Pistacia vera L. - ACS Publications - American Chemical Society

Identification of Phenolic Compounds in Red and Green Pistachio (Pistacia vera L.) Hulls (Exo- and Mesocarp) by HPLC-DAD-ESI-(HR)-MSn. Sevcan Erşanâ€...
1 downloads 0 Views 1MB Size
Subscriber access provided by UNIV OF NEBRASKA - LINCOLN

Article

Identification of Phenolic Compounds in Red and Green Pistachio (Pistacia vera L.) Hulls (Exo- and Mesocarp) by HPLC-DAD-ESI-(HR)-MS

n

Sevcan Er#an, Özlem Güçlü Üstünda#, Reinhold Carle, and Ralf M. Schweiggert J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01745 • Publication Date (Web): 11 Jun 2016 Downloaded from http://pubs.acs.org on June 12, 2016

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 free 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 accessible to all readers and 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.

Journal of Agricultural and Food Chemistry 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 35

Journal of Agricultural and Food Chemistry

Identification of Phenolic Compounds in Red and Green Pistachio (Pistacia vera L.) Hulls (Exo- and Mesocarp) by HPLC-DAD-ESI-(HR)-MSn

Sevcan Erşan,†, ‡ Özlem Güçlü Üstündağ,‡ Reinhold Carle,†,§ and Ralf M. Schweiggert†*



Chair of Plant Foodstuff Technology and Analysis, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstrasse 25, 70599 Stuttgart, Germany ‡

§

Department of Food Engineering, Faculty of Engineering, Yeditepe University, 26 Ağustos Yerleşimi, Kayışdağı Cad., 34755, Istanbul, Turkey

Biological Science Department, King Abdulaziz University, P. O. Box 80257, Jeddah 21589, Saudi Arabia

*

To whom correspondence should be addressed: Phone +49 711 459 22995, Fax: +49 711 459 24110, Email: [email protected]

ACS Paragon Plus Environment

1

Journal of Agricultural and Food Chemistry

Page 2 of 35

1

ABSTRACT

2

Phenolic constituents of the non-lignified red and green pistachio hulls (exo- and

3

mesocarp) were assessed by HPLC-DAD-ESI-MSn as well as by HR-MS. A total of 66

4

compounds was identified in the respective aqueous methanolic extracts. Among them,

5

gallic acid, monogalloyl glucoside, monogalloyl quinic acid, penta-O-galloyl-β-D-

6

glucose, hexagalloyl hexose, quercetin 3-O-galactoside, quercetin 3-O-glucoside,

7

quercetin 3-O-glucuronide, and (17:1)-, (13:0)-, (13:1)-anacardic acids were detected at

8

highest signal intensity. The main difference between red and green hulls was the

9

presence of anthocyanins in the former ones. Differently galloylated hydrolyzable

10

tannins, anthocyanins, and minor anacardic acids were identified for the first time.

11

Pistachio hulls were thus shown to be a source of structurally diverse and potentially

12

bioactive phenolic compounds. They therefore represent a valuable by-product of

13

pistachio processing having potential for further utilization as raw material for the

14

recovery of pharmaceutical, nutraceutical, and chemical products.

15 16

Keywords: Pistacia vera L., waste, by-product, gallotannin, flavonoid, quercetin

17

glycoside, anthocyanin, anacardic acid, mass spectrometry, high resolution mass

18

spectrometry, bioactive.

2 ACS Paragon Plus Environment

Page 3 of 35

Journal of Agricultural and Food Chemistry

19

INTRODUCTION

20

Pistachio (Pistacia vera L.) seeds are an important commercial crop, whose annual

21

production has doubled during the past decade to reach a worldwide production of

22

approx. 1 million tons in 2013. Top producing countries are Iran followed by the USA

23

and Turkey.1 Although the fruit of the pistachio tree is botanically considered a drupe,

24

pistachio seeds are often regarded as “nuts”, being most commonly consumed as roasted

25

and salted snack food. In addition, a certain amount of pistachio seeds is used as food

26

ingredient, e.g., in pastry, ice-cream, chocolate, confectionary production, and

27

mortadella. Pistachio drupes consist of an edible seed characterized by light-green

28

cotyledons (kernels), a mauvish seed coat (testa) covered by a creamy lignified shell

29

(endocarp), and a green to yellow-red colored outer hull (exo- and mesocarp) depending

30

on the degree of ripeness.2 According to their final use, they are processed to separate

31

either the non-lignified hull for snack pistachios or the entire hull, shell, and seed coat to

32

obtain the isolated kernels.3,4 In both cases, significant amounts of waste accrue having

33

no or low commercial value, which need to be disposed at the processor’s expense.

34

Therefore, valorization of pistachio by-products is of great interest, so far being

35

hampered by missing identification of valuable target compounds which merit utilization.

36

Extracts of pistachio outer hull, the main by-product of pistachio processing, have been

37

shown to exert antioxidant,5–8 antimicrobial,8–10 antimutagenic8 and cytoprotective6 as

38

well as potential antitumor11 and anticancer activities.12 Despite such potent bioactivities,

39

the phytochemicals in the pistachio hull remain to be comprehensively characterized.

40

Only a limited number of previous studies has aimed at elucidating the phenolic profiles 3 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 4 of 35

41

of pistachio hulls (either ripe exocarp or green exo- and mesocarp), suggesting phenolic

42

acids (gallic, protocatechuic, hydroxybenzoic, p-coumaric, and vanillic acids) and

43

flavonoids (quercetin glycosides, isorhamnetin glycosides, naringenin, eriodictyol, and

44

catechin) represent the main phenolic constituents, as only being identified by HPLC-

45

DAD-FLD or HPLC-DAD-MS,5,6 together with (13:0)-, (13:1)-, (15:0)-, (15:1)-, and

46

(17:1)-anacardic acids.13 Since pistachio belongs to the Anacardiaceae, a wide array of

47

phenolic compounds is to be expected in pistachio hull.14 For instance, flavonol

48

glycosides, gallotannins, and specific phenolic lipids have been reported in other

49

members of the Anacardiaceae such as mango (Mangifera indica L.),15–17 Brazilian

50

pepper (Schinus terebinthifolius Raddi),18 and cashew (Anacardium occidentale L.).19

51

Thus, we sought to provide a comprehensive analysis of the phenolic constituents present

52

in pistachio hull using highly sensitive tools such as HR-MSn, particularly, aiming at the

53

further utilization of pistachio hull as a source of potent phenolic bioactives.

54

Pistachio drupes are harvested at different maturity stages based on their desired

55

properties and final use. A high portion of pistachio drupes are harvested red colored at

56

full maturity, and preferentially used for the production of snack food, because their fully

57

developed taste and high shell splitting ratio are important quality traits. However, green

58

drupes, i.e. early-harvested drupes, are desired to produce intensely green colored kernels

59

for pastry and confectionary industry of high market value. Thus, both green and red

60

pistachio hulls, either in fresh or dried form depending of agricultural practices of the

61

country, can be obtained in high amounts as by-products of pistachio processing.

62

Particularly, dried pistachio hulls accrue in large amounts from numerous processors,

63

because drying allows the off-season processing of the otherwise highly perishable

4 ACS Paragon Plus Environment

Page 5 of 35

Journal of Agricultural and Food Chemistry

64

pistachio drupes. Hitherto, no attention has been given to their compositional differences,

65

and a lack of unambiguous sample descriptions in previous studies often hampers their

66

comparative consideration.5–8 Therefore, in this study, we additionally sought to compare

67

the phenolic profiles of red and green pistachio hull as a further reference for their

68

utilization as a phenolic source.

69

For the above mentioned purposes, dried red and green hulls were obtained from a

70

commercial pistachio processor to be extracted with aqueous methanol yielding phenolic-

71

rich samples. Subsequently, these were screened for bioactive phenolic compounds by

72

HPLC-DAD-ESI-MSn and HPLC-HR-MS.

73

MATERIALS AND METHODS

74

Reagents

75

Gallic acid, protocatechuic acid, penta-O-galloyl-β-D-glucose, and (15:0)-anacardic acid

76

were obtained from Sigma–Aldrich Chemie (Steinheim, Germany). β-Glucogallin (1-O-

77

galloyl β-D-glucopyranose) was from PhytoLab (Vestenbergsgreuth, Germany), quercetin

78

3-O-glucuronide, quercetin 3-O-glucoside, quercetin 3-O-galactoside, myricetin 3-O-

79

galactoside and cyanidin 3-O-β-D-galactopyranoside were from Extrasynthèse (Genay

80

Cedex, France). HPLC grade methanol from VWR (Darmstadt, Germany), analytical

81

grade formic acid from Merck (Darmstadt, Germany), and deionized water were used

82

throughout the study.

83 84

Samples and sample preparation

5 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 6 of 35

85

Dried red and green pistachio drupes cv. ‘Uzun’ (Figure 1) were obtained from a

86

pistachio processor (Gaziantep, Turkey). Both red and green pistachio drupes were

87

harvested between August and September in 2013 in Gaziantep region of Turkey,

88

traditionally sun-dried to decrease their moisture contents to 4.6% ± 0.2% as determined

89

according to AOAC Official Method 934.0120 and stored for a year by the producer as

90

usual in Turkey. Commonly, moisture content of pistachio drupes during commercial

91

storage was reported to range from 40-50% in fresh form to 3-5% in dried form21.

92

Average air temperatures were between 3.4 °C and 28.6 °C for this region during the year

93

the drupes were stored with a relative humidity of 29.0-78.9%.22

94

Pistachio drupes were sampled in triplicate (each 500.0 g ± 0.1 g). Hulls and seeds were

95

separated manually and weighted. Proportions of hulls and seeds of the whole pistachio

96

drupe were approx. 20.5% and 37.4% on dry weight basis, respectively. Pistachio hulls

97

were subsequently ground using an A11 laboratory mill (IKA, Staufen, Germany).

98

Ground samples were stored at -20 °C until analyses.

99 100

Extraction of phenolics

101

Ground red and green pistachio hulls (1.00 g ± 0.01 g) were combined with 5 mL of

102

acidified (0.1% HCl, v/v) aqueous methanol (80%, v/v) and subjected to probe sonication

103

at 70% amplitude for 30 s. After centrifugation (1233 x g, 3 min), the supernatant was

104

collected and the solid residues were re-extracted four times as described above. The

105

combined methanolic extracts were evaporated to dryness in vacuo at 30 °C. Then, the

106

dried extract was dissolved in 1 mL of 50% aqueous methanol containing 1% (v/v)

6 ACS Paragon Plus Environment

Page 7 of 35

Journal of Agricultural and Food Chemistry

107

formic acid and membrane-filtered (0.45 µm, regenerated cellulose) into amber vials

108

prior to HPLC analyses.

109 110

HPLC-DAD-ESI-MSn analyses

111

An 1100 series HPLC system with G1322A degasser, a G1312A pump module, a

112

G1313A autosampler, a G1316A column thermostat and a G1315A diode array detector

113

(Agilent, Waldbronn, Germany) was equipped with a 250 mm × 4.6 mm i.d., 5 µm

114

particle size, 100 Å pore size Kinetex C18 core-shell reversed-phase column fitted with

115

4.6 mm x 2 mm i.d. SecurityGuard Ultra C18 guard column both from Phenomenex

116

(Aschaffenburg, Germany). Mobile phases were water for eluent A and methanol for

117

eluent B both containing 1% (v/v) formic acid. Chromatographic separation was achieved

118

at 35 °C column temperature, 1 mL/min flow rate, and using the following gradient

119

profile: isocratic at 2% B for 10 min, from 2-37% B in 27 min, isocratic at 37% B for 5

120

min, from 37-40% B in 18 min, from 40-60% B in 10 min, from 60-100% B in 20 min,

121

isocratic at 100% B for 14 min, from 100-2% B in 1 min followed by isocratic

122

conditioning at 2% B for 7 min prior to the next run. Total run time was 112 min and

123

injection volume was 5 µL.

124

For multi-stage mass spectrometry, the above described LC system was interfaced with

125

an Esquire 3000+ ion-trap mass spectrometer (Bruker Daltonics, Bremen, Germany),

126

which had been fitted with an ESI source operated in positive ion mode for anthocyanins

127

and negative ion mode for all other analyses. Ion scan rate was in the range of m/z 100-

128

2000 at a scan speed of m/z 13,000/s. Nitrogen was used both as drying and nebulizing

129

gas at a flow rate of 11 mL/min and at pressure of 60 psi, respectively. Nebulizer 7 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 8 of 35

130

temperature was 365 °C. The potential on the capillary was set at ±2287 V for both

131

negative and positive ion modes. Helium at a pressure of 4 x 10-6 mbar was used for

132

collision induced dissociation (CID) at a fragmentation amplitude of 1.2 V. Ion

133

chromatograms were analyzed using Esquire Control software.

134 135

HPLC-ESI-HR-MS analyses

136

HPLC-HR-MS analyses were performed using micrOTOF-Q mass spectrometer (Bruker

137

Daltonics, Bremen, Germany) coupled to an Agilent 1200 series HPLC system operated

138

according to the above mentioned parameters. HR-MS was operated in negative ESI

139

mode with +2200 V capillary voltages. Ion scan rate was in the range of m/z 250-3000 at

140

a scan speed of m/z 12,900/s (at m/z 996.8). Nitrogen was used both as nebulizing and

141

drying gas at a pressure of 3.0 bar and a flow rate of 8 L/min, respectively. Drying gas

142

was heated to 300 °C. Instrument calibration was carried out according to the

143

manufacturer’s instructions using sodium formate. DataAnalysis 3.4 software was used to

144

generate molecular formulas based on accurate mass measurements.

145

RESULTS AND DISCUSSION

146

Analysis of phenolic compounds by HPLC-DAD-ESI-MSn and HPLC-ESI-HR-MS

147

A total of 66 phenolic compounds was detected in pistachio hull extracts, representative

148

structures are presented in Figure 2. Monitoring was performed at different wavelengths

149

as shown in Figure 3. All analytical data obtained for the examined compounds are listed

150

in Table 1, including their retention times, UV absorption maxima, ESI-MSn

8 ACS Paragon Plus Environment

Page 9 of 35

Journal of Agricultural and Food Chemistry

151

fragmentation pattern, and the respective high resolution mass-to-charge (m/z) signals.

152

The identification of the examined 66 compounds led to their allocation into three

153

structurally related groups, i.e., gallotannins, flavonoids, and anacardic acids

154 155

Gallotannins

156

Gallotannins represent a subgroup of hydrolyzable tannins, more specifically being esters

157

of at least one gallic acid molecule with polyols such as sugars, shikimic acids or quinic

158

acids.23 While gallic acid, 2, was tentatively identified by comparing the obtained

159

retention time, UV absorption and mass spectra to those of an authentic standard, a total

160

of 30 related gallic acid derivatives and gallotannins was identified in pistachio hull

161

extracts, mainly based on the formation of characteristic product ions at m/z 169 ([gallic

162

acid-H]-) and 125 ([gallic acid-CO2-H]-) as well as due to the specific neutral loss of a

163

dehydrated galloyl moiety (152 Da). The identification was corroborated by their UV

164

absorption spectra and high resolution MS data.

165 166

Galloyl hexoses

167

Compound 1 with a parent ion [M-H]- at m/z 331 revealed a daughter ion [M-H-162]- at

168

m/z 169 upon CID fragmentation, indicating the loss of a hexose moiety. It was identified

169

as 1-O-galloyl β-D-glucopyranose (β-glucogallin) after comparing its retention time, UV

170

absorption and mass spectra with those of an authentic standard. Glucogallin has already

171

been reported in many plants including other members of Anacardiaceae, such as

9 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 10 of 35

172

Brazilian pepper18 and mango,24 and is considered a primary metabolite and galloyl donor

173

for gallotannin biosynthesis.25,26

174

The parent ion [M-H]- at m/z 493 of compound 8 formed daughter ions [M-H-162]- at m/z

175

313 and [M-H-162-162]- at m/z 169. In agreement with its UV and high resolution MS

176

data, compound 8 was tentatively identified as a galloyl dihexose. Similar galloyl

177

dihexoses have been previously found in plant parts of other Anacardiaceae, namely in

178

sumac (Rhus coriaria L.).27 Compounds 6 and 7 exhibited both UV absorption spectra

179

and high resolution mass signals identical to those of compound 8 (Table 1). However,

180

their different fragmentation patterns indicated that they might represent distinct isomers

181

of galloyl dihexoses as reported previously.28 For instance, while predominant CID

182

daughter ions [M-H-180]- at m/z 313 and [M-H-324]- at m/z 169 were observed for

183

compounds 6 and 8, those of compound 7 were observed at m/z 271 ([M-H-162-60]-), at

184

m/z 211 ([M-H-162-120]-) and at m/z 313 ([M-H-180]-) (Table 1).24

185

Two further minor compounds, 14 and 16, were tentatively assigned as digalloyl hexoses

186

(m/z 483) due to loss of a dehydrated galloyl (152 Da) and a hexose (162 Da) moiety,

187

ultimately yielding gallic acid (m/z 169) as daughter ion. The loss of dehydrated galloyl

188

units (152 Da) may indicate depsidically linked gallic acids due to previously reported

189

predominance of galloyl fission in these types of linkages.29

190

The pseudo-molecular ions [M-H]- of three compounds, 31, 37 and 39, exhibited

191

sequential losses of galloyl moieties (152 Da) from their parent ions at m/z 787, 939 and

192

1091, respectively. These compounds were tentatively identified as tetra- , penta- , and

193

hexagalloyl hexose, respectively, based on their retention order, UV absorption maxima,

194

high resolution MS data, and MSn fragmentation pattern as compared with literature.30,31 10 ACS Paragon Plus Environment

Page 11 of 35

Journal of Agricultural and Food Chemistry

195

The identity of compound 37 was further confirmed as penta-O-galloyl-β-D-glucose after

196

comparing its analytical data with that of the corresponding authentic standard.

197

Hexagalloyl hexose, 39, was the gallotannin having the highest degree of galloylation

198

that was detected as a separate peak, although several unresolved peaks eluting after 40

199

min (Figure 3) may be attributed to the presence of higher degrees of galloylated tannins.

200

The chromatographic separation of such highly galloylated gallotannins was previously

201

shown to be most intricate due to the increased number of possible gallotannin isomers

202

with the increase in the number of galloyl units.32 In agreement, our extracted ion

203

chromatograms containing the respective traces of penta-, hexa-, hepta-, octa-, and

204

nonagalloyl tannins are shown in Figure 4 to illustrate the increasing complexity of these

205

compounds and their related mass signals. In addition, characteristic doubly charged

206

pseudo-molecular ions [M-2H]2− and corresponding fragment ions [M-n × 152-2H]2−

207

with (n = 1–4) were observed in the region of highly galloylated gallotannins.18,33

208 209

Galloyl quinic acids

210

By analogy to differently galloylated hexoses, quinic acid was found to be galloylated to

211

different degrees. Compounds 4, 19, and 28 were tentatively identified as mono-, di- and

212

trigalloyl quinic acids due to sequential losses of galloyl moieties (152 Da) from their

213

parent ions at m/z 343, 495 and 647, respectively, and the formation of a final product ion

214

at m/z 191 (deprotonated quinic acid).34,35 HR-MS measured exact molecular masses

215

were also in good agreement with calculated masses of the respective galloyl quinic

216

acids, corroborating their identification (Table 1).

11 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 12 of 35

217

An additional quinic acid derivative (compound 29) was also tentatively identified due to

218

the formation of a putative product ion at m/z 191 (deprotonated quinic acid) upon CID

219

experiments. Compounds with different degrees of galloyl quinic acids were also

220

reported in Pistacia lentiscus L.,34 Myrtus communis L.,36 green tea (Camellia sinensis

221

L.), and tara (Caesalpinia spinosa (Molina) Kuntze).35

222 223

Galloyl shikimic acids

224

Three compounds, 9, 10 and 12, with identical UV absorption spectra, identical high

225

resolution MS data, and identical pseudo-molecular ions [M-H]- at m/z 325 were

226

characterized by the loss of 156 Da, yielding a daughter ion at m/z 169. In agreement with

227

our analytical data, these compounds were identified as monogalloyl shikimic acids. Two

228

compounds, 23 and 24, with pseudo-molecular ions [M-H]- at m/z 477 produced daughter

229

ions at m/z 325 due to the neutral loss [M-H-152]- of a putative galloyl moiety. Further

230

fragmentation of m/z 325 yielded spectra similar to the above mentioned monogalloyl

231

shikimic acids, 9, 10, and 12. In agreement with their chemical formula (Table 1),

232

compounds 23 and 24 were tentatively identified as the isomers of digalloyl shikimic

233

acids. Despite their uncommon presence in plants, differently galloylated shikimic acids

234

were previously reported in other plants,37–40 including Brazilian pepper from the

235

Anacardiaceae.18 As shikimic acid has been reported as a precursor of gallate synthesis,41

236

galloylated shikimic acids may represent intermediates for the biosynthesis of higher

237

molecular weight gallotannins.

238

The presence of gallotannins has previously been reported in other Anacardiaceae such as

239

mango30 and sumac.27 However, this is the first detailed report on pistachio hull 12 ACS Paragon Plus Environment

Page 13 of 35

Journal of Agricultural and Food Chemistry

240

gallotannins. Behgar et al.42 previously reported the total “tannin content” of pistachio

241

hull as determined by an unspecific protein precipitation based radial diffusion assay.

242

Other gallic acid derivatives

243 244

Two peaks, 13 and 20, exhibited parent ions [M-H]- at m/z 321. Their CID fragmentations

245

resulted in product ions at m/z 169 and 125 characteristic of gallic acid. Thus, these

246

compounds were tentatively identified as digallic acids, although their differences on

247

linkages between the two galloyl moieties remain unknown despite their slightly different

248

UV absorption maxima (Table 1). The pseudo-molecular ion [M-H]- of compound 32 at

249

m/z 473 tentatively indicated the presence of a trigallic acid due to sequential loss of two

250

galloyl moieties, yielding product ions specific for gallic acid. We were unable to provide

251

evidence that these three compounds, 13, 20, and 32, represented depsides, although

252

depsidically linked gallic acids were previously found in tanoak acorns (Notholithocarpus

253

densiflorus (Hook. & Arn. ) Manos, Cannon & S. H. Oh)28 and Anacardiaceae such as

254

sumac,27 mango peel31 as well as in Rhus chinensis Mill. leaves, a traditional Chinese

255

herb.43

256

Compound 15 exhibited a parent ion [M-H]- at m/z 183 with a corresponding

257

demethylated product ion [M-H-15]- at m/z 168, thus being tentatively assigned as methyl

258

gallate in agreement with previous reports.31 Similarly, compound 36 with a parent ion at

259

m/z 335 was identified as methyl digallate due to loss of a digalloyl moiety during CID.

260

In addition, a product ion [M-H-152]- at m/z 183 (methyl gallate) was observed in the

261

MSn spectra. Noteworthy, methyl gallate, 15, and methyl digallate, 36, may represent

262

potential artefacts, resulting from methanolysis of depsidically linked gallotannins in the 13 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 14 of 35

263

course of extraction and analysis.29,35 In agreement, an increase in both peak area and

264

height of compound 15 and 36 was observed upon re-analysis of methanolic extracts kept

265

at room temperature for 24 h (data not shown).

266

Compound 26 produced a parent ion [M-H]- at m/z 319 and its HR-MS measured exact

267

molecular mass revealed a good fit to luteic acid, a digallic acid with an additional C-C

268

bond between its benzene rings (Table 1). Although compound 17, 26 and 44 had

269

common MSn product ions at m/z 139, which may indicate the formation methyl

270

pyrogallol fragments upon CID, the identities of compounds 17 and 44 yet remain

271

unknown. Luteic acid, a molecule present in the structure of myrobalanitannin, has been

272

reported in the fruits of Terminalia chebula Renz. as an intermediate of ellagic acid

273

biosynthesis.44

274

Five compounds, 3, 11, 18, 21, and 22, were tentatively identified as gallic acid

275

derivatives due to the formation of a deprotonated gallic acid at m/z 169 as MSn daughter

276

ion, although their further characterization remains pending. The yet unidentified gallic

277

acid derivative, compound 3, may contain nitrogen due to its even-numbered mass-to-

278

charge ratio at m/z 296 (Table 1).

279 280

Flavonoids

281 282

Flavonols

283

A total of 17 flavonols was tentatively identified in red pistachio hull extracts, including

284

quercetin, myricetin and kaempferol derivatives. Quercetin derivatives, 38, 40-43, 45-47,

14 ACS Paragon Plus Environment

Page 15 of 35

Journal of Agricultural and Food Chemistry

285

50 and 51, were the major flavonol constituents of the hulls under investigation,

286

according to their highly characteristic UV absorption maxima at ca. 350 nm, common

287

fragment ions at m/z 301 (deprotonated quercetin), and a characteristic fragment of the

288

quercetin aglycone at m/z 179. The major quercetin derivatives, quercetin 3-O-

289

galactoside, 40, quercetin 3-O-glucuronide, 41, and quercetin 3-O-glucoside, 42, were

290

identified by comparing their retention times, UV absorption and MS data to those of

291

authentic standards (Figure 3). In addition to quercetin hexosides, a quercetin pentoside,

292

47, was tentatively identified. Moreover, several types of galloylated quercetin glycosides

293

were tentatively identified according to their analytical data shown in Table 1, such as

294

quercetin galloyl hexosides, 38, 43, and 45, quercetin galloyl deoxyhexose, 46, quercetin

295

galloyl hexuronide, 50, and quercetin galloyl pentoside, 51.

296

Four myricetin derivatives, 30, 33-35, were tentatively assigned based on their

297

characteristic fragment ion at m/z 317 (myricetin aglycone) and their characteristic

298

secondary fragments at m/z 299 and 271. Compound 33 was tentatively identified as

299

myricetin hexuronide due to its parent ion [M-H]- at m/z 493 and the derived, previously

300

reported29 predominant daughter ion at m/z 317, indicating the loss of an uronic acid

301

moiety in agreement with its high resolution MS data. Compounds 34 and 35 were

302

tentatively identified as myricetin hexosides based on the analytical data presented in

303

Table 1. Compound 34 was further identified as myricetin 3-O-galactoside after

304

comparison of its analytical data with that of an authentic standard. The parent ion [M-H]-

305

of compound 30 at m/z 631 was characterized by the sequential loss of 162 Da (hexose)

306

and 152 Da (galloyl moiety), thus being identified as myricetin galloyl hexoside in

307

agreement with its high resolution MS data. Moreover, three kaempferol derivatives,

15 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 16 of 35

308

namely two hexosides and one pentoside, 49, 52 and 53, were identified in trace amounts.

309

HR-ESI-MS accurate mass measurements were in agreement with all proposed flavonols

310

(Table 1).

311

Flavonols of pistachio hull have been recently investigated using HPLC-DAD.5

312

According to this study, quercetin rutinoside represented the major flavonol accompanied

313

by lower amounts of quercetin, quercetin galactoside, quercetin glucoside, kaempferol

314

and isorhamnetin glycosides. The presence of these flavonol derivatives was confirmed

315

by our study, except for quercetin rutinoside and isorhamnetin derivatives which were not

316

detected in our samples. In further contrast, in our samples, quercetin galactoside,

317

quercetin glucuronide and quercetin glucoside were the major flavonols accompanied by

318

low amounts of myricetin and kaempferol derivatives (Table 1).

319 320

Anthocyanins

321

Compound 26 was the main peak observed in the chromatogram recorded at 520 nm

322

(Figure 3), whose parent ion [M]+ at m/z 449 exhibited a characteristic daughter ion [M-

323

162]+ at m/z 287, the cyanidin aglycone. After comparing its retention time, UV/Vis

324

absorption and mass spectra with those of an authentic standard, compound 25 was

325

tentatively identified as cyanidin 3-O-β-D-galactopyranoside. Minor amounts of a

326

putative cyanidin pentoside, 27, were also tentatively identified based on the formation of

327

a cyanidin aglycone at m/z 287 after the loss of a pentose (132 Da) upon CID

328

fragmentation. The presence of cyanidin 3-O-galactoside in P. vera seed coat (named as

329

“skin” in the study)45,46 and cyanidin 3-O-glucoside in leaves of P. lentiscus34 has

330

previously been reported. However, this is the first report on the occurrence and 16 ACS Paragon Plus Environment

Page 17 of 35

Journal of Agricultural and Food Chemistry

331

identification of cyanidin derivatives in red pistachio hull, although a previous study

332

reported

333

spectrophotometrically.47 Interestingly, 7-O-methylated anthocyanins have not been

334

observed in our study, although their presence in mango,48 cashew apple,49 Brazilian

335

pepper,18 and sumac27 has been proposed to be a chemotaxonomic marker of the

336

Anacardiaceae. Noteworthy, our samples underwent drying and storage prior to analyses,

337

and thus, further studies on freshly collected pistachio fruits should be done. On the other

338

hand, it is worth mentioning that P. vera has occasionally been classified in its own

339

family Pistaciaceae rather than in the Anacardiaceae, which may be supported by the

340

aforementioned lack of 7-O-methylated anthocyanins.

total

anthocyanin

contents

of

pistachio

hulls

as

determined

341 342

Anacardic acids

343

A total of 11 anacardic acids 54-60, 62-64, 66, with different lengths of alkyl chains

344

(C13, C15 and C17) and saturation degrees (fully saturated or mono-, di-, or

345

triunsaturated) were identified in pistachio hull extracts (Table 1). They eluted late at 88-

346

94 min (Figure 3) due to their lipophilic alk(en)yl side chain. In the following, the

347

compounds are named according to the length of their side chain and the number of

348

double bonds in the side chain (Figure 2). All of them produced similar UV absorption

349

spectra with maximum absorbance at 250 and 311 nm. Their CID mass spectra had a

350

product ion [M-44]- in common, indicating a CO2 loss from the phenolic carboxyl group.

351

Furthermore, characteristic product ions at m/z 106 or m/z 107 have been reported to

352

occur due to the elimination of the phenol group, while product ions at m/z 119 and 149

353

were described to result from fragmentation at the allyl position of unsaturated anacardic 17 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 18 of 35

354

acids.50,51 Compound 63 was assigned as (15:0)-anacardic acid after comparing its

355

retention time, UV absorption and mass spectra including the characteristic loss of CO2

356

(44 Da), and the product ion at m/z 106 with those of an authentic standard. The

357

identification of further anacardic acids was based on UV and mass spectra including

358

accurate mass measurements and the corresponding molecular formulas that were

359

consistent with previously published data of Jerz et al.50 As shown in Figure 3, the most

360

abundant representatives were (13:0)-, 59, (13:1)-, 58, (15:0)-, 63, (15:1)-, 60, and (17:1)-

361

anacardic acids, 64, being in agreement with an earlier study on phenolic acids of P.

362

vera.13 Their elution occurred later with increasing chain length and decreasing degree of

363

saturation as previously shown.50 Based on the oxidative degradation of these major

364

compounds, Yalpani et al.13 determined the localization of the double bond of the

365

unsaturated (13:1)-, (15:1)-, and (17:1)-anacardic acids to be at the 8 position of the alkyl

366

chain for monounsaturated anacardic acids from green pistachio hull (named as “outer

367

green shell” in their study). We assume that this allocation of the double bonds may also

368

be valid for our results. Besides confirming these major compounds, our study is the first

369

report of the occurrence of six minor anacardic acids, namely (16:1)-, (13:2)-, (11:0)-,

370

(15:3)-, (17:2)-, and (17:0)-anacardic acids. In contrast to pistachio kernels,51 cardanols,

371

decarboxylated derivatives of anacardic acids, were not detected in pistachio hulls.

372

Interestingly, anacardic acids are currently considered to be chemotaxonomic markers of

373

the Anacardiaceae,14 consistently occurring in cashew nuts and shells,50 as well as in

374

mango.52 These findings might be of interest for the above mentioned discussion on the

375

assignment of the genus Pistacia.

376

18 ACS Paragon Plus Environment

Page 19 of 35

377

Journal of Agricultural and Food Chemistry

Minor compound

378

Compound 5 with a parent ion [M-H]- at m/z 153 was tentatively identified as

379

protocatechuic acid after comparison of its analytical data with those of an authentic

380

standard. Protocatechuic acid has been reported in pistachio hull before.5,6

381 382

Comparison of red and green hulls

383

When red and green type pistachio hulls were compared (Figure 3), virtually identical

384

phenolic profiles were observed, except for the anthocyanins that only occurred in red

385

hulls. These findings indicate that green and red pistachio hulls may be utilized without

386

separation as a source of phenolic compounds. However, further research on the quantity

387

and contribution of each class of phenolic compounds to the biological activity of red and

388

green pistachio hull extracts should be performed to determine technologically optimal

389

recovery strategies for phenolics from different types of pistachio hulls.

390 391

In conclusion, the complex phenolic profiles of dried red and green P. vera hulls were

392

characterized in this study to provide basic knowledge for their future utilization. Apart

393

from anthocyanins that are characteristic of red hulls, phenolic constituents of pistachio

394

hulls may largely be grouped in three major phenolic classes: gallotannins, flavonoids

395

and anacardic acids. The identity of the gallotannins of pistachio hull was elucidated for

396

the first time, revealing the presence of galloyl hexoses with up to nine galloyl units, and

397

galloyl quinic and shikimic acids with up to three galloyl units. Pistachio hulls also

398

contained glycosides of flavonoids such as quercetin glycosides and a cyanidin 3-O-β-D19 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 20 of 35

399

galactopyranoside. Furthermore, anacardic acids, a distinct class of polar phenolic lipids,

400

were identified and may be useful as chemotaxonomic markers. In brief, a wide range of

401

phenolic compounds was present in pistachio hulls, ranging from simple (gallic acid) to

402

very complex ones (gallotannins) and from polar/mid-polar (gallotannins and flavonoids)

403

to amphiphilic (anacardic acids) ones. Thus, pistachio hull represents an interesting

404

source for the production of multifunctional phenolic extracts. Further research on the

405

extraction and isolation of these compounds and the determination of their relation with

406

attributed biological functions should be encouraged.

407

ACKNOWLEDGEMENTS

408

We thank Joachim Trinkner (University of Stuttgart, Germany) for conducting the

409

HPLC-ESI-HR-MS analyses.

410

Funding

411

TUBITAK 2211/C and 2214/A Programs are acknowledged for financial support of one

412

of the authors (S.E.).

413

Notes

414

The authors declare no competing financial interest.

415

ABBREVIATIONS

416

(11:0)-anacardic acid, undecylsalicylic acid; (13:0)-anacardic acid, tridecylsalicylic acid;

417

(13:1)-anacardic

acid,

tridecenylsalicylic

acid;

(13:2)-anacardic

acid, 20

ACS Paragon Plus Environment

Page 21 of 35

Journal of Agricultural and Food Chemistry

418

tridecadienylsalicylic acid; (15:0)-anacardic acid, pentadecylsalicylic acid; (15:1)-

419

anacardic

420

pentadecatrienylsalicylic acid; (16:1)-anacardic acid, hexadecenylsalicylic acid; (17:0)-

421

anacardic acid, heptadecylsalicylic acid; (17:1)-anacardic acid, heptadecenylsalicylic

422

acid; (17:2)-anacardic acid, heptadecadienylsalicylic acid.

acid,

pentadecenylsalicylic

acid;

(15:3)-anacardic

acid,

21 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

423

REFERENCES

424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466

(1) (2)

(3) (4) (5)

(6)

(7)

(8)

(9)

(10) (11) (12)

(13) (14)

(15)

Page 22 of 35

FAO. FAOSTAT database of the Food and Agriculture Organization of the United Nations. (http://faostat.fao.org/) (accessed Feb 28, 2016). Seeram, N. P.; Zhang, Y.; Bowerman, S. Phytochemicals and Health Aspects of Pistachio (Pistacia vera L.). In Tree Nuts: Composition, Phytochemicals, and Health Effects; Alasalvar, C., Shahidi, F., Eds.; CRC Press: Boca Raton, 2008; pp 295–303. Bloch, F.; Brekke, J. E. Processing of pistachio nuts. Econ. Bot. 1960, 14, 129– 144. Payne, T. J. Nuts. In Processing Fruits: Science and Technology; Barrett, D. M., Somogyi, L., Ramaswamy, H., Eds.; CRC Press: Boca Raton, 2004; pp 765–808. Barreca, D.; Laganà, G.; Leuzzi, U.; Smeriglio, A.; Trombetta, D.; Bellocco, E. Evaluation of the nutraceutical, antioxidant and cytoprotective properties of ripe pistachio (Pistacia vera L., variety Bronte) hulls. Food Chem. 2016, 196, 493–502. Garavand, F.; Madadlou, A.; Moini, S. Determination of phenolic profile and antioxidant activity of pistachio hull using HPLC-DAD-ESI-MS as affected by ultrasound and microwave. Int. J. Food Prop. 2015, doi:0.1080/10942912.2015.1099045. Goli, A. H.; Barzegar, M.; Sahari, M. A. Antioxidant activity and total phenolic compounds of pistachio (Pistachia vera) hull extracts. Food Chem. 2005, 92, 521– 525. Rajaei, A.; Barzegar, M.; Mobarez, A. M.; Sahari, M. A.; Esfahani, Z. H. Antioxidant, anti-microbial and antimutagenicity activities of pistachio (Pistachia vera) green hull extract. Food Chem. Toxicol. 2010, 48, 107–112. Castillo-Juárez, I.; Rivero-Cruz, F.; Celis, H.; Romero, I. Anti-Helicobacter pylori activity of anacardic acids from Amphipterygium adstringens. J. Ethnopharmacol. 2007, 114, 72–77. Kubo, I.; Muroi, H.; Himejima, M. Structure-antibacterial activity relationships of anacardic acids. J. Agric. Food Chem. 1993, 14, 1016–1019. Kubo, I.; Ochi, M.; Vieira, P. C.; Komatsu, S. Antitumor agents from the cashew (Anacardium occidentale) apple juice. J. Agric. Food Chem 1993, 41, 1012–1015. Alam-Escamilla, D.; Estrada-Muñiz, E.; Solís-Villegas, E.; Elizondo, G.; Vega, L. Genotoxic and cytostatic effects of 6-pentadecyl salicylic anacardic acid in transformed cell lines and peripheral blood mononuclear cells. Mutat. Res. Genet. Toxicol. Environ. Mutagen. 2015, 777, 43–53. Yalpani, M.; Tyman, J. H. P. The phenolic acids of Pistachia vera. Phytochemistry 1983, 22, 2263–2266. Schulze-Kaysers, N.; Feuereisen, M. M.; Schieber, A. Phenolic compounds in edible species of the Anacardiaceae family – a review. RSC Adv. 2015, 5, 73301– 73314. Berardini, N.; Fezer, R.; Conrad, J.; Beifuss, U.; Carle, R.; Schieber, A. Screening of mango (Mangifera indica L.) cultivars for their contents of flavonol O- and xanthone C-glycosides, anthocyanins, and pectin. J. Agric. Food Chem. 2005, 53, 1563–1570. 22 ACS Paragon Plus Environment

Page 23 of 35

467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512

Journal of Agricultural and Food Chemistry

(16)

(17)

(18)

(19)

(20)

(21) (22) (23) (24)

(25)

(26)

(27)

(28)

(29)

Engels, C.; Gänzle, M. G.; Schieber, A. Fractionation of gallotannins from mango (Mangifera indica L.) kernels by high-speed counter-current chromatography and determination of their antibacterial activity. J. Agric. Food Chem. 2010, 58, 775– 780. Geerkens, C. H.; Matejka, A. E.; Carle, R.; Schweiggert, R. M. Development and validation of an HPLC method for the determination of alk(en)ylresorcinols using rapid ultrasound-assisted extraction of mango peels and rye grains. Food Chem. 2015, 169, 261–269. Feuereisen, M. M.; Hoppe, J.; Zimmermann, B. F.; Weber, F.; Schulze-Kaysers, N.; Schieber, A. Characterization of phenolic compounds in Brazilian pepper (Schinus terebinthifolius Raddi) exocarp. J. Agric. Food Chem. 2014, 62, 6219– 6226. Paramashivappa, R.; Kumar, P. P.; Vithayathil, P. J.; Rao, A. S. Novel method for isolation of major phenolic constituents from cashew (Anacardium occidentale L.) nut shell liquid. J. Agric. Food Chem. 2001, 49, 2548–2551. AOAC Official Method 934.01. Moisture in Animal Feed. In Official Methods of Analysis of the Association of Official Analytical Chemists; AOAC International: Gaithersburg, MD, 2002; Vol. 1. MEB. Antepfıstığı Yetiştiriciliği; T.C. Milli Eğitim Bakanlığı: Ankara, Türkiye, 2010. TUIK. Turkey’s Statistical Yearbook 2013; Turkish Statistical Institute: Ankara, Türkiye, 2014. Khanbabaee, K.; van Ree, T. Tannins: Classification and definition. Nat. Prod. Rep. 2001, 18, 641–649. Krenek, K. A.; Barnes, R. C.; Talcott, S. T. Phytochemical composition and effects of commercial enzymes on the hydrolysis of gallic acid glycosides in mango (Mangifera indica L. cv. “Keitt”) pulp. J. Agric. Food Chem. 2014, 62, 9515– 9521. Gross, G. G. Synthesis of mono-, di-and trigalloyl-β-ᴅ-glucose by β-glucogallindependent galloyltransferases from oak leaves. Z. Naturforsch. C 1983, 38, 519– 523. Niemetz, R.; Niehaus, J. U.; Gross, G. G. Biosynthesis and Biodegradation of Complex Gallotannins. In Plant Polyphenols 2: Chemistry, Biology, Pharmacology, Ecology; Gross, G. G., Hemingway, R. W., Yoshida, T., Eds.; Springer US: New York, 2012; pp 63–82. Abu-Reidah, I. M.; Ali-Shtayeh, M. S.; Jamous, R. M.; Arráez-Román, D.; SeguraCarretero, A. HPLC-DAD-ESI-MS/MS screening of bioactive components from Rhus coriaria L. (Sumac) fruits. Food Chem. 2015, 166, 179–191. Meyers, K. J.; Swiecki, T. J.; Mitchell, A. E. Understanding the native Californian diet: Identification of condensed and hydrolyzable tannins in tanoak acorns (Lithocarpus densiflorus). J. Agric. Food Chem. 2006, 54, 7686–7691. Regazzoni, L.; Arlandini, E.; Garzon, D.; Santagati, N. A.; Beretta, G.; Maffei Facino, R. A rapid profiling of gallotannins and flavonoids of the aqueous extract of Rhus coriaria L. by flow injection analysis with high-resolution mass spectrometry assisted with database searching. J. Pharm. Biomed. Anal. 2013, 72, 202–207. 23 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558

(30)

(31)

(32) (33)

(34)

(35)

(36)

(37)

(38) (39)

(40)

(41) (42)

(43)

Page 24 of 35

Berardini, N.; Carle, R.; Schieber, A. Characterization of gallotannins and benzophenone derivatives from mango (Mangifera indica L. cv. “Tommy Atkins”) peels, pulp and kernels by high-performance liquid chromatography/electrospray ionization mass spectrometry. Rapid Commun. Mass Spectrom. 2004, 18, 2208– 2216. Geerkens, C. H.; Schweiggert, R. M.; Steingass, H.; Boguhn, J.; Rodehutscord, M.; Carle, R. Influence of apple and citrus pectins, processed mango peels, a phenolic mango peel extract, and gallic acid as potential feed supplements on in vitro total gas production and rumen methanogenesis. J. Agric. Food Chem. 2013, 61, 5727–5737. Mueller-Harvey, I. Analysis of hydrolysable tannins. Anim. Feed Sci. Technol. 2001, 91, 3–20. Luo, F.; Fu, Y.; Xiang, Y.; Yan, S.; Hu, G.; Huang, X.; Huang, G.; Sun, C.; Li, X.; Chen, K. Identification and quantification of gallotannins in mango (Mangifera indica L.) kernel and peel and their antiproliferative activities. J. Funct. Foods 2014, 8, 282–291. Romani, A.; Pinelli, P.; Galardi, C.; Mulinacci, N.; Tattini, M. Identification and quantification of galloyl derivatives, flavonoid glycosides and anthocyanins in leaves of Pistacia lentiscus L. Phytochem. Anal. 2002, 13, 79–86. Clifford, M. N.; Stoupi, S.; Kuhnert, N. Profiling and characterization by LC-MSn of the galloylquinic acids of green tea, tara tannin, and tannic acid. J. Agric. Food Chem. 2007, 55, 2797–2807. Taamalli, A.; Iswaldi, I.; Arráez-Román, D.; Segura-Carretero, A.; FernándezGutiérrez, A.; Zarrouk, M. UPLC-QTOF/MS for a rapid characterisation of phenolic compounds from leaves of Myrtus communis L. Phytochem. Anal. 2014, 25, 89–96. Bravo, L.; Goya, L.; Lecumberri, E. LC/MS characterization of phenolic constituents of mate (Ilex paraguariensis, St. Hil.) and its antioxidant activity compared to commonly consumed beverages. Food Res. Int. 2007, 40, 393–405. Lee, M.-W.; Tanaka, T.; Nonaka, G.-I.; Nishioka, I. Dimeric ellagitannins from Alnus japonica. Phytochemistry 1992, 31, 2835–2839. Ishimaru, K.; Nonaka, G. I.; Nishioka, I. Gallic acid esters of proto-quercitol, quinic acid and (-)-shikimic acid from Quercus mongolica and Q. myrsin aefolia. Phytochemistry 1987, 26, 1501–1504. Nonaka, G.; Nishioka, I.; Nishizawa, M.; Yamagishi, T.; Kashiwada, Y.; Dutschman, G. E.; Bodner, A. J.; Kilkuskie, R. E.; Cheng, Y. C.; Lee, K. H. AntiAIDS agents, 2: Inhibitory effects of tannins on HIV reverse transcriptase and HIV replication in H9 lymphocyte cells. J. Nat. Prod. 1990, 53, 587–595. Harbowy, M. E.; Balentine, D. A.; Davies, A. P.; Cai, Y. Tea chemistry. Crit. Rev. Plant Sci. 1997, 16, 415–480. Behgar, M.; Ghasemi, S.; Naserian, A.; Borzoie, A.; Fatollahi, H. Gamma radiation effects on phenolics, antioxidants activity and in vitro digestion of pistachio (Pistachia vera) hull. Radiat. Phys. Chem. 2011, 80, 963–967. Tian, F.; Li, B.; Ji, B.; Zhang, G.; Luo, Y. Identification and structure-activity relationship of gallotannins separated from Galla chinensis. LWT - Food Sci. Technol. 2009, 42, 1289–1295. 24 ACS Paragon Plus Environment

Page 25 of 35

559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593

Journal of Agricultural and Food Chemistry

(44) (45)

(46)

(47)

(48)

(49)

(50)

(51)

(52)

Nierenstein, M.; Potter, J. The distribution of myrobalanitannin. Biochem J. 1945, 39, 390–392. Tomaino, A.; Martorana, M.; Arcoraci, T.; Monteleone, D.; Giovinazzo, C.; Saija, A. Antioxidant activity and phenolic profile of pistachio (Pistacia vera L., variety Bronte) seeds and skins. Biochimie 2010, 92, 1115–1122. Bellomo, M. G.; Fallico, B. Anthocyanins, chlorophylls and xanthophylls in pistachio nuts (Pistacia vera) of different geographic origin. J. Food Compos. Anal. 2007, 20, 352–359. Nadernejad, N.; Ahmadimoghadam, A.; Hosseinifard, J.; Pourseyedi, S. Phenylalanin ammonia-lyase activity, total phenolic and flavonoid content in flowers, leaves, hulls and kernels of three pistachio (Pistacia vera L.) cultivars. Am. J. Agric. Environ. Sci. 2012, 12, 807–814. Berardini, N.; Schieber, A.; Klaiber, I.; Beifuss, U.; Carle, R.; Conrad, J. 7-Omethylcyanidin 3-O-β-D-galactopyranoside, a novel anthocyanin from mango (Mangifera indica L. cv. “Tommy Atkins”) peels. Z. Naturforsch. B 2005, 60, 801–804. Schweiggert, R. M.; Vargas, E.; Conrad, J.; Hempel, J.; Gras, C. C.; Ziegler, J. U.; Mayer, A.; Jimenez, V.; Esquivel, P.; Carle, R. Carotenoids, carotenoid esters, and anthocyanins of yellow- , orange- , and red-peeled cashew apple (Anacardium occidentale L.). Food Chem. 2016, 200, 274–282. Jerz, G.; Murillo-Velásquez, J. A.; Skrjabin, I.; Gök, R.; Winterhalter, P. Anacardic Acid Profiling in Cashew Nuts by Direct Coupling of Preparative HighSpeed Countercurrent Chromatography and Mass Spectrometry (prepHSCCC-ESI/APCI-MS/MS). In Recent Advances in the Analysis of Food and Flavors; Toth, S., Mussinan, C., Eds.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012; Vol. 1098, pp 145–165. Saitta, M.; Giuffrida, D.; La Torre, G. L.; Potortì, A. G.; Dugo, G. Characterisation of alkylphenols in pistachio (Pistacia vera L.) kernels. Food Chem. 2009, 117, 451–455. Knödler, M.; Berardini, N.; Kammerer, D. R.; Carle, R.; Schieber, A. Characterization of major and minor alk(en)ylresorcinols from mango (Mangifera indica L.) peels by high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. Rapid Commun. Mass Spectrom. 2007, 21, 945–951.

25 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 26 of 35

FIGURE CAPTIONS Figure 1.

Photograph of dried (A) green and (B) red Pistacia vera L. drupes. Background was cut off without further manipulation. Note that the green color has been lost upon drying and storage due to its instability.

Figure 2.

Representative structures of phenolic compounds detected at high signal intensity in aqueous methanolic extracts of Pistacia vera L. hulls

Figure 3.

HPLC separation of phenolic compounds from (A) red and (B) green Pistacia vera L. hulls at 280 nm. (C) Chromatogram of phenolic compounds from red hull monitored at different wavelengths. Peak assignments are shown in Table 1.

Figure 4.

Extracted ion chromatograms indicating the putative presence of (A) penta- (m/z 939), (B) hexa- (m/z 1091), (C) hepta- (m/z 1243), (D) octa(m/z 1395), and (E) nona- (m/z 1547) galloyl hexoses.

26 ACS Paragon Plus Environment

Page 27 of 35

Journal of Agricultural and Food Chemistry

Table 1. HPLC Retention Times, UV/Vis Spectra, and MS Data of Pistachio (Pistacia vera L.) Hull Phenolics a

HPLCUV/Vis abs. max (nm) 274

HR-ESI(-)-MS [M-H]m/z exp. (theo.)

molecular formula

HPLC-ESI-MSn experiment m/z

331.0684 (331.0671)

C13H16O10

[331]: 169(100), 271(56), 193(31), 211(31), 183(25), 315(10)

gallic acidb gallic acid derivative (1)

272 276

nac 296.0788 (296.0789)

na na

11.2 13.4 14.5

galloyl quinic acid protocatechuic acidb galloyl dihexose (1)

275 259 274

343.0689 (343,0671) na 493.1195 (493.1194)

C14H16O10 na C19H26O15

7

16.5

galloyl dihexose (2)

274

493.1209 (493.1194)

C19H26O15

8

17.2

galloyl dihexose (3)

274

493.1205 (493.1199)

C19H26O15

9

18.0

galloyl shikimic acid (1)

274

325.0571 (325.0565)

C14H14O9

10

19.6

galloyl shikimic acid (2)

274

325.0568 (325.0565)

C14H14O9

11

20.2

gallic acid derivative (2)

276

na

na

12

20.8

galloyl shikimic acid (3)

274

325.0576 (325.0565)

C14H14O9

13

21.1

digallic acid (1)

280

321.0261 (321.0252)

C14H10O9

14

21.5

digalloyl hexose (1)

280

483.0805 (483.0780)

C20H20O14

15

22.3

methyl gallate

272

na

na

16

23.8

digalloyl hexose (2)

288

483.0794 (483.0780)

C20H20O14

17

25.0

gallic acid derivative (3)

274

509.0972 (509.0996)

C15H26O19

compound no.

tR (min)

compound identity

1

6.0

1-O-galloyl β-Dglucopyranose (βglucogallin)b

2 3

6.8 10.0

4 5 6

[331→169]:125(100) [169]: 125(100) [296]:169(100), 125(15), 195(5), 107(5), 171(2) [296→169]:125(100) [343]: 191(100), 169(17), 125(12) [153]: 109(100) [493]: 313(100), 169(39), 283(29), 331(10) [493→313]: 283(100), 169(44), 223(43), 135(39), 241(15) [493]: 271(100), 313(25), 211(20), 331(20), 169(7) [493→271]: 211(100), 125(4) [493]: 313(100) [493→313]: 169(100), 125(92), 189(30), 242(18) [325]: 169(100), 125(22), 139(9), 193(8) [325→169]: 125(100) [325]: 169(100), 125(15), 281(7), 111(4), 173(3) [325→169]: 124(100), 125(28) [571]: 285(100), 169(7) [571→285]: 133(100), 169(49), 170(10) [325]: 169(100), 125(15), 137(3), 173(3) [325→169]: 125(100) [321]: 169(100) [321→169]: 125(100) [483]: 331(100), 271[(16), 169(15) [483→331]: 169(100), 313(60), 271(51), 193(25), 123(15), 241(12), 195(12), 211(10) [183]: 168(100), 124(10) [183→168]: 124(100) [483]: 331(100), 169(27), 332(17), 271(11) [483→331]: 169(100), 241(53), 125(15) [509]: 267(100), 429(11) [509→267]: 139(100)

27 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

18

26.0

gallic acid derivative (4)

274

423.0931 (423.0933)

C19H20O11

19

26.4

digalloyl quinic acid

274

495.0776 (495.0780)

C21H20O14

20

27.3

digallic acid (2)

275

321.0246 (321.0252)

C14H10O9

21

28.1

gallic acid derivative (5)

268

403.1254 (403.1246)

C17H24O11

22

29.5

gallic acid derivative (6)

274

467.1197 (467.1195)

C21H24O12

23

30.6

digalloyl shikimic acid (1)

275

477.0688 (477.0675)

C21H18O13

24

31.0

digalloyl shikimic acid (2)

276

477.0703 (477.0675)

C21H18O13

25

31.6

cyanidin 3-O-β-Dgalactopyranosideb

278, 517

na

na

26

33.2

luteic acid

278

319.0133 (319.0096)

C14H8O9

27 28

33.7 33.8

cyanidin pentoside trigalloyl quinic acid

520 273

na 647.0873 (647.0890)

na C28H24O18

29

34.5

quinic acid derivative (1)

276

523.1455 (523.1157)

C24H28O13

30

35.3

myricetin galloyl hexoside

270, 354

631.0934 (631.0941)

C28H24O17

31

35.8

tetragalloyl hexose

278

787.1010 (787.0999)

C34H28O22

32

36.7

trigallic acid

274

473.0373 (473.0362)

C21H14O13

33

36.9

myricetin hexuronide

357, 252

493.0640 (493.0624)

C21H18O14

34

37.1

myricetin 3-O-galactoside

359, 252

479.0848 (479.0831)

C21H20O13

35

37.4

myricetin hexoside

357, 252

479.0884 (479.0831)

C21H20O13

36

37.9

methyl-digallate

274

335.0414 (335.0409)

C15H12O9

37

38.3

penta-O-galloyl-β-D

280

939.1130 (939.1109)

C41H32O26

Page 28 of 35

[423]: 313(100), 169(70), 125(36), 211(24) [423→313]: 169(100), 313(39), 295(17) [495]: 343(100), 191(29), 344(16), 271(4) 169(3) [495→343]: 191(100) [321]: 169(100), 125(16) [321→169]: 125(100) [403]: 169(100), 151(55), 313(37), 125(36), 271(21), 211(18), 179(15), [403→169]: 124(100), 125(34), 107(34) [467]: 313(100), 169(17), 295(7) [467→313]: 169(100), 153(10), 191(5) [477]: 325(100), 169(47), 326(33) [477→325]: 169(100) [477]: 325(100), 169(47) [477→325]: 169(100), 125(11), 281(10) d [449]: 287(100) [449→287]: 137(100) [319]: 239(100), 139(15), 240(14) [319→239]: 139(100), 124(12) d [419]: 287(100) [647]: 495(100), 343(22), 191(2) [647→495]: 343(100), 191(46) [523]: 209(100), 371(45), 505(30), 313(19), 169(14), 191(14) [523→209]: 191(100), 165(21), 151(18), 123(2) [631]: 479(100), 317(10) [631→479]: 316(100), 317(69), 325(15), 271(11) [787]: 617(100) [787→617]: 465(100), 589(55), 221(41), 277(34), 449(30), 600(26), 296(20), 235(18), 466(12) [473]: 321(100), 169(25) [473→321]: 169(100), 125(6) [493]: 317(100), 299(4), 151(3), 137(2) [493→317]: 227(100), 151(23), 137(18) [479]: 317(100), 271(20), 179(19), 287(15) [479→317]: 215(100), 271(99), 164(24), 270(22), 287(22), 242(17) [479]: 316(100), 317(57), 271(12) [479→316]: 271(100), 179(40), 317(35), 180(29), 255(26), 137(23) [335]: 183(100), 253(5) [335→183]: 168(100) [939]: 769(100), 617(18), 787(12)

28 ACS Paragon Plus Environment

Page 29 of 35

Journal of Agricultural and Food Chemistry

glucoseb [939→769]: 617(100), 387(43), 601(40), 465(28), 323(21), 259(19), 403(19), 725(17), 245(12), 573(12), 386(11) [615]: 463(100), 301(32)

38

38.9

quercetin galloyl hexoside (1)

257, 354

615.1031 (615.0992)

C28H24O16

39

40.6

hexagalloyl hexose

263, 353

1091.1423 (1091.1213)

C48H36O30

40

41.0

quercetin 3-O-galactosideb

252, 352

463.0922 (463.0882)

C21H20O12

41

41.2

quercetin 3-Oglucuronideb

255, 355

477.0715 (477.0675)

C21H18O13

42

41.6

quercetin 3-O-glucosideb

260, 355

463.0915 (463.0877)

C21H20O12

43

42.1

quercetin galloyl hexoside (2)

254, 358

615.1031 (615.0986)

C28H24O16

44

42.7

gallic acid derivative (7)

279

347.0409 (347.0406)

C16H12O9

45

42.9

quercetin galloyl hexoside (3)

276, 358

615.1098 (615.0986)

C28H24O16

[615→301]: 179(100), 151(99) [347]: 267(100), 139(5), 249(4), 269(4), 124(2) [347→267]: 139(100), 249(28), 83(16), 140(10), 205(8) [615]: 301(100), 179(5), 463(3)

46

43.3

quercetin galloyl deoxyhexose

274, 355

599.1075 (599.1037)

C28H24O15

[615→301]: 151(100), 170(32), 107(21), 179(20), [599]: 463(100), 301(36)

47

43.7

quercetin pentoside

254, 355

433.0810 (433.0771)

C20H18O11

48

44.1

unknown (1)

275, 357

na

na

49

45.0

kaempferol hexoside

272, 350

447.0963 (447.0927)

C21H20O11

50

45.4

quercetin galloyl hexuronide

276, 352

629.0852 (629.0779)

C28H22O17

[599→463]: 301(100), 255(7) [433]: 301(100), 255(6), 242(4), 193(4) [433→301]: 271(100), 179(42), 151(41), 255(11), 243(9), [629]: 327(100), 459(15), 477(15), 328(11) [629→327]: 113(100), 169(49), 283(40), 175(38), 170(37), 177(35) [447]: 285(100), 255(28), 327(19) [447→285]: 255(100), 223(6), 256(5) [629]: 477(100), 301(26)

51

46.3

quercetin galloyl pentoside

277, 352

585.0910 (585.0881)

C27H22O15

[629→477]: 301(100), 323(5), 175(3) [585]: 301(100)

[615→463]: 301(100), 179(8), 229(7), 253(6), 272(6), 151(2) [1091]: 939(100), 769(16) [1091→939]: 769(100), 770(13), 617(10) [463]: 301(100), 179(8) [463→301]: 151(100), 229(58), 271(43), 343(30), 254(26), 258(24), 181(16) [477]: 301(100), 179(7), 151(2) [477→301]: 179(100), 152(20), 180(11), 121(8), 256(7), 273(6), 229(6) [463]: 301(100) [463→301]: 179(100), 271(52), 152(39), 272(32), 255(31), 151(27), 203(21) [615]: 301(100), 313(15), 315(8), 273(6), 463(3)

[585→301]: 151(100), 179(45), 165(44), 121(9)

29 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

52

46.9

kaempferol hexoside

270, 350

447.0952 (447.0927)

C21H20O11

53

48.9

kaempferol pentoside

270, 350

417.0907 (417.0822)

C20H18O10

54

88.4

(16:1)-anacardic acid

238, 310

359.2521 (359.2586)

C23H36O3

55

88.5

(13:2)-anacardic acid

238, 310

315.1968 (315.1966)

C20H28O3

56

88.9

(11:0)-anacardic acid

238, 308

291.1960 (291.1966)

C18H28O3

57

89.1

(15:3)-anacardic acid

238, 310

341.2112 (341.2122)

C22H30O3

58

89.6

(13:1)-anacardic acid

246, 310

317.2115 (317.2117)

C20H30O3

59

90.7

(13:0)-anacardic acid

248, 312

319.2289 (319.2279)

C20H32O3

60

91.0

(15:1)-anacardic acid

240, 310

345.2429 (345.2434)

C22H34O3

61

91.2

unknown (2)

260

455.3510 (455.3405)

C30H48O3

62

91.4

(17:2)-anacardic acid

238, 310

371.2599 (371.2592)

C24H36O3

63

92.0

(15:0)-anacardic acidb

242, 311

347.2623 (347.2592)

C22H36O3

64

92.2

(17:1)-anacardic acid

248, 312

373.2743 (373.2743)

C24H38O3

65

92.4

unknown (3)

260

373.2748 (373.2748)

C24H38O3

66

93.1

(17:0)-anacardic acid

238, 312

375.2951 (375.2977)

C24H40O3

Page 30 of 35

[447]: 285(100), 284(92), 255(63), 163(18), 151(17) [447→285]: 255(100), 197(13), 240(10), 198(8), 213(7), 227(6), 169(5) [417]: 284(100), 255(37) [417→284]: 255(100), 160(5), 165(4), 227(4), 195(3), 151(3) [359]: 315(100), 341(43), 161(21), 315(17), 107(13), 343(13), 317(11) [359→315]: 108(100) [315]: 271(100), 107(6) [315→271]: 107(100) [291]: 247(100) [291→247]: 106(100) [341]: 297(100) [341→297]:149(100) [317]: 273(100) [317→273]: 107(100) [319]: 275(100) [319→275]: 106(100) [345]: 301(100), 119(4) [345→301]: 119(100) [455]: 418(100) [455→418]: 434(100), 395(66), 375(60), 399(15) [371]: 327(100) [371→327]: 327(100), 119(18), 107(10) [347]: 303(100) [373→303]: 106(100) [373]: 329(100) [373→329]: 106(100) [373]: 329(100) [373→329]: 119(100) [375]: 331(100) [375→331]: 106(100)

a

tR: retention time. Verified by reference standards. c na: not available. d Positive ionization mode was used for the identification of anthocyanins. e All compounds were detected in both red and green pistachio hulls, except for the anthocyanins, 25 and 27, which were only found in red hulls. b

30 ACS Paragon Plus Environment

Page 31 of 35

Journal of Agricultural and Food Chemistry

Figure 1 79x74mm (600 x 600 DPI)

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Figure 2 117x80mm (600 x 600 DPI)

ACS Paragon Plus Environment

Page 32 of 35

Page 33 of 35

Journal of Agricultural and Food Chemistry

Figure 3 156x136mm (600 x 600 DPI)

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Figure 4 120x86mm (600 x 600 DPI)

ACS Paragon Plus Environment

Page 34 of 35

Page 35 of 35

Journal of Agricultural and Food Chemistry

TOC Graphical Abstract 44x23mm (600 x 600 DPI)

ACS Paragon Plus Environment