Synthesis and Antioxidant Activity of Nitrohydroxytyrosol and Its Acyl

Article pubs.acs.org/JAFC

Synthesis and Antioxidant Activity of Nitrohydroxytyrosol and Its Acyl Derivatives Mariana Trujillo,†,∥ Elena Gallardo,†,∥ Andrés Madrona,† Laura Bravo,§ Beatriz Sarriá,§ José A. González-Correa,# Raquel Mateos,*,§ and José Luis Espartero*,† †

Department of Organic and Pharmaceutical Chemistry, Faculty of Pharmacy, University of Seville, Seville, Spain Department of Metabolism and Nutrition, Institute of Food Science, Technology and Nutrition (ICTAN), Spanish National Research Council (CSIC), Madrid, Spain # Department of Pharmacology, Faculty of Medicine, University of Malaga, Malaga, Spain §

ABSTRACT: A series of nitroderivatives has been synthesized from hydroxytyrosol, the natural olive oil phenol, to increase the assortment of compounds with putative effects against Parkinson’s disease. Nitrohydroxytyrosyl esters were obtained from nitrohydroxytyrosol using a chemoselective one-step, high-yield, transesterification procedure. The antioxidant activity of these new series of nitrocatechols was evaluated using FRAP, ABTS, and ORAC assays and compared to that of free hydroxytyrosol. The nitro functional group induced a significant increase in the antioxidant activity of nitrohydroxytyrosol compared to hydroxytyrosol. Regarding nitroester derivatives, variable antioxidant activity was observed depending on the acyl side-chain length; shorter chains maintained or even enhanced the antioxidant activity compared to nitrohydroxytyrosol, decreasing the activity with longer side chains in keeping with their lipophilic nature. Therefore, it may be concluded that nitroester derivatives of hydroxytyrosol, which may be obtained by a simple, high-yield reaction, have elevated antioxidant activity and thus present potential bioactivity. KEYWORDS: nitrocatechol, nitroester derivatives, hydroxytyrosol, antioxidant activity, Parkinson’s disease

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INTRODUCTION The phenolic fraction of virgin olive oil has proved to have antioxidant, hypocholesterolemic, antithrombotic, and antihypertensive properties in addition to improving endothelial function and vascular response, among other beneficial effects,1 confirming its positive influence on cardiovascular health. However, to date, only the beneficial effect of preventing lowdensity lipoproteins (LDL) oxidation has been accepted as a health claim. 2 Recently, within the framework of the PREDIMED study, olive oil consumption, specifically extravirgin olive oil, has been associated with reduced risk of cardiovascular disease and mortality in individuals at high cardiovascular risk,3 although no significant associations were found for cancer and all-cause mortality. Nevertheless, the number of studies showing olive oil anticancer and neuroprotective activities is increasing.1,4 Among the phenolic compounds in extra-virgin or virgin olive oil, hydroxytyrosol (HTy) is considered one of the most representative olive oil phenols, present mainly as the secoiridoid derivative, together with minor amounts of the free form and the acetylated derivative hydroxytyrosyl acetate.5 Hydroxytyrosyl acetate is of special interest because it is transported across the small intestinal epithelial cell barrier more efficiently than HTy,6 also showing a higher hepatic bioavailability.7 Moreover, hydroxytyrosyl acetate presents higher in vitro and ex vivo antioxidant activity than HTy,8 thus having the potential to protect against oxidative damage in HepG2 cells,9 DNA oxidative injury in blood cells,10 ironinduced oxidative stress in human cervical cells,11 and hypoxiareoxygenation injury in rat brain slices.12 © XXXX American Chemical Society

On the other hand, for years nitrocatechols such as entacapone and tolcapone with high cathecol-O-methyltransferase (COMT) inhibitory activity have been widely used in a combinatory therapy with levodopa in the clinical treatment of Parkinson’s disease.13,14 Whereas entacapone has been extensively used, tolcapone has been restricted to certain indications due to its hepatotoxicity.13 However, despite its catecholic structure, entacapone is a poor substrate for COMT, and its major urinary conjugates are rapidly excreted in humans.15 Taking into account the broad biological activity described for HTy and its acetylated derivative hydroxytyrosyl acetate, including their therapeutic potential against neurodegenerative diseases,1,4,12 the aims of the present study were to synthesize nitroester derivatives of HTy (3−10) with variable acyl sidechain lengths (Figure 1) from 2 to 16 carbon atoms and to evaluate their potential bioactivity by measuring the antioxidant activity by FRAP, ABTS, and ORAC assays. Hydroxytyrosol recovered from olive oil wastewaters was used as starting material for these purposes.

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MATERIALS AND METHODS

Materials. All solvents and reagents were of analytical grade unless stated otherwise. 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), anhydrous sodium sulfate, hexane, tetrahydrofuran (THF), p-toluenesulfonic acid, diethyl ether, potassium persulfate, Received: July 24, 2014 Revised: September 26, 2014 Accepted: September 29, 2014

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dx.doi.org/10.1021/jf503543x | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

Figure 1. Chemical structures of hydroxytyrosol (1), nitrohydroxytyrosol (2), and nitrohydroxytyrosyl esters (acetate (3), butyrate (4), hexanoate (5), octanoate (6), decanoate (7), laurate (8), myristate (9), and palmitate (10)). phenolic OHs), 7.46 (s, 1H, H7), 6.73 (s, 1H, H4), 4.20 (t, J = 6.5 Hz, 2H, H1), 3.07 (t, 2H, H2), 2.20 (t, J = 7.2 Hz, 2H, H2′), 1.48 (m, 2H, H3′), 0.81 (t, J = 7.4 Hz, 3H, H4′); 13C NMR (125 MHz, DMSO-d6) δ 172.5 (C1′), 151.2 (C5), 144.2 (C6), 139.7 (C8), 126.3 (C3), 118.5 (C4), 112.2 (C7), 63.2 (C1), 35.3 (C2′), 31.9 (C2), 17.8 (C3′), 13.3 (C4′); HRMS (FAB) m/z calcd for C12H15NO6Na [M + Na]+ 292.0797, found 292.0796 (0.4 ppm). Data for Nitrohydroxytyrosyl Hexanoate (5): 80% yield; obtained as a syrup; 1H NMR (500 MHz, DMSO-d6) δ 10.10 (bs, 2H, phenolic OHs), 7.46 (s, 1H, H7), 6.73 (s, 1H, H4), 4.20 (t, J = 6.5 Hz, 2H, H1), 3.07 (t, 2H, H2), 2.20 (t, J = 7.4 Hz, 2H, H2′), 1.45 (m, 2H, H3′), 1.21 (m, 4H, H4′ and H5′), 0.82 (t, J = 7.4 Hz, 3H, H6′); 13C NMR (125 MHz, DMSO-d6) δ 172.7 (C1′), 151.2 (C5), 144.1 (C6), 139.7 (C8), 126.2 (C3), 118.4 (C4), 112.2 (C7), 63.2 (C1), 33.4 (C2′), 31.8 (C2), 30.5 (C4′), 24.0 (C3′), 21.7 (C5′), 13.7 (C6′); HRMS (FAB) m/z calcd for C14H19NO6Na [M + Na]+ 320.1110, found 320.1107 (1.0 ppm). Data for Nitrohydroxytyrosyl Octanoate (6): 72% yield; obtained as a colorless liquid; 1H NMR (500 MHz, DMSO-d6) δ 10.10 (bs, 2H, phenolic OHs), 7.47 (s, 1H, H7), 6.73 (s, 1H, H4), 4.20 (t, J = 6.5 Hz, 2H, H1), 3.07 (t, 2H, H2), 2.20 (t, J = 7.4 Hz, 2H, H2′), 1.44 (m, 2H, H3′), 1.21 (m, 8H, H4′−H7′), 0.82 (t, J = 7.4 Hz, 3H, H8′); 13C NMR (125 MHz, DMSO-d6) δ 172.7 (C1′), 151.2 (C5), 144.2 (C6), 139.7 (C8), 126.3 (C3), 118.4 (C4), 112.2 (C7), 63.2 (C1), 33.5 (C2′), 31.9 (C2), 31.1 (C6′), 28.4−28.3 (C4′ and C5′), 24.4 (C3′), 22.0 (C7′), 13.9 (C8′); HRMS (CI) m/z calcd for C16H24NO6 [M + H]+ 326.1604, found 326.1601 (0.8 ppm). Data for Nitrohydroxytyrosyl Decanoate (7): 81% yield; obtained as a white solid; mp 65−66 °C; 1H NMR (500 MHz, DMSO-d6) δ 10.33 and 9.84 (2 bs, 2H, phenolic OHs), 7.47 (s, 1H, H7), 6.74 (s, 1H, H4), 4.20 (t, J = 6.5 Hz, 2H, H1), 3.07 (t, 2H, H2), 2.21 (t, J = 7.4 Hz, 2H, H2′), 1.45 (m, 2H, H3′), 1.22 (m, 12H, H4′−H9′), 0.84 (t, J = 6.9 Hz, 3H, H10′); 13C NMR (125 MHz, DMSO-d6) δ 172.6 (C1′), 151.1 (C5), 144.1 (C6), 139.7 (C8), 126.2 (C3), 118.4 (C4), 112.2 (C7), 63.2 (C1), 33.4 (C2′), 31.8 (C2), 31.2 (C8′), 28.7−28.3 (C4′− C7′), 24.2 (C3′), 22.0 (C9′), 13.8 (C10′); HRMS (FAB) m/z calcd for C18H27NO6Na [M + Na]+ 376.1736, found 376.1736 (0.0 ppm). Data for Nitrohydroxytyrosyl Laurate (8): 58% yield; obtained as a white solid; mp 76−77 °C; 1H NMR (500 MHz, DMSO-d6) δ 10.10 (bs, 2H, phenolic OHs), 7.47 (s, 1H, H7), 6.73 (s, 1H, H4), 4.20 (t, J = 6.5 Hz, 2H, H1), 3.07 (t, 2H, H2), 2.21 (t, J = 7.4 Hz, 2H, H2′), 1.44 (m, 2H, H3′), 1.22 (m, 16H, H4′−H11′), 0.84 (t, J = 6.9 Hz, 3H, H12′); 13 C NMR (125 MHz, DMSO-d6) δ 172.6 (C1′), 151.2 (C5), 144.1 (C6), 139.7 (C8), 126.2 (C3), 118.4 (C4), 112.2 (C7), 63.2 (C1), 33.4 (C2′), 31.8 (C2), 31.2 (C10′), 28.9−28.3 (C4′−C9′), 24.3 (C3′), 22.0 (C11′), 13.9 (C12′); HRMS (FAB) m/z calcd for C20H31NO6Na [M + Na]+ 404.2049, found 404.2045 (1.0 ppm). Data for Nitrohydroxytyrosyl Myristate (9). 60% yield; obtained as a white solid; mp 84−85 °C; 1H NMR (500 MHz, DMSO-d6) δ 10.11 (bs, 2H, phenolic OHs), 7.46 (s, 1H, H7), 6.73 (s, 1H, H4), 4.20 (t, J = 6.6 Hz, 2H, H1), 3.07 (t, 2H, H2), 2.20 (t, J = 7.4 Hz, 2H, H2′), 1.44 (m, 2H, H3′), 1.22 (m, 20H, H4′−H13′), 0.84 (t, J = 6.9 Hz, 3H, H14′); 13 C NMR (125 MHz, DMSO-d6) δ 172.7 (C1′), 151.2 (C5), 144.1 (C6), 139.7 (C8), 126.2 (C3), 118.4 (C4), 112.2 (C7), 63.2 (C1), 33.4 (C2′), 31.8 (C2), 31.2 (C12′), 29.0−28.3 (C4′−C11′), 24.3 (C3′), 22.0 (C13′), 13.9 (C14′); HRMS (FAB) m/z calcd for C22H35NO6Na [M + Na]+ 432.2362, found 432.2361 (0.2 ppm).

methanol, acetone, sodium nitrite, hydrogen chloride, iron(III) chloride hexahydrate, sodium hydrogen phosphate, potassium dihydrogen phosphate, acetic acid, sodium acetate trihydrate, hexadeuterated dimethyl sulfoxide (DMSO-d6), ethyl acetate, and methyl esters (butyrate, hexanoate, octanoate, decanoate, laurate, myristate, and palmitate) were from Aldrich (Madrid, Spain). Fluorescein, methylated β-cyclodextrin (RMCD), 2,2′-azinobis(3ethylbenzothiazoline-6-sulfonic acid) diammonium salt (98%) (ABTS), 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), and 2,4,6-tri-(2-pyridyl)-1,3,5-triazine (TPTZ) were from Sigma (Madrid, Spain). HTy was recovered with 95% purity from olive oil wastewaters16 and further purified by column chromatography. NMR spectra were recorded on a Bruker AVANCE 500 spectrophotometer operating at 500.13 MHz (1H) and 125.75 MHz (13C). Chemical shifts are given in parts per million, with the residual solvent signals (2.49 ppm for 1H and 39.5 ppm for 13C) as references. Samples were dissolved (10−20 mg/mL) in DMSO-d6, and spectra were recorded at 303 K. High-resolution CI or FAB mass spectra (HRMS) were obtained on a Micromass AUTOSPECQ spectrometer. Synthesis Procedures. Synthesis of Nitrohydroxytyrosol (2). HTy (1, 154 mg, 1 mmol) was added to 0.1 M acetate buffer (pH 3.8) (200 mL) followed by sodium nitrite (138 mg, 2 mmol). After 30 min at room temperature, the mixture was extracted with ethyl acetate (6 × 50 mL), and the combined organic layers were dried over anhydrous sodium sulfate and taken to dryness to give a yellow residue (180 mg). The resulting residue was purified by column chromatography (ethyl acetate/hexane 3:1) to obtain pure nitrohydroxytyrosol (2) (160.2 mg, 80.5% yield) as a yellow solid, previously described by Napolitano et al.:17 mp 197 °C (dec); 1H NMR (500 MHz, DMSO-d6) δ 10.05 (bs, 2H, phenolic OHs), 7.43 (s, 1H, H7), 6.75 (s, 1H, H4), 4.65 (bs, 1H, alcoholic OH), 3.56 (t, J = 6.8 Hz, 2H, H1), 2.90 (t, 2H, H2); 13C NMR (125 MHz, DMSO-d6) δ 150.9 (C5), 143.7 (C6), 139.7 (C8), 127.8 (C3), 118.5 (C4), 112.0 (C7), 61.0 (C1), 36.0 (C2); HRMS (CI) m/z calcd for C8H10NO5 [M + H]+ 200.0559, found 200.0566 (3.5 ppm). General Method for Esterification. p-Toluenesulfonic acid (10 mg) was added to a solution of nitrohydroxytyrosol (2, 199 mg, 1 mmol) in 16 mL of a 1:1 mixture of THF and the corresponding alkyl ester (acetate, butyrate, hexanoate, octanoate, decanoate, laurate, myristate, or palmitate). The resulting mixture was stirred at 60−65 °C until completion of the reaction (TLC). The solvent was evaporated, and the resulting residue was purified by column chromatography using silica gel as stationary phase and different hexane/diethyl ether mixtures as eluent to yield pure nitrohydroxytyrosyl ester derivatives (3−10), the chemical structures of which are depicted in Figure 1. Data for Nitrohydroxytyrosyl Acetate (3): 96% yield; obtained as a white solid; mp 107−109 °C; 1H NMR (500 MHz, DMSO-d6) δ 10.11 (bs, 2H, phenolic OHs), 7.46 (s, 1H, H7), 6.74 (s, 1H, H4), 4.18 (t, J = 6.6 Hz, 2H, H1), 3.06 (t, 2H, H2), 1.94 (s, 3H, H2′);13C NMR (125 MHz, DMSO-d6) δ 170.2 (C1′), 151.2 (C5), 144.2 (C6), 139.8 (C8), 126.2 (C3), 118.4 (C4), 112.2 (C7), 63.4 (C1), 31.8 (C2), 20.6 (C2′); HRMS (CI) m/z calcd for C10H11NO6 [M]•+ 241.0586, found 241.0591 (1.9 ppm). Data for Nitrohydroxytyrosyl Butyrate (4): 70% yield; obtained as a syrup; 1H NMR (500 MHz, DMSO-d6) δ 10.34 and 9.85 (2 bs, 2H, B



Article

Table 1. 1H NMR Data (500.13 MHz, DMSO-d6, 303 K) for Compounds 1−10a 1 phenethyl unit 1 3.49 (t) (J1′,2′ = 7.2) 2 2.52 (t) 4 6.57 (d) (J4′,8′ = 2.0) 7 6.60 (d) (J7′,8′ =8.0) 8 6.42 (dd) acyl chain 2′ 3′ 4′ 5′ 6′

2

3

4

5

6

7

8

9

10

3.56 (t) (J1′,2′ = 6.8) 2.90 (t) 6.75 (s)

4.18 (t) (J1′,2′ = 6.6) 3.06 (t) 6.74 (s)

4.20 (t) (J1′,2′ = 6.5) 3.07 (t) 6.73 (s)

4.20 (t) (J1′,2′ = 6.5) 3.07 (t) 6.73 (s)

4.20 (t) (J1′,2′ = 6.5) 3.07 (t) 6.73 (s)

4.20 (t) (J1′,2′ = 6.5) 3.07 (t) 6.74 (s)

4.20 (t) (J1′,2′ = 6.5) 3.07 (t) 6.73 (s)

4.20 (t) (J1′,2′ = 6.6) 3.07 (t) 6.73 (s)

4.20 (t) (J1′,2′ = 6.5) 3.07 (t) 6.73 (s)

7.43 (s)

7.46 (s)

7.46 (s)

7.46 (s)

7.47 (s)

7.47 (s)

7.47 (s)

7.46 (s)

7.47 (s)

1.94 (s)

2.20 (t) (3J = 7.2) 1.48 (m) 0.81 (t) (3J = 7.4)

2.20 (t) (3J = 7.4) 1.45 (m) 1.21 (m)

2.20 (t) (3J = 7.4) 1.45 (m) 1.21 (m)

2.21 (t) (3J = 7.4) 1.45 (m) 1.22 (m)

2.21 (t) (3J = 7.4) 1.44 (m) 1.22 (m)

2.20 (t) (3J = 7.4) 1.44 (m) 1.22 (m)

2.20 (t) (3J = 7.4) 1.44 (m) 1.22 (m)

1.21 (m) 0.82 (t) (3J = 7.4)

1.21 (m) 1.21 (m)

1.22 (m) 1.22 (m)

1.22 (m) 1.22 (m)

1.22 (m) 1.22 (m)

1.22 (m) 1.22 (m)

1.21 (m) 0.82 (t) (3J = 7.4)

1.22 (m) 1.22 (m)

1.22 (m) 1.22 (m)

1.22 (m) 1.22 (m)

1.22 (m) 1.22 (m)

1.22 (m) 0.84 (t) (3J = 6.9)

1.22 (m) 1.22 (m)

1.22 (m) 1.22 (m)

1.22 (m) 1.22 (m)

1.22 (m) 0.84 (t) (3J = 6.9)

1.22 (m) 1.22 (m)

1.22 (m) 1.22 (m)

1.22 (m) 0.84 (t) (3J = 6.9)

1.22 (m) 1.22 (m)

7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16′ a

1.22 (m) 0.84 (t) (3J = 6.9)

Chemical shifts (δ in parts per million) and coupling constants (J in hertz).

Data for Nitrohydroxytyrosyl Palmitate (10): 55% yield; obtained as a white solid; mp 90−91 °C; 1H NMR (500 MHz, DMSO-d6) δ 10.12 (bs, 2H, phenolic OHs), 7.47 (s, 1H, H7), 6.73 (s, 1H, H4), 4.20 (t, J = 6.5 Hz, 2H, H1), 3.07 (t, 2H, H2), 2.20 (t, J = 7.4 Hz, 2H, H2′), 1.44 (m, 2H, H3′), 1.22 (m, 24H, H4′−H15′), 0.84 (t, J = 6.9 Hz, 3H, H16′); 13C NMR (125 MHz, DMSO-d6) δ 172.6 (C1′), 151.2 (C5), 144.1 (C6), 139.7 (C8), 126.2 (C3), 118.4 (C4), 112.2 (C7), 63.2 (C1), 33.4 (C2′), 31.8 (C2), 31.2 (C14′), 29.0−28.3 (C4′−C13′), 24.3 (C3′), 22.0 (C15′), 13.9 (C16′); HRMS (FAB) m/z calcd for C24H39NO6Na [M + Na]+ 460.2675, found 460.2689 (3.0 ppm). Ferric Reducing Antioxidant Power (FRAP) Assay. The FRAP assay was carried out according to the procedure described by Pulido et al.18 The antioxidant potential of the synthesized compounds was estimated from their ability to reduce the ferric tripyridyltriazine (TPTZ−Fe(III)) complex to its stable ferrous form (TPTZ−Fe(II)). Briefly, the FRAP reagent contained 2.5 mL of a 10 mM TPTZ solution in 40 mM HCl, plus 2.5 mL of 20 mM FeCl3·6H2O and 25 mL of 0.3 M acetate buffer to a final pH of 3.6. This reagent was freshly prepared and warmed to 37 °C prior to use. Nine hundred microliters of FRAP reagent was mixed with 90 μL of distilled water and 30 μL of either a standard, methanol (as appropriate reagent blank), or a test sample (ranging from 50 to 400 μM for esters with shorter acyl chain (≤6) and from 100 to 1000 μM for esters with longer acyl chain (≥8)). All compounds were dissolved in methanol. Once the mixture was shaken, readings at the absorption maximum at 595 nm were taken every 20 s, and the reaction was monitored up to 30 min at 37 °C, using a UV−visible Varian (Cary 50 BIO) spectrophotometer equipped with a thermostated autocell-holder. The reading at 30 min was selected in each case for the calculation of FRAP

values. Methanolic solutions of Trolox were used for calibration. The FRAP values are expressed as millimolar TEAC (Trolox equivalent). All analyses were run in triplicate. ABTS Assay. The free radical scavenging capacity was measured using the ABTS decoloration method19 with some modifications. The method is based on the capacity of different components to scavenge the ABTS radical cation (ABTS•+) compared to a standard antioxidant (Trolox). Briefly, ABTS was dissolved in a 2.45 mM potassium persulfate solution and stored in the dark at room temperature for 12− 16 h, to set a 7 mM concentration of ABTS radical cation (ABTS•+) solution. The ABTS•+ stock solution was diluted with methanol to get an absorbance of 0.70 ± 0.02 at 730 nm. After the addition of 0.1 mL of sample dissolved in methanol (ranging from 50 to 400 μM for esters up to butyrate and from 100 to 500 μM for the rest of the compounds), methanol as a blank, or Trolox standard to 3.9 mL of diluted ABTS•+ solution, absorbance readings were taken every 20 s at 30 °C over 6 min, using a UV−visible spectrophotometer. The percentage inhibition of absorbance was plotted against time, and the area under the curve (0−360 s) was calculated. Methanolic solutions of known concentrations of Trolox were used for calibration. Results are expressed in millimolar TEAC (Trolox equivalent). Each value is the average of three determinations. Oxygen Radical Scavenging Capacity (ORAC) Assay. The oxygen radical scavenging capacity was measured by the lipophilic ORAC assay according to the method developed by Huang et al.20 with some modifications. The assay is based on the fluorescence decay of a reference substance (fluorescein) after the addition of a peroxyl radical (AAPH), which acts as an initiator of the oxidative reaction. Nitrohydroxytyrosol (2) and its derivatives (3−10) from 5 to 25 μM C



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Table 2. 13C NMR Chemical Shifts (Parts per Million; 125.76 MHz, DMSO-d6, 303 K) for Compounds 1−10 1 phenethyl unit 1 62.5 2 38.4 3 130.1 4 116.2 5 144.8 6 143.2 7 115.3 8 119.3 acyl chain 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16′

2

3

4

5

6

7

8

9

61.0 36.0 127.8 118.5 150.9 143.7 112.0 139.7

63.4 31.8 126.2 118.4 151.2 144.2 112.2 139.8

63.2 31.9 126.3 118.5 151.2 144.2 112.2 139.7

170.2 20.6

172.5 35.3 17.8 13.3

63.2 31.8 126.2 118.4 151.2 144.1 112.2 139.7

63.2 31.9 126.3 118.4 151.2 144.2 112.2 139.7

63.2 31.8 126.2 118.4 151.1 144.1 112.2 139.7

63.2 31.8 126.2 118.4 151.2 144.1 112.2 139.7

63.2 31.8 126.2 118.4 151.2 144.1 112.2 139.7

63.2 31.8 126.2 118.4 151.2 144.1 112.2 139.7

172.7 33.4 24.0 30.5 21.7 13.7

172.7 33.5 24.4 28.4−28.3 28.4−28.3 31.1 22.0 13.9

172.6 33.4 24.2 28.7−28.3 28.7−28.3 28.7−28.3 28.7−28.3 31.2 22.0 13.8

172.6 33.4 24.3 28.9−28.3 28.9−28.3 28.9−28.3 28.9−28.3 28.9−28.3 28.9−28.3 31.2 22.0 13.9

172.7 33.4 24.3 29.0−28.3 29.0−28.3 29.0−28.3 29.0−28.3 29.0−28.3 29.0−28.3 29.0−28.3 29.0−28.3 31.2 22.0 13.9

172.6 33.4 24.3 29.0−28.3 29.0−28.3 29.0−28.3 29.0−28.3 29.0−28.3 29.0−28.3 29.0−28.3 29.0−28.3 29.0−28.3 29.0−28.3 31.2 22.0 13.9

and Trolox standard (6.25, 12.5, 25, 50, 75, and 100 μM) were dissolved in 7% methylated β-cyclodextrin (RMCD) in acetone/water (1:1, v/v) solution. Then, 25 μL of either Trolox, test sample, or solvent as blank was added to a 96-well microplate followed by the addition of 150 μL of fluorescein work solution (8.5 × 10−5 mM) prepared in 75 mM phosphate buffer (pH 7.4). The microplate reader (Bio-Tek, Winooski, VT, USA) was programmed to record every 2 min for 120 min at 485 and 528 nm excitation and emission wavelengths, respectively, the fluorescence after the addition of 30 μL of AAPH (153 mM) as peroxyl radical generator, which was also prepared in 75 mM phosphate buffer (pH 7.4). Trolox was used for calibration, and values are expressed as millimolar TEAC (Trolox equivalent). All analyses were run in quadruplicate. Statistical Analysis. Results are expressed as means ± standard deviation (SD) of three measurements for the ABTS and FRAP assays and of four measurements for the ORAC assay. The data were subjected to a one-way analysis of variance (ANOVA) using the program SPSS 19.0. The level of significance was set at p < 0.05.

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each derivative by 2D heteronuclear correlation experiments (HSQC and HMBC spectra) and were in good agreement with the proposed structures. In addition, the study by HRMS spectrometry of the molecular ion for compounds 3−10 confirmed their calculated molecular masses and elemental compositions, with a mass deviation ranging between 0.0 and 3.0 ppm (see Materials and Methods). Antioxidant Activity Evaluation of Nitrohydroxytyrosol (2) and Its Acyl Derivatives (3−10). Results of the reducing power by the FRAP assay and the radical scavenging activity using ABTS and ORAC assays of nitrohydroxytyrosol (2) and its acyl derivatives (3−10) are summarized in Figure 2 and Table 3, expressed as millimolar TEAC (Trolox equivalent).

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RESULTS Preparation and Characterization of Nitrohydroxytyrosyl Esters (3−10). Hydroxytyrosol recovered from olive oil wastewaters (OOWW) was used as starting material for the synthesis of new compounds. Slight modifications to the procedure described by Napolitano et al.17 allowed increased yield of nitrohydroxytyrosol (2) formation from 60 to 80%. These modifications consisted in the use of a higher concentration of reactants and a shorter reaction time. Nitrohydroxytyrosol (2) was obtained as a yellow solid and further esterified following the procedure described by our research group21,22 to directly obtain the nitroester derivatives 3−10 (Figure 1) with moderate to excellent yields. Synthetic nitrohydroxytyrosyl esters (3−10) were characterized by NMR and HR-MS spectroscopy. 1H and 13C NMR chemical shifts (Tables 1 and 2, respectively) were unequivocally assigned for

Figure 2. Graphical representation of data from FRAP, ABTS, and ORAC assays of hydroxytyrosol (1), nitrohydroxytyrosol (2), and nitrohydroxytyrosyl esters (3−10). D



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Table 3. Reducing Power Evaluated Using the FRAP Assay and Radical Scavenging Activity Assessed Using ABTS and ORAC Assays of Hydroxytyrosol (1), Nitrohydroxytyrosol (2), and Nitrohydroxytyrosyl Esters (3−10)a compd 1b 2 3 4 5 6 7 8 9 10

FRAP assay (mM) 1.39 2.51 3.02 2.58 2.21 1.67 0.68 0.48 0.35 0.21

± ± ± ± ± ± ± ± ± ±

0.05e 0.04b 0.05a 0.03b 0.03c 0.02d 0.06f 0.05g 0.02h 0.03i

ABTS assay (mM) 0.84 2.13 2.42 2.12 1.86 1.74 1.68 1.50 1.35 1.36

± ± ± ± ± ± ± ± ± ±

0.02g 0.07b 0.08a 0.06b 0.05c 0.05d 0.06d 0.05e 0.04f 0.06f

± ± ± ± ± ± ± ± ± ±

DISCUSSION

Natural dietary antioxidants have the potential to be therapeutic and/or preventative agents against free radicals by combating oxidative stress. Reactive oxygen species accumulation in cellular components is the major factor causing molecular injury, which ultimately leads to cell aging as well as other agerelated degenerative diseases.23 Phenolic compounds in virgin olive oil have been widely studied, focusing on their antioxidant activity.24 Among these compounds, there is evidence that HTy (1) presents relevant bioactivity, which highlights its potential preventing oxidative stress.25 Recently, another compound also naturally present in virgin olive oil, hydroxytyrosyl acetate, is gaining interest due to its higher bioavailability,6,7 biological activity, and particularly antioxidant activity compared to HTy.8−12 Olive oil phenols have also shown neuroprotective properties,1,4,12 which prompted the hypothesis that nitrocatechol derivatives of HTy might present interesting biological activities with potential as effective and safer therapeutic alternatives to the nitrocatechols currently used in the treatment of Parkinson’s disease.13−15 In the present work, the synthesis of nitrohydroxytyrosol and up to eight derivatives is presented together with the characterization of their antioxidant activity. After nitrohydroxytyrosol (2) was obtained, a chemoselective transesterification procedure21,22 was applied to esterify it, yielding nitrohydroxytyrosyl acetate (3) as well as other more lipophilic nitrohydroxytyrosyl ester derivatives such as butyrate, hexanoate, octanoate, decanoate, laurate, myristate, and palmitate, labeled 4, 5, 6, 7, 8, 9, and 10, respectively (Figure 1). Subsequently, in the present work the antioxidant activity characterization of nitrohydroxytyrosol (2) and its acyl derivatives (3−10) was evaluated using complementary methods (FRAP, ABTS, and ORAC) and comparatively studied against results previously published for free HTy.8 As expected, the synthesis of nitroester derivatives (3−10) took place in good to excellent yield. The esterification reaction used to obtain the nitroester derivatives (3−10) had been previously set up21,22 and presented significant advantages with respect to other methods used to synthesize lipophilic ester phenolic derivatives.26,27 According to the results obtained, the antioxidant activity of nitro derivatives of HTy (2−10) depended on the length of the acyl side chain. The addition of a nitro functional group to the HTy chemical structure, yielding nitrohydroxytyrosol (2), induced a positive, significant increase in the antioxidant activity (Table 3). The enhanced antioxidant capacity of nitrohydroxytyrosol (2) is in accordance with the stabilization of the phenoxyl radical by ortho substitutions with electrondonating groups described by other authors.28,29 As mentioned, the length of the linear acyl side chain of nitroester derivatives (3−10) induced variable effects on antioxidant activity. The shorter acyl side chains (from two to four carbon atoms) stabilized the phenoxyl radical, thus enhancing or maintaining the antioxidant activity of their precursor (2), whereas longer acyl side chains (six or more carbon atoms) induced a negative effect on the antioxidant activity, probably due to steric hindrance. These results disagree with the polar paradox that assumes that there is a linear relationship between the hydrophobic character and antioxidant capacity of a compound.30 Nevertheless, they are in accordance with results previously reported for hydroxytyrosyl esters,8 homovanillyl esters,31 chlorogenate esters,32 rosmarinate

ORAC assay (mM) 1.92 2.48 2.61 2.51 2.22 2.14 1.94 1.88 1.63 1.56

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0.04d 0.04b 0.05a 0.04b 0.06c 0.04c 0.05d 0.04d 0.04e 0.05e

The data represent the mean ± standard deviation of three determinations for FRAP and ABTS assays and four determinations for ORAC assay. Results are expressed as mM Trolox equivalent (TEAC, mM). All values within a column with different letters are significantly different, p < 0.05. bFRAP and ABTS values of HTy (1) have been previously published by Mateos et al.8 and are included for comparative purposes. a

FRAP Assay. Nitrohydroxytyrosol (2) exhibited higher reducing activity than HTy (1). The antioxidant activity of the new nitrohydroxytyrosyl esters (3−10) in comparison with their precursor nitrohydroxytyrosol (2) varied depending on the length of the acyl side chain. Thus, nitrohydroxytyrosyl acetate (3) presented significantly higher antioxidant activity than nitrohydroxytryrosol (2), whereas nitrohydroxytyrosyl butyrate (4) showed similar activity, but the more lipophilic compounds hexanoate (5) and octanoate (6) presented lower activity and decanoate, laurate, myristate, and palmitate (7−10) presented activity even lower than that of HTy (1). ABTS Assay. The radical scavenging activities of nitrohydroxytyrosol (2) and its acyl derivatives, nitrohydroxytyrosyl esters (3−10), showed a tendency similar to the reducing activity. The nitro functional group induced a positive effect on antioxidant activity because nitrohydroxytyrosol (2) as its acyl derivatives (3−10) showed higher antioxidant activity than their precursor HTy (1). Within the nitro derivatives, nitrohydroxytyrosol (2) showed lower activity than nitrohydroxytyrosyl acetate (3), similar to that of butyrate derivative (4) and higher than the rest of the tested ester derivatives (5− 10). ORAC Assay. The oxygen radical scavenging capacities of nitrohydroxytyrosyl esters (3−10) and their precursors, nitrohydroxytyrosol (2) and HTy (1), were in agreement with the results from the FRAP and ABTS analyses. Nitrohydroxytyrosol (2) showed higher antioxidant capacity than HTy (1), whereas its acyl derivatives (3−10) showed different behaviors related to the side-chain length; that is, the shorter chain of nitrohydroxytyrosyl acetate (3) and butyrate (4) was associated with higher and similar activity, respectively, whereas the longer acyl chains (5−10) were related with significantly lower antioxidant capacities with respect to the nitro derivative precursor (2). Almost all nitro derivatives (2− 7) showed potency higher than or equal to that of HTy (1) except laurate (8), myristate (9), and palmitate (10) derivatives. E



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esters,33 and other lipophilic HTy derivatives such as alkyl hydroxytyrosyl ethers.34 In fact, this nonlinear phenomenon called the cutof f ef fect, coined by Laguerre et al.,32 has been observed in both biological and physicochemical systems.35 Three possible mechanisms of actions have been proposed to explain this effect: the reduced mobility, internalization, and selfaggregation hypotheses,36 but no definitive conclusions have been yet reached.37 Moreover, a nonlinear association between biological activity and the lipophilic nature of homologous series of molecules has already been described. Ester derivatives of gallic acid were cytotoxic to L1210 leukemia cells,38 whereas hydroxytyrosyl ethers showed antiplatelet effects in blood from healthy human volunteers39 and after oral administration to rats.40 Additionally, cytotoxic activity of hydroxytyrosol alkyl ether derivatives against A549 lung cancer cells and MRC5 nonmalignant lung fibroblasts was recently described.41 In all of the aforementioned studies, biological activity increased up to medium length of the acyl or alkyl side chain, whereas the most lipophilic compounds showed lower biological activities. In conclusion, a series of nitroderivatives has been synthesized from natural olive oil phenols to increase the assortment of compounds with a putative effect against Parkinson’s disease. Compounds with shorter acyl side lengths showed higher antioxidant activities compared to HTy, whereas this activity progressively decreased further with the length of the acyl side chain in good accordance with the cutoff theory. Experiments to test their neuroprotective potential will follow.

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(5) Mateos, R.; Espartero, J. L.; Trujillo, M.; Rios, J. J.; LeonCamacho, M.; Alcudia, F.; Cert, A. Determination of phenols, flavones, and lignans in virgin olive oils by solid-phase extraction and highperformance liquid chromatography with diode array ultraviolet detection. J. Agric. Food Chem. 2001, 49, 2185−2192. (6) Mateos, R.; Pereira-Caro, G.; Saha, S.; Cert, R.; RedondoHorcajo, M.; Bravo, L.; Kroon, P. A. Acetylation of hydroxytyrosol enhances its transport across differentiated CaCo-2 cell monolayers. Food Chem. 2011, 125, 865−872. (7) Mateos, R.; Goya, L.; Bravo, L. Metabolism of olive oil phenols hydroxytyrosol, tyrosol, and hydroxytyrosyl acetate by human hepatoma HepG2 cells. J. Agric. Food Chem. 2005, 53, 9897−9905. (8) Mateos, R.; Trujillo, M.; Pereira-Caro, G.; Madrona, A.; Cert, A.; Espartero, J. L. New lipophilic tyrosyl esters. Comparative antioxidant evaluation with hydroxytyrosyl esters. J. Agric. Food Chem. 2008, 56, 10960−10966. (9) Pereira-Caro, G.; Mateos, R.; Sarria, B.; Cert, R.; Goya, L.; Bravo, L. Hydroxytyrosyl acetate contributes to the protective effects against oxidative stress of virgin olive oil. Food Chem. 2012, 131, 869−878. (10) Grasso, S.; Siracusa, L.; Spatafora, C.; Renis, M.; Tringali, C. Hydroxytyrosol lipophilic analogues: enzymatic synthesis, radical scavenging activity and DNA oxidative damage protection. Bioorg. Chem. 2007, 35, 137−152. (11) Bouallagui, Z.; Bouaziz, M.; Lassoued, S.; Engasser, J. M.; Ghoul, M.; Sayadi, S. Hydroxytyrosol acyl esters: biosynthesis and activities. Appl. Biochem. Biotechnol. 2011, 163, 592−599. (12) Gonzalez-Correa, J. A.; Navas, M. D.; Lopez-Villodres, J. A.; Trujillo, M.; Espartero, J. L.; De La Cruz, J. P. Neuroprotective effect of hydroxytyrosol and hydroxytyrosol acetate in rat brain slices subjected to hypoxia-reoxygenation. Neurosci. Lett. 2008, 446, 143− 146. (13) Gordin, A.; Kaakkola, S.; Teravainen, H. Clinical advantages of COMT inhibition with entacapone − a review. J. Neural Transm. 2004, 111, 1343−1363. (14) Bonifacio, M. J.; Palma, P. N.; Almeida, L.; Soares-da-Silva, P. Cathecol-O-methyltransferase and its inhibitors in Parkinson diseases. CNS Drugs 2007, 13, 352−379. (15) Lautala, P.; Kivimaa, M.; Salomies, H. Glucuronidation of entacapone, nitecapone, tolcapone and some other nitrocatechols by rat liver microsomes. Pharm. Res. 1997, 14, 1444−1448. (16) Fernández-Bolaños, J.; Heredia, A.; Rodríguez, G.; Rodríguez, R.; Jimén ez, A.; Gillen, R. Methods for obtaining purified hydroxytyrosol from products and byproducts derived from the olive tree. U.S. 6849,770 B2, 2005. (17) Napolitano, A.; Panzella, L.; Savarese, M.; Sacchi, R.; Giudicianni, I.; Paolillo, L.; d’Ischia, M. Acid-induced structural modifications of unsaturated fatty acids and phenolic olive oil constituents by nitrite ions: a chemical assessment. Chem. Res. Toxicol. 2004, 17, 1329−1337. (18) Pulido, R.; Bravo, L.; Saura-Calixto, F. Antioxidant activity of dietary polyphenols as determined by a modified ferric reducing/ antioxidant power assay. J. Agric. Food Chem. 2000, 48, 3396−3402. (19) Re, R.; Pellegrini, N.; Proteffente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant activity applying an improved ABTS radical cation decolouration assay. Free Radical Biol. Med. 1999, 26, 1231− 1237. (20) Huang, D.; Ou, B.; Hampsch-Woodill, M.; Flanagan, J. A.; Deemer, E. K. Development and validation of oxygen radical absorbance capacity assay for lipophilic antioxidants using randomly methylated β-cyclodextrin as the solubility enhancer. J. Agric. Food Chem. 2002, 50, 1815−1821. (21) Alcudia, F.; Cert, A.; Espartero, J. L.; Mateos, R.; Trujillo, M. Method of preparing hydroxytyrosyl esters, esters thus obtained and use of same. PCT WO 2004/005237, 2004. (22) Trujillo, M.; Mateos, R.; Collantes de Teran, L.; Espartero, J. L.; Cert, R.; Jover, M.; Alcudia, F.; Bautista, J.; Cert, A.; Parrado, A. Lipophilic hydroxytyrosyl esters. Antioxidant activity in lipid matrices and biological systems. J. Agric. Food Chem. 2006, 54, 3779−3785.

AUTHOR INFORMATION

Corresponding Authors

*(R.M.) Mail: Department of Metabolism and Nutrition, Instituto de Ciencia y Tecnologı ́a de Alimentos y Nutrición (ICTAN-CSIC), C/José Antonio Nováis 10, Ciudad Universitaria, Madrid, Spain. E-mail: [email protected]. *(J.L.E.) Mail: Department of Organic and Pharmaceutical Chemistry, Faculty of Pharmacy, University of Seville, C/Prof. Garcia Gonzalez 2, E41012 Seville, Spain. E-mail: [email protected]. Author Contributions ∥

M.T. and E.G. contributed equally to this work.

Funding

This work was supported by Grant P09-AGR-5098 from Junta de Andaluciá (Spain). E.G. thanks Junta de Andaluciá for a predoctoral fellowship. Notes

The authors declare no competing financial interest.

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Synthesis and Antioxidant Activity of Nitrohydroxytyrosol and Its Acyl

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