Antioxidant and Biological Activities of Hydroxytyrosol and Homovanillic Alcohol Obtained from Olive Mill Wastewaters of Extra-Virgin Olive Oil Production

Article abstract of DOI:10.1021/acs.jafc.0c05230

Some constituents of the Mediterranean diet, such as extra-virgin olive oil (EVOO) contain substances such as hydroxytyrosol (HT) and its metabolite homovanillic alcohol (HA). HT has aroused much interest due to its antioxidant activity as a radical scavenger, whereas only a few studies have been made on the HA molecule. Both chemical synthesis and extraction techniques have been developed to obtain these molecules, with each method having its advantages and drawbacks. In this study, we report the use of tyrosol from olive mill wastewaters as a starting molecule to synthesize HT and HA, using a sustainable procedure characterized by high efficiency and low cost. The effects of HT and HA were evaluated on two cell lines, THP-1 human leukemic monocytes and L-6 myoblasts from rat skeletal muscle, after treating the cells with a radical generator. Both HT and HA efficiently inhibited ROS production. In particular, HT inhibited the proliferation of the THP-1 leukemic monocytes, while HA protected L-6 myoblasts from cytotoxicity.

Full text of DOI:10.1021/acs.jafc.0c05230

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Article
Antioxidant and Biological Activities of Hydroxytyrosol and
Homovanillic Alcohol Obtained from Olive Mill Wastewaters of
Extra-Virgin Olive Oil Production
Alessandra Ricelli,* Fabio Gionfra, Zulema Percario, Martina De Angelis, Ludovica Primitivo,
Veronica Bonfantini, Roberto Antonioletti, Simonetta Maria Bullitta, Luciano Saso, Sandra Incerpi,
and Jens Zacho Pedersen
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ABSTRACT: Some constituents of the Mediterranean diet, such as extra-virgin olive oil (EVOO) contain substances such as
hydroxytyrosol (HT) and its metabolite homovanillic alcohol (HA). HT has aroused much interest due to its antioxidant activity as
a radical scavenger, whereas only a few studies have been made on the HA molecule. Both chemical synthesis and extraction
techniques have been developed to obtain these molecules, with each method having its advantages and drawbacks. In this study, we
report the use of tyrosol from olive mill wastewaters as a starting molecule to synthesize HT and HA, using a sustainable procedure
characterized by high efficiency and low cost. The effects of HT and HA were evaluated on two cell lines, THP-1 human leukemic
monocytes and L-6 myoblasts from rat skeletal muscle, after treating the cells with a radical generator. Both HT and HA efficiently
inhibited ROS production. In particular, HT inhibited the proliferation of the THP-1 leukemic monocytes, while HA protected L-6
myoblasts from cytotoxicity.
KEYWORDS: antioxidant, sustainable, olive mill wastewaters, tyrosol, hydroxytyrosol, homovanillic alcohol
the two adjacent hydroxyl groups, to the conjugation and the
resonance effects, and also to the ability to improve the
stability of the corresponding phenoxyl radicals formed by the
hydrogen-transfer reaction.10−13 The role of HT in promoting
health benefits by lowering oxidative stress in biological
systems such as human cells and plasma has also been
investigated.14−16 Recently, the ability of HT and of some of
its analogues to fit into the catalytic domain of COX-2
regulating the anti-inflammatory response has been reported.17
HT can undergo further metabolization in the organism; its
major identified metabolite in humans is the methylated
derivative homovanillic alcohol (HA), 4-(2-hydroxyethyl)-2-
methoxyphenol, produced by the activity of catechol-O-
methyltransferase that is also naturally present in EVOO.18
Despite the scarcity of studies on HA, those conducted have
shown that this molecule exerts an antioxidant activity
comparable to that of HT in simple chemical systems. In
fact, HT and HA both inhibit H2O2-induced oxidative damage
in kidney cells18 and can protect other cells in culture against
peroxide-induced renal epithelial injury.17,19−21
1. INTRODUCTION
Many studies have focused on the beneficial health effects of
the Mediterranean diet and, in particular, some of its
constituents such as extra-virgin olive oil (EVOO) that has
been reported to prevent cardiovascular and neurodegener-
ative diseases when consumed daily within the context of a
balanced diet.1,2 Among the variety of bioactive components
found in olives, several phenolic compounds such as
hydroxytyrosol (HT) 4-(2-hydroxyethyl)-1,2-benzenediol
seem to have key roles. These phenols are powerful
hydrogen-donating antioxidants and scavengers of reactive
oxygen and nitrogen species. Several reports have demon-
strated a role for specific phenolic compounds through their
selective interactions within signaling cascades, such as
tyrosine kinase, PI3-kinase/Akt, as well as the protein kinase
C and mitogen-activated protein kinase pathways, which
regulate cell survival following exposure to oxidative stress.3,4
The concentrations of phenolic compounds such as
oleuropein (OLE) and HT differ widely among olive
products, depending on factors such as the cultivar, soil
composition, the climate, and the degree of ripeness of the
olives.5 OLE is the main phenolic compound present in Olea
europaea fruits and leaves; it is a hydroxytyrosol ester with an
α-glucosylated elenolic acid and it is responsible for the bitter
taste of unripened olives.6
Received: August 18, 2020
Revised: November 22, 2020
Accepted: November 27, 2020
The structure of HT contains a catechol moiety, and HT
has been investigated and successfully tested for its free-radical
scavenging activity.7−9 This is mainly due to the presence of
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was evaporated at low pressure and the purification by diethyl ether
was repeated. The product was constituted by 2-bromo-4- (2-
hydroxyethyl)-phenol (4.49 g, 20 mmol, 98%) as a yellow oil (2).
An amount of 2.157 g of 2-bromo-4-(2-hydroxyethyl)-phenol (9.9
mmol) (2) was dissolved in 14.6 mL of dimethyl carbonate (DMC).
The mixture was put under stirring while heating at 100 °C, the
copper complex (0.867 g CuBr, 6 mmol) together with 55.63 mL of
CH3ONa/CH3OH 25% w/v were added, and then the mixture was
allowed to reflux for 15 h. The monitoring of the reaction was
accomplished by TLC using n-hexane/ethyl acetate 1:1 v/v as a
mobile phase. Once the methoxylation was complete, the mixture
was initially cooled to RT and then placed in an ice bath. HCl 6 N
was added to obtain a weakly acidic pH, and then the solvent was
eliminated at low pressure. The aqueous residue was extracted using
diethyl ether, the extract was washed by a saturated NaCl solution,
and then the organic extract was dried over anhydrous sodium
sulfate. The solvent was removed at low pressure obtaining a pale-
yellow oil constituted by a mixture of 4-(2-hydroxyethyl)-2-
methoxyphenol (3) and its methyl carbonate derivative (4).
The mixture of 4-(2-hydroxyethyl)-2-methoxyphenol (3) and its
methyl carbonate derivative (4) was subjected to basic hydrolysis by
adding 100 mL of K2CO3/CH3OH 1% w/v and stirring at RT for 12
h. The monitoring of the reaction was accomplished by TLC using n-
hexane/ethyl acetate 1:1 v/v as a mobile phase. Once the reaction
was complete, the solvent was eliminated at low pressure and the
aqueous residue was extracted using cold ethyl acetate. The extract
was washed using a saturated NaCl solution, the organic extract was
then dried over anhydrous Na2SO4, and the solvent was eliminated at
low pressure. Any remaining dibrominated residue was removed by
column chromatography (n-hexane: ethyl acetate 1:1 v/v). The
product obtained (3) was homovanillic alcohol (1.43 g, 8.46 mmol,
86%).
An amount of 0.3 g (1.79 mmol) of homovanillic alcohol (3) was
heated for 12 h at 50 °C in 1 mL of acetic anhydride containing 0.1
mL of acetic acid. The monitoring of the reaction was accomplished
by TLC using n-hexane/ethyl acetate 1:1 v/v as a mobile phase.
Once the acetylation reaction was complete, the mixture was diluted
by 1 mL of cold distilled water and extracted (four times) using 0.5
mL of ethyl acetate. The organic extract was washed three times
using 0.15 mL of a saturated NaHCO3 solution, then twice by 0.2
mL of a saturated NaCl solution, and then dried over anhydrous
Na2SO4. The organic solvent was eliminated at low pressure at 40
°C, and the product obtained was 4-(2-acetoxyethyl)-3-methoxy-1-
acetoxy-benzene (5), (0.306 g, 1.20 mmol, 67%).
An amount of 0.239 g 4-(2-acetoxyethyl)-1,2-dihydroxybenzene
(5) was dissolved in 1 mL of CH2Cl2 at −20 °C in an inert
atmosphere and 0.2 mL of BBr3 was added dropwise. The reaction
was monitored by TLC using n-hexane/ethyl acetate 1:1 v/v as a
mobile phase. Once the reaction of demethylation/deacetylation was
complete, quenching was obtained by adding crushed ice directly
into the solution.
The results of the research on the characteristic properties
of these compounds have led to a strong interest in their
production in a pure form. For this purpose, both neo-
syntheses and strategies based on extraction have been
developed. Among the first group, the method published by
Deffieux et al.22 for HT is elegant and convenient; however,
the starting substrate, eugenol, is rather expensive and in itself
displays interesting biological properties.23 Also, the bio-
synthetic methods reported for HT usually require expensive
substrates and/or have a low efficiency.24,25
Other synthetic strategies, such as the one described in
Capasso et al.26 have been applied, starting from compounds
that are not present in the olives. Few studies have been
published describing the synthesis of HA; to the best of our
knowledge, these studies report only the biocatalyzed
synthesis of the monoglucuronide form of HA.27
The HT extraction has been based on olive mill wastewater
(OMW) because it is very rich in this substance. Moreover,
OMW is not only inexpensive but also constitutes a polluting
material, the disposal of which is a subject to the EU
regulations for waste management and is becoming signifi-
cantly costly.28 However, the extraction of HT from OMW
involves drawbacks such as the need to use large amounts of
organic solvents. Diethyl ether, methyl isobutyl ketone, methyl
ethyl ketone, and ethyl acetate are the solvents most
frequently used.29 Finally, the high reactivity of HT makes
its extraction extremely difficult. OMW is also rich in tyrosol
(TY),14 a precursor of HT deprived of the catechol moiety,
which unlike HT has low reactivity and can be easily obtained
from OMW by decantation.
In this study, we used TY as a starting molecule to
synthesize HT and HA. We evaluated the toxic effects of HT
and HA on two cell lines, L-6 myoblasts and THP-1 human
leukemic monocytes, and treated the cells with a radical
generator, cumene hydroperoxide (CH). The results show
that both HT and HA from OMW could be obtained by
simple and environmental friendly synthesis pathways with
high yields (>42% for HT and >80% for HA), and they had
significant roles as antioxidants, similar to that of commer-
cially available compounds.
2. MATERIALS AND METHODS
2.1. Materials and Cell Lines. Organic solvents and reagents,
thin-layer chromatography (TLC) plates 0.25 mm F254, and silica
gel (F230-400 mesh) were purchased from Merck/Sigma-Aldrich
(Darmstadt, Germany).
The reaction mixture was extracted by ethyl acetate five times, and
then the organic phase was washed by a saturated NaCl solution.
After checking that the pH was less than 4, the solution was dried
over anhydrous Na2SO4. The organic solvent was eliminated at low
pressure at 40 °C, and the product obtained was 4-(2-acetoxyethyl)-
1,2-dihydroxybenzene (6), (0.186 g, 0.94 mmol, 78%).
An amount of 0.186 g of 4-(2-acetoxyethyl)-1,2-dihydroxybenzene
(6) was dissolved in 0.95 mL of CH2Cl2 and added to 2.85 mL of
HCl 2 M. The reaction was allowed to continue at RT for 24 h
under magnetic stirring.
Roswell Park Memorial Institute medium (RPMI-1640), Dulbec-
co′s modified Eagle medium (DMEM), streptomycin (100 mg/mL),
penicillin (100 U/mL), D-glucose, cumene hydroperoxide, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and
phosphate-buffered saline (PBS) one tablet/L buffer without calcium
and magnesium were provided from Sigma-Aldrich (St. Louis, MO).
2′,7′-Dichlorodihydrofluorescein diacetate (H2DCF-DA) was ob-
tained from Molecular Probes (Eugene, OR). Sterile plasticware for
cell culture was obtained from Falcon Brand (San Diego, CA); fetal
bovine serum was from GIBCO (Grand Island, NY).
The monitoring of the reaction was accomplished by TLC using
n-hexane/ethyl acetate 1:1 v/v as a mobile phase. Once the
deacetylation reaction was complete, the organic solvent was
eliminated at low pressure at 40 °C, and the product obtained was
hydroxytyrosol (7), (0.140 g, 0.90 mmol, 96%).
2.3. Cells in Culture. L-6 myoblasts and human leukemic
monocytes THP-1 were obtained from American Type Culture
Collection (Rockville, MD). L-6 were seeded in 75 mL tissue culture
flasks and grown in Dulbecco’s modified Eagle’s medium containing
2.2. Organic Synthesis. An amount of 3 g of tyrosol (1) was
dissolved in 86.9 mL of methanol (21 mmol) and then 4.6 g of N-
bromosuccinimide (NBS) was added to the mixture. The reaction
was proceeded at room temperature (RT) until the bromuration was
completed (about 3 h). The monitoring of the reaction was
accomplished by TLC using n-hexane/ethyl acetate 1:1 v/v as a
mobile phase. Once the bromuration was complete, the solvent was
eliminated at low pressure, and then 20 mL of diethyl ether was
added to obtain a partial purification by decantation. The mixture
B
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Figure 1. THP-1 cell images after pretreatment with baicalein, hydroxytyrosol, homovanillic alcohol, and the radical generator cumene
hydroperoxide. Magnification 400×.
4.5 g/L glucose, supplemented with 10% fetal bovine serum, 100 μg/
mL streptomycin, and 100 U/mL penicillin in a humidified
atmosphere with 5% CO2 at 37 °C. Cells reached confluency after
5 days (approximately 6 × 106 cells/flask) and were kept in culture
as myoblasts by continuously passaging at preconfluent stages.1
Human leukemic monocytes THP-1 were grown in suspension in
an RPMI-1640 medium supplemented with 10% fetal bovine serum,
100 μg/mL streptomycin, and 100 U/mL penicillin in a humidified
atmosphere with 5% CO2 at 37 °C.1,30 THP-1 monocytes were
passaged twice a week by 1:4 dilutions and reseeded in 25 mL tissue
culture flasks.
density of approximately 5 × 104 cells/dish as specified under
Materials and Methods section. The following day, the cells were
treated with HT and HA at a final concentration of 1 μM and 10
μM. Cell counts using a Neubauer chamber were made every 24 h up
to confluence after mild trypsinization.31
THP-1 cells were seeded in 12-well plates (1 × 105 cells/well) in 1
mL of the growth medium supplemented as reported in Materials
and Methods section. After 24 h, the cells were treated with HT and
HA at the same concentrations specified in the previous paragraph
and counted up to 96 h from seeding using a Neubauer chamber.
2.5. Intracellular ROS Determination. The method used was a
standard assay based on the intracellular fluorescent probe 2′,7′-
dichlorodihydrofluorescein diacetate (H2DCF-DA).30 THP-1 mono-
2.4. Proliferation Studies. L-6 cells were seeded in 60 × 15
mm2 Petri dishes in 4 mL of the complete growth medium at a
C
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Figure 2. L-6 cell images after pretreatment with baicalein, hydroxytyrosol, homovanillic alcohol, and the radical generator cumene
hydroperoxide. Magnification 400×.
cytes were resuspended and centrifuged at 180g for 10 min; the
supernatant was discarded and cells were washed twice with 5 mL of
PBS−glucose at 37 °C to remove the serum that might affect the
action of the fluorescent probe. After the final centrifugation, the
pellet was resuspended in PBS−glucose and H2DCF-DA was added
at 10 μM final concentration (from a stock solution of 10 mM in
dimethyl sulfoxide (DMSO)). The cells were then left to incorporate
the fluorescent probe for 30 min in the dark at 37 °C. At the end of
the incubation, cells were washed twice, centrifuged at 180g for 5
min, and the final cell pellet was resuspended in PBS−glucose. Before
the experiments, cells recovered at RT for 1 h. ROS production was
measured in a Perkin-Elmer Multilabel Counter Victor3V Wallac
1420 by measuring the intracellular DCF fluorescence at the
excitation and emission wavelengths of the probe (498 and 530
nm, respectively). We measured the ability of HT and HA to buffer
ROS production in cells exposed to the radical generator cumene
hydroperoxide (CH; 200 μM) at different times (30 min, 1, 3, and
24 h). Before addition of CH, the cells were preincubated with
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different concentrations of HT or HA (0.01, 0.1, 1, and 10 μM) at
37 °C for 10 min.
Scheme 1. Synthesis of 2-Bromo-4-(2-hydroxyethyl)-phenol
(2)
2.6. Cell Images after Pretreatment with Baicalein,
Hydroxytyrosol, Homovanillic Alcohol, and the Radical
Generator Cumene Hydroperoxide. The protocol applied was
the same as that used for ROS determination and the images were
obtained after 3 and 24 h because these are the times when the effect
of the treatments is best appreciated on the cell morphology (Figures
1 and 2).
L-6 myoblasts and THP-1 monocytes were seeded in 24-multiwell
plates, 105 cells/mL or well and 4 × 105/mL or well, respectively, in
a complete medium. After 24 h, the cells were pretreated with either
baicalein or hydroxytyrosol or homovanillic alcohol at 10 μM for 10
min, and then cumene hydroperoxide was given. For both the cell
types, the images were taken at 3 and 24 h at magnification 400×.
2.7. MTT Assay. Cell viability and the potential cytotoxic effect of
HT and HA were assessed by the MTT assay. L-6 cells were seeded
in 96-wells plates at 1 × 104 cells/well in 200 μL of DMEM
containing 10% serum. The day after seeding, the medium was
discarded and 100 μL of the new medium containing cumene
hydroperoxide (27.5 μM)30 with or without HT and HA at different
concentrations was added to each well, depending on the specific
experimental condition, as described above. Then, an MTT solution
(5 mg/mL in PBS) was added at a final concentration of 10% with
respect to the total volume, and incubation was carried out at 37 °C
for 3−4 h in the dark. During the incubation, there was a conversion
of the yellow MTT to purple formazan by the mitochondrial
succinate dehydrogenase of living cells. Then, a lysis buffer (DMSO
containing ammonia)32 was added and further incubation at 37 °C
for 30 min in the dark was carried out. Cells were then resuspended,
and the optical density was read with an ELISA-reader at 550−570
nm. Results are reported as mean standard deviation (SD) of two
experiments carried out in quintuplicate.
of only the monohalogenated product through an electrophilic
aromatic substitution. The conversion of TY to 2-bromotyr-
osol (2) under these reaction conditions reaches yields higher
than 95%.
The brominated TY (2) is the substrate of the
methoxylation reaction that is conducted using DMC as a
solvent (Scheme 2). The use of DMC as a solvent is a safe
Scheme 2. Synthesis of 4-(2-Hydroxyethyl)-2-
methoxyphenol (HA) (3)
2.8. Statistical Analysis. All data obtained from cultured cells
were analyzed by one-way analysis of variance (ANOVA) followed
by post hoc Bonferroni’s multiple comparison test. In some cases,
Student’s t-test was applied. Analyses were carried out using the
Prism 4.0 statistics program (GraphPad, San Diego, CA). Differences
were considered significant at p < 0.05.
3. RESULTS AND DISCUSSION
and environmentally friendly replacement for dimethylforma-
mide since it does not produce inorganic salts in either
acylation or alkylation reactions. Moreover, DMC is
synthesized through a clean process.37
However, in these conditions, the reaction led not only to
the formation of HA (3) but also to its side-chain methyl
carbonate (4). This compound is obtained through the
reaction between the alkoxylated HA and dimethyl carbonate
(DMC). The reaction is a nucleophilic acyl substitution via a
base-catalyzed alkyl cleavage bimolecular mechanism37
(Scheme 2).
In the course of this study, the synthesis of homovanillic
alcohol (HA) and hydroxytyrosol (HT) from tyrosol (TY), a
byproduct from the production of olive oil, has been
examined.33 The effect of HA and HT on ROS production
and on cell proliferation in THP-1 and L-6 cell lines was also
evaluated. THP-1 cells are leukemic monocytes from
peripheral blood; the monocytes defend the body against
infections by killing foreign cells through the production of
large amounts of ROS.
L-6 cells from rat skeletal muscle are able to differentiate
from myoblasts in myotubes when they reach confluency. The
contraction of skeletal muscles requires low levels of ROS, but
the presence of ROS at high levels may result in contractile
dysfunction and fatigue.34−36
The synthesis of HA and HT was accomplished using a
sequence of simple reactions of bromination and alkoxylation
from TY. The approach is to oxyfunctionalize the aromatic
rings of natural substances to increase their biological activity;
in particular, the strategy adopted involves the methoxylation
of aromatic bromides.
Scheme 7 represents the proposed reaction mechanism of a
coupling reaction mediated by copper(I) salt.
The mechanism involves the oxidative addition of copper(I)
to the Ar−Br bond, the coordination of the methoxide, and
the formation of a copper complex (III). The reaction ends
with a reductive elimination, which provides the product of
coupling Ar−OCH3 and brings the copper to the previous
state of oxidation (+1). Other methods for the etherification
of aryl bromides are reported in the literature, such as through
the catalysis using an expensive palladium complex.
3.1. HA Synthesis. NBS allows TY (1) bromination in the
ortho-phenolic position (Scheme 1). To avoid the further
bromination of TY (1) in the second activated position of the
ring, the addition of NBS is carried out in three aliquots to
keep the reagent as a limiting factor. Given that the second
bromination is slower, this procedure allows the obtainment
To transform the HA side-chain methyl carbonate (4) in
HA (3), the mixture of these compounds was subjected to a
basic hydrolysis reaction by a solution of K2CO3 (Scheme 3).
HA (3) was used both as such in the subsequent biological
tests on THP-1 cells and as an intermediate for the synthesis
of HT (7), which, in turn, was used in the biological tests.
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Scheme 3. Hydrolysis of Carbonate Derivative (4)
Scheme 7. Reaction Mechanism of a Coupling Reaction
Mediated by Copper(I) Salt
3.2. HT Synthesis. The transformation of HA (3) into
HT (7) requires the protection of hydroxyl groups through
acetylation. The reaction was accomplished using acetic
anhydride and acetic acid, which do not require tedious
work-up (Scheme 4).
leukemic monocytes are very sensitive to ROS due to their
nature of cells responsible for the immune defense against
infections, thus representing the killing machine of the body.
HT and HA were tested in a wide concentration range (0.01−
10 μM) to investigate their effect on ROS production. The
experiments were carried out at a time course of 30 min, 1, 3,
and 24 h (Figures 3 and 4), either alone or in combination
with CH, a known radical generator. Baicalein (BAI), a very
effective antioxidant compound, was used to compare its
scavenger action with respect to HT and HA.30,38
Scheme 4. Synthesis of 4-(2-Acetoxyethyl)-3-methoxy-1-
acetoxy-benzene (5)
In THP-1 cells, HT (0.1 μM) was able to inhibit ROS
generation in the presence of CH (Figure 3) at 30 min (p <
0.05), although the most significant effect was at 10 μM at all
time points, being more effective at 24 h (p < 0.001). The HT
molecule is strongly reactive toward ROS, and therefore
capable of a very prompt action; the more effective
concentrations were 1 and 10 μM. On the other hand,
because of its high reactivity, this molecule could undergo a
lowering of the active concentration in contexts where there is
a high presence of ROS, so with the prolongation of the
incubation period, a greater HT concentration is necessary to
obtain a significant effect.
The acetylated product (5) was subjected to a demethy-
lation reaction obtaining the product (6) shown in Scheme 5,
Scheme 5. Synthesis of 4-(2-Acetoxyethyl)-1,2-
dihydroxybenzene (6)
HA is able to inhibit ROS generation in THP-1 human
leukemic monocytes (Figure 4); the inhibition started after 30
min of incubation (p < 0.05 with HA 1 μM; p < 0.001 with
HA 10 μM) and was more efficient at 24 h in cells treated
with HA at 10 μM (p < 0.001). The efficacy of HT and HA in
inhibiting the generation of ROS is similar to that shown by
baicalein at the same concentration (10 μM). A lower
inhibition of ROS production shown by HA compared to HT
could be due to the presence of a methoxy group, which
confers rigidity and lowers the reactivity to the HA molecule.
In addition, the HA molecule is hydrolyzed in the cells, giving
rise to HT, thus the antioxidant effect observed following the
treatment with HA is, at least in part, due to the HT formed
by the demethylation of HA.39
thanks to simultaneous selective deacetylation of the acetoxy
group on the aromatic ring. The hydrolysis on the product (6)
was accomplished using 2 M HCl in CH2Cl2, leading to the
formation of hydroxytyrosol (7) (Schemes 6 and 7).
3.3. Effect of HT and HA on ROS Production in THP-1
Human Leukemic Monocytes. The THP-1 human
Scheme 6. Synthesis of Hydroxytyrosol (HT) (7)
3.4. Effect of HT and HA on ROS Generation in L-6
Cells. L-6 myoblasts require low levels of ROS for the
contraction of skeletal muscles but the presence of ROS at
high levels results in contractile dysfunction.35,36,40
As shown in Figure 5, HT significantly inhibits the
generation of ROS in L-6 cells stimulated with cumene
hydroperoxide for 1 h (p < 0.05 with HT 10 μM) and up to
24 h (p < 0.001 with HT at 1 and 10 μM).
F
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Figure 3. Dose-response effect of hydroxytyrosol (HT) on ROS production in THP-1 cells after 30 min, 1, 3, and 24 h from stimulation with
cumene hydroperoxide (CH). The results are mean SD of four to five different experiments carried out in quadruplicate. *p < 0.01, at least vs
CH.
Figure 4. Dose-response effect of homovanillic alcohol (HA) on ROS generation in THP-1 cells after 30 min, 1, 3, and 24 h from stimulation
with cumene hydroperoxide (CH). The data are mean SD of two different experiments carried out in quadruplicate. *p < 0.05, at least vs CH.
The inhibition by HT at 10 μM of the production of ROS
is similar to that of baicalein at the same concentration in cells
stimulated with CH. In the untreated cells, baicalein showed
an effect by itself on basal ROS production; a similar effect
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Figure 5. Effect of hydroxytyrosol (HT) at different concentrations on ROS generation in L-6 cells after 30 min, 1, 3, and 24 h from stimulation
with cumene hydroperoxide (CH). The data are mean SD of two different experiments carried out in quadruplicate. *p < 0.05, at least vs CH.
Figure 6. Dose-response effect of homovanillic alcohol (HA) on ROS production in L-6 myoblasts after 30 min, 1, 3, and 24 h from stimulation
with cumene hydroperoxide (CH). The data are mean SD of two different experiments carried out in quadruplicate. *p < 0.01, at least vs CH.
was observed in THP-1 monocytes. We do not know at
present why, but it could depend on some stress of the cells
due to the experimental procedure or a basal activity of ROS
production.
H
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Figure 7. Dose-response effect of HT and HA on the proliferation of THP-1 human leukemic monocytes (upper and center panels). The THP-1
cells growing in suspension were seeded in 24-well plates with 1 mL of the complete medium. Cells were stimulated with either HT or HA 24 h
from seeding and then counted every 24 h up to optimal density (96 h). Results are mean SD of three different experiments carried out in
duplicate. *p < 0.05 vs control, as from Student’s t-test. The cytotoxicity/proliferation (MTT) assay (lower panel) was carried out with either HT
or HA in L-6 myoblasts. The concentration of cumene hydroperoxide (CH) was 27.5 μM throughout the experiments. Results are mean SD of
two different experiments carried out in quintuplicate. *p < 0.001 vs control and either HT or HA alone; °p < 0.001 vs CH.
HA is able to inhibit ROS both in cells stimulated with CH
as well as in those not stimulated at 24 h (Figure 6).
In the first group, ROS inhibition is evident after 24 h of
incubation (p < 0.05) with HT 10 μM. In the cells stimulated
with CH, ROS inhibition takes place after 30 min (p < 0.05
HA 1 μM; p < 0.001 HA 10 μM) and remains at the same
level until 24 h.
The lower concentration of ROS in the cellular environ-
ment of muscle cells compared to that of leukemic monocytes
might explain the lower efficacy of HT on L-6 cells in the first
3 h of incubation. HT demonstrates the same efficacy on
THP-1 cells and L-6 cells after 24 h of incubation in the
presence of CH, when radical generation is also remarkable in
L-6 cells (Figures 5 and 6).
The rigidity and the lower reactivity of the HA with respect
to the HT molecule, in this case, are advantageous as they
allow a greater resistance and durability. In this way, HA can
be available even after long incubation times (24 h in this
case).
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Article
3.5. Proliferation Curves and Cell Viability/Cytotox-
icity Assay. The effect of HT and HA on the proliferation of
THP-1 monocytes and L-6 myoblasts was tested at
concentrations from 1 to 40 μM. A significant effect was
evident only in THP-1 cells at 20 μM HT at 72 h, so for the
sake of simplicity, only THP-1 is reported in the graphic
(Figure 7, upper and center panels). The proliferation of L-6
myoblasts was not significantly affected by either HT or HA
and it is not shown.
The possible cytotoxic effect of HT and HA as well as their
effect on cell viability were assessed by the MTT assay on L-6
myoblasts (Figure 7, lower panel). No cytotoxic effect was
found for either HT or HA. HA (80 μM) was able to protect
the cells from oxidative stress but the other concentrations did
not protect the L-6 myoblasts from cumene hydroperoxide, in
good agreement with previously published data.16
Roberto Antonioletti − Institute of Molecular Biology and
Pathology-CNR, I-00185 Roma, Italy
Simonetta Maria Bullitta − Institute for the Animal
Production System in the Mediterranean Environment-CNR,
I-07100 Sassari, Italy
Luciano Saso − Dept Physiology and Pharmacology,
University “Sapienza”, I- 00185 Rome, Italy
Sandra Incerpi − Dept Sciences, University Roma Tre, I-
00146 Roma, Italy
Jens Zacho Pedersen − Dept Biology, University Tor
Vergata, I-00133 Rome, Italy; orcid.org/0000-0002-
5758-9544
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.0c05230
Funding
In this study, tyrosol, a phenol abundant in olives and a
polluting waste product from the oil industry,41 has been
transformed into value-added products. In particular, tyrosol
was transformed into HA and HT. These phenolic molecules
are present in plants and have demonstrated remarkable
biological activity in the considered system. Hydroxytyrosol
and homovanillic alcohol are metabolites of dopamine and
both endogenous and exogenous derived products may be
found in the plasma from both sources and modulate cell
activities.42
The value of tyrosol, a product with low biological activity
and high polluting power that must be disposed of according
to special procedures as required by law, has thus been
enhanced. The procedures used in the synthesis are
characterized by high efficiency and low cost or in one
word, are sustainable; moreover, they can become part of the
circular economy of olive oil production.43
The yield was high for both HA and HT, being >80 and
>42%, respectively. Our data show that HT and HA derived
from tyrosol of OMW have antioxidant and biological
activities in cells in culture similar to that of the compounds
commercially available or derived from other more conven-
tional sources.
In particular, HA gave full protection from oxidative insult
given by cumene hydroperoxide at the MTT assay, whereas
HT (20 μM) was capable of killing tumor cell THP-1
monocytes.
Financial support “CAL” from the Department of Sciences,
University Roma Tre to S.I. is gratefully acknowledged.
Notes
The authors declare no competing financial interest.
ABBREVIATION USED
■
ROS, reactive oxygen species; EVOO, extra-virgin olive oil;
OLE, oleuropein; HT, hydroxytyrosol; DPPH, 2,2-diphenyl-1-
picryhydrazyl; HA, homovanillic alcohol; OMW, olive mill
wastewater; TY, tyrosol; DMC, dimethyl carbonate; TLC,
thin-layer chromatography; NBS, N-bromosuccinimide; CH,
cumene hydroperoxide; PBS, phosphate-buffered saline; RT,
room temperature
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AUTHOR INFORMATION
■
Corresponding Author
Alessandra Ricelli − Institute of Molecular Biology and
Pathology-CNR, I-00185 Roma, Italy; orcid.org/0000-
0002-1151-6120; Email: alessandra.ricelli@cnr.it
Authors
Fabio Gionfra − Dept Sciences, University Roma Tre, I-
00146 Roma, Italy
Zulema Percario − Dept Sciences, University Roma Tre, I-
00146 Roma, Italy
Martina De Angelis − Institute of Molecular Biology and
Pathology-CNR, I-00185 Roma, Italy; Dept Chemistry,
University “Sapienza”, I-00185 Roma, Italy
Ludovica Primitivo − Institute of Molecular Biology and
Pathology-CNR, I-00185 Roma, Italy; Dept Chemistry,
University “Sapienza”, I-00185 Roma, Italy
Veronica Bonfantini − Dept Chemistry, University
“Sapienza”, I-00185 Roma, Italy
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