Oleocanthalic Acid, a Chemical Marker of Olive Oil Aging and

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Bioactive Constituents, Metabolites, and Functions

OLEOCANTHALIC ACID, A CHEMICAL MARKER OF OLIVE OIL AGING AND EXPOSURE TO HIGH STORAGE TEMPERATURE WITH POTENTIAL NEUROPROTECTIVE ACTIVITY ANNIA TSOLAKOU, PANAGIOTIS DIAMANTAKOS, ILIANA KALABOKI, ANTONIO MENA-BRAVO, FELICIANO PRIEGO-CAPOTE, IHAB ABDALLAH, AMAL KADDOUMI, ELENI MELLIOU, and PROKOPIOS MAGIATIS J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00561 • Publication Date (Web): 14 Jun 2018 Downloaded from http://pubs.acs.org on June 17, 2018

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Journal of Agricultural and Food Chemistry

OLEOCANTHALIC ACID, A CHEMICAL

1

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MARKER OF OLIVE OIL AGING AND EXPOSURE

3

TO HIGH STORAGE TEMPERATURE WITH

4

POTENTIAL NEUROPROTECTIVE ACTIVITY ANNIA TSOLAKOU†, PANAGIOTIS DIAMANTAKOS†, ILIANA

5 6

KALABOKI†, ANTONIO MENA-BRAVO‡, FELICIANO PRIEGO-CAPOTE‡,

7

IHAB M ABDALLAH§, AMAL KADDOUMI§, ELENI MELLIOU†, PROKOPIOS

8

MAGIATIS†,*

9 10

Department of Pharmacognosy and Natural Products Chemistry, Faculty of

11

Pharmacy, National and Kapodistrian University of Athens, Panepistimiopolis

12

Zografou, 15771 Athens, Greece, Department of Analytical Chemistry, University of

13

Cordoba, Spain, Department of Drug Discovery and Development, Harrison School

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of Pharmacy, Auburn University, 720 S. Donahue Dr. Auburn, AL 36849, USA

15 16 17

* Corresponding author. (Tel.: +30 210 7274052. E-mail: [email protected])

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University of Athens

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University of Cordoba

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§

Auburn University

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Abstract

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Investigation of olive oils stored for a period of 24 months under appropriate

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conditions (25oC, dark place, air-tight container) led to the identification of a new

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major phenolic ingredient, which was named oleocanthalic acid. The structure of the

26

new compound was elucidated using 1D and 2D NMR in combination with MS/MS.

27

The new compound is an oxidation product of oleocanthal and is found in fresh oils in

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very low concentrations. The concentration of oleocanthalic acid increased with

29

storage time while oleocanthal concentration decreased. A similar increase of the

30

oleocanthalic acid/oleocanthal ratio was achieved after exposure of olive oil to 60oC

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for 14 days. Although the presence of an oxidized derivative of decarboxymethylated

32

ligstroside aglycon had been reported, it is the first time that its structure is

33

characterized. The isolated compound could induce the expression of amyloid-β

34

major transport proteins as well as tight junctions expressed at the blood-brain barrier

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suggesting oleocanthalic acid could be beneficial against Alzheimer’s disease.

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Keywords: Olive oil, phenolics, stability, NMR, LC-MS/MS, oleocanthal, oleocanthalic acid, Alzheimer’s Disease

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INTRODUCTION

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Olive oil, since 2012 in the European Union, is officially recognized as a food that

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under specific conditions can bear a health claim related to protection of blood lipids

42

from oxidative stress.1 The factor determining if an olive oil can have on its label the

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above health claim is the concentration of hydroxytyrosol and its derivatives

44

including all the secoiridoid-phenolics coming from oleuropein and ligstroside. A

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series of compounds like oleocanthal (1), oleacein, oleuropein and ligstroside

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aglycons

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oleuropeindials, ligstrodials, oloekoronal, oleomissional) have been recognized as

48

belonging to the class of phenolics that are measured in order to support the health

49

claim.

dialdehydic,

monoaldehydic

and

enolic

forms

(known

also

as

50

We have developed2-5 an analytical method based on 1D qNMR spectroscopy that

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is able to measure the concentration of all the above ingredients in one experiment

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and since 2012 we have analyzed more than 4000 samples with the above method.2-7

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A critical question raised by most olive oil producers is what happens to the major

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phenolic ingredients during storage and which parameters play the most important

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role and consequently which are the levels of phenolic compounds in olive oil that

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could guarantee that an olive oil will qualify for the health claim during all its shelf-

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life.

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There is a number of previous works that have tried to investigate the chemical

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composition and product stability (or shelf-life) parameters.8-11 It is known for over a

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decade that storing extra virgin olive oil (EVOO) at room temperature leads to

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chemical transformations of the complex phenolic compounds, through hydrolytic or

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oxidation mechanisms.12 Previous stability studies of EVOO13,14 have indicated the

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presence of oxidized products coming from the known phenolic derivatives, such as

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oleocanthal and oleacein, however their precise structure has not been elucidated as

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well as the factors that determine their formation.

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To provide more answers to the above questions, we selected randomly 29 samples

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of olive oil from different varieties, geographic origin and harvest time and we

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monitored the levels of the phenolic compounds related to the health claim for up to

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24 months under three different storage temperatures in air tight containers. The

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complete results of this study are a subject of a separate publication but interestingly

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during the monitoring period, we noticed the appearance or increase of a new

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compound. This fact directed our investigation towards its isolation and structure

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elucidation by 1D, 2D NMR and MS/MS. In addition, we investigated the main

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factors related to its formation and especially the role of temperature and time of olive

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oil storage as well as the presence or not of oxygen in the headspace, aiming to show

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that this compound can be potentially used as a new marker of aging and heat

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exposure of olive oil during storage.

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Moreover, based on the previously described in vitro and in vivo protective activity

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of oleocanthal on Alzheimer’s disease15-19 we investigated the activity of the new

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compound on the expression of amyloid-β (Aβ) transport proteins, mainly P-

81

glycoprotein (P-gp) and low-density lipoprotein receptor-related protein 1 (LRP1), as

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well as tight junctions.

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MATERIALS AND METHODS

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Chemicals and standards

85

All solvents used for extraction and isolation were of analytical grade and

86

purchased from Merck (Darmstad, Germany). Acetonitrile and formic acid used for

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analysis were of LC–MS grade from Scharlab (Barcelona, Spain). Deionized water

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(18 MΩ cm) from a Millipore Milli-Q water purification system (Bedford, MA, USA)

89

was used to prepare the aqueous mobile phase. Oleocanthal and oleacein were

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isolated as previously described using normal phase column chromatography and

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preparative TLC performed on an EVOO extract obtained after liquid-liquid

92

extraction.2

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Instruments

94

1

H and

13

C NMR spectra were recorded at 400 MHz using a NMR spectrometer

95

(Bruker DRX 400). LC–MS/MS analysis was performed on a system consisting of an

96

Agilent (Palo Alto, CA, USA) 1200 Series liquid chromatograph coupled to an

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Agilent 6540 UHD Accurate-Mass QTOF hybrid mass spectrometer equipped with a

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dual electrospray ionization (ESI) source.

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Olive oil samples origin and storage conditions

100

For this study we used 29 EVOO samples of different total phenolic concentration,

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which were monitored periodically, regarding their phenolic content, for a period up

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to two years. The samples were stored in dark glass bottles, with 5% headspace. The

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bottles were placed either inside a dark cabinet, with average temperature 25oC, or in

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a refrigerator at 4oC or at -18 oC and three replicates of each sample were analyzed

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every 6 months. Information about origin, and variety of the analyzed oils are

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provided in Supplementary Table 1.

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NMR Quantitative analysis of phenolic compounds in olive oil

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Direct measurements of the phenolic compounds were made using the protocol of

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Karkoula et al.2 More specifically, olive oil (5.0 g) was mixed with cyclohexane (20

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mL) and acetonitrile (25 mL). The mixture was homogenized using a vortex mixer for

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30 s and centrifuged at 4000 rpm for 5 min. A part of the acetonitrile phase (25 mL)

112

was collected, mixed with 1.0 mL of a syringaldehyde internal standard solution (0.5

113

mg/mL) in acetonitrile, and evaporated under reduced pressure using a rotary

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evaporator (Buchi, Flawil, Switzerland). The residue of the above procedure was

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dissolved in CDCl3 (750 µL) and an accurately measured volume of the solution (550

116

µL) was transferred to a 5 mm NMR tube. Typically, 32 scans were collected into

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32K data points over a spectral width of 0−16 ppm with a relaxation delay of 1 s and

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an acquisition time of 1.7 s. Prior to Fourier transformation (FT), an exponential

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weighting factor corresponding to a line broadening of 0.3 Hz was applied. The

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spectra were phased corrected and integrated automatically using TOPSPIN.

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Contained oleocanthal was quantified by integrating the peak of the aldehydic proton

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at 9.23 ppm.

123

Oleocanthalic acid (2) isolation

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An EVOO sample (400 g), two years after its production, was extracted with 4:5

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cyclohexane:acetonitrile (4 L) and the acetonitrile phase was evaporated under

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reduced pressure using a rotary evaporator. The extract was then submitted to column

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chromatography using normal phase silica gel. The elution was made for the first 20

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fractions with 95:5 cyclohexane:EtOAc, the next 20 with the same solvents 90:10, the

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next with ratio 80:20, 70:30, 60:40 and 50:50. The final 20 fractions were eluted with

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100% EtOAc. The fractions 82-125 containing the oleocanthalic acid (80 mg) were

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identified with spotting on normal phase silica gel TLC plates, developed with 100%

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EtOAc. (Rf values: Oleocanthalic acid = 0.15, Oleacein = 0.56, Oleocanthal = 0.67, in

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cyclohexane/EtOAc 40:60)

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LC–QTOF MS/MS confirmatory analysis

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Samples of pure oleocanthal, oleacein and oleocanthalic acid were reconstituted in

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acetonitrile to obtain a concentration around 500 µg mL-1 and, then, aliquots were

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diluted in acetonitrile up to 10 µg mL-1. These aliquots were injected (5 µL) without

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any additional pretreatment into the LC–MS/MS system. This system allows the

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simultaneous spraying of a mass reference solution that enabled continuous

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calibration of detected m/z ratios. The analytical column was a reversed phase C18

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Mediterranea Sea (50 × 0.46 mm i.d., 3 µm particle size) from Teknokroma

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(Barcelona, Spain). A pre-column, 40 × 3.0 mm i.d., from Phenomenex (Torrance,

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CA, USA) was inserted prior to the analytical column for preservation. The mobile

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phases were: mobile phase A, 0.1% formic acid in water, and B, 0.1% formic acid in

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acetonitrile. The gradient program, at 0.8 mL min−1 constant flow rate, was as follows:

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initially, the mobile phase was 80:20 A/B, and the first gradient was from 20% to

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45% phase B in 3 min, followed by a ramp of 9 min from 45 to 70% phase B; finally,

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a new gradient was applied in 3 min to reach 100% phase B. The total analysis time

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was 15 min, and 5 additional min were required to re-establish and equilibrate the

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initial conditions. The column temperature was constant at 20 ºC during the analysis.

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The operating conditions were as follows: gas temperature, 300 °C; drying gas,

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nitrogen at 10 L min−1; nebulizer pressure, 50 psi; sheath gas temperature, 360 °C;

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sheath gas flow, nitrogen at 12 L min−1; capillary voltage, 3000 V in negative

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ionization mode; nozzle voltage, 1000 V; skimmer, 65 V; octopole radiofrequency

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voltage, 750 V; fragmentor voltage, 130 V. Data were acquired in centroid mode in

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high resolution (2 GHz). Full scan was carried out at 1 spectrum s−1 within the m/z

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range 50–1200 with subsequent activation of the three most intense precursor ions per

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scan (only single or double charged ions were allowed) by tandem mass spectrometry

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(MS/MS) using a collision energy of 15 eV. MS/MS scanning was carried out at 1

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spectrum s−1 within the m/z range 50–1200.

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MassHunter Workstation software (version 7.00 Qualitative Analysis, Agilent

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Technologies, Santa Clara, CA, USA) was used for processing the raw LC–QTOF

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data files. Targeted extraction of molecular features was carried out by searching the

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molecular formula of oleocanthal, oleacein and oleocanthalic acid. The isotopic

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distribution of valid molecular features should be defined by two or more ions with a

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peak spacing tolerance of m/z 0.0025 plus 10 ppm in terms of mass accuracy. As mass

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acquisition was in negative ionization mode precursor ions were mainly [M–H]– ions,

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but other adducts (HCOO– and Cl–) were also taken into account together with the

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neutral loss by dehydration. The three target phenols were tentatively confirmed by

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identifying the structure of representative product ions detected in MS/MS spectra.

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Mass accuracy errors in MS and MS/MS acquisition were set at 5 and 10 ppm,

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respectively.

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Accelerated EVOO ageing

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Pure oleocanthal (50 mg) was dissolved in EVOO (100 mL) with zero phenolic

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concentration and placed in an oven at 60 oC, in an open vial exposed to atmospheric

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O2 for 14 days. The samples were analyzed using the above described qNMR method

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every 2 days.

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Cell culture

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The immortalized mouse brain endothelial cell line, bEnd3 was obtained from

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ATCC (Manassas, VA). bEnd3 cells, passage 25–35, were cultured in DMEM

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supplemented with 10% fetal bovine serum (FBS), penicillin G (100 IU/ml),

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streptomycin (100 µg/ml), 1% w/v nonessential amino acids, and glutamine 2 mM.

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Cultures were maintained in a humidified atmosphere (5%CO2/95% air) at 37°C and

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media was changed every other day.

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Western blot analysis

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bEnd3 cells were seeded in 10 mm dishes for western blot analysis and allowed to

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grow to 70% confluency before treatment with oleocanthalic acid in a humidified

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atmosphere (5%CO2/95% air) at 37°C. On day of treatment, cells were treated with 0,

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1, 2.5, 5 and 10 µM oleocanthalic acid, dissolved in DMSO, for 72 h. At the end of

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treatment period, cells were lysed with RIPA buffer containing complete mammalian

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protease inhibitors followed by centrifugation at 21 000g for 1 h at 4°C. The

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supernatant was collected as protein extract and stored at −80 °C until the time of

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analysis. Protein concentrations were determined by the BCA method. For Western

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blot analysis, 25 µg of protein was resolved on TGX stain-free acrylamide 10% gels

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(Bio-Rad, Hercules, CA) and electro-transferred onto a 0.45 µm polyvinylidene

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difluoride (PVDF) membrane. Membranes were blocked with 2% BSA and incubated

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overnight with monoclonal antibodies for P-gp (C-219; BioLegend, San Diego, CA),

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LRP1 (Abcam, Cambridge, MA), claudin-5 (Clone 4C3C2; Invitrogen, Carlsbad,

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CA), and ZO1 (Invitrogen). Proteins were normalized to total protein. For detection,

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the membranes were washed free of primary antibodies and incubated with HRP-

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labeled secondary IgG anti-mouse antibody for P-gp, claudin-5, and ZO1, and anti-

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rabbit antibody for LRP1 (all from Santa Cruz Biotechnology, Dallas, TX). The bands

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were visualized using a Pierce chemiluminescence detection kit (Thermo Scientific;

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Waltham, MA). Quantitative analysis of the immunoreactive bands was performed

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using ChemiDoc V3 (Bio-Rad), and band intensity was measured by densitometric

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analysis.

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RESULTS AND DISCUSSION

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It is known for over a decade that storing EVOO in room temperature leads to

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chemical decomposition of the complex phenolic compounds, through hydrolytic or

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oxidation mechanisms.12 The percentage of phenolic loss within 21 months of storage

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has been reported to be between 43-73% in different EVOO samples.12 This

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observation has led to the investigation of the reasons for the transformations taking

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place and the impact of the storage conditions. For example, storage container

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studies,19,20 pointed the tin can followed by glass vessels as the best storage medium

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for olive oil. In other stability of EVOO studies13,14 there are indications of the

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presence of oxidized products, coming from known phenolic derivatives, such as

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oleocanthal and oleacein.

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Investigation of olive oils stored for long periods up to 24 months under

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appropriate conditions (25 oC, dark place, air tight container) led to the discovery of a

220

new major phenolic ingredient, which was named oleocanthalic acid (2). The

221

structure of the new compound was elucidated using 1D and 2D NMR in combination

222

with MS/MS. The new compound is an oxidation product of oleocanthal (1) and was

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detected in fresh oils in very low concentrations. The concentration of oleocanthalic

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acid, as demonstrated below, increased with storage time while oleocanthal

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concentration decreased and in several cases after 24 months it was the major

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phenolic ingredient found in the olive oil.

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Structure elucidation of oleocanthalic acid (2)

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The 1H-NMR spectrum of oleocanthalic acid (2) (Figure 1) showed significant

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similarities with the spectrum of oleocanthal (1) but also showed two major

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differences. The first one was that 2 presented only one aldehydic signal at 9.25 ppm,

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slightly deshielded in comparison with 1 and corresponding to the conjugated

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aldehyde of position 1 while the second aldehyde group at position 3 was absent

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(Figure 2). In addition, the peaks corresponding to protons H-4 had a significant

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change in their chemical shift indicating that the main point of differentiation was the

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functional group at position 3. Thorough investigation with COSY, HMQC and

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HMBC experiments led to the assignment of each proton and carbon peaks and

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mainly showed that the aldehyde group at position 3 of oleocanthal had been replaced

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by another functional group with a carbonyl observed at 177 ppm. More specifically

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H-5 showed two J3 correlations with two carbonyls: one at 172.3 ppm and one at 177

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ppm instead of 199 ppm that was in the case of 1. The chemical shift of the new

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carbon signal was compatible with a carboxyl group that was confirmed by the

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increase of the molecular weight by 16 amu corresponding to the addition of an

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oxygen atom. The stereochemistry of the double bond at positions 8 and 9 was found

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to be E as in the case of oleocanthal based on the NOESY correlation between the H-9

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and the aldehyde H-1. The absolute configuration at C-5 was the same as in 1 as both

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compounds showed the same optical rotation. Based on the above observations it can

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be confirmed that the previously reported14,21-24 but undescribed oxidized derivative of

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oleocanthal is the carboxylic derivative for which we propose the name oleocanthalic

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acid. The complete description of the proton and carbon signals and the comparison

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with the corresponding chemical shifts of oleocanthal are reported in Table 1.

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Confirmatory analysis of purified phenols by LC–QTOF MS/MS

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To confirm the presence of oleocanthalic acid as well as oleocanthal and oleacein

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in olive oil samples included in the present study, the pure compounds were analyzed

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by LC–QTOF MS/MS using the method described in the Experimental Section.

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Oleocanthal and oleacein are characterized by the dialdehydic structure with the

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difference of esterification to tyrosol or hydroxytyrosol. The hypothesis here is that

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oleocanthal is partially oxidized to form oleocanthalic acid. Therefore, the molecular

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formula of oleocanthal, C17H20O5, is slightly altered by addition of an oxygen atom to

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give C17H20O6 for oleocanthalic acid, which also fits the molecular formula of

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oleacein. Chromatographically, oleacein and oleocanthalic acid are properly

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separated, which avoids the possibility of coelution. Extracted ion chromatograms

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(EIC) for [M–H]– ions from purified fractions of oleacein with m/z 319.1181 (1.9

263

ppm), oleocanthal with m/z 303.1242 (1.3 ppm) and oleocanthalic acid with m/z

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319.1181 (1.9 ppm) showed three peaks at 6.6, 7.8 and 8.3 min (Figure 3),

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respectively. The pH of the mobile phases (around pH 3.0 by use of formic acid as

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ionization agent) ensures that oleocanthalic acid is not as carboxylate ion, which

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would explain its elution after oleocanthal due to a higher retention with the reverse

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phase. The presence of a unique peak in the three purified fractions proved the

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efficiency of the isolation process.

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MS/MS fragmentation with TOF detection enabled to obtain high-resolution

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information to proceed with identification and confirmation of the candidate

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compounds. Fragmentation of the precursor ion m/z 303.1238 (oleocanthal) generated

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several representative product ions as a result of the activation of the molecule at the

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weakest bond. Thus, the ion detected at m/z 137.0597 (C8H9O2) corresponded to the

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tyrosol moiety by b-phenyl ester fragmentation via McLafferty rearrangement. Two

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other fragments were detected at m/z 139.0390 (C7H7O3) and m/z 123.0445 (C7H7O2),

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which were assigned to the dialdehydic moiety, released after separation of the

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tyrosol, and its main fragment, respectively, as shows Figure 4a. The fifth ion at m/z

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59.0141 (C2H3O2) fits the acetoxy fragment associated to the ester bound.

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Fragmentation of oleacein is quite similar to that presented for oleocanthal as

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Figure 4b illustrates. The fragmentation of oleacein led to two main ion products at

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m/z 139.0403 (C7H7O3) and at m/z 59.0144 (C2H3O2) corresponding to the

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dialdehydic moiety and the acetoxy fragment released after separation of

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hydroxytyrosol by analogy to oleocanthal. Besides, one ion at m/z 123.0450 was

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clearly identified as the main fragment of hydroxytyrosol when this is activated by

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MS/MS, which allowed confirming the identity of oleacein.

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Despite oleacein and oleocanthalic acid have the same molecular formula, the

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fragmentation mechanism was completely different as can be elucidated in Figure 4c.

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The oxidation of one of the aldehydic groups alters the activation process, which is

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clearly simplified. In fact, the MS/MS spectrum of oleocanthalic acid is clearly

291

dominated by two main product ions at 199.0611 (C9H11O5) and 111.0086 (C5H3O3)

292

m/z, while other minor fragments are also detected. Partial oxidation of the

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dialdehydic structure alters completely the fragmentation mechanism by tandem mass

294

spectrometry. This profile is indicative of a simple fragmentation that leads to these

295

two main product ions without any other chemical alteration. The product ion m/z

296

199.0611 was formed by fragmentation of oleocanthalic acid through the ester bond,

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particularly, this ion fit the deacetoxy monoaldehydic acid moiety resulting after

298

oxidation of the dialdehydic structure. The product ion m/z 111.0086 is a sub-

299

fragment of the previous product ion that gives the most intense signal of the MS/MS

300

spectrum. This ion is formed by release of the C4H6O fragment and loss of the

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hydroxyl group (Figure 5).

302

Monitoring of phenolics concentration by qNMR

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During the “cabinet” storage, our results using qNMR confirmed previous works,

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with a loss of 42.8±16.1% of oleocanthal content of the samples after 18 (measured

305

for 29 samples) or up to 70% after 24 months (measured for 5 samples)

306

(Supplementary Table 2). The studied samples contained oleocanthal with initial

307

concentration ranging from 0 up to 469 mg/Kg. Supplementary figures 1-4 show the

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evolution of selected samples, with the reduction of oleocanthal and the increase of

309

the oleocanthalic acid. Figure 6a shows the increase of the oleocanthalic

310

acid/oleocanthal ratio during storage for up to 24 months.

311

The samples stored at 4oC or -18oC did not show any increase of the oleocanthalic

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acid/oleocanthal ratio even at 24 months showing that the formation of oleocanthalic

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acid in mainly dependent by the temperature of storage.

314

Accelerated aging and oleocanthal oxidation

315

A similar increase of the oleocanthalic acid/oleocanthal ratio, mimicking the two-

316

years storage period, was achieved after exposure of olive oil to 60oC for 14 days in

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open vials showing that oleocanthalic acid/oleocanthal ratio can be potentially used as

318

a marker of exposure to high temperatures and aging of olive oil.

319

More specifically, we dissolved pure oleocanthal to a sample of EVOO with

320

naturally zero phenolic content and PV=8 meq O2/Kg , stored it for 14 days at 60o C

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and measured its oleocanthal and oleocanthalic acid concentration every two days.

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The result of this heat treatment was the oxidation of oleocanthal and the formation of

323

its oxidized product, the oleocanthalic acid. A percentage of the contained oleocanthal

324

was oxidized to oleocanthalic acid, as shown in Figure 6b, while the oleocanthalic

325

acid concentration was increasing steadily. These results were obtained by

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quantitative 1H-NMR spectra analysis and integration of the aldehydic proton peaks

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as shown in Figure 7. As it can be seen in Figure 7, the whole region of the aldehydic

328

protons remains the same except the appearance of the peak of oleocanthalic acid and

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the decrease of the peaks of oleocanthal. It is clear that under these specific conditions

330

there is no observable formation of other related oxidized compounds. The same

331

happens in the whole 1H-NMR spectrum. The only new signals are those belonging to

332

oleocanthalic acid. However, we cannot exclude the formation of other oxidation

333

products that are not extracted with acetonitrile (or other polar solvents) from olive oil

334

(e.g highly lipophilic oxidized derivatives). It should be noted that oxidation is only

335

one of the decomposition procedures for oleocanthal. The other major one is the

336

hydrolysis reaction leading from oleocanthal to free tyrosol. This reaction was also

337

observed in most cases of the 29 studied olive oil samples during the 24 months

338

storage. Oleocanthalic acid is also susceptible to hydrolysis of the ester bond exactly

339

in the same way as oleocanthal. For this reason, the oleocanthalic acid/oleocanthal

340

ratio is independent of the hydrolytic procedure but dependent only from the oxidative

341

pathway.

342

Heat exposure and oxygen

343

In order to investigate the role of several factors leading to the formation of

344

oleocanthalic acid we set up a series of experiments including heating in the presence

345

or not of atmospheric oxygen. In addition, to examine if there is a link between olive

346

oil peroxide value and the formation of oleocanthalic acid, we applied steady heat

347

without the presence of oxygen, in samples with different peroxide levels.

348

In a first experiment, pure oleocanthal diluted in zero phenolics olive oil with PV

349

=8 meq O2/Kg was exposed to 60oC for 20 days either in an open vial or in an air tight

350

vial without headspace. As shown in Supplementary Figure 5, the absence of oxygen

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led to a much smaller amount of oleocanthalic acid in comparison with the open vial,

352

showing the crucial role of oxygen. In the air tight closed vial only 25% of

353

oleocanthal was oxidized to oleocanthalic acid while in the open vial 80% was

354

oxidized.

355

Interestingly, heat exposure of oleocanthal diluted in organic solvent failed to give

356

oleocanthalic acid. More specifically, when pure oleocanthal was diluted in

357

dichloromethane and heated at 60oC for 14 days in half – filled, closed vials, the

358

formation of oleocanthalic acid could not be observed even in traces. The above

359

procedure showed that the oxidation of oleocanthal to oleocanthalic acid is not a

360

direct reaction with atmospheric oxygen but requires the presence of other

361

constituents of olive oil which act as intermediate oxidizing agents. The headspace

362

oxygen when combined with increased heat exposure increases the formation of lipid

363

peroxides that in turn play a role in the oxidation of oleocanthal. It was confirmed by

364

heating air tight closed bottles, containing olive oil with low peroxides count. Even in

365

that case, we monitored the oxidation of a small proportion of oleocanthal to

366

oleocanthalic acid.

367

The role of peroxides was confirmed in a third experiment, where pure oleocanthal

368

was added in two olive oil samples, in air tight vials; one with low peroxides (PV = 8

369

meq O2/Kg) and one with high peroxides (PV = 30 meq O2/Kg) and heated at 60oC

370

for 14 days. The results as shown in Figure 8 showed that the oxidation of oleocanthal

371

to oleocanthalic acid was accelerated by the presence of lipid peroxides. Interestingly,

372

the peroxide value was reduced in parallel to oleocanthalic acid formation. There

373

seems to be a combination of available atmospheric oxygen and lipid peroxides

374

present in olive oil samples, which accelerates the formation of oleocanthalic acid. It

375

should be mentioned that the two oils used in this experiment were carefully selected

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from the same variety having zero phenolic content but the first one was fresh with

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low PV and the second one was one year old and had naturally developed a high PV

378

value. In this way we were able to study the behavior of oleocanthal when added to

379

those oils which presented high similarity but significant difference in the PV values.

380

The fact that the oleocanthalic acid formation was accelerated in the high PV oil and

381

that the PV was reduced in parallel with the oleocanthalic acid formation provides

382

strong evidence that the lipid peroxides are implicated in the reaction mechanism. The

383

aldehyde oxidation to carboxylic acid with hydrogen peroxide or hydroperoxides is a

384

well-studied reaction with a well known mechanism.25,26 However, it has not been

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studied in the case of lipid or alkyl peroxides found in olive oil.

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Potential activity against Alzheimer’s disease: in vitro studies

387

Recently, we have reported oleocanthal as a potential molecule against

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Alzheimer’s disease (AD).15-19 In vitro and in vivo studies have demonstrated

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oleocanthal to increase Aβ clearance across the blood-brain barrier (BBB) caused by

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its effect on up-regulating Aβ major transport proteins P-gp and LRP1 expressed in

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the endothelial cells of the BBB. Besides, oleocanthal induced the tight junction

392

claudin-5, which could be responsible for BBB increased intactness in AD mice

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brains. The isolation of oleocanthalic acid in the current study prompted us to

394

investigate its activity on the induction of P-gp, LRP1 and tight junctions’

395

expressions. As shown in Figures 9a-d, and consistent with our findings with 1,15-19 2

396

significantly increased the expression of P-gp, LRP1, ZO1 and claudin-5, however to

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a variable degree with the effect is more pronounced at the lower range of examined

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concentrations. Oleocanthalic acid significantly induced P-gp by 48 and 15% at 1.0

399

and 2.5 µM, respectively, but has no effect at higher concentrations (Fig. 9a). LRP1,

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on the other hand, while the effect was only significant at 2.5 µM, oleocanthalic acid

401

increased LRP1 expression approximately by 50% in the concentration range 1-10

402

µM (Fig. 9b).

403

increased ZO1 expression by 47 and 75%, and claudin-5 by 11% at 1 and 2.5 µM,

404

respectively (Fig. 9b&c). This finding suggests that, at low concentrations,

405

oleocanthalic acid could be beneficial against AD and Aβ related pathology.

406

Concerning the structure activity relationships for the effect on up-regulation of Aβ

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major transport proteins in vitro it seems that the aldehyde group of oleocanthal at

408

position 3 is not critical and can be replaced by other groups such as the carboxyl

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group. While further confirmatory in vitro and in vivo studies in AD models are

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necessary, this interesting bioactivity of oleocanthalic acid suggests that an older oil

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could maintain a part of its protective activity against AD despite oleocanthal loss.

For its effect on tight junctions, oleocanthalic acid significantly

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FUNDING SOURCES

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The authors would like to thank the European Regional Development fund for

414

financial support through the project “ARISTOIL”

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REFERENCES

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oleocanthal and oleacein levels in olive oil by quantitative 1H NMR. Establishment of

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Diamantakos, P; Velkou, A.; Killday, K.B.; Gimisis, Th., Melliou, E.;

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Magiatis, P. Oleokoronal and oleomissional: new major phenolic ingredients of extra

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Magiatis, P.; Melliou, E.; Killday, B. A new ultra rapid screening method for

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olive oil health claim evaluation using selective pulse NMR spectroscopy. In

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Ben Mansour, A.; Gargouri, B.; Melliou, E.; Magiatis, P.; Bouaziz, M. Oil

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Phenolic Compounds during Long-Term Storage (18 Months) at Temperatures of 5-

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Brkić Bubola, K.; Koprivnjak, O.; Sladonja, B.; Belobrajić, I. Influence of

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Croatian virgin olive oils. Grasas Aceites 2014, 65, e034.

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11. Krichene, D.; .Allalout, A.; Mancebo-Campos, V.; Salvador, M.D.; Zarrouk,

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M.; Fregapane, G. Stability of virgin olive oil and behaviour of its natural antioxidants

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under medium temperature accelerated storage conditions. Food Chem. 2010, 121,

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12. Gómez-Alonso, S.; Mancebo-Campos, V.; Salvador, M.D.; Fregapane, G.

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Evolution of major and minor components and oxidation indices of virgin olive oil

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during 21 months storage at room temperature. Food Chem. 2007, 100, 36-42.

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13. Kotsiou, K.; Tasioula-Margari, M. Monitoring the phenolic compounds of Greek extra-virgin olive oils during storage. Food Chem. 2016, 200, 255-262.

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14. Rios, J.J.; Gil, M.J.; Gutierrez-Rosales, F. Solid-phase extraction gas

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chromatography-ion trap-mass spectrometry qualitative method for evaluation of

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ligstroside aglycons and their oxidation products. J Chromatogr. A 2005, 1093, 167-

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15. Abuznait, A.H.; Qosa, H.; Busnena, B.A.; El Sayed, K.A.; Kaddoumi, A.

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Olive-oil-derived oleocanthal enhances β-amyloid clearance as a potential

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neuroprotective mechanism against Alzheimer's disease: in vitro and in vivo studies.

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16. Qosa, H.; Batarseh, Y.S.; Mohyeldin, M.M.; El Sayed, K.A.; Keller. J.N.;

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17. Batarseh, Y.S.; Mohamed, L.A.; Al Rihani, S.B.; Mousa, Y.M.; Siddique,

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A.B.; El Sayed, K.A.; Kaddoumi, A. Oleocanthal ameliorates amyloid-β oligomers'

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toxicity on astrocytes and neuronal cells: In vitro studies. Neurosci. 2017, 352, 204-

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18. Batarseh, Y.S.; Kaddoumi, A. Oleocanthal-rich extra-virgin olive oil enhances

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donepezil effect by reducing amyloid-β load and related toxicity in a mouse model of

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Alzheimer’s disease. J. Nutr.Biochem. in press.

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19. Méndez; A.I.; Falqué, E. Effect of storage time and container type on the quality of extra-virgin olive oil. Food Control 2007, 18, 521-529.

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20. Dabbou, S.; Gharbi, I.; Dabbou, S.; Brahmi, F.; Nakbi, A.; Hammami, M.

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Impact of packaging material and storage time on olive oil quality. Afr. J. Biotechn.

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22. Tasioula-Margari,

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identification of phenolic compounds in virgin olive oil by HPLC-DAD and HPLC-

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MS. Antioxidants 2015, 4, 548-562.

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23. Armaforte, E.; Mancebo-Campos, V.; Bendini, A.; Salvador, M.D.;

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Fregapane, G.; Cerretani, L. Retention effects of oxidized polyphenols during

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2401–2406.

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24. Rovellini, P.; Cortesi, N. Liquid chromatography – mass spectrometry in the

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their oxidized products. Riv. Ital. Sost. Grasse, 2002, 77, 1–14.

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25. Sato, Κ.; Hyodo, M.; Takagi, J.; Aoki, M.; Noyori, R. Hydrogen peroxide

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26. Shaikh, T.M.; Hong, F.E. Efficient method for the oxidation of aldehydes and

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Figure captions

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Figure 1. 1H-NMR spectrum of the isolated oleocanthalic acid.

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Figure 2. Comparison between the aldehydic and aromatic protons region of

508

oleocanthal (up) and oleocanthalic acid (bottom).

509

Figure 3. Extracted ion chromatograms (EIC) for [M–H]– ions from purified fractions

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of oleacein (m/z 319.1181, 1.1 ppm) (a), oleocanthalic acid (m/z 319.1181, 0.8

511

ppm)(b) and oleocanthal (m/z 303.1238, 1.0 ppm) (c).

512

Figure 4. MS/MS fragmentation spectra provided by LC–QTOF analysis of:

513

oleocanthal (a), oleacein, (b), oleocanthalic acid (c).

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Figure 5. Fragmentation scheme of oleocanthalic acid to confirm the detected

515

compound in aged oils.

516

Figure 6. Comparison of the oleocanthalic acid/oleocanthal ratio change with time

517

with (a) normal aging at 25 oC (top), and (b) “accelerated” ageing process in open

518

vials at 60 oC (bottom). The results are expressed as molar ratio measured by the

519

integration in the 1H-NMR spectrum of the aldehyde peaks in comparison with

520

internal standard. In the normal aging the values are the mean values of the 29 studied

521

samples.

522

Figure 7. 1H- NMR spectra of the accelerated ageing process, measured every two

523

days starting from day 2 of the experiment (bottom) until day 14 (top), showing the

524

oxidation of oleocanthal to oleocanthalic acid, in olive oil with PV=8 heated at 60oC

525

in open vial.

526

Figure 8. Changes in oleocanthal to oleocanthalic acid ratio, caused by different

527

peroxide value of the olive oil sample that was used for the accelerated ageing

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experiment for 14 days at 60oC in closed air-tight vials. PV = 30 (up). PV = 8

529

(bottom).

530

Figure 9 a-d. Representative western blot and densitometry analysis of P-gp (a),

531

LRP1 (b), ZO1 (c), and claudin-5 (d) expressions in bEnd3 cells treated for 72 h with

532

increasing concentrations of oleocanathalic acid. Data are presented as mean ± SD of

533

3 independent experiments. *P