Preparation and antioxidant activity of tyrosyl and homovanillyl ethers

Article abstract of DOI:10.1016/j.foodchem.2011.05.098

Preparation of tyrosyl and homovanillyl lipophilic derivatives was carried out as a response to the food industry's increasing demand for new synthetic lipophilic antioxidants. Tyrosyl and homovanillyl ethers were synthesized in high yields by a three-step procedure starting from tyrosol (Ty) and homovanillic alcohol (HMV). The antioxidant activity of these new series of alkyl tyrosyl and homovanillyl ethers was evaluated by the Rancimat test in a lipophilic food matrix and by the FRAP, ABTS and ORAC assays and compared to free Ty and HMV as well as two antioxidants widely used in the food industry, butylhydroxytoluene (BHT) and α-tocopherol. The results pointed out the higher activity of homovanillyl series in comparison with tyrosyl series with all the assayed methods. However, while both synthetic series were less antioxidant than BHT and α-tocopherol in a lipophilic matrix after their Rancimat test evaluation, homovanillyl alkyl ethers showed the best reducing power and radical scavenging activity of all evaluated compounds. This batch of synthetic lipophilic compounds, derived from biologically active compounds such as Ty and HMV, provide interesting and potentially bioactive compounds.

Full text of DOI:10.1016/j.foodchem.2011.05.098

Food Chemistry 129 (2011) 1169–1178
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Preparation and antioxidant activity of tyrosyl and homovanillyl ethers
A. Madrona a, G. Pereira-Caro b, L. Bravo c, R. Mateos c, J.L. Espartero a,
⇑
a Departamento de Química Orgánica y Farmacéutica, Facultad de Farmacia, Universidad de Sevilla, E-41012 Sevilla, Spain
b IFAPA, Centro Venta del Llano, Bailen-Motril, Km 18.5, E-23620 Mengíbar, Jaén, Spain
c CSIC, ICTAN-Instituto del Frío, C/José Antonio Novais 10, Ciudad Universitaria, E-28040 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Preparation of tyrosyl and homovanillyl lipophilic derivatives was carried out as a response to the food
industry’s increasing demand for new synthetic lipophilic antioxidants. Tyrosyl and homovanillyl ethers
were synthesized in high yields by a three-step procedure starting from tyrosol (Ty) and homovanillic
alcohol (HMV). The antioxidant activity of these new series of alkyl tyrosyl and homovanillyl ethers
was evaluated by the Rancimat test in a lipophilic food matrix and by the FRAP, ABTS and ORAC assays
and compared to free Ty and HMV as well as two antioxidants widely used in the food industry, buty-
Received 4 October 2010
Received in revised form 29 March 2011
Accepted 20 May 2011
Available online 26 May 2011
Keywords:
Olive
Hydroxytyrosol
Tyrosol
lhydroxytoluene (BHT) and
ies in comparison with tyrosyl series with all the assayed methods. However, while both synthetic series
were less antioxidant than BHT and -tocopherol in a lipophilic matrix after their Rancimat test evalua-
a-tocopherol. The results pointed out the higher activity of homovanillyl ser-
a
tion, homovanillyl alkyl ethers showed the best reducing power and radical scavenging activity of all
evaluated compounds. This batch of synthetic lipophilic compounds, derived from biologically active
compounds such as Ty and HMV, provide interesting and potentially bioactive compounds.
Ó 2011 Elsevier Ltd. All rights reserved.
Homovanillic alcohol
Alkyl ethers
Antioxidant activity
Lipophilic antioxidants
Rancimat
ABTS
FRAP
ORAC
1. Introduction
effect against oxidative injury in cell models (Giovannini et al.,
1999) and improves the intracellular antioxidant defence systems
It is well known that lipid peroxidation decreases food’s nutri-
tive value and deteriorates its flavour and taste. Food industry at-
tempts to prevent food oxidation using food additives, in order to
improve its quality. In this sense, numerous phenolic antioxidants
have the potential to be used in hydrophilic food matrices. Among
natural polyphenols, olive oil phenols and, particularly, hydroxyty-
rosol, (2-(30,40-dihydroxyphenyl)ethanol, HTy, 1a), has the capacity
to protect against oxidative stress by scavenging radical species
(Goya, Mateos, & Bravo, 2007; Rietjents, Bast & Haenen, 2007)
and by inducing antioxidant enzymes (Martín et al., 2010). Fur-
thermore, several studies have demonstrated that HTy has cardio-
protective effects (De la Torre-Carbot et al., 2010; Rietjents, Bast,
Vente & Haenen, 2007), anti-inflammatory (Bitler, Viale, Damaj, &
Crea, 2005) and antiplatelet aggregation activity (Dell’Agli et al.,
2008), largely related to its antioxidant properties.
(Di Benedetto et al., 2007). In fact, one of the tyrosyl secoiridoid
derivatives present in olive oil, oleocanthal, has shown anti-inflam-
matory activity similar to ibuprofen (Beauchamp et al., 2005). Fur-
thermore, recent studies suggest that specifically mono-phenols as
Ty or p-coumaric acid as well as o-diphenolic compounds inhibit
homocysteine-induced endothelial cell adhesion, regardless their
antioxidant activity, playing a key role in the control of several
inflammation-associated processes (Manna, Napoli, Cacciapuoti,
Porcelli, & Zappia, 2009).
Taking into account the high potential effectiveness as antioxi-
dants of these virgin olive oil polyphenols and the great interest in
the use of phytochemicals as biological ingredients for functional
foods with enhanced nutritional value, HTy (1a) has already been
used as a bioactive ingredient in tomato juice (Larrosa, Espín, &
Tomás-Barberán, 2003) and fish products (Pazos, Alonso, Sánchez,
& Medina, 2008), showing good results. To our knowledge, no
applications has been studied for Ty (1b) or HMV (1c), which are
present in the phenolic fraction of virgin olive oil, and may be
effectively recovered from olive oil wastewaters, similarly to HTy
(Fernández-Bolaños et al., 2005).
In contrast, Ty (1b), another natural olive oil phenol, has signif-
icantly lower antioxidant capacity than HTy (Mateos, Domínguez,
Espartero, & Cert, 2003) due to the absence of the ortho-diphenolic
group in its chemical structure. Nevertheless, Ty exerts a protective
In response to the lack of antioxidants to be used in lipidic
foods, new lipophilic derivatives have been investigated in the last
⇑
Corresponding author.
E-mail address: jles@us.es (J.L. Espartero).
0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.05.098

1170
A. Madrona et al. / Food Chemistry 129 (2011) 1169–1178
years. In this sense, several chemical (Alcudia, Cert, Espartero,
Mateos, & Trujillo, 2004; Appendino, Minassi, Daddario, Bianchi,
& Tron, 2002; Bernini, Mincione, Barontini, & Crisante, 2008; Cap-
asso, Sannino, De Martino, & Manna, 2006; Gordon, Paiva-Martins,
& Almeida, 2001; Palozza et al., 2008; Tofani, Balducci, Gasperi,
Incerpi, & Gambacorta, 2010; Torregiani, Seu, Minassi, & Appendino,
2005; Trujillo et al., 2006) or enzymatic (Alcudia et al., 2004;
Bouallagui et al., 2011; Buisman et al., 1998; Grasso, Siracusa,
Spatafora, Renis, & Tringali, 2007; Lucas et al., 2010; Mateos
et al., 2008; Torres de Pinedo, Peñalver, & Morales, 2007; Torres
de Pinedo, Peñalver, Pérez-Victoria, Rondón, & Morales, 2007;
Torres de Pinedo, Peñalver, Rondon, & Morales, 2005) synthesis
reactions of lipophilic esters derivatives of hydroxytyrosol, homo-
vanillic alcohol and/or tyrosol esters have been reported and
recently reviewed (Chillemi, Sciuto, Spatafora, & Tringali, 2010;
hydrogen phosphate and potassium dihydrogen phosphate were
from Panreac (Madrid, Spain). Tyrosol (1b), homovanillic alcohol
(1c) and the alkyl iodides (methyl, ethyl, n-propyl, n-butyl, n-hexyl,
n-octyl and n-dodecyl iodides) were from Aldrich (Steinheim,
Germany). Fluorescein, methylated b-cyclodextrin (RMCD), 2,
20-azobis(2-amidinopropane) dihydrochloride (AAPH), 2,20-azino
bis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt
(98%), 2,4,6-tri-(2-pyridyl)-1,3,5-triazine (TPTZ) were from Sigma
(Madrid, Spain).
NMR spectra were recorded on a Bruker Avance 500 spectro-
photometer operating at 500.13 MHz (1H) and 125.75 MHz (13C).
Samples were dissolved (0.1 mmol/ml) in hexadeuterated methyl-
sulfoxide (DMSO-d6), and spectra were recorded at 303 K. Chemi-
cal shifts are given in ppm with the residual solvent signals
(2.49 ppm for 1H and 39.5 ppm for 13C) as references. Elemental
analyses were made on a Leco CHNS-932 apparatus. High-resolu-
tion EI, CI and FAB mass spectra were obtained on a Micromass
AUTOSPECQ spectrometer.
Fernández-Bolanos, Lopez, Fernández-Bolanos,
&
Rodríguez-
Gutiérrez, 2008). The antioxidant effects of the new series of esters
derivatives, containing lipophilic acyl chains of different length
with increasing lipophilicity, have been tested using different
methods. Remarkable antioxidant capacity has been observed
when the new compounds were tested in cell lines (Bouallagui
et al., 2011; Grasso et al., 2007; Tofani et al., 2010) and in food
matrices, such as oils and oil-in-water emulsions (Lucas et al.,
2010; Mateos et al., 2008; Medina, Lois, Alcántara, Lucas, &
Morales, 2009; Torres de Pinedo, Peñalver, & Morales, 2007; Torres
de Pinedo, Peñalver, Pérez-Victoria et al., 2007; Trujillo et al.,
2006). Having been evaluated using different methods, it may be
concluded that these new esters derivatives possess slightly higher
or comparable antioxidant activity than their respective
precursors.
Recently, a new group of lipophilic hydroxytyrosyl derivatives,
hydroxytyrosyl ethers (Madrona et al., 2009), with linear alkyl side
chains of variable length, have been synthesised by our group.
These new derivatives of HTy (1a) showed comparable or even
higher antioxidant capacity than free HTy (Pereira-Caro et al.,
2009) and higher bioavailability at both intestinal (Pereira-Caro
et al., 2010) and hepatic levels (Pereira-Caro, Bravo, Madrona,
Espartero, & Mateos, 2010). Taking into account the enhanced anti-
oxidant properties of alkyl hydroxytyrosyl ethers in comparison
with their precursor HTy (1a) and the promising biological activity
described for Ty (1b), the synthesis of alkyl tyrosyl derivatives
could be an interesting alternative to meet the food industry needs.
Moreover, homovanillyl ethers have a great potential as phyto-
chemicals, considering that their precursor homovanillic alcohol
(HMV, 1c) presents biological activity against oxidative kidney cell
injury (Incani et al., 2010), and is one of the identified metabolites
after human virgin olive oil intake (Caruso, Visioli, Patelli, Galli, &
Galli, 2001; Miró-Casas et al., 2003; Visioli et al., 2000; Vissers,
Zock, Roodenburg, Leenen, & Katan, 2002). Bearing this in mind,
in the present work the synthesis of tyrosyl (4h–n) and homovan-
illyl ethers (4o–u) and their oxidative stability in lipid matrices
spiked with these new synthetic compounds (4h–u) by Rancimat
test, was carried out. Moreover, reducing power by FRAP assay
and radical scavenging activity by ABTS and ORAC assays of tyrosyl
and homovanilly ethers (4h–u) were also assessed.
2.2. Synthetic procedures
2.2.1. General procedure for alkylation of 2b and 2c
A mixture of 2b (Ajao, Bird, & Chauhan, 1985) or 2c (Battersby,
Le Count, Garratt, & Thrift, 1961) (1 mmol), KOH (335 mg) and the
corresponding alkyl iodide (3 mmol) in methylsulfoxide (12 ml)
was stirred at room temperature until completion of reaction (thin
layer chromatography, TLC). 3 M HCl (25 ml) was added and the
mixture was extracted with CHCl3 (3 ꢀ 25 ml). The organic phase
was further washed with 2% NaHSO3 (25 ml) and water (25 ml),
dried over anhydrous Na2SO4, filtered and evaporated. The crude
products were purified by flash column chromatography over silica
gel yielding the desired products 3h–u.
1-(Benzyloxy)-4-(2-methoxyethyl)benzene (3h): colourless liquid
(85% yield); 1H-NMR d ppm 7.37 (m, 5H, Ph), 7.12 (d, J = 8.7 Hz, 2H,
H5), 6.90 (d, J = 8.7 Hz, 2H, H4), 5.05 (s, 2H, CH2Ph in pos. 6), 3.46 (t,
J = 6.9 Hz, 2H, H1), 3.21 (s, 3H, H1 ), 2.71 (t, J = 6.9 Hz, 2H, H2); 13C-
0
NMR d ppm 156.6 (C6), 137.2 (Cipso, Bn group), 131.1 (C3), 129.7
(C4), 128.3–127.5 (C2–C4, Bn group), 114.5 (C5), 72.9 (C1), 69.1
0
(CH2Ph in pos. 6), 57.7 (C1 ), 34.4 (C2). Elem. Anal. Calcd for
C16H18O2: C, 79.31; H, 7.49; found: C, 79.27; H, 7.27; HRMS,
242.1302 (2 ppm).
1-(Benzyloxy)-4-(2-ethoxyethyl)benzene (3i): colourless liquid
(89% yield); 1H-NMR d ppm 7.37 (m, 5H, Ph), 7.12 (d, J = 8.7 Hz,
2H, H5), 6.90 (d, J = 8.7 Hz, 2H, H4), 5.05 (s, 2H, CH2Ph in pos. 6),
0
3.49 (t, J = 7.1 Hz, 2H, H1), 3.39 (q, J = 7.0 Hz, 2H, H1 ), 2.71 (t,
J = 7.1 Hz, 2H, H2), 1.07 (t, J = 7.0 Hz, 3H, H2 ); 13C-NMR d ppm
0
156.6 (C6), 137.2 (Cipso, Bn group), 131.1 (C3), 129.5 (C4), 128.3–
127.5 (C2–C4, Bn group), 114.9 (C5), 71.1 (C1), 69.1 (CH2Ph in pos.
0
0
6), 65.1 (C1 ), 34.7(C2), 15.0 (C2 ). Elem. Anal. Calcd for C17H20O2:
C, 79.65; H, 7.86; found: C, 79.46; H, 7.95; HRMS, 256.1465
(0.7 ppm).
1-(Benzyloxy)-4-(2-propoxyethyl)benzene (3j): colourless liquid
(89% yield); 1H-NMR d ppm 7.37 (m, 5H, Ph), 7.12 (d, J = 8.7 Hz,
2H, H5), 6.90 (d, J = 8.7 Hz, 2H, H4), 5.05 (s, 2H, CH2Ph in pos. 6),
0
3.49 (t, J = 7.1 Hz, 2H, H1), 3.31 (t, J = 6.6 Hz, 2H, H1 ), 2.71 (t,
0
0
J = 7.1 Hz, 2H, H2), 1.47 (m, 2H, H2 ) 0.83 (t, J = 7.4 Hz, 3H, H3 );
13C-NMR d ppm 156.6 (C6), 137.2 (Cipso, Bn group), 131.1 (C3),
0
2. Materials and methods
129.6 (C4), 128.3–127.5 (C2–C4, Bn group), 114.4 (C5), 71.5 (C1 ),
0
0
71.0 (C1), 69.1 (CH2Ph in pos. 6), 34.6 (C2), 22.3 (C2 ), 10.4 (C3 ).
Elem. Anal. Calcd for C18H22O2: C, 79.96; H, 8.20; found: C, 80.17;
H, 8.18; HRMS, 270.1630 (3.8 ppm).
2.1. Materials
All solvents and reagents were of analytical grade unless other-
wise stated. -Tocopherol, 2,6-di-tert-butyl-4-methylphenol (BHT)
and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
(Trolox) were from Aldrich (Madrid, Spain). Benzyl bromide was
from Fluka (Steinheim, Germany). Sodium hydroxide, sodium
1-(Benzyloxy)-4-(2-butoxyethyl)benzene (3k): colourless liquid
(87% yield); 1H-NMR d ppm 7.37 (m, 5H, Ph), 7.12 (d, J = 8.7 Hz,
2H, H5), 6.90 (d, J = 8.7 Hz, 2H, H4), 5.05 (s, 2H, CH2Ph in pos. 6),
0
3.49 (t, J = 7.0 Hz, 2H, H1), 3.34 (t, J = 6.5 Hz, 2H, H1 ), 2.71
0
0
(t, J = 7.0 Hz, 2H, H2), 1.44 (m, 2H, H2 ) 1.27 (m, 2H, H3 ), 0.84

A. Madrona et al. / Food Chemistry 129 (2011) 1169–1178
1171
(t, J = 7.4 Hz, 3H, H4 ); 13C-NMR d ppm 156.6 (C6), 137.2 (Cipso, Bn
ppm 149.0 (C5), 146.1 (C6), 137.2 (Cipso, Bn group), 131.9 (C3),
128.3–127.5 (C2–C4, Bn group), 120.5 (C8), 113.7 (C7), 113.0 (C4),
0
group), 131.1 (C3), 129.6 (C4), 128.3–127.5 (C2–C4, Bn group),
0
0
114.4 (C5), 71.1 (C1), 69.5 (C1 ), 69.1 (CH2Ph in pos. 6), 34.6 (C2),
71.5 (C1 ), 71.1 (C1), 70.0 (CH2Ph in pos. 6), 55.5 (OMe in pos. 5),
0
0
0
0
0
31.2 (C2 ), 18.7 (C3 ), 13.6 (C4 ). Elem. Anal. Calcd for C19H24O2: C,
80.24; H, 8.51; found: C, 80.21; H, 8.68; HRMS, 284.1775
(0.5 ppm).
35.1 (C2), 22.4 (C2 ), 10.6 (C3 ). Elem. Anal. Calcd for C19H24O3: C,
75.97; H, 8.05; found: C, 75.97; H, 8.08; HRMS, 300.1724
(ꢁ0.5 ppm).
1-(Benzyloxy)-4-(2-hexyloxyethyl)benzene (3l): colourless liquid
(73% yield); 1H-NMR d ppm 7.37 (m, 5H, Ph), 7.12 (d, J = 8.7 Hz,
2H, H5), 6.89 (d, J = 8.7 Hz, 2H, H4), 5.05 (s, 2H, CH2Ph in pos. 6),
1-(Benzyloxy)-2-methoxy-4-(2-butoxyethyl)benzene (3r): colour-
less liquid (76% yield); 1H-NMR d ppm 7.37 (m, 5H, Ph), 6.90 (d,
J = 8.1 Hz, 1H, H7), 6.85 (d, J = 1.9 Hz, 1H, H4), 6.69 (dd, 1H, H8),
5.02 (s, 2H, CH2Ph in pos. 6), 3.74 (s, 3H, OMe in pos. 5), 3.51 (t,
0
3.49 (t, J = 6.9 Hz, 2H, H1), 3.33 (t, J = 6.5 Hz, 2H, H1 ), 2.70
0
0
0
0
(t, J = 6.9 Hz, 2H, H2), 1.44 (m, 2H, H2 ) 1.22 (m, 6H, H3 –H5 ), 0.84
J = 6.9 Hz, 2H, H1), 3.36 (t, J = 6.6 Hz, 2H, H1 ), 2.71 (t, J = 6.9 Hz,
(t, J = 7.1 Hz, 3H, H6 ); 13C-NMR d ppm 156.6 (C6), 137.2 (Cipso, Bn
group), 131.1 (C3), 129.6 (C4), 128.3–127.5 (C2–C4, Bn group),
2H, H2), 1.45 (m, 2H, H2 ), 1.29 (m, 2H, H3 ), 0.85 (t, J = 7.4 Hz, 3H,
0
0
0
H4 ); 13C-NMR d ppm 149.0 (C5), 146.1 (C6), 137.2 (Cipso, Bn group),
0
0
114.4 (C5), 71.0 (C1), 69.8 (C1 ), 69.0 (CH2Ph in pos. 6), 34.6 (C2),
131.9 (C3), 128.3–127.5 (C2–C4, Bn group), 120.5 (C8), 113.8 (C7),
0
0
0
0
0
0
31.0 (C4 ), 29.1 (C2 ), 25.2 (C3 ), 22.0 (C5 ), 13.8 (C6 ). Elem. Anal.
Calcd for C21H28O2: C, 80.73; H, 9.03; found: C, 80.73; H, 9.02;
HRMS, 312.2091 (0.5 ppm).
113.1 (C4), 71.0 (C1), 70.0 (CH2Ph in pos. 6), 69.5 (C1 ), 55.5 (OMe
0
0
0
in pos. 5), 35.1 (C2), 31.2 (C2 ), 18.8 (C3 ), 13.6 (C4 ). Elem. Anal. Calcd
for C20H26O3: C, 76.40; H, 8.33; found: C, 76.45; H, 8.08; HRMS,
314.1881 (ꢁ0.3 ppm).
1-(Benzyloxy)-4-(2-octyloxyethyl)benzene (3m): colourless liquid
(75% yield); 1H-NMR d ppm 7.37 (m, 5H, Ph), 7.12 (d, J = 8.7 Hz, 2H,
H5), 6.89 (d, J = 8.7 Hz, 2H, H4), 5.05 (s, 2H, CH2Ph in pos. 6), 3.49 (t,
1-(Benzyloxy)-2-methoxy-4-(2-hexyloxyethyl)benzene (3s): col-
ourless liquid (86% yield); 1H-NMR d ppm 7.37 (m, 5H, Ph), 6.89
(d, J = 8.1 Hz, 1H, H7), 6.85 (d, J = 1.8 Hz, 1H, H4), 6.69 (dd, 1H,
H8), 5.02 (s, 2H, CH2Ph in pos. 6), 3.74 (s, 3H, OMe in pos. 5), 3.51
0
J = 6.9 Hz, 2H, H1), 3.33 (t, J = 6.5 Hz, 2H, H1 ), 2.70 (t, J = 6.9 Hz, 2H,
0
0
0
H2), 1.44 (m, 2H, H2 ) 1.22 (m, 10H, H3 –H7 ), 0.84 (t, J = 7.1 Hz,3H,
H8 ); 13C-NMR d ppm 156.6 (C6), 137.2 (Cipso, Bn group), 131.1
(t, J = 7.0 Hz, 2H, H1), 3.34 (t, J = 6.5 Hz, 2H, H1 ), 2.71 (t, J = 7.0 Hz,
0
0
0
0
0
(C3), 129.6 (C4), 128.3–127.5 (C2–C4, Bn group), 114.4 (C5), 71.0
2H, H2), 1.45 (m, 2H, H2 ), 1.23 (m, 6H, H3 –H5 ), 0.83 (t, J = 7.0 Hz,
3H, H6 ); 13C-NMR d ppm 149.0 (C5), 146.1 (C6), 137.2 (Cipso, Bn
0
0
0
(C1), 69.8 (C1 ), 69.0 (CH2Ph in pos. 6), 34.6 (C2), 31.1 (C6 ), 29.1
0
0
0
0
0
0
(C2 ), 28.7 (C4 ), 28.6 (C5 ), 25.6 (C3 ), 22.0 (C7 ), 13.8 (C8 ). Elem. Anal.
Calcd for C23H32O2: C, 81.13; H, 9.47; found: C, 81.42; H, 9.75;
HRMS, 340.2378 (7.1 ppm).
group), 131.9 (C3), 128.3–127.5 (C2–C4, Bn group), 120.5 (C8),
0
113.7 (C7), 113.0 (C4), 71.1 (C1), 70.0 (CH2Ph in pos. 6), 69.9 (C1 ),
0
0
0
55.5 (OMe in pos. 5), 35.1 (C2), 29.1 (C2 ), 29.1 (C4 ), 25.3 (C3 ),
0
0
1-(Benzyloxy)-4-(2-dodecyloxyethyl)benzene (3n): colourless li-
quid (70% yield); 1H-NMR d ppm 7.37 (m, 5H, Ph), 7.12 (d,
J = 8.7 Hz, 2H, H5), 6.89 (d, J = 8.7 Hz, 2H, H4), 5.05 (s, 2H, CH2Ph
22.0 (C5 ), 13.8 (C6 ). Elem. Anal. Calcd for C22H30O3: C, 77.16; H,
8.83; found: C, 77.27; H, 8.95; HRMS, 342.2190 (ꢁ1.4 ppm).
1-(Benzyloxy)-2-methoxy-4-(2-octyloxyethyl)benzene (3t): col-
ourless liquid (84% yield); 1H-NMR d ppm 7.37 (m, 5H, Ph), 6.89
(d, J = 8.1 Hz, 1H, H7), 6.85 (d, J = 1.9 Hz, 1H, H4), 6.69 (dd, 1H,
H8), 5.02 (s, 2H, CH2Ph in pos. 6), 3.74 (s, 3H, OMe in pos. 5), 3.51
0
in pos. 6), 3.48 (t, J = 6.9 Hz, 2H, H1), 3.33 (t, J = 6.5 Hz, 2H, H1 ),
0
0
2.70 (t, J = 6.9 Hz, 2H, H2), 1.44 (m, 2H, H2 ) 1.22 (m, 18H, H3 –
H11 ), 0.83 (t, J = 6.8 Hz, 3H, H12 ); 13C-NMR d ppm 156.6 (C6),
137.2 (Cipso, Bn group), 131.1 (C3), 129.6 (C4), 128.3–127.5 (C2–C4,
0
0
0
(t, J = 6.9 Hz, 2H, H1), 3.34 (t, J = 6.5 Hz, 2H, H1 ), 2.70 (t,
0
0
0
0
Bn group), 114.4 (C5), 71.0 (C1), 69.8 (C1 ), 69.0 (CH2Ph in pos. 6),
J = 6.9 Hz, 2H, H2), 1.45 (m, 2H, H2 ), 1.22(m, 10H, H3 –H7 ), 0.84
(t, J = 7.1 Hz, 3H, H8 ); 13C-NMR d ppm 149.0 (C5), 146.1 (C6),
0
0
0
0
0
0
34.6 (C2), 31.2 (C10 ), 29.0 (C2 ), 29.0–28.5 (C4 , C9 ), 25.6 (C3 ), 22.0
0
0
(C11 ), 13.8 (C12 ). Elem. Anal. Calcd for C27H40O2: C, 81.77; H,
10.17; found: C, 81.62; H, 10.10; HRMS, 396.3012 (4.1 ppm).
1-(Benzyloxy)-2-methoxy-4-(2-methoxyethyl)benzene (3o): col-
ourless liquid (85% yield); 1H-NMR d ppm 7.37 (m, 5H, Ph), 6.90
(d, J = 8.1 Hz, 1H, H7), 6.85 (d, J = 2.0 Hz, 1H, H4), 6.69 (dd, 1H,
H8), 5.02 (s, 2H, CH2Ph in pos. 6), 3.74 (s, 3H, OMe in pos. 5), 3.49
137.2 (Cipso, Bn group), 131.9 (C3), 128.3–127.5 (C2–C4, Bn group),
120.5 (C8), 113.7 (C7), 113.1 (C4), 71.0 (C1), 70.0 (CH2Ph in pos. 6),
0
0
0
69.8 (C1 ), 55.5 (OMe in pos. 5), 35.1 (C2), 31.1 (C6 ), 29.1 (C2 ),
0
0
0
0
0
28.7 (C4 ), 28.6 (C5 ), 25.6 (C3 ), 22.0 (C7 ), 13.8 (C8 ). Elem. Anal.
Calcd for C24H34O3: C, 77.80; H, 9.25; found: C, 77.54; H, 9.35;
HRMS, 370.2510 (0.6 ppm).
0
(t, J = 7.0 Hz, 2H, H1), 3.22 (s, 3H, H1 ), 2.71 (t, J = 7.0 Hz, 2H, H2);
1-(Benzyloxy)-2-methoxy-4-(2-dodecyloxyethyl)benzene
(3u):
13C-NMR d ppm 149.0 (C5), 146.1 (C6), 137.2 (Cipso, Bn group),
131.9 (C3), 128.3–127.5 (C2–C4, Bn group), 120.5 (C8), 113.8 (C7),
113.0 (C4), 72.9 (C1), 70.0 (CH2Ph in pos. 6), 57.7 (C1 ), 55.5 (OMe
colourless liquid (64% yield); 1H-NMR d ppm 7.37 (m, 5H, Ph),
6.89 (d, J = 8.2 Hz, 1H, H7), 6.85 (d, J = 1.9 Hz, 1H, H4), 6.68 (dd,
1H, H8), 5.02 (s, 2H, CH2Ph in pos. 6), 3.74 (s, 3H, OMe in pos. 5),
0
0
in pos. 5), 34.8 (C2). Elem. Anal. Calcd for C17H20O3: C, 74.97; H,
7.40; found: C, 74.64; H, 7.15; HRMS, 272.1408 (ꢁ1.6 ppm).
1-(Benzyloxy)-2-methoxy-4-(2-ethoxyethyl)benzene (3p): colour-
less liquid (86% yield); 1H-NMR d ppm 7.37 (m, 5H, Ph), 6.90 (d,
J = 7.9 Hz, 1H, H7), 6.86 (d, J = 1.9 Hz, 1H, H4), 6.69 (dd, 1H, H8),
5.02 (s, 2H, CH2Ph in pos. 6), 3.74 (s, 3H, OMe in pos. 5), 3.52 (t,
3.51 (t, J = 7.0 Hz, 2H, H1), 3.34 (t, J = 6.5 Hz, 2H, H1 ), 2.71 (t,
0
0
0
J = 7.0 Hz, 2H, H2), 1.45 (m, 2H, H2 ), 1.22 (m, 18H, H3 –H11 ), 0.84
(t, J = 7.0 Hz, 3H, H12 ); 13C-NMR d ppm 149.0 (C5), 146.1 (C6),
0
137.2 (Cipso, Bn group), 131.9 (C3), 128.3–127.5 (C2–C4, Bn group),
120.5 (C8), 113.8 (C7), 113.1 (C4), 71.0 (C1), 70.0 (CH2Ph in pos. 6),
69.8 (C1 ), 55.5 (OMe in pos. 5), 35.1 (C2), 31.2 (C10 ), 29.1 (C2 ),
0
0
0
0
0
0
0
0
0
0
0
0
J = 7.0 Hz, 2H, H1), 3.41 (q, J = 6.9 Hz, 2H, H1 ), 2.71 (t, J = 7.0 Hz,
28.9–28.6 (C4 , C5 , C6 , C7 , C8 , C9 ), 25.6 (C3 ), 22.0 (C11 ), 13.8
2H, H2), 1.08 (t, J = 6.9 Hz, 3H, H2 ); 13C-NMR d ppm 149.0 (C5),
(C12 ). Elem. Anal. Calcd for C28H42O3: C, 78.83; H, 9.92; found: C,
0
0
146.1 (C6), 137.2 (Cipso, Bn group), 131.9 (C3), 128.3–127.5 (C2–C4,
78.73; H, 9.59; HRMS, 426.3148 (3.3 ppm).
Bn group), 120.5 (C8), 113.8 (C7), 113.0 (C4), 70.8 (C1), 70.0 (CH2Ph
0
0
in pos. 6), 65.1 (C1 ), 55.5 (OMe in pos. 5), 35.1 (C2), 15.0 (C2 ). Elem.
Anal. Calcd for C18H22O3: C, 75.50; H, 7.74; found: C, 76.00; H, 7.64;
HRMS, 286.1574 (1.8 ppm).
2.2.2. General procedure for cleavage of benzyl protecting groups
A catalytic amount of palladium over charcoal (Pd–C) was
added to a solution of the corresponding protected ethers (3h–u,
1 mmol) in THF (20 ml) and the mixture was hydrogenated at
4 bar with magnetic stirring. After 24 h at room temperature the
catalyst was filtered off over Celite and solvent was evaporated
in vacuum. Further purification by column chromatography
yielded the desired compounds in each case (4h–u).
1-(Benzyloxy)-2-methoxy-4-(2-propoxyethyl)benzene (3q): col-
ourless liquid (83% yield); 1H-NMR d ppm 7.37 (m, 5H, Ph), 6.90
(d, J = 8.1 Hz, 1H, H7), 6.86 (d, J = 1.8 Hz, 1H, H4), 6.69 (dd, 1H,
H8), 5.02 (s, 2H, CH2Ph in pos. 6), 3.74 (s, 3H, OMe in pos. 5), 3.51
0
(t, J = 7.0 Hz, 2H, H1), 3.32 (t, J = 6.7 Hz, 2H, H1 ), 2.71 (t, J = 7.0 Hz,
2H, H2), 1.48 (m, 2H, H2 ), 0.83 (t, J = 7.3 Hz, 3H, H3 ); 13C-NMR d
0
0

1172
A. Madrona et al. / Food Chemistry 129 (2011) 1169–1178
4-(2-Methoxyethyl)phenol (4h): colourless liquid (82% yield);
(C6), 129.6 (C3), 120.8 (C8), 115.2 (C7), 113.0 (C4), 73.1 (C1), 57.7
1H-NMR d ppm 9.09 (s, 1H, phenolic OH), 6.98 (d, J = 8.3 Hz, 2H,
H5), 6.64 (d, J = 8.3 Hz, 2H, H4), 3.44 (t, J = 7.0 Hz, 2H, H1), 3.21 (s,
(C1 ), 55.5 (OMe in pos. 5), 34.9 (C2). Elem. Anal. Calcd for
10H14O3: C, 65.91; H, 7.74; found: C, 66.33; H, 8.05; HRMS,
0
C
3H, H1 ), 2.66 (t, J = 7.0 Hz, 2H, H2); 13C-NMR d ppm 155.4 (C6),
182.0949 (3.3 ppm).
0
0
129.5 (C4), 128.9 (C3), 114.9 (C5), 73.1 (C1), 57.6 (C1 ), 34.4 (C2).
2-Methoxy-4-(2-ethoxyethyl)phenol (4p): colourless liquid (80%
Elem. Anal. Calcd for C9H12O2: C, 71.03; H, 7.95; found: C, 71.14;
H, 7.60; HRMS, 152.0828 (6.1 ppm).
yield); 1H-NMR
d ppm 8.62 (s, 1H, phenolic OH), 6.77 (d,
J = 1.9 Hz, 1H, H4), 6.65 (d, J = 7.9 Hz, 1H, H7), 6.58 (dd, 1H, H8),
3.72 (s, 3H, OMe in pos. 5), 3.49 (t, J = 7.2 Hz, 2H, H1), 3.41 (q,
4-(2-Ethoxyethyl)phenol (4i): colourless liquid (81% yield); 1H-
NMR d ppm 9.11 (s, 1H, phenolic OH), 6.99 (d, J = 8.5 Hz, 2H, H5),
6.64 (d, J = 8.5 Hz, 2H, H4), 3.46 (t, J = 7.2 Hz, 2H, H1), 3.39(q,
0
J = 7.0 Hz, 2H, H1 ), 2.67 (t, J = 7.2 Hz, 2H, H2), 1.08 (t, J = 6.9 Hz,
3H, H2 ); 13C-NMR d ppm 147.2 (C5), 144.7 (C6), 129.6 (C3), 120.8
0
0
0
J = 6.9 Hz, 2H, H1 ), 2.65 (t, J = 7.2 Hz, 2H, H2), 1.07 (t, J = 6.9 Hz,
(C8), 115.2 (C7), 113.0 (C4), 71.0 (C1), 65.1 (C1 ), 55.5 (OMe in pos.
3H, H2 ); 13C-NMR d ppm 155.4 (C6), 129.5 (C4), 128.9 (C3), 114.9
5), 35.1 (C2), 15.0 (C2 ). Elem. Anal. Calcd for C11H16O3: C, 67.32;
0
0
0
0
(C5), 71.1 (C1), 65.1 (C1 ), 34.7 (C2), 15.0 (C2 ). Elem. Anal. Calcd
for C10H14O2: C, 72.26; H, 8.49; found: C, 72.16; H, 8.13; HRMS,
166.0992 (ꢁ1.1 ppm).
H, 8.22; found: C, 67.21; H, 8.30; HRMS, 196.1106 (3.3 ppm).
2-Methoxy-4-(2-propyloxyethyl)phenol (4q): colourless liquid
(83% yield); 1H-NMR d ppm 8.66 (s, 1H, phenolic OH), 6.78 (d,
J = 1.8 Hz, 1H, H4), 6.64 (d, J = 7.9 Hz, 1H, H7), 6.58 (dd, 1H, H8),
3.72 (s, 3H, OMe in pos. 5), 3.49 (t, J = 7.2 Hz, 2H, H1), 3.31 (t, J =
4-(2-Propoxyethyl)phenol (4j): colourless liquid (87% yield); 1H-
NMR d ppm 9.09 (s, 1H, phenolic OH), 6.99 (d, J = 8.4 Hz, 2H, H5),
6.64 (d, J = 8.4 Hz, 2H, H4), 3.47 (t, J = 7.2 Hz, 2H, H1), 3.30 (t,
0
0
6.7 Hz, 2H, H1 ), 2.67 (t, J = 7.2 Hz, 2H, H2), 1.48 (m, 2H, H2 ), 0.84
J = 6.5 Hz, 2H, H1 ), 2.66 (t, J = 7.2 Hz, 2H, H2), 1.47 (m, 2H, H2 ),
(t, J = 7.3 Hz, 3H, H3 ); 13C-NMR d ppm 147.2 (C5), 144.7 (C6),
0
0
0
0.82 (t, J = 7.4 Hz, 3H, H3 ); 13C-NMR d ppm 155.4 (C6), 129.5 (C4),
129.6 (C3), 120.9 (C8), 115.2 (C7), 113.0 (C4), 71.5 (C1 ), 71.3 (C1),
0
0
0
0
0
0
128.9 (C3), 114.9 (C5), 71.4 (C1 ), 71.3 (C1), 34.7 (C2), 22.3 (C2 ),
55.5 (OMe in pos. 5), 35.2 (C2), 22.5 (C2 ), 10.4 (C3 ). Elem. Anal.
Calcd for C12H18O3: C, 68.54; H, 8.63; found: C, 68.35; H, 8.65;
HRMS, 210.1252 (ꢁ1.9 ppm).
0
10.4 (C3 ). Elem. Anal. Calcd for C11H16O2: C, 73.30; H, 8.95; found:
C, 72.91; H, 8.96; HRMS, 180.1152 (0.9 ppm).
4-(2-Butoxyethyl)phenol (4k): yellowish liquid (86% yield); 1H-
NMR d ppm 9.09 (s, 1H, phenolic OH), 6.99 (d, J = 8.5 Hz, 2H, H5),
6.64 (d, J = 8.4 Hz, 2H, H4), 3.46 (t, J = 7.2 Hz, 2H, H1), 3.34 (t, J =
2-Methoxy-4-(2-butyloxyethyl)phenol (4r): colourless liquid
(81% yield); 1H-NMR d ppm 8.62 (s, 1H, phenolic OH), 6.77 (d,
J = 1.9 Hz, 1H, H4), 6.65 (d, J = 7.9 Hz, 1H, H7), 6.58 (dd, 1H, H8),
3.72 (s, 3H, OMe in pos. 5), 3.49 (t, J = 7.1 Hz, 2H, H1), 3.35 (t,
0
0
6.5 Hz, 2H, H1 ), 2.65 (t, J = 7.2 Hz, 2H, H2), 1.44 (m, 2H, H2 ), 1.28
(m, 2H, H3 ), 0.84 (t, J = 7.4 Hz, 3H, H4 ); 13C-NMR d ppm 155.4
J = 6.5 Hz, 2H, H1 ), 2.67 (t, J = 7.1 Hz, 2H, H2), 1.45 (m, 2H, H2 ),
0
0
0
0
1.29 (m. 2H, H3 ), 0.85 (t, J = 7.3, 3H, H4 ); 13C-NMR d ppm 147.2
(C5), 144.7 (C6), 129.6 (C3), 120.8 (C8), 115.1 (C7), 113.0 (C4), 71.2
0
0
0
(C6), 129.5 (C4), 128.9 (C3), 114.9 (C5), 71.3 (C1), 69.5 (C1 ), 34.7
(C2), 31.2 (C2 ), 18.8 (C3 ), 13.7 (C4 ). Elem. Anal. Calcd for
12H18O2: C, 74.29; H, 9.34; found: C, 74.24; H, 9.38; HRMS,
0
0
0
0
0
C
(C1), 69.5 (C1 ), 55.4 (OMe in pos. 5), 35.1 (C2), 31.3 (C2 ), 18.8
0
0
194.1294 (6.6 ppm).
(C3 ), 13.7 (C4 ). Elem. Anal. Calcd for C13H20O3: C, 69.61; H, 8.99;
found: C, 69.04; H, 9.30; HRMS, 224.1418 (2.5 ppm).
4-(2-Hexyloxyethyl)phenol (4l): yellowish liquid (85% yield); 1H-
NMR d ppm 9.08 (s, 1H, phenolic OH), 6.98 (d, J = 8.5 Hz, 2H, H5),
6.64 (d, J = 8.5 Hz, 2H, H4), 3.46 (t, J = 7.1 Hz, 2H, H1), 3.33 (t,
2-Methoxy-4-(2-hexyloxyethyl)phenol (4s): colourless liquid
(86% yield); 1H-NMR d ppm 8.61 (s, 1H, phenolic OH), 6.77 (d,
J = 1.9 Hz, 1H, H4), 6.64 (d, J = 7.9 Hz, 1H, H7), 6.58 (dd, 1H, H8),
3.72 (s, 3H, OMe in pos. 5), 3.49 (t, J = 7.1 Hz, 2H, H1), 3.34 (t,
0
0
J = 6.5 Hz, 2H, H1 ), 2.65 (t, J = 7.1 Hz, 2H, H2), 1.44 (m, 2H, H2 ),
1.23 (m, 6H, H3 –H5 ), 0.84 (t, J = 7.2 Hz, 3H, H6 ); 13C-NMR d ppm
0
0
0
0
0
0
155.4 (C6), 129.5 (C4), 128.9 (C3), 114.9 (C5), 71.3 (C1), 69.8 (C1 ),
J = 6.5 Hz, 2H, H1 ), 2.66 (t, J = 7.1 Hz, 2H, H2), 1.46 (m, 2H, H2 ),
0
0
0
0
0
0
0
0
34.7 (C2), 31.0 (C4 ), 29.1 (C2 ), 25.3 (C3 ), 22.0 (C5 ), 13.8 (C6 ). Elem.
Anal. Calcd for C14H22O2: C, 75.63; H, 9.97; found: C, 75.80; H,
10.28; HRMS, 222.1623 (1.4 ppm).
1.24 (m. 6H, H3 -H5 ), 0.84 (t, J = 7.1 Hz, 3H, H4 ); 13C-NMR d ppm
147.2 (C5), 144.7 (C6), 129.6 (C3), 120.8 (C8), 115.1 (C7), 113.0
0
0
(C4), 71.2 (C1), 69.2 (C1 ), 55.4 (OMe in pos. 5), 35.1 (C2), 29.1 (C2 ),
0
0
0
0
4-(2-Octyloxyethyl)phenol (4m): colourless liquid (85% yield);
1H-NMR d ppm 9.08 (s, 1H, phenolic OH), 6.98 (d, J = 8.5 Hz, 2H,
H5), 6.64 (d, J = 8.5 Hz, 2H, H4), 3.46 (t, J = 7.1 Hz, 2H, H1), 3.33 (t,
29.1 (C4 ), 25.3 (C3 ), 22.1 (C5 ), 13.8 (C6 ). Elem. Anal. Calcd for
C15H24O3: C, 71.39; H, 9.59; found: C, 71.33; H, 9.59; HRMS,
252.1725 (-0.2 ppm).
0
0
J = 6.5 Hz, 2H, H1 ), 2.65 (t, J = 7.1 Hz, 2H, H2), 1.44 (m, 2H, H2 ),
2-Methoxy-4-(2-octyloxyethyl)phenol (4t): colourless liquid (79%
yield); 1H-NMR d ppm 8.65 (s, 1H, phenolic OH), 6.77 (d, J = 1.7 Hz,
1H, H4), 6.64 (d, J = 7.9 Hz, 1H, H7), 6.57 (dd, 1H, H8), 3.72 (s, 3H,
OMe in pos. 5), 3.48 (t, J = 7.1 Hz, 2H, H1), 3.34 (t, J = 6.5 Hz, 2H,
1.22 (m, 10H, H3 –H7 ), 0.84 (t, J = 7.1 Hz, 3H, H8 ); 13C-NMR d
ppm 155.4 (C6), 129.5 (C4), 128.9 (C3), 114.9 (C5), 71.3 (C1), 69.8
(C1 ), 34.7 (C2), 31.2 (C6 ), 29.1 (C2 ), 28.7 (C4 ), 28.6 (C5 ), 25.6
0
0
0
0
0
0
0
0
0
0
0
0
0
(C3 ), 22.0 (C7 ) 13.8 (C8 ). Elem. Anal. Calcd for C16H26O2: C,
76.75; H, 10.47; found: C, 76.45; H, 10.42; HRMS, 250.1926
(2.7 ppm).
H1 ), 2.66 (t, J = 7.1 Hz, 2H, H2), 1.45 (m, 2H, H2 ), 1.22 (m. 10H,
H3 -H7 ), 0.84 (t, J = 7.0 Hz, 3H, H8 ); 13C-NMR d ppm 147.2 (C5),
144.7 (C6), 129.6 (C3), 120.8 (C8), 115.1 (C7), 112.9 (C4), 71.3 (C1),
0
0
0
0
0
0
4-(2-Dodecyloxyethyl)phenol (4n): colourless liquid (89% yield);
1H-NMR d ppm 9.08 (s, 1H, phenolic OH), 6.97 (d, J = 8.5 Hz, 2H,
H5), 6.64 (d, J = 8.5 Hz, 2H, H4), 3.45 (t, J = 7.1 Hz, 2H, H1), 3.32 (t,
69.9 (C1 ), 55.4 (OMe in pos. 5), 35.1 (C2), 31.2 (C6 ) 29.2 (C2 ), 28.7
0
0
0
0
0
(C4 ), 28.6(C5 ), 25.7 (C3 ), 22.0 (C7 ), 13.8 (C8 ). Elem. Anal. Calcd
for C17H28O3: C, 72.82; H, 10.06; found: C, 72.78; H, 10.03; HRMS,
280.2033 (ꢁ1.9 ppm).
0
0
J = 6.5 Hz, 2H, H1 ), 2.65 (t, J = 7.1 Hz, 2H, H2), 1.44 (m, 2H, H2 ),
1.22 (m, 18H, H3 –H11 ), 0.84 (t, J = 7.0 Hz, 3H, H12 ); 13C-NMR d
ppm 155.4 (C6), 129.5 (C4), 128.9 (C3), 114.8 (C5), 71.3 (C1), 69.8
2-Methoxy-4-(2-dodecyloxyethyl)phenol (4u): colourless liquid
(72% yield); 1H-NMR d ppm 8.62 (s, 1H, phenolic OH), 6.77 (d,
J = 1.9 Hz, 1H, H4), 6.64 (d, J = 7.9 Hz, 1H, H7), 6.57 (dd, 1H, H8),
3.72 (s, 3H, OMe in pos. 5), 3.48 (t, J = 7.1 Hz, 2H, H1), 3.34 (t,
0
0
0
0
0
0
0
0
0
0
0
(C1 ), 34.7 (C2), 31.2 (C10 ), 29.1 (C2 ), 28.9–28.6 (C4 , C5 , C6 , C7 , C8 ,
0
0
0
0
C9 ), 25.6 (C3 ), 22.0 (C11 ) 13.8 (C12 ). Elem. Anal. Calcd for
C20H34O2: C, 78.38; H, 11.18; found: C, 77.99; H, 11.02; HRMS,
306.2546 (4.2 ppm).
0
0
J = 6.5 Hz, 2H, H1 ), 2.66 (t, J = 7.1 Hz, 2H, H2), 1.45 (m, 2H, H2 ),
1.22 (m. 18H, H3 -H11 ), 0.84 (t, J = 7.0 Hz, 3H, H12 ); 13C-NMR d
ppm 147.2 (C5), 144.7 (C6), 129.6 (C3), 120.8 (C8), 115.1 (C7),
0
0
0
2-Methoxy-4-(2-methoxyethyl)phenol (4o): colourless liquid
(78% yield); 1H-NMR d ppm 8.63 (s, 1H, phenolic OH), 6.76 (d,
J = 1.9 Hz, 1H, H4), 6.65 (d, J = 7.9 Hz, 1H, H7), 6.58 (dd, 1H, H8),
3.72 (s, 3H, OMe in pos. 5), 3.46 (t, J = 7.1 Hz, 2H, H1), 3.22 (s, 3H,
0
113.0 (C4), 71.2 (C1), 69.8 (C1 ), 55.4 (OMe in pos. 5), 35.1 (C2),
31.2 (C10 ) 29.1 (C2 ), 28.9–28.6 (C4 ,C5 , C6 , C7 , C8 , C9 ), 25.6 (C3 ),
22.0 (C11 ), 13.8 (C12 ). Elem. Anal. Calcd for C21H36O3: C, 74.95; H,
10.98; found: C, 74.92; H, 10.76; HRMS, 336.2669 (1.4 ppm).
0
0
0
0
0
0
0
0
0
0
0
H1 ), 2.68 (t, J = 7.1 Hz, 2H, H2); 13C-NMR d ppm 147.2 (C5), 144.7
0

A. Madrona et al. / Food Chemistry 129 (2011) 1169–1178
1173
2.3. Evaluation of oxidative stability by the Rancimat Method
UV–Visible Varian (Cary 50 BIO, Holland) spectrophotometer,
equipped with a thermostatted auto-cell-holder. The percentage
inhibition of absorbance versus time was plotted, and the area be-
low the curve (0–360 s) was calculated. Methanolic solutions of
known concentrations of Trolox were used for calibration. Results
are expressed in mM TE (Trolox Equivalent). Each value is the aver-
age of three determinations.
The oxidative stability was evaluated by an accelerated auto-
mated test using the Rancimat equipment (Model 743, Metrohm
Co. Basel, Switzerland). A lipid matrix was obtained from virgin ol-
ive oil (VOO) of ‘Arbequina’ variety by purification through alu-
mina (Yoshida, Kondo, & Kajimoto, 1992), according to the ‘free
solvent’ procedure. The purified matrix, free of antioxidants, was
stored at ꢁ18 °C under nitrogen atmosphere. Absence of polyphe-
nols and tocopherols were checked by solid phase extraction and
HPLC analysis with UV detector at 280 nm and C18 Column (Tec-
2.6. ORAC assay
The oxygen radical scavenging capacity was measured by the
lipophilic ORAC assay according to the method developed by
Huang (Huang, Ou, Hampsch-Woodill, Flanagan, & Deemer, 2002)
with some modifications. This 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
knokroma, 5
l
m, 25 cm ꢀ 4,6 mm i.d.) (Mateos et al., 2001) and
by HPLC analysis on a silica gel column (Lichrorb SI, 5
l
m
25 cm ꢀ 4.0 cm i.d.) using a UV–Visible detector at 292 nm (Paquot
& Hautfenne, 1992), respectively. The fatty acid composition of the
matrix was C16:0 (15.0%), C16:1 (1.4%), C17:0 (0.1%), C17:1 (0.2%),
C18:0 (1.8%), C18:1 (70.2%), C18:2 (10.0%), C18:3 (0.5%), C20:0
(0.4%), C20:1 (0.3%) and C22:0 (0.1%). Aliquots of the glyceridic ma-
trix were spiked with increasing amounts of the new prepared
compounds, ranging from 0.2 to 2.2 mmol/kg and then subjected
to accelerated oxidation in a Rancimat apparatus at 80 °C. Results
are expressed as induction time (IT) in hours corresponding to
the stability of the lipid matrix evaluated. All determinations were
carried out in duplicate.
reaction. Ty (1b) and its derivatives (4h–n) from 5 to 50
(1c) and its derivatives (4o–u) from 5 to 40 M and Trolox stan-
dard (6.25, 12.5, 25, 50, 75 and 100 M) were dissolved in 7%
methylated b-cyclodextrin (RMCD) in acetone/water (1:1, v/v)
solution. Then, 25 l of either trolox or test sample or solvent as
lM, HMV
l
l
l
blank were added to a 96-well microplate followed by the addition
of 150
l
l of fluorescein work solution (8.5 ꢀ 10ꢁ5 mM) prepared in
75 mM phosphate buffer (pH 7.4). The microplate reader (Bio-Tek,
Winooski, VT, USA) was programmed to record every two minutes
for 120 min at 485 and 528 nm excitation and emission wave-
2.4. Ferric Reducing Antioxidant Power (FRAP) Assay
lengths, respectively, the fluorescence after the addition of 30 ll
of AAPH (153 mM) as peroxyl radical generator, prepared also in
75 mM phosphate buffer (pH 7.4). Limit of quantification was set
The FRAP assay was carried out according to the procedure de-
scribed by Benzie and Strain (Benzie & Strain, 1996), with some
modifications (Pulido, Bravo, & Saura-Calixto, 2000). The antioxi-
dant potential of the synthesized compounds was estimated from
their ability to reduce the ferric tripyridyltriazine (TPTZ–FeIII) com-
plex to its stable ferrous form (TPTZ–FeII complex). 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 FeCl3ꢂ6H2O and 25 ml of 0.3 M
acetate buffer to a final pH of 3.6. This reagent was freshly pre-
at 5
lM. Each value is the average of four determinations. Final re-
sults were calculated according the following equation:
ðAUCSample ꢁ AUCBlank
Þ
½Troloxꢃ
ORACValue
¼
ꢂ
ðAUCTrolox ꢁ AUCBlank
Þ
½Sampleꢃ
where AUCSample is the area under curve in the presence of the
tested compounds; AUCBlank is the area under curve of control;
AUCTrolox is the area under curve in the presence of Trolox, and
[Trolox] and [Sample] are the molar concentrations of Trolox and
tested compounds, respectively. ORAC values are expressed as
micromoles of Trolox/micromoles of antioxidant compound.
pared and warmed to 37 °C prior to its use. 900
mixed with 90 l of distiled water and 30 l of either test sample
(ranging from 500 to 10000 M for Ty (1b) and its derivatives
(4h–n) and from 100 to 800 M for HMV (1c) and its derivatives
ll of FRAP was
l
l
l
l
(4o–u)), standard, or methanol (as appropriate reagent blank),
and 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,
2.7. Statistical analysis
Results are expressed as means standard deviation (SD) of
three measurements for the analytical determination. The data
were subjected to a one-way analysis of variance (ANOVA) using
Statistix 8.0. The level of significance was p < 0.05.
Holland) spectrophotometer, equipped with
a thermostatted
auto-cell-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 mM
TE (Trolox Equivalent). All analyses were run in triplicate.
3. Results
2.5. ABTS assay
3.1. Preparation and characterisation of tyrosyl (4h–n) and
homovanillyl (4o–u) alkyl ethers
The free-radical scavenging capacity was measured using the
ABTS decolouration method (Re et al., 1999) with some modifica-
tions. The method is based on the capacity of different components
to scavenge the ABTS radical cation (ABTSÅ+) compared to a stan-
To carry out the synthesis of the new compounds, commercial
tyrosol (1b) and homovanillic alcohol (1c) were used. Pure tyrosol
and homovanillic alcohol were transformed into their benzyl
derivatives, 2b and 2c, respectively, by reaction with benzyl bro-
mide/potassium carbonate in acetone (Ajao et al., 1985; Battersbay
et al., 1961). Further alkylation of the remaining free hydroxylic
group with the corresponding alkyl iodides yielded the intermedi-
ate compounds, 3h–u, in good to excellent yields. In the final step,
desired alkyl tyrosyl and homovanillyl ethers, 4h–u, were obtained
in good overall yield by hydrogenolytical cleavage of the protecting
benzyl groups (Scheme 1). Synthesized compounds were charac-
terised by MS spectroscopy, as well as by their elemental analyses.
dard antioxidant (Trolox). Briefly, ABTS was dissolved in
a
2.45 mM K2S2O8 solution and stored in the dark at room tempera-
ture for 12–16 h, to get a 7 mM concentration of ABTS radical cat-
ion (ABTSÅ+) stock solution. The ABTSÅ+ 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 (ranging from 200 to 2000
lM for
Ty (1b) and its derivatives (4h–n) and from 100 to 800
lM for
HMV (1c) and its derivatives (4o–u)), methanol as a blank, or Trol-
ox standard to 3.9 ml of diluted ABTSÅ+ solution, absorbance read-
ings were taken every 20 s at 30 °C over 6 min, using
a

1174
A. Madrona et al. / Food Chemistry 129 (2011) 1169–1178
Scheme 1. Synthesis of new alkyl ethers.
Fig. 1. Induction times (ITs) of lipid matrices spiked with the assayed compounds: (Panel A) tyrosol (1b), homovanillic alcohol (1c), tyrosyl derivatives (4h–n), homovanillyl
derivatives (4o–u), -tocopherol and BHT; (Panel B) tyrosol (1b) and tyrosyl derivatives (4h–n); (Panel C) homovanillic alcohol (1c) and homovanillyl derivatives (4o–u).
a
Their structures were unambiguously determined by NMR spec-
troscopy (see Experimental part).
98 °C; although higher temperatures than 98 °C (100–120 °C) have
been also reported to reduce the analysis time (Mateos, Uceda,
Aguilera, Escuderos, & Beltrán, 2006). However, taking into account
the low activity previously reported for free Ty (1b) (Mateos et al.,
2003), and in order to find greater differences in the activity of the
new synthesized compounds, an experimental temperature of
80 °C was selected for this determination. Fig. 1 (Panel A) shows
the induction times (IT) corresponding to purified matrices of olive
oil spiked with different concentrations of the new ethers (4h–u),
3.2. Antioxidant activity in lipid matrices
The efficacy of the new synthesized compounds as antioxidants
in food in comparison with two widely used synthetic antioxidants
(a-tocopherol and BHT) was evaluated using the Rancimat method.
The most standard temperature used for Rancimat analysis is

A. Madrona et al. / Food Chemistry 129 (2011) 1169–1178
1175
Table 2
Ty (1b) or HMV (1c), BHT and
a-tocopherol. Results indicate that
Radical-scavenging capacity evaluated by the ABTS assay of tyrosol (1b), homovanillic
alcohol (1c), tyrosyl ethers (4h–n), homovanillyl ethers (4o–u), -tocopherol and
BHT. Each value is the mean of triplicate measurements standard deviations. Results
are expressed as mM TE (Trolox Equivalents). All values within a column with
different superscript letters are significantly different, p < 0.05.
all new prepared ethers maintain or even overcome the activity
of their respective free biophenols (1b or 1c) (Panel B and C).
Besides, as expected, the homovanillyl alkyl ether series (4o–u)
showed a better protecting capacity against oxidation in lipid
matrices than the tyrosyl alkyl ether series (4h–n) although it
a
Compound
mM TE
Compound
mM TE
did not reach the activity showed by
(Panel A).
a-tocopherol or BHT
Ty (1b)
0.39 0.01e
0.30 0.01c,d
0.37 0.01e
0.31 0.01d
0.29 0.01b,c
0.30 0.01c,d
0.30 0.01c,d
0.18 0.01a
0.27 0.01b
1.01 0.02f
HMV (1c)
1.29 0.01e
1.37 0.01g
1.32 0.01f
1.49 0.02h
1.33 0.01f
1.27 0.01e
1.06 0.01c
1.13 0.01d
0.27 0.01a
1.01 0.02b
Methyl_Ty (4h)
Ethyl_Ty (4i)
Propyl_Ty (4j)
Butyl_Ty (4k)
Hexyl_Ty (4l)
Octyl_Ty (4m)
Dodecyl_Ty (4n)
BHT
Methyl_HMV (4o)
Ethyl_HMV (4p)
Propyl_HMV (4q)
Butyl_HMV (4r)
Hexyl_HMV (4s)
Octyl_HMV (4t)
Dodecyl_HMV (4u)
BHT
3.3. Ferric-reducing antioxidant power (FRAP)
The reducing capacities of 4h–u in comparison with their pre-
cursors, 1b and 1c, and two synthetic antioxidants widely used,
a-tocopherol and BHT, were determined by the FRAP assay. The re-
sults summarised in Table 1 are expressed as mM TE (Trolox equiv-
alent). The reducing capacities of Ty (1b) and its ethers (4h–n) was
significantly lower than those shown by HMV (1c) and its deriva-
a-Tocopherol
a-Tocopherol
tives (4o–u) and the references
a-tocopherol and BHT. In fact,
HMV and its derivatives showed to be the most active compounds
studied. Exceptions were the higher homologous ethers of this ser-
ie, compounds 4t and 4u, which showed lower reducing capacities
than BHT. Comparison of the results obtained for the two synthetic
series of ethers (Table 1) with their respective free phenols, Ty (1b)
and HMV (1c), revealed a contradictory trend. While tyrosyl ethers
(4h–n) showed significantly lower reducing activities than their
natural precursor, Ty (1b), homovanillyl ethers (4o–u) showed a
variable behaviour depending on side-chain length, with higher
activity than the parent compound HMV (1c) for those less lipo-
philic ethers (methyl (4o), ethyl (4p) and propyl (4q)), similar for
the butyl homovanillyl ether (4r) and lower activity for the more
lipophilic hexyl, octyl and, specially, dodecyl homovanillyl ethers
(4s–u).
reported in Table 3. Also, slopes and intercepts of linear function
obtained after plotting net AUC versus the concentration are
summarised in Table 3. As expected, HMV and its derivatives (1c,
4o–u) exerted higher protection than Ty and its derivatives
(1b, 4h–m) against the loss of fluorescence provoked by free radi-
cals. Regarding the commonly used antioxidants, BHT possessed
the lowest ORAC value of all assayed compounds, while a-tocoph-
erol showed similar radical scavenging activity in comparison with
Ty (1b) and its derivatives (4h–m) but lower than HMV (1c) and its
derivatives (4o–u). Furthermore, the behaviour observed within
each series was similar to that described above for the ferric reduc-
ing activity and for radical-scavenging activities by ABTS assay.
4. Discussion
3.4. The ABTS assay
An increasingly strict regulation of the use of additives in foods,
particularly within the European Union, is leading towards signif-
icant reductions in the number of antioxidants available for use
as food preservatives. In particular, it cannot be ruled out a limita-
tion in the number of the already few lipophilic antioxidants avail-
able for use in foods because concerns on the safety of current
synthetic compounds such as BHA (butylated hydroxyanisole),
BHT (butylated hydroxytoluane), TBHQ (tertiary butylhydroqui-
none) and PG (propyl gallate) may lead to them being banned. Cur-
rently available natural lipophilic antioxidants are tocopherols (e.g.
vitamin E), beta-carotene, in some cases, tocotrienols, and phos-
pholipids (e.g. lecithin) in combination with phenolic antioxidants.
For this reason, there is major interest by the food industry for the
development of new lipophilic antioxidants for use as functional
ingredients for incorporation into lipid food matrices such as fats
and oils, processed foods, margarines that contain animal fats, milk
fat, or frozen fish or fish oil among others (Chillemi et al., 2010;
Pokorny, 1991, 2007). This is a challenge for food scientists since
most phytochemicals with antioxidant activity are of hydrophilic
nature. In order to find useful alternatives to synthetic additives,
two series of potentially biologically active compounds, tyrosyl
and homovanillyl ethers with linear alkylic side chains of variable
length from 1 to 12 carbon atoms, 4h–u (Scheme 1) have been syn-
thesized, starting from two characteristic phenolic compounds of
virgin olive oil as Ty (1b) and homovanillic alcohol (1c) by a
straight three-steps procedure, similar to that previously described
(Madrona et al., 2009) for the synthesis of hydroxytyrosyl ether
derivatives (4a–g). Thus, protection of the phenolic OH by reaction
with BnBr and K2CO3 in acetone, followed by the introduction of
the alkyl side chain after reacting with the corresponding alkyl io-
dide in presence of KOH, and cleavage of protecting benzyl group
by hydrogenolysis, constituted the main steps involved in the
The radical-scavenging activities of the evaluated compounds
(4h–u), Ty (1b), HMV (1c), BHT and a-tocopherol are summarised
in Table 2. Results are expressed as mM TE (Trolox equivalent). The
order of the scavenging activities towards the ABTS radical was
HMV and its derivatives (1c, 4o–u) > a-tocopherol > tyrosol and
its derivatives (1b, 4h–m) > BHT > dodecyl tyrosyl ether (4n). In
addition, trends on the antioxidant activity observed within each
series were similar to those described above for the ferric reducing
activity.
3.5. The ORAC assay
The antioxidant capacity of Ty (1b), HMV (1c) and their deriva-
tives (4h–u), BHT and
a-tocopherol determined by ORAC assay is
Table 1
Reducing antioxidant power evaluated by the FRAP assay of tyrosol (1b), homovanillic
alcohol (1c), tyrosyl derivatives (4h–n), homovanillyl derivatives (4o–u), -tocoph-
a
erol and BHT. Each value is the mean of triplicate measurements standard
deviations. Results are expressed as mM TE (Trolox Equivalents). All values within
a column with different superscript letters are significantly different, p < 0.05.
Compound
mM TE
Compound
mM TE
Ty (1b)
0.31 0.03c
0.16 0.01b
0.18 0.03b
0.16 0.02b
0.16 0.02b
0.15 0.02a,b
0.11 0.02a
0.13 0.02a,b
1.32 0.05e
0.80 0.04d
HMV (1c)
1.44 0.02f,g
1.47 0.03g
1.60 0.03h
1.61 0.03h
1.41 0.03e,f
1.37 0.02d
1.05 0.02c
0.87 0.02b
1.32 0.05d
0.80 0.04a
Methyl_Ty (4h)
Ethyl_Ty (4i)
Propyl_Ty (4j)
Butyl_Ty (4k)
Hexyl_Ty (4l)
Octyl_Ty (4m)
Dodecyl_Ty (4n)
BHT
Methyl_HMV (4o)
Ethyl_HMV (4p)
Propyl_HMV (4q)
Butyl_HMV (4r)
Hexyl_HMV (4s)
Octyl_HMV (4t)
Dodecyl_HMV (4u)
BHT
a-Tocopherol
a-Tocopherol

1176
A. Madrona et al. / Food Chemistry 129 (2011) 1169–1178
Table 3
Radical-scavenging capacity evaluated by ORAC assay of tyrosol (1b), homovanillic alcohol (1c), tyrosyl ethers (4h–n), homovanillyl ethers (4o–u),
values are expressed as mol Trolox/ mol tested compound. Linear range used to plot Net AUC versus concentration, slopes and intercepts of the linear functions are also
indicated. Each value is the mean of quadruplicate measurements standard deviations. All values with different superscript letters are significantly different, p < 0.05.
a-tocopherol and BHT. ORAC
l
l
Compound
ORAC value
Conc. Range (
lM)
Slope
Intercept
R2
Ty (1b)
0.79 0.04f
0.57 0.06b,c
0.47 0.05b
0.64 0.05c,d
0.69 0.05d,e
0.62 0.02c,d
0.69 0.03e
0.68 0.03e
1.15 0.04i,j
1.36 0.01l
1.25 0.04k
1.33 0.04k,l
1.09 0.04h,i
1.18 0.02j
1.04 0.05h
0.92 0.02g
0.12 0.01a
0.63 0.06c,d,e
5–20
5–20
5–20
5–20
5–20
5–20
20–40
20–50
5–40
5–40
5–40
5–40
5–40
10–40
10–40
10–40
50–400
12.5–100
0.2945
0.2622
0.2239
0.1630
0.2517
0.2357
0.3352
0.2217
0.4597
0.4692
0.4758
0.5334
0.4014
0.4337
0.2039
0.2125
0.0425
0.2174
1.6529
0.6876
0.5062
2.0843
1.4854
1.2597
2.0080
5.1970
2.1122
3.2223
2.4999
2.4077
2.3145
2.5589
3.9922
3.2015
2.9380
3.7231
0.9908
0.9988
0.9936
0.9980
0.9980
0.9986
0.9945
0.9998
0.9920
0.9903
0.9968
0.9981
0.9992
0.9961
0.9994
0.9998
0.9989
0.9963
Methyl_Ty (4h)
Ethyl_Ty (4i)
Propyl_Ty (4j)
Butyl_Ty (4k)
Hexyl_Ty (4l)
Octyl_Ty (4m)
Dodecyl_Ty (4n)
HMV (1c)
Methyl_HMV (4o)
Ethyl_HMV (4p)
Propyl_HMV (4q)
Butyl_HMV (4r)
Hexyl_HMV (4s)
Octyl_HMV (4t)
Dodecyl_HMV (4u)
BHT
a-Tocopherol
preparation of tyrosyl and homovanillyl alkyl ethers with good
yield.
Then, their potential antioxidant activities were evaluated by
different methods and compared with their precursors Ty (1b)
and HMV (1c), and others two controls traditionally used as food
most potent antioxidant should be the most polar one. Besides,
these results were in disagreement with those previously reported
for the hydroxytyrosyl series (Pereira-Caro et al., 2009), whose al-
kyl derivatives (4a–g) resulted less actives than free hydroxytyro-
sol and no relationship was found between the length and nature
of the alkyl side chain and the stability of lipid matrices.
antioxidants: butylated hydroxytoluene (BHT) and
a-tocopherol.
The selected methods to evaluate their antioxidant capacity were,
as it was explained above, the Rancimat test and the FRAP, ABTS
and ORAC assays.
On the other hand, the reducing and radical-scavenging capac-
ities of the new synthesized compounds (4h–u) depicted in Tables
1–3, were significantly higher for homovanillyl derivatives (4o–u)
in comparison with tyrosyl ones (4h–n). This great difference of
activity between both series was somehow surprising taking into
account that 3-methoxy-4-hydroxyphenolic series presents the
ortho-diphenolic structure blocked by a methoxy group that could
affect their antioxidant activity as indeed occurred in the Rancimat
analysis, where HTy (1a) was substantially more antioxidant than
Ty (1b) and HMV (1c). Furthermore, FRAP and ABTS activities of
HMV (1c) (1.44 and 1.29 mM Trolox Equivalent, respectively) were
somewhat higher than that described for HTy (1.39 and 0.84 mM
Trolox Equivalent, respectively), previously reported by our group
(Pereira-Caro et al., 2009). However, ORAC activity of HMV (1c)
was significantly lower than in the case of HTy (1.15 and 2.28 as
An overall evaluation of the described results showed the
higher antioxidant capacity for homovanillic alcohol (1c) and
its alkyl ether derivatives (4o–u) in comparison with Ty (1b)
and its derivatives (4h–n). In the particular case of the Rancimat
test (Fig. 1), comparison of IT values with those previously re-
ported for HTy (1a) and its alkyl derivatives (4a–g) (Pereira-Caro
et al., 2009) pointed out the higher influence of the ortho-
diphenolic structure on the antioxidant capacity present in the
hydroxytyrosyl series, in comparison with the monophenolic
(1b, 4h–n) and 3-methoxy-4-hydroxyphenolic series (1c, 4o–u),
respectively. However, the higher activity described above for
homovanillyl ethers in comparison with tyrosyl derivatives indi-
cated the positive influence of the methoxy group in ortho posi-
tion to the hydroxylic group, in agreement with the described
stabilization of the phenoxyl radical by ortho substitutions with
electron-donating groups as alkyl or methoxy groups, among
lmol Trolox/lmol tested compound, respectively).
Recently, the radical-scavenging activity of HTy (1a), Ty (1b),
and HMV (1c), as well as some hydroxytyrosyl and HMV esters
determined by the DPPH method has been reported (Grasso
et al., 2007). Apart from the higher antioxidant capacity of lipo-
philic hydroxytyrosyl analogues in comparison with the homovan-
illyl series or free Ty (1b), substitution of the hydroxylic function in
the aromatic ring by a methoxy group provoked a drastic decrease
in the antioxidant activity, being HMV (1c) and its esters even less
active than free Ty (1b). These results agreed with that reported by
Torres de Pinedo, Peñalver, & Morales, 2007; Torres de Pinedo,
Peñalver, Pérez-Victoria et al., 2007, where ortho-diphenolic alco-
hols (protocatechuyl alcohol, hydroxytyrosol, dihydrocaffeoyl alco-
hol and caffeoyl alcohol) showed better radical-scavenging activity
evaluated by DPPH assay than did the corresponding mono-
methoxylated cathecolic alcohols (vanillyl alcohol, homovanillyl
alcohol, dihydroconifeyl alcohol and coniferyl alcohol). However,
these same authors evaluated the activity of new synthetic lipo-
philic antioxidants derivatives of HTy in comparison with HMV
derivatives by ABTS assay observing as methoxylated-cathecolic
antioxidants were significantly better radical scavengers than
ortho-diphenolic lipophilic antioxidants. This behaviour is
others (Chimi, Cillard, Cillard,
& Rahmani, 1991; Pokorny,
1987). Finally, it is important to remark that, in spite of the
higher activity of homovanillyl series (1c, 4o–u) versus tyrosyl
series (1b, 4h–n) showed in the Rancimat test, it did not reach
the activity of the references evaluated, BHT and
a-tocopherol,
significantly more active than the two synthetic series (4h–u)
and their references (1b and 1c), due to their chemical structures
with methyl group in position para to the functional hydroxylic
group in the chromanol ring of vitamin E and phenolic group of
BHT, fundamental to the hydrogen donating ability implied in
the lipidic peroxidation evaluated by this test.
Regarding the activity showed within each series by the Ranci-
mat test (Fig. 1b and c), all the alkyl derivatives were slightly more
active than their respective precursors, Ty (1b) or HMV (1c), and, in
the specific case of homovanillyl series (1c, 4o–u), a direct relation-
ship between lipophilic nature and antioxidant activity can been
observed (Fig. 1c). In this sense, the results obtained here were
contrary to the polar paradox by which in non-polar medium the

A. Madrona et al. / Food Chemistry 129 (2011) 1169–1178
1177
extensive to hydroxycinnamic acids (caffeic, ferulic and chlorogen-
References
ic acids) after their evaluation by ABTS assay, where substitution of
the 3-hydroxyl group of caffeic acid by a methoxyl group in ferulic
acid enhanced the antioxidant capacity (Rice-Evans, Miller, &
Paganga, 1996). In line with this results, Gómez-Ruiz, Leake, and
Ames (2007) found that ferulic acid was more active than caffeic
acid evaluated by ABTS assay while their scavenging activity eval-
uation by ORAC assay showed a contrary tendency (Dávalos,
Gómez-Cordovés, & Bartolomé, 2004; Gómez-Ruiz et al., 2007),
confirming the results presented in the present work.
In this sense, although ABTS, ORAC and DPPH have been classified
as radical-scavenging activity methods in which free radicals react
with a H-atom donor such as phenolics compounds, DPPH and ORAC
seems to be more selective than ABTS in the reaction with H-donors
and it does not react with aromatic acids containing only one OH-
group (Von Gadov, Joubert, & Hansmann, 1997), while ABTS is re-
duced in the presence of compounds with aromatic OH-groups,
independently of their real antioxidant power (Roginsky & Lissi,
2005).
Ajao, J. F., Bird, C. W., & Chauhan, Y. P. (1985). The evaluation of a biogenetically
based approach to the synthesis of octahydro-1h-benzofuro[3, 2-
e]isoquinolines. Tetraedron Letters, 41, 1367–1372.
Alcudia, F., Cert, A., Espartero, J. L., Mateos, R., & Trujillo, M. (2004). Method of
preparing hydroxytyrosol esters, esters thus obtained and use of same. PCT WO
2004/005237.
Appendino, G., Minassi, A., Daddario, N., Bianchi, F.,
& Tron, G. C. (2002).
Chemoselective esterification of phenolic acids and alcohols. Organic Letters, 4,
3839–3841.
Battersby, A. R., Le Count, D. J., Garratt, S.,
& Thrift, R. I. (1961). Synthetic
applications of 1,2-dihydroisoquinolines: Synthesis of ( )-coreximine.
Tetraedron, 14, 46–53.
Beauchamp, G. K., Keast, R. S. J., Morel, D., Lin, J., Pika, J., Han, Q., et al. (2005).
Ibuprofen-like activity in extra-virgin olive oil. Nature, 437, 45–46.
Benzie, I. F. F., & Strain, J. J. (1996). The Ferric reducing ability of plasma (FRAP) as a
measure of ‘‘antioxidant power’’: The FRAP assay. Analytical Biochemistry, 239,
70–76.
Bernini, R., Mincione, E., Barontini, M., & Crisante, F. (2008). Convenient synthesis of
hydroxytyrosol and its lipophilic derivatives from tyrosol or homovanillyl
alcohol. Journal of Agricultural and Food Chemistry, 56, 8897–8904.
Bitler, C. M., Viale, T. M., Damaj, B., & Crea, R. (2005). Hydrolyzed olive vegetation
water in mice has anti-inflammatory activity. Journal of Nutrition, 135,
1475–1479.
On the other hand, the behaviour of the radical scavenging
and reducing activities of these new lipophilic synthetic com-
pounds (4h–u) in comparison with their precursors (1b and
1c) was antagonistic. While tyrosyl ethers (4h–n) were less ac-
tive than their reference Ty (1b), homovanillyl ethers (4o–u)
showed a non-linear evolution of antioxidant capacity with the
enhanced lipophilic nature, with a parabolic relationship be-
tween the alkyl chain length and the antioxidant activity. This
behaviour agrees with that previously described for tyrosyl and
hydroxytyrosyl esters (Mateos et al., 2008) and hydroxytyrosyl
ethers (Pereira-Caro et al., 2009). It is interesting to remark that
the characteristic non-linear evolution of antioxidant capacity of
homovanillyl ethers (4o–u) with the enhanced lipophilic nature
are partially out of the popular polar paradox and in accordance
with the results reported by Laguerre et al., after the evaluation
of chlorogenate esters (Laguerre et al., 2009) and rosmarinate es-
ters (Laguerre et al., 2010) of variable acylic chain length. Be-
sides, in order to explain the non-linear hypothesis of the
antioxidant activity, evaluation of surface-active properties of
lipophilic antioxidants Ty (1b) and hydroxytyrosol fatty acid es-
ters in oil-in-water emulsion was carried out (Lucas et al., 2010),
providing a useful tool in the rational design of antioxidants
used in oil-in-water emulsions.
In conclusion, these results provide a batch of new derivatives
of Ty (1b) and HMV (1c) in which the enhanced lipophilic nature
is attractive for their possible commercialisation as lipophilic
derivatives in the food industry. Besides, despite the variable
antioxidant activity observed after their evaluation by the Ranc-
imat test and the FRAP, ABTS and ORAC assays, where homovan-
illyl ethers (4o–u) showed the best activity as reducing agents
and radical scavengers, it is important to remark on the interest-
ing biological activity described for their precursors, Ty (1b) and
HMV (1c), potentially extrapolated to their alkyl derivatives
(4h–u) presented herein.
Bouallagui, Z., Bouaziz, M., Lassoued, S., Engaser, J. M., Ghoul, M., & Sayadi, S. (2011).
Hydroxytyrosol acyl esters: Biosynthesis and activities. Applied Biochemistry and
Biotechnology, 163, 592–599.
Buisman, G. J. H., Van Helteren, C. T. W., Kramer, G. F. H., Veldsink, J. W., Derksen, J.
T. P., & Cuperus, F. P. (1998). Enzymatic esterifications of functionalized phenols
for the synthesis of lipophilic antioxidants. Biotechnology Letters, 20, 131–136.
Capasso, R., Sannino, F., De Martino, A.,
& Manna, C. (2006). Production of
triacetylhydroxytyrosol from olive mill waste waters for use as stabilized
bioantioxidant. Journal of Agricultural and Food Chemistry, 54, 9063–9070.
Caruso, D., Visioli, F., Patelli, R., Galli, C., & Galli, G. (2001). Urinary excretion of olive
oil phenols and their metabolites in humans. Metabolism Clinical and
Experimental, 50, 1426–1428.
Chillemi, R., Sciuto, S., Spatafora, C., & Tringali, C. (2010). Hydroxytyrosol lipophilic
analogues: Synthesis, radical scavenging activity and human cell oxidative
damage protection. In V. R. Preedy & R. R. Watson (Eds.), Olives and olive oil in
health and disease prevention (pp. 1233–1243). New York: Academic Press.
Chimi, H., Cillard, J., Cillard, P., & Rahmani, M. (1991). Peroxyl radical scavenging
activity of some natural phenolic antioxidants. Journal of the American Oil
Chemists Society, 68, 307–312.
Dávalos, A., Gómez-Cordovés, C., & Bartolomé, B. (2004). Extending applicability of
the oxygen radical absorbance capacity (ORAC-Fluorescein) assay. Journal of
Agricultural and Food Chemistry, 52, 48–54.
De la Torre-Carbot, K., Chavez-Servin, J. L., Jaúregui, O., Castellote, A. I., Lamuela-
Raventós, R. M., Nurmi, T., et al. (2010). Elevated circulating LDL phenol levels in
men who consumed virgin rather than refined olive oil are associated with less
oxidation of plasma LDL. Journal of Nutrition, 140, 501–508.
Dell’Agli, M., Maschi, O., Galli, G. V., Fagnani, R., Dal Cero, E., Caruso, D., et al. (2008).
Inhibition of platelet aggregation by olive oil phenols via cAMP-
phosphodiesterase. British Journal of Nutrition, 99, 945–951.
Di Benedetto, R., Vari, R., Scazzocchio, B., Filesi, C., Santangelo, C., Giovannini, C.,
et al. (2007). Tyrosol, the major extra virgin olive oil compound, restored
intracellular antioxidant defences in spite of its weak antioxidative
effectiveness. Nutrition, Metabolism and Cardiovascular Diseases, 17, 535–545.
Fernández-Bolaños, J., Heredia, A., Rodríguez, G., Rodríguez, R., Jiménez, A., & Gillen,
R. (2005). Methods for obtaining purified hydroxytyrosol from products and by-
products derived from the olive tree. US 6849,770 B2.
Fernández-Bolanos, J. G., Lopez, O., Fernández-Bolanos, J., & Rodríguez-Gutiérrez, G.
(2008). Hydroxytyrosol and derivatives: Isolation, synthesis, and biological
properties. Current Organic Chemistry, 12, 442–463.
Giovannini, C., Straface, E., Modesti, D., Coni, E., Cantafora, A., & De Vincenzi, M.
(1999). Tyrosol, the major olive oil biophenol, protects against oxidized LDL-
induced injury in Caco2 cells. Journal of Nutrition, 129, 1269–1277.
Gómez-Ruiz, J. A., Leake, D. S., & Ames, J. M. (2007). In vitro antioxidant activity of
coffee compounds and their metabolites. Journal of Agricultural and Food
Chemistry, 55, 6962–6969.
Gordon, M. H., Paiva-Martins, F., & Almeida, M. (2001). Antioxidant activity of
hydroxytyrosol acetate compared with that of other olive oil polyphenols.
Journal of Agricultural and Food Chemistry, 49, 2480–2485.
Acknowledgements
Goya, L., Mateos, R., & Bravo, L. (2007). Effect of the olive oil phenol hydroxytyrosol
on human hepatoma HepG2 cells. Protection against oxidative stress induced
by tert-butylhydroperoxide. European Journal of Nutrition, 46, 70–78.
Grasso, S., Siracusa, L., Spatafora, C., Renis, M., & Tringali, C. (2007). Hydroxytyrosol
lipophilic analogues: Enzymatic synthesis, radical scavenging activity and DNA
oxidative damage protection. Bioorganic Chemistry, 35, 137–152.
Huang, D., Ou, B., Hampsch-Woodill, M., Flanagan, J. A., & Deemer, E. K. (2002).
Development and validation of oxygen radical absorbance capacity assay for
lipophilic antioxidants using randomly methylated b-cyclodextrin as the
solubility enhancer. Journal of Agricultural and Food Chemistry, 50, 1815–1821.
Incani, A., Deiana, M., Corona, G., Vafeiadou, K., Vauzour, D., Dessi, M. A., et al.
(2010). Involvement of ERK, Akt and JNK signalling in H2O2-induced cell injury
This work was supported by projects AGL2007-66373/ALI and
CSD2007-00063 as part of the Consolider-Ingenio Program from
the Spanish Ministry of Science and Innovation (CICYT), Grant
RTA2007-000036-00-00 from the Research National Institute of
Food, Agriculture and Technology (INIA) and
a
contract
(110105090014) with CSIC-IFAPA. G.P.-C. is a predoctoral fellow
of the National Research Institute of Food, Agriculture and Tech-
nology (INIA).

1178
A. Madrona et al. / Food Chemistry 129 (2011) 1169–1178
and protection by hydroxytyrosol and its metabolite homovanillic alcohol.
Molecular Nutrition and Food Research, 54, 788–796.
Pereira-Caro, G., Madrona, A., Bravo, L., Espartero, J. L., Alcudia, F., Cert, A., et al.
(2009). Antioxidant activity evaluation of alkyl hydroxytyrosyl ethers, a new
class of hydroxytyrosyl derivatives. Food Chemistry, 115, 86–91.
Pereira-Caro, G., Mateos, R., Saha, S., Madrona, A., Espartero, J. L., Bravo, L., et al.
(2010). Transepithelial Transport and Metabolism of New Lipophilic Ether
Derivatives of Hydroxytyrosol by Enterocyte-like Caco-2/TC7 Cells. Journal of
Agricultural and Food Chemistry, 58, 11501–11509.
Laguerre, M., Giraldo, L. J., Lecomte, J., Figueroa-Espinoza, M.C., Baréa, B., Weiss, J.,
Decker, E.A., & Villeneuve, P. (2010). Relationship between hydrophobicity and
antioxidant ability of ’Phenolipids’ in emulsion: a parabolic effect of the chain
length of rosmarinate esters. Journal of Agricultural and Food Chemistry, 58,
2869-2876 (b).
Laguerre, M., Giraldo, L. J., Lecomte, J., Figueroa-Espinoza, M. C., Baréa, B., Weiss, J.,
et al. (2009). Chain length affects antioxidant properties of chlorogenate esters
in emulsion: The cutoff theory behind the polar paradox. Journal of Agricultural
and Food Chemistry, 57, 11335–11342.
Pokorny, J. (1987). Major factors affecting the antioxidant of lipids. In H. Chan (Ed.),
Autoxidation of unsaturated lipids (pp. 141–206). London: Academic Press.
Pokorny, J. (1991). Natural antioxidants for food use. Trends in Food Science and
Technology, 16, 223–227.
Larrosa, M., Espín, J. C., & Tomás-Barberán, F. A. (2003). Antioxidant capacity of
tomato juice functionalised with enzymatically synthesised hydroxytyrosol.
Journal of the Science of Food and Agriculture, 83, 658–666.
Lucas, R., Comelles, F., Alcantara, D., Maldonado, O. S., Curcuroze, M., Parra, J. L., et al.
(2010). Surface-active properties of lipophilic antioxidants tirosol and
hydroxytyrosol fatty acid esters: A potencial explanation for the nonlinear
hipótesis of the antioxidant activity in oil-in-water emulsions. Journal of
Agricultural and Food Chemistry, 58, 8021–8026.
Pokorny, J. (2007). Are natural antioxidants better – and safer – than synthetic
antioxidants? European Journal of Lipid Science and Technology, 109, 629–642.
Pulido, R., Bravo, L., & Saura-Calixto, F. (2000). Antioxidant activity of dietary
polyphenols as determined by a modified ferric reducing/antioxidant power
assay. Journal of Agricultural and Food Chemistry, 48, 3396–3402.
Re, R., Pellegrini, N., Proteffente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999).
Antioxidant activity applying an improved ABTS radical cation decolouration
assay. Free Radical Biology and Medicine, 26, 1231–1237.
Madrona, A., Pereira-Caro, G., Mateos, R., Rodriguez, G., Trujillo, M., Fernandez-
Bolanos, J., et al. (2009). Synthesis of hydroxytyrosyl alkyl ethers from olive oil
waste waters. Molecules, 14, 1762–1772.
Rice-Evans, C. A., Miller, N. J., & Paganga, G. (1996). Structure-antioxidant activity
relationship of flavonoids and phenolic acids. Free Radical Biology and Medicine,
20, 933–956.
Manna, C., Napoli, D., Cacciapuoti, G., Porcelli, M., & Zappia, V. (2009). Olive oil
phenolic compounds inhibit homocysteine-induced endothelial cell adhesion
regardless of their different antioxidant activity. Journal of Agricultural and Food
Chemistry, 57, 3478–3482.
Martín, M. A., Ramos, S., Granado-Serrano, A. B., Rodríguez-Ramiro, I., Trujillo, M.,
Bravo, L., et al. (2010). The olive oil phenol Hydroxytyrosol induces antioxidant/
detoxificant enzymes activity and Nrf2 translocation via ERKs and PI3K/AKT
pathways in HepG2 cells. Molecular Nutrition and Food Research, 54, 1–11.
Mateos, R., Domínguez, M. M., Espartero, J. L., & Cert, A. (2003). Antioxidant effect of
Rietjens, S. J., Bast, A., & Haenen, G. R. M. M. (2007). New insights into controversies
on the antioxidant potential of the olive oil antioxidant hydroxytyrosol. Journal
of Agricultural and Food Chemistry, 55, 7609–7614.
Rietjens, S. J., Bast, A., Vente, J., & Haenen, G. R. M. M. (2007). The olive oil
antioxidant hydroxytyrosol efficiently protects against the oxidative stress-
induced impairment of the NO response of isolated rat aorta. American Journal of
Physiology Heart and Circulatory Physiology, 292, 1931–1936.
Roginsky, V., & Lissi, E. A. (2005). Review of methods to determine chain-breaking
antioxidant activity in food. Food Chemistry, 92, 235–254.
phenolic compounds,
a
-tocopherol, and other minor components in virgin olive
Tofani, D., Balducci, V., Gasperi, T., Incerpi, S., & Gambacorta, A. (2010). Fatty acid
hydroxytyrosyl esters: Structure/antioxidant activity relationship by ABTS and
in cell-culture DCF assays. Journal of Agricultural and Food Chemistry, 58,
5292–5299.
Torregiani, E., Seu, G., Minassi, A., & Appendino, G. (2005). Cerium(III) chloride-
promoted chemoselective esterification of phenolic alcohols. Tetrahedron
Letters, 46, 2193–2196.
oil. Journal of Agricultural and Food Chemistry, 51, 7170–7175.
Mateos, R., Espartero, J. L., Trujillo, M., Ríos, J. J., León-Camacho, M., Alcudia, F., et al.
(2001). Determination of phenols, flavones, and lignans in virgin olive oils by
solid-phase extraction and high-performance liquid chromatography with
diode array ultraviolet detection. Journal of Agricultural and Food Chemistry,
49, 2185–2192.
Mateos, R., Trujillo, M., Pereira-Caro, G., Madrona, A., Cert, A., & Espartero, J. L.
(2008). New lipophilic tyrosyl esters. Comparative antioxidant evaluation with
hydroxytyrosyl esters. Journal of Agricultural and Food Chemistry, 56,
10960–10966.
Mateos, R., Uceda, M., Aguilera, M. P., Escuderos, M. E., & Beltrán, G. (2006).
Relationship of rancimat method values at varying temperatures for virgin olive
oils. European Food Research and Technology, 223, 246–252.
Torres de Pinedo, A., Peñalver, P., & Morales, J. C. (2007). Synthesis and evaluation of
new phenolic-based antioxidants: Structure–activity relationship. Food
Chemistry, 103, 55–61.
Torres de Pinedo, A., Peñalver, P., Pérez-Victoria, I., Rondón, D., & Morales, J. C.
(2007). Synthesis of new phenolic fatty acid esters and their evaluation as
lipophilic antioxidants in an oil matrix. Food Chemistry, 105, 657–665.
Torres de Pinedo, A., Peñalver, P., Rondon, D., & Morales, J. C. (2005). Efficient lipase-
catalyzed synthesis of new lipid antioxidants based on a catechol structure.
Tetrahedron, 61, 7654–7660.
Trujillo, M., Mateos, R., Collantes de Teran, L., Espartero, J. L., Cert, R., Jover, M., et al.
(2006). Lipophilic hydroxytyrosyl esters. Antioxidant activity in lipid matrices
and biological systems. Journal of Agricultural and Food Chemistry, 54,
3779–3785.
Visioli, F., Galli, C., Bornet, F., Mattei, A., Patelli, R., Galli, G., et al. (2000). Olive oil
phenolics are dose-dependently absorbed in humans. FEBS Letters, 468,
159–160.
Medina, I., Lois, S., Alcántara, D., Lucas, R.,
& Morales, J. C. (2009). Effect of
lipophilization of hydroxytyrosol on its antioxidant activity in fish oils and fish
oil-in-water emulsions. Journal of Agricultural and Food Chemistry, 57,
9773–9779.
Miró-Casas, E., Covas, M. I., Farré, M., Fito, M., Ortuño, J., Weinbrenner, T., et al.
(2003). Hydroxytyrosol disposition in humans. Clinical Chemistry, 49, 945–952.
Palozza, P., Simone, R., Picci, N., Buzzoni, L., Ciliberti, N., Natangelo, A., et al. (2008).
Design, synthesis, and antioxidant potency of novel a-tocopherol analogues in
isolated membranes and intact cells. Free Radical Biology
& Medicine, 44,
1452–1464.
Vissers, M. H., Zock, P. L., Roodenburg, A. J., Leenen, R., & Katan, M. B. (2002). Olive
oil phenols are absorbed in humans. Journal of Nutrition, 132, 409–417.
Von Gadov, A., Joubert, E., & Hansmann (1997). Comparison of the antioxidant
activity of aspalathin with that of other plant phenols of Roobies tea (Aspalathus
linearis), -tocopherol, BHT, and BHA. Journal of Agricultural and Food Chemistry,
44, 632–638.
Yoshida, H., Kondo, I., & Kajimoto, G. (1992). Participation of free fatty acids in the
oxidation of purified soybean oil during microwave heating. Journal of American
Oil Chemists Society, 69, 1136–1140.
Paquot, C., & Hautfenne, A. (1992). Determination of tocopherols and tocotrienols in
vegetable oils and fats by HPLC. Method 2, 432. Standard methods for the analysis of
oils, fats and derivatives. IUPAC, revised and enlarged (7th ed.). Oxford, UK:
Blackwell Scientific Publications.
Pazos, M., Alonso, A., Sánchez, I., & Medina, I. (2008). Hydroxytyrosol prevents
oxidative deterioration in foodstuffs rich in fish lipids. Journal of Agricultural and
Food Chemistry, 56, 3334–3340.
Pereira-Caro, G., Bravo, L., Madrona, A., Espartero, J. L., & Mateos, R. (2010). Uptake
and metabolism of new synthetic lipophilic derivatives, hydroxytyrosyl ethers,
by human hepatoma HepG2 cells. Journal of Agricultural and Food Chemistry, 58,
798–806.