Protective activity of hydroxytyrosol metabolites on erythrocyte oxidative-induced hemolysis

Article abstract of DOI:10.1021/jf4016202

The capacity of important hydroxytyrosol metabolites (homovanillyl alcohol, hydroxytyrosol acetate, homovanillyl alcohol acetate, hydroxytyrosol 3′ and 4′-O-glucuronides, and homovanillyl alcohol 4′-O-glucuronide) to protect red blood cells (RBCs) from oxidative injury induced by the radical initiator 2,2′-azo-bis(2-amidinopropane) dihydrochloride (AAPH) or by the natural radical initiator H₂O₂ was evaluated. In the presence of AAPH, all compounds showed to protect RBCs from hemolysis in a dose-dependent manner, exccept for the homovanillyl alcohol glucuronide, with the order of activity being at 20 μM hydroxytyrosol > hydroxytyrosol glucuronides = hydroxytyrosol acetate = homovanillyl alcohol = homovanillyl acetate > homovanillyl alcohol glucuronide. At 10 μM, hydroxytyrosol, hydroxytyrosol acetate, and hydroxytyrosol glucuronides still protected hemoglobine from oxidation and from morphological RBC changes. In the presence of H₂O₂, hydroxytyrosol showed to significantly protect RBCs from oxidative hemolysis in a dose-dependent manner, but the hydroxytyrosol glucuronides showed only a limited protection that was independent of the concentration used.

Full text of DOI:10.1021/jf4016202

Article
pubs.acs.org/JAFC
Protective Activity of Hydroxytyrosol Metabolites on Erythrocyte
Oxidative-Induced Hemolysis
Fatima Paiva-Martins,*,† Aníbal Silva,† Vasco Almeida,† Mafalda Carvalheira,† Cristina Serra,†
́
Jose Enrique Rodrígues-Borges,† Joao Fernandes,‡ Luis Belo,‡ and Alice Santos-Silva‡
́
̃
†Centro de Investigaca
Alegre, 687, 4169-007 Porto, Portugal
‡Servico
̧
o em Química and Departamento de Química, Faculdade de Cien
̂
cias, Universidade do Porto, Rua do Campo
̃
̧
de Bioquímica, Faculdade de Farmac
́
ia, Universidade do Porto, Rua Aníbal Cunha, 164, 4050-047 Porto, Portugal, and
Instituto de Biologia Molecular e Celular, IBMC, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal
ABSTRACT: The capacity of important hydroxytyrosol metabolites (homovanillyl alcohol, hydroxytyrosol acetate, homovanillyl
alcohol acetate, hydroxytyrosol 3′ and 4′-O-glucuronides, and homovanillyl alcohol 4′-O-glucuronide) to protect red blood cells
(RBCs) from oxidative injury induced by the radical initiator 2,2′-azo-bis(2-amidinopropane) dihydrochloride (AAPH) or by the
natural radical initiator H2O2 was evaluated. In the presence of AAPH, all compounds showed to protect RBCs from hemolysis in
a dose-dependent manner, exccept for the homovanillyl alcohol glucuronide, with the order of activity being at 20 μM
hydroxytyrosol > hydroxytyrosol glucuronides = hydroxytyrosol acetate = homovanillyl alcohol = homovanillyl acetate >
homovanillyl alcohol glucuronide. At 10 μM, hydroxytyrosol, hydroxytyrosol acetate, and hydroxytyrosol glucuronides still
protected hemoglobine from oxidation and from morphological RBC changes. In the presence of H2O2, hydroxytyrosol showed
to significantly protect RBCs from oxidative hemolysis in a dose-dependent manner, but the hydroxytyrosol glucuronides showed
only a limited protection that was independent of the concentration used.
KEYWORDS: polyphenols, erythrocytes, olive oil, hydroxytyrosol, homovanillyl alcohol, hydroxytyrosol acetate, glucuronides
Hy is well absorbed in the small intestine12 and, together with
its acetate, glucuronide, and sulfate conjugates and homo-
INTRODUCTION
■
Antioxidants have received particular attention because of their
vanillyl alcohol and its glucuronide and sulfate conjugates, it can
potential to modulate oxidative stress associated with chronic
be found in urine and plasma after olive oil consumption
disease. The lower incidence of coronary heart disease and of
some cancers in the Mediterranean area led to the hypothesis
(Figure 1).13−15
Until recently, studies on the bioavailability for secoiridoids,
except for oleuropein, were inexistent. However, an important
body of evidence has shown that OL aglycones are bioavailable.
After the consumption of VOO, the concentration of Hy rises
more than expected if only the Hy existent in the oil was
absorbed, and it is proportional to the total phenol content and
not to the Hy content of the oil.12 The first study on the
bioavailability of 3,4-DHPEA-EDA and 3,4-DHPEA-EA
showed that both secoiridoid compounds are well absorbed
by Caco-2 cells and by the rat intestine and are not only
metabolized to reduced forms and conjugated with glucuronic
acid but also hydrolyzed with the production of hydroxytyrosol
and its metabolites.16 Actually, it is believed that olive oil
consumption could reduce oxidative damage due to its richness
in oleic acid, as well as due to its minor components,
particularly the phenolic compounds. However, which
components have a major role in this protection is still
unknown. Therefore, the potential health benefits of Hy and its
derivatives may be attributed to both parental compounds and
their phase I and phase II metabolites.
that a diet rich in fruits, vegetables, and grains has a beneficial
effect on health. The major fat component of the so-called
“Mediterranean diet” is virgin olive oil (VOO),1 and several
studies have suggested that phenolic compounds, although
considered among the minor constituents of VOO, may
contribute to the healthy nature of this diet.2−8
The classes of phenols present in olives are phenolic alcohols,
such as hydroxytyrosol (3,4-dihydroxyphenylethanol, 3,4-
DHPEA, or Hy) and tyrosol (4-hydroxyphenylethanol, 4-
HPEA, or Ty), secoiridoids, flavonoids, and lignans. Secoir-
idoids are the most representative class in olives; the glycoside
oleuropein (OL), one of the most important secoiridoids,
represents up to 14% of the dry weight of olives. During VOO
extraction, secoiridoid glycosides such as oleuropein are
hydrolyzed. Therefore, the major phenolic compounds found
in VOO are the dialdehydic form of elenolic acid linked to Hy
(3,4-DHPEA-EDA), the OL aglycone (3,4-DHPEA-EA), and
the dialdehydic form of elenolic acid linked to Ty (4-HPEA-
EDA). These compounds account for up to 80% of the total
phenolic fraction.9−11
Although many studies have investigated the in vitro
antioxidant properties of the minor VOO phenolics OL and
especially of Hy, as well as their protective effects against cell
injury, the biological properties of these phenols in vivo will
depend on the extent to which they are absorbed and
metabolized. Bioavailability studies have demonstrated that
Received: April 15, 2013
Revised: June 18, 2013
Accepted: June 19, 2013
Published: June 19, 2013
© 2013 American Chemical Society
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Figure 1. Metabolic pathways of the main polyphenolic compounds found in olive oil. Discontinuous lines indicate that more than a pathway is
possible for hydroxytyrosol obtained from secoiridoids. UCT, glucuronosyltransferase; ACT, O-acetyltransferase; COMT, catechol methyl
transferase; SF, sulfotransferase; Hy, hydroxytyrosol; HyAc, hydroxytyrosol acetate; Hy-4′-O-GlcA and Hy-3′-O-GlcA, hydroxytyrosol glucuronides;
HVA, homovanillyl alcohol; HVAAc, homovanillyl alcohol acetate; HVA-GlcA, homovanillyl alcohol glucuronide.
Human red blood cells (RBC) are particularly useful in the
evaluation of the antioxidant properties of several compounds.
RBCs, the most abundant blood cells, are particularly
susceptible to endogenous oxidative damage because of their
specific role as oxygen carriers. In the normal metabolism of
RBCs, around 0.3% of the oxygen molecule is shifted from its
normal role with the production of superoxide anion.16 Healthy
subjects are equipped with efficient RBC antioxidant
endogenous systems. However, if reactive oxygen species
radicals from the plasma to the RBCs. The capacity of
hydroxytyrosol 3′ and 4′ O-glucuronides was also evaluated
against the oxidative injury induced by the physiological
oxidizing agent in erythrocytes, the H2O2. Our aim was,
therefore, to evaluate the antioxidant capacity of these
compounds that might be important in protecting cells from
oxidative damage in oxidative stress conditions, such as
cardiovascular diseases, diabetes, and neoplasic disorders.
−
(ROS), such as H2O2 and O2 , are overproduced within the
MATERIALS AND METHODS
■
erythrocyte, an “oxidative stress” condition will develop,
inducing oxidative damage on erythrocyte constituents, which
may lead ultimately to hemolysis.17 When the capacity of
protective hemoglobin-scavenging mechanisms has been
saturated, levels of cell-free hemoglobin increase in the
plasma.18 Whenever the hemoglobin is released from
erythrocytes, it is potentially dangerous because it can be
converted into oxidized forms, powerful promoters of oxidative
processes in blood.18,19 Moreover, the production and
modulation of NO blood concentrations20,21 and ascorbate
recycling22 by RBCs are also very important for the body
defense against the development of oxidative stress in the
cardiovascular system.
In this work, the capacity of some of the main hydroxytyrosol
(Hy) phase I and phase II metabolites, hydroxytyrosol acetate
(HyAc), hydroxytyrosol 3′ and 4′ O-glucuronides (Hy-GlcA),
homovanillyl alcohol (HVA), homovanillyl acetate (HVAAc),
and homovanillyl alcohol 4′-O-glucuronide (HVA-GlcA), to
protect RBCs from oxidative injury induced by the water-
soluble radical initiator 2,2-azobis(2-amidinopropane) dihydro-
chloride (AAPH) was evaluated. At 37 °C, azo-initiator
compounds react with molecular oxygen producing peroxyl
radicals at a constant rate that mimetizes the attack of free
All chemicals were obtained from chemical suppliers and used without
further purification, unless otherwise noted. All reactions were
monitored by thin layer chromatography (TLC) on precoated Silica-
Gel 60 plates F254, and detected by heating with Mostain [500 mL of
10% H2SO4, 25 g of (NH4)6Mo7O2·4H2O, 1 g of Ce(SO4)2·4H2O].
Products were purified by flash chromatography with Silica Gel 60
(200−400 mesh). NMR spectra were recorded on 400 MHz NMR
equipment, at room temperature for solutions in CDCl3, D2O, or
CD3OD. Chemical shifts are referred to the solvent signal and are
expressed in ppm. 2D NMR experiments (COSY, TOCSY, ROESY,
and HMQC) were carried out when necessary to assign the
corresponding signals of the compounds.
Phenolic Compounds. Hydroxytyrosol (1) was synthesized from
3,4-dihydroxyphenylacetic acid (Sigma-Aldrich Quimica-S.A. Madrid,
Spain) according to the procedure of Baraldi et al.23 Hydroxytyrosol
acetate (2) and homovanillyl alcohol acetate were synthesized from
hydroxytyrosol and homovanillyl alcohol (3), respectively, according
to the procedure of Bernini et al.24
Synthesis of Hydroxytyrosol Metabolites. The synthesis of
glucuronides was performed according to the method described by
Lucas et al.25 with some modifications (Figure 2).
2-[3′-Hydroxy-4′-(methyl-2,3,4-tri-O-acetyl-β-D-glucopyranosy-
luronate) phenyl] EtOAc (8) and 2-[4′-Hydroxy-3′-(methyl-2,3,4-tri-
O-acetyl-β-D-glucopyranosyluronate)phenyl] Ethyl Acetate (10). To
a solution of trichloroacetimidate 7 (990 mg, 2 mmol) and
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purified by flash column chromatography (hexane−ethyl acetate 1:3)
to afford 2-[3′-metoxy-4′-(methyl-2,3,4-tri-O-acetyl-β-D-
1
glucopyranosyluronate)phenyl]ethyl acetate, 11 (471 mg, 32%). H
NMR (400 MHz, CDCl3) δ 7.06 (d, J = 8.0 Hz, 1H, Harom), 6.74 (d, J
= 1.9 Hz, 1H, Harom), 6.72 (dd, 1H, Harom), 5.29 (m, 3H), 4.99 (d, 1H,
J = 7.2 Hz), 4.25 (t, 1H, J = 7.1 Hz), 3.80 (s, 3H), 3.74 (s, 3H), 2.88
(t, 2H, J = 7.1 Hz), 2.08 (s, 3H), 2.04 (s, 6H), 2.02 (s, 3H); 13C NMR
(75 MHz, CDCl3) δ 171.0, 170.1 (CO), 169.4, 169.3, 167.0, 150.6,
144.4, 134.8, 121.1, 120.8, 113.4, 100.8, 72.6, 71.9, 71.1, 69.3, 64.8,
56.01, 52.9, 34,8, 21.0, 20.6, 20.5.
2-[3-Metoxy-4′-β-D-glucopyranosyluronic acid)phenyl] Ethanol
(4). A solution of the compound 11 (200 mg, 0.38 mmol) in methanol
(6 mL) was stirred at room temperature under argon with a solution
of Na2CO3 (70 mg, 0.65 mmol) in H2O (1.5 mL). After 24 h, glacial
acetic acid water was added to adjust the pH to 6. The solvents were
then removed, and residue was purified by flash column chromatog-
raphy [CH2Cl2/CH3OH (1:1)] affording compound 4 (87 mg, 67%).
1H NMR (500.13 MHz, CD3OD) δ 7.10 (d, 1H, J = 8.2 Hz), 6.85 (d,
1H, J = 1.9 Hz), 6.73 (dd, 1H), 4.81 (d, 1H, J = 7.2 Hz), 3.81 (s, 3H),
3.68 (t, 2H, J = 7.1 Hz), 3.64 (s, 3H), 3.48 (m, 3H), 2.73 (t, 2H, J =
7.0 Hz); 13C NMR (100 MHz, CD3OD) δ 176.2, 150.9, 146.6, 136.0,
122.7, 119.3, 114.7, 103.7, 77.6, 76.5, 74.7, 73.5, 64.3, 56.9, 39.9. MS:
m/z = 343.33 [M − H]+, 175.07 [M − 167]+, 167.27 [M − 175]+,
152.27 [M − H − 175 − CH3]+, 113.02 [M − H − 175 − CO2 −
H2O]+. Spectral data were in accordance with those found in the
literature.26
Figure 2. Synthesis of hydroxytyrosol 3′ and 4′-O-β-D-glucuronides (5
and 6) and homovanillyl alcohol-4′-O-β-D-gucuronide (4).
Preparation of Erythrocyte Suspensions. Blood was obtained
from healthy, nonsmoker volunteers (two women and two man aged
23−50 years old) by venipuncture, and collected into tubes containing
ethylenediaminetetraacetic acid (EDTA), as an anticoagulant. Samples
were immediately centrifuged at 400g for 10 min; plasma and buffy
coat were carefully removed and discarded. RBCs were washed three
times with phosphate buffered saline solution (PBS; 125 mM NaCl
and 10 mM sodium phosphate buffer, pH 7.4) at 4 °C and, finally,
resuspended in PBS, to obtain RBC suspensions at 2% hematocrit.
RBC suspensions were used on the day they were prepared.
Induced Hemolysis. RBC suspensions were prepared at 2%
hematocrit, and the assays were performed by using AAPH at final
concentration of 60 mM or H2O2 at 7.5 mM. In all sets of experiments
(n = 4, in quadruplicates), a negative control (RBCs in PBS) was used,
as well as phenolic compound controls (RBCs in PBS, with each
phenolic compound). Controls and sample tests were run in duplicate.
Incubations of RBC suspensions were carried out at 37 °C for 4 h,
under gentle shaking, in the presence of each individual compound or
in the presence of the phenolic compound plus the radical initiator.
Phenolic compounds were incubated 15 min with RBCs before the
addition of the radical initiator (AAPH or H2O2), and they were tested
at concentrations of 10, 20, 40, and 80 μM. Hemolysis was determined
spectrophotometrically, according to Ko et al.27 After the incubation
period (2 or 4 h), an aliquot of the RBC suspensions was diluted with
20 volumes of saline and centrifuged (1200g for 10 min). The
absorption (A) of the supernatant was read at 540 nm. The absorption
(B), corresponding to a complete hemolysis, was acquired after
centrifugation of a RBC suspension that was previously treated with 20
volumes of ice-cold distilled water. The percentage of hemolysis was
then calculated (A/B × 100).
Induced Morphological Changes. To study the morphological
changes of RBC suspensions by optical microscopy, aliquots (50 μL)
were taken from test tubes containing 10 and 80 μM of phenolic
compounds, with and without AAPH, and controls at the end of the
incubations. The samples were diluted (1:50) and then mounted in a
slide with a coverslip. By using the same volume of the RBC
suspensions, it was possible to compare in a rough way the number of
RBC per microscopic field with the RBC lysis quantified previously by
spectrophotometry.
Protective Effect of Phenolic Compounds against AAPH-
Induced Hemoglobin Oxidation. To clarify the nature of the
hemoglobin linked to RBC membranes and the concentration of oxy-
hemoglobin in hemolysates,28 visible absorption spectral scans (450−
650 nm) were performed.29
hydroxytyrosol acetate 2 (550 mg, 2.8 mmol) in anhydrous CH2Cl2 (6
μL) at −10 °C was added TMSOTf (95 μL, 0.52 mmol) dropwise.
After 2 h, TLC (hexane−EtOAc 1:1) showed the formation of a new
product and complete consumption of the glycosyl donor. The
reaction was neutralized with triethylamine and concentrated in
vacuum. The resulting residue was purified by flash column
chromatography (hexane−ethyl acetate 1:1) to afford a 1:1
1
regioisomeric mixture of 9 and 10 (445 mg, 31%). H NMR (400
MHz, CDCl3) δ 6.88 (d, 2H, Harom, J = 8.0 Hz), 6.82 (d, 2H, Harom, J =
1.8 Hz), 6.66 (dd, 2H, Harom), 5.37−5.25 (m, 6H), 5.00 (d, 1H, J =
10.4 Hz), 4.99 (d, 1H, J = 10.4 Hz), 4.23 (t, 2H, J = 7.2 Hz), 4.22 (t,
2H, J = 7.2 Hz), 4.17 (d, 2H, J = 9.2 Hz), 3.73 (s, 6H, CH3O), 2.84−
2.80 (m, 4H, 2 CH2), 2.11−2.03 (m, 24H, CH3CO); δ 13C NMR (75
MHz, CDCl3) 171.9, 170.9.0, 170.7, 170.3, 167.7, 148.4, 147.1, 144.8,
143.7, 136.4, 130.9, 126.7, 121.6, 119.3, 119.0, 118.0, 117.5, 102.5,
102.4, 78.3, 73.4, 72.3, 69.9, 65.8, 65.7, 54.1, 35.4, 35.2, 21.9, 21.6,
21.5, 21.4. Spectral data were in accordance with those found in the
literature.26
2-[3′-Hydroxy-4′-β-D-glucopyranosyluronic acid)phenyl] Ethanol
(5) and 2-[4′-Hydroxy-3′-β-D-glucopyranosyluronic acid) phenyl]
Ethanol (6). A solution of the regioisomeric mixture of 9 and 10 (200
mg, 0.39 mmol) in methanol (6 mL) was stirred at room temperature
under argon with a solution of Na2CO3 (70 mg, 0.65 mmol) in H2O
(1.5 mL). After 24 h, glacial acetic acid water was added to adjust the
pH to 6. The solvents were then removed, and residue was purified by
flash column chromatography [CH2Cl2/CH3OH (1:1)] affording
compounds 5 and 6 (116 mg, 90%) as a 1:1 regioisomeric mixture
1
(determined by HPLC and NMR). H NMR (400 MHz, D2O) δ
7.12−6.61 (m, 6H, Harom), 4.71 (d, 1H, J = 7.0 Hz), 4.70 (d, 1H, J =
7.0 Hz), 3.69 (t, 2H, J = 7.2 Hz), 3.68 (t, 2H, J = 7.2 Hz), 3.66−3.49
(m, 8H), 2.70 (t, 2H, J = 7.0 Hz), 2.69 (t, 2H, J = 7.2 Hz); 13C NMR
(75 MHz, D2O) δ 177.0 (CO), 148.8, 147.3, 146.9, 145.5, 136.8,
132.3, 125.9, 121.7, 120.9, 120.3, 118.1, 117.4, 105.4, 105.2, 77.4, 74.8,
74.7, 73.6, 64.5, 64.4, 39.8, 39.6. Spectral data were in accordance with
those found in the literature.26
2-[3′-Metoxy-4′-(methyl-2,3,4-tri-O-acetyl-β-D-glucopyranosylur-
onate) phenyl]ethyl Acetate (9). To a solution of trichloroacetimidate
7 (990 mg, 2 mmol) and homovanillyl alcohol acetate 8 (590 mg, 2.8
mmol) in anhydrous CH2Cl2 (6 mL) at −10 °C was added BF3·OEt2
(70 μL) dropwise (Figure 2). After 6 h, TLC (hexane−ethyl acetate
1:1) showed the formation of a new product and complete
consumption of the glycosyl donor. The reaction was neutralized
with NEt3 and concentrated in vacuum. The resulting residue was
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Statistical Analysis. The results obtained for the four independent
hemolysis experiments (blood obtained each time from a different
donor), performed in quadruplicate, are expressed as mean SEM.
Statistical differences between groups of experiments with different
antioxidant compounds were analyzed by two-way analysis of variance
with posthoc testing using Tukey’s test. A p value lower than 0.05 was
accepted as being statistically significant.
RESULTS AND DISCUSSION
■
Increasing evidence has supported the hypothesis that a
number of nutrients or non-nutrient dietary components,
labeled as “antioxidants”, might have a beneficial role regarding
the course of chronic diseases. In particular, it has been claimed
that olive oil polyphenols may play a major role on the
protective effects of olive oil consumption against oxidative
damage. However, little research has been addressed to the
study of the antioxidant profile of the most significant olive oil
phenolic compounds metabolites in biological systems. In this
work, we addressed that issue by studying the protective
properties of some of the most important olive oil phenolic
compounds metabolites, the hydroxytyrosol metabolites, upon
human RBC under AAPH and H2O2-induced oxidative stress.
This biological model has been extensively studied both as a
source of free radicals and as a target for oxidative damage.
After 4 h of incubation, hydroxytyrosol, hydroxytyrosol
acetate, hydroxytyrosol glucuronides, homovanillyl alcohol, and
homovanillyl alcohol acetate protected RBCs in a significant
way from oxidative-AAPH induced hemolysis at the concen-
tration of 80 μM (Figures 3 and 4). At lower concentrations
Figure 4. Inhibition of hemolysis of RBCs (mean
SEM) at 2%
hematocrit, incubated for 4 h with hydroxytyrosol derivative
compounds at 10, 20, 40, and 80 μM and with 60 mM AAPH. Hy,
hydroxytyrosol; HyAc, hydroxytyrosol acetate; Hy-GlcA, hydroxytyr-
osol glucuronides. Different letters within a concentration indicate
samples that were significantly different (p < 0.05).
ytyrosol agree with those acquired in a similar system by Manna
et al.30 and Paiva-Martins et al.31
According to the literature, the concentration of hydroxytyr-
osol metabolites in the plasma rises after the consumption of
VOO, with Tmax around 1−2 h of ingestion.14,15 Therefore, we
also evaluated the protective effect of hydroxytyrosol
metabolites against RBC-induced hemolysis after 2 h of
incubation (Figure 5). At this time of incubation, Hy, HyAc
Figure 5. Inhibition of hemolysis of RBCs (mean
SE) at 2%
hematocrit, incubated for 2 h with hydroxytyrosol derivative
compounds at 10, 20, 40, and 80 μM and with 60 mM AAPH. Hy,
hydroxytyrosol; HyAc, hydroxytyrosol acetate; Hy-GlcA, hydroxytyr-
osol glucuronides. Different letters within a concentration indicate
samples that were significantly different (p < 0.05).
Figure 3. Inhibition of hemolysis of RBCs (mean
SEM) at 2%
hematocrit, incubated for 4 h with with homovanillyl derivative
compounds at 10, 20, 40, and 80 μM and with 60 mM AAPH. HVA,
homovanillyl alcohol; HVAAc, homovanillyl alcohol acetate; HVA-
GlcA, homovanillyl alcohol glucuronide. Different letters within a
concentration indicate samples that were significantly different (p <
0.05).
and Hy-GlcA showed a quite similar protective activity at all
concentrations tested and were able to significantly protect
RBCs against the oxidative hemolysis, even at 10 μM.
The RBCs morphology before and after exposure to AAPH
in the absence and presence of hydroxytyrosol and hydrox-
ytyrosol glucuronides at 80 and 10 μM is illustrated in Figure 6.
At these concentrations, the compounds were still able to
protect RBC from hemolysis induced by AAPH. By using the
same volume of the RBC suspensions, it was also possible to
observe that the cellular density in the hydroxytyrosol
glucuronides samples was lower than that in the hydroxytyrosol
samples for the same concentration but still higher than the
observed in the samples containing only AAPH. This
observation was in accordance with the hemolysis study.
It was also investigated the nature of hemoglobin in the
hemolysate of samples containing AAPH and several
concentrations of hydroxytyrosol metabolites. When perform-
ing spectral scans (450−650 nm) of lysed RBC suspensions28
(20−60 μM), all compounds, except HVA-GlcA, protected
RBCs from oxidative hemolysis in a dose-dependent manner.
Indeed, the homovanyllyl alcohol glucuronide presented the
worst antioxidant performance among the six tested com-
pounds, showing just a slight activity, independent of
concentration. The ranking activity order at 20 μM was:
hydroxytyrosola > hydroxytyrosol glucuronidesb = hydroxytyr-
osol acetateb = homovanillyl alcoholbc = homovanillyl actatec >
homovanillyl glucuronidec. At the lowest concentration tested
(10 μM), the hydroxytyrosol direct metabolites still had an
important protective activity (Figure 4) being the ranking
activity order hydroxytyrosola > hydroxytyrosol acetateb
hydroxytyrosol glucuronidesb. The data obtained for hydrox-
=
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Figure 6. Optical microscopic evaluation of the erythrocyte morphology. (A) RBCs plus phenolic compounds at 80 μM and AAPH after 4 h of
incubation. (B) RBCs plus phenolic compounds at 10 μM and 60 mM AAPH after 4 h of incubation (original magnification ×400). (C) Control just
with RBCs. (D) Control with RBCs plus AAPH. Hy, hydroxytyrosol; Hy-GlcA, hydroxytyrosol glucuronides.
in the presence of phenolic compounds (lyses after 4 h of
incubation, without AAPH), we did not observe any change in
the oxy-hemoglobin peaks (540 and 578 nm) nor in its
concentration29 (data not shown), as compared to the control
assay (RBCs without phenolic compounds). Furthermore, the
oxy-hemoglobin disappeared in the presence of AAPH, but this
was partially reversed by the addition of hydroxytyrosol,
hydroxytyrosol acetate, and hydroxytyrosol glucuronides
(Figure 7). Hydroxytyrosol glucuronides were apparently less
effective in this protection at 80 μM than the other two
compounds but showed a protection similar to that of
hydroxytyrosol and hydroxytyrosol acetate at 20 and 10 μM.
The capacity of hydroxytyrosol and hydroxytyrosol glucur-
onides to protect RBC from oxidative hemolysis induced by
H2O2, a physiological oxidizing agent in erythrocytes, was also
evaluated. In this case, cells are attacked not only from the
outside but also from the inside as H2O2 can easily cross the
cell membrane. As expected, hydroxytyrosol protected RBC
from oxidative injury in a dose-dependent manner. However, in
the case of its glucuronides, the small protection observed was
not dose dependent. In fact, the activity was very similar for all
concentrations studied and similar to that of hydroxytyrosol for
the lower concentrations tested (10 and 20 μM). Because
glucuronides have not been described as crossing membranes, it
can be hypothesized that some sort of saturation of specific
RBC transporters could occur, thus leading to the same
protection at higher concentrations. Alternatively, these
glucuronides may have activity only after hydrolysis to
hydroxytyrosol, which can then be absorbed by the cells.
Some studies propose that glucuronides may act not only as
detoxifing metabolites but also as bioactive agents, being
precursors of more hydrophobic aglycones. Accordingly,
aglycones may be assumed to emerge in the target site by the
action of glucuronidases under oxidative stress.32 The
cardiovascular system and central nervous system seem to be
the major targets of phenol glucuronides circulating in the
human blood.32 Therefore, the activity of glucuronides will be
dependent on the availability of glucuronidases in a specific
tissue or cell. It is also important to note that when RBCs are
prepared, their content remains intact in the erythrocyte
structure, but the exogenous antioxidant defenses are washed
out during the manipulation, and there are no interactions with
other blood cells and epithelium cells. These results are in
agreement with those found for the hydroxytyrosol glucur-
onides in the protection of hemoglobin from the AAPH-
induced injury (Figure 8). Indeed, because hemoglobin is
located inside the RBC, it can only be protected from the
oxidative injury if the compounds can cross the RBC
membrane.
The present results suggest that hydroxytyrosol metabolites
may play an important role in the protective activity of olive oil
phenolic compounds. If 50 g of extra virgin olive oil with a high
concentration in polyphenols is consumed, this would
correspond to an intake of up to 32 mg per day.9−11 This
dose would lead to a relatively low plasma concentration (up to
5−10 μM),15 but it is known that regular low doses, for
example, of aspirin can confer cardiovascular health benefits.33
In fact, hydroxytyrosol and its metabolites have been found not
only in the plasma but also attached to lipoproteins,34 other
blood proteins, and cells.31 Therefore, it is possible that the
regular low lifetime intake of olive oil results in an overall
protective effect. This phenomenon has been demonstrated by
clinical trials showing that short-term consumption of olive oil
in humans (50 mL/day) can change several oxidative stress
markers,35,36 although the concentrations of phenols are lower
than those required to show in vitro biological activity. In fact,
it should be taken in mind that food components are different
than drugs and their actions on human physiology are usually
moderate. While medicines are habitually employed for limited
timeframes, food and its macro- and microcomponents are
ingested throughout a lifetime, during which even modest
effects, like those found for hydroxytyrosol glucuronides in the
presence of H2O2, may become noteworthy.
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AUTHOR INFORMATION
Corresponding Author
*Tel.: +351220402556. Fax: +351220402659. E-mail:
mpmartin@fc.up.pt.
■
Notes
The authors declare no competing financial interest.
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