Comprehensive Characterization of Extractable and Nonextractable

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Comprehensive characterization of extractable and nonextractable phenolic compounds by HPLC-ESI-QTOF of a grape/pomegranate pomace dietary supplement Iza Pérez-Ramírez, Rosalia Reynoso-Camacho, Fulgencio Saura Calixto, and Jara Pérez-Jiménez J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05901 • Publication Date (Web): 27 Dec 2017 Downloaded from http://pubs.acs.org on December 28, 2017

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Journal of Agricultural and Food Chemistry

TOC Graphics 254x190mm (96 x 96 DPI)

ACS Paragon Plus Environment


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Comprehensive Characterization of Extractable and Non-extractable Phenolic

2

Compounds by HPLC-ESI-QTOF of a Grape/Pomegranate Pomace Dietary

3

Supplement

4 5

Iza F Pérez-Ramírez†,‡, Rosalía Reynoso-Camacho†, Fulgencio Saura-Calixto‡, Jara

6

Pérez-Jiménez‡*

7 8

†

9

Autónoma de Querétaro, Cerro de las Campanas s/n, 76010 Querétaro, Qro., México.

Research and Graduate Studies in Food Science, Facultad de Química, Universidad

10

‡

11

Nutrition (ICTAN-CSIC), José Antonio Novais 10, 28040, Madrid, Spain.

12

*Corresponding author: Tel: (+34) 91 549 23 00; Fax: (+34) 91 549 36 27; E-mail:

13

[email protected].

Department of Metabolism and Nutrition, Institute of Food Science, Technology and


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Abstract

15

Grape and pomegranate are rich sources of phenolic compounds and their derived

16

products could be used as ingredients for the development of functional foods and

17

dietary supplements. However, the profile of non-extractable or macromolecular

18

phenolic compounds in these samples has not been evaluated. Here, we a

19

comprehensive characterization of extractable and non-extractable phenolic compounds

20

of a grape/pomegranate pomace dietary supplement using high-performance liquid

21

chromatography–quadrupole time-of-flight (HPLC-ESI-QTOF) and matrix-assisted

22

laser desorption/ionization MALDI-TOF techniques. The main extractable phenolic

23

compounds were several anthocyanins (principally malvidin 3-O-glucoside) as well as

24

gallotannins and gallagyl derivatives; some phenolic compounds are reported in grape

25

or pomegranate for the first time. Additionally, there was a high proportion of non-

26

extractable phenolic compounds, including vanillic acid, and dihydroxybenzoic acid.

27

Unidentified polymeric structures were detected by MALDI-TOF MS analysis. This

28

study shows that mixed grape and pomegranate pomaces are a source of different

29

classes of phenolic compounds, including a high proportion of non-extractable phenolic

30

compounds.

31

Keywords: Grape; pomegranate; pomaces; phenolic compounds; ellagitannins; mass

32

spectrometry.



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INTRODUCTION

34

Grapes (Vitis vinifera L.) are the largest fruit crop in the world, with annual production of

35

around 75 million tons, mainly used to make wine.

36

granatum L.) annual production is some 1.5 million tons, and is consumed fresh or used in

37

the elaboration of juices and jams. Interestingly, pomegranate production has grown

38

steadily in recent years,

39

associated with its consumption. Indeed, both these fruits contain high levels of phenolic

40

compounds, known for their health-related properties, and in fact the two present

41

complementary profiles: red grape is rich in polymeric flavanols or proanthocyanidins,

42

while the most characteristic phenolic compounds in pomegranate are ellagitannins.

43

Furthermore, both contain other flavonoids such as anthocyanins or flavonols, as well as

44

phenolic acids. 3, 4 The complementary profile of phenolic compounds in both fruits makes

45

especially interesting to prepare products combining both of them, since they would

46

provide a whole diversity of phenolic compounds. Indeed, phenolic compounds have been

47

suggested to exhibit class-specific effects, such as those attributed to the microbial-derived

48

metabolites of ellagitannins present in pomegranate

49

proanthocyanidins found in grape. 6

50

A huge amount of grape and pomegranate pomace is generated during the industrial

51

processing of these fruits, which is mainly comprised of remnant pulp, seeds and peel.

52

Mass spectrometry (MS) coupled to liquid chromatography (LC) has been used to identify

53

phenolic acids and flavonoids in grape pomace.

54

some specific phenolic compounds have been measured by high-performance liquid

55

chromatography (HPLC)–diode-array detection (DAD),

56

analysis, including that of low-molecular-weight (< 1200 Da) ellagitannins and gallotannins

2

1

Meanwhile, pomegranate (Punica

probably due to consumer awareness of the health benefits

3, 7

5

or to those derived from

Regarding pomegranate pomace, only

8

while detailed HPLC-MS

3 ACS Paragon Plus Environment

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has been performed on other pomegranate byproducts such as outer skin, mesocarp and

58

divider membranes.

59

phenolic compounds (low-molecular-weight phenolic compounds that are free in the food

60

matrix), while they do not include evaluation of non-extractable phenolic compounds or

61

macromolecular antioxidants, i.e., low-molecular-weight phenolic compounds that are

62

strongly associated with the food matrix or high-molecular-weight phenolic compounds.

63

This phenolic compound fraction represents about 57% of total phenolic compounds in

64

fruits and vegetables.

65

fraction of non-extractable ellagitannins has been reported. 11

66

In recent years, matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF)

67

MS has increasingly been used for the characterization of proanthocyanidins.

68

studies have used this technique to characterize either proanthocyanidins or anthocyanins in

69

grape pomaces.

70

pomace, which also contains high-molecular-weight ellagitannins. These compounds have

71

only been characterized once using MALDI-TOF MS, in the particular case of blackberry.

72

15

73

Grape and pomegranate pomaces can be used as functional ingredients or for the

74

development of dietary supplements to increase phenolic compounds consumption. Indeed,

75

their inclusion in a single product, combining their complementary phenolic composition,

76

could lead to synergies between the beneficial effects reported for both grape and

77

pomegranate, for instance in the modulation of metabolic syndrome.

78

aim of this study was to provide a comprehensive characterization of the phenolic

79

composition, including non-extractable phenolic compounds, using HPLC-ESI-QTOF and

9

13, 14

Nevertheless, those studies focus on the so-called extractable

10

Indeed, in the case of pomegranate, the existence of a specific

12

A few

Nevertheless, it has not been used to characterize pomegranate

16, 17

Therefore, the



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80

MALDI-TOF MS techniques, of a dietary supplement made with grape and pomegranate

81

pomaces which has been used in a clinical trial.

82 83

MATERIALS AND METHODS

84

Chemicals

85

The Folin-Ciocalteu reagent was from Panreac (Castellar del Vallés, Barcelona, Spain).

86

ABTS

87

tetramethylchroman-2-carboxylic acid (Trolox), anthralin or dithranol (1,8-dihydroxy-9,10-

88

dihydroanthracen-9-one), NaCl, 2,5-dihydroxybenzoic acid, gallic acid, ellagic acid, p-

89

hydroxybenzoic acid, vanillic acid, cafeic acid, p-coumaric acid, ferulic acid, punicalagin,

90

procyanidin B2, quercetin, myricetin and kaempferol were all obtained from Sigma-Aldrich

91

(St. Louis, MO). 2,4,6-Tris(2-pyridyl)-S-triazine (TPTZ) was from Fluka Chemicals

92

(Madrid, Spain). All the reagents used for the preparation of phenolic compounds fractions

93

and spectrophotometric determinations were of analytical grade; while for MS analysis,

94

they were of MS grade.

95

Condensed tannin concentrate from Mediterranean carob pods (Ceratonia siliqua L) was

96

supplied by Nestlé Ltd. (Vers-chez-les Blancs, Switzerland).

97

Sample preparation

98

Pomegranate (Punica granatum L., cv ‘Mollar de Elche’) juice pomace (obtained by

99

pressing the aryls, after previous removal of the outer skin) was obtained from Vitalgrana

100

S.A. (Catral, Alicante); whereas grape (Vitis vinifera L., cv ‘Tempranillo’) wine pomace

101

was obtained from Roquesan Wineries (Quemada, Burgos). Both pomace samples were

102

collected in situ at the moment of wine devatting or pomegranate pressing. Then, they were

103

transported at -20 ºC, freeze-dried and ground in a mill (ZM200, Retsch; Haan, Germany)

(2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic

acid),

6-hydroxy-2,5,7,8-


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104

to a particle size of 0.5 mm. Then, in order to obtain the dietary supplement, samples were

105

mixed in a 50/50 ratio, and stored at -20 ºC in vacuum-sealed packages, protected from

106

light. This product was further packed in individual doses and employed as dietary

107

supplement (consumed in water solution) in a clinical trial in subjects with abdominal

108

obesity, registered with code NCT02710461. 18

109

Methods

110

Obtaining phenolic compounds fractions

111

Usually, extractable phenolic compounds, hydrolyzable phenolic compounds and non-

112

extractable proanthocyanidins provide the overall profile for the content of phenolic

113

compounds in a sample.

114

profile of phenolic compounds, as discussed above, additional treatments were applied to

115

the original sample

116

in order to ensure proper evaluation of the concentrations of these compounds. The whole

117

procedure was applied to three different replicates of the sample. Further determinations

118

were performed in triplicate in each extract and are reported on a dry weight (dw) basis.

119

The moisture content was determined by drying at 105°C in an oven to constant weight.

120

The results are expressed as mean values + standard deviation.

121

Extractable phenolic compounds. The sample (0.5 g) was subjected to successive extraction

122

with 20 mL of methanol/water (50:50 v/v, pH 2) and then with 20 mL of acetone/water

123

(70:30, v/v). Then the two supernatants were combined, which corresponded to the

124

extractable phenolic compounds fraction. 19

125

Hydrolyzable phenolic compounds. The residue from the extractable phenolic compounds

126

extraction was treated with 20 mL of methanol and 2 mL of concentrated sulfuric acid at 85

11

19

Nevertheless, since pomegranate is characterized by a specific

to obtain extractable ellagitannins and non-extractable ellagitannins,



20, 21

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127

ºC for 20 h.

After several washings with distilled water, an aliquot of the extract was

128

adjusted to pH 5.5

129

Non-extractable proanthocyanidins. The residues were treated with butanol/HCl (97.5:2.5,

130

v/v) with 0.1% FeCl3 at 100 ºC for 1 h. 22, 23

131

Extractable ellagitannins. The sample (150 mg) was subjected to successive extraction with

132

5 mL of water/HCl (98.65:1.35, v/v) and then with 4 mL of methanol/DMSO/acidified

133

water (40:40:20, v/v/v). Then the two supernatants were combined, which corresponded to

134

the extractable ellagitannins fraction. 11

135

Non-extractable ellagitannins. The residue from the extractable ellagitannins extraction was

136

treated with 6.7 mL of water and 3.3 mL of HCl at 90 ºC for 24 h. Then the pH was

137

adjusted to 2.5, and the samples were washed with 10 mL of water. 11

138

Determination of extractable phenolic compounds, hydrolyzable phenolic compounds, and

139

non-extractable proanthocyanidin content

140

The extractable phenolic compounds, hydrolyzable phenolic compounds and non-

141

extractable proanthocyanidins fractions were used for spectrophotometric determinations of

142

total phenolic compounds content (corresponding to the sum of the three fractions). Briefly,

143

the Folin-Ciocalteu assay

144

compound fractions; the results were expressed as g of gallic acid equivalents/100 g dw.

145

The non-extractable proanthocyanidin content was determined in the hydrolyzates by

146

measuring the sum of absorbance at 450 and 555 nm; the results were expressed as mg of

147

non-extractable proanthocyanidins/100 g dw, by using a standard curve from a polymeric

148

proanthocyanidin concentrate.

149

spectrophotometer (Perkin-Elmer; Waltham, MA, U.S.A.).

24

was applied to the extractable and hydrolyzable phenolic

25

All these measurements were carried out in a Lambda 12


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Characterization of phenolic compounds by HPLC-ESI-QTOF analysis

151

For HPLC-MS analysis, the extractable and hydrolyzable phenolic compound fractions

152

were concentrated (6:1) with a N2 stream; whereas the extractable and non-extractable

153

ellagitannins fractions were injected directly. Chromatographic analysis was based on

154

procedures previously described.

155

Agilent Technologies, Santa Clara, CA) was coupled with DAD (Agilent G1315B) and a

156

QTOF mass analyzer (Agilent G6530A) with an atmospheric pressure electrospray

157

ionization (ESI). The column used was a 250 mm x 4.6 mm i.d., 5 µm, Gemini C18

158

(Phenomenex, Torrance, CA).

159

Gradient elution was performed with a binary system consisting of: 0.1% formic acid in

160

acetonitrile (solvent A) and 0.1% aqueous formic acid (solvent B). The following gradient

161

was applied at a flow rate of 0.4 mL/min: 0 min, 5% B; 20 min, 30% B; 30 min, 55% B; 38

162

min, 90% B; 40 min, 90% B; followed by a re-equilibration step. The injection volume was

163

10 µL and the column temperature was 25 ºC. Data were acquired using both negative and

164

positive ion mode with a mass range of 100-1,200 Da, and using a source temperature of

165

325°C and a gas flow of 10 L/h. Peak identity was established by comparison with the

166

retention times of commercial standards when available. Also, the molecular formula

167

proposed by the MassHunter Workstation software version 4.0 for the different signals

168

obtained in the MS experiments were compared with previously reported phenolic

169

compounds, especially in grape and pomegranate, and a maximum error of 8 ppm was

170

accepted. For MS/MS experiments, the auto MS/MS acquisition mode was used; the main

171

fragments were compared with the fragmentation patterns reported for phenolic

172

compounds.

26

For separation, the HPLC apparatus (Agilent 1200,



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173

A relative quantitation was performed based on UV signals, using the calibration curve of a

174

commercial standard for each class of phenolic compounds, as previously reported.

175

Hydroxybenzoic acids and gallotannins were monitored at 280 nm and quantitated with

176

gallic acid; hydroxycinnamic acids and ellagitannins were monitored at 320 nm and

177

quantitated with ellagic acid and punicalagin, respectively; flavanols were monitored at 214

178

nm and quantitated with the procyanidin B2 dimer; flavonols were monitored at 365 nm

179

and quantitated with kaempferol; and anthocyanins were monitored at 520 nm and

180

quantitated with malvidin chloride.

181

Characterization of phenolic compounds by MALDI-TOF MS analysis

182

A fivefold concentrated extractable phenolic compounds fraction was obtained, following

183

the procedure described above, but with a fivefold reduction in the solvent volume. In

184

contrast, a threefold concentrated extractable ellagitannin fraction was obtained, following

185

the procedure described above, but with a threefold increase in sample amount. Then, both

186

fractions were concentrated to dryness with a N2 stream, and re-dissolved in 200 µL of the

187

mixture of solvents mentioned above.

188

For the extractable phenolic compounds fraction, the best results were obtained with the

189

matrix 2,5-dihydroxybenzoic acid (10 mg) with Na (NaCl, 1 mg) solved in aqueous 1%

190

trifluoroacetic acid. While for the extractable ellagitannin fraction, the matrix chosen was

191

anthralin or dithranol, where 10 mg was solved in dichloromethane. In both cases, the

192

matrix and the extract were mixed (1:1, v:v), vortexed, and then deposited (2 µL) on the

193

target plate. Once the solvent was dried (at room temperature), the crystals were analyzed.

194

An AutoFLEX III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics; Bremen,

195

Germany) equipped with a pulsed N2 laser (337 nm) controlled by the Flexcontrol 1.1

10


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196

software package was used to obtain MS data in positive mode. The accelerating voltage

197

was 20 kV and the reflectron voltage 21 kV. The spectra registered were the sum of 500

198

shots with a frequency of 200 Hz. These conditions were previously reported as adequate

199

for proanthocyanidin analysis. 26

200

Evaluation of in vitro antioxidant capacity

201

Antioxidant capacity was determined by ferric reducing/antioxidant power (FRAP) and

202

ABTS assays in both extractable and hydrolyzable phenolic compound fractions.

203

Additionally, the ABTS assay was carried out on the non-extractable proanthocyanidin

204

fraction (the butanol media do not allow the application of the FRAP assay to this fraction).

205

FRAP reagent, freshly prepared and warmed to 37°C, was mixed with distilled water and

206

the test sample, standard or appropriate blank reagent. Readings after 30 min, at 595 nm, in

207

a Lambda 12 spectrophotometer were selected to calculate the FRAP values.

208

ABTS assays, after the addition of the sample or Trolox standard to the ABTS•+ solution,

209

absorbance readings were taken at 595 nm every 20 s for 6 min using a DU-640

210

spectrophotometer (Beckman Instruments Inc.; Fullerton, CA). The percentage inhibition of

211

absorbance was plotted against time and the area under the curve (0 to 6 min) was

212

calculated. 29

213

RESULTS AND DISCUSSION

214

Total phenolic compounds content and associated antioxidant capacity

215

In this study, extractable phenolic compounds, i.e., those soluble in aqueous-organic

216

solvents, were obtained by two different procedures in order to get extractable phenolic

217

compounds and extractable ellagitannins. While those remaining in the residues of those

218

extractions, called non-extractable phenolic compounds or macromolecular antioxidants

219

were obtained as non-extractable proanthocyanidins, hydrolyzable phenolic compounds and

27, 28

For



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220

non-extractable ellagitannins. Altogether, these fractions contain the whole phenolic

221

compounds profile of the grape/pomegranate pomace dietary supplement.

222

The total phenolic compounds content of the grape/pomegranate dietary supplement,

223

measured

224

proanthocyanidins, and hydrolyzable phenolic compounds, is shown in Table 1. The

225

extractable phenolic compounds values were slightly lower than those previously reported

226

for a product derived from grape pomace, 30 indicating a lower content of these compounds

227

in pomegranate pomace. The most notable result, however, is that macromolecular

228

antioxidants, consisting mainly of non-extractable proanthocyanidins, represented the major

229

(∼ 80%) phenolic compounds fraction of the grape/pomegranate dietary supplement. This

230

agrees with previous results for a grape pomace product, where most phenolic compounds

231

were present as non-extractable proanthocyanidins,

232

parts of the pomegranate (outer skin, mesocarp, divider membrane) where most of the

233

phenolic compounds belonged to the non-extractable fraction.

234

contribution of macromolecular antioxidants to total phenolic compounds content of

235

pomegranate

236

macromolecular antioxidants reach the colon, different metabolites are produced by

237

microbiota action. These metabolites are further absorbed, and have been associated with

238

beneficial effects on gastrointestinal health and cardiovascular diseases. 31

239

The antioxidant capacity of the different phenolic compounds fractions of the

240

grape/pomegranate dietary supplement was evaluated by FRAP and ABTS assays (Table

241

1). FRAP could not be measured in the non-extractable proanthocyanidin fraction due to

242

the interference of the butanol medium. 32 For all the fractions and both methods, the results

as

the

pomace

sum

had

of

not

extractable

previously

phenolic

30

compounds,

non-extractable

as well as with a study of different

been

reported.

9

Nevertheless, the

Interestingly,

when


Page 13 of 42


243

show that this dietary supplement provides between five and nine times the mean

244

antioxidant capacity of fruits commonly consumed.

245

antioxidants contributed less to the total antioxidant capacity than to total phenolic

246

compounds content, they were still responsible for about half the antioxidant capacity of the

247

grape/pomegranate pomace dietary supplement. This, together with the health-related

248

properties associated with this class of dietary antioxidants, 31 make the search for products

249

rich in both extractable phenolic compounds and macromolecular antioxidants extremely

250

interesting.

251

Phenolic compounds profile by HPLC-ESI- QTOF analysis

252

Extractable fraction

253

A total of seventy-nine different compounds were found in the extractable fraction

254

(extractable phenolic compounds and extractable ellagitannins) of the grape/pomegranate

255

dietary supplement, from which fifty-eight compounds were identified as phenolic

256

compounds by analyzing their exact mass (confirmation of elemental composition with 8 ppm); however, MS/MS fragments are characteristics of this compound. 2This compound was identified according to its fragmentation pattern, since its molecular formula has not been reported. n.a.,



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Table 3. HPLC-ESI-QTOF Profile of the Extractable Ellagitanin Fraction of a Grape/Pomegranate Pomaces Dietary Supplement. no.

retention proposed compound time (min) anthocyanins 1 10.59 cyanidin 3-O-glucoside 2 8.51 cyanidin 3,5-Odiglucoside 3 17.42 delphinidin 3-O-(6′′-pcoumaroyl-glucoside)

ionization

experimental mass

MS/MS ions

calculated mass

ppm

formula

amount (mg/100 g dw)

ESI+

449.1101

449.1084

-3.82

C21H21O11

10.9 ± 0.2

ESI+

611.1620

611.1612

-1.29

C27H31O16

1.4 ± 0.1

ESI+

611.1413

611.1401

-2.00

C30H27O14

4.1 ± 0.4

ESI+

465.1048

121, 2872 164, 261, 289, 3672 121, 159, 217, 307, 405, 481, 5772 114, 121, 3032

465.1033

-3.22

C21H21O12

14.3 ±1.3

ESI+

535.1462

535.1452

-1.93

C25H27O13

1.2 ± 0.1

malvidin 3-O-(6′′coumaroyl-glucoside) malvidin 3-O-glucoside pyruvic acid (visitin A) malvidin 3-O-glucoside pelargonidin 3-Oglucoside

ESI+

639.1734

639.1714

-3.16

C21H31O14

15.0 ± 0.9

ESI+

561.1241

ESI+

4

9.67

delphinidin 3-Oglucoside malvidin 3-O-(6′′acetylhexoside)

6

16.39

7

19.99

8

16.73

9 10

12.53 11.59

12

11.15

petunidin 3-O-glucoside

13

18.72

54

18.14

petunidin 3-O-(6′′ coumaroyl-glucoside) malvidin 3-O-(6′′caffeoyl-glucoside)

561.1244

0.59

C26H25O14

1.1 ± 0.1

493.1360

121, 241, 309, 390, 448, 453, 505, 5212 121, 236, 331, 397, 4652 121, 190, 273, 341, 399, 4932 259, 331, 4192

493.1346

-2.84

C23H25O12

35.6 ± 3.3

ESI+

433.1148

271, 3442

433.1135

-3.07

C21H21O10

2.3 ± 0.1

ESI+

479.1204

121, 159, 234, 279, 317, 3742

479.1190

-3.44

C22H23O12

10.7 ± 1.0

ESI+

625.1571

215, 346, 4752

625.1557

-2.19

C31H29O14

4.3 ± 0.4

ESI+

655.1660

3312

639.1663

0.45

C21H31O14

1.7 ± 0.1


Page 35 of 42


flavonols 55 29.99

kaempferol

ESI-

285.0408

15 16 56

23.77 17.83 27.33

myricetin myricetin 3-O-glucoside quercetin 3-O-glucoside

ESIESIESI-

317.0300 479.0834 301.0365

17

21.39

ESI-

18

21.82

quercetin 3-Oglucuronide syringetin 3-Ogalactoside

flavanols 19 13.05 20 15.08

(epi)catechin (epi)catechin

22 9.37 (epi)gallocatechin 23 14.05 procyanidin B2 dimer 24 13.85 procyanidin dimer 25 7.99 prodelphinidin dimer hydroxybenzoic acids 26 6.67 gallic acid 28 29.45 vanillic acid O-hexoside hydroxycinnamic acids 32 13.65 dicaffeoylquinic acid gallotannins 36 8.26 monogalloyl hexoside ellagitannins 57 15.31 brevifolin carboxylic acid 37 19.89 ellagic acid 38 18.84 ellagic acid O-

113, 161, 178, 197, 269 113, 161 113, 316 113, 161, 197, 219

285.0405

-1.18

C15H10O6

1.6 ± 0.01

317.0303 479.0831 301.0354

0.91 -0.60 -3.75

C15H10O8 C21H20O13 C15H10O7

0.7 ± 0.01 1.2 ± 0.01 3.8 ± 0.1

477.0677

113, 301, 435

477.0675

-3.21

C21H18O13

1.2 ± 0.03

ESI-

507.1142

111, 113, 263, 345, 447

507.1144

0.42

C23H24O13

0.8 ± 0.1

ESIESI-

289.0731 289.0717

289.0718 289.0718

-4.61 0.21

C15H14O6 C15H14O6

8.7 ± 0.1 6.4 ± 0.1

ESIESIESIESI-

305.0665 577.1359 577.1362 609.1250

111, 179, 205 111, 161, 170, 205 111, 125, 161 133, 289 125, 301, 407 111, 161

305.0667 577.1351 577.1351 609.1250

0.58 -1.30 -0.78 -0.03

C15H14O7 C30H26O12 C30H26012 C30H26014

2.8 ± 0.5 6.2 ± 0.2 0.4 ± 0.0 0.4 ± 0.1

ESIESI-

169.0147 329.2331

111, 113, 161 113, 161, 219

169.0142 329.2333

-2.67 0.75

C7H6O5 C18H34O5

9.6 ± 0.9 0.1 ± 0.01

ESI+

517.1359

517.1347

-3.58

C25H24O12

8.3 ± 0.2

ESI-

331.0663

111, 161

331.0671

2.32

C13H16010

10.6 ± 0.3

ESI-

291.0145

113, 161

291.0146

0.48

C13H8O8

2.3 ± 0.1

ESIESI-

300.9999 447.0579

113 113, 299

300.0000 447.0579

-3.01 -2.23

C14H6O8 C20H16O12

79.8 ± 1.1 11.0 ± 0.1 34



39 40

15.89 18.34

41

7.43

43

7.09

45 46 47

14.51 20.81 17.20

deoxyhexoside ellagic acid O-hexoside ellagic acid O-pentoside gallagyl-hexoside (punicalin) gallagyl-hexoside (punicalin) derivative

ESIESI-

463.0521 433.0413

ESI-

781.0545

ESI-

1101.0701

113, 300 111, 160, 262, 345 111, 368, 480, 601, 781, 956 111, 129, 161, 197, 286, 427, 550, 601, 781, 955 111, 179, 205 113, 301, 433

Page 36 of 42

463.0518 433.0412

-0.62 -0.12

C20H16O13 C19H14O12

34.8 ± 0.6 11.7 ± 0.6

781.0545

-0.01

C34H22O22

47.5 ± 1.4

n.a.

n.a.

n.a.

14.2 ± 0.8

galloyl-HHDP-hexoside ESI633.0736 633.0736 -0.41 C27H22O18 3.6 ± 0.1 1 galloyl-HHDP-hexoside ESI633.0550 633.0550 28.91 C27H22O18 0.6 ± 0.1 galloyl-HHDP-DHHDPESI951.0753 113, 301, 509 951.0745 -0.61 C41H28O27 7.5 ± 0.1 hexoside (granatin B) 8.79 HHDP-gallagyl – 111, 161, 301, ESI1083.0609 1083.0593 -1.51 C48H28O30 7.9 ± 0.2 hexoside (punicalagin I) 601 11.01 HHDP-gallagyl – ESI1083.0589 111, 301, 577, 1083.0593 -0.59 C48H28O30 53.7 ± 1.8 hexoside (punicalagin II) 601, 781, 956 12.45 HHDP-gallagyl – ESI1083.0600 1083.0593 -0.68 C48H2803 94.2 ± 3.8 111, 301, 541, hexoside (punicalagin 601, 781, 956 III) 8.53 unknown ESI663.1325 111, 161 n.a. n.a. n.a. 5.9 ± 0.3 Data are expressed as mean ± standard error (n = 3). DHHDP, dehydro-hexahydroxydiphenic acid; HHDP, hexahydroxydiphenic acid. n.a., non applicable. 1This compound showed a higher mass error than the accepted in this study (>8 ppm); however, MS/MS fragments are characteristics of this compound, 2MS/MS fragments obtained from the analysis of EP fraction.

51 52 53

58

1


Page 37 of 42


Table 4. HPLC-ESI-QTOF Profile of the Hydrolyzable Phenolic Compounds and the Non-extractable Ellagitannin Fractions of a Grape/Pomegranate Pomaces Dietary Supplement. no.

retention proposed compound time (min) hydrolyzable phenolic compounds fraction flavanones 59 24.17 pinocembrin hydroxybenzoic acids 60 12.48 methyl gallate 61

17.39

vanillic acid

hydroxycinnamic acids 62 23.13 ferulic acid

hydroxyciynnamaldehydes 63 27.38 ferulaldehyde hydroxyphenylpropanoic acids 64 15.34 3-(3,4-dihydroxyphenyl)2-methoxy propionic acid ellagitannins 37 19.88 ellagic acid 44 galloyl-HHDP-glucoside 11.93 (lagerstannin C) non-extractable ellagitannins fraction hydroxybenzoic acids 65 11.77 hydroxybenzoic acid 66 9.73 dihydroxybenzoic acid

ionization

experimental mass

MS/MS ions

calculated mass

ppm

formula

amount (mg/100 g dw)

ESI-

255.0668

111, 113, 161

255.0663

-2.02

C15H12O4

0.4 ± 0.04

ESI-

183.0295

183.0299

2.16

C8H8O5

52.1 ± 5.6

ESI-

167.0348

167.0350

1.08

C8H8O4

40.7 ± 6.7

ESI-

193.0512

101, 111, 113, 133, 161, 179

193.0506

-2.93

C10H10O4

20.1 ± 4.7

ESI-

177.0557

111, 161

177.0557

0.10

C10H10O3

61.8 ± 8.9

ESI-

211.0623

111, 113, 126

211.0612

5.20

C10H12O5

3.6 ± 1.0

ESI-

300.999

300.9990

-3.01

C14H6O8

7.9 ± 0.2

ESI-

649.0685

101,185, 229 113, 361, 481, 557

549.0683

-0.83

C27H22O19

5.2 ± 1.7

ESIESI-

137.0243 153.0197

101, 111, 124, 165 111, 113, 126, 161

113 109, 113, 125

137.0244 153.0193

0.85 -2.39

C7H6O3 C7H6O4

4.8 ± 0.7 74.3 ± 9.0 36



61

17.28 vanillic acid ESI167.0353 113, 135 167.0350 Data are expressed as mean ± standard error (n = 3). HHDP, hexahydroxydiphenic acid.

Page 38 of 42

-1.89

C8H8O4

2.2 ± 0.3


Page 39 of 42


Figure 1 254x190mm (96 x 96 DPI)





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Comprehensive Characterization of Extractable and Nonextractable

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