Identification of hydroxytyrosyl oleate, a derivative of hydroxytyrosol with anti-inflammatory properties, in olive oil by-products

Article abstract of DOI:10.1016/j.foodchem.2018.12.007

Hydroxytyrosyl esters with short, medium and long acyl chains were evaluated for their ability to reduce nitric oxide (NO) production by lipopolysaccharide-stimulated RAW264.7 macrophages. Among the compounds tested, C18 esters, namely hydroxytyrosyl stea

Full text of DOI:10.1016/j.foodchem.2018.12.007

Accepted Manuscript
Identification of hydroxytyrosyl oleate, a derivative of hydroxytyrosol with anti-
inflammatory properties, in olive oil by-products
Pierluigi Plastina, Cinzia Benincasa, Enzo Perri, Alessia Fazio, Giuseppina
Augimeri, Mieke Poland, Renger Witkamp, Jocelijn Meijerink
PII:
S0308-8146(18)32101-0
DOI:
https://doi.org/10.1016/j.foodchem.2018.12.007
FOCH 23965
Reference:
To appear in:
Food Chemistry
Received Date:
Revised Date:
Accepted Date:
24 July 2018
16 November 2018
4 December 2018
Please cite this article as: Plastina, P., Benincasa, C., Perri, E., Fazio, A., Augimeri, G., Poland, M., Witkamp, R.,
Meijerink, J., Identification of hydroxytyrosyl oleate, a derivative of hydroxytyrosol with anti-inflammatory
properties, in olive oil by-products, Food Chemistry (2018), doi: https://doi.org/10.1016/j.foodchem.2018.12.007
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Identification of hydroxytyrosyl oleate, a derivative of hydroxytyrosol with anti-inflammatory
properties, in olive oil by-products
1
,
2
2
1
1,3
Pierluigi Plastina, * Cinzia Benincasa, Enzo Perri, Alessia Fazio, Giuseppina Augimeri, Mieke
4
4
4
Poland, Renger Witkamp, and Jocelijn Meijerink
1
Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036
Arcavacata di Rende (CS), Italy
2
Council for Agricultural Research and Economics - Research Centre for Olive, Citrus and Tree
Fruit, C.da Li Rocchi, 87036 Rende (CS) – Italy
3
Health Center, University of Calabria, 87036 Arcavacata di Rende (CS), Italy
4
Division of Human Nutrition and Health, Wageningen University, 6700 AA Wageningen, The
Netherlands
*
Corresponding author. Tel.: +39 0984 493128; fax: +39 0984 493107
E-mail: pierluigi.plastina@unical.it
1

Abstract
Hydroxytyrosyl esters with short, medium and long acyl chains were evaluated for their ability to
reduce nitric oxide (NO) production by lipopolysaccharide-stimulated RAW264.7 macrophages.
Among the compounds tested, C18 esters, namely hydroxytyrosyl stearate (HtySte) and
hydroxytyrosyl oleate (HtyOle), were found to decrease NO production in a concentration-
dependent manner, while the other compounds, including the parent hydroxytyrosol, were
ineffective in the tested concentration range (0.5-5 μM). Further study of the potential immune-
modulating properties of HtyOle revealed a significant and concentration-dependent suppression of
prostaglandin E production. At a transcriptional level, HtyOle inhibited the expression of inducible
2
NO synthase, cyclooxygenase-2 and interleukin-1β. Moreover, HtyOle was identified for the first
time in olive oil by-products by means of high performance liquid chromatography coupled with
mass spectrometry. By contrast, HtyOle was not found in intact olives. Our results suggest that
HtyOle is formed during oil processing and represents a significant form in which hydroxytyrosol
occurs.
KEYWORDS: COX-2; Hydroxytyrosyl ester; Inflammation; Macrophages; NO; olive oil; olive
mill waste water; PGE2
Abbreviations used
Acetonitrile (ACN); Cyclooxygenase-2 (COX-2); Dulbecco’s modified Eagle’s medium (DMEM);
extra virgin olive oil (EVOO); high performance liquid chromatography (HPLC); Hydroxytyrosol
(Hty); Hydroxytyrosyl Acetate (HtyAce); Hydroxytyrosyl Laurate (HtyLau); Hydroxytyrosyl
Palmitate (HtyPal); Hydroxytyrosyl Stearate (HtySte); Hydroxytyrosyl Oleate (HtyOle); inducible
nitric oxide synthase (iNOS); interleukin-1β (IL-1β); Limit of quantitation (LOQ); Limit of
detection (LOD) lipopolysaccharide (LPS); nitric oxide (NO); nuclear factor-kappa B (NF-kB);
oleic acid (OA); olive mill waste water (OMWW); olive oil (OO); prostaglandin E (PGE ); real
2
2
2

time polymerase chain reaction (RT-PCR); Solid phase extraction (SPE); stearic acid (SA);
Trifluoroacetic acid (TFA); Tyrosyl Oleate (TyOle).
3

1
.
Introduction
Hydroxytyrosol [2-(3,4-dihydroxyphenyl)ethanol, Hty, Table 1] is one of the major phenolic
components of olive and olive oil, present in the esterified form, mainly as secoiridoid derivatives
(oleuropein and its aglycone), as well as in the free form. The amount of free Hty increases during
olive ripening and olive oil processing as a result of endogenous β-glucosidase hydrolytic activity
releasing Hty from the secoiridoids (Gambacorta et al., 2007; Robles-Almazan et al., 2018). In line
with the behavior of other hydrophilic phenolics, Hty is found in greater amounts in aqueous by-
products of oil processing, including pomace and olive mill waste water (OMWW), compared to
olive oil itself. Hydroxytyrosol displays a wide range of potential health benefits, including
antioxidant, cardio-protective, anticancer, and anti-inflammatory effects (Bernini, Merendino,
Romani, & Velotti, 2013; Lucas et al., 2010; Toteda et al., 2017; Zhang, Cao, & Zhong, 2009).
However, its pharmacokinetic properties are unfavorable, as the compound shows low oral
bioavailability and fast elimination in humans (Robles-Almazan et al., 2018). These features and its
hydrophilic character limit its use as a dietary supplement as well as an additive in foods (Mateos,
Martínez-López, Arévalo, Amigo-Benavent, Sarriá, & Bravo-Clemente, 2016; Navarro & Morales,
2
017). In order to improve these properties, derivatives with different acyl chains to increase
lipophilicity have been synthesized. Some of these compounds display enhanced antioxidant
activities compared to Hty, in both cell-free and cell-based models (Bernini, Crisante, Barontini,
Tofani, Balducci, & Gambacorta, 2012; Bernini, Gilardini Montani, Merendino, Romani, & Velotti,
2
015; Bouallagui, Bouaziz, Lassoued, Engasser, Ghoul, & Sayadi, 2011; Candiracci, Madrona,
Espartero, Zappia, & Piatti, 2016; Grasso, Siracusa, Spatafora, Renis, & Tringali, 2007; Mateos,
Trujillo, Pereira-Caro, Madrona, Cert, & Espartero, 2008; Procopio, Celia, Nardi, Oliverio, Paolino,
&
Sindona, 2011; Sun, Zhou, & Shahidi, 2018; Tofani, Balducci, Gasperi, Incerpi, & Gambacorta,
010; Zhou, Sun, & Shahidi, 2017). Next to this, some Hty esters, including conjugates of Hty with
n-3 fatty acids, have been synthesized with the aim of stabilizing oil matrices and emulsions
2
(Akanbi & Barrow, 2018; Almeida, Losada-Barreiro, Costa, Paiva-Martins, Bravo-Díaz, &
4

Romsted, 2016; Medina, Lois, Alcántara, Lucas, & Morales, 2009; Torres de Pinedo, Peñalver,
Pérez-Victoria, Rondón, & Morales, 2007; Trujillo et al., 2006). By contrast, investigations on the
anti-inflammatory properties of Hty esters have focused only on hydroxytyrosyl acetate (HtyAce).
This compound has been reported to inhibit cyclooxygenase-2 (COX-2) protein expression and
nuclear factor-kappa B (NF-kB) activation in lipopolysaccharide (LPS)-stimulatedmurine peritoneal
macrophages (ꢀꢁaꢂicio-ꢃoꢄoꢅ ꢃꢆncꢇeꢈ-ꢉidalgoꢅ ꢊonꢈꢆleꢈ-ꢋenꢌumeaꢅ Maꢍaꢅ ꢉeꢂnꢆndeꢈ-ꢋolaꢎosꢅ ꢏ
ꢀlaꢂcꢐn-de-la-Lastra, 2015), and in dextran sulfate sodium (DSS)-induced acute colitis in mice
(ꢃꢆncꢇeꢈ-Fidalgo et al., 2015). Moreover, dietary supplementation of HtyAce induced preventive
effect in collagen-induced arthritis (Rosillo, Sánchez-Hidalgo, González-Benjumea, Fernández-
Bolaños, Lubberts, & Alarcón-de-la-Lastra, 2015) and inhibition of pristane-induced systemic lupus
erythematous (Aparicio-Soto, Sánchez-Hidalgo, Cárdeno, González-Benjumea, Fernández-Bolaños,
&
Alarcón-de-la-Lastra, 2017) in mice, and down-regulation of the production of mediators
involved in joint erosion in human synovial cells (Rosillo et al., 2017). In order to expand the
knowledge on the biological activities of Hty fatty esters as potential ingredients in functional foods
with improved quality, we evaluated the anti-inflammatory properties of a series of Hty esters with
short (C2), medium (C12) and long (C16 and C18) acyl chains by lipopolysaccharide (LPS)-
stimulated murine macrophage cell line. In addition, we identified that the most promising
compound, namely hydroxytyrosyl oleate (HtyOle), is present in olive oil by-products.
2
.
Materials and methods
2
.1
Chemicals
Hydroxytyrosol was synthesized by reduction of 3,4-dihydroxyphenylacetic acid with LiAlH4
Sigma-Aldrich (Milan, Italy)] as previously reported (Fazio, Caroleo, Cione, & Plastina, 2017).
[
Tyrosol, t-butanol, stearic acid (SA), oleic acid (OA), trifluoroacetic acid (TFA) and fatty acid
®
methyl esters were from Sigma-Aldrich (Milan, Italy). Novozym 435 was from Novozymes. n-
Hexane, acetone, and ethyl acetate (analytical grade) were purchased from Carlo Erba Reagenti
5

(
Milan, Italy). Methanol and formic acid (LC/MS grade) were supplied from Sigma-Aldrich
Riedel-de Haën, Laborchemikalien, Seelze). Aqueous solutions were prepared using ultrapure
(
waꢄeꢂꢅ wiꢄꢇ a ꢂesisꢄiviꢄꢍ of 18.2 MΩ cmꢅ obꢄained fꢂom a Milli-Q plus system (Millipore, Bedford,
MA, USA). Dulbecco’s modified Eagle’s medium (DMEM)ꢅ feꢄal bovine seꢂumꢅ sꢄꢂeꢁꢄomꢍcin and
penicillin were acquired from Lonza (Verviers, Belgium). Bacterial lipopolysaccharides (LPS, E.
coli O111:B4) was obtained from Sigma-Aldrich (Schnelldorf, Germany). Griess reagents, nitrite
standard and PGE EIA kits were purchased from Cayman Chemical (Ann Arbor, MI, USA). Solid
2
phase extraction (SPE) Discovery DSC C8 columns (500 mg, 3 mL) were from Supelco
(Bellefonte, PA, USA).
2
.2
Instrumentation
1
13
H NMR and C NMR spectra were recorded at 25 °C on a Bruker AC 300 spectrometer in CDCl
3
solutions at 300 MHz and 75 MHz, respectively, with TMS as the internal standard. HPLC analysis
were performed using an Agilent Technologies 1200 series liquid chromatography system equipped
with a G1379B degasser, a G1312A pump, and a G1329A autosampler. Chromatographic
separation was achieved by injecting 10 µL of methanol/water extracts in a Discovery C-18 column
(3 µm particle size; 150 mm length; 4.6 mm i.d., Merck KGaA, Darmstadt, Germany) and a mobile
phase consisted of an aqueous formic acid (0.1%) solution (A) and methanol (B). The binary
gradient for the chromatographic run was as follows: the concentration of B was increased from
3
0% to 100% in 10 minutes, hold for 5 minutes, and ramp down to the original composition in 10
minutes. Flow rate was set at 400 µL/min. MS/MS analyses were performed by means of an API
000 Q-Trap (MSD Sciex Applied Biosystem) mass spectrometer. The compound was monitored in
4
negative ion mode using multiple reaction monitoring (MRM). The instrumental parameters, such
as entrance potential (EP), declustering potential (DP), collision energy (CE) and collision exit
potential (CXP) were, therefore, optimized for the transitions monitored.
2
.3
Synthesis of (hydroxyl)tyrosyl esters
6

The synthesis of hydroxytyrosyl acetate (HtyAce) was achieved based on a previously reported
enzymatic procedure with modifications (Fazio et al., 2017). Briefly, hydroxytyrosol (1.6 mmol),
was stirred in an orbital shaker at 50 °C in the presence of ethyl acetate (2 mL), as the acyl donor
®
and solvent, and Novozym 435 (immobilized Candida antarctica lipase B, CALB, 200 mg), as the
catalyst. The synthesis of the other esters was carried out using hydroxytyrosol or tyrosol (1.6
mmol), the appropriate fatty acid methyl ester (3.2 mmol) as acyl donor, in the presence of CALB
(200 mg), in t-butanol as the solvent (2 mL) in an orbital shaker at 50 °C. In both procedures, the
enzyme was filtered off after 24 h, and the solvent evaporated under reduced pressure.
Subsequently, products were purified by column chromatography (SiO , using n-hexane-acetone as
2
the eluent). Characterization data of the purified compounds were in agreement with those available
in the literature (Mateos et al., 2008; Trujillo et al., 2006). The purity of Hty esters was assessed by
HPLC and found ꢄo be as ≥98% in all cases.
2
.4
Cell culture
RAW264.7 mouse macrophages (obtained from the American Type Culture Collection,
Teddington, UK) were grown at 37 °C in a 5% CO ꢇumidified aiꢂ aꢄmosꢁꢇeꢂe in Dulbecco’s
2
modified Eagle’s medium (DMEM)ꢅ suꢁꢁlemenꢄed wiꢄꢇ feꢄal bovine seꢂum (10%)ꢅ sꢄꢂeꢁꢄomꢍcin
5
−1
and penicillin. Cells were seeded into 96-well cell culture plates (2.5 × 10 cells mL ) for nitrite,
5
viability/toxicity measurements and analysis of PGE by EIA, or in 6-well plates (5 × 10 cells
2
−
1
mL ) for RNA extraction, purification and quantitative reverse transcription real-time PCR and for
extraction and determination of HtyOle in cell culture medium. After overnight incubation, adherent
-1
cells were incubated with LPS (0.5 mg mL ) in the presence (or absence) of the test compounds for
4
8h (for nitrite and viability/toxicity measurements) or 24 h (for PGE production and RNA
2
extraction).
2
.5
Assays for viability and cytotoxicity
7

Cell viability and potentialcytotoxic effects of compounds were determinedby an XTT assay and by
measuring LDH leakage, respectively. To measure XTT conversion, the XTT Cell Proliferation Kit
II fromRoche Applied Science (Almere, The Netherlands) wasused. Briefly, RAW264.7 cells were
incubated for 48 h with the compounds and LPS. After incubation, supernatants were removed (for
LDH determination) and 100 μL of fꢂesꢇ medium suꢁꢁlemenꢄed wiꢄꢇ sodium 30-[1-
(phenylaminocarbonyl)-3,4-tetrazolium]bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate
(XTT) (final concentration= 0.45 mM) and N-methyldibenzopyrazine methylsulfate (1.25 mM),
was added to the cells. After incubation at 37°C, the amount of formazan accumulated in the
medium was measured at 450 nm on a plate reader (Multiskan Ascent, ThermoLabsystem, Breda,
The Netherlands). LDH leakage was measured using a Cytotoxicity Detection Kit (Roche Applied
Science, Almere, The Netherlands). LDH was measured in culꢄuꢂe suꢁeꢂnaꢄanꢄs (100 μL)ꢅ wꢇicꢇ
were ꢂemoved and mixed wiꢄꢇ enꢈꢍme ꢂeagenꢄs (diaꢁꢇoꢂase/NꢀD mixꢄuꢂeꢅ 250 μL) and dꢍe
solutions (iodotetrazoliumchloride and sodium lactate, 11.25 mL). After 30 min of incubation at
2
5°C, the absorbance was measured at 492 nm.
2
.6
Nitric oxide (NO) and prostaglandin E (PGE ) quantification
2 2
Nitrite accumulated in the culture medium, following 48 h of incubation, was measured as an
indicator of NO production as described previously (Wang et al., 2017). ꢋꢂieflꢍꢅ 100 μL of the cell
culꢄuꢂe medium weꢂe mixed wiꢄꢇ 100 μL of ꢊꢂiess ꢂeagenꢄs and incubaꢄed aꢄ ꢂoom ꢄemꢁeꢂaꢄuꢂe foꢂ
1
0 min. Absorbance was measured at 540 nm using an ELISA plate reader. PGE production in the
2
cell culture was measured using a PGE EIA kit following manufacturer’s instructions.
2
2
.7
RNA extraction, purification and quantitative reverse transcription real-time PCR
After incubating for 24 h, medium was removed and total RNA was extracted using TRIzol
Inviꢄꢂogenꢅ ꢋꢂedaꢅ Tꢇe Neꢄꢇeꢂlands). RNꢀ (1 μg/sample) was reverse transcribed to give
(
complementary DNA using the reverse transcription system from Promega (Leiden, The
8

Netherlands). Complementary DNA was amplified by PCR using platinum Taq DNA polymerase
(
Invitrogen) and SYBR green (Molecular Probes, Leiden, The Netherlands) on an iCycler apparatus
Bio-Rad, Veenendaal, The Netherlands). The following primer pairs were used for amplification of
(
iNOS: 5'-GTTCTCAGCCCAACAATACAAGA-3' (forward) e 5'-GTGGACGGGTCGATGTCAC-
(reverse); COX-2: 5'-GGAGAGACTATCAAGATAGT-3' (forward) and 5'-
ATGGTCAGTAGACTTTTACA-3' (reverse); IL-1β: 5'-TGCAGAGTTCCCCAACTGGTACATC-
' (forward) and 5'-GTGCTGCCTAATGTCCCCTTGAATC-3' (reverse). Samples were analyzed
3
'
3
in duplicate, and mRNA expression levels of the different genes were normalized to RPS27A2.
Primer pairs for RPS27A2 were 5'-GGTTGAACCCTCGGACACTA-3' (forward) and 5'-
GCCATCTTCCAGCTGCTTAC-3' (reverse).
2
.8
Olive sampling and oil extraction
Olea europaea L. fruits (Cassanese cultivar) were hand-collected in the experimental olive grove of
the Research Center for Olive, Citrus and Fruit Trees located in Rende (Italy) in the crop year
2
017/2018 in two different periods, October and January. Extra virgin olive oils (EVOO) and the
corresponding by-products, including pomace and olive mill waste water (OMWW), were produced
by crushing 10 kg of olives by means of Oliomio 50 hammer mill [Toscana Enologica Mori, Val di
Pesa (FI)]. Olive oils, stored in dark glass bottles, and wastes were kept in the refrigerator until
analysis.
2
.9
Extraction of phenolics from matrices
A previously reported method allowing isolation and identification of TyOle from the fruits of
Cassanese cv. was used (Bianco, Melchioni, Ramunno, Romeo, & Uccella, 2004). Each matrix (100
g, olive fruits were stoned first) was homogenized with a 1/1 MeOH-acetone (v/v) solution (100
mL) containing 0.5% sodium metabisulfite. The resulting mixture was sonicated, centrifuged and
the supernatant collected. The extraction procedure was repeated five more times. After the last
9

centrifugation, all the collected supernatants were concentrated in vacuo. The residue was than
dissolved in acetone and centrifuged once again to remove proteins and sugars. The supernatant was
concentrated in vacuo and the residue dissolved in acidic water (pH 2). A further centrifugation
allowed the separation of phenolic-containing phase from the aqueous phase. The crude sample was
dissolved in a n-hexane-CH CN mixture and stirred for 5 h under N .
3
2
2
.10 Preparation of standard solutions
The assay of HtyOle was achieved by an external standard calibration method: a standard stock
-1
solution was prepared by dissolving HtyOle in methanol (5.7 µg mL ). Aliquots of this solution
were further diluted with methanol/water/0.1% formic acid (70/30; v/v) to obtain calibration
-1
standards in a concentration range of 0.03-2 µg mL . Assay performance was checked by recovery
tests of spiked solutions (Table S2 in supporting information). Limit of quantitation (LOQ) and
limit of detection (LOD) were calculated from procedural blanks. More specifically, LOD was
defined as 10 times the standard deviation of the signal from reagent blanks, after correction for
sample weight and dilution; LOQ was defined as 3 times LOD.
2
.11 LC-MS/MS analysis of HtyOle levels in cell culture medium
The extraction of HtyOle was achieved using a SPE procedure already described in literature
Balvers, Verhoeckx, Plastina, Wortelboer, Meijerink, & Witkamp, 2010) with some modifications.
Cell culture medium was transferred to a 2-mL Eppendorf tube and phenylmethanesulfonyl fluoride
PMSF) was added ꢄo a final concenꢄꢂaꢄion of 100 μM. The samples were vortexed and
(
(
trifluoroacetic acid (TFA) was added to a final concentration of 0.1%. The tubes were vortexed and
centrifuged for 5 min at 14,000 rpm. Supernatants were subsequently loaded on activated SPE C8
columns, washed with 4 mL 20% v/v ACN in MQ containing 0.1% TFA, and eluted with 4 mL
8
0% v/v ACN in MQ containing 0.1% TFA, evaporated to dryness using a vacuum concentrator
(Eppendorf concentrator 5301). Dried extracts were stored at −20 °C prior to analysis or
1
0

immediately analyzed for hydroxytyrosyl oleate (HtyOle), with the LC-MS method described
above.
2
.12 Statistical analysis
All experiments were performed in duplicate in at least three independent experiments. Data from
all experiments using RAW264.7 macrophages are expressed as percentage of the LPS-treated
controls (set at 100 %). All data are presented as means ± standard deviation. Statistical differences
between treatments were evaluated by one-way ANOVA followed by Dunnett’s ꢁosꢄ ꢇoc ꢄesꢄ. P
values <0.05 (*) and <0.01 (**) were considered as statistically significant.
3
. Results
.1 Effect of Hty esters with different chain lengths on LPS-induced nitric oxide production
3
A series of hydroxytyrosyl esters characterized by short (C2), medium (C12) and long (C16 and
C18) acyl chains, were tested for their abilities to reduce the level of nitric oxide (NO) by
lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The molecular structures of the
compounds tested as well as their corresponding logP values, the partition coefficient between n-
octanol and water, used as an estimation of lipophilicity, are reported in Table 1. No effects of the
compounds on cell viability were found for the concentration ranges tested (Table S1 in supporting
information). Both C18 esters, namely hydroxytyrosyl stearate (HtySte) and hydroxytyrosyl oleate
(HtyOle), significantly reduced NO release in a concentration-dependent way. In particular, HtySte
and HtyOle (Figure 1, panels E and F, respectively) gave a reduction of approximately 40% at 2.5
µM and 50% at 5 µM, with HtyOle being already effective at 1 µM. By contrast, hydroxytyrosyl
acetate (HtyAce) and other Hty esters having a chain length shorter than eighteen carbons, as well
as the parent Hty, did not inhibit NO production in the concentration range tested (Figure 1). The
fatty acid precursors of HtySte and HtyOle, stearic acid (SA) and oleic acid (OA), respectively, did
not produce any suppression of NO production in the concentration range tested (Figure 1, panels G
1
1

and H, respectively). The analogue tyrosyl oleate (TyOle, Table 1) was not effective in reducing
NO up to 5 µM (data not shown).
3
.2
HtyOle reduces LPS-induced prostaglandin E (PGE ) production
2 2
Anti-inflammatory potential of HtyOle was further investigated by measuring its effects on
prostaglandin E (PGE ) production. Hydroxytyrosyl oleate was found to significantly inhibit the
2
2
release of PGE by LPS-stimulated RAW264.7 cells at all concentrations tested, and the maximum
2
of reduction (by approximately 75%) was found at 2.5 μM (Figure 2 A).
3
.3
HtyOle attenuates LPS-induced iNOS, COX-2, and IL-1β gene expression
In order to investigate whether inhibition elicited by HtyOle was regulated at the gene expression
level, we used quantitative RT-PCR to determine inducible nitric oxide synthase (iNOS) (Figure 2
B) and cyclooxygenase-2 (COX-2) (Figure 2 C) mRNA expression. A concentration of 5 µM of
HtyOle gave a significant reduction of approximately 40% and 55% of iNOS and COX-2 gene
expression, respectively. Moreover, HtyOle at 5 µM reduced of more than 50% interleukin-1β (IL-
1
3
3
β) mRNA expression, as shown in Figure 2 D.
.4
HPLC/MS analysis
.4.1 Method validation
To determine the best instrumental conditions for HytOle, a standard solution of the analyte was
introduced directly into the ion source of the mass spectrometer by direct infusion (FIA) at a flow
-1
rate of 10 L min . The instrumental parameters, optimized to maximize the reading, are reported
in the experimental section. The mass spectrum obtained for HtyOle in product ion scan (PIS) mode
-
-
showed a pseudo-molecular ion at m/z 417.1 ([M-H] ) and ionic fragments at m/z 281.2 ([oleate] ),
-
2
36.8, 187.2, 135.5 ([Hty-H-H O] ), 96.8 and 58.9 (Figure 3). The MRM transitions chosen and
2
monitored for the assay of HtyOle weꢂe 471.1 → 281.2 and 417.1 → 135.5. The first transition
1
2

(417.1 → 281.2) coꢂꢂesꢁonds ꢄo ꢄꢇe bꢂeakdown of ꢄꢇe deꢁꢂoꢄonaꢄed moleculaꢂ ion of HꢄꢍOle ꢄo give
rise to the product ion at m/z 281 (oleate anion), and was used for the quantitation. The other
transiꢄion (417.1 → 135.5) leading ꢄo ꢄꢇe ion m/ꢈ 135 was used ꢄo confiꢂm ꢄꢇe deꢄecꢄion of HꢄꢍOle.
Subsequently, to determine the best chromatographic conditions, the same standard solution was
injected into the HPLC-MS system through the chromatographic column. The optimal instrumental
parameters were as follows: source temperature, 400 °C; gas (1), 40 psi; gas (2), 30 psi; dwell time,
-1
4
00 ms; flow rate, 400 L min . HtyOle eluted at 12.0 min (Figure 4). Calibration curve,
constructed using a least-squares linear regression analysis, was linear with a correlation coefficient
2
-1
R =0.999 (Figure S1 in supporting info). Spiked solutions at 0.25 and 1 µg mL gave good
recoveries (92 and 106%, respectively, Table S2 in supporting info), and satisfactory precision
(relative standard deviation (RDS) 0.08 and 0.01%, respectively, Table S2 in supporting info) with
-1
LOQ and LOD values of 0.079 and 0.026 g g , respectively. All data were obtained from three
independent injections.
3
.4.2 Identification and quantification of HtyOle in olive oil by-products and in cell culture media
We first explored the possibility that HtyOle could be present in table olives and extra virgin olive
oil (EVOO). However, in our experiments, carried out on the fruits of Cassanese cultivar and
EVOO obtained from the same cultivar, HtyOle was below the detection limits. Next, we explored
the possibility that HtyOle could be present in olive oil by-products. Indeed, HtyOle was identified
in both pomace and olive mill waste water (OMWW), as reported in Figure 5. Its amount in the two
-1
matrices was quantified as 4.3 and 2.6 µg g , respectively. The amount of hydroxytyrosol (Hty) in
-1
the free form in the pomace residue and OMWW was quantified as 74 and 436 µg g , respectively.
Other compounds, including oleuropein and tyrosol, were also detected, in line with reported
literature (Ciriminna, Meneguzzo, Fidalgo, Ilharco & Pagliaro, 2016).
1
3

The quantification of HtyOle extracted from cell culture media under the experimental set-up was
assessed using LC-MS. HtyOle concentrations decreased with a 21% reduction at 2 h, 31%
reduction at 24 h, to reach a 90% decrease after 48 h.
4
.
Discussion
So far, hydroxytyrosyl oleate is mainly known as a semi-synthetic lipophilic derivative of
hydroxytyrosol. The compound has been shown before to possess antioxidant activities (Mateos et
al., 2008; Torres de Pinedo et al., 2007; Trujillo et al., 2006). Furthermore, esterification generates
a molecule with advantageous technological and pharmacokinetic properties compared to
hydroxytyrosol itself (Bouallagui et al., 2011; Tofani et al., 2010; Zhou et al., 2017). To the best of
our knowledge, we are the first showing that HtyOle is also found in by-products of olive oil
processing. In our study, levels of HtyOle were below detection limits in olive fruit. At the same
time, HtyOle has recently been identified in extra virgin olive oil (EVOO) and olive oil (OO) and its
-1
amount has been quantified as approximately 5 and 1 µg g , respectively (Lee et al., 2016).
Our results also demonstrate for the first time that HtyOle possesses potent anti-inflammatory
properties in vitro, acting on multiple targets. Of the Hty esters tested in the present study, we found
that only compounds with a chain length of eighteen carbons, namely HtySte and HtyOle, had the
ability to inhibit NO formation by LPS-stimulated RAW264.7 macrophages. By contrary, esters
with shorter chain length, as well as parent Hty, stearic and oleic acids, were ineffective. In line
with our findings, Hty and its acetate (HtyAce) were reported to inhibit NO at much higher
concentrations than those tested in our work (Aparicio-Soto et al., 2015). NO is a free radical
produced in the nitric oxide synthase (NOS)-catalyzed oxidative deamination of L-arginine. When
produced at low concentrations by constitutively expressed isoforms of the enzyme (nNOS or
NOS1 and eNOS or NOS3), NO plays a role as a signaling molecule in various physiological
processes. During inflammation, microbial products such as LPS, inflammatory cytokines or
interferon-γ prompt the up-regulation of inducible isoform of the enzyme (iNOS or NOS2) that
produces high concentrations of NO, acting as a key mediator in several inflammatory disorders
1
4

(Bogdan, 2001). The differences found among the different Hty esters in their ability to reduce NO
release can be rationalized on the basis of differences in lipophilicity, since this feature plays an
important role in cell-uptake and metabolic stability of compounds (Bayrasy et al., 2013).
Lipophilicity can be estimated by theoretical or experimental evaluation of logP, the partition
coefficient between n-octanol and water. In line with our hypothesis, logP values available in
literature of HtySte and HtyOle are the highest among those of the Hty esters investigated (Tofani et
al., 2010). The catechol moiety seems to be a crucial structural feature for the observed bioactivity,
since TyOle, bearing a tyrosyl group linked to oleic acid chain (Table 1), was found to be
ineffective in our model despite its structural relation with HtyOle and its high logP value. This is in
line with the usually higher bioactivity exerted by hydroxytyrosol and its derivatives with respect to
tyrosol and its conjugates (Zhou et al., 2017).
Inhibition of COX-2-mediated eicosanoid production has been reported, by us and others, to play a
major role in the reduction of pro-inflammatory mediators, including NO (Chang et al., 2006;
Meijerink et al., 2015; Wang et al., 2017). Reduction of eicosanoids is considered an effective
strategy for suppressing inflammation and, therefore, COX-2 represents an important therapeutic
target in many inflammatory disorders. Our data showed that HtyOle strongly inhibited the release
of PGE by LPS-stimulated RAW264.7 cells at all concentrations tested. The expression of iNOS
2
and COX-2 is known to be highly induced under inflammatory conditions and as such
predominantly regulated at the transcriptional level. Hydroxytyrosyl oleate caused a reduction of
mRNA expression of both iNOS and COX-2 and additionally of IL-1β. The latter is an inducible
cytokine that is not produced by healthy cells, and is implicated in pain, inflammation and
autoimmune disorders. Its crucial role in mediating NF-kB activity and COX-2 transcription under
inflammatory conditions has been demonstrated in vivo using IL-1β knock-out mice (Laflamme,
Lacroix, & Rivest, 1999; Ren & Torres, 2009). Our results suggest that inhibition elicited by
HtyOle could be regulated at the mRNA level. However, HtyOle affected mRNA expressions only
at the highest concentration, while it had a concentration-dependent effect on NO release and was
1
5

effective at all the concentrations tested on PGE production. Therefore, the anti-inflammatory
2
effects elicited by HtyOle may be only partly regulated at mRNA level and other mechanisms
maybe implicated. Our results indicate that the effects elicited by HtyOle are not due to its
hydrolysis to hydroxytyrosol and oleic acid, which could occur through lipase activity, since both
compounds were found to be ineffective in inhibiting NO. Our data on the stability of HtyOle in cell
culture medium support this hypothesis, since HtyOle was almost retained at 24h. Besides, during
the inflammatory cascade, the inflammatory markers measured here, all have their specific time
points of physiological activity. NO, for instance, is a late marker, known to peak around 48 hours
after LPS-induction. As such HtyOle-elicited effects are likely induced at initiation of the
inflammatory cascade. Our 24 hour data, showing a HtyOle induced reduction of PGE levels and
2
of iNOS, COX-2 and IL-1β gene exꢁꢂession suꢁꢁoꢂꢄ ꢄꢇis view.
In our experiments carried out on EVOO, we could not detect any HtyOle. This discrepancy with
the data reported by Lee and coworkers (Lee et al., 2016) could be due to many factors, including
differences in cultivars, ripening stage, geographical origin and production procedures. By contrast,
we successfully identified and quantified for the first time HtyOle in pomace residue and olive mill
waste water (OMWW), the by-products of olive oil production that represent a rich source of
hydroxytyrosol and other phenolic compounds. Although olive fruit is rich in phenolics, only 2% of
the total phenolic content of the olive fruit is found in the oil (Ciriminna, Meneguzzo, Fidalgo,
Ilharco & Pagliaro, 2016). The main fractions are found in the OMWW and in pomace
(approximately 53% and 45%, respectively). In the past decades, the research aiming at the
valorization of olive oil by-products and the market of the phenolics extracted there from and to be
used as active ingredients of several valued functional, cosmetic and nutraceutical products have
dramatically increased (Ciriminna et al., 2016). Recently, a method for the lipophilization of
hydroxytyrosol-enriched fractions from OMWW yielding HtyOle has been reported (Bernini et al.,
2
017). However, the authors did not investigate the presence of HtyOle in the starting material used.
Interestingly, we did not find HtyOle in intact olives, while its structural related analogue TyOle has
1
6

been isolated and identified in the fruits of Cassanese cultivar (Bianco et al., 2004). This could
suggest that, despite HtyOle precursors, namely oleic acid and Hty, are present in the olive fruits,
HtyOle is formed when the drupes are stoned through the activation of an appropriate esterase.
Important factors, including cultivars, ripening stage, geographical origin and production
procedures, probably play a role in the formation of HtyOle while its distribution among oil,
pomace and OMWW depends on the relative solubility of HtyOle or of its precursors. Another
possible explanation is that this compound is formed in pomace and in OMWW independently, as a
result of esterase activity coupling oleic acid and hydroxytyrosol present in high amounts in those
matrices. Indeed, the mechanism of formation of HtyOle and, in particular, how and when this
compound is formed remains unclear. In conclusion, in this work we have demonstrated that
hydroxytyrosyl oleate has in vitro anti-inflammatory properties and can be considered as a booster
of the parent hydroxytyrosol. Moreover, HtyOle occurs in olive oil by-products while it is not
detectable in intact olives. Taken together, these findings suggest that HtyOle represents a
significant form in which hydroxytyrosol occurs.
Conflict of interest
The authors declaꢂe no conflicꢄ of inꢄeꢂesꢄ.
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2
3

Table 1: Structures of hydroxytyrosol and (hydroxyl)tyrosyl esters
Acyl
a
Compound
Structure
LogP
chain
OH
OH
b
Hty
-
0.021
HO
b
HtyAce
HtyLau
HtyPal
HtySte
HtyOle
C2
n = 0
n = 10
n = 14
n = 16
0.961
6.275
8.400
9.462
b
C12:0
C16:0
C18:0
C18:1
OH
OH
O
n
O
b
b
b
R = OH 8.944
c
TyOle
C18:1
R = H
9.2
a
b
c
Partition coefficient between n-octanol and water. Tofani et al., 2010. PubChem BioAssay Tools
Figure captions
Figure 1. Concentration–response graphs of lipopolysaccharide (LPS)-stimulated nitric oxide
reduction induced by hydroxytyrosol (Hty, panel A), a series of its esters (B-F), stearic acid (SA,
panel G) and oleic acid (OA, panel H) by RAW264.7 macrophages. Compounds were tested in a
concentration range of 0.5-5 µM. RAW264.7 macrophages were seeded at a density of 250 000
-1
-1
cells mL and incubated for 48 h with (or without) LPS (0.5 µg mL ) in the presence (or absence)
of compounds. Data are expressed as percentage, where LPS stimulation (containing vehicle) was
set at 100 %. Values are means of three separate experiments (each done in duplicate), ± standard
deviation. Mean values were significantly different from the control (*P< 0.05, **P< 0.01).
2
4

Figure 2. Effects of hydroxytyrosyloleate (HtyOle) on lipopolysaccharide (LPS)-stimulated
prostaglandin E (PGE ) production (A), and mRNA expression of inducible nitric oxide synthase
2
2
(iNOS) (B), cyclooxygenase-2 (COX-2) (C) and interleukin-1β (IL-1β) (D) at 24 h by RAW264.7
-1
macrophages. RAW264.7 cells were seeded at a density of 500 000 cells mL and incubated for 24
h with (or without) LPS in the presence (or absence) of compounds. Total RNA was isolated and
reverse transcribed to give complementary DNA before performing quantitative real-time PCR.
iNOS, IL1β and COX-2 fold increase normalized to RPS27A2 is expressed as percentage, where
LPS stimulation (containing vehicle) was set at 100 %. Values are means of three separate
experiments performed in duplicate, ± standard deviation. Mean values were significantly different
from that of the control (*P< 0.05, **P< 0.01, ***P< 0.001).
Figure 3. HtyOle mass spectrum obtained in negative product ion scan (PIS) mode at -25 eV
collision energy.
Figure 4. HPLC-MS/MS chromatogram of HtyOle. The transitions monitored in negative multiple
reaction monitoring (MRM) mode were 471.1 → 281.2 and 417.1 → 135.5.
Figure 5. Multiple reaction monitoring (MRM) chromatograms of: blank solution (A); extract of
olive mill waste water (OMWW, B) and of olive pomace (C).
2
5

A
C
E
B
D
F
**
**
**
**
**
G
H
2
6

B
**
**
*
***
***
C
D
*
**
2
7

[
HtyOle - H] -
[
Hty – H – H
2
O] -
2
8

2
9