Screening of Anthocyanins and Anthocyanin-Derived Pigments in Red

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Screening of anthocyanins and anthocyanin-derived pigments in red wine grape pomace using LC/DAD-MS and MALDI-TOF techniques Joana Oliveira, Mara Alhinho da Silva, Natercia Teixeira, Victor De Freitas, and Erika Salas J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 27 Apr 2015 Downloaded from http://pubs.acs.org on April 27, 2015

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Journal of Agricultural and Food Chemistry

Screening of anthocyanins and anthocyanin-derived pigments in red wine grape pomace using LC/DAD-MS and MALDI-TOF techniques. Joana Oliveira1*, Mara Alhinho da Silva1, Natércia Teixeira1, Victor de Freitas1, Erika Salas2*

1

REQUIMTE – Laboratório Associado para a Química Verde, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugal. 2

Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, Ciudad Universitaria s/n, C.P. 31170, Chihuahua, México.

*

Author

to

whom

correspondence

should

be

addressed,

[email protected];

[email protected] Fax: (52) 6142366000 Tel: (52) 6142366000 ext. 4286

1

REQUIMTE

2

Facultad de Ciencias Químicas

1

ACS Paragon Plus Environment


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1

ABSTRACT

2

Two phenolic extracts were made from a red wine grape pomace (GP) and fractionated first

3

by sequential liquid-liquid extraction with organic solvents. The aqueous fraction was

4

fractionated by low pressure chromatography on Toyopearl HW-40 gel and on C18.

5

Different fractions were obtained by sequential elution with aqueous/organic solvents, and

6

then analysed by liquid chromatography and mass spectrometry (LC-DAD/MS and

7

MALDI-TOF). Over 50 anthocyanin based pigments were detected by LC-DAD/MS in GP,

8

mainly

9

methylpyranoanthocyanins. The presence of oligomeric malvidin-3-O-coumaroylglucoside-

10

based anthocyanins were also detected in GP using both mass spectrometry techniques LC-

11

DAD/MS and MALDI-TOF.

pyranoanthocyanins

including

A

and

B-type

Vitisins

and

12

red

grape

pomace

(GP);

malvidin-3-O-coumaroylglucoside;

13

Keywords:

14

pyranoanthocyanins; anthocyanin oligomers; LC/DAD-MS; MALDI-TOF.

15

2


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16

Introduction

17

Grape pomace, GP (skins and seeds) is the main waste product produced by the wine

18

industry obtained from the grapes/must pressing during the winemaking process and

19

corresponds to about 30% of the grapes initial weight. Similarly to wines, GP is a very

20

complex matrix containing different classes of polyphenolic compounds (phenolic

21

acids, flavanols, flavones, anthocyanins, etc)1-4 which means that GP can be used as a

22

good source of high-valuable compounds, since these compounds are not fully extracted

23

from grapes into wine. The higher or lower amounts of polyphenolic compounds present

24

in GP are directly correlated to the grape variety and more importantly to the wine-

25

making process. For example, due to the lack of maceration of grape skins during the

26

rose wine production, it is expected that the GP produced herein presents higher

27

concentrations of polyphenolic compounds when compared to the GP obtained by

28

pressing the grape must after the end of alcoholic fermentation in red wines.

29

Moreover, there is a current trend in the food and cosmetics industries to find pigments

30

from natural sources, and GP could be the ideal source as is inexpensive and contains

31

considerable amounts of anthocyanins.5,

32

distilleries that use it to produce mainly alcohol. With this management plan the referred

33

potential bioactive compounds are being lost in the process. These materials can be easily

34

transformed, with adequate processing steps, from a low-value resource into a very

35

interesting high-value one in terms of consumer choice and acceptance in the food,

36

cosmetic and pharmaceutical industries.

37

Furthermore, many works have been published related to the beneficial effects of the

38

grape pomace extracts.7-10 The majority of those studies use white grape pomace and

39

therefore the anthocyanin content and identity of anthocyanin-derived compounds in red

40

grape pomace is known in a lesser extent.4, 5, 11, 12 Bearing this, the aim of this work is to

41

identify the anthocyanins and anthocyanin-derived pigments present in a red grape

6

Nowadays, wine companies sell the GP to

3



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42

pomace from the Douro Demarcated Region from North of Portugal using mass

43

spectrometry techniques such as, LC-DAD/MS and MALDI-TOF. LC-DAD/MS is very

44

complementary to MALDI-TOF analysis. On one hand LC-DAD/MS provides the

45

means to make peak assignments and acquire quantitative data by relying on UV-VIS

46

data and mass spectra data. On the other hand MALDI-TOF analysis is the ideal

47

technique for the analysis of complex mixtures as it produces only a singly charged

48

molecular ion for each parent molecule. Another important difference between ESI-MS

49

and MALDI-TOF is that sometimes when analysing highly concentrated anthocyanin

50

solutions (even pure standards) you can detect stacking in ESI-MS, which is the double

51

(or sometimes even triple) of the anthocyanin m/z. Stacking has not been reported to

52

occur in MALDI-TOF analysis.

53 54

Materials and methods

55

Red grape pomace.

56

Red GP (wine seeds and skins) was provided by Gran Cruz – Sociedade Comercial de

57

Vinhos, Lda. (a Douro and Port wine company from the North of Portugal) and was

58

obtained as a result of the pressing of the red wine must after the end of the alcoholic

59

fermentation. The red wine must was made from the main red varieties produced in the

60

Douro Region (Touriga Nacional, Touriga Franca, Tinta Roriz, Tinta Barroca and Tinto

61

Cão).

62 63

Red grape pomace extracts.

64

A red GP extract (Extract 1) was obtained by the extraction of 250 g of red GP with 1

65

L of water/methanol/acetone 3.5/1.5/5 solvent acidified with HCl (0.01 M). The

66

solution was sonicated in an ice bath for 30 min and then the solids filtered using glass

4


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67

wool. A similar extract (Extract 2) was obtained but in this case the solution was

68

sonicated for 18 h.

69 70

Purification of the grape pomace extracts.

71

Organic solvents were removed from the previous extracts 1 and 2, by evaporation in a

72

rotoevaporator under vacuum. and the resulting aqueous extract (200 mL) was

73

submitted to a liquid-liquid extraction procedure with 2 x 200 mL hexane. After

74

rotoevaporation of the hexane, the residue was analysed by HPLC-DAD-MS. The

75

proteins present in the aqueous fraction were precipitated in cold methanol (1:50) and

76

then removed by centrifugation. The aqueous fractions (Extract 1 and 2) without

77

proteins were lyophilized and then analysed by LC/DAD-MS and MALDI-TOF.

78 79

Fractionation of the grape pomace extracts.

80

1 g of GP Extract 1 was applied on top of a C18 (20 g) column and different fractions

81

were obtained by elution with water acidified with formic acid (1%), ethyl ether, ethyl

82

acetate and aqueous solution with increasing percentages of methanol (10%, 30%, 60%

83

and 100%, v/v) acidified with formic acid. Additionally, 100 mg of the same extract

84

were fractionated by TSK Toyopearl 40-HW(S) column chromatography using different

85

aqueous solvents with increasing percentages of methanol (0, 10, 20, 30 and 100%,

86

v/v). The obtained fractions were analysed by LC/DAD-MS and MALDI-TOF.

87

1 g of Extract 2 dissolved in 200 mL of water was submitted to a liquid-liquid

88

extraction procedure with ethyl acetate (3 x 200 mL) and isoamyl alcohol (2 x 200

89

mM). All the fractions were analysed by LC/DAD-MS.

90 91

LC/DAD-MS

5



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92

The anthocyanin and anthocyanin-derived compounds composition of each fraction was

93

evaluated by LC/DAD-MS. An Accela series liquid chromatograph, equipped with a

94

150x4.6 mm i.d., 5 µm LicroCART® reversed-phase C18 column was used and

95

detection was carried out between 200-600 nm using an Accela PDA detector. The mass

96

detection was performed using a LTQ Orbitrap XL mass spectrometer (Thermo Fischer

97

Scientific, Bremen, Germany) controlled by LTQ Tune Plus 2.5.5 and Xcalibur 2.1.0.

98

Solvents were A: H2O/HCOOH (99:1), and B: HCOOH/H2O/CH3CN (1:69:30). The

99

gradient was performed using an Accela 600 Pump and consisted of 20-85% B for 70

100

min at a flow rate of 0.3 mL/min. The column was washed with 100% B for 10 min and

101

then stabilized with the initial conditions for another 10 min. The capillary voltage of

102

the electrospray ionization (ESI) was set to 3100 V and the capillary temperature was

103

275 ºC. The sheath gas flow rate (nitrogen) was set to 5 (arbitrary unit as provided by

104

the software settings). The capillary voltage was 49 V and the tube lens voltage 250 V.

105

Spectra were recorded in positive ion mode between m/z 50 and 2000. The mass

106

spectrometer was programmed to do a series of three scans: a full scan mass, a zoom

107

scan of the most intense ion in the first scan (SIM – Selected Ion Monitoring), and a

108

MS-MS of the most intense ion using relative collision energy of 30 and 60 V.

109 110

MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight) analysis.

111

The different GP fractions were analysed by MALDI-TOF using an UltrafleXtreme

112

mass spectrometer (Bruker, Bremen, Germany) operating in positive reflectron ion

113

detection mode with laser SmartBeamIII and under FlexCompass 1.4 software control

114

(Bruker Daltonics, Germany). For each sample the laser power was adjusted to 55%, the

115

detector gain 4.0x (2410V) and the mass range between 200 and 3500.

6


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116

For the sample preparation, a tIAA (trans-3-indolacrylic acid) matrix (≥98.5%, Sigma-

117

Aldrich, USA) 50 mg/mL was prepared in an aqueous solution of 70% (v/v) acetone.

118

Moreover, each grape pomace fraction was prepared in the same solvent in a

119

concentration of 5 mg/mL. The polyphenolic fractions eluted from the Toyopearl

120

column were mixed with the matrix solution at volumetric ratios of (1:2). The

121

analyte:matrix mixture was first deionized with Dowex 50X8-400 cation exchange resin

122

(Supelco), equilibrated in 70% aq. acetone (v/v). Positive ion mode was used for the

123

detection of anthocyanins. Two microliters from each sample (after mixing with the

124

matrix and the resin) was applied onto a stainless steel target plate (MTP 394 target

125

plate ground steel BC, Bruker Daltonik GmbH, Germany) and fully air-dried. For each

126

assay, three sample spot replicates were analysed and samples were spotted in triplicate.

127 128

Results and discussion

129 130

GP anthocyanic profile by LC/DAD-MS

131

According to the LC/DAD analysis of the red GP Extract 1 without using any previous

132

fractionation methodology it was possible to observe in the chromatogram recorded at

133

520 nm, the presence of twelve peaks (Figure 1A). The main compound was identified

134

by its UV-Visible spectra, retention time, ion mass (m/z 639) in the positive ion mode

135

and fragmentation pattern (MS2 331) and was attributed to the malvidin-3-O-

136

coumaroylglucoside (peak 43) (Figure 1 and Table 1). The area of malvidin-3-O-

137

coumaroylglucoside represents around 65% of the total area. The chromatographic

138

profile (Figure 1) of the GP extract is quite different from one that is usually observed

139

in grapes and young red wines where generally, malvidin-3-O-glucoside is the main

140

anthocyanin present with the coumaroyl-derivative being the second most intense

7



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141

especially in the Douro region.13 The appearance of this chromatographic profile can be

142

due to the specificities of the winemaking process or it can be explained by the lower

143

solubility of the coumaroyl-derivative in aqueous solution, slowing or limiting its

144

diffusion to the hydroalcoholic solution during the fermentation/winemaking process.

145

Additionally to the malvidin-3-O-coumaroylglucoside pigment, other peaks were

146

detected in the same GP extract corresponding to the four non-acylated anthocyanins-3-

147

O-glucosides mainly present in grapes and red wines (delphinidin, petunidin, peonidin

148

and malvidin) and to some of their acetylated, coumaroylated and caffeoylated

149

derivatives (Figure 1 and Table 1). The presence of the A and B-type vitisins

150

compounds were also observed (peaks 10 and 13, respectively) (Figure 1 and Table 1).

151

Moreover, the detection of a compound (peak 40) (Figure 1) with the same ion mass as

152

the malvidin-3-O-coumaroylglucoside (m/z 639) (peak 43) in the positive ion mode was

153

observed. Similarly to what was reported in the literature by Monagas et al. (2003) and

154

Núñez et al. (2004)14, 15 in grapes and wines from Vitis vinifera this can be attributed to

155

the occurrence of the cis isomer of the malvidin-3-O-(6-p-coumaroyl)-glucoside (40) in

156

GP that is present in smaller amounts when compared with the trans isomer.

157

In order to obtain fractions with different chemical compositions and therefore better

158

characterize the GP extract polyphenolic composition, a fractionation methodology was

159

developed. First, the anthocyanin and anthocyanin-derived compounds from Extract 1

160

were separated in a reverse phase C-18 resin and eluted with solvents that present

161

different polarities, namely, ethyl ether, ethyl acetate, water and aqueous solutions with

162

increasing percentages of methanol. With the less polar solvent (ethyl ether) it was

163

possible to remove the chlorophylls and the hydrophobic fractions but due to its lack of

164

solubility in water or methanolic solvents this fraction was not analyzed by LC/DAD-

165

MS. No additional peaks were detected in those fractions obtained from the

8


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166

fractionation of the GP with C-18 resin or with Toyopearl HW-40(S) gel when

167

compared to direct analysis of the GP extract (Extract 1). The main exception was the

168

fraction eluted with 60% (v/v) methanol where it was possible to detect by LC-MS in

169

positive ion mode the presence of the malvidin-3-O-coumaroylglucoside dimer (49)

170

(m/z 1277) and the malvidin-3-O-glucoside trimer (4) (m/z 1477) (Figure 2) both

171

already reported in the literature to be present in red grapes and wines.16-18 The

172

possibility of detecting anthocyanins self-assembling by LC-MS made us think that

173

these signals detected in fraction 60% (v/v) methanol could be due to the auto-

174

association

175

respectively, leading to the formation of the dimer and the trimer. To confirm that dimer

176

and trimer are isolated compounds and not products formed during the anthocyanins

177

auto-association, MALDI-TOF analysis was used and the results are discussed below.

178

However, the high complexity of the red GP was possible to recognize after the

179

LC/DAD-MS analysis of GP Extract 2 where the solid/solvent contact time was

180

increased to 18 hours. In this extract more than fifty different anthocyanin and

181

anthocyanin-derived compounds were detected and the most of them identified (Table

182

1). In this extract the five anthocyanins-monoglucosides (delphinidin, cyanidin,

183

petunidin, peonidin and malvidin), two acetylglucoside derivatives (petunidin and

184

malvidin), five coumaroyl-derivatives and three caffeoyl-derivatives (delphinidin,

185

peonidin and malvidin) were identified based on their ion mass, fragmentation patterns

186

(Table 1) and UV-Visible spectra (data not shown). The presence of these anthocyanins

187

in GP is not surprising since they are present in red grapes and therefore are also

188

expected to be present in the resulting red GP.19, 20 Additionally, in the mass spectrum in

189

the positive ion mode was detected an ion mass at m/z 655 (peak 5) and two fragments

190

m/z 493 (- 162 a.m.u.) and m/z 331 (- 2 x 162 a.m.u). The fragmentation pattern is in

of

malvidin-3-O-coumaroylglucoside

and

malvidin-3-O-glucoside,

9



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191

agreement with the presence of the malvidin aglycone (m/z 331) and two glucose

192

moieties (- 2 x 162 a.m.u) like is observed in malvidin-3,5-diglucoside (malvin). This is

193

also corroborated by the retention time that is smaller (RT 39.13 min) than the one

194

observed for malvidin-3-glucoside (RT 42.92 min) as is expected for a diglucoside

195

(more polar) in a reverse phase C-18 column. The presence of malvin is not expected to

196

occur in Vitis vinifera grapes and only in other Vitis sp. Even though its occurrence in

197

Vitis vinifera-based GP has never been reported in the literature, Baldi et al. and Heier

198

et al. have detected small amounts of anthocyanin-3,5-diglucosides in Vitis vinifera L.

199

grapes and wines.20, 21 Moreover, the same ion mass (m/z 655) was observed later in the

200

mass chromatogram (RT 65.43 min) but in this case the fragmentation pattern was m/z

201

331 (corresponding to the loss of a caffeoylglucoside moiety, - 324 a.m.u.) and by this

202

was attributed to the malvidin-3-O-caffeoylglucoside (32). Furthermore, seven A-type

203

vitisins

204

coumaroylated and caffeoylated) were also detected in red GP and identified by

205

comparison with their known fragmentation patterns (Table 1) reported in the

206

literature.22-24 Similarly, five B-type vitisins and three methylpyranoanthocyanins were

207

also identified based on their fragmentation patterns (Table 1).25,

208

fragmentation pattern of A and B-type vitisins is the loss of their sugar moiety that can

209

be in their non-acylated (glucose – 162 a.m.u.) or acylated (acetylglucose – 204 a.m.u.,

210

coumaroylglucose – 308 a.m.u. or caffeoylglucose – 324 a.m.u.) forms giving origin to

211

the respective A and vitisin aglycones (m/z 399 and 355, respectively for the malvidin-

212

derived compounds). The presence of these anthocyanin-derived pigments, although

213

expected, has never been reported in the literature for grape pomace.

214

Besides, anthocyanin-derived compounds containing a catechin moiety were also

215

detected in GP. These compounds belong to the family of the malvidin-methylmethine-

(petunidin,

peonidin

and

malvidin-based,

non-acylated,

26

acetylated,

The typical

10


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216

catechin pigments and were identified by their ion mass (m/z 809) and characteristic

217

fragmentation patterns (m/z 519 and 357) according to the data reported in the

218

literature27. These ion mass and fragmentation patterns were observed for two

219

compounds (15 and 21) (Table 1) and two explanations can be given for this. First, the

220

malvidin-methylmethine-catechin pigment has an asymmetric carbon in the

221

methylmethine group and this way two enantiomers are possible to be present. Another

222

explanation is the possibility of formation of a similar compound but in this case with (-

223

)-epicatechin instead of the (+)-catechin. As (+)-catechin and (-)-epicatechin are isomers

224

the same ion mass and fragmentation patterns are expected to occur in (-)-epicatechin-

225

based pigments. The unequivocal identification of these compounds is not possible to

226

achieve through LC-MS spectrometry and only using NMR spectroscopy that structure

227

would be confirmed. The coumaroyl-derivative of this compound was also found to

228

occur in GP (42) (Table 1). Moreover, the presence of two pyranomalvidin compounds

229

with a phenol moiety were also detected and identified by LC/DAD-MS in the GP

230

extract, namely, the pyranomalvidin-3-O-glucoside-phenol (46) and the respective

231

coumaroyl-derivative (50). The ion mass detected at 82.85 min with m/z 813 and

232

fragment

233

coumaroylglucoside-pyrogallol (48) (Figure 3A, Table 1) with a structure similar to B-

234

type portisins but that has never been described in the literature. However, this is only a

235

hypothetical structure since no full characterization by NMR has been performed on the

236

compound due to its small concentration in GP. On the other hand, the ion mass m/z 671

237

detected in GP extract with a fragment at m/z 331 was attributed to the malvidin-3-O-

238

galloylglucoside (41) (Figure 3B, Table 1). The galloylated-derivative of anthocyanins

239

has never been described in the literature. However, this identification is only based on

240

the fragmentation pattern of the molecule as the full characterization of the compound

m/z

505

could

be

identified

as

a

vinylpyranomalvidin-3-O-

11



Page 12 of 26

241

by NMR was not possible. Additionally, small amounts of the malvidin-3-O-caftaric

242

acid (11) (Figure 3C, Table 1) were also detected in the GP extract. Moreover, some

243

compounds with known fragmentation patterns, namely loss of glucose or RDA (Retro-

244

Dials Alder) fragmentation that indicates the presence of a flavanol moiety were also

245

observed nevertheless, the identity of these compounds was not achieved. Their ion

246

masses are not reported in the literature and using only mass spectrometry it is not

247

possible to fully identify these anthocyanin-derived compounds. Additionally, the small

248

amounts of the compounds present in the GP extract makes its full characterization by

249

NMR nearly impossible to accomplish. These compounds are indicated in Table 1 as

250

“unknown”.

251 252

MALDI-TOF

253

The use of the mass spectrometry MALDI-TOF technique to characterize the

254

oligomeric nature of the GP extract was of crucial importance since the presence of the

255

oligomeric anthocyanins ion masses (m/z 1277, 1477, 1623 and 1915) in the ESI-MS

256

could be due to a self-assembling mechanism that sometimes is possible to observe

257

when the concentration of the sample is high in this mass spectrometry technique. In

258

MALDI-TOF stacking has not been reported to occur.

259

Figures 4a and 4b show the MALDI-TOF analysis of the GP extract (Extract 1). It can

260

be observed that the ion at m/z 639 is the most intense, which is in agreement with

261

Figure 1, malvidin-3-O-coumaroylglucoside (compound 43).

262

Figure 4b shows a series of masses that could correspond to oligomeric coumaroylated

263

anthocyanins: the ion at m/z 1131 might be a mono coumaroylated malvidin-3-O-

264

glucoside dimer, the ion at m/z 1277 could correspond to the malvidin-3-O-

265

coumaroylglucoside dimer (compound 49, Table 1), the ion at m/z 1623 could be a

12


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266

mono coumaroylated malvidin-3-O-glucoside trimer (compound 23, Table 1), the ion at

267

m/z 1769 might be di-coumaroylated malvidin-3-O-glucoside trimer and finally the ion

268

at m/z 1915 could correspond to a trimer of malvidin 3-O-coumaroylglucoside. This

269

latter in its non-acylated glucoside form has already been detected in a young Port

270

wine.18

271

MALDI-TOF-TOF fragmentation of the ion at m/z 1915 gave fragments at m/z 1607 (-

272

308), m/z 1461 and m/z 331 (Figure 5). The loss of 308 mass units corresponds to a

273

glucose acylated with a coumaric acid, the loss of 146 mass units corresponds to a

274

coumaric acid moiety, therefore it can be concluded that these fragmentations confirms

275

the suspected nature of the trimer.

276

MALDI-TOF has already been used in the analysis of oligomeric food polyphenols,28

277

mainly procyanidins, however it is not commonly used in anthocyanin analysis since

278

monomeric anthocyanins have a molecular weight of around 500 mass units, which is a

279

difficult mass range for MALDI-TOF analysis. However, oligomeric pigments

280

containing both anthocyanins and flavanols have already been detected by MALDI-TOF

281

to occur in cranberry fruit.28, 29 Nevertheless, to our knowledge, this is the first study

282

where MALDI-TOF is used for the analysis of oligomeric anthocyanins.

283

One of the late eluting fractions (obtained with 100% organic solvent in low pressure

284

chromatography with Toyopearl HW-40(S) gel) was particularly enriched in less

285

hydrophilic pigments. This fraction was mainly composed of coumaroyl derivatives of

286

anthocyanins (monomers and oligomers).

287 288

Grape pomace is an interesting source of anthocyanin based pigments (both native and

289

derived), as over 50 different anthocyanin based compounds were detected in this study.

290

Oligomeric anthocyanins with coumaroylated derivatives of malvidin were detected by

13



Page 14 of 26

291

means of mass spectrometry for the first time in this work, and it remains still unknown

292

the influence of oligomeric anthocyanins on wine color; these oligomeric anthocyanins

293

(malvidin-3-O-glucoside trimer) have already been detected in young port wine,

294

however, the less hydrophilic nature of oligomeric coumaroylated anthocyanins arises

295

the question of their extractability in wine. Moreover, recently the equilibrium forms of

296

malvidin-3-O-glucoside trimer present in grape skins were studied in aqueous solution

297

at different pH values through UV-Visible spectroscopy. It was observed that the

298

reactivity of this compound is strongly dominated by acid-base chemistry, with the

299

hydration reaction accounting less than 10% of the overall reactivity.30 This points to

300

that polymerization may be a natural stabilization mechanism for the red color of

301

anthocyanins.

302 303

Abbreviations Used

304

GP – Grape Pomace

305

RT – Retention Time

306 307

Acknowledgements

308

The authors thank Msc. Silvia Maia for the LC-DAD/MS and MALDI-TOF analysis.

309 310

References

311

1.

312

Chem. 1999, 65, 1-8.

Lu, Y.; Yeap Foo, L., The polyphenol constituents of grape pomace. Food

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2.

Amico, V.; Napoli, E. M.; Renda, A.; Ruberto, G.; Spatafora, C.; Tringali, C.,

314

Constituents of grape pomace from the Sicilian cultivar `Nerello Mascalese'. Food

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Chem. 2004, 88, 599-607.

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3.

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of bioactive compounds: extraction, characterization, and biotechnological applications

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of phenolics. J. Agric. Food Chem. 2013, 61, 8987-9003.

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de Sa, M.; Justino, V.; Spranger, M. I.; Zhao, Y. Q.; Han, L.; Sun, B. S.,

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Boonchu,

T.;

Utama-Ang,

N.,

Optimization

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extraction

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Monagas, M.; Núñez, V.; Bartolomé, B.; Gómez-Cordovés, C., Anthocyanin-

Núñez, V.; Monagas, M.; Gomez-Cordovés, M. C.; Bartolomé, B., Vitis vinifera

Vidal, S.; Meudec, E.; Cheynier, V.; Skouroumounis, G.; Hayasaka, Y., Mass

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anthocyanin-derived pigments in red wines. J. Agric. Food Chem. 2001, 49, 4836-4840.

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new color-stable anthocyanins occurring in some red wines. J. Agric. Food Chem. 1997,

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compounds. Tetrahedron Lett. 2009, 50, 3933-3935.

Oliveira, J.; da Silva, M. A.; Jorge Parola, A.; Mateus, N.; Brás, N. F.; Ramos,

Segade, S. R.; Vazquez, E. S.; Rodriguez, E. I. V.; Martinez, J. F. R., Influence

Baldi, A.; Romani, A.; Mulinacci, N.; Vincieri, F. F.; Casetta, B., HPLC/MS

Heier, A.; Blaas, W.; Droß, A.; Wittkowski, R., Anthocyanin analysis by

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changes during ageing. Vitis 1993, 32, 111-118.

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of oligomeric food polyphenols. Phytochemistry 2005, 66, 2248-2263.

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Ionization

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Christian, G. K.; Martha, M. V.; Jess, D. R., Matrix-Assisted Laser DesorptionTime-of-Flight

Mass

Spectrometry

of

anthocyanin-poly-flavan-3-ol

Oliveira, J.; Bras, N. F.; da Silva, M. A.; Mateus, N.; Parola, A. J.; de Freitas,

400 401

This work received financial support from FEDER funds through COMPETE,

402

POPH/FSE, QREN and FCT (Fundação para a Ciência e Tecnologia) from Portugal by

403

one PhD SFRH/BD/70053/2010 and one post-doctoral SFRH/BPD/65400/2009

404

scholarship. E. Salas thanks the Consejo Nacional de Ciencia y Tecnologia

405

(CONACYT,

Mexico)

for

providing

grant

number

204199.

18


Page 19 of 26


FIGURE CAPTION

Figure 1 – A) Chromatogram at 520 nm obtained from the LC/DAD-MS from the red GP Extract 1; B) Chromatogram at 520 nm obtained from the LC/DAD-MS from the isoamilic alcohol fraction and C) aqueous fraction of the red GP Extract 2. *, unidentified peak. Figure 2 – MS-ESI analysis performed in the positive-ion mode of a GP fraction (60% (v/v) methanol) showing the presence of anthocyanin dimers and trimers: A, full mass spectrum (m/z 1277) of the malvidin-3-O-coumaroylglucoside dimer (49) and B, full mass spectrum (m/z 1477) of the malvidin-3-O-glucoside trimer (4). Figure 3 – Hypothetic structures of the compounds A: 48 (vinylpyranomalvidin-3-Ocoumaroylglucoside-pyrogallol); B: 41 (malvidin-3-O-galloylglucoside) and C: 11 (malvidin-3-O-caftaric acid) detected by LC-MS in the positive ion mode in the GP extract 2. Figure 4– A) MALDI-TOF analysis in the positive ion mode of the red GP extract (Extract 1); B) Same analysis from m/z 1000 to 2000. Figure 5 – MALDI-TOF-TOF fragmentation of the ion at m/z 1915.

19



Page 20 of 26

Table 1 – Molecular Ion in the Positive Ion Mode and Respective Fragments MS2 and MS3 Obtained by API-LC-ESI-MS/MS of Several Anthocyanins and AnthocyaninDerived Compounds Detected in Red GP (Extract 2). [M+]

Compound

Retention time (min)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

29.52 35.52 36.50 33.65 39.13 41.54 42.92 45.09 47.32 48.36 48.53 48.65 49.10 51.53 52.95 53.28 54.07 54.49 54.67

465 449 479 1477 655 463 493 657 487 561 625 547 517 801 809 603 663 521 627

20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

55.18 56.89 58.30 58.79 59.08 59.60 59.98 60.04 61.79 62.24 63.23 65.65 65.43 65.88 66.52 66.83 67.15 67.33 67.95 68.69 69.42 70.74 70.77 73.19 73.99 76.75 78.44 82.59 82.85 83.01 85.43

531 809 693 1623 723 927 479 535 679 611 649 625 655 707 663 663 693 595 625 663 639 671 955 639 677 1315 609 909 813 1277 755

[MS2]

[MS3]

303 287 317 493 301 331 495 325 399 331 385 355 331 519 399 369 317 303 369 519 385 399 619 303 331 355 303 341 301 331 399 369 339 369 287 317 355 331 331 665 331 369 447 601 505 447

331

357

357

357

449

Identity Delphinidin-3-O-glucoside Cyanidin-3-O-glucoside Petunidin-3-O-glucoside Malvidin-3-O-glucoside-trimer Malvidin-3,5-O-diglucoside Peonidin-3-O-glucoside Malvidin-3-O-glucoside Unknown Pyranopeonidin-3-O-glucoside Carboxypyranomalvidin-3-O-glucoside Malvidin-3-O-caftaric acid Carboxypyranopetunidin-3-O-glucoside Pyranomalvidin-3-O-glucoside Malvidin-3-O-coumaroylglucoside-5-O-glucoside Malvidin-3-O-glucoside-(8,8)-methylmethyne-catechin Carboxypyranomalvidin-3-O-acetylglucoside Methylpyranomalvidin-3-O-caftaric acid Petunidin-3-O-acetylglucoside Delphinidin-3-O-caffeoylglucoside Methylpyranomalvidin-3-O-glucoside Malvidin-3-O-glucoside-(8,8)-methylmethyne-epicatechin Carboxypyranopetunidin-3-O-coumaroylglucoside Mono coumaroyl Malvidin-3-O-glucoside-trimer Carboxypyranomalvidin-3-O-caffeoylglucoside Unknown Unknown Malvidin-3-O-acetylglucoside Pyranomalvidin-3-O-caffeoylglucoside Delphinidin-3-O-coumaroylglucoside Pyranopetunidin-3-O-coumaroylglucoside Peonidin-3-O-caffeoylglucoside Malvidin-3-O-caffeoylglucoside Carboxypyranomalvidin-3-O-coumaroylglucoside Unknown Methylpyranopeonidin-3-O-caffeoylglucoside Carboxypyranopeonidin-3-O-caffeoylglucoside Cyanidin-3-O-coumaroylglucoside Petunidin-3-O-coumaroylglucoside Pyranomalvidin-3-O-coumaroylglucoside Malvidin-3-O-cis-coumaroylglucoside Malvidin-3-O-galloylglucoside Malvidin-3-O-coumaroylglucoside-(8,8)-methylmethyne-catechin Malvidin-3-O-trans-coumaroylglucoside Methylpyranomalvidin-3-O-coumaroylglucoside Unknown Pyrano-Malvidin-3-O-glucoside-phenol Unknown Vinylpyrano-Malvidin-3-O-coumaroylglucoside-pyrogallol Malvidin-3-O-coumaroylglucoside dimer Pyrano-Malvidin-3-O-coumaroylglucoside-phenol

20


Page 21 of 26


Figure 1 43

A

130000

Absorbance at 520 nm (uAU)

110000

90000

70000

50000

30000

7 32 38 40

13 10000

1

0 0

10

20

30

27 29

10

6

3

40

50

60

70

80


43

B


1600000 1400000 1200000 1000000 800000

38

600000

28 400000 200000

6 3 0

10

20

30

37

32 31

39 42 40

45

47

27

7

40

50

50

60

70

80


7

C

550000


500000 450000

10

400000

13

350000 300000 250000

20 15 16

200000 150000

3

100000

1 50000

0

10

20

30

6

*

27 44 24 28 23 33 39 36

*

42 40

50

60

70

80


21



Page 22 of 26

Figure 2 1277.33

100

A

90

Relative Abundance

80 70 60 50 40 30

331.08 639.17

20

447.11

755.19

10

1770.46 1916.50

969.24 1157.31 1459.35

987.25

2105.92

2419.93 2554.65

2826.07

2400

2800

0 400

600

800

1000

1200

1400

1600

1800

2000

2200

2600

3000

m/z

1477.38

100

B

90

Relative Abundance

80 70 60 50 40 360.14

1447.37 1417.36

30 20

331.08 181.05

10

299.06

1315.33 470.18 493.13

1519.39 677.14

1153.28 1285.32

758.17 873.24

1624.42

1780.46

1938.61

0 200

400

600

800

1000

1200

1400

1600

1800

2000

m/z

22


Page 23 of 26


Figure 3 A

B

C

23



Page 24 of 26

Figure 4A x105 639

3.0

2.5

Intensity

2.0

755 1.5 331 535

1.0

781

1277

0.5 493

969

1173 1607

1769

1915

0.0 500

1000

1500

2000

2500

3000

m/z

Figure 4B x104 1277

4

Intensity

3

2 1301 1247.5 1131 1915. 1769 1623

1

0 1100

1200

1300

1400

1500

1600

1700

1800

1900

m/z

m/z

24


Page 25 of 26


Figure 5

OMe

1461

OH

800 HO

O OMe O HO

O

OH OH O

O

OH OMe

1607 OH

600

-146

OH

-308

O

O

OMe

Intensity

O HO

O

OH OH O

O

OH OMe

400

OH OH O

O OMe O HO

O

OH O

OH

OH O

200 OH

331

0

200

400

600

800

1000

1200

1400

1600

1800

m/z

25



Page 26 of 26

Graphic for Table of Contents

26


Screening of Anthocyanins and Anthocyanin-Derived Pigments in Red

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