES, PTER and PIN derivatives except PIN-GlcA (11) and PIN-SO3- (12) showed moderate 418 toxicity for macrophages when incubated for 24 h at concentrations from 100 to 1 μM (Figure 5). 419 Toxicity of RES and its metabolites to RAW macrophages has previously been reported in the 420 literature.50-51 421 Next, we checked the anti-inflammatory capacity of RES, PTER and their metabolites. To do so, 422 we measured cell viability on RAW macrophages after lipopolysacharide (LPS) challenge and 423 after treatment with the different compounds. In general, the co-treatment experiments showed 424 that compounds didn´t alleviate the LPS-induced cytotoxicity (Figure 6A and 6B, left) compared 425 17 with the LPS control. Only PIN glucuronate (11), PIN sulfate (12) and RES glucuronate (5) at 426 100 M had a moderate protective effect under these experimental conditions. This result can be 427 clearly explained by the intrinsic toxicity of RES, PTER and their metabolites to macrophages. 428 Thus, the only non-cytotoxic compounds were the ones exerting some anti-inflammatory effect 429 (see Figures 5, 6A and 6B, left). 430 Surprisingly, the pretreatment experiments showed mild anti-inflammatory activity for almost all 431 compounds at 1 and 10 μM concentration (Figure 6A and 6B, right), and also at 100 μM for the 432 three glucuronated metabolites screened (5, 7 and 11). The rest of the compounds didn´t show 433 anti-inflammatory activity at the highest concentration tested due to their toxicity to RAW 434 macrophages (see Figure 5). 435 Finally, the inhibition of IL-6 and NO inflammatory markers on RAW macrophages were 436 assayed for the metabolites showing the best results on the previous experiments (compounds 5, 437 7 and 11). RES and PTER were included as controls and LPS was used as inflammatory trigger. 438 Only PIN glucuronate (11) showed a promising inhibitory activity, both on IL-6 (ca. 20% 439 inhibition) and NO (ca. 85% inhibition) values (see Figure 7). PTER showed a moderate NO 440 inhibition (ca. 20%) and very mild IL-6 amelioration. RES glucuronate (5) exhibited a mild IL-6 441 inhibition (ca. 5-10%), whereas no inhibition was observed in the production of NO. Both RES 442 and PTER glucuronate (1 and 7, respectively) showed no inhibition under these experimental 443 conditions. TNF- was also measured but no inhibition was observed for PTER or RES 444 metabolites. 445 RES and PTER have been reported in the literature to inhibit both IL-6 and NO to certain 446 extent.50,52 In any case, it has been described that RES is toxic to RAW macrophages and that 447 stimulation with LPS reduces its toxicity via a mechanism that involves activation of toll like 448 receptor 4.53 In our case, the lack of activity could be explained based on the high observed 449 toxicity of our metabolites at the concentration used in the ELISA experiments (see Figure 5). 450 Other explanations could be the lower activation produced by LPS in this set of experiments 451 18 compared to previous assays carried out in our laboratory (data not shown), which could be due 452 to differences in cells conditions (passage number, cell cycle, etc). 453 In summary, 3-methyl-4´-glucuronate-resveratrol (11) prevented neuronal death via attenuation 454 of ROS levels and increased REDOX activity in SH-SY5Y cells. This metabolite was also able 455 to ameliorate LPS-mediated inflammation on macrophages via inhibition of IL-6 and NO 456 production. Thus, RES and PTER metabolites could be playing and active role on the 457 neuroprotective and anti-inflammatory activities reported for this family of compounds. 458 459 Abbreviations used 460 H2DCFDA , 2′,7′-dichlorodihydrofluorescein diacetate; DMSO, dimethyl sulfoxide; DMEM, 461 Dubelco minimal essential medium; FBS, fetal bovine serum; MTT, 3-(4,5-dimethyl-2-thiazolyl)- 462 2,5-diphenyl-2H-tetrazolium bromide; PBS, phosphate-buffered saline; ROS, reactive oxygen 463 species; REDOX, reduction-oxidation activity; RES, trans-resveratrol; PTER, Pterostilbene; PIN, 464 Pinostilbene; SD, standard deviation; sulf, sulfate; LPS, lipopolysacharide; IL-6, interleukin 6; 465 NO, nitric oxide; H2O, water; H2O2, oxygen peroxide solution; MeOH, methanol; CH2Cl2; 466 methylene chloride; THF, tetrahydrofurane ;NEt3, triethylamine; CD3OD, deuterated methanol; 467 CDCl3, deuterated chloroform; D2O, deuterated water; HRMS, high resolution mass 468 spectrometry; ES, electrospray; Calcd., calculated; KF, potassium fluoride; K2CO3, potassium 469 carbonate; KOH, potassium hydroxide; RP-C18, reversed-phase high performance liquid 470 chromatography column packet with octadecyl carbon chain (C18)-bonded silica; BF3 ∙OEt2, 471 Boron trifluoride diethyl etherate; DMF dimethylformamide, TBDMSCl, tert-butyl di-methyl 472 silyl chloride; EtOAc, ethyl acetate; Na2SO4, sodium sulfate anhydrous; MHz, Mega hertz; TLC, 473 thin layer chromatography; ppm, parts per million; 2D, bi-dimimentional; NMR, nuclear 474 magnetic resonance; COSY, correlated spectroscopy; TOCSY, total correlated spectroscopy ; 475 ROESY, rotating-frame overhauser spectroscopy; HMQC, Heteronuclear Multiple-Quantum 476 Correlation; ELISA, Enzyme-linked immunosorbent assays; GlcAc, glucuronic acid; SO3-, 477 sulfate. 478 19 AUTHOR INFORMATION 479 Corresponding Authors 480 *P.P: phone, +34-958181621; E-mail: [email protected] 481 *J.C.M: phone, +34-958181644; E-mail: [email protected] 482 ORCID 483 Juan Carlos Morales: 0000-0003-2400-405X 484 Pablo Peñalver: 0000-0002-2884-282X 485 486 ACKNOWLEDGEMENTS 487 This work was supported by the Junta de Andalucía (P11-FQM-7316 and P12-FQM-1553) and 488 FEDER funds from the EU are gratefully acknowledged. PP´s work was supported by Junta de 489 Andalucía (P11-FQM-7316). 490 491 Appendix A. Supplementary data 492 1H and 13C-NMR spectra of the new PTER and PIN derivatives synthesized can be found at: 493 494 NOTES 495 The authors declare no competing financial interest. 496 497 REFERENCES 498 (1) Tabner, B. J.; El-Agnaf, O. M.; Turnbull, S.; German, M. J.; Paleologou, K. E.; Hayashi, Y.; 499 Cooper, L. J.; Fullwood, N. J.; Allsop, D. Hydrogen peroxide is generated during the very early 500 stages of aggregation of the amyloid peptides implicated in Alzheimer disease and familial British 501 dementia. J. Biol. Chem. 2005, 280, 35789-35792. 502 (2) Uttara, B.; Singh, A. V.; Zamboni, P.; Mahajan, R. T. Oxidative stress and neurodegenerative 503 diseases: a review of upstream and downstream antioxidant therapeutic options. Curr. 504 Neuropharmacol. 2009, 7, 65-74. 505 20 (3) Rosini, M.; Simoni, E.; Milelli, A.; Minarini, A.; Melchiorre, C. 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Effect of RES and PTER metabolites (100, 10 and 1 μM) on REDOX activity in SH-SY5Y 686 neuronal cells; A) RES metabolites; B) PTER metabolites. Values (%) are expressed as the mean ± SD (n 687 = 2-4, in quatriplicate). Symbols: a, means significant difference (p < 0.05) compared to control cells; b, 688 means significant difference (p < 0.05) compared to H2O2 treatment alone. 689 33 690 Figure 5. Toxicity of RES and PTER metabolites (100, 10 and 1 μM) (24 h co-treatment with LPS) in 691 RAW 264.7 macrophages. Values (%) are expressed as the mean ± SD (n = 2-4, in quatriplicate). Symbols: 692 a, means significant difference (p < 0.05) compared to control cells (- DMSO); b, means significant 693 difference (p < 0.05) compared to DMSO treatment alone. 694 34 695 696 Figure 6. Effect of RES and PTER metabolites (100, 10 and 1 μM) (6 h pretreatment, 24 h cotreatment 697 with LPS) in RAW 264.7 macrophages; A) RES metabolites; B) PTER metabolites. Values (%) are 698 35 expressed as the mean ± SD (n = 2-4, in quatriplicate). Symbols: a, means significant difference (p < 0.05) 699 compared to control cells; b, means significant difference (p < 0.05) compared to H2O2 treatment alone. 700 701 702 Figure 7. Effect of RES, PTER and their more promising metabolites on the pro-inflamatory makers 703 inhibition (10 μM, 24 h cotreatment with LPS) in RAW 264.7 macrophages; A) IL-6; B) NO Values (%) 704 are expressed as the mean ± SD. Symbols: (*) means significant difference (p < 0.05) compared to LPS 705 treatment alone. 706 707