Hydroxytyrosol and tyrosol sulfate metabolites protect against the oxidized cholesterol pro-oxidant effect in Caco-2 human enterocyte-like cells

Food & Function Cite this: DOI: 10.1039/c0xx00000x www.rsc.org/xxxxxx Dynamic Article Links ► ARTICLE TYPE This journal is © The Royal Society of Chemistry [year] [journal], [year], [vol], 00–00 | 1 Hydroxytyrosol and tyrosol sulfate metabolites protect against oxidized cholesterol pro-oxidant effect in Caco-2 human enterocyte-like cells. Angela Atzeria, Ricardo Lucasb, Alessandra Incania, Pablo Peñalverc, Alberto Zafra-Gómezd, M. Paola Melisa, Roberto Pizzalae, Juan C. Moralesb,c* and Monica Deianaa* Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX 5 DOI: 10.1039/b000000x Abstract The aim of this study was to investigate the ability of sulfate metabolites of hydroxytyrosol (HT) and tyrosol (TYR) to act as antioxidants counteracting the pro-oxidant effect of oxidized cholesterol in intestinal cells. For this purpose, we synthesized sulfate metabolites of HT and TYR using chemical 10 methodology and examined their antioxidant activity in Caco-2 monolayers in comparison with the parent compounds. Exposure to oxidized cholesterol led to ROS production, oxidative damage, as indicated by MDA increase, a decrease of reduced glutathione concentration and an enhancement of glutathione peroxidase activity. All the tested compounds were able to counteract the oxidizing action of oxidized cholesterol; HT and TYR sulfate metabolites showed an efficiency in protecting intestinal cells 15 comparable to that of the parent compounds, strengthening the assumption that the potential beneficial effect of the parent compounds is retained, although extensive metabolisation occurs, being the resulting metabolites able to exert a biological action themselves. Keywords: 20 sulfate metabolites, oxidative stress, oxidized cholesterol, olive oil phenolic compounds CREATED USING THE RSC ARTICLE TEMPLATE (VER. 3.0) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS ARTICLE TYPE www.rsc.org/xxxxxx | XXXXXXXX 2 | Journal Name, [year], [vol] , 00–00 This journal is © The Royal Society of Chemistry [year] Introduction Polyphenols present in extra virgin olive oil, the principal fat component of the Mediterranean diet, have been demonstrated to exert many potentially beneficial biological effects due to, at least in part, their ability to protect against oxidative stress mediated damage 1-2. Hydroxytyrosol (3,4-dihydroxyphenylethanol; HT) and tyrosol (4-hydroxyphenylethanol; TYR) (Fig. 1) are the most biologically active phenolic alcohols present in the oil, where they exist in simple forms or as conjugates (such as oleuropein); they are structurally identical 5 except that HT has an extra hydroxyl group in the meta position. 10 Fig. 1 Structure of tyrosol (TYR), hydroxytyrosol (HT) and their sulfate metabolites 3-5 The literature on phenolic absorption and metabolism after olive oil ingestion is extensive 3-4; however, there are still several issues to be clarified. Some complex olive oil polyphenols, among the secoiridoids, are relatively stable under gastric conditions and reach the 15 intestine where they may be directly absorbed or metabolised under absorption5; most of the complex olive oil polyphenols however seems to undergo gastrointestinal biotransformation (see de Bock for an excellent review ) 6, increasing the relative amount of simple phenols, mainly TYR and HT, entering the small and large intestine 7. At this location the concentration of simple phenols may be quite high, in the high M range 7, and they might exert direct protective effects by scavenging reactive species and/or preventing their formation 8. However, in the process of crossing enterocytes olive oil phenolic compounds are subjected to classical phase I/II 20 biotransformation and to an important first pass metabolism, resulting in almost undetectable concentrations of free HT and TYR in body fluids 9. Sulfated and glucuronidated HT and TYR are the predominant metabolites found in human plasma and urine 10-11, and they have also been shown to concentrate in the intestinal epithelium, since glucuronidation and sulfation are the major pathways of phase II xenobiotic metabolism in the human intestine 12-13. The potential health benefits of HT, TYR and their derivatives are likely to be due to both 25 parental compounds and their phase I and phase II major metabolites. Glucuronidated metabolites of HT showed a more efficient radical scavenging potency than HT itself 11 and the ability to protect renal cells 14 and erythrocytes from oxidative injury was also better than the parent compound 15. However no data are available up-to-date concerning the antioxidant activity of sulfate metabolites of HT and TYR. In this study we investigated the possible protective effect of the sulfate metabolites of TYR and HT, in comparison with the parent 30 compounds, against the oxidative damage to intestinal mucosa due to oxidized cholesterol exposure in the human colon adenocarcinoma cell line, Caco-2. After confluence, these cells spontaneously undergo full differentiation in vitro with enterocyte-like features 16. Caco-2 cells have been recognized as a suitable model for evaluating the effect of nutrient components, for both normal dietary constituents and toxicants, as oxidizing agents 17. Large amounts of lipid oxidation products become available, of both exogenous and endogenous origin, at the level of the intestinal mucosa. Dietary oxysterols, derived from cholesterol degradation and oxidation after prolonged storage or 35 cooking of foods rich in cholesterol, have recently been shown to contribute to the onset and further development of oxidative stress and inflammation related intestinal diseases 18-19. The major oxysterols found in food may contribute to oxidative unbalance of the intestinal epithelium by inducing the generation of reactive oxygen species (ROS) 18, 20. Our purpose was to investigate the ability of sulfate metabolites of HT and TYR to act as antioxidants counteracting the oxysterols induced changes of the cellular redox state and compare them with their parent compounds. For this purpose, we synthesized sulfate metabolites of HT and TYR using chemical methodology 40 and evaluate their uptake/stability in Caco-2 monolayers. Cytotoxicity, production of MDA and ROS species, levels of glutathione and glutathione peroxidase activity were measured to assess the protecting effect of the sulfate metabolites in comparison with the (their) parent olive (remove) phenolic compounds. CREATED USING THE RSC ARTICLE TEMPLATE (VER. 3.0) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS ARTICLE TYPE www.rsc.org/xxxxxx | XXXXXXXX 2 | Journal Name, [year], [vol] , 00–00 This journal is © The Royal Society of Chemistry [year] Materials and methods Preparation of oxidized cholesterol Oxidized cholesterol was prepared essentially as previously described 21. In brief, samples of 2 ml (2ml aliquots) of pure cholesterol (Sigma Aldrich, St. Louis, MO) solution (5 mg/ml in EtOH) were dried down into round-bottomed glass test tubes under vacuum and 5 heated in a 140 °C oil bath in air for 3 h. The oxidized mixture was separated and analysed by GC-MS for the determination of the relative oxysterols. Briefly, 2 L of the mixture was diluted in 2 mL of EtOH (1: 1000 dilution). After evaporating under a nitrogen stream, 50 L of pyridine (Sigma Aldrich) and 50 L of N,O-Bis(trimethylsilyl)trifluoroacetamide with trimethylchlorosilane (BSTFA:TMCS; 99: 1) (Sigma Aldrich) were added, mixed and kept at 60 °C for 45 min. The derivatized sample (1.5 L) was injected into an Agilent (Waldbronn, DE) GC-MS in splitless mode and the column temperature was programmed starting from 70 °C to 250 °C 10 (10 °C/minute), after up to 290 °C (5 °C/min) maintained for 10 min, ending at 300 °C (2 °C/min) for 5 min (duration about 45 min total). The helium flow was set at 1.3 mL/min. The specific parameters of the instrument were as published by Calderon-Santiago et al. 22 Spectra were acquired in scan mode. GS-MS analysis demonstrated that the mixture contained 58.9% of cholesterol, 13.5% of 7-ketocholesterol, 9.54% of 7- hydroxycholesterol, 5.6% of 6-hydroxycholesterol, 5.87% of 7-hydroxycholesterol, 4.5% of cholesta-4,6-dien-3-ol and 2.1% of 315 keto-4-cholestene. Synthesis of sulfate metabolites General methods. HT and HT acetate were obtained from Seprox Biotech (Madrid, Spain), TYR from Sigma Aldrich. All other chemicals obtained from commercial sources were used without further purification, unless otherwise noted. All reactions were monitored by TLC on precoated 20 Silica-Gel 60 plates F254, and detected by heating with Mostain (500 mL of 10% H2SO4, 25g of (NH4)6Mo7O24•4H2O, 1g Ce(SO4)2•4H2O). Products were purified by flash chromatography with Merck Silica gel 60 (200-400 mesh). High resolution mass spectra were obtained on an ESI/quadrupole AutoSpec-Q mass spectrometer. NMR spectra were recorded on 300 or 500 MHz spectrometers, at room temperature for solutions in CDCl3, or D2O. Chemical shifts are referred to the solvent signal. Metabolites were purified by chromatography with Reverse Phase-C18 Silica gel. Data were processed using manufacturer software, raw data were 25 multiplied by shifted exponential window function prior to Fourier transform, and the baseline was corrected using polynomial fitting. General procedure for the microwave-assisted O-sulfation. Microwave based sulfation reactions were performed using a microwave synthesizer in sealed reaction vessels. Phenolic derivatives (1.0 equiv), sulfur trioxide–trimethylamine complex, SO3.NMe3, (5 equiv per OH; being this complex previously washed with H2O, MeOH, and CH2Cl2 and dried under high vacuum) and a magnetic stirrer bar were placed in a 2-5 mL microwave reaction vial and fitted with a 30 septum, which was then pierced with a needle. The closed vial was then evacuated in high vacuum for 2 h. The mixture was dissolved in dry CH3CN (2.0 mL) and NEt3 (0.3-1.0 mL) was then added. Reaction mixture was subjected to microwave radiation for 20–40 min (depending on the compound) at 100 ºC (50-60W average power). MeOH (1 mL) and CH2Cl2 (1 mL) were added, and the solution was layered on the top of a Sephadex LH-20 chromatography column which was eluted with CH2Cl2/MeOH (1:1) to obtain the corresponding triethylammonium salt as a white powder (94–98% yield). 35 Triethylamine, 4-(2-(butyryloxy)ethyl)phenyl sulfate salt (7). Tyrosol butyrate 6 23-24 (70 mg, 0.33 mmol) and SO3.NMe3 (233 mg, 1.7 mmol) were submitted under sulfation conditions for 20 min. TLC (Ethyl acetate: MeOH, 10:1) showed the formation of a major product and complete consumption of the initial material. Solvents were removed and the crude extract was purified by sephadex LH-20 (CH2Cl2:MeOH, 1:1) to afford 7 (92 mg, 98%) as a white powder. 1H-NMR (300 MHz, D2O) : 6.72, 7.18 (2d, 4H, J = 8.7 Hz, Harom), 40 4.24 (t, 2H, CH2OAc), 3.10 (q, 6H, -CH2CH3), 2.88 (t, 2H, CH2Ar), 2.19 (t, 2H, J = 7.5Hz, CH2), 1.45 (m, 2H, CH2), 1.18 (t, 9H, CH2CH3), 0.74 (t, 3H, CH3); 13C-NMR (125 MHz, D2O) δ: 177.1 (CO), 150.0, 136.2, 130.3 (2x CHarom), 121.3 (2x CHarom), 65.4 (CH2OAc), 46.5, 36.0 (CH2Ar), 34.0, 18.3, 12.6, 8.0 (CH3). ESI-HRMS (ES-) Calcd. for C12H15O6S (M-H) 287.0589, Found: 287.0594. Potassium 4-(2-hydroxyethyl)phenyl sulfate (5). Compound 7 (97 mg, 0.32 mmol) was dissolved in MeOH (10 mL) and K2CO3 (90 45 mg, 0.66 mmol) was added. The reaction mixture was stirred at room temperature for 24 h and then neutralized with IR 120 H+ resin. Solvent was then removed in vacuum and the crude extract was purified by an RP-C18 column eluted with H2O: MeOH (from 100:0 to 70:30). Fractions containing the desired product were concentrated and freeze-dried affording compound 5 as a white solid (68 mg, 94%). 1H-NMR (400 MHz, D2O) : 7.18, 6.86 (2d, 4H, J = 8.4 Hz, Harom), 3.78, 2,78 (2t, 4H, J = 6.7 Hz, CH2OH, CH2Ar); 13CNMR (75 MHz, D2O) δ: 157 (Cq), 130.3 (2x CHarom), 129.8 (Cq), 117.1 (2x CHarom), 63.0 (CH2OH), 36.9 (CH2Ar). ESI-HRMS (ES-) 50 Calcd. for C8H9O5S (M-H) 217.0171, Found: 217.0171. 3-((tert-butyldimethylsilyl)oxy)-4-hydroxyphenethyl acetate (9) and 4-((tert-butyldimethylsilyl)oxy)-3-hydroxyphenethyl acetate (10). To a solution of hydroxytyrosol acetate 8 23-24 (223 mg, 1.13 mmol) in DMF (anhydrous, 3 mL) cooled in an ice-water bath under argon were added sequentially tert-butyldimethylsilyl-trifluoromethanesulfonate (TBDMSOTf, 287 L, 1.25 mmol, 1.10 equiv) and 55 diisopropylethylamine ( i-Pr2Net, 265 L, 1.52 mmol, 1.35 equiv). The mixture was allowed to stir for 30 min at 0 ºC, and TLC (hexane: Journal Name Cite this: DOI: 10.1039/c0xx00000x www.rsc.org/xxxxxx Dynamic Article Links ► ARTICLE TYPE This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol] , 00–00 | 3 ethyl acetate 3:1) at that point indicated that the reaction was complete. The pale yellow reaction mixture was diluted with EtOAc (100 mL), cast into a separatory funnel, and washed with water (2x50 mL), brine (50 mL), and the organic phase was dried (Na2SO4). Filtration and concentration in vacuum afforded the crude extract that was purified by flash column chromatography (hexane:ethyl acetate from 15:1 to 10:1) to afford 9 and 10 (314 mg, 90%, powder) like a regioisomeric mixture in ratio ~ 1:1 . 1H NMR (400 MHz, CDCl3) δ 6.88 (d, 1H, J = 8.1 Hz, Harom), 6.83 (s, 1H, Harom), 6.77 (d, 1H, J = 8.4 Hz, Harom), 6.73 (d, 1H, J = 8.4 Hz, Harom), 5 6.71(s, 1H, Harom), 6.62 (d, 1H, J = 8.1 Hz, Harom ), 5.52, 5.45 (2s, 2H, 2xOH), 4.26 (t, 2H, J = 6.7 Hz, CH2OAc), 4.24 (t, 2H, J = 6.4 Hz, CH2OAc), 2.86 (t, 2H, J = 6.7 Hz, CH2Ar), 2,84 (t, 2H, J = 6.4 Hz, CH2Ar), 2.07-2.05 (2s, 6H, CH3C=O), 1.05, 1.03 (2s, 18H, C(CH3)3 x2), 0.30, 0.29 (2s, 12H, -Si(CH3)2 x2); 13C-NMR (125 MHz, CDCl3) δ: 171.1, 171.0 (C=O), 147.1, 145.9, 142.3, 141.0, 131.8, 129.6 (Cqarom), 122.4, 120.2, 118.6, 117.7, 115.4, 114.8 (CHarom), 65.2, 65.0 (CH2OAc), 34.5 (2x CH2Ar), 25.7 (C(CH3)3), 21.0, 20.9 (CH3C=O), 18.2 (C(CH3)3), -4.2 (Si(CH3)2); HRMS (ES+) Calcd. for C16H26O4NaSi (M+Na) 333.1498, Found: 10 333.1508. Triethylamine, 5-(2-acetoxyethyl)-2-((tert-butyldimethylsilyl)oxy)phenyl sulfate salt (11) and triethylamine, 4-(2-acetoxyethyl)-2- ((tert-butyldimethylsilyl)oxy)phenyl sulfate salt (12). Regioisomeric mixture of compounds 9 and 10 (157 mg, 0.506 mmol) and SO3.NMe3 (351 mg, 2.52 mmol) were submitted under sulfation conditions for 2x20 min. TLC (Ethyl acetate:MeOH 10:1) showed the 15 formation of a major product and complete consumption of the starting material. Solvents were removed and crude was purified by sephadex LH-20 in a solvent mixture of CH2Cl2: MeOH 1:1 to afford 11 and 12 (231 mg, 94%, powder) like a regioisomeric mixture in ratio ~ 1:1. 1H-NMR (400 MHz, CDCl3) δ: 7.50 (d, 1H, J = 7.9 Hz, Harom), 7.46 (s, 1H, Harom), 6.82 (d, 1H, J = 8.2 Hz, Harom), 6.78 (d, 1H, J = 8.2 Hz, Harom), 6.73 (d, 1H, J = 7.9 Hz, Harom), 6.72 (s, 1H, Harom), 4.20 (t, 4H, J = 7.08 Hz, CH2OAc), 3.10-3.00 (dq, 12H, CH2CH3), 2.83 (t, 2H, J = 7.1 Hz, CH2Ar), 2.82 (t, 2H, J = 7.05 Hz, CH2Ar), 2.04, 2.03 (2s, 6H, CH3C=O), 1.26 (t, 18H, CH2CH3), 20 1.00, 0.99 (2s, 18H, C(CH3)3 x2), 0.21, 0.20 (2s, 12H, -Si(CH3)2 x2); 13C-NMR (125 MHz, CDCl3) δ: 171.0, 170.9 (C=O), 146.8, 145.6, 143.9, 142.8, 134.0, 130.7 (Cqarom), 124.5, 122.4, 121.9, 121.8, 121.6, 121.0 (CHarom), 65.1 (CH2OAc), 46.3 (CH2CH3), 34.5, (CH2Ar), 25.7 (C(CH3)3), 21.0 (CH3C=O), 18.8 (C(CH3)3), 8.8 (CH2CH3), -4.2 (Si(CH3)2); ESI-HRMS (ES-) Calcd. for C16H25O7SiS (MH) 389.1090, Found: 389.1092. 25 Potassium 2-hydroxy-4-(2-hydroxyethyl)phenyl sulfate (3) and potassium 2-hydroxy-5-(2-hydroxyethyl)phenyl sulfate (4). Regioisomeric mixture of 11 and 12 (231 mg, 0.47 mmol), potassium fluoride (KF,55 mg, 0.94 mmol) and potassium carbonate (K2CO3, 130 mg, 0.94mmol) were dissolved in MeOH (10 mL). The reaction mixture was stirred at room temperature for 18 h and solvent was then removed in vacuum. The crude extract was purified by column chromatography with RP-C18 silica gel eluting with H2O: MeOH (from 100:0 to 70:30). Fractions containing the desired product were concentrated and freeze-dried affording compounds 3 and 4 (115 30 mg, 90%, white powder) like a regioisomeric mixture in ratio ~ 1:1. 1H-NMR (300 MHz, D2O) : 7.21 (d, 2H, J = 8.0 Hz, Harom), 7.17 (s, 1H, Harom), 6.98 (d, 1H, J = 8.5 Hz, Harom), 6.88 (d, 1H, J = 8.5 Hz, Harom), 6.80 (s, 1H, Harom), 6.70 (d, 1H, J = 8.0 Hz, Harom), 3.76-3.69 (m, 4H, CH2OAc), 2.73-2.70 (m, 4H, CH2Ar); 13C-NMR (125 MHz, D2O) δ: 149.4, 147.4, 139.0, 138.4, 137.8, 130.7, 127.6, 123.0, 122.6, 120.0, 118.2, 117.6, 62.6, 62.4 (CH2OAc), 37.4, 36.9 (CH2Ar). HRMS-ESI (ES-) Calcd. for C8H9O6S (M-H) 233.0120, Found: 233.0126. 35 Cell culture Cell culture materials were purchased from Invitrogen (Milano, Italy). Caco-2 cells were obtained from the European Collection of Cell Cultures (ECACC, Salisbury UK) and grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 2.5% of heatinactivated bovine serum and 100U/mL penicillin and 100 μg/mL streptomycin, at 37 °C under a humidified atmosphere of 95% air and 40 5% CO2. For experimental studies Caco-2 cells, at passage 45–60, were plated at a density of about 1 × 105/mL and used 21 days post seeding Cytotoxic activity The cytotoxic effect caused by the exposure to increasing concentrations of oxidized cholesterol and the protective effect of HT, TYR, and sulfate metabolites was assessed on Caco-2 cells, seeded in 24-well plates, by the Neutral Red method 25. Cells were exposed to 45 oxidized cholesterol (0–175 μg/mL) in complete medium and incubated for 24 h. In order to assess the protective effect of the phenolic compounds cells were pretreated with the tested compounds (2,5-10 µM in water solution, 30 min) prior to oxidized cholesterol exposure (100 μg/mL for 24 h). After incubation, the medium was removed; a Neutral Red Solution (Sigma Aldrich) (0.033% in medium) was then added to the wells to determine cell viability. After 30 min of incubation, the Neutral red solution was carefully removed and cells quickly rinsed with PBS. The incorporated dye was then solubilised in Neutral Red Solubilisation Solution, acetic acid/ethanol /water 50 CREATED USING THE RSC ARTICLE TEMPLATE (VER. 3.0) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS ARTICLE TYPE www.rsc.org/xxxxxx | XXXXXXXX 4 | Journal Name, [year], [vol] , 00–00 This journal is © The Royal Society of Chemistry [year] (1/45/50, v/v/v), and the absorbance was measured at 540 nm. Determination of MDA MDA levels were determined in the medium of treated cells by the TBARS test with HPLC quantification, using the method described by Templar et al. 26, with some modifications. Briefly, 100 μL of 10% trichloroacetic acid (TCA, Sigma Aldrich) was added to 400 μL of 5 the medium, samples were mixed and left at room temperature. After 20 min, 200 μL of 2-thiobarbituric acid (TBA, Sigma Aldrich) (0.6%) were added; samples were incubated at 90 °C for 45 min and then centrifuged at 5000 x g for 15 min at 4 °C. Aliquots of the supernatant were injected into an Agilent 1100 HPLC system (Agilent Technologies, Santa Clara, CA) equipped with a diode-array detector (HPLC–DAD) and separation was achieved using a Varian (Middelburg, The Netherlands) Inertsil 5 ODS-2, 150 × 4.6 mm column; the mobile phase was a mixture of KH2PO4 50 mM pH 7/MeOH (65/35, v/v) at a constant flow rate of 1 mL/min. The formation 10 of the MDA–TBA adduct was revealed measuring its absorbance at 532 nm. A standard curve was prepared using a 1,1,3,3,- tetraethoxypropane (TEP, Sigma Aldrich) solution in PBS (0.05–10 μM). ROS production in Caco-2 cells Intracellular ROS production was monitored by adding the 2',7'-dichlorodihydrofluorescein diacetate (H2-DCF-DA, Sigma Aldrich), according to Dinicola et al. 27, in Caco-2 cells exposed to oxidized cholesterol (75 μg/mL) in complete medium and incubated for 0-180 15 min and in cells pretreated with the tested phenolic compounds (5-25 µM in water solution, 30 min), prior to exposure to oxidized cholesterol (75 μg/mL for 30 min), with some modifications. After incubation the culture medium was replaced with PBS and cells were loaded with 10 µM H2-DCF-DA for 30 min. After incubation, PBS and H2-DCF-DA were removed and the cells were washed twice. Increase in cell fluorescence was measured at excitation and emission wavelengths of 490 and 520 nm, respectively, using an Infinite 200 auto microplate reader (Tecan, Salzburg, Austria), at 25 °C. 20 Glutathione (GSH) level and Glutathione peroxidase (GPx) activity To assess the changes in the levels of GSH and GPx activity, cells were seeded in plate dishes and exposed to oxidized cholesterol (75 μg/mL) in complete medium for 0-24 h. In order to assess the effect of HT, TYR, HT-S and TYR-S, cells were pretreated with the tested compounds (5-25 µM in water solution, 30 min) prior to exposure to oxidized cholesterol 75 μg/mL for 30 min to determine GSH level and for 18 h to measure GPx activity. At the end of incubation time, cells were washed with PBS, and subsequently scraped into 25 500 μL of 5% metaphosphoric acid. Samples were then sonicated and centrifuged at 10000 × g for 20 min at 4 ºC; supernatants were collected and used to determine GSH level and GPx activity, using Glutathione Assay Kit and Glutathione peroxidase Assay Kit (Cayman Chemical Company, Ann Arbor, USA) according to the manufacturer’s instructions. Statistical analysis Data are expressed as means ± S.D (n=12 for each sample/condition). The statistical evaluation of the results was performed by analysis 30 of variance (ANOVA) followed by a Bonferroni post-hoc test using GraphPad InStat version 3.05 (GraphPad Software, San Diego, CA, USA). Journal Name Cite this: DOI: 10.1039/c0xx00000x www.rsc.org/xxxxxx Dynamic Article Links ► ARTICLE TYPE This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol] , 00–00 | 5 Results Synthesis of HT and TYR sulfate metabolites has been carried out using a protection-deprotection strategy together with the use of microwaves in the critical sulfation step. In the case of TYR, the primary alcohol was acylprotected using immobilized lipase Novozym 435® and vinyl butyrate in tert-butyl methyl ether (98 % yield) (Scheme 1). Next, microwave-assisted sulfation was performed by treatment with SO3.NMe3 complex and triethylamine in acetonitrile at 100 ºC (98% yield). Final deprotection with K2CO3 in MeOH 5 afforded tyrosol sulfate 5 in good yield (94%). 10 Scheme 1 Preparation of tyrosol sulfate 5 15 Mono-sulfated hydroxytyrosol derivatives 3 and 4 were prepared as a regioisomeric mixture using a synthetic strategy similar to that of TYR. In this case, hydroxytyrosol acetate 8 was mono-silyl protected to avoid disulfated products difficult to separate from the monosulfated derivatives (Scheme 2). 20 25 Scheme 2 Preparation of hydroxytyrosol sulfate metabolites 3 and 4 Random TBDMS-protection of HT acetate 8 and subsequent chromatographic separation afforded a 1:1 regioisomeric mixture of the two possible mono-phenolic compounds 9 and 10. The same sulfation reaction conditions used before were applied to give 94% yield of a 1:1 mixture of isomers 11 and 12. Finally, acetyl and silyl deprotection was carried out in one step using KF and K2CO3 in MeOH to obtain a 30 mixture of mono-sulfated hydroxytyrosol derivatives 3 and 4 (90% yield). To investigate the potential protective effect of the HT and TYR sulfate metabolites, in comparison with their parent compounds, against the pro-oxidant effect of oxidized cholesterol in intestinal cells, Caco-2 monolayers were treated for 24 h with a mixture obtained from the oxidation of cholesterol at 140 °C for 3 h. In these oxidizing conditions half of the initial cholesterol was turned into oxidation products (reported in the methods section). Treatment with growing concentration of the mixture induced production of MDA and cell 35 death (Fig. 2), indicating the presence of oxidative cell injury. 40 CREATED USING THE RSC ARTICLE TEMPLATE (VER. 3.0) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS ARTICLE TYPE www.rsc.org/xxxxxx | XXXXXXXX 6 | Journal Name, [year], [vol] , 00–00 This journal is © The Royal Society of Chemistry [year] 5 10 15 Fig.2 Values of MDA (2A) and cell viability (2B) measured in Caco-2 cells after 24 h incubation with different concentrations of oxidized cholesterol. * = p<0.05 versus control 20 Pretreatment with the phenolic compounds significantly inhibited the increase of MDA (Fig. 3) in Caco-2 cells treated with 75 g/mL of the oxidizing mixture, the highest amount able to induce a significant production of MDA but not cell death. MDA reduction was significant from 2.5 M for HT-S and from 5 M for HT; TYR and its sulfate metabolite exerted the same efficacy starting from 2.5 M. 25 30 35 40 45 50 Fig.3 Values of MDA measured in Caco-2 cells after 24 h incubation with 75 g/mL oxidized cholesterol and pretreated (30 min) with TYR, HT, HT sulfate metabolites 3-4 (HT-S) and TYR sulfate metabolite 5 (TYR-S) (2.5-10 M). * = p<0.05 versus control, a = p<0.05 versus oxidized cholesterol treated (Ox). A B 0 50 100 150 200 250 300 Control 25 50 75 100 125 150 175 MDA (% of the control) Oxidized cholesterol (µg/ml) * * * * * 0 40 80 120 Control 25 50 75 100 125 150 175 Cell viability (% of the control) Oxidized cholesterol (µg/ml) * * * * 0 40 80 120 160 Control Ox Ox + 2,5 Ox + 5 Ox + 10 MDA (% of the control) Compound (M) HT HT-S * a aa aa 0 40 80 120 160 Control Ox Ox + 2,5 Ox + 5 Ox + 10 MDA (% of the control) Compound (M) TYR TYR-S * a a a a a a Journal Name Cite this: DOI: 10.1039/c0xx00000x www.rsc.org/xxxxxx Dynamic Article Links ► ARTICLE TYPE This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol] , 00–00 | 7 Next we examined the ability of HT, TYR and their corresponding sulfate metabolites to protect cells against death in Caco-2 cells treated with 100 g/mL of oxidized cholesterol, the lowest amount able to induce a significant cell death (Fig. 4). We observed that HT preserved cell viability from the concentration of 5 M whereas HT sulfates 3-4 showed protection at the 10 M concentration. In the case of TYR and its sulfate derivative, both improved cell viability at 10 M concentration. 5 10 15 20 25 Fig.4 Cell viability measured in Caco-2 cells after 24 h incubation with 100g/mL oxidized cholesterol and pretreated (30 min) with TYR, HT or their sulfate metabolites (2.5-10 M). * = p<0.05 versus control, a=p<0. 05 versus oxidized cholesterol treated (Ox). 30 35 40 45 0 20 40 60 80 100 120 Control OX OX + 2,5 OX + 5 OX + 10 Cell viability (% of the control) Compound (M) HT HT-S * aa a *** 0 20 40 60 80 100 120 Control OX OX + 2,5 OX + 5 OX + 10 Cell viability (% of the control) Compound (M) TYR TYR-S aa ** ** * CREATED USING THE RSC ARTICLE TEMPLATE (VER. 3.0) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS ARTICLE TYPE www.rsc.org/xxxxxx | XXXXXXXX 8 | Journal Name, [year], [vol] , 00–00 This journal is © The Royal Society of Chemistry [year] 5 10 15 20 Fig.5 ROS level, expressed as % of the control samples, in Caco-2 cells, treated with 75 g/ml oxidized cholesterol for different incubation times (5A) or pretreated (30 min) with TYR, HT or their sulfate metabolites (5-25 M) and treated with oxidized cholesterol for 30 min (5B), exposed to 2',7'- dichlorodihydrofluorescein diacetate H2-DCF-DA (10 mM) for 30 min. * = p<0.05 versus control, a = p<0.05 versus oxidized cholesterol treated (Ox), °= p<0.05 versus 0 min 25 The oxidizing action of the mixture of oxidized cholesterol was then investigated monitoring the alteration of the cellular redox status with time: after 30 min of incubation a significant production of ROS was observed in the cells treated with the oxidized cholesterol in comparison with the control (Fig. 5A). Next, we measured ROS production at 30 min, after pretreatment with the different phenolic compounds (5, 10, 25 M). We observed that ROS production was significantly lower from the concentration of 10M for all the tested 30 compounds, except for TYR-S (Fig. 5B). After 30 min of incubation with the oxidized cholesterol, ROS production was associated with a significant reduction of Caco-2 cellular GSH, around 40% of the initial value, as shown in Figure 6A. The level of GSH increased with time, reaching the control value at 3/6 h, to decrease again thereafter. Pretreatment with both HT and TYR preserved the control level of GSH at all the tested concentrations and their sulfate metabolites exerted a comparable efficacy (Fig. 6B). Interestingly, none of the tested compounds was able to alter GSH level 35 when incubated alone (25M) in the experimental conditions used (p>0.5 versus control). With respect to the considered parameters, we did not observe any concentration dependent protective effect of the tested phenolic compounds, nor significant differences among parent compounds and metabolites. 40 45 B A 0 50 100 150 200 250 Control Ox Ox + 5 Ox + 10 Ox + 25 ROS level (% of the control) Compound (M) HT HT-S * a,* a * *a,* a,* 0 50 100 150 200 Control Ox Ox + 5 Ox + 10 Ox + 25 ROS level (% of the control) Compound (M) TYR TYR-S * aaa * ** 0 50 100 150 0 30 60 180 ROS level (%of the control) Incubation time (min)