Synthesis and antioxidant evaluation of isochroman-derivatives of hydroxytyrosol: Structure-activity relationship

Article abstract of DOI:10.1016/j.foodchem.2014.10.036

Isochroman-derivatives of the natural olive oil phenol hydroxytyrosol (HT) have been synthesised via Oxa-Pictet-Spengler reaction in high yields. Lipophilicity and antioxidant activity were determined to establish the structure-activity relationship of isochromans compared to HT, BHT and α-tocopherol. Antioxidant capacity was tested in two different media: bulk oils, using the Rancimat test, and brain homogenates, by measuring malondialdehyde (MDA) levels as a lipoperoxidation biomarker. In addition, other antioxidant assays (FRAP, ABTS and ORAC) were carried out. Rancimat and MDA results show that antioxidant activity was related with lipophilicity, directly in brain homogenates and inversely in the oils, in agreement with the polar paradox. Free o-diphenolic groups positively determined the activity in the oils, whereas reducing and radical-scavenging activities were related to the number of free hydroxyl moieties. BHT and α-tocopherol showed lower antioxidant activity than isochromans and HT. We conclude that HT-isochromans present significant potential as bioactive compounds.

Full text of DOI:10.1016/j.foodchem.2014.10.036

Accepted Manuscript
Synthesis and antioxidant evaluation of isochroman-derivatives of hydroxytyr-
osol: structure-activity relationship
Raquel Mateos, Andrés Madrona, Gema Pereira-Caro, Vanessa Domínguez,
Rosa Mª Cert, Juan Parrado, Beatriz Sarriá, Laura Bravo, José Luis Espartero
PII:
S0308-8146(14)01598-2
DOI:
http://dx.doi.org/10.1016/j.foodchem.2014.10.036
FOCH 16563
Reference:
Food Chemistry
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Revised Date:
Accepted Date:
8 August 2014
3 October 2014
6 October 2014
Please cite this article as: Mateos, R., Madrona, A., Pereira-Caro, G., Domínguez, V., Cert, R.M., Parrado, J.,
Sarriá, B., Bravo, L., Espartero, J.L., Synthesis and antioxidant evaluation of isochroman-derivatives of
hydroxytyrosol: structure-activity relationship, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/j.foodchem.
2014.10.036
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ACCEPTED MANUSCRIPT
Synthesis and antioxidant evaluation of isochroman-derivatives of hydroxytyrosol:
structure-activity relationship
Raquel Mateos1,*, Andrés Madrona2, Gema Pereira-Caro3, Vanessa Domínguez2, Rosa Mª Cert4, Juan
Parrado5, Beatriz Sarriá1, Laura Bravo1, José Luis Espartero2,*
1) Department of Metabolism and Nutrition, Institute of Food Science, Technology and Nutrition (ICTAN),
Spanish National Research Council (CSIC), C/ José Antonio Nováis 10, 28040 Madrid, Spain.
2) Department of Organic and Pharmaceutical Chemistry, Faculty of Pharmacy, University of Seville, C/ Prof.
García González 2, Seville, Spain.
3) IFAPA. Alameda del Obispo, Avda. Menéndez-Pidal s/n, Córdoba, Spain.
4) Instituto de la Grasa (CSIC), Avda. Padre García Tejero 4, Sevilla, Spain.
5) Department of Biochemistry, Faculty of Pharmacy, University of Seville. C/ Prof. García González 2, E-
41012 Sevilla, (Spain).
Running title: Synthesis and antioxidant evaluation of isochromans.
* Corresponding authors:
Raquel Mateos. Department of Metabolism and Nutrition, Instituto de Ciencia y Tecnología de Alimentos y
Nutrición (ICTAN-CSIC). C/ José Antonio Nováis 10, Ciudad Universitaria, 28040 Madrid, Spain. E-mail:
raquel.mateos@ictan.csic.es
José Luis Espartero. Department of Organic and Pharmaceutical Chemistry, School of Pharmacy, University
of Seville, 41012 Seville, Spain. E-mail: jles@us.es
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ABSTRACT
Isochroman-derivatives of the natural olive oil phenol hydroxytyrosol (HT) have been synthesized via Oxa-
Pictet-Spengler reaction in high yields. Lipophilicity and antioxidant activity were determined to establish
the structure-activity relationship of isochromans compared to HT, BHT and α-tocopherol. Antioxidant
capacity was tested in two different media: bulk oils, using the Rancimat test, and brain homogenates, by
measuring malondialdehyde (MDA) levels as a lipoperoxidation biomarker. In addition, other antioxidant
assays (FRAP, ABTS and ORAC) were carried out. Rancimat and MDA results show that antioxidant activity
was related with lipophilicity, directly in brain homogenates and inversely in the oils, in agreement with the
polar paradox. Free o-diphenolic groups positively determined the activity in the oils, whereas reducing and
radical-scavenging activities were related to the number of free hydroxyl moieties. BHT and α-tocopherol
showed lower antioxidant activity than isochromans and HT. We conclude that HT-isochromans present
significant potential as bioactive compounds.
Keywords: Olive oil isochromans, hydroxytyrosol, antioxidant activity, lipophilic phenolic antioxidants, polar
paradox.
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1. INTRODUCTION
The beneficial effects on health of mono and polyunsaturated fatty acids are well known, particularly their
effects on cardiovascular function (Schwingshackl & Hoffmann, 2012; Kromhout & De Goede, 2014). During
food processing, transport or storage, oxidative degradation of unsaturated fatty acids takes place having
negative repercussions on food quality. Therefore, avoiding such oxidation represents a challenge for the
food industry as well as the cosmetic industry (Sanches-Silva et al., 2014; Liu, Jin & Zhang, 2014). Oxidation
may be counteracted to a certain extent by adding antioxidants that may prevent or delay oxidative
reactions. Nowadays, most antioxidants used to control lipid oxidation are synthetic, such as butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertiary butyl hydroquinone (TBHQ), and propyl
gallate (EEC/2006/52, 2006). However, certain synthetic antioxidants have shown potential carcinogenic
effects; besides, consumers prefer natural ingredients over synthetic compounds. The use of natural
antioxidants, such as phenolic compounds, is a safer and healthier alternative to synthetic compounds.
Most natural phenols are hydrophilic polyhydroxy derivatives, which reduces their application, particularly
under hydrophobic conditions (Liu et al., 2014). Other lipophilic phenolic compounds such as phenolic acid
esters or phenolic acid lactones, among others, are also natural antioxidants although present in foods in
small amounts, requiring time-consuming and not cost-profitable procedures for their isolation and
purification, which limits their potential industrial application. Therefore, the synthesis of lipophilic
phenolic compounds is an ongoing challenge.
Among the phenolic compounds naturally present in our diet, hydroxytyrosol HT (1), as secoiridoid
derivative, in free form or as an acetate ester (Mateos et al. 2001), is one of the most abundant and
representative olive oil phenols. The phenolic fraction in virgin and/or extra virgin olive oil has well
established beneficial health effects. In fact, EFSA has recently issued a positive opinion on HT and other
olive oil phenols’ capacity to protect low-density lipoproteins (LDL) against oxidation (EFSA, 2011). Another
minor group of phenolic compounds related to HT that have been identified in olive oil are 6,7-
dihydroxyisochromans, in particular 1-phenyl-6,7-dihydroxyisochroman and 1-(4'-hydroxy-3'-
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methoxyphenyl)-6,7-dihydroxyisochroman (4) (Bianco, Coccioli, Guiso, & Marra, 2001). These compounds
have shown antiplatelet activity (Togna, Togna, Franconi, Marra, & Guiso, 2003), cytoprotective properties
(Zeh, Lorenz, Kreutzmann, & Schonfeld, 2008), in addition to antioxidant activity, reducing oxygen/nitrogen
reactive species (Schönfeld, Kruska, & Reiser, 2009), and anti-inflammatory activity (Trefiletti, Togna,
Latina, Marra, Guiso, & Togna, 2011, Togna, Latina, Trefiletti, Guiso, Moschini, & Togna, 2013). All these
effects confirm dihydroxyisochromans’ contribution to the beneficial health effects of the phenolic fraction
of olive oil.
Taking into account the bioactivity already attributed to isochromans and the increasing interest in
obtaining new lipophilic phenolic compounds with lower hydrophilic/lipophilic balance (HLB) than HT in
order to enhance its solubility in more lipophilic media, the aim of the present study was to synthesize a
batch of isochromans from HT, via oxa-Pictet-Spengler reaction. Lipophilicity and antioxidant activity of
isochromans were determined by complementary tests (Rancimat, FRAP, ABTS, ORAC and MDA assays).
Comparison with natural (HT, α-tocopherol) and synthetic (BHT) antioxidants was also carried out.
2. MATERIALS AND METHODS
2.1. Materials
All solvents and reagents were of analytical grade unless otherwise stated. Cumene hydroperoxide (CH), p-
toluenesulphonic acid, hexadeuterated methylsulfoxide (DMSO-d6), sucrose, trichloroacetic acid,
chloroform, sodium chloride, n-octanol, iron (III) chloride hexahydrate, potassium persulphate,
phenylmethylsulphonyl fluoride, ethylenediaminetetraacetic acid tetrasodium salt hydrate (EDTA),
chymostatin, leupeptin, microbial pepstatin A, α-tocopherol, 2,6-di-tert-butyl-4-methylphenol (BHT), 6-
hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), fluorescein, randomized methylated β-
cyclodextrin (RMCD), 2,2’-azobis(2-amidinopropane) dihydrocloride (AAPH), 2,2’-azino bis-(3-
ethylbenzothiazoline-6-sulphonic acid) diammonium salt (98%), 2,4,6-tri-(2-pyridyl)-1,3,5-triazine (TPTZ)
and 2-thiobarbituric acid (TBA) were from Sigma-Aldrich (Madrid, Spain). Sodium hydroxide, methanol,
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sodium hydrogen phosphate and potassium dihydrogen phosphate were from Panreac (Madrid, Spain).
Neutral alumina, type 507C, grade I, was acquired from Fluka AG (Buchs, Switzerland). Acetone, 3,4-
dihydroxybenzaldehyde,
3,4-dimethoxybenzaldehyde,
3,4-dioctyloxybenzaldehyde,
3-hydroxy-4-
methoxybenzaldehyde, 4-hydroxy-3-methoxybenzaldehyde were purchased from Acros Organics (Geel,
Belgium). HT was recovered with 95% purity from olive oil wastewaters following a patented industrial
procedure (Fernández-Bolaños, Heredia, Rodriguez, Rodriguez, Jimenez, & Guillen, 2005).
NMR spectra were recorded on a Bruker Avance 500 spectrophotometer operating at 500.13 MHz (1H) and
125.75 MHz (13C). Samples were dissolved (0.1 mmol/mL) in DMSO-d6 and spectra were recorded at 303 K.
Chemical 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 carried out on a Leco CHNS-932 apparatus. High-resolution EI mass
spectra were obtained on a Micromass AUTOSPECQ spectrometer.
2.2. Isochromans synthesis
Isochromans (2-7) were synthesized by means of the oxa-Pictet-Spengler reaction from HT (1) and the
corresponding carbonylic compounds, using an acid catalyst (Guiso, Marra, & Cavarischia, 2001, Guiso,
Bianco, Marra, & Cavarischia, 2003) (Figure 1). Briefly, 1 mmol of HT (1, 154 mg) was dissolved in 4 mL of
anhydrous methyl alcohol and mixed with 2 mmol of carbonyl compound (100% molar excess) in the
presence of 10 mg of p-toluenesulphonic acid as acid catalyst. The mixture was heated under reflux for 2
hours. After reaction completion (TLC plate), the mixture was cooled and the solvent evaporated under low
pressure. Finally, the products were purified using silica gel column chromatography.
Data for 1,1-dimethyl-6,7-dihydroxyisochroman (2): using acetone as carbonyl compound and purification
through silica gel column chromatography with ether/hexane (2:3, v/v) as eluent, a white solid was
obtained with a yield of 98%. M.p. 130-2ºC [130-2ºC] (Guiso et al., 2003). NMR data (see Tables 1 and 2).
Elem. Anal. Calcd. for C11H14O3: C, 68.02%; H, 7.27%; found: C, 67.84%; H, 7.41%. HRMS (El) m/z found
194.093926 (1.9 ppm).
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Data for 1-(3’,4’-dihydroxyphenyl)-6,7-dihydroxyisochroman (3): using 3,4-dihydroxybenzaldehyde as
carbonyl compound and purification through silica gel column chromatography with
dichloromethane/methanol (20:1, v/v) as eluent, a pink solid was obtained with a yield of 84.3%. M.p. 204-
6ºC [177-9ºC] (Lorenz, Zeh, Martens-Lobenhoffer, Schmidt, Wolf, & Horn, 2005). NMR data (see Tables 1
and 2). Elem. Anal. Calcd. for C15H14O5: C, 65.69%; H, 5.15%; found: C, 65.34%; H, 5.34%. HRMS (El) m/z
found 274,083643 (1.8 ppm).
Data
for
1-(4’-hydroxy-3’-methoxyphenyl)-6,7-dihydroxyisochroman
(4):
using
4-hydroxy-3-
methoxybenzaldehyde as carbonyl compound and purification through silica gel column chromatography
with dichloromethane/methanol (30:1, v/v) as eluent, a slightly pink solid was obtained with a yield of
72.6%. M.p.: 165-7ºC [154-6ºC] (Lorenz et al., 2005). NMR data (see Tables 1 and 2). Elem. Anal. Calcd. for
C16H16O5 x ¾ H2O: C, 63.67%; H, 5.84%, found: C, 63.72%; H, 5.52%. HRMS (El) m/z found 288,100202 (1.5
ppm).
Data
for
1-(3’-hydroxy-4’-methoxyphenyl)-6,7-dihydroxyisochroman
(5):
using
3-hydroxy-4-
methoxybenzaldehyde as carbonyl compound and purification through silica gel column chromatography
with dichloromethane/methanol (30:1, v/v) as eluent, an orange solid was obtained with a yield of 82.3%.
M.p.: 180-2ºC. NMR data (see Tables 1 and 2). Elem. Anal. Calcd. for C16H16O5 x H2O: C, 62.74%; H, 5.92%.
Found: C, 62.03%; H, 5.84%. HRMS (El) m/z found 288,100311 (1.9 ppm).
Data for 1-(3’,4’-dimethoxyphenyl)-6,7-dihydroxyisocroman (6): using 3,4-dimethoxybenzaldehyde as
carbonyl compound and purification through silica gel column chromatography with
dichloromethane/methanol (30:1, v/v) as eluent, a white solid was obtained with a yield of 77.5%. M.p.:
169-71ºC [156-8ºC] (Lorenz et al., 2005). NMR data (see Tables 1 and 2). Elem. Anal. Calcd. for C17H18O5: C,
67.54%; H, 6.00%; found: C, 67.35%; H, 6.18%. HRMS (El) m/z found 302,115349 (0.2 ppm).
Data for 1-(3’,4’-dioctiloxiphenyl)-6,7-dihydroxyisocroman (7): using 3,4-dioctyloxybenzaldehyde as
carbonyl compound and purification through silica gel column chromatography with ether/hexane (1:4,
v/v) as eluent, a white solid was obtained with a yield of 88.1%. M.p.: 93-5ºC. NMR data (see Tables 1 and
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2). Elem. Anal. Calcd. for C31H46O5: C, 74.66%; H, 9.30%; found: C, 74.61%; H, 9.98%. HRMS (El) m/z found
498,334416 (0.2 ppm).
2.3. Determination of compound polarity
Two procedures were used to determine the polarity of the synthesized isochromans (2-7) in comparison
with their precursor HT (1) and antioxidants, α-tocopherol (8) and BHT (9): the retention factor (Rf) and
partition coefficient (logP). In addition, theoretical values of logP were determined by ChemBioDraw Ultra
(version 13.0).
Retention factor (Rf) values were determined by silica gel thin layer chromatography (TLC) using
chloroform/methanol (20:1, v/v) as eluent, as described by Porter et al (Porter, Black, & Drolet, 1989).
Partition coefficient (logP) was determined by the ‘shake-flask method’ based on the quantification of
substance distribution between n-octanol and water (EEC/92/69, 1992). Briefly, 0.01 M stock solution of
each compound was diluted with n-octanol to yield different concentrations and absorbances were
measured at their corresponding maxima wavelengths using a UV-vis spectrophotometer (HT (1) at 282 nm,
isochromans (2-6) at 286 nm and isochroman (7) at 284 nm). 50 mL of each compound dissolved in n-
octanol were placed in a separatory funnel and extracted three times with 50 mL of water by shaking 10
min and resting 30 min to equilibrate phases. Subsequently, phases were separated and analysed
spectrophotometrically. Partition coefficient was determined by the following equation: log P = [X]o/[X]w
where [X]o represents the analyte concentration in the organic phase (n-octanol) and [X]w the analyte
concentration in the aqueous phase (water). All measurements were carried out in triplicate.
2.4. Evaluation of lipid oxidative stability by the Rancimat method
Lipid oxidative stability was evaluated by an accelerated automated test using the Rancimat equipment
(Model 743, Metrohm Co. Basel, Switzerland). The lipid matrix was obtained from commercial sunflower oil
by purification through alumina (Yoshida, Kondo, & Kajimoto, 1992) following the ‘free solvent’ procedure.
The purified matrix, free of antioxidants, was stored at -18°C under nitrogen atmosphere. Absence of
polyphenols and tocopherols was checked by solid phase extraction and HPLC analysis with UV detector at
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280 nm and C18 Column (Tecknokroma, 5 µm, 25 cm x 4.6 mm i.d.) (Mateos et al., 2001) and by HPLC
analysis on a silica gel column (Lichrorb SI, 5 µm 25 cm x 4.0 cm i.d.) using a UV-vis detector at 292 nm
(Paquot & Hautfenne, 1992), respectively. The fatty acid composition of the matrix was C16:0 (5.7%), C16:1
(0.1%), C18:0 (3.6%), C18:1 (27.4%), C18:2 (61.4%), C18:3 (0.1%), others (1.7%). Aliquots of the glyceridic
matrix were spiked with increasing amounts of compounds, ranging from 0.2 mmol/kg to 2.0 mmol/kg and
then subjected to accelerated oxidation in the Rancimat apparatus at 90°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.
2.5. Ferric Reducing Antioxidant Power (FRAP) Assay
The FRAP assay was carried out according to the procedure described by Pulido, Bravo, and Saura-Calixto
(2000). The antioxidant potential of the synthesized compounds was estimated from their ability to reduce
the ferric tripyridyltriazine (TPTZ-Fe+3) complex to its stable ferrous form (TPTZ-Fe+2 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 FeCl3x6H2O
and 25 mL of 0.3 M acetate buffer was added to obtain a final pH of 3.6. This reagent was freshly prepared
and warmed to 37 ºC prior to its use. 900 μL of FRAP reagent were mixed with 90 μL of distilled water and
30 μL of either test sample, standard (Trolox), or methanol, which was the reagent blank since standards
were dissolved in methanol, and shaken. Readings 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, Holland) spectrophotometer,
equipped with a thermostated 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. FRAP values are
expressed as mM TE (Trolox Equivalent). All analyses were run in triplicate.
2.6. ABTS assay
Free-radical scavenging capacity was measured using the ABTS decoloration method (Re, Pellegrini,
Proteggente, Pannala, Yang, & Rice-Evans, 1999) with some modifications. The method is based on the
capacity of different components to scavenge the ABTS radical cation (ABTS•+) compared to a standard
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antioxidant (Trolox). Briefly, ABTS was dissolved in a 2.45 mM K2S2O8 solution in the dark at room
temperature for 12-16 hours to obtain a 7 mM concentration of ABTS radical cation (ABTS•+) stock solution.
The ABTS•+ stock solution was diluted with methanol to obtain an absorbance of 0.70 ± 0.02 at 730 nm.
After the addition of 0.1 mL of sample (dissolved in methanol), methanol (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 Varian (Cary 50 BIO, Holland) spectrophotometer equipped with a thermostatted auto-cell-holder.
The percentage of inhibition of absorbance versus time was plotted, 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 as mM TE (Trolox Equivalent). Each value is the average of three determinations.
2.7. ORAC assay
The oxygen radical scavenging capacity was measured using the lipophilic ORAC assay according to the
method developed by Huang, Ou, Hampsch-Woodill, Flanagan, and 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 reaction. Briefly, HT (1),
isochromans (2-7) and Trolox standards were dissolved in 7% methylated β-cyclodextrin (RMCD) in
acetone/water (1:1, v/v) solution. Then, 25 µL of either test sample, solvent (blank), or Trolox were added
to a 96-well microplate followed by the addition of 150 µL of fluorescein work solution (8.5 x 10-5 mM)
prepared in 75 mM phosphate buffer (pH 7.4). After the addition of 30 µL of AAPH (153 mM) as peroxyl
radical generator, also prepared in 75 mM phosphate buffer (pH 7.4), fluorescence was recorded every 2
min on a microplate reader (Bio-Tek, Winooski, VT, USA) for 120 min at 485 and 528 nm excitation and
emission wavelengths, respectively. Each value is the average of four determinations. Results are expressed
as mM TE (Trolox Equivalent).
2.8. Oxidative stress model in brain homogenates
Three months old Wistar rats were housed in a pathogen-free room under controlled conditions (19–23°C,
50–60% humidity and 12 h light/darkness cycles) with access to food and water ad libitum.
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The study was approved by the Animal Ethics Committee of the University of Seville (Seville, Spain).
Protocols for animal handling and experimentation were in strict conformation with the Spanish law for the
protection of experimental animals (RD 53/2013, BOE nº 34 Sec I page 11370, February 8th, 2013).
Rats were sacrificed by decapitation and brains were isolated and homogenized in three volumes of 50 mM
Tris/HCl buffer, pH 7.5, containing 1 mM sodium chloride and 0.25 M sucrose in addition to a mixture of
protease inhibitors (0.2 mM phenylmethylsulphonyl fluoride, 50 mM EDTA and 1 μg/mL of chymostatin,
leupeptin and pepstatin A) to prevent proteolysis. After centrifugation (1000 g, 5 min, 4°C), supernatants
were collected for later experiments. Protein content was determined using the Bradford assay.
A volume of brain homogenates containing 4 mg protein/mL was incubated in the absence and presence of
three doses of antioxidant (0.2, 1 and 5 mM) for 30 min at 37°C. Oxidation was initiated by adding CH to
obtain a final concentration of 2 mM and the mixture was maintained for 30 min at 37°C. Proteins were
precipitated by adding two volumes of 10% (v/v) ice-cold trichloroacetic acid to the samples and
centrifuging at 8000 g for 5 min. MDA quantification was carried out in supernatants.
2.9. Determination of malondialdehyde (MDA)
In order to quantify lipid peroxidation in brain homogenates (Esterbauer & Cheeseman, 1990),
malondialdehyde was determined using the thiobarbituric acid reactive substances (TBARS) assay. Samples
were treated with TBA reagent (20 mM TBA in 50% (v/v) glacial acetic acid) and then heated at 100°C for 1
h. TBARS were quantified at 532 nm on a spectrophotometer (Perkin-Elmer C-532001, Waltham, MA, USA).
Results are expressed as percentage referred to MDA controls. Brain homogenates treated with 2 mM CH
in the absence of any antioxidant were used as positive control.
2.10. Statistical Analysis
Prior to statistical analysis, data were tested for homogeneity of variance using the Levene test. Multiple
comparisons were carried out using one-way ANOVA followed by Bonferroni tests when variances were
homogeneous and Tamhane test when not. The level of significance was established at p < 0.05. The
statistical package SPSS (version 19.0) was used.
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3. RESULTS
3.1. Preparation and characterization of isochromans from HT
Oxa-Pictet-Spengler reaction allowed to easily synthetize isochroman compounds (2-7) from HT (1) after
the reaction with a carbonyl compound and acid catalyst (Guiso et al., 2001; Guiso et al., 2003).
Isochromans (2-7) were synthetized in good to excellent yields (represented in Figure 1). Compounds were
characterized by NMR and HR-MS spectrometry. 1H and 13C NMR chemical shifts (Tables 1 and 2,
respectively) were unequivocally assigned to each compound by 2D homo- and heteronuclear correlation
experiments (COSY, NOESY, HSQC and HMBC spectra) and are completely in agreement with the proposed
structures.
3.2. Polarity determination of compounds
Values of partition coefficient (LogP) and retention factor (Rf) (summarized in Table 3) allowed to
characterize isochromans’ (2-7) polarity in comparison with their precursor, HT (1), and antioxidants α-
tocopherol (8) and BHT (9). The relative order of Rf was as follows: 1 ≈ 3 < 4 ≈ 5 < 6 ≈ 2 < 7 < 8 < 9.
Regarding experimental partition coefficient (LogPexp), the compounds showed the following behavior: 1 < 2
≈ 3 < 4 ≈ 5 < 6 < 7 < 9 < 8. Theoretical partition coefficient (LogPtheor) values were almost coincident with
LogPexp values, showing the following order: 1 < 2 < 3 < 4 ≈ 5 < 6 < 9 < 7 < 8.
3.3. Antioxidant activity in lipid matrices
The efficacy of isochromans (2-7) as antioxidants in sunflower oil in comparison with HT (1), α-tocopherol
(8) and BHT (9) was evaluated using the Rancimat method at 90°C in air saturation conditions. Results were
expressed as the induction time (IT) corresponding to the stability of the lipid matrix. Considering that
minimal differences in fatty acid composition of lipid matrixes critically determine stability and
consequently induction time, the antioxidant activity of the references (1, 8 and 9) was analysed in order to
carry out a more accurate comparison with that of isochromans (2-7), although the former data had been
published previously (Pereira-Caro et al., 2009). Figure 2 shows the IT values of purified matrices of
sunflower oil spiked with increasing concentrations of the studied compounds (1-9). Attending to the
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present results, HT (1) and isochromans (2-7) showed better activity against induced oxidation in lipid
matrices than α-tocopherol (8) and BHT (9). Regarding isochromans (2-7) and their precursor HT (1), a
direct relationship between their polar nature and antioxidant capacity in lipid matrices was observed,
except for compound 3, which showed the highest antioxidant power of all the evaluated compounds in
spite of being less polar than HT (1) and compound (2).
3.4. Ferric-reducing antioxidant power (FRAP)
Results of the reducing power of isochromans (2-7) evaluated by the FRAP assay (expressed as mM TE
Trolox equivalent) in comparison with HT (1), α-tocopherol (8) and BHT (9) are summarized in Table 4. The
antioxidant activity of isochromans (2-7) varied depending on the number of free hydroxyl groups
contained in the molecule, observing a direct relationship with the antioxidant power. Thus, HT (1) showed
lower activity than isochromans (2-5), similar to that of compound (6) but slightly higher than that of
compound (7). Regarding the two commercial food antioxidants, α-tocopherol (8) and BHT (9), both
showed lower antioxidant potency than isochromans (2-7), except compound (7), which was less active
than α-tocopherol (8).
3.5. The radical-scavenging activity by ABTS and ORAC assays
The radical-scavenging activity of isochromans (2-7) in comparison with HT (1), α-tocopherol (8) and BHT
(9) determined by ABTS and ORAC assays are shown in Table 4. Results regarding the free radical
scavenging capacity evaluated by ORAC assay were similar to those of the ferric reducing activity, as
isochromans (2-6) showed higher antioxidant capacity than HT (1) except for the most lipophilic compound
(7), which showed similar activity as HT (1). According to both assays, α-tocopherol (8) and BHT (9) had
lower activity than isochromans (2-7), except for compounds 6 and 7, with similar values to those of α-
tocopherol (8) according to the ABTS assay.
3.5. Antioxidant activity evaluation in tissue homogenates
The oxidative damage induced by cumene hydroperoxide (CH) was reverted after treatment with 0.2, 1 and
5 mM of HT (1) and isochromans (2-7) in a dose-dependent manner (Figure 2). An association between the
12

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compounds’ lipophilic nature and their antioxidant capacity was observed. Surprisingly, although α-
tocopherol (8) is highly lipophilic, it showed little protection against oxidation whereas BHT (9) was as
active as the most lipophilic isochroman derivatives (6 and 7).
4. DISCUSSION
The fact that HT is easily recovered from alperujo, an abundant by-product obtained during olive oil
production (Fernandez-Bolanos et al., 2005), together with the high bioactivity and antioxidant power it
presents, turns HT into an interesting substrate to obtain phenolic derivatives with a modified
hydrophilic/lipophilic balance (HLB) and enhanced solubility in lipophilic media. Moreover, the HT
derivatives may represent an alternative that answers the food industry’ demand for this type of
antioxidants. Isochromans are HT derivatives naturally present in the phenolic fraction of virgin and/or
extra-virgin olive oil (Bianco et al., 2001); in addition, they may be easily obtained from HT by means of the
Oxa-Pictet-Spengler reaction in high to excellent yields (Guiso et al., 2001; Guiso et al., 2003). In the present
work a wide range of derivatives have been studied: compound (4), naturally occurring in olive oil (Bianco
et al., 2001), in addition to other isochromans which have been previously synthesized, (2) (Guiso et al.,
2003), (3) and (6) (Lorenz et al., 2005), together with two new derivatives, namely 1-(3’-hydroxy-4’-
methoxyphenyl)-6,7-dihydroxyisochroman (5) and 1-(3’,4’-dioctyloxyphenyl)-6,7-dihydroxyisochroman (7),
which to our knowledge have been synthesized for the first time, in order to establish a structure-
1
antioxidant activity association. H and 13C NMR chemical shifts, as indicated in Tables 1 and 2, in addition
to COSY, NOESY, HSQC and HMBC spectra, allowed us to unequivocally identify the chemical structure of
isochromans (2-7). It is noteworthy that 13C NMR data of compounds (3) and (6), in particular C10 and C1’,
are exchanged in the literature (Lorenz et al., 2005).
Lipophilicity of isochromans (2-7) was evaluated by means of estimating their retention factor (Rf) and both
experimental (LogPexp) and theoretical (LogPtheor) partition coefficients (Table 3). Rf results provided only a
guidance of lipophilicity as these values are not constant and can vary by using different eluents in TLC.
13

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Bearing this in mind, after observing the correspondence between theoretical and experimental partition
coefficients, LogPexp values were taken as a basis to discuss the results observed in the present work.
HT (1) showed the highest polar nature among all the evaluated compounds, presenting a LogP value in
agreement with data previously published (Grasso, Siracusa, Spatafora, Renis, & Tringali, 2007). Compound
(2) was the second most hydrophilic compound. Among isochromans (3-7), compound (3) showed the most
hydrophilic nature due to the presence of two free o-catechol groups in its chemical structure in contrast to
the other compounds. The higher degree of hydroxylation of compound (3) compared to HT (1) did not
result in increased polarity, probably due to its higher carbon skeleton density in comparison with HT (1).
The succeeding compounds in polarity ranking were the regioisomers (4) and (5), showing similar
lipophilicity since the only difference between these compounds is the methyl group in position 3’
(compound 4) or 4’ (5). The double substitution of 3’ and 4’ positions in the phenyl moiety by two methyl
groups in compound (6) or two octyl groups in compound (7) enhanced their lipophilic character,
particularly that of compound (7). Finally, standards BHT (8) and α-tocopherol (9) showed the highest
lipophilic values of all the evaluated compounds in agreement with their chemical structure.
Regarding the in vitro antioxidant activity of isochromans (2-7), which was evaluated by complementary
methods (Rancimat, FRAP, ABTS and ORAC assays) and compared with HT (1) and α-tocopherol (8) and BHT
(9), isochromans (2-7) and HT (1) showed higher antioxidant capacity than α-tocoferol (8) and BHT (9) with
minor changes depending on their chemical structure and the medium in which they were dissolved.
Lipid matrix stability after spiking with isochromans (2-7) and references (1, 8 and 9) was evaluated using
the Rancimat test (Figure 2). Results partially agreed with the polar paradox (Porter et al., 1989), which
indicates that polar antioxidants are more efficient in bulk oil than lipophilic antioxidants, whereas non-
polar antioxidants are more efficient in emulsions or cell membrane systems. From this point of view, a
direct association was observed between hydrophilicity and antioxidant activity, with some exceptions
related to the chemical structure of the compounds. Compound (3), which was more lipophilic than HT (1)
and compound (2), showed the highest antioxidant capacity among all the evaluated products due to the
14

ACCEPTED MANUSCRIPT
double free o-diphenolic moieties present in its chemical structure. However, isochromans 4, 5, 6 and 7,
which contain two o-diphenolic groups partially substituted by one (4 and 5) and two methyl groups (6) or
two octyl groups (7), showed an antioxidant activity similar to molecules containing one o-diphenolic group
[(1) and (2)], which is modulated by their lipophilic nature. Similarly, α-tocopherol (8) was more effective
than BHT (9) in spite of having a more lipophilic nature than (9) due to its polyunsaturated chemical
structure, which favours radical stabilization.
On the other hand, the reducing and radical-scavenging capacities of compounds (2-7), depicted in Table 4,
showed an association between the number of free hydroxyl groups and the antioxidant activity of the
compounds, regardless the o-diphenolic unit was partially substituted or not. Thus, isochromans (4 and 5)
with two o-diphenolic groups and four hydroxyl groups (three free and one substituted by a methyl group)
presented higher antioxidant activity than HT (1) and isochromans (2) in contrast to results obtained by the
Rancimat analysis. The present outcome is in agreement with previous studies in which FRAP and ABTS
activities of the homovanillic alcohol (1.44 and 1.29 mM Trolox Equivalent, respectively), with an hydroxyl
group substituted with a methyl group in position 3, was significantly higher than that of HT (1.39 and 0.84
mM Trolox Equivalent, respectively) (Madrona, Pereira-Caro, Bravo, Mateos, & Espartero, 2011).
Furthermore, these results are in line with those reported by Lorenz et al., (2005) who studied the radical
.-
scavenging activity for 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide O2 and peroxinitrite (ONOO-) of
isochromans (3), (4) and (6), observing an association between the degree of hydroxylation and the
antioxidant activity, being molecule (3) the most active of the tested compounds, followed by (4) and (6).
To complete the characterization of the antioxidant activity of isochromans (2-7), the protective effects
against oxidative stress induced by cumene hydroperoxide (CH) was assessed in homogenate brain using
malondialdehyde (MDA) as an index of lipoperoxidation. Brain homogenate is a substrate widely used as an
oxidative stress model because of the high oxygen demand of this organ, the abundance of redox-active
metals (iron and copper), the high levels of polyunsaturated fatty acids susceptible to oxidation, and also
because of the scarcity of endogenous antioxidant defences (Wang & Michaelis, 2010). Non physiological
15

ACCEPTED MANUSCRIPT
doses of HT and isochromans were used to compare the results obtained with this model with those
observed in lipid matrices in order to further investigate the polar paradox. In the present study, the
oxidative damage induced by CH was reverted in a dose and lipophilic dependent manner. This outcome
contrasts with that observed in bulk oils, but was in agreement with the polar paradox, where lipophilic
antioxidants are more efficient in emulsions or cell membrane systems, as brain homogenates.
It may be concluded that isochromans are antioxidants with significant bioactive potential that can be
easily obtained from HT (1), being more efficient than α-tocopherol (8) and BHT (9). Isochromans (2-7) are
excellent antioxidants in bulk oils as well as in biological systems, in agreement with the polar paradox, also
showing high reducing and radical-scavenging activities.
5. ACKNOWLEDGEMENTS
This research was supported by Grant P09-AGR-5098 from Junta de Andalucía (Spain) and AGL2010-18269
and Consolider-Ingenio CSD2007-00063 from Spanish Ministry of Science and innovation.
The authors have declared no conflict of interest.
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Figure Legends
Figure 1. Chemical reactions for the synthesis of isochromans (2-7) from hydroxytyrosol (1) and chemical
structures of synthesized compounds: 1,1-dimethyl-6,7-dihydroxyisochroman (2); 1-(3’,4’-
dihydroxyphenyl)-6,7-dihydroxyisochroman (3); 1-(4’-hydroxy-3’-methoxyphenyl)-6,7-dihydroxyisochroman
(4);
1-(3’-hydroxy-4’-methoxyphenyl)-6,7-dihydroxyisochroman
(5);
1-(3’,4’-dimethoxyphenyl)-6,7-
dihydroxyisochroman (6); 1-(3’,4’-dioctyloxyphenyl)-6,7-dihydroxyisochroman (7).
Figure 2. Panel A: Induction time (IT) of sunflower oil lipid matrices spiked with antioxidants; Panel B:
Inhibitory capacity of the test compounds at three concentrations (0.2, 1 and 5 mM) against lipid
peroxidation in brain homogenates. The compounds evaluated were hydroxytyrosol (1), 1,1-dimethyl-6,7-
dihydroxyisochroman (2); 1-(3’,4’-dihydroxyphenyl)-6,7-dihydroxyisocroman (3); 1-(4’-hydroxy-3’-
methoxyphenyl)-6,7-dihydroxyisocroman (4); 1-(3’-hydroxy-4’-methoxyphenyl)-6,7-dihydroxyisocroman (5);
1-(3’,4’-dimethoxyphenyl)-6,7-dihydroxyisocroman (6); 1-(3’,4’-dioctyloxyphenyl)-6,7-dihydroxyisocroman
(7), α-tocopherol (8) and BHT (9). Values represent means ± SD. Different letters indicate statistically
significant differences (p < 0.05) among groups.
20

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Figure 1.
O
HO
HO
HO
HO
+
R
R'
O
OH
carbonylic
compound
R
R'
(1)
(2-7)
HO
HO
HO
R3'
R4'
O
O
HO
(3)
(4)
(5)
(6)
H
Me
H
H
H
Me
Me
(2)
Me
R3'O
(7) nOct nOct
OR4'
21

ACCEPTED MANUSCRIPT
Figure 2.
Panel A
1
2
3
4
5
6
7
8
9
100
80
60
40
20
0
0
0,5
1
1,5
2
Concentration (mmol/Kg)
Panel B
a a a
100
0.2 mM
1 mM
5 mM
b
b
b
b
80
60
40
20
0
b
c,d
c
d
e
d,e
c
c
f
c,d
d,e
f
d
d
e
e
e
e
g
f
f
f,g
g
Control
1
2
3
4
5
6
7
8
9

ACCEPTED MANUSCRIPT
1
Table 1. H NMR (500.13 MHz, DMSO-d6, 303k) for isochromans (2-7). Chemical shifts
(δ) are expressed in ppm and coupling constants (J) in Hz.
2
3
4
5
6
7
• Isochroman unit
1
5.32 (s)
5.40 (s)
5.36 (s)
5.45 (s)
5.42 (s)
3eq
3.74 (t)
3J3,4 = 5.5
3.94 (ddd)
3.98 (ddd)
3.95 (ddd)
3.99 (ddd)
3.97 (ddd)
2J3a,3e = 11.4
3J3e,4a = 5.4
3J3e,4e = 4.0
2J3a,3e = 11.1
3J3e,4a = 5.4
3J3e,4e = 3.8
2J3a,3e = 11.1
3J3e,4a = 5.5
3J3e,4e = 3.9
2J3a,3e = 11.1
3J3e,4a = 5.4
3J3e,4e = 3.7
2J3a,3e = 11.2
3J3e,4a = 5.5
3J3e,4e = 3.7
3ax
3.67 (ddd)
3.69 (ddd)
3.68 (ddd)
3.71 (m)
3.69 (ddd)
3J3a,4a = 9.2
3J3a,4a = 9.7
3J3a,4a = 9.5
3J3a,4a = 9.6
3J3a,4e = 4.0
3J3a,4e =3.8
3J3a,4e = 4.0
3J3a,4e = 4.0
4eq
2.51 (t)
2.51 (dt)
2J4a,4e = 15.9
2.52 (dt)
2J4a,4e = 15.6
2.52 (dt)
2J4a,4e = 15.8
2.53 (dt)
2J4a,4e = 15.9
2.52 (dt)
2J4a,4e = 16.0
4ax
5
2.77 (ddd)
6.48 (s)
2.82 (ddd)
6.49 (s)
2.78 (ddd)
6.49 (s)
2.83 (ddd)
6.50 (s)
2.81 (ddd)
6.49 (s)
6.40 (s)
6.48 (s)
8
6.05 (s)
6.05 (s)
6.04 (s)
6.05 (s)
6.03 (s)
• 1-Substituent
1’
1.34 (s)
2’
5’
6’
6.57 (d)
6.78 (d)
6.61 (d)
6.82 (d)
6.79 (d)
4J2´,6´ = 2.0
4J2´,6´ = 1.9
4J2´,6´ = 2.0
4J2´,6´ = 1.9
4J2´,6´ = 1.9
6.66 (d)
3J5´,6´ = 8.0
6.71 (d)
3J5´,6´ = 8.0
6.84 (d)
3J5´,6´ = 8.2
6.89 (d)
3J5´,6´ = 8.1
6.87 (d)
3J5´,6´ = 8.2
6.53 (dd)
6.62 (dd)
3.70 (s)
6.66 (dd)
6.75 (dd)
3.69 (s)
6.72 (dd)
• 3’-Substituent
1’’
3.86 (ddt)
(sist. ABX2)
2J = 9.5
3J = 6.3
2’’
3’’ − 7’’
8’’
1.66 (m)
1.25 (m)
0.85 (t)
• 4’-Substituent
1’’’
3.74 (s)
3.74 (s)
3.91 (t)
3J = 6,3
2’’’
3’’’ − 7’’’
8’’’
1.66 (m)
1.25 (m)
0.85 (t)

ACCEPTED MANUSCRIPT
Table 2. 13C NMR (125.75 MHz, DMSO-d6, 303k) for isochromans (2-7). Chemical shifts
(δ) are expressed in ppm.
2
3
4
5
6
7
• Isochroman unit
1
3
73.3
58.8
78.1
62.8
78.3
63.1
77.9
62.9
78.2
63.2
78.1
63.1
4
5
28.2
27.5
27.5
27.5
27.5
27.5
114.9
143.4
143.4
112.3
133.3
123.0
114.9
143.9
143.1
113.4
128.3
123.9 a
115.0
143.9
143.1
113.3
128.3
123.9
114.9
143.9
143.2
113.3
128.1
123.9
115.0
143.9
143.2
113.3
128.0
123.9 a
114.9
143.9
143.2
113.3
128.0
123.9
6
7
8
9
10
• 1-Substituent
1’
2’
29.7
133.9 a
115.8
144.8
144.8
114.8
119.7
133.9
112.6
147.2
146.1
114.8
121.2
135.6
115.7
146.2
147.2
111.6
119.5
135.4 a
112.1
148.5
148.3
111.2
120.8
135.5
114.4
148.2
148.3
113.3
121.1
3’
4’
5’
6’
• 3’-Substituent
1’’
55.6
55.5
68.5
25.5-31.1
22.0
2’’ − 6’’
7’’
8’’
13.8
• 4’-Substituent
1’’’
55.6
55.4
68.5
25.5-31.1
22.0
2’’’ − 6’’’
7’’’
8’’’
13.8
a Data interchanged in literature (Lorenz et al., 2005).

ACCEPTED MANUSCRIPT
Table 3. Retention factor (Rf) and partition coefficient (LogPexp) values obtained for
hydroxytyrosol (1), isochromans (2-7), α-tocopherol (8) and BHT (9). Theoretical
partition coefficient (LogPtheor) values were calculated using ChemBioDraw Ultra
Software.
Compound
Rf
LogPexp
LogPtheor
1
2
3
4
5
6
7
8
9
0.12 ± 0.01a
0.36 ± 0.02c
0.10 ± 0.01a
0.24 ± 0.02b
0.28 ± 0.02b
0.32 ± 0.02c
0.69 ± 0.02d
0.93 ± 0.02e
0.97 ± 0.02f
0.14 ± 0.02a
1.26 ± 0.07b
1.34 ± 0.04b
1.46 ± 0.03c
1.47 ± 0.02c
2.62 ± 0.06d
3.64 ± 0.05e
10.0 ± 0.06g*
5.10 ± 0.06f*
0.96
1.56
1.95
2.22
2.22
2.48
8.30
9.98
5.54
* For comparative purposes, LogP of α-tocopherol (8) and BHT (9) taken from The
Drugbank 4.0 (http://www.drugbank.ca) and The Human Metabolome Database
(HMDB, http://www.hmdb.ca), respectively, were included for comparative purposes.

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Table 4. Antioxidant capacity of the studied compounds (1-9)*.
FRAP
ABTS
ORAC
Compound
1†
(mM TE)
(mM TE)
(mM TE)
1.39 ± 0.05 d,e
0.84 ± 0.02 f
1.92 ± 0.04 e
2
3
1.46 ± 0.02 d
4.20 ± 0.05 a
2.18 ± 0.02 b
2.22 ± 0.02 b
1.53 ± 0.02 c
1.18 ± 0.02 f
0.80 ± 0.04 g
1.12 ± 0.01 c
2.06 ± 0.02 a
1.37 ± 0.02 b
1.38 ± 0.01 b
1.03 ± 0.01 d
0.94 ± 0.01 e
1.01 ± 0.02 d
2.26 ± 0.04 c
4.86 ± 0.11a
3.51 ± 0.14 b
3.70 ± 0.06 b
2.15 ± 0.12 c,d
1.99 ± 0.06 d,e
0.63 ± 0.06 f
4
5
6
7
8†
1.32 ± 0.05 e
0.27 ± 0.01 g
0.12 ± 0.01 g
9†
* Reducing antioxidant power was determined by the FRAP assay, and free radical-
scavenging capacity by the ABTS and ORAC methods. FRAP and ABTS data is the mean
of triplicate measurements ± standard deviation; ORAC data represents the mean of
quadruplicate measurements ± standard deviations. Results are expressed as mM TE
(Trolox Equivalents). Values within a column with different superscripts are
significantly different, p < 0.05.
†FRAP, ABTS and ORAC values of HT (1), α-tocopherol (8) and BHT (9) have been
published by Madrona (Madrona et al., 2011) and are included for comparative
purposes.

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HIGHLIGHTS
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Isochromans are obtained from hydroxytyrosol (HT) via Oxa-Pictet Spengler reaction
Most isochromans are better antioxidants than HT, α-tocopherol and BHT
Antioxidant activity of isochromans agrees with the polar paradox
Isochromans’ o-diphenolic groups in oils positively determine the antioxidant power
Hydroxyl groups are responsible for isochromans’ reducing and radical-scavenging power
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