Comprehensive study of phenolic compounds profile and antioxidant

Subscriber access provided by Washington University | Libraries

Bioactive Constituents, Metabolites, and Functions

Comprehensive study of phenolic compounds profile and antioxidant activity of eight pistachio cultivars, their residual cakes and virgin oils Rosa Maria Ojeda-Amador, M. Desamparados Salvador Moya, Giuseppe Fregapane, and Sergio Gómez-Alonso J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06509 • Publication Date (Web): 01 Mar 2019 Downloaded from http://pubs.acs.org on March 1, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 41

Journal of Agricultural and Food Chemistry

1

Comprehensive study of phenolic compounds profile and antioxidant

2

activity of eight pistachio cultivars, their residual cakes and virgin oils

3

M.

Ojeda-Amadora,

4

Rosa

María

Desamparados

5

Fregapanea, Sergio Gómez-Alonsoa,b*,

6

aDepartamento

7

Universidad de Castilla-La Mancha, Ciudad Real, Spain

8

bInstituto

9

La Mancha, Ciudad Real, Spain

Salvadora,

Giuseppe

de Tecnología de Alimentos, Facultad de Ciencias Químicas,

Regional de Investigación Científica Aplicada, Universidad de Castilla-

10 11

*Corresponding author: Sergio Gómez Alonso: [email protected]

12 13 14

Other authors e-mails:

15

Rosa M. Ojeda-Amador: [email protected]

16

María Desamparados Salvador, [email protected]

17

Giuseppe Fregapane, [email protected]

18 19 20 21 22 23 24 25 26 27 28 29 30

ACS Paragon Plus Environment


31

Abstract

32

Phenolic compounds of eight pistachio (Pistacia vera L.) cultivars, their residual

33

cakes and virgin oils (screw pressing) were studied using HPLC-DAD-ESI-

34

MS/MS. A total of 25 compounds were identified and quantified for pistachio nuts

35

and residual cakes, being reported for the first time the presence of five flavonols,

36

six flavanols and one gallotannin. Total phenolics in pistachio nuts showed a

37

concentration from 1359 mg/kg (Kastel) to 4507 mg/kg (Larnaka). Flavanols were

38

the most abundant phenolics, about 90%, resulting procyanidin B1 and

39

gallocatechin the main ones, depending on the cultivar. Other phenolic groups

40

such as anthocyanins (from 54 to 218 mg/kg), flavonols (from 76 to130 mg/kg),

41

flavanones (from 12 to 71 mg/kg) and gallotannins (from 4 to 46 mg/kg) were also

42

identified. Residual cakes presented the same phenolic profile, but with a

43

concentration almost double because of the concentration effect caused by the

44

oil separation. Virgin pistachio oils showed a very low phenolic content, being

45

eriodyctiol the only compound identified.

46 47

Keywords: pistachio; by-product; residual cake; virgin oil; phenolic compounds;

48

antioxidant activity.

49 50

Abbreviations: High Performance Liquid Chromatography (HPLC), Diode Array

51

Detector (DAD), Electrospray Ionization (ESI), Mass Spectrometry (MS), 2,2-

52

diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH), oxygen radical absorbance

53

capacity (ORAC), 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH), Trolox

54

Equivalent (TE), Retention time (Rt), ultraviolet-visible (UV-vis), Fresh weigh

55

(FW), Procyanidin B1 (PB1), Procyanidin B2 (PB2), Analysis of Variance

56

(ANOVA) and Principal Component Analysis (PCA).

57 58 59


Page 2 of 41

Page 3 of 41


60 61

1. INTRODUCTION

62

Pistacia vera L., is a plant of the Anacardiaceae family known since prehistoric

63

times. It was originated in western Asia and its cultivation spreads to the

64

Mediterranean basin through Iran, which has been the major crop producer for a

65

long time1. The top worldwide producer in 2014 was Iran (415,000 tons), followed

66

by United States (230,000 tons) and Turkey (80,000 tons)2. In Spain orchards

67

have been planted since 1996, contributing in 2016 with more than 15,000 ha

68

with a production over 1,000 tons, being placed 80% of the cultivation in the

69

region of Castilla-La Mancha3.

70

Pistachios are rich in protein and fat, with a balanced content of mono- (~70%)

71

and polyunsaturated (~20%) fatty acids, which could result in the reduction of

72

both, LDL levels and therefore the risk of coronary heart disease4. Moreover, they

73

present a high content of bioactive compounds, such as tocopherols, phytosterols

74

and phenolic compounds5,6, being this kind of nuts among the top fifty foods with

75

a high antioxidant potential7. Some bioactive compounds present in pistachios

76

have been reported to present a rapid accessibility in the stomach, thereby

77

contributing to the beneficial relationship between pistachio consumption and

78

health-related outcomes8.

79

Since few years ago, several studies have reported the chemical composition of

80

pistachios9,10 not only because of their properties but also for their geographic

81

origin differentiation11. However, there are very few publications related to the

82

comprehensive study of pistachios phenolic profile, which include anthocyanins,

83

flavonoids, flavanones, phenolic acids, flavanols and isoflavones12-14.



84

The most important use of pistachio nuts has traditionally been as snack, but it

85

has been also employed in a wide range of manufactured formulations into the

86

food industry, such as ice-creams, bakery, desserts and cold cuts15. Nowadays,

87

virgin edible oils as foodstuffs are emerging as an alternative related to the

88

interest of people about consuming beneficial healthy products and less-

89

processed foods16. Within these kind of oils, stand out those obtained from nuts,

90

which have recently appeared in the market. Virgin tree nut oils are very

91

appreciated in gastronomy because of their typical aroma, taste and nutritional

92

characteristic, which depends on the extraction technology and processing

93

conditions, as roasting17. The by-product obtained after pressing, may be used

94

as a partially defatted product, rich in proteins and carbohydrates, for applications

95

such as specialty baking, energy bars and animal feeds. Moreover, recent

96

evidences about their high content in bioactive substances as phenolic

97

compounds10,18 could make it possible the development of new applications as

98

functional ingredients.

99

To the best of our knowledge, no data related to differences in the phenolic profile

100

of diverse Pistacia vera L. cultivars grown under the same agronomical conditions

101

are reported in bibliography, even though this is one of the most important factor

102

that affects phenolic composition of fruits and vegetables as olive19 and walnuts20.

103

In this sense, this study aims to complete the information about phenolic profile

104

and antioxidant activity in pistachio nuts from eight different cultivars grown under

105

the same agronomical conditions, employing a HPLC-DAD-ESI-MS/MS, as well

106

as in the virgin oil and the by-product obtained after pressing. On the other hand,

107

a complete characterization of phenolic profile of residual cakes from pistachios

108

has been carried out.


Page 4 of 41

Page 5 of 41


109

2. MATERIALS AND METHODS

110

2.1. Experimental pistachio orchards: the study was carried out during the crop

111

season 2014/2015 in an experimental pistachio orchard where different pistachio

112

cultivars were grown. Eight Pistacia vera L. varieties (Aegina, Avdat, Kastel,

113

Kerman, Larnaka, Mateur, Napoletana and Sirora; 10–15 kg each) were provided

114

by the regional research centre ‘Centro de Mejora Agraria El Chaparrillo-IRIAF’,

115

located in Ciudad Real (Spain; 39⁰ 00' 17" N, 3⁰ 57' 44" W, elevation 628 m.a.s.l).

116

The soil at the experimental site is a shallow clay-loam (Alfisol Xeralf Petrocalcic

117

Palexeralfs) with a depth of 1.3 m and a discontinuous petrocalcic horizon

118

between 0.75 and 0.85 m. The trees were spaced 5 m × 5 m apart (400 trees

119

ha−1). This area shows a Mediterranean continental climate, with an average

120

annual rainfall about 400 mm, mostly distributed outside of a 4-month (June-

121

September) summer drought period. The total rainfall in Ciudad Real was 324

122

mm during the 2014/2015 hydrological years (Oct-Sep). All agronomical

123

treatments applied to the experimental pistachio orchards were identical and no

124

irrigation treatment was applied.

125

2.2. Virgin pistachio oil and by-product extraction: the oil was extracted using

126

a screw press (Komet Screw Vegetable Oil Expeller CA59G-CA563, IBG

127

Monforts Oekotec GmbH & Co. KG, Mönchengladbach, Germany) equipped with

128

a 6 mm diameter nozzle and operating at a screw speed of 30 rpm. The screw

129

press was first run empty for 10–15 min to raise the screw-press barrel

130

temperature to the minimum required for extracting the oil (about 50 ⁰C) using

131

the electrical resistance ring attached around the press barrel10. The resulting

132

crude oil (approximately 1.0–1.2 L of each variety) was centrifuged at 5000 rpm

133

to remove the residual plant material. Oil samples were stored in amber bottles,



134

without headspace, to protect them from light. The residual cakes were placed in

135

labelled pouches and vacuum-packed to prevent any oxidative degradation. All

136

samples were stored in the dark at 4 ⁰C until analysed. The extraction yield of the

137

screw press process was ranged between 52.7-72.7%, depending on the variety.

138

2.3. Phenolic extracts preparation: 0.4 g of ground pistachio nut or residual

139

cake were first mix two times with n-hexane (6 + 4 mL) for removing fat, being

140

discarded this fraction after centrifugation. Then, a double extraction of the solid

141

residue obtained from the n-hexane extraction was performed in 20 mL

142

MeOH:H2O:HCOOH (80:20:0.1; 10 + 10 mL), with 2 min vortex, followed by 5 min

143

ultrasound and centrifugation at 2000 g for 10 min. The combined hydro-

144

methanolic extracts were filtered with a 0.45 µm nylon syringe filter and stored at

145

-20 ºC until use.

146

Virgin oil (5 g) was dissolved in n-hexane (10 mL) and 10 ml of

147

MeOH:H2O:HCOOH (80:20:0.1) were added. The mixture was vortexed for 2 min,

148

followed by 5 min of ultrasound and then centrifuged at 2000 g for 10 min. Finally,

149

the polar fraction was separated and filtered with a 0.45 µm nylon syringe filter

150

and stored at -20 ºC until use.

151

2.4. Antioxidant activity was evaluated by measuring the radical scavenging

152

effect of the methanolic extract toward the synthetic radical 2,2-diphenyl-1-(2,4,6-

153

trinitrophenyl)hydrazyl (DPPH), as reported previously21,22. Briefly, 100 µl of the

154

polar extract, prepared according to point 2.3, was added to a methanolic DPPH

155

solution (2.9 ml, 6·× 10-5 M) and stored in the dark for 30 min. The decrease in

156

absorbance of the resulting solution was then measured at 515 nm using an

157

Agilent 8453 spectrophotometer. A calibration curve was constructed using

158

Trolox as the external standard and results are expressed as of the assays are


Page 6 of 41

Page 7 of 41


159

expressed as mmol Trolox Equivalents per kg of pistachio (kernel, cake or oil),

160

mmol TE/kg.

161

Furthermore, the oxygen radical absorbance capacity (ORAC) was assessed23

162

by preincubation of 20 µl of polar extract and 120 µl of fluorescein (70 nM) at

163

37°C for 15 min. Then, 60 µl of 24 mM 2,2’-azobis(2-amidinopropane)

164

dihydrochloride (AAPH) was added and the mixture incubated at 37°C,

165

measuring fluorescence every minute for 80 min, with a pre-measurement

166

agitation at maximum intensity for 10 s. The experiment was carried out in

167

Nunclon black 96-well flat-bottom plates (Sigma-Aldrich, Madrid, Spain) and

168

measurements acquired using a plate reader with emission and excitation filters

169

set at 528 and 485 nm, respectively (Synergy HT, Bio-Tek; Vermot, USA). The

170

fluorescence curves were normalized with respect to the blank curve (without

171

antioxidant). The calibration curve was prepared using Trolox as the external

172

standard and results of the assays are expressed as mmol Trolox Equivalents

173

per kg of pistachio (kernel, cake or oil), mmol TE/kg.

174

2.5. Analysis of phenolic compounds by HPLC-DAD-MSn. Aliquots (1.75 mL)

175

of phenolic extracts were evaporated to dryness in a rotary evaporator at 35 °C

176

under vacuum, and then redissolved in 200 µL of methanol/water (20:80, v/v) by

177

sonicating (5 min.) and vortexed (2 min) before injecting 20 µL in the HPLC

178

system.

179

Individual phenolic compounds were determined by an HPLC-DAD-ESI-MS/MS

180

method adapted from conditions previously described24. The analysis was

181

performed on an Agilent 1100 series system (Agilent, Waldbronn, Germany)

182

equipped with a photodiode array detector (DAD) and an LC/MSD Trap VL



183

electrospray ionization mass spectrometry (ESI-MS/MS), both coupled with an

184

Agilent ChemStation for data processing.

185

Separation of phenolic compounds was achieved on a narrow-bore column

186

Zorbax Eclipse XDB-C18 (2.1 x 150 mm; 3.5 µm particle; Agilent), with pre-

187

column Zorbax Eclipse XDB-C8 (2.1 x 12.5 mm; 5 µm particle; Agilent), both

188

thermostated at 40 ⁰C by using a ternary gradient at 0.19 mL/min flow

189

rate. Eluents were (A) acetonitrile/water/formic acid (3:88.5:8.5 v/v/v), (B)

190

acetonitrile/water/formic acid (50:41.5:8.5 v/v/v), and (C) methanol/water/formic

191

acid (90:1.5:8.5 v/v/v). The linear solvent´s gradient was as follows: zero min, 98

192

% A 2 % B and 0% C; 8 min, 98 % A 2% B and 0% C; 40 min, 70 % A 17 % B

193

and 13 % C; 54 min, 50 % A 30% B and 20 % C; 54.5 min, 30 % A 40 % B and

194

30 % C; 59 min, 0 % A 50 % B and 50 % C; 60 min, 0 % A 50 % B and 50 % C;

195

67 min, 98 % A 2 % B and 0 % C. For identification, an Ion Trap ESI-MS/MS

196

detector was used in negative ion mode, setting the following parameters: dry

197

gas N2, 8 L/min; drying temperature, 325 °C; nebulizer, N2, 50 psi; scan range,

198

50-1,200 m/z. Identification was based on spectroscopic data (UV-Vis and

199

MS/MS) obtained from authentic standard or data previously reported in

200

literature. Quantification was made using the DAD chromatograms recorded at

201

280 nm (gallotannins, flavanols and flavanones) 360nm (flavonols) and 520 nm

202

(anthocyanins) and calibration curves for the analysed compounds were

203

prepared from pure standards, when available. Table 1 shows the UV-vis spectral

204

characteristic and m/z of molecular and fragmented ions of different phenolic

205

compounds, as well as the standard employed for it quantification.

206

2.6. Chemicals and solvents: catechin, epicatechin, gallocatechin and

207

kaempferol-3-O-galactoside standards were purchased from Sigma Aldrich


Page 8 of 41

Page 9 of 41


208

Chemical Co. (Tres Cantos, Madrid, Spain) and procyanidin B1, procyanidin B2,

209

cyanidin-3-O-galactoside, cyanidin-3-O-glucoside, quercetin, quercetin-3-O-

210

glucoside, quercetin-3-O-rutinoside, quercetin-3-O-rhamnoside and eriodictyol-

211

7-O-glucoside from Extrasynthese (Genay, France). Myricetin-3-O-galactoside

212

and quercetin-3-O-glucuronide were isolated from Petit Verdot red grape skins24.

213

Moreover, DPPH, AAPH, Trolox, procyanidin B3, apigenin-7-O-glucoside,

214

luteolin, daidzein, caffeic acid, gallic acid, coumaric acid, protocatechuic acid,

215

syringic acid and vanillic acid standards (Sigma Aldrich Chemical Co., Tres

216

Cantos, Madrid, Spain) where analysed in order to compare their retention time

217

(Rt), ultraviolet-visible (UV-vis) and mass spectra characteristics with those of the

218

compound present in pistachio samples.

219

Methanol for extraction of phenolics was acquired from Sigma-Aldrich Chemical

220

Co. (Tres Cantos, Madrid, Spain). The eluent for the mobile phases were HPLC-

221

MS grade acetonitrile, methanol and formic acid from (Fisher Scientific, Madrid,

222

Spain). Water for mobile phase was of Milli-Q quality (Merk-Millipore, Darmstadt,

223

Germany).

224

2.7. Statistical analysis: Analysis of Variance (ANOVA) and Principal

225

Component Analysis (PCA) were performed using XLStat 19.5 statistical

226

software (Addinsoft, Paris, France). One-way ANOVA and post hoc Duncan tests

227

were carried. Means were considered statistically different at p < 0.05.

228 229

3. RESULTS AND DISCUSSION

230

3.1. Determination of individual phenolic



231

The identification of phenolic compounds was performed by comparison of the

232

mass spectra (m/z of molecular ions and fragment ions), together with the UV-

233

visible spectra and retention times with those of authentic standards, whenever

234

possible, and also with data found in bibliography. The quantification was done

235

using the standard, when available, or a representative compound of the family.

236

Quantitative results of phenolic compound analysis are expressed on fresh

237

weight (FW) for nuts and by-product (about 5% of moisture) and as mg/kg for oils.

238

Figure 1 shows characteristic chromatograms of phenolic compounds from

239

pistachio nuts at 520, 360 and 280 nm. Information about compounds

240

identification, HPLC retention time and mass and UV-Vis spectra data are

241

summarised in Table 1. Numbers assigned to each compound in table 1,

242

correspond with compounds numbers in figure 1 and tables 2, 3 and 4. A total of

243

26 compounds (compounds 1-26 in table 1) were found in the phenolic profile of

244

pistachio nuts and by-products and 25 of them were identified (three

245

anthocyanins, ten flavonols, one gallotannin, nine flavanols and two flavanones).

246

On the other hand, 8 different compounds (compounds 24 and 27-33 in table 1),

247

all of them at very low concentration, were detected in virgin pistachio oils, but it

248

was only possible to identify one of them, the flavanone eriodictyol. The same

249

qualitative phenolic profile was followed by all the varieties, but significant

250

quantitative differences were observed between them.

251

3.2. Identification and quantification of phenolics in pistachio nuts

252

Table 2, shows the phenolic composition of pistachio nuts. Differences were

253

found among cultivars, being Larnaka variety that with a significantly higher

254

phenolic concentration (4893 mg/kg) and appearing Avdat (4225 mg/kg) as the

255

second one. Mateur and Napoletana showed an intermediate content with a


Page 10 of 41

Page 11 of 41


256

concentration exceeding 3300 mg/kg, whilst Aegina (2240 mg/kg), Kastel (1683

257

mg/kg), Kerman (1936 mg/kg) and Sirora (1980 mg/kg) belongs to the group with

258

the lowest phenolic content. These results match with those reported by Ojeda-

259

Amador et al.10 who found the highest total polyphenols content in Larnaka variety

260

(9550 mg/kg) and the lowest one in Kastel (6168 mg/kg), expressed as gallic acid

261

equivalent when measured by Folin-Ciocalteu method, among eight pistachio

262

varieties.

263

-

Flavanols

264

The major phenolic group in pistachio nuts was the flavanols one, with a

265

concentration ranging from 1522 mg/kg to 4478 mg/kg, where Kastel and Larnaka

266

presented the lowest and highest contents, respectively. This group was

267

constituted by eight different compounds, contributing about 90% of the total

268

amount of phenolics in the nut, with catechin, epicatechin, gallocatechin,

269

procyanidin B1, procyanidin B2 and a proanthocyanidin dimer as principal

270

components. Catechin, which has been determined as one of the most powerful

271

antioxidant among flavanols25, presented the highest content also in Larnaka

272

cultivar with 1016 mg/kg almost seven-fold the concentration of Kastel (155

273

mg/kg) and Kerman (177 mg/kg) varieties. Similar values were obtained by

274

Gültekin-Özgüven et al.26 who stated a concentration of catechin between 343-

275

1545 mg/kg in pistachios from Turkey. Epicatechin, has been previously found

276

in pistachio skins6,13,27; in our work we found epicatechin concentrations from 42

277

mg/kg (Avdat) to 630 mg/kg (Larnaka) in nuts. Similar to our findings, Gültekin-

278

Özgüven et al.26, identified epicatechin in Turkish pistachio nuts with a

279

concentration from 48 to 281 mg/kg, whilst Tomaino et al.13 reported a content of

280

105 mg/kg in the skin of Bronte pistachios, having reported Grace et al.27 a total



281

content of 151 mg/kg in pistachio skin, as well as Liu et al.6 for Kerman cultivar

282

(229 mg/kg). A recent study14 stated that, in addition to variety, rootstock also

283

affects epicatechin content in Kerman pistachios, ranging that from 255 mg/kg to

284

426 mg/kg depending on the rootstock studied. Gallocatechin, has the major

285

concentration in Napoletana cultivar with 692 mg/kg, presenting Kastel the

286

smallest concentration for this compound (283 mg/kg). It is the first time that

287

gallocatechin have been identified in pistachio nuts, but not in nuts; Figueroa et

288

al.28 found a concentration up to 1600 mg/kg of gallocatechin in different walnut

289

genotypes. Catechin gallate was also reported for the first time in pistachio as a

290

minor monomeric flavanol, 31.9 mg/kg in Larnaka cultivar and 3.6 mg/kg in

291

Kastel.

292

Procyanidin B1 (PB1) resulted the most abundant compound in pistachio nuts,

293

reaching values from 227 in Kastel to 1170 mg/kg in Larnaka. This kind of

294

procyanidin has been previously described in pistachio skins, with a

295

concentration of 635 mg/kg27. Procyanidin B2 (PB2) presented a concentration

296

from 190 mg/kg (Aegina) to 301 mg/kg (Larnaka), being reported for the first time

297

in pistachios, but stating Figueroa et al.28 a concentration from 110 mg/kg to 208

298

mg/kg in walnuts cultivars. A proanthocyanidin dimer (compound 2), whose

299

mass spectra showed a molecular ion [M-H]- at m/z 593 with MS2 fragmentation

300

at m/z 423 and 305 and an UV-Vis spectra characteristic of flavanols was found

301

and tentatively identified as a dimer of catechin/epicatechin (procyanidin unit) and

302

gallocatechin/epigallocatechin (prodelphinidin unit). This is the first time that this

303

kind of proanthocyanidin compound is reported in pistachio nuts, but a

304

proanthocyanidin with the same molecular ion was found in malt29. Compound 2

305

reaches its highest concentration in Mateur (558 mg/kg), Avdat (548 mg/kg) and


Page 12 of 41

Page 13 of 41


306

Larnaka (546 mg/kg) cultivars. Kerman (202 mg/kg) and Sirora (231 mg/kg)

307

varieties showed the lowest contents. Another proanthocyanidin, tentatively

308

identified as a galloylated procyanidin dimer (compound 6), and not

309

previously reported in pistachios, was also found, presenting the highest content

310

Larnaka (146 mg/kg) and Avdat (142 mg/kg) cultivars and the lowest one in Sirora

311

(37 mg/kg). Finally, a procyanidin dimer (compound 8), whose UV-Vis and

312

MS/MS spectra was the same than those of PB1, PB2 and PB3 but with a

313

different retention time, appeared within a range of 15-66 mg/kg (Kastel and

314

Larnaka respectively).

315

-

Anthocyanins

316

Other phenolic families such as anthocyanins were also present in pistachio

317

cultivars contributing about 3.5% of the total phenolics, with Larnaka and Avdat

318

as the varieties with the highest concentrations (218 mg/kg and 179 mg/kg

319

respectively) among the eight cultivars. These compounds present only in the

320

skin of pistachios6,13 are responsible of the purple tonality so characteristic of this

321

kind of nuts. Three compounds were identified into this group (table 2), being

322

cyanidin-3-O-galactoside the major one which concentrations ranging from 44

323

mg/kg in Kerman to 214 mg/kg in Larnaka. These data agree with those

324

previously reported by Bellomo et al.30, who found a concentration of 145 mg/kg

325

(as cyanidin-3-O-galactoside) in a turkey pistachio variety. Tomaino et al.13 also

326

reported cyanidin-3-O-galactoside in a concentration of 5865 mg/kg and 35.6

327

mg/kg of cyanidin-3-O-glucoside (expressed as cyanidin-3-O-glucoside) in

328

pistachio skins from Bronte variety, having found our research group a range

329

between 1.4-8.9 mg/kg in pistachio whole nut for the last compound. Kerman

330

variety presented the highest content of cyanidin-3-O-glucoside, despite being



331

the variety with the lowest total anthocyanin content. Another minor anthocyanin

332

was also found and identified as a peonidin-3-O-hexoside (it could be

333

galactoside or glucoside) according to its UV-Vis and MS/MS spectra. This

334

compound accounted up to 1 mg/kg in Avdat cultivar. This is not the first time that

335

peonidin derivatives have been identified in pistachio nuts, peonidin-3-O-

336

glucoside was found by Wu et al.31

337

-

Flavonols

338

Flavonols were the third group in importance, being formed by eleven

339

compounds and accounting about 2.5% of the total phenols. This family was

340

constituted with glycosides of quercetin, myricetin and kaempferol as well as free

341

quercetin. Kerman and Mateur varieties presented the highest content of this

342

phenolic group surpassing 115 mg/kg as total flavonol content. Into this family,

343

myricetin-3-O-galactoside (peak 13, figure 1B) showed the highest content

344

among flavonols in all varieties, appearing six different statistical groups with

345

Mateur (60 mg/kg) presenting the highest content and Kastel the lowest one (16

346

mg/kg). To the best of our knowledge, this is the first time this compound has

347

been identified and quantified as a component of pistachio nut. Assignment was

348

done by comparison of mass spectral data ([M-H]- at m/z 479 with MS2

349

fragmentation at m/z 317), UV-visible spectrum (maximum at 258 nm and 359

350

nm) and retention time (21.1 min) with that of the pure standard. Concerning

351

published data, Erşan et al.32 identified myricetin-3-O-galactoside in pistachio

352

hulls (exo- and mesocarp) but they did not quantify it, whilst Grace et al.27 found

353

another myricetin hexoside, myricetin-3-O-glucoside, both in skin (63 mg/kg) and

354

kernel (2 mg/kg) of pistachios. Regarding quercetin and its glycosylated

355

derivatives, the compounds that appears in a higher concentration were


Page 14 of 41

Page 15 of 41


356

quercetin-3-O-galactoside (4.9 mg/kg, Aegina 15.1 mg/kg, Avdat), quercetin-

357

3-O-glucoside (4.6 mg/kg, Aegina and Napoletana

358

quercetin-3-O-glucuronide (2.8 mg/kg, Aegina – 8.2 mg/kg Kerman) and

359

quercetin-3-O-rhamnoside (2.6 mg/kg, Napoletana – 17.6 mg/kg Kerman);

360

whilst quercetin-3-O-pentoside,

361

rhamnosil-glucoside-5-glucoside and quercetin aglycone contribute with

362

about 2 mg/kg each one to the total content of phenolics. Barreca et al.33 and

363

Erşan et al.32 also identified these compounds (with the exception of quercetin-3-

364

O-rhamnoside and quercetin-3-O-rhamnosil-glucoside-5-glucoside) in extracts of

365

pistachio hull, and Grace et al.27 found quercetin-3-O-galactoside, quercetin-3-O-

366

glucoside, quercetin-3-O-rutinoside and quercetin aglycone in pistachio nuts but

367

this is the first time that quercetin-3-O-glucuronide, quercetin-3-O-pentoside and

368

quercetin-3-O-rhamnoside have been identified (tentatively identification for

369

quercetin-3-O-pentoside, according to their UV-Vis and MS/MS spectra) in

370

pistachio nuts. Moreover, compound 22 (table 1, figure 1B) presented the same

371

UV-Vis and MS/MS spectra than the pure standards of kampferol-3-O-

372

galactoside and kaempferol-3-O-glucoside, however their retention times differed

373

to that of compound 22 so it cannot be assigned; therefore we tentatively

374

identified compound 22 as a kaempferol-hexoside (as previously tentatively

375

identified by Erşan et al.32 in pistachio hulls) with a range of concentration

376

between 4.8 mg/kg (Mateur) and 40.0 mg/kg (Napoletana). Some authors have

377

previously reported the presence of kaempferol aglycone13,34 in pistachios and

378

very recently Noguera-Artiaga et al.14 reported for the first time the presence of a

379

kaempferol glycoside in pistachios (Kerman cultivar, 17 mg/kg) which they

380

assigned as kaempferol-3-O-glucoside. However, our results using pure

11.9 mg/kg, Kerman),

quercetin-3-O-rutinoside, quercetin-3-O-



381

standards (kaempferol-3-O-glucoside and kaempferol-3-O-galactoside) do not

382

support the identity they assigned to this flavanol14. Finally, a compound

383

(compound 26, table 1, Rt = 28.6 min) with [M-H]- at m/z 547, fragment ion at m/z

384

385 and a UV-Vis spectra similar to that of flavonols (maximum at 257 nm and

385

359 nm) has been found, however its identification was not possible and it

386

appears in table 2 as Rt 28.6. This compound presents a very similar

387

concentration for all varieties (about 12 mg/kg) except for Sirora, which contained

388

an amount of 20.5 mg/kg and formed a statistically-separated group with the

389

maximum content.

390

-

Gallotannins

391

Pentagalloyl glucose was the only component of the gallotannis family

392

tentatively identified in pistachio nuts according to their UV-Vis and MS/MS

393

spectra. Its concentration ranged from 4 mg/kg in Kastel to 46 mg/kg in Avdat

394

cultivar. Erşan et al.32 also identified a pentagalloyl glucose (penta-O-galloyl-β-D-

395

glucose) in pistachio hulls but this is the first time that this gallotannin has been

396

found in pistachio nuts.

397

-

Flavanones

398

To conclude, pistachio nuts also contain compounds from flavanone´s family,

399

representing from 0.7% to 2.1% of the total amount of phenolics. They were

400

represented by two compounds, eriodictyol and eriodictyol-7-O-glucoside,

401

being the last one that presenting the higher concentration with Mateur (65.2

402

mg/kg) and Kastel (8.2 mg/kg) as the varieties with the maximum and minimum

403

values. Comparing with bibliography, very close values were published by

404

Rodríguez-Bencomo et al.35 with 22 mg/kg of eriodictyol and 41 mg/kg of

405

eriodictyol-7-O-glucoside in pistachios of Uzun cultivar (Turkey). Grace et al.27


Page 16 of 41

Page 17 of 41


406

only identified eriodictyol-7-O-glucoside in pistachios with a concentration of 59

407

mg/kg in skin and 4 mg/kg kernel, whilst Ballistreri et al.12 only identified

408

eriodictyol in pistachio nuts with a concentration of 21 mg/kg. Tomaino et al.13

409

also identified both compounds in the skin (63 mg/kg, eriodictyol; 366 mg/kg,

410

eriodictyol-7-O-glucoside) and kernel (9 mg/kg, eriodictyol; 32 mg/kg, eriodictyol-

411

7-O-glucoside) of pistachio from Bronte variety. Martorana et al.34 found both

412

compounds, eriodictyol and eriodictyol-7-O-glucoside, in the skin (245 mg/kg and

413

1121 mg/kg, respectively) and kernel (112 mg/kg and 275 mg/kg, respectively) of

414

pistachios from Bronte variety, being these concentrations higher than those

415

reported in the present work and the other authors.

416

Published literature have reported the presence of different phenolic acids in

417

pistachio nuts26 such as syringic, gallic, proto-catechuic, caffeic, ferulic, o-

418

coumaric and p-coumaric acids. In this sense, pure standards of these

419

compounds were injected into our HPLC-DAD-ESI-MS/MS system and no mass

420

spectra/fragmentation, retention time neither UV-vis spectra coincided with any

421

of the signals of our pistachio samples.

422

3.3. Individual phenolic compounds in residual cakes

423

The same 26 phenolic compounds found in pistachio nuts appeared in the

424

resulting residual cake, but in this case with a higher concentration. This means

425

that the extrusion process doesn´t change the natural profile of phenolic

426

compounds presented in the raw material. Furthermore, their concentrations

427

were increased in the by-product due to oily phase removal (~20% oil in residual

428

cake) and the low solubility of the phenolic compounds in the extracted oil. This

429

fact makes it possible to think in novel and potential applications of the residual

430

cakes as functional ingredients and rich sources of bioactive compounds. In this



431

sense, flavanols were the major phenolic compounds determined in by-products

432

and all of them were found in higher concentration than in the nut. This group

433

represents from 88 to 92% of the total phenolic compounds content, with

434

catechin (215 mg/kg, Kastel; 1580 mg/kg, Larnaka) and procyanidin B1 (402

435

mg/kg, Kastel; 1945 mg/kg, Larnaka) as the compounds with the highest

436

concentrations, depending on the variety. Moreover, epicatechin (229 mg/kg,

437

Mateur; 823 mg/kg, Larnaka), gallocatechin (641 mg/kg, Kerman; 1099 mg/kg,

438

Aegina) and proanthocyanidin dimer (compound 2) (312 mg/kg, Kerman; 845

439

mg/kg, Larnaka) were also major compounds in these by-products. Whereas, the

440

galloylated procyanidin dimer (compound 6) and the procyanidin dimer

441

(compound 8) resulted as minor flavanols in the residual cakes with a range

442

between 71 mg/kg, Napoletana

268 mg/kg, Mateur; and 19 mg/kg, Napoletana

443

120 mg/kg, Mateur; respectively. Finally, catechin gallate was the flavanol with

444

the smallest concentration, from 9 mg/kg in Napoletana to 65 mg/kg in Larnaka.

445

Some differences among varieties are shown (tab. 3), for example Aegina (1099

446

mg/kg), Kastel (678 mg/kg), Kerman (641 mg/kg), Napoletana (788 mg/kg) and

447

Sirora (780 mg/kg) contained gallocatechin as principal phenolic. However,

448

Avdat, Larnaka and Mateur contained procyanidin B1 as principal component

449

with a concentration from 1390 mg/kg (Avdat) to 1945 mg/kg (Larnaka).

450

Moreover, epicatechin (4% Mateur, 14% Napoletana) and proanthocyanidin

451

dimer (10% Kerman, 14% Mateur) also represented a very important percentage

452

in residual cakes. The comparison of flavanols levels revealed four statistical

453

different groups among cultivars, again with Larnaka as the variety with the

454

highest content (7089 mg/kg). Comparing with bibliography, just one study

455

related to phenolic compounds in pistachio residual cakes (screw press


Page 18 of 41

Page 19 of 41


456

extraction) from Kerman variety have been published; in that case three flavanols

457

were identified with catechin as the principal component (65.5 mg/kg) followed

458

by procyanidin dimer (15 mg/kg) and epicatechin (7.7 mg/kg)18. These results

459

partially agree with our findings, although they reported a much lower

460

concentration than our research.

461

Anthocyanins were found as the second major group of flavonoids in residual

462

cakes, with cyanidin-3-O-galactoside as the mayor compound. Larnaka cakes

463

showed the highest concentrations of this compound (291 mg/kg) and Kastel and

464

Kerman the lowest (72 and 75 mg/kg). The other two anthocyanins were found in

465

minor amounts. Cyanidin-3-O-glucoside ranged from 1.9 to 4.9 mg/kg in all

466

varieties except for Kerman, which overpassed 12.8 mg/kg, while peonidin-3-O-

467

hexoside showed the lowest concentration in all varieties. Martínez et al.18 also

468

identified cyanidin-3-O-galactoside (21 mg/kg) and cyanidin-3-O-glucoside (0.9

469

mg/kg) as components of pistachio flours obtained after oil extraction although

470

the contents were lower than that obtained in our research.

471

Among flavonols, myricetin-3-O-galactoside was the most abundant in all

472

samples, ranging from 38 mg/kg for Aegina cultivar to 98 mg/kg for Mateur. Other

473

flavonols like quercetin-3-O-galactoside and quercetin-3-O-glucoside were

474

present in halfway concentration, between 6-20 mg/kg. Finally, kaempferol-

475

hexoside, quercetin aglycone and the rest of glycosides of quercetin were

476

minor flavonol in most cakes. The total amount of flavonols contained in this

477

matrix was between 2.4% (Avdat) and 5.7% (Kerman) of the total phenolic

478

compounds. Martinez et al.18 reported free quercetin and myricetin as well as

479

quercetin-3-O-glucoside and a quercetin-3-O-hexoside in the by-products from

480

Kerman variety but in a concentration smaller than 5 mg/kg for every compound.



481

Pentagalloyl glucose, the only gallotannin found, increased its concentration in

482

the residual cake respect to nut, as the other phenolic compounds. Pentagalloyl

483

glucose concentration was from 22 mg/kg to 96 mg/kg in Kerman and Larnaka

484

varieties, respectively and four different statistical groups appeared. Martínez et

485

al.18 found gallic acid in the residual cake but not a pentagalloyl glucose.

486

Eriodictyol-7-O-glucoside with a concentration ranging from 16 mg/kg (Kastel)

487

to 124 mg/kg (Larnaka) and eriodictyol from 6.9 mg/kg (Kerman) to 20.0 mg/kg

488

(Aegina) were the only flavanones found. Previous studies also found similar

489

contents of eriodictyol (11.3 mg/kg) in by-products from Kerman variety18, but the

490

level eriodictyol hexosides found in that study (0.80 mg/kg Approx.) were much

491

lower than the contents of eriodictyol-7-O-glucoside determined among the eight

492

varieties of our study.

493

To conclude, the total concentration of phenolic compounds in pistachio by-

494

products goes from 7774 mg/kg for Larnaka to 3217 mg/kg for Kerman varieties

495

which corresponded to the statistical group with the lowest and highest content

496

respectively.

497

3.4. Individual phenolic compounds in virgin oils

498

Due to the polarity characteristics of phenolic compound their transference from

499

fruit to oil during the extraction process was very low. Concentrations below 0.4

500

mg/kg were found in every sample of virgin oil facing contents between 4507 and

501

1507 mg/kg in nuts. Furthermore, pistachio nuts which presented the highest

502

content of phenolics did not lead to the oils with the highest concentration. Due

503

to their low concentration, phenolic compounds have apparently no effect on the

504

sensory characteristics of these pistachio oils; and the sensory attributes bitter


Page 20 of 41

Page 21 of 41


505

and/or astringent, commonly associated to flavan-3-ols36, the major phenolic

506

compounds found in pistachio nuts, were not detected, resulting “sweet” virgin

507

pistachio oils as described by trained assessors10.

508

Eight different peaks were detected in virgin pistachio oil chromatograms

509

acquired at 280 nm, but only one of them was identified as a phenolic compound,

510

eriodictyol, and the other seven remain unknown (Table 4). The unknown

511

family presented the highest concentration with a percentage accounting about

512

98 % depending on the variety, eriodictyol was found in very low concentration

513

(from 0.01 to 0.06 mg/kg). Saber-Tehrani et al.37 and Sonmezdag et al.38 studied

514

the phenolic composition of virgin pistachio oils and also found eriodictyol among

515

other compounds such as gallic acid, protocatechuic acid, catechin and rutin, all

516

of them in a very low concentration. We could not identify those compounds in

517

our oils despite having the pure standards of all of them.

518

On the other hand, tocopherols - a lipophilic phenolic compound group - have

519

been reported to be present in this virgin pistachio oils, being γ-tocopherol the

520

main homolog with concentrations ranging between 548 mg/kg (Larnaka) and

521

719 mg/kg (Kastel), but also with the presence of α- and β-tocopherols and δ-

522

and γ-tocotrienols (1.2–2.8% each of the total amount)10.

523

3.5. Antioxidant activity of pistachio nuts, residual cakes and virgin oils

524

from different pistachio varieties

525

Figure 2.a., shows the antioxidant activity of pistachio nuts and by-products from

526

different varieties, evaluated by the DPPH and ORAC methods. Both methods

527

have different basis, while DPPH test measures the capacity of the compound to

528

scavenge the stable radical 2,2-Diphenyl-1-picrylhydrazyl by donating a



Page 22 of 41

529

hydrogen atom or an electron, ORAC method evaluates the capacity of

530

compounds to inhibit peroxylradical-induced oxidation of fluorescein initiated by

531

thermal

532

(AAPH). This difference in the basis of these methods led to changes in the

533

relative order of the samples antioxidant activity regarding to the method used.

534

Among pistachio nuts, Larnaka presented the highest Trolox Equivalent

535

Antioxidant Capacity (TEAC) both DPPH (35 mmol TE/kg) and ORAC (330 mmol

536

TE/kg), belonging to a different statistical group. Moreover, Mateur and Avdat

537

presented both a TEAC of 28 mmol TE/kg (DPPH) and 282 mmol TE/kg and 168

538

mmol TE/kg (ORAC) respectively, being the cultivars with the second highest

539

antioxidant activity. Kastel and Kerman were the varieties with the smallest TEAC

540

values with 13 mmol TE/kg and 10 mmol TE/kg (DPPH) and 89 mmol TE/kg and

541

61 mmol TE/kg (ORAC) respectively. These results partially match with those

542

obtained from the total phenolic content when analysed by HPLC, being Larnaka,

543

Mateur and Avdat the varieties with a highest concentration and Kastel and

544

Kerman the ones with the smallest content. These results were similar to those

545

obtained by Rodríguez-Bencomo et al.35, who studied the antioxidant activity, by

546

DPPH method, of a Turkish pistachio cultivar (Uzun) which resulted in 8 mmol

547

TE/kg. Moreover, Wu et al.31 developed a database regarding ORAC in foods,

548

obtaining a concentration of 76 mmol TE/kg for pistachio nuts, with results very

549

close to our results for Kastel (61 mmol TE/kg) and Kerman (89 mmol TE/kg)

550

cultivars.

551

Regarding by-products, the concentration of phenolic compounds respect their

552

corresponding nuts, due to the extraction of the oil, observed previously have a

553

reflection in their antioxidant activity values. Varieties followed the same trend

decomposition

of

2,2’-azobis(2-amidino-propane)


dihydrochloride

Page 23 of 41


554

than nuts, being Larnaka the cultivar with the highest TEAC. However, while

555

according to the DPPH test Larnaka showed higher antioxidant activity than

556

Mateur, in the ORAC test no significant differences were found between these

557

two cultivars. Kerman presented the lowest antioxidant activity with 19 mmol

558

TE/kg and 156 mmol TE/kg for DPPH and ORAC respectively.

559

Finally, all virgin pistachio oils presented a very low TEAC, less than 0.1 mmol

560

TE/kg for DPPH and 1 mmol TE/kg for ORAC, being Kerman that presenting the

561

highest TEAC in both assays with 0.1 mmol TE/kg (DPPH) and 0.8 mmol TE/kg

562

(ORAC).

563

3.6. PCA biplot of phenolic compounds in pistachio nuts from different

564

varieties

565

Throughout all the results and discussion section of this work, it has been shown

566

that genetic differences among eight Pistacia vera L. cultivars grown exactly

567

under the same agronomical and climatic conditions led to statistically significant

568

differences in phenolic compounds content and antioxidant activity. Literature

569

regarding phenolic composition of most of the cultivars presented in this work is

570

scarce and most of available information correspond to Kerman and Napoletana

571

(also known as Bianca)12,14,30. Some studies have stated the differentiation of

572

many pistachio cultivars through DNA analysis techniques such as microsatellite

573

markers; however, as for phenolic compounds, we have not found any

574

information about many of the varieties included on this article, being Kerman,

575

Mateur, Aegina, and Sirora those of our cultivars found in literature39-41..

576

Principal Component Analysis (PCA) of the phenolic composition depicted in

577

Figure 3 explained 62.39 % of the observed variance.



Page 24 of 41

578

Attending to the principal component 1 (F1, 39.70 %), samples are divided in two

579

groups. Avdat, Larnaka and Mateur varieties appeared in the positive part of the

580

F1, which is mainly related to pentagalloyl glucose (factor loading: 0.969); the

581

flavanols procyanidin B1 (0.953), catechin (0.924), catechin gallate (0.908) and

582

procyanidin dimer (0.848), the flavanone eriodictyol-7-O-glucoside (0.831) and

583

the anthocyanin cyanidin-3-O-galactoside (0.889). The rest of the samples –

584

Aegina, Kastel, Kerman, Napoletana and Sirora – belongs to the negative part of

585

the F1 which is related to the quercetin derivative compounds but with

586

kaempferol-hexoside as the component with the highest negative contribution (-

587

0.722).

588

Regarding the second component (F2, 22.69 %), Kerman was clearly separated

589

in the uppers part of the positive side of the PCA graphic and presented the

590

highest differences with Aegina and Napoletana varieties, appearing these ones

591

in the lowest part of the graphic. Kastel, Sirora, Mateur, Avdat and Larnaka had

592

values close to zero for F2. This second component is positively related with

593

quercetin-3-O-rhamnoside

594

quercetin-3-O-glucoside (0.854) and negative connected with quercetin-3-O-

595

pentoside (-0.817) and eriodictyol (-0.787).

596

Considering both components together, pistachio varieties were clearly divided

597

into four groups according to its phenolic composition. Kerman in the left upper

598

side of the graph, Kastel and Sirora in the middle left part, Mateur, Avdat and

599

Larnaka in the middle right part, and finally Aegina and Napoletana in the bottom

600

left side. Since phenolic compounds are related with sensory characteristics of

601

food, these qualitative and quantitative differences in the phenolic compounds

602

profile of nuts, which were also observable in by products, could be responsible

(0.847),

cyanidin-3-O-glucoside


(0.883)

and

Page 25 of 41


603

of a part of the differences in the sensorial properties found among pistachio

604

varieties42. Cultivar groups found in our PCA study did not agree with those

605

expected from data regarding genetic similarity of cultivars. According to the

606

results of microsatellites presented by Fares et al.40, Kerman and Mateur cultivars

607

are very close, but according to the phenolic composition found in our work they

608

have a clearly different profile. On the other hand, the dendogram presented by

609

Motalebipour et al.41, positioned Sirora and Kerman as close cultivars, while

610

Aegina showed some differences. However, in PCA analysis (figure 3), Sirora is

611

closer to Aegina than to Kerman, which seems that phenolic compositional

612

differences must be due to genetic differences located in DNA regions different

613

from those corresponding to the SSR markers used by Motalebipour et al.41 and

614

Fares et al.40

615

To conclude, 25 phenolic compounds were identified and quantified in pistachio

616

nuts and by-products, being reported for the first time five flavonols (myricetin-3-

617

O-galactoside, quercetin-3-O-glucuronide, quercetin-3-O-pentoside, quercetin-3-

618

O-rhamnoside and kaempferol-hexoside), six flavanols (procyanidin B2,

619

gallocatechin,

620

procyanidin dimer and catechin gallate) and one gallotannin (pentagalloyl

621

glucose). In this sense, flavanols were the most abundant phenolic compounds,

622

accounting about 90% of the total phenolics and procyanidin B1 and

623

gallocatechin resulted as the major phenolic components depending on the

624

variety. Moreover, other families such as anthocyanins, flavonols, flavanones and

625

gallotannins were found in all cultivars. To the best of our knowledge, this is the

626

most comprehensive study of phenolics in pistachio nuts and the first report

627

regarding phenolic profile characterization of different pistachio cultivars (growing

proanthocyanidin

dimer,

procyanidin


dimer,

galloylated


628

under the same conditions), their residual cakes and virgin pistachio oils. The

629

high phenolic compounds and reduced fat contents of these residual cakes open

630

new ways to their use as healthy ingredients by the food industry as snacks or in

631

other applications which should be explored.

632 633

Acknowledgement

634

The authors acknowledge their indebtedness to the “Centro de Mejora Agraria,

635

El Chaparrillo-IRIAF” for providing the pistachio samples used in this research

636

work.

637

Founding sources

638

This research project was supported by the Junta de Comunidades de Castilla-

639

La Mancha and the European Regional Development Fund (FEDER; ref. POII-

640

2014-003-P).

641 642 643 644 645 646 647 648 649 650 651 652 653


Page 26 of 41

Page 27 of 41


654

REFERENCES

655

(1) Couceiro, J.F.; Guerrero, J.; Gijón, M.C.; Moriana, A.; Pérez, D.; Rodríguez

656

de Francisco, M. El cultivo del pistacho, 2nd Edition. Publisher: Madrid, Spain,

657

2017.

658

(2) FAOSTAT, Food and Agriculture Organization: FAOSTAT data. 2014.

659

Available at http://faostat.fao.org

660

(3) MAGRAMA (2016). Anuario de Estadística-Avance 2014. Madrid.

661

(4) Kris-Etherton, P.M.; Zhao, G.X.; Binkoski, A.E.; Coval, S.M.; Etherton, T.D.

662

The effects of nuts on coronary heart disease risk. Nutr. Rev. 2001, 59, 103-111.

663

(5) Bulló, M.; Juanola-Falgarona, M.; Hernández-Alonso, P.; Salas-Salvadó, J.

664

Nutrition attributes and health effects of pistachio nuts. Br. J. Nutr. 2015, 113,

665

S79-93.

666

(6) Liu, Y.; Blumberg, J.B.; Chen, C.-Y.O. Quantification and bioaccessibility of

667

California pistachio bioactives. J. Agric. Food Chem., 2014, 62, 1550-1556.

668

(7) Halvorsen, B.L.; Carlsen, M.H.; Phillips, K.M., Bøhn, S.K.; Holte, K.; Jacobs,

669

D.R. Jr.; Blomhoff, R. Content of redox-active compounds (i.e. antioxidants) in

670

foods consumed in the United States. Am. J. Clin. Nutr. 2006, 84, 95–135.

671

(8) Mandalari, G.; Bisignano, C.; Filocamo, A.; Chessa, S.; Sarò, M.; Torre, G.;

672

Faulks, R.M.; Dugo, P. Bioaccessibility of pistachio polyphenols, xanthophylls,

673

and tocopherols during simulated human digestion. Nutrition. 2013, 29, 338–344.

674

(9) Kucukoner, E.; Yurt, B. Some chemical characteristics of Pistacia vera

675

varieties produced in Turkey. Eur. Food Res. Technol. 2003, 217, 308–310.



676

(10) Ojeda-Amador, R.M.; Fregapane, G.; Salvador, M.D. Composition and

677

properties of virgin pistachio oils and their by-products from different cultivars.

678

Food Chem. 2018, 240, 123-130.

679

(11) Anderson, K.A.; Smith, B.W. Use of profiling to differentiate geographic

680

growing origin of raw pistachios. J. Agric. Food Chem. 2005, 53, 410–418.

681

(12) Ballistreri, G.; Arena, E.; Fallico, B. Influence of ripeness and drying process

682

on the poliphenols and tocopherols of Pistacia vera L. Molecules. 2009, 14, 4358-

683

4369.

684

(13) Tomaino, A.; Martorana, M.; Arcoraci, T.; Monteleone, D.; Giovinazzo C.;

685

Saija, A. Antioxidant activity and phenolic profile of pistachio (Pistacia vera L.,

686

variety Bronte) seeds and skins. Biochimie. 2010, 92, 1115-1122.

687

(14) Noguera-Artiaga, L.; Pérez-López, D.; Burgos-Hernández, A.; Wojdyło, A.;

688

Carbonell-Barrachina, A.A. Phenolic and triterpenoid composition and inhibition

689

of α-amylase of pistachio kernels (Pistacia vera L.) as affected by rootstock and

690

irrigation treatment. Food Chem. 2018, 261, 240-245.

691

(15) Saitta, M.; Giuffrida, D.; Di Bella, G.; La Torre, G.L.; Dugo, G. Compounds

692

with antioxidant properties in pistachio (Pistacia vera L.) seeds. In Nuts and seeds

693

in health and disease prevention; Preedy, V.R., Watson, R.R., Patel, V.B., Eds.;

694

Publisher: London, United Kingdom, 2011, pp 909–918.

695

(16) Matthaus, B. Virgin oils - The return of a long known product. Eur. J. Lipid

696

Sci. Technol. 2008, 110, 595-596.


Page 28 of 41

Page 29 of 41


697

(17) Kamal-Eldin, A.; Moreau, R.A. Tree nut oils. In Gourmet and Health-

698

Promoting Specialty Oils; Moreau, R.A., Kamal-Eldin, A., Eds.; Publisher:

699

EE.UU., 2010; Vol. 3, pp. 127-150.

700

(18) Martínez, M.L.; Fabani, M.P.; Baroni, M.V.; Magrini Huaman, R.N.; Ighani,

701

M.; Maestri, D.M.; Wunderlin, D.; Tapia, A.; Feresin, G. Argentinian pistachio oil

702

and flour: a potential novel approach of pistachio nut utilization. J. Food Sci.

703

Technol. 2016, 53, 2260-2269.

704

(19) Gómez-Rico, A.; Salvador, M.D.; Fregapane, G. Virgin olive oil and olive fruit

705

minor constituents as affected by irrigation management based on SWP and TDF

706

as compared to ETc in medium-density young olive orchards (Olea europaea L.

707

cv. Cornicabra and Morisca). Food Res. Int. 2009 42, 1067-1076.

708

(20) Slatnar, A.; Mikulic-Petkovsek, M.; Stampar, F.; Veberic, R.; Solar, A.

709

Identification and quantification of phenolic compounds in kernels, oil and

710

bagasse pellets of common walnut (Juglans regia L.). Food Res. Int. 2015, 67,

711

255-263.

712

(21) Brand-Williams, W.; Cuvelier, M.E.; Berset, C. Use of a free radical method

713

to evaluate antioxidant activity. Lebensm.-Wiss. Technol. 1995, 28, 25-30.

714

(22) Sánchez-Moreno, C.; Larrauri. J.A.; Saura-Calixto, F. A procedure to

715

measure the antiradical efficiency of polyphenols. J. Sci. Food Agric. 1998, 76,

716

270-276.

717

(23) Dávalos, A.; Gómez-Cordovés, C.; Bartolomé, B. Extending Appliability of

718

the Oxigen Radical Absorbance Capacity (ORAC-Fluorescein) Assay. J. Agric.

719

Food Chem. 2004, 52, 48-54.



720

(24) Castillo-Muñoz, N.; Gómez-Alonso, S.; García-Romero, E.; Gómez, V.;

721

Velders, A.H.; Hermosín-Gutiérrez, I. Flavonol 3-O-Glycosides Series of Vitis

722

vinífera Cv. Petit Verdot Red Wine Grapes. J. Agric. Food Chem. 2009, 57, 209-

723

219.

724

(25) Saitta, M.; La Torre, G.L.; Potorti, A.G.; Di Bella, G.; Dugo, G. Polyphenols

725

of pistachio (Pistacia vera L.) oil samples and geographical differentiation by

726

principal component analysis. J. Am. Oil Chem. Soc. 2014, 91, 1595-1603.

727

(26) Gültekin-Özgüven, M.; Davarci, F.; Pasli, A.A.; Demir, N.; Özçelik, B.

728

Determination of phenolic compounds by ultra high liquid chromatography-

729

tandem mass spectrometry: Applications in nuts. LWT- Food Sci. Technol. 2015,

730

64, 42-49.

731

(27) Grace, M.H.; Esposito, D.; Timmers, M.A.; Xiong, J.; Yousef, G.;

732

Komarnytsky, S.; Lila, M.A. In virtro lipolytic, antioxidant and anti-inflammatory

733

activities of roasted pistachio kernel and skin constituents. Food Funct. 2016, 7,

734

4285-4298.

735

(28) Figueroa, F.; Marhuenda, J.; Zafrilla, P.; Villaño, D.; Martínez-Cachá, A.;

736

Tejada, L.; Cerdá, B.; Mulero, J. High-performance liquid chromatography-diode

737

array detector determination and availability of phenolic compounds in 10

738

genotypes of walnuts. Int. J. Food Prop. 2017, 20, 1074-1084.

739

(29) Friedrich, W.; Eberhardt, A., Galensa. Investigation of proanthocyanidins by

740

HPLC with electrospray ionization mass spectrometry. Eur. Food Res. Technol.

741

2000, 211, 56-64.


Page 30 of 41

Page 31 of 41


742

(30) Bellomo, M.G.; Fallico, B. Anthocyanins, chlorophylls and xanthophylls in

743

pistachio nuts (Pistacia vera) of different geographic origin. J. Food Compos.

744

Anal. 2007, 20, 352-359.

745

(31) Wu, X.; Prior, R.L. Identification and characterization of anthocyanins by

746

high-performance liquid chromatography-electrospray ionization-tandem mass

747

spectrometry in common foods in the United States: Vegetables, nuts, and grains.

748

J. Agric. Food Chem. 2005, 53, 3101-3113.

749

(32) Erşan, S.; Güçlü Üstündağ, O.; Carle, R.; Schweiggert, M. Identification of

750

phenolic compounds in red and green pistachio (Pistacia vera L.) hulls (Exo- and

751

mesocarp) by HPLCDAD-ESI-(HR)-MSn. J. Agric. Food Chem. 2016, 5334-5344.

752

(33) Barreca, D.; Laganà, G.; Leuzzi, U.; Smeriglio, A.; Trombetta, D.; Belloco, E.

753

Evaluation of the nutraceutical, antioxidant and cytoprotective properties of ripe

754

pistachio (Pistacia vera L., variety Bronte) hulls. Food Chem. 2016, 196, 493-502.

755

(34) Martorana, M.; Arcoraci, T.; Rizza, L.; Cristani, M.; Bonina, F.P.; Saija, A.;

756

Trombetta, D.; Tomaino, A. In virtro antioxidant and in vivo photoprotective effect

757

of pistachio (Pistacia vera L., variety Bronte) seed and skin extracts. Fitoterapia.

758

2013, 85, 41-48.

759

(35) Rodríguez-Bencomo, J.J.; Kelebek, H.; Sonmezdag, A.S.; Rodríguez-Alcalá,

760

L.M.; Fontecha, J.; Selli, S. Characterization of the aroma-active, phenolic, and

761

lipid profiles of the pistachio (Pistacia vera L.) nut as affected by the single and

762

double roasting process. J. Agric. Food Chem. 2015, 63, 7830-7839.

763

(36) Vidal, S.; Francis, L.; Guyot, S.; Marnet, N.; Kwiatkowski, M.; Gawel, R.;

764

Cheynier, V.; Waters, E.J. The mouth-feel properties of grape and apple

765

proanthocyanidins in a wine-like medium. J. Sci. Food Agric. 2003, 83, 564-573.



766

(37) Saber-Tehrani, M.; Givianrad, M.H.; Aberoomand-Azar, P.; Waqif-Husain,

767

S.; Jafari Mohammadi, S.A. Chemical composition of Iran´s Pistacia atlantica

768

cold-pressed oil. J. Chem. 2013, 1-6.

769

(38) Sonmezdag, A.; Kelebek, H.; Selli, S. Pistachio oil (Pistacia vera L. cv.

770

Uzun): Characterization of key odorants in a representative aromatic extract by

771

GC-MS-olfactometry and phenolic profile by LC-ESI-MS/MS. Food Chem. 2018,

772

240, 24-31.

773

(39) Hormaza, J.I.; Pinney, K.; Polito, V.S. Genetic diversity of pistachio (Pistacia

774

vera, Anacardiaceae) germplasm based on randomly amplified polymorphic DNA

775

(RAPD) markers. Econ. Bot. 1998, 52, 78-87.

776

(40) Fares, K.; Guasmi, F.; Touil, L.; Triki, T.; Ferchichi, A. Genetic diversity of

777

pistachio tree using inter-simple sequence repeat markers ISSR supported by

778

morphological and chemical markers. Biotechnol. 2009, 8, 24-34.

779

(41) Motalebipour, E.Z.; Kafkas, S.; Khodaeiaminjan, M.; Çoban, N.; Gözel, H.

780

Genome survey of pistachio (Pistacia vera L.) by next generation sequencing:

781

development of novel SSR markers and genetic diversity in Pistacia species.

782

BMC Genomics. 2016, 17, 1-14.

783

(42) Tsantili, E.; Takidelli, C.; Christopoulos, M.V.; Lambrinea, E.; Rouskas, D.;

784

Roussos, P.A. Physicial, compositional and sensory differences in nuts among

785

pistachio (Pistacia vera L.) varieties. Sci. Hortic. 2010, 125, 562-568.

786 787 788


Page 32 of 41

Page 33 of 41


789

FIGURES AND TABLES CAPTIONS

790 791

Table 1.- Chromatographic, UV-Vis and mass (negative ionization mode) spectral characteristics of phenolic compounds found in this study.

792

RT (retention time)

793 794 795 796 797 798

Table 2. Content of individual phenolic compounds from the groups of anthocyanins, flavanols, flavanones, flavonols and gallotannins (mg/kg, FW) in pistachio nuts from different cultivars. Values in the same row with different lower-case letters (a – f) are significantly different at p

Comprehensive study of phenolic compounds profile and antioxidant

Recommend Documents