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

2

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

14

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])

18

†

University of Athens

19

‡

University of Cordoba

20

§

Auburn University

21

ACS Paragon Plus Environment


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Abstract

23

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

24

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

25

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

28

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

31

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

35

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

36 37

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

38


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INTRODUCTION

40

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

41

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

43

above health claim is the concentration of hydroxytyrosol and its derivatives

44

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

45

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

46

aglycons

47

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

51

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

52

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

53

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

54

phenolic ingredients during storage and which parameters play the most important

55

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

56

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

57

life.

58

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

59

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

60

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

61

chemical transformations of the complex phenolic compounds, through hydrolytic or

62

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



63

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

64

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

65

well as the factors that determine their formation.

66

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

67

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

68

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

69

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

70

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

71

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

72

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

73

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

74

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

75

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

76

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

77

exposure of olive oil during storage.

78

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

79

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

80

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

82

well as tight junctions.

83

MATERIALS AND METHODS

84

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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87

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

88

(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

90

isolated as previously described using normal phase column chromatography and

91

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

92

extraction.2

93

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

97

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

98

dual electrospray ionization (ESI) source.

99

Olive oil samples origin and storage conditions

100

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

101

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

102

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

103

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

104

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

105

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

106

provided in Supplementary Table 1.

107

NMR Quantitative analysis of phenolic compounds in olive oil

108

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

109

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

110

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



111

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

114

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

115

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

117

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

118

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

119

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

120

spectra were phased corrected and integrated automatically using TOPSPIN.

121

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

122

at 9.23 ppm.

123

Oleocanthalic acid (2) isolation

124

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

125

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

126

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

127

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

128

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

129

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

130

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

131

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

132

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

133

cyclohexane/EtOAc 40:60)

134

LC–QTOF MS/MS confirmatory analysis


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135

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

136

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

137

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

138

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

139

simultaneous spraying of a mass reference solution that enabled continuous

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

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

152

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

153

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

154

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

155

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

156

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

157

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

158

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



159

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

160

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

161

MassHunter Workstation software (version 7.00 Qualitative Analysis, Agilent

162

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

163

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

164

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

165

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

166

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

167

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

168

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

169

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

170

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

171

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

172

respectively.

173

Accelerated EVOO ageing

174

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

175

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

176

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

177

every 2 days.

178

Cell culture

179

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

180

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

181

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

182

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

184

media was changed every other day.

185

Western blot analysis

186

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

187

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

188

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

189

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

190

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

191

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

192

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

193

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

194

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

195

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

196

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

197

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

198

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

199

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

200

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

201

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

202

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

203

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

204

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

205

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

206

analysis.



207

RESULTS AND DISCUSSION

208

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

209

chemical decomposition of the complex phenolic compounds, through hydrolytic or

210

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

211

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

212

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

213

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

214

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

215

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

216

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

217

oleocanthal and oleacein.

218

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

219

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

223

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

224

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

225

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

226

phenolic ingredient found in the olive oil.

227

Structure elucidation of oleocanthalic acid (2)

228

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

229

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

230

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


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231

slightly deshielded in comparison with 1 and corresponding to the conjugated

232

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

233

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

234

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

235

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

236

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

237

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

238

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

239

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

240

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

241

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

242

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

243

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

244

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

245

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

246

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

247

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

248

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

249

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

250

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

251

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

252

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

253

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

254

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



255

Oleocanthal and oleacein are characterized by the dialdehydic structure with the

256

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

257

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

258

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

259

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

260

oleacein. Chromatographically, oleacein and oleocanthalic acid are properly

261

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

262

(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

264

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

265

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

266

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

267

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

268

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

269

efficiency of the isolation process.

270

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

271

information to proceed with identification and confirmation of the candidate

272

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

273

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

274

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

275

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

276

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

277

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

278

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

279

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


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280

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

281

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

282

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

283

dialdehydic moiety and the acetoxy fragment released after separation of

284

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

285

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

286

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

287

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

288

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

289

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

290

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

293

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,

297

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

301

hydroxyl group (Figure 5).

302

Monitoring of phenolics concentration by qNMR



303

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

304

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

308

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

312

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

313

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

317

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

321

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

322

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

326

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


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327

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

329

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



351

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


Page 16 of 36

Page 17 of 36


376

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

377

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

385

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

386

Potential activity against Alzheimer’s disease: in vitro studies

387

Recently, we have reported oleocanthal as a potential molecule against

388

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

389

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

390

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

391

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

393

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

397

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

398

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,



400

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

407

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

409

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

410

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

411

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

For its effect on tight junctions, oleocanthalic acid significantly

412

FUNDING SOURCES

413

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

414

financial support through the project “ARISTOIL”

415


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Page 19 of 36


416

REFERENCES

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Karkoula, E.; Skantzari, A.; Melliou, E.; Magiatis, P. Quantitative

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

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

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ACS Chem Neurosci. 2013, 4, 973-982.

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

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

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

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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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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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505

Figure captions

506

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

507

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

510

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).

514

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



528

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

Oleocanthalic Acid, a Chemical Marker of Olive Oil Aging and

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