Comparative study of different cocoa (Theobroma cacao L.) clones in terms of their phytoprostanes and phytofurans contents

Accepted Manuscript Comparative study of different cocoa (Theobroma cacao L.) clones in terms of their phytoprostanes and phytofurans contents Daniel León-Perez, Sonia Medina, Julián Londoño-Londoño, Marina CanoLamadrid, Ángel Carbonell-Barrachina, Thierry Durand, Alexandre Guy, Camille Oger, Jean-Marie Galano, Federico Ferreres, Claudio JiménezCartagena, José Restrepo-Osorno, Ángel Gil-Izquierdo PII: DOI: Reference:

S0308-8146(18)32176-9 https://doi.org/10.1016/j.foodchem.2018.12.072 FOCH 24031

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

20 February 2018 11 December 2018 14 December 2018

Please cite this article as: León-Perez, D., Medina, S., Londoño-Londoño, J., Cano-Lamadrid, M., CarbonellBarrachina, A., Durand, T., Guy, A., Oger, C., Galano, J-M., Ferreres, F., Jiménez-Cartagena, C., Restrepo-Osorno, J., Gil-Izquierdo, A., Comparative study of different cocoa (Theobroma cacao L.) clones in terms of their phytoprostanes and phytofurans contents, Food Chemistry (2018), doi: https://doi.org/10.1016/j.foodchem. 2018.12.072

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

Comparative study of different cocoa (Theobroma cacao L.) clones in terms of their

2

phytoprostanes and phytofurans contents

3 4

Daniel León-Pereza,b, Sonia Medinaa,c,*, Julián Londoño-Londoñob, Marina Cano-

5

Lamadridd, Ángel Carbonell-Barrachinad, Thierry Durande, Alexandre Guye, Camille Ogere,

6

Jean-Marie Galanoe, Federico Ferreresa, Claudio Jiménez-Cartagenab, José Restrepo-

7

Osornof, Ángel Gil-Izquierdo a,*

8

aResearch

9

Science and Technology, CEBAS (CSIC), P.O. Box 164, 30100 Campus University

Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food

10

Espinardo, Murcia, Spain

11

bInstituto

12

cFaculty

13

Caldas, Antioquia, Colombia.

14

dDepartment

15

Beniel, km 3.2, 03312 Orihuela, Alicante, Spain.

16

eInstitut

17

Montpellier, ENSCM, Montpellier, France.

18

f

19

*Corresponding authors at: Quality, Safety and Bioactivity of Plant Foods Group,

20

Department of Food Science and Technology, CEBAS-CSIC, P.O. Box 164, 30100

21

Espinardo, Murcia, Spain.

de Ciencia y Tecnología Alimentaria - INTAL, Itagüi, Antioquia, Colombia.

of Engineering, Food Engineering Program, Corporación Universitaria Lasallista,

of Agro-Food Technology, Miguel Hernández University of Elche, Carretera

des Biomolécules Max Mousseron (IBMM), UMR 5247, CNRS, Université de

National Federation of Cocoa – FEDECACAO, Colombia.

1

22

*E-mails

23

[email protected]

of

the

corresponding

authors:

[email protected],

2

24

Abstract

25

Cocoa has been widely discussed as a bioactive food rich in sensory stimulation and

26

health benefits. However, no information has been provided concerning phytoprostanes

27

(PhytoPs) and phytofurans (PhytoFs) in cocoa. These compounds are of interest because

28

they play a role in the regulation of immune function. The present study included 31 cocoa

29

clones. The PhytoPs and PhytoFs were quantified by UHPLC-QqQ-MS/MS. The total

30

PhytoPs and PhytoFs contents ranged from 221.46 to 1589.83 ng g-1 and from 1.18 to

31

13.13 ng g-1, respectively. The profiles of the PhytoPs and PhytoFs identified in the cocoa

32

beans showed significant differences among the clones analysed. The results indicate that

33

dry fermented cocoa beans are rich in PhytoPs and PhytoFs, which may represent an

34

additional benefit of the consumption of foods derived from cocoa.

35

Highlights

36



First report of the occurrence of phytoprostanes and phytofurans in cocoa beans.

37



The profiles of phytoprostanes and phytofurans varied according to the clone.

38



Content of PUFAs were related to the amounts of phytoprostanes and phytofurans.

39

Keywords

40

Mass spectrometry; phytofurans; phytoprostanes; oxidative stress; cocoa beans; PUFAs

41

Abbreviations

42

BHA, Butylated hydroxyanisole; ALA, α-linolenic acid; FAMEs, fatty acids methyl esters;

43

GC-MS, Gas chromatography–mass spectrometry; IsoPs, isoprostanes; LOD, limit of

44

detection; LOQ, limit of quantification; MRM, multiple reaction monitoring; Nfr2, nuclear

45

factor erythroid-2; OS, oxidative stress; PCA, principal components analysis; PhytoFs,

46

phytofurans; PhytoPs, phytoprostanes; PUFA, polyunsaturated fatty acids; PVDF,

47

polyvinylidene fluoride; ROS, reactive oxygen species; RSD, relative standard deviation;

48

SD, standard deviation; SPE, solid-phase extraction; UHPLC-ESI-QqQ-MS/MS, ultra-high 3

49

performance

50

spectrometry.

liquid

chromatography-electrospray

ionization-triple

quadrupole

mass

51 52

1. Introduction

53

The interest in the consumption of foods that provide health benefits and possess

54

attractive sensorial characteristics has always stimulated research, much of which has

55

focused on bioactive compounds that promote health or provide sensory stimulation

56

(Marseglia, Sforza, Faccini, Bencivenni, Palla, & Caligiani, 2014). Accordingly, cocoa and

57

its derivatives have been well studied with this approach. Cocoa has been recognized as

58

an important source of polyphenols (Demidchik, 2015), flavonoids (Patras, Milev,

59

Vrancken, & Kuhnert, 2014), catechins, procyanidins (Cádiz-Gurrea, Lozano-Sanchez,

60

Contreras-Gámez, Legeai-Mallet, Fernández-Arroyo, & Segura-Carretero, 2014), amino

61

acids (Marseglia, Palla, & Caligiani, 2014), polypeptides, and oligopeptides (Marseglia,

62

Sforza, Faccini, Bencivenni, Palla, & Caligiani, 2014). To this list may include compounds

63

that add sensory properties to the product, known as “single-origin” (Torres-Moreno,

64

Tarrega, Costell, & Blanch, 2012), which are of interest to both researchers and

65

consumers. Transversally, these studies may repeatedly denote homogeneity, but the

66

increased understanding of food chemistry that they have provided has underpinned new

67

guidelines and motivated consumption. In addition, nowadays, the focus on food

68

characteristics related to geographic origin is often prioritized. Furthermore, the study of

69

compounds or metabolites not reported so far (“unknown”) is facilitated by the use of

70

analytical tools with higher resolution and sensitivity.

71

According to these perspectives, the study of novel compounds like phytoprostanes

72

(PhytoPs) and phytofurans (PhytoFs) is of interest because it is known that they play a role

73

in the regulation of immune function (Barden, Croft, Durand, Guy, Mueller, & Mori, 2009) 4

74

and display anti-inflammatory and apoptosis-inducing activities, among others, similar to

75

other prostanoids (Durand, Bultel-Poncé, Guy, El Fangour, Rossi, & Galano, 2011).

76

These metabolites are prostaglandin–like compounds (plant oxylipins) derived from the

77

lipid peroxidation of polyunsaturated fatty acids (PUFAs), such as α-linolenic acid (ALA,

78

C18:3 n-3), via a nonenzymatic free-radical-catalyzed pathway (Jahn, Galano, & Durand,

79

2010). In the biosynthesis of IsoPs which are oxidative stress (OS) markers in mammals -,

80

a carbon radical undergoes a 5-exo-trig cyclization (Baldwin rules (Baldwin, 1976; Baldwin,

81

Thomas, Kruse, & Silberman, 1977)) to form the cyclopentane ring of isofurans (IsoFs)

82

(Jahn, Galano, & Durand, 2008; Morrow, Hill, Burk, Nammour, Badr, & Roberts, 1990).

83

Meanwhile, the PhytoPs - like PhytoFs - are oxylipins which may be formed from ALA, but

84

they require a high oxygen tension (higher than 21%) to attack the same carbon radical as

85

PhytoPs and form compounds containing a tetrahydrofuran ring. So, the IsoFs are

86

characterized by a tetrahydrofuran ring and two side chains. These bioactive compounds

87

act as endogenous mediators capable of protecting cells from damage under various

88

conditions related to OS (Loeffler, Berger, Guy, Durand, Bringmann, Dreyer, et al., 2005).

89

In the scientific literature, PhytoPs and PhytoFs have been reported in numerous natural

90

or unprocessed products, as also in processed products (as shown Supplementary Table

91

S1), but, so far, these compounds have not been reported in cocoa beans. For this reason,

92

the objective of the present work was to assess, for the first time, the concentrations of

93

PhytoPs and PhytoFs in cocoa bean from different clones. Hence, a comparative study of

94

the content of PhytoPs and PhytoFs by UHPLC-MS/MS was developed in cocoa beans

95

obtained from the same farm, the same period of flowering and ripeness of the fruits. In

96

addition, the same process of fermentation and drying was assured. Only the variety of the

97

genetic material of the cocoas was varied. The profile of these metabolites was studied in

98

conjunction with the fatty acids levels. The value of these matrices as a source of these 5

99

largely unknown oxylipins needs to be addressed in order to plan nutritional trials devoted

100

to shed some light on their biological activity.

101

2. Materials and methods

102

2.1. Chemicals and reagents

103

The PhytoPs - 9-F1t-PhytoP, 9-epi-9-F1t-PhytoP, ent-16-F1t-PhytoP, ent-16-epi-16-F1t-

104

PhytoP, 9-D1t-PhytoP, 9-epi-9-D1t-PhytoP, ent-16-B1-PhytoP, 16-B1-PhytoP, ent-9-L1-

105

PhytoP, and 9-L1-PhytoP - were synthesized according to our published procedures (El

106

Fangour, Guy, Despres, Vidal, Rossi, & Durand, 2004; El Fangour, Guy, Vidal, Rossi, &

107

Durand, 2005; Pinot, Guy, Fournial, Balas, Rossi, & Durand, 2008). The PhytoFs - ent-16-

108

(RS)-9-epi-ST-Δ14-10-PhytoF, ent-9-(RS)-12-epi-ST-Δ10-13-PhytoF, ent-9-(S)-12-epi-ST-

109

Δ10-13-PhytoF,

110

PhytoF - were synthesized according to our previous reports (Cuyamendous, Leung,

111

Durand, Lee, Oger, & Galano, 2015) (Figure 1). Hexane, sodium hydroxide (NaOH),

112

anhydrous sodium sulfate, and water for LC-MS were obtained from Panreac (Castellar

113

Del Vallés, Barcelona, Spain). Fatty acid methyl ester (FAME) Mix Supelco GLC-50, GLC-

114

100, CLA isomers (c9 t11 CLA and t10 c12 CLA), nonanoic acid (C9:0), heptadecanoic

115

acid

116

tris(hydroxymethyl)methane)

117

(Steinheim, Germany). Methylene chloride was purchased from Labscan Ltd (Dublin,

118

Ireland). All LC–MS grade solvents, methanol, and acetonitrile were purchased from J.T.

119

Baker (Phillipsburg, New Jersey, USA). The SPE cartridges (Strata X-AW 33µ, 100 mg/3

120

mL) were acquired from Phenomenex (Torrance, CA, USA), and the Sep-Pak® classic

121

C18 cartridges from Waters (Milford, MA).

122

(C17:0),

ent-9-(R)-12-epi-ST-Δ10-13-PhytoF,

boron

trifluoride were

(BF3),

and

obtained

and

Bis–Tris from

ent-16-(RS)-13-epi-ST-Δ14-9-

(bis(2-hydroxyethyl)

Sigma–Aldrich

Chemie

aminoGmbH

2.2. Geographical origin of the samples

6

123

Cocoa (Theobromoa cacao) trees were cultivated, and their cobs fermented and extracted,

124

at the “Cannes” (Maceo) experimental farm, located 132 km from Medellín, Department of

125

Antioquia, Colombia (Supplementary Material). Cannes is situated 1000 m above sea level

126

(6°31'16.62"N and -74°49'25.57"W), the annual average temperature is 23°C, the annual

127

precipitation is 2700 mm, and the mean relative humidity is 80%. The study included 31

128

different clones of cocoa beans grown on the experimental farm, which were classified

129

according to their denomination on the farm (three criollos, three criollos modernos, four

130

forasteros, two forasteros albinos, ten regionals, and nine trinitarios) (Table 1). For the

131

harvest of samples, the period of cocoa grown known as “traviesa” (crossbred) was

132

selected (from May to June of 2016); this was after the first flowering of the year, but

133

before the main harvest. The fruit ripeness was determined by an expert farmer; healthy

134

and representative cobs were selected from each clone batch grown on the experimental

135

farm. The extraction of the cocoa beans was done the same day for all the samples. The

136

beans were separated inside plastic meshes and labelled for subsequent identification.

137

The meshes with the beans were arranged in fermentation drawers. The fermentation

138

process was developed in a place covered in the same area of cocoa crop and was

139

performed at room temperature spontaneously and in wooden drawers designed in three

140

columns by three rows with a capacity of 150 kg per drawer. The three central horizontal

141

drawers were used, and labelled as drawer 1, drawer 2 and drawer 3. The monitoring and

142

evolution was developed under the guidance of a farmer and a FEDECACAO (National

143

Federation of Cocoa-Colombia) technician with expertise in the cocoa fermentation

144

process. The fermentation was performed with beans that were healthy and free of

145

impurities such as leaves or insects, for six days, guaranteeing the death of the embryo in

146

each bean.

7

147

A natural drying process was performed on wooden platforms in a marquee dryer. The

148

meshes were opened and the beans were carefully extended, ensuring that the clones

149

were not mixed. The drying was completed on the seventh day, as indicated by the

150

cracking of the beans and the, development of a kidney shape and brown colour. Samples

151

(18 cocoa beans in triplicate) were processed and analysed up to one month after

152

collection on the farm. The samples were stored at -80°C before being extracted for further

153

analyses.

154

2.3. Processing of cocoa beans

155

The PhytoPs and PhytoFs from the cocoa beans samples were isolated using a dispersive

156

liquid–liquid extraction followed by an SPE, according to a procedure described by Collado

157

and colleagues (Collado-González, Medina, et al., 2015) with modifications. Each sample

158

was ground in a coffee mill to obtain a semi-fine cocoa powder (< 1000 µm),

159

homogenized, and weighed (about 2 g), before being added to a mortar together with 10

160

mL of BHA dissolved in methanol (1 g L-1). Then, the homogenized sample was

161

transferred to a centrifuge tube and centrifuged for 10 min at 4°C and 2000g. The

162

supernatant was filtered in a C18 Sep-Pak (previously activated with 10 mL of methanol

163

and 10 mL of water). Exactly 1 mL of the extract was diluted with 10 mL of hexane, 2 mL

164

of methanol, and 2 mL of BIS–TRIS buffer (0.02 M, HCl, pH = 7); between each addition

165

the mixture was stirred vigorously. The emulsion obtained was subjected to SPE using a

166

Strata X-AW cartridge (100 mg/3 mL); prior to this, the cartridge was conditioned and

167

equilibrated with 2 mL of hexane, 2 mL of methanol, and 2 mL of milliQ water. After

168

removal of the non-subject compounds, the cartridge was washed with 2 mL of hexane, 2

169

mL of milliQ water, 2 mL of a solution of methanol/milliQ water (1/3, v/v), and 2 mL of

170

acetonitrile. The target compounds were eluted with 1 mL of methanol and dried using a

171

SpeedVac concentrator (Savant SPD121P, Thermo Scientific, MA, USA). The dry extracts 8

172

were reconstituted with 200 μL of mobile phase (solvent A, a mixture of milliQ water/0.01%

173

(v/v) acetic acid, and solvent B, a solution of methanol/0.01% (v/v) acetic acid, in a

174

proportion of 90:10 v/v, respectively). The samples were filtered through 0.45-μm PVDF

175

filters (Millipore, MA, USA) and 20 μL of each sample were analysed. The samples were

176

analysed in triplicate.

177

Stock solutions of PhytoFs and PhytoPs were prepared in methanol/water (50:50, v/v), to

178

facilitate the ionization process in the mass spectrometer, at a concentration of 1000 nM

179

for each compound and stored in Eppendorf tubes at −80 °C. Twelve successive dilutions

180

were prepared for the calibration curve (with R2 value higher than 0.99).

181

2.4. Measurements

182

2.4.1. Phytofurans and phytoprostanes analyses

183

The identification and quantification of PhytoFs and PhytoPs were performed using a

184

UHPLC coupled to a 6460 triple quadrupole-MS/MS (Agilent Technologies, Waldbronn,

185

Germany), with a BEH C18 analytical column (2.1 × 50 mm, 1.7 μm) (Waters, Milford, MA).

186

The column temperature was 6 °C. The mobile phases consisted of water/acetic acid

187

(99.99:0.01, v/v) (A) and methanol/acetic acid (99.99:0.01, v/v) (B) and 20 µL of the diluted

188

extract was injected at a flow of 0.2 mL min-1. The following gradient program was used:

189

60% B at 0 min, 62% B at 2 min, 62.5% B at 4 min, 65% B at 8 min, and 60% B at 8.01

190

min. The acquisition time was 8.01 min for each sample, with a post-run of 1.5 min for

191

column equilibration. Analysis was performed in the negative mode by multiple reaction

192

monitoring (MRM). The MS parameters fragmentor (ion optics: capillary exit voltage),

193

collision energy, and preferential MRM transition of the corresponding analytes were

194

optimized for each analyte (Table 2). The source parameters used were gas flow: 8 L min-

195

1;

gas temperature: 325 °C; sheath gas temperature: 350 °C; jetstream gas flow: 12 L min-

196

1;

nebulizer: 30 psi; capillary voltage: 3000 V; and nozzle voltage: 1750 V. Data acquisition 9

197

and processing were performed using MassHunter software version B.04.00 (Agilent

198

Technologies). The elution gradient and mass spectrometric parameters were as

199

described previously (Collado-González, Medina, et al., 2015). The quantification of

200

PhytoPs and PhytoFs detected in cocoa clones was performed using authentic standard

201

according to standard curve freshly prepared as mentioned in the previous section 2.3.

202

2.4.2. Fatty acid profile

203

The analysis of fatty acids was performed by methylating these compounds in situ,

204

following the procedure described by Taber, Morrow, & Jackson Roberts (1997) and later

205

improved by Trigueros & Sendra (2015). The cocoa beans were ground, homogenized,

206

and weighed. A sample of approximately 40 mg was transferred into a test tube. 60 µL of

207

C17:0 in n-hexane solution as internal standard (-20 mg mL-1 solution in HPLC grade n-

208

hexane) was added. Then, 100 mL of methylene chloride and 1 mL of 0.5 M NaOH in

209

methanol were added, and the tubes were heated in a water bath at 90 ºC for 10 min. One

210

milliliter of BF3 in methanol was added and the mixture was left at room temperature (25

211

ºC) for 30 min. One milliliter of distilled water and 600 mL of hexane were added, and then

212

the fatty acids methyl esters (FAMEs) were extracted by vigorous shaking for about 1 min.

213

Following centrifugation, aliquots were dried with anhydrous sodium sulfate and the top

214

layer was transferred into a vial flushed with nitrogen, which was stored at -20 ºC until

215

analysis. The FAMEs were analysed using GC-MS with a SupraWax-280 column, 100%

216

polyethylene glycol (Teknokroma S. Co. Ltd., 165 Barcelona, Spain; 30 m × 0.25 mm ×

217

0.25 µm film thickness). The analyses were carried out using helium as carrier gas at a

218

flow rate of 1.1 mL min-1 and a program as follows: initial temperature 80 ºC, hold for 2

219

min; rate of increase of 8.0 ºC min-1 to 160 ºC; rate of 4 ºC min-1 from 160 to 220 ºC, hold

220

for 13 min; and rate of 10 ºC min-1 from 220 to 260 ºC, hold for 6 min. The injector and

10

221

detector temperatures were held at 230 and 260 ºC, respectively; 0.5 µL of the extract was

222

injected. The analyses were run in duplicate.

223

2.4.3. Statistical analysis

224

To determine the effect of each factor on each individual PhytoP, PhytoF, and PUFA, an

225

analysis of variance (ANOVA, p ⩽ 0.05), Pearson's correlation analysis of the different

226

variables studied and multiple range test (Tukey’s test) were carried out using the SPSS

227

program, version 23.0 (SPSS Inc., Chicago, IL, USA). Mean values were compared by the

228

LSD (Least Significant Difference) test when significant interactions between factors were

229

found.

230

3. Results and discussion

231

Oxidative stress is a complex chemical and physiological phenomenon that accompanies

232

practically all the biotic and abiotic stresses in plants (Hasanuzzaman, Hossain, da Silva,

233

& Fujita, 2012). It develops as a result of the overproduction and accumulation of reactive

234

oxygen species (ROS) (Demidchik, 2015).

235

In this study, we selected the Cannes experimental farm because it is located in an area of

236

cocoa production in the region of Antioquia, Colombia. This farm has a rich clone

237

plantation; the cocoa trees are the same age and there are many different clones. In

238

addition, the abiotic stresses (such as drought, salinity, cold, high light/UV-B, heat, air

239

pollution, heavy metals, mechanical wounding, and nutritional deficiency (Nakabayashi &

240

Saito, 2015)) influencing the beans were considered homogeneous for all samples, as was

241

the biotic stress experienced during fermentation. These variables, although influencing

242

the formation of the target metabolites, were assumed to be constant and, although they

243

may have exerted some influence on the results obtained, the impact of the genetic

244

resource (clones) was assumed to be far superior. However, as far as we know, this is the

245

first report where different classes of PhytoPs have been detected and quantified in 11

246

fermented and dried cocoa beans of different clones; and as a result it was determined

247

statistically different PhytoP and PhytoF levels were found among the different cocoa

248

beans clones (Table 3 and 4). They were independent of a classification like criollo (and

249

criollo moderno), regional, forastero, or trinitario cocoa. The results of statistical tests are

250

shown in Supplementary Table S2.

251

PhytoPs and PhytoFs were detected in all the cocoa beans samples analysed, except for

252

some specific samples in which they were below the LOD (Tables 3 and 4) and with

253

respect to recovery percentages were in the range of 85 and 123 % for all the analytes

254

(PhytoPs and PhytoFs) according to our recent published validated method (Domínguez-

255

Perles et al., 2018). The identification of these free PhytoPs was confirmed according to

256

their pseudomolecular ions, the characteristic MS/MS fragmentation product ions

257

(quantification and confirmation transitions), and their corresponding retention times

258

(Collado-González, Medina, et al., 2015). PhytoPs and PhytoFs are formed non-

259

enzymatically as regio- and stereoisomeric mixtures, so the analytical conditions employed

260

in our study did not allow enantiomeric separation; therefore, they are quantified in the

261

cocoa beans samples as racemic mixtures of PhytoPs and PhytoFs (Figure 1). It is

262

important to note that ent-16-epi-16-F1t-PhytoP + ent-16-F1t-PhytoP are epimers at position

263

C16; ent-9-(RS)-12-epi-ST-Δ10-13-PhytoF + ent-9-(S)-12-epi-ST-Δ10-13-PhytoF + ent-9-

264

(R)-12-epi-ST-Δ10-13-PhytoF are a racemic mixture at position C9 (50% 9R and 50% 9S);

265

and 16-B1t-PhytoP + ent-16-B1t-PhytoP and 9-L1-PhytoP + ent-9-L1-PhytoP represent

266

racemic mixtures that are not separable in our chromatographic conditions (Collado-

267

González, Medina, et al., 2015).

268

The total PhytoPs and PhytoFs amounts in the cocoa beans ranged from 221.46 to

269

1589.83 and 1.18 to 13.13 ng g-1, respectively, being Criollo-2 and Regional-8 the clones

270

with the highest concentration of PhytoPs and PhytoFs, respectively (Table 3 and 4). 12

271

Moreover, the greatest proportion of the PhytoPs profile of the samples was represented

272

by the series F1t-, while the series B1-, L1-, and D1t- were detected at lower levels,

273

corresponding to ranges of 209.46 to 1558.44, 3.04 to 46.35, 2.11 to 26.34, and 1.82 to

274

21.79 ng g-1, respectively. These results are consistent with those reported previously for

275

Passiflora edulis Sims shell, macroalgae, and red wine (Barbosa, et al., 2015; Marhuenda,

276

et al., 2015; Medina, et al., 2017). In relation to PhytoFs, there are few studies of these

277

compounds. Cuyamendous, Leung, Durand, Lee, Oger, & Galano (2015) described for the

278

first time the synthesis of PhytoFs and quantified the compound ent-16(RS)-13-epi-ST-Δ14-

279

9-PhytoF in nuts (pine nuts and walnuts) and seeds (flaxseeds and chia seeds). Yonny, et

280

al. (2016) studied PhytoFs in melon leaves, but they did not find ent-16(RS)-13-epi-ST-Δ14-

281

9-PhytoF; conversely, in cocoa beans, this PhytoF was the most abundant (Table 3).

282

Accordingly, the profile of the individual series differed significantly among the samples of

283

the cocoa clones analysed. Furthermore, differences in genotype could also affect the

284

apparent PhytoPs content and series in plant samples (Carrasco-Del Amor, et al., 2016;

285

Collado-González, Medina, et al., 2015; Collado-González, Moriana, Girón, Corell,

286

Medina, Durand, et al., 2015). On this matter, a study of different types of nuts (walnut,

287

macadamia, and pecan) was reported by Carrasco-Del Amor and colleagues (Carrasco-

288

Del Amor, et al., 2017). This work showed that the PhytoP profile varied according to the

289

nut type, and possibly according to the given genotype within specie. This could explain

290

the results in our study - where the qualitative and quantitative PhytoP profiles varied

291

greatly among the cocoa clones, but they were also affected by factors such as the

292

growing conditions and chemical composition with respect to ALA, OA, LA and other lipids,

293

and tocopherols. These compounds varied tremendously within the species, according to

294

environmental variability, geographical origin, temperature, and irrigation (Zhu, Taylor,

295

Sommer, Wilkinson, & Wirthensohn, 2015). Further studies are needed to investigate if the

13

296

cultivation of the same cocoa clones in different areas has a real effect on the production

297

of specific PhytoPs and PhytoFs.

298

The importance of PhytoPs and PhytoFs is related not only to plant physiology but

299

also to their biological effects in humans, since they are part of our diet and their presence

300

has already been reported in human biofluids (Barden, Croft, Durand, Guy, Mueller, &

301

Mori, 2009). They can reach the gastrointestinal tract and interact with the gut microflora.

302

In fact, there is evidence that these bioactive lipid derivatives can modify the functions of

303

the immune systems (Durand, Bultel-Poncé, Guy, El Fangour, Rossi, & Galano, 2011) and

304

neuronal systems (Minghetti, Salvi, Lavinia Salvatori, Antonietta Ajmone-Cat, De Nuccio,

305

Visentin, et al., 2014). Furthermore, there is similarity between the plant and mammalian

306

stress responses. In this sense, Medina and colleagues (Medina, et al., 2017) tentatively

307

proposed that the effect of PhytoPs on human physiological mechanisms could be related

308

to different metabolic processes. For instance, PhytoPs can activate the stress-sensing

309

mammalian

310

Zimmermann, Schwaiger, Vouk, Mayerhofer, et al., 2014). This transcription factor is an

311

emerging regulator of cellular resistance to oxidants and it controls and induces

312

expression of the antioxidant response. Accordingly, Nrf2 activation contributes to several

313

beneficial bioactivities of natural products. In this sense, the benefits of fruit consumption

314

may be due to the interactions of different bioactive compounds (flavonoids, other

315

polyphenols, and PhytoPs, among others), since they could activate the Nrf2 pathway and

316

increase expression of the antioxidant response (Waltenberger, Mocan, Šmejkal, Heiss, &

317

Atanasov, 2016).

318

In relation to cocoa or its derivatives, the capacity of this food has been hypothesized to

319

activate the Nrf2 factor, but it is associated with flavan-3-ol(-) - epicatechin (Shah, Li,

320

Ahmad, Kensler, Yamamoto, Biswal, et al., 2010). Accordingly, the results of the present

transcription

factor

Nrf2

(nuclear

factor

erythroid-2)

(Heiss,

Tran,

14

321

study show the presence and diverse concentrations of the different series of PhytoPs in

322

cocoa beans, and can formulate the contribution of PhytoPs in favor of the reduction of the

323

risk of ischemic heart disease. As previously stated by Carrasco-del Amor and colleagues

324

(2015) and Marhuenda and colleagues (2015), this study also supports the importance of

325

PhytoPs and PhytoFs as relevant OS biomarkers in plants.

326

Furthermore, the FAME profile was determined with the objective of comparing it with the

327

concentrations and profiles of PhytoPs and PhytoFs; as mentioned above, ALA is the

328

precursor of both PhytoPs and PhytoFs, so some relationship between this fatty acid and

329

the oxylipins under study here was expected. Other fatty acids from cocoa beans were

330

measured in order to find possible correlations with PhytoPs and PhytoFs synthesis. The

331

result of this analysis was expressed as g Kg-1and it is presented in Table 5. The most

332

important fatty acids, for types of cocoa beans, were C16:0, and C18:0 and C18:1.

333

Pearson correlations were made between the totals and each of the series of PhytoPs,

334

PhytoFs, and FAMEs. The results showed medium linear correlations, with a level of

335

significance of p<0.05 between the total PhytoPs and C18:1 (r=0.357); total PhytoFs and

336

C16:1 and C20:0 (r=0.406 and r=-0.477, respectively); 9-F1t-PhytoP and C18:1 (r=0.433);

337

9-epi-9-D1t-PhytoP and C14:0 (r=-0.439); ent-16-B1-PhytoP + 16-B1-PhytoP and C16:1 (r=-

338

0.395); ent-9-L1-PhytoP + 9-L1-PhytoP and C16:1 (r=-0.391); ent-9-(RS)-12-epi-ST-Δ10-13-

339

PhytoF + ent-9-(S)-12-epi-ST-Δ10-13-PhytoF + ent-9-(R)-12-epi-ST-Δ10-13-PhytoF and

340

C14:0 (r=0.648); ent-16-(RS)-13-epi-ST-Δ14-9-PhytoF and C14:0, C16:0; C18:0, and C18:3

341

(r=0.438, r=0.424, r=-0.416, and r=0.485, respectively). Although the correlation of the

342

results is positive in this preliminary analysis, it should not be attributed solely to this

343

correlation among the series of the PhytoPs, PhytoFs and FAMEs. In future assays, it

344

should be considered other physicochemical phenomena linked to the biotic and abiotic

345

factors that can occur both in the harvest and postharvest periods of the cocoa fruits.

15

346

According to previous studies, the formation of these compounds develops from the attack

347

of ROS on ALA. But, as we indicated previously in the results, only ent-16-(RS)-13-epi-ST-

348

Δ14-9-PhytoF showed a positive correlation, in accordance with Cuyamendous and

349

collaborators (Cuyamendous, Leung, Durand, Lee, Oger, & Galano, 2015). Notably,

350

flaxseeds showed the highest C18:1 level but had a relatively low ent-16-(RS)-13-epi-ST-

351

Δ14-9-PhytoF level compared to walnuts and chia seeds, that contained less C18:1.

352

However, pine nuts had the lowest ALA level and the lowest ent-16-(RS)-13-epi-ST-Δ14-9-

353

PhytoF level; but, it was also established in the same study that the relative concentrations

354

of ent-16-(RS)-13-epi-ST-Δ14-9-PhytoF do not necessarily express the relative C18:1

355

concentrations in walnuts and chia seeds.

356

4. Conclusions

357

The study of PhytoPs and PhytoFs can be considered recent, and it has been carried out

358

in several matrices; such as rice, walnuts, almonds, melon plants (leaves), macroalgae,

359

wines and musts, vegetables oils, among others. However, this is the first identification

360

and quantification of these compounds in dry fermented cocoa beans.

361

The results obtained show dry fermented cocoa beans to be a rich source of PhytoPs and

362

PhytoFs. In fact, the individual concentrations of the PhytoPs and PhytoFs reported in the

363

present study are higher than in other matrices studied. Moreover, the concentrations of

364

the PhytoPs and PhytoFs determined in the different cocoa clones showed significant

365

differences among

366

regional, forastero, and trinitario. One well known characteristic of cocoa is the quantity

367

and quality of its fatty acids and fats, which implies their theoretical correlation with the

368

profiles of the studied compounds. But, taking into account the lack of correlation with ALA

369

and other fatty acids, the variation in the concentrations of PhytoPs and PhytoFs may be

370

attributed to the biotic and abiotic stressors to which the plants are exposed. Also, this

them, but unrelated to

the classification: criollo, criollo moderno,

16

371

variation may be due to other characteristics of the matrix, such as the presence of

372

antioxidants and equivalent molecules, followed by variations in the genetic resource, the

373

origin of the cocoa crop, and the fermentation and drying processes. This information may

374

be very useful for cocoa industries and for the development of the cocoa growing regions.

375

Acknowledgements

376

We are grateful to Dr. David Walker (native English speaker) for his review of the English

377

grammar and style of the current report. The authors are also grateful to the Special

378

Cooperation Agreement 4600003348 celebrated between the Department of Antioquia -

379

Secretary of Agriculture and Rural Development, the University Corporation Lasallista, the

380

National Cocoa Association of Colombia, and the University of Antioquia, whose objective

381

is to join forces to identify, characterize, and evaluate the genetic resource of fine

382

Colombian aroma cocoa and form the CECE (elite collection of special cocoa) for the

383

Department of Antioquia. We also thank the farmer Mr. Evelio González, for his assistance

384

in his experimental farm (Cannes). This work was partially funded by the “Fundación

385

Séneca de la Región de Murcia” Grupo de Excelencia 19900/GERM/15. This work was

386

funded by the Spanish Research Council (CEBAS-CSIC) and the CNRS, by “Projet

387

International de Coopération Scientifique” (PICS-2015-261141) and the Spanish project

388

AGL2017-83386-R from the Spanish Ministry of Science, Innovation and Universities.

389 390

Conflict of interest

391

The authors declare no conflict of interest.

17

392 393

References

394

Baldwin, J. E. (1976). Rules for ring closure. Journal of the Chemical Society, Chemical

395

Communications(18), 734-736.

396

Baldwin, J. E., Thomas, R. C., Kruse, L. I., & Silberman, L. (1977). Rules for ring closure:

397

ring formation by conjugate addition of oxygen nucleophiles. The Journal of Organic

398

Chemistry, 42(24), 3846-3852.

399

Barbosa, M., Collado-González, J., Andrade, P. B., Ferreres, F., Valentão, P., Galano, J.-

400

M., Durand, T., & Gil-Izquierdo, Á. (2015). Nonenzymatic α-Linolenic Acid Derivatives from

401

the Sea: Macroalgae as Novel Sources of Phytoprostanes. Journal of Agricultural and

402

Food Chemistry, 63(28), 6466-6474.

403

Barden, A. E., Croft, K. D., Durand, T., Guy, A., Mueller, M. J., & Mori, T. A. (2009).

404

Flaxseed Oil Supplementation Increases Plasma F1-Phytoprostanes in Healthy Men. The

405

Journal of Nutrition, 139(10), 1890-1895.

406

Cádiz-Gurrea, M. L., Lozano-Sanchez, J., Contreras-Gámez, M., Legeai-Mallet, L.,

407

Fernández-Arroyo, S., & Segura-Carretero, A. (2014). Isolation, comprehensive

408

characterization and antioxidant activities of Theobroma cacao extract. Journal of

409

Functional Foods, 10, 485-498.

410

Carrasco-Del Amor, A. M., Aguayo, E., Collado-González, J., Guy, A., Galano, J.-M.,

411

Durand, T., & Gil-Izquierdo, Á. (2016). Impact of packaging atmosphere, storage and

412

processing conditions on the generation of phytoprostanes as quality processing

413

compounds in almond kernels. Food Chemistry, 211, 869-875.

414

Carrasco-Del Amor, A. M., Aguayo, E., Collado-González, J., Guy, A., Galano, J.-M.,

415

Durand, T., & Gil-Izquierdo, Á. (2017). Impact of processing conditions on the 18

416

phytoprostanes profile of three types of nut kernels. Free Radical Research, 51(2), 141-

417

147.

418

Carrasco-Del Amor, A. M., Collado-Gonzalez, J., Aguayo, E., Guy, A., Galano, J. M.,

419

Durand, T., & Gil-Izquierdo, A. (2015). Phytoprostanes in almonds: identification,

420

quantification, and impact of cultivar and type of cultivation. RSC Advances, 5(63), 51233-

421

51241.

422

Collado-González, J., Medina, S., Durand, T., Guy, A., Galano, J.-M., Torrecillas, A.,

423

Ferreres, F., & Gil-Izquierdo, A. (2015). New UHPLC–QqQ-MS/MS method for quantitative

424

and qualitative determination of free phytoprostanes in foodstuffs of commercial olive and

425

sunflower oils. Food Chemistry, 178, 212-220.

426

Collado-González, J., Moriana, A., Girón, I. F., Corell, M., Medina, S., Durand, T., Guy, A.,

427

Galano, J.-M., Valero, E., Garrigues, T., Ferreres, F., Moreno, F., Torrecillas, A., & Gil-

428

Izquierdo, A. (2015). The phytoprostane content in green table olives is influenced by

429

Spanish-style processing and regulated deficit irrigation. LWT - Food Science and

430

Technology, 64(2), 997-1003.

431

Collado-González, J., Pérez-López, D., Memmi, H., Gijón, M. C., Medina, S., Durand, T.,

432

Guy, A., Galano, J.-M., Fernández, D. J., Carro, F., Ferreres, F., Torrecillas, A., & Gil-

433

Izquierdo, A. (2016). Effect of the season on the free phytoprostane content in Cornicabra

434

extra virgin olive oil from deficit-irrigated olive trees. Journal of the Science of Food and

435

Agriculture, 96(5), 1585-1592.

436

Cuyamendous, C., Leung, K. S., Durand, T., Lee, J. C. Y., Oger, C., & Galano, J. M.

437

(2015). Synthesis and discovery of phytofurans: metabolites of [small alpha]-linolenic acid

438

peroxidation. Chemical Communications, 51(86), 15696-15699.

19

439

Demidchik, V. (2015). Mechanisms of oxidative stress in plants: From classical chemistry

440

to cell biology. Environmental and Experimental Botany, 109, 212-228.

441

Domínguez-Perles, R., Abellán, A., León, D., Ferreres, F. ,Guy, A., Oger, C., Galano, J.-

442

M., Durand, T., Gil-Izquierdo, A. (2018). Sorting out the phytoprostane and phytofuran

443

profile in vegetable oils. Food Research International, 107, 619-628.

444

Durand, T., Bultel-Poncé, V., Guy, A., El Fangour, S., Rossi, J. C., & Galano, J. M. (2011).

445

Isoprostanes and phytoprostanes: Bioactive lipids. Biochimie, 93(1), 52-60.

446

El Fangour, S., Guy, A., Despres, V., Vidal, J.-P., Rossi, J.-C., & Durand, T. (2004). Total

447

Synthesis of the Eight Diastereomers of the Syn-Anti-Syn Phytoprostanes F1 Types I and

448

II. The Journal of Organic Chemistry, 69(7), 2498-2503.

449

El Fangour, S., Guy, A., Vidal, J.-P., Rossi, J.-C., & Durand, T. (2005). A Flexible

450

Synthesis of the Phytoprostanes B1 Type I and II. The Journal of Organic Chemistry,

451

70(3), 989-997.

452

Hasanuzzaman, M., Hossain, M. A., da Silva, J. A. T., & Fujita, M. (2012). Plant Response

453

and Tolerance to Abiotic Oxidative Stress: Antioxidant Defense Is a Key Factor. In B.

454

Venkateswarlu, A. K. Shanker, C. Shanker & M. Maheswari (Eds.), Crop Stress and its

455

Management: Perspectives and Strategies,

456

Netherlands.

457

Heiss, E. H., Tran, T. V. A., Zimmermann, K., Schwaiger, S., Vouk, C., Mayerhofer, B.,

458

Malainer, C., Atanasov, A. G., Stuppner, H., & Dirsch, V. M. (2014). Identification of

459

Chromomoric Acid C-I as an Nrf2 Activator in Chromolaena odorata. Journal of Natural

460

Products, 77(3), 503-508.

(pp. 261-315). Dordrecht: Springer

20

461

Jahn, U., Galano, J.-M., & Durand, T. (2008). Beyond Prostaglandins—Chemistry and

462

Biology of Cyclic Oxygenated Metabolites Formed by Free-Radical Pathways from

463

Polyunsaturated Fatty Acids. Angewandte Chemie International Edition, 47(32), 5894-

464

5955.

465

Jahn, U., Galano, J.-M., & Durand, T. (2010). A cautionary note on the correct structure

466

assignment of phytoprostanes and the emergence of a new prostane ring system.

467

Prostaglandins, Leukotrienes and Essential Fatty Acids, 82(2–3), 83-86.

468

Loeffler, C., Berger, S., Guy, A., Durand, T., Bringmann, G., Dreyer, M., von Rad, U.,

469

Durner, J., & Mueller, M. J. (2005). B(1)-Phytoprostanes Trigger Plant Defense and

470

Detoxification Responses. Plant Physiology, 137(1), 328-340.

471

Marhuenda, J., Medina, S., Díaz-Castro, A., Martínez-Hernández, P., Arina, S., Zafrilla, P.,

472

Mulero, J., Oger, C., Galano, J.-M., Durand, T., Ferreres, F., & Gil-Izquierdo, A. (2015).

473

Dependency of Phytoprostane Fingerprints of Must and Wine on Viticulture and Enological

474

Processes. Journal of Agricultural and Food Chemistry, 63(41), 9022-9028.

475

Marseglia, A., Palla, G., & Caligiani, A. (2014). Presence and variation of γ-aminobutyric

476

acid and other free amino acids in cocoa beans from different geographical origins. Food

477

Research International, 63, 360-366.

478

Marseglia, A., Sforza, S., Faccini, A., Bencivenni, M., Palla, G., & Caligiani, A. (2014).

479

Extraction, identification and semi-quantification of oligopeptides in cocoa beans. Food

480

Research International, 63, 382-389.

481

Medina, S., Collado-González, J., Ferreres, F., Londoño-Londoño, J., Jiménez-Cartagena,

482

C., Guy, A., Durand, T., Galano, J.-M., & Gil-Izquierdo, A. (2017). Quantification of

483

phytoprostanes – bioactive oxylipins – and phenolic compounds of Passiflora edulis Sims

484

shell using UHPLC-QqQ-MS/MS and LC-IT-DAD-MS/MS. Food Chemistry, 229, 1-8. 21

485

Minghetti, L., Salvi, R., Lavinia Salvatori, M., Antonietta Ajmone-Cat, M., De Nuccio, C.,

486

Visentin, S., Bultel-Poncé, V., Oger, C., Guy, A., Galano, J.-M., Greco, A., Bernardo, A., &

487

Durand, T. (2014). Nonenzymatic oxygenated metabolites of α-linolenic acid B1- and L1-

488

phytoprostanes protect immature neurons from oxidant injury and promote differentiation

489

of oligodendrocyte progenitors through PPAR-γ activation. Free Radical Biology and

490

Medicine, 73(Supplement C), 41-50.

491

Morrow, J. D., Hill, K. E., Burk, R. F., Nammour, T. M., Badr, K. F., & Roberts, L. J. (1990).

492

A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-

493

cyclooxygenase, free radical-catalyzed mechanism. Proceedings of the National Academy

494

of Sciences of the United States of America, 87(23), 9383-9387.

495

Nakabayashi, R., & Saito, K. (2015). Integrated metabolomics for abiotic stress responses

496

in plants. Current Opinion in Plant Biology, 24(Supplement C), 10-16.

497

Patras, M. A., Milev, B. P., Vrancken, G., & Kuhnert, N. (2014). Identification of novel

498

cocoa flavonoids from raw fermented cocoa beans by HPLC–MSn. Food Research

499

International, 63, 353-359.

500

Pinciroli, M., Dominguez-Perles, R., Abellan, A., Guy, A., Durand, T., Oger, C., Galano, J.

501

M., Ferreres, F., & Gil-Izquierdo, A. (2017). Comparative Study of the Phytoprostanes and

502

Phytofurans Content of Indica and Japonica Rice’s (Oryza sativa L.) Flours. Journal of

503

Agricultural and Food Chemistry, 65(40), 8938-8947.

504

Pinot, E., Guy, A., Fournial, A., Balas, L., Rossi, J.-C., & Durand, T. (2008). Total

505

Synthesis of the Four Enantiomerically Pure Diasteroisomers of the Phytoprostanes

506

E1Type II and of the 15-E2t-Isoprostanes. The Journal of Organic Chemistry, 73(8), 3063-

507

3069.

22

508

Shah, Z. A., Li, R.-c., Ahmad, A. S., Kensler, T. W., Yamamoto, M., Biswal, S., & Doré, S.

509

(2010). The flavanol (−)-epicatechin prevents stroke damage through the Nrf2/HO1

510

pathway. Journal of Cerebral Blood Flow & Metabolism, 30(12), 1951-1961.

511

Taber, D. F., Morrow, J. D., & Jackson Roberts, L. (1997). A nomenclature system for the

512

isoprostanes. Prostaglandins, 53(2), 63-67.

513

Torres-Moreno, M., Tarrega, A., Costell, E., & Blanch, C. (2012). Dark chocolate

514

acceptability: influence of cocoa origin and processing conditions. Journal of the Science

515

of Food and Agriculture, 92(2), 404-411.

516

Trigueros, L., & Sendra, E. (2015). Fatty acid and conjugated linoleic acid (CLA) content in

517

fermented milks as assessed by direct methylation. LWT - Food Science and Technology,

518

60(1), 315-319.

519

Waltenberger, B., Mocan, A., Šmejkal, K., Heiss, E. H., & Atanasov, A. G. (2016). Natural

520

Products to Counteract the Epidemic of Cardiovascular and Metabolic Disorders.

521

Molecules (Basel, Switzerland), 21(6),

522

Yonny, M. E., Rodríguez Torresi, A., Cuyamendous, C., Réversat, G., Oger, C., Galano,

523

J.-M., Durand, T., Vigor, C., & Nazareno, M. A. (2016). Thermal Stress in Melon Plants:

524

Phytoprostanes and Phytofurans as Oxidative Stress Biomarkers and the Effect of

525

Antioxidant Supplementation. Journal of Agricultural and Food Chemistry, 64(44), 8296-

526

8304.

527

Zhu, Y., Taylor, C., Sommer, K., Wilkinson, K., & Wirthensohn, M. (2015). Influence of

528

deficit irrigation strategies on fatty acid and tocopherol concentration of almond (Prunus

529

dulcis). Food Chemistry, 173, 821-826.

530

23

531

Figure captions

532

Figure 1. Chemical structures of phytoprostanes and phytofurans (named according to the

533

((Collado-González, et al., 2016;) Taber/Roberts nomenclature).

534 Phytoprostanes Series F HO

Phytofurans

HO

O

O

CH3

OH

OH

HO

9-F1t-PhytoP

HO

HO

H

OH

Ent-16-(RS)-9-epi-ST-Δ14-10-PhytoF H HO

OH

9-epi-9-F1t-PhytoP

CH3 O

O

OH

HO

Series D

OH H

OH

OH

Ent-16-epi-16-F1t-PhytoP

Ent-9-(RS)-12-epi-ST-Δ10-13-PhytoF H HO

CH3 O

CH3

CH3

HO

HO

OH

Ent-9-epi-9-D1t-PhytoP

H OH

OH

OH

Ent-9-D1t-PhytoP

Ent-9-(S)-12-epi-ST-Δ10-13-PhytoF

Series B

H

O

O

HO

CH3

O

O CH3

CH3

HO

O H OH

Ent-16-B1-PhytoP

Ent-9-(R)-12-epi-ST-Δ10-13-PhytoF

Series L

H

O CH3

O

O

9-L1-PhytoP

OH

OH

OH

OH

OH CH3

O

OH

535 536

O

HO

O CH3

O

OH

OH

HO

OH

OH

16-B1-PhytoP

O

OH

OH

O

O

OH

O

OH

HO

O

O

OH

OH

Ent-16-F1t-PhytoP

CH3 HO

O

HO

O

CH3

CH3

HO

O

CH3

OH

H

HO

H OH

Ent-9-L1-PhytoP

Ent-16-(RS)-13-epi-ST-Δ14-9-PhytoF

Figure 1. Chemical structures of phytoprostanes and phytofurans (named according to the ((Collado-González, et al., 2016) Taber/Roberts nomenclature).

537 538

Highlights

539



First report of the occurrence of phytoprostanes and phytofurans in cocoa beans.

540



The profiles of phytoprostanes and phytofurans varied according to the clone. 24

541



Content of PUFAs were related to the amounts of phytoprostanes and phytofurans.

542 543 544 Samples

Variety

Grain size

Criollo moderno - 1

Criollo moderno

Big

Criollo moderno - 2

Criollo moderno

Big

Criollo moderno - 3

Criollo moderno

Middle

Criollo - 1

Criollo

Big

Criollo - 2

Criollo

Big

Criollo - 3

Criollo

Big

Regional - 1

Regional

Middle

Regional - 2

Regional

Small

Regional - 3

Regional

Middle

Regional - 4

Regional

Big

Regional - 5

Regional

Big

Regional - 6

Regional

Middle

Regional - 7

Regional

Small

Regional - 8

Regional

Middle

Regional - 9

Trinitario

Big

Regional - 10

Regional

Middle

Regional - 11

Regional

Big

Albino - 1

Forastero

Middle

Albino - 2

Forastero

Middle

CCN-51

Forastero

Middle

FMT-1

Forastero

Middle

FTU-6

Forastero

Middle

CAU-37

Forastero

Small

EET-8

Trinitario

Big

EET-96

Trinitario

Middle

FMAC-11

Trinitario

Middle

FMAC-12

Trinitario

Middle

ICS-1

Trinitario

Big

ICS-39

Trinitario

Big

ICS-95

Trinitario

Small

TSH-565

Trinitario

Small

25

545 546

Table 1 Cacao clones evaluated and their respective characteristics. Small bean size: (<1.4 g/ grain); middle (1.4 – 1.6 g/ grain); big (≥ 1.6 g/ grain).

547

26

Compound

Retention time (min)

ESI mode

MRM transition (m/z)

Fragmentor (V)

Collision energy (V)

Phytoprostanes Ent-16-epi-16-F1t-PhytoPX

1.583

Negative

327.1 > 283.2Z 327.1 > 225.1Y

80 80

15 15

9-F1t-PhytoP

1.631

Negative

327.2 > 273.1 327.2 > 171.0

110 110

15 15

Ent-16-F1t-PhytoPX

1.712

Negative

327.2 > 283.2 327.2 > 225.1

80 80

10 10

9-epi-9-F1t-PhytoP

1.785

Negative

327.2 > 272.8 327.2 > 171.0

110 110

10 10

9-D1t-PhytoP

1.791

Negative

325.2 > 307.3 325.2 > 134.7

100 100

4 4

9-epi-9-D1t-PhytoP

2.022

Negative

325.2 > 307.2 325.2 > 134.9

100 100

7 7

16-B1-PhytoP

2.620

Negative

307.2 > 223.2 307.2 > 235.1

100 100

10 100

9-L1-PhytoP

3.079

Negative

307.2 > 185.1 307.2 > 196.7

110 110

7 7

Phytofurans Ent-9-(RS)-12-epi-ST-Δ10-13-PhytoF

0.906

Negative

344.0 > 300.0 344.0 > 255.9

110 110

10 10

Ent-16-(RS)-9-epi-ST-Δ14-10-PhytoF

1.501

Negative

343.9 > 209.0 343.9 > 201.1

90 90

12 12

Ent-16-(RS)-13-epi-ST-Δ14-9-PhytoF

1.523

Negative

343.0 > 171.1 343.0 > 97.2

90 90

22 22

Table 2 UHPLC-QqQ-MS/MS parameters for the quantification and confirmation of phytoprostanes and phytofuranes. Z Quantification transition. Y Confirmation transition. X Coeluting diastereoisomers quantified together.

27

Samples Criollo moderno - 1 Criollo moderno - 2 Criollo moderno - 3 Criollo - 1 Criollo - 2

ent-16-(RS)-9-epiST-Δ14-10-PhytoF 1.13

± 0.08

ent-9-(RS)-12-epi-ST-Δ10-13-PhytoF + ent-9-(S)-12-epi-ST-Δ10-13-PhytoF + ent-9-(R)-12-epi-ST-Δ10-13-PhytoF 0.99 ± 0.05

ND 1.09

ND

± 0.06

1.18

1.51 1.26

ND

ND

ent-16-(RS)-13-epiST-Δ14-9-PhytoF

1.55

ND

± 0.07

0.97

±

± ± ±

0.22

3.31

±

Criollo - 3

ND

ND

ND

ND

ND

ND

1.48

± 0.13

Regional - 3

0.99

± 0.05

Regional - 4

1.49

± 0.07

Regional - 5

0.95

± 0.08

Regional - 6

1.88

± 0.18

Regional - 7

2.70

± 0.39

Regional - 9

1.08

± 0.15

Regional - 10

1.33

± 0.28

Regional - 11

1.09

± 0.00

Albino - 1

1.20

± 0.03

Albino - 2

1.56

± 0.21

CCN-51

1.80

± 0.11

FMT-1

1.39

± 0.03

FTU-6

1.00

± 0.17

CAU-37

1.38

± 0.13

±

0.10

ND 1.55

±

0.04

ND 2.21

ND

Regional - 8

EET-8

1.12

± ±

1.20

0.36

2.19

±

0.07

1.11

±

0.05

1.00

±

0.09

ND ND

0.21

7.49

±

0.30

1.74

±

0.30

2.38

±

0.37

ND

2.24

±

0.24

ND

2.13

±

0.35

2.79

±

0.35

3.29

±

0.07

3.06

±

0.26

1.01

±

0.13

2.06

±

0.06

±

±

0.10

0.08

ND 1.10

±

0.13

ND 1.58

ND

±

0.31

ND

EET-96

1.05

± 0.03

FMAC-11

1.38

± 0.20

1.08

±

FMAC-12

1.01

± 0.02

1.14

±

1.38

ND

± ±

0.80

0.11

1.82

±

0.18

0.06

1.60

±

0.47

ND

ND

ND

ICS-39

ND

ND

ND

ICS-95

1.18

± 0.19

1.58

± 0.09

ND 1.30

0.17

2.14

ICS-1

TSH-565

0.12

±

0.18

ND 1.37

0.62

2.96

ND 2.94

0.34

ND

Regional - 1 Regional - 2

0.15

ND ±

0.12

2.23

±

0.32

g-1)

Table 3. Individual series of PhytoFs levels (ng in cacao beans. The values are represented as means ± SD (standard deviation). ND: not detected or not quantified

28

Criollo moderno - 1

244.96

±

22.95

107.97

±

12.88

ent-16-epi-16-F1tPhytoP + ent-16-F1tPhytoP 25.67 ± 2.34

Criollo moderno - 2

320.64

±

36.90

66.70

±

11.43

9.30

±

0.45

1.33

±

0.05

2.47

±

0.36

3.99

±

0.26

2.56

±

0.18

Criollo moderno - 3

449.68

±

57.87

371.10

±

65.50

12.38

±

1.14

2.37

±

0.25

2.93

±

0.36

7.80

±

1.25

5.18

±

0.65

Criollo - 1

572.81

±

25.46

318.40

±

51.67

24.91

±

1.71

1.14

±

0.06

1.67

±

0.22

9.86

±

1.14

6.28

±

0.79

Criollo - 2

1134.08

±

212.70

406.01

±

28.62

18.35

±

3.31

4.88

±

0.42

5.08

±

0.80

13.37

±

0.58

8.07

±

0.54

1.35

±

0.19

2.01

±

0.37

8.90

±

0.41

5.42

±

0.46

5.42

±

0.20

3.66

±

0.05

Samples

9-F1t-PhytoP

9-epi-9-F1t-PhytoP

9-D1t-PhytoP

9-epi-9-D1tPhytoP

ent-16-B1-PhytoP + 16-B1-PhytoP

ent-9-L1-PhytoP + 9-L1-PhytoP

1.60

±

0.15

2.50

±

0.22

7.05

±

0.32

4.55

±

0.26

Criollo - 3

670.14

±

34.95

302.28

±

70.04

16.99

±

0.71

Regional - 1

251.02

±

15.05

63.70

±

7.28

7.67

±

1.13

Regional - 2

649.34

±

94.65

386.37

±

52.60

12.72

±

1.95

6.16

±

0.68

4.94

±

0.71

13.97

±

2.28

8.34

±

1.16

Regional - 3

320.25

±

65.76

518.81

±

121.36

8.63

±

1.70

5.23

±

0.68

6.15

±

1.28

11.65

±

2.37

7.04

±

1.32

Regional - 4

435.83

±

103.97

223.81

±

17.68

63.36

±

10.96

2.93

±

0.34

3.25

±

0.58

7.42

±

2.10

5.80

±

0.13

Regional - 5

610.16

±

9.62

251.89

±

20.07

24.05

±

2.64

1.49

±

0.08

2.74

±

0.24

6.80

±

0.35

3.96

±

0.14

Regional - 6

147.10

±

31.39

24.78

±

1.11

37.58

±

7.80

1.54

±

0.06

1.52

±

0.21

5.40

±

0.46

3.53

±

0.38

Regional - 7

452.57

±

36.50

448.04

±

3.81

16.67

±

2.02

1.95

±

0.15

4.66

±

0.22

9.71

±

0.37

6.03

±

0.35

Regional - 8

275.30

±

20.37

22.54

±

2.38

23.23

±

1.02

1.82

±

0.20

3.50

±

0.43

2.41

±

0.19

Regional - 9

328.78

±

37.50

278.46

±

34.32

9.21

±

0.96

2.20

±

0.35

5.13

±

0.60

7.08

±

0.98

4.51

±

0.46

Regional - 10

385.29

±

16.71

200.45

±

17.10

42.61

±

2.20

2.45

±

0.30

4.72

±

0.23

9.18

±

0.41

5.31

±

0.30

Regional - 11

641.25

±

14.30

381.23

±

28.75

20.01

±

1.53

1.39

±

0.10

3.34

±

0.51

11.56

±

0.12

7.13

±

0.29

Albino - 1

650.20

±

107.60

657.92

±

157.03

29.53

±

0.44

3.07

±

0.76

7.53

±

2.19

13.66

±

2.11

8.44

±

1.61

Albino - 2

205.59

±

23.80

37.82

±

34.57

6.21

±

0.53

1.26

±

0.12

1.49

±

0.06

3.29

±

0.02

2.28

±

0.10

CCN-51

668.81

±

61.69

752.70

±

66.74

14.39

±

1.59

8.92

±

1.25

10.90

±

1.52

15.59

±

1.44

9.18

±

0.95

FMT-1

723.22

±

104.58

449.17

±

74.52

18.59

±

3.42

4.27

±

0.61

5.39

±

1.08

16.71

±

2.51

9.74

±

1.48

ND

ND

ND

FTU-6

434.26

±

63.81b

200.98b

±

33.88

21.93

±

0.91

3.63

±

0.39

4.40

±

0.20

7.36

±

1.39

4.69

±

0.87

CAU-37

210.29

±

12.83

781.51

±

100.28

14.25

±

2.68

3.67

±

0.27

7.77

±

2.52

46.35

±

2.50

26.34

±

1.09

EET-8

749.60

±

23.52

290.92

±

32.21

20.99

±

0.38

1.47

±

0.19

3.01

±

0.31

11.26

±

0.99

7.11

±

0.29

EET-96

745.53

±

6.34

236.05

±

24.00

21.99

±

3.17

1.93

±

0.03

2.75

±

0.26

8.15

±

0.17

5.41

±

0.17

FMAC-11

1030.26

±

164.26

448.78

±

75.07

26.04

±

3.90

2.66

±

0.34

6.45

±

1.24

20.26

±

3.37

12.34

±

2.08

29

FMAC-12

32.88

±

12.69

4.94

±

0.54

3.04

±

0.49

2.11

±

0.23

ICS-1

247.78

±

39.44

149.60

±

2.54

12.88

±

1.17

2.88

±

0.03

3.15

±

0.75

6.13

±

0.62

3.55

±

0.33

ICS-39

114.52

±

2.18

384.91

±

12.78

3.57

±

0.01

16.52

±

0.66

5.27

±

1.66

11.09

±

0.87

6.71

±

0.48

ICS-95

784.45

±

184.41

520.64

±

66.11

22.60

±

3.63

1.55

±

0.25

2.66

±

0.38

19.78

±

1.55

11.83

±

0.89

TSH-565

827.97

±

13.07

264.18

±

8.27

30.99

±

1.39

2.03

±

0.22

3.44

±

0.09

12.08

±

0.93

6.75

±

0.17

p-value

261.93 ± 26.74

⩽ 0.05

⩽ 0.05

⩽ 0.05

ND

⩽ 0.05

ND

⩽ 0.05

⩽ 0.05

⩽ 0.05

Table 4 Individual series of PhytoPs levels (ng g-1) in cacao beans. The values are represented as means ± SD (standard deviation). ND: not detected or not quantified

30

548 Myristic acid

Palmitic acid

Palmitic acid

Stearic acid

Oleic acid

Isomer

Linoleic acid

α-Linolenic acid

Gadoleic acid

C14:0

C16:0

C16:1

C18:0

C18:1

C18:1

C18:2

C18:3

C20:0

Criollo moderno - 1

0.13

48.06

0.51

51.04

51.07

0.75

7.98

0.40

1.80

Criollo moderno - 2

0.24

105.06

1.24

114.15

114.80

1.93

13.30

0.67

4.27

Criollo moderno - 3

0.24

92.34

1.21

110.41

121.60

2.11

11.94

0.86

3.60

Criollo - 1

0.54

90.18

0.83

135.60

127.94

2.04

12.56

0.88

4.51

Criollo - 2

0.11

92.45

0.89

104.09

113.62

2.07

9.75

0.73

3.28

Criollo - 3

0.49

106.09

0.97

128.48

130.84

1.56

13.62

0.92

4.61

Regional - 1

0.21

99.18

0.74

106.02

114.11

1.55

13.53

0.84

3.64

Regional - 2

0.13

77.79

0.93

83.44

89.37

1.47

10.92

0.72

2.44

Regional - 3

0.13

102.95

0.96

146.62

133.79

2.24

13.17

0.76

5.05

Regional - 4

0.13

78.45

1.01

94.56

94.82

1.45

10.21

0.58

3.36

Regional - 5

0.11

73.67

0.79

77.58

92.88

1.96

10.14

0.66

2.73

Regional - 6

0.18

86.97

0.72

111.82

108.42

1.44

12.22

0.75

3.49

Regional - 7

0.18

77.35

0.67

101.38

96.95

1.25

8.70

0.46

3.43

Regional - 8

0.33

79.24

1.31

72.74

84.95

1.20

9.02

0.71

2.45

Regional - 9

0.15

78.32

1.11

104.83

98.65

1.90

11.37

0.61

3.60

Regional - 10

0.17

66.16

0.74

79.17

81.47

1.34

8.20

0.52

2.57

Regional - 11

0.15

78.73

0.93

119.17

107.22

1.98

14.12

0.79

4.03

Albino - 1

0.15

78.73

0.93

119.17

107.22

1.98

14.12

0.79

4.03

Albino - 2

0.12

89.27

0.87

107.04

116.61

1.66

10.89

0.69

3.67

CCN-51

0.09

93.43

0.91

106.23

114.75

1.69

9.91

0.87

3.66

FMT-1

0.15

57.34

0.41

69.10

72.32

0.85

5.73

0.42

2.04

FTU-6

0.17

70.59

1.09

88.17

85.30

1.68

8.88

0.57

2.92

CAU-37

0.07

30.29

0.21

36.27

36.64

0.47

4.35

0.24

1.09

EET-8

0.17

103.32

0.67

151.59

138.63

1.58

12.33

0.72

4.74

EET-96

0.14

73.89

0.72

102.46

99.76

1.62

10.00

0.62

3.10

FMAC-11

0.11

99.56

1.01

117.90

124.88

1.94

14.75

0.84

3.64

FMAC-12

0.16

88.01

0.91

92.59

99.55

1.40

8.30

0.55

3.13

Sample

549

ICS-1

0.15

72.71

0.70

94.49

91.99

1.35

12.10

0.62

3.03

ICS-39

0.14

58.91

0.52

75.29

72.40

1.01

9.06

0.49

2.36

ICS-95

0.40

103.33

0.67

97.73

116.13

1.42

11.90

0.86

3.98

TSH-565

0.26

84.24

0.82

102.16

103.55

1.28

9.77

0.62

3.86

Table 5 FAMEs levels in cacao beans. The values are represented as g

Kg-1

550 551

Declaration of interests

552 31

553 554

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

555 556 557 558

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

559 560 561 562 563

32