Identification of Anthocyanins from Four Kinds of Berries and Their

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Identification of anthocyanins from four kinds of berries and their inhibition activity to #-glycosidase and protein tyrosine phosphatase 1B by HPLC-FT-ICR MS/MS Ting Xiao, Zhenghong Guo, Baoshan Sun, and Yuqing Zhao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02550 • Publication Date (Web): 12 Jul 2017 Downloaded from http://pubs.acs.org on July 15, 2017

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Journal of Agricultural and Food Chemistry

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Identification of anthocyanins from four kinds of berries and their inhibition

2

activity to α-glycosidase and protein tyrosine phosphatase 1B by HPLC-FT-ICR

3

MS/MS Ting Xiaoa, Zhenghong Guob, Baoshan Suna, d, Yuqing Zhaoa, c*

4 5

a.

6

b.

7

c.

8

Education, Shenyang Pharmaceutical University, Shenyang 110016, China.

9

d.

Shenyang Pharmaceutical University, Shenyang 110016, China. Bijie Municipal Hospital of Traditional Chinese Medicine, Bijie 551700, China. Key Laboratory of Structure-based Drug Design and Discovery of Ministry of

Pólo Dois Portos, Instituto National de Investigação Agrária e Veterinária, I.P.,

10

Quinta da Almoinha, 2565-191 Dois Portos, Portugal.

11

*

12

School of functional food and wine, Shenyang Pharmaceutical University, No.103,

13

Wenhua Road, Shenhe District, Shenyang 110016, Liaoning, P. R.China. Tel:

14

+86-24-23986521, Fax: +86-24-23986521, email: [email protected] *(Y. Zhao)

15

E-mail address of authors:

16

[email protected]

17

[email protected] (B. Sun)

Address for correspondence:

(T.

Xiao);

[email protected]

18 19 20 21 22 23 24 25 26 27 28

ACS Paragon Plus Environment

(Z.

Guo);


29

Abstract: The polyphenolic profiles of four berries (blueberry, bilberry, mulberry, and

30

cranberry) in China were investigated using Fourier transform-ion cyclotron

31

resonance mass spectrometry (FT-ICR MS). Thirty-nine polyphenols including 26

32

anthocyanins, 9 flavonoids, and 4 phenolic acids were identified accurately. Cyanidin

33

aglycones are common in four berries, and malvidin aglycones are the main

34

compounds found in bilberry and cranberry. The anthocyanin level in blueberry are

35

the highest with 739.6 ± 17.14 mg/g DW and presented the strongest antioxidant

36

capacity in DPPH, ABTS, FRAP, and ORAC assay. In α-glycosidase, the inhibition

37

activity was in the following order: mulberry > bilberry > blueberry > cranberry. For

38

the PTP1B inhibition assay, blueberry demonstrated the highest inhibitory effect with

39

IC50 3.06 ± 0.02 µg/mL, followed by bilberry, mulberry, and cranberry. Molecular

40

docking results showed that cyanidin aglycones had the highest inhibition activity to

41

PTP1B.

42

Keywords: Anthocyanins; HPLC-FT-ICR MS/MS; berries; antioxidant capacity;

43

α-glycosidase and protein tyrosine phosphatase 1B (PTP1B) inhibitors; molecular

44

docking;

45 46 47 48 49 50 51 52


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1. Introduction

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Clinical and epidemiological studies have revealed that polyphenol-rich diets

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may decrease the risk of many chronic or age-related diseases, such as diabetes

56

mellitus, cardiovascular diseases, neurodegenerative disease, and other degenerative

57

disorders.1,2 Fruits and vegetables are rich in phenolic compounds. These compounds

58

consist of water-soluble (phenolic acids, flavonoids, anthocyanins, and quinones) or

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water-insoluble compounds (condensed tannins) and have a conjugated aromatic

60

system and hydroxyl groups.3 Many physiological benefits of phenolic acids,

61

flavonoids, and anthocyanins have been attributed to their antioxidant and free radical

62

scavenging properties. 4

63

Anthocyanins are the largest group of water-soluble polyphenolic pigment in the

64

plant kingdom with a positive charge in acidic solution. They have strong antioxidant,

65

aging resistance, anti-inflammatory, antidiabetic, and anti-cancer properties and help

66

in controlling obesity.5 They are also responsible for most red, violet, and blue colors

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of fruits and vegetables.6 Anthocyanins are glycosides of anthocyanidins, which have

68

different degrees and positions of hydroxylation and methoxylation. Among the more

69

than 560 natural anthocyanins in nature, the most common anthocyanins are cyanidin,

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delphinidin, malvinidin, pelargonidin, peonidin and petunidin. The most common

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glycosides are galactose, glucose, xylose, arabinose, and rhamnose, which are

72

conjugated to anthocyanin skeletons via the C3 hydroxyl group. 7

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HPLC-MS has been proven to be a powerful and reliable analytical approach for

74

rapid identification of chemical constituents in berries by means of the high sensitivity

75

and functional versatility of MS. They include simple quadrupole (Q), ion-trap mass

76

spectrometers (ITMS), and higher resolution for identification instruments, such as

77

triple quadrupole (qQq), quadrupole-time-of-flight (Q-TOF), and Oribitrap systems.8

78

This method combined with electrospray ionization (ESI) and atmospheric-pressure

79

chemical ionization (APCI), show high sensitive and great ionization stability, which

80

indispensable for analysis of polyphenols and semi-quantitative determination of



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anthocyanins. qQq is capable of performing multiple reaction ion monitoring (MRM),

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but it has low mass resolution, which is insufficient for infering the molecular formula

83

of an unknown compound.9 ITMS could perform MSn experiments for compounds

84

distinguishable in the MS2 spectra, but the mass resolution obtained is weaker than

85

qQq.9 For Q-TOF and Oribitrap mass spectrometry, in spite of the high resolution, it

86

cannot determine anthocyanins with similar structures. Therefore, mass spectrometry

87

is necessary for anthocyanins with less chromatographic resolution.

88

Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS)

89

combined with ESI, provides sub-femtomolar sensitivity and infusion mode for minor

90

or trace compounds in complex mixtures.4 First, it can provide accurate mass

91

measurement and significant structural information about minor compounds,

92

especially unknown compounds in complex mixtures, with no reference substance.10

93

Second, it can isolate individual species for MS/MS with high front-end resolution

94

and analyze isobaric species with mass less than that of an electron. Thus FT-ICR MS

95

is an ideal method for profiling anthocyanins, which cannot be chromatographiclly

96

separated, without the need for separation.11 recently, it has been applied for the

97

analysis of fruit samples.12 Several anthocyanins were quantified and detected in

98

berries using HR-mass spectrometry, such as HPLC coupled to ESI-ToF or

99

ESI-Orbitrap. For example, 31 and 25 anthocyanins were identified from Chilean

100

berries and blueberries, respectively, by HPLC-HR-ESI-ToF-MS;6,13 10 and 12

101

anthocyanins were identified from raspberries and blueberry extracts,respecrively, by

102

high-resolution Exactive Orbitrap Mass Spectrometer.14 Furthermore, anthocyanins

103

were detected from different kinds of berries by low-resolution QTrap MS/MS. 15, 16

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As a popular health food around the world, berries are gaining more and more

105

attention, because of their chemopreventive, antioxidant, anti-inflammatory,

106

anti-microbial,

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hepatoprotective.17 Blueberries (Vaccinium uliginosum L.), cranberries (Vaccinium

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oxycoccos L.), and bilberries (Vaccinium myrtillus L.) belong to the family Ericaceae

109

within the genus Vaccinium. Many of these species are present in North America and

anti-radiation,

cardioprotective,


gastroprotective,

and

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Northern Europe. In China, they are distributed mainly in Changbai Mountains,

111

Daxingan and Xiaoxingan Mountains. Mulberries (Morus alba L.) belong to the

112

family of Moraceae, in the genus Morus. They are widely cultivated in China, Korea,

113

and Japan.18 These berries are rich sources of bioactive compounds, such as phenolic

114

acids, flavonoids, and anthocyanins, which have antiproliferative, antioxidant,

115

anti-inflammatory, and antimicrobial properties.

116

Type 2 diabetes mellitus, characterized by high glucose levels and aberrant

117

insulin secretion, has affected a significant part of the world’s population. The World

118

Health Organization (WHO) estimated that around 692 million people worldwide will

119

suffer from diabetes by 2030, and associated healthcare costs will rise to $235 billion

120

in Europe.19 With intensive studies on mechanisms and development of potentially

121

effective therapeutic approaches, α-glycosidase and protein tyrosine phosphatase 1B

122

(PTP1B) are considered effective measures for regulating type 2 diabetes, but

123

single-approach methods have not yet been developed.20 Thus, combined therapeutic

124

strategies may be alternative methods to treat this disease. α-Glycosidases are

125

important enzymes involved in carbohydrate dissolution and absorption in the small

126

intestine. Inhibition of these enzymes may reduce absorption and disintegration of

127

polysaccharides and postprandial blood glucose levels. As a negative regulator of

128

insulin, the overexpression of PTP1B may inhibit expression of insulin in the insulin

129

signaling pathway.21 Berries provide valuable sources for treatment of diabetes

130

mellitus because of their active compounds.

131

The key features of FT-ICR MS analysis include high mass accuracy

132

measurements and the ability to resolve isobaric species with mass less than that of an

133

electron. Thus, anthocyanins, without reference substances and perfect separation in

134

chromatographic columns, can be detected and indentified. In this study,

135

HPLC-FT-ICR MS was used to analyze the polyphenolic profiles of four berries

136

(blueberry, bilberry, mulberry, and cranberry) in China for the first time, and the

137

anthocyanins were determined by HPLC. PTP1B has become a highly plausible

138

candidate for the therapeutic inhibitors of type 2 diabetes, so we speculated that



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anthocyanins from the berries may be the novel PTP1B inhibitors. Molecular docking

140

was used to predict the inhibition activities of anthocyanins on PTP1B for the first

141

time. Total anthocyanin, phenols, and flavonoids were analyzed, and the

142

inter-relationships between polyphenolic profiles with the antioxidants were measured

143

by DPPH radical, total antioxidant capacity assay (ABTS), and the ferric reducing

144

antioxidant power (FRAP) assay. In addition, the α-glycosidase was also tested in this

145

study.

146

2. Materials and methods

147

2.1 Materials and reagents

148

Blueberries (Vaccinium uliginosum L.) and bilberries (Vaccinium myrtillus L.)

149

were purchased from Yichun of Heilongjiang, China. Mulberries (Morus alba L.)

150

were purchased from Bijie of Guizhou, China. The voucher specimens (NO.VU001,

151

VM001, and MA001, respectively) have been deposited in the herbarium of Shenyang

152

Pharmaceutical University. Cranberries (Vaccinium oxycoccos L.) were from Tianjin

153

JianFeng

154

cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside (purity higher than 90% by

155

HPLC) were isolated from black raspberry in our previous work. Gallic acid,

156

protocatechuic acid, cholrogenic acid, catechin and cafferic acid

157

purity higher than 95% by HPLC) were purchased from national institutes for food

158

and drug control (China). α-Glycosidase was from recombinant Saccharomyces

159

cerevisiae (expressed in unspecified

160

4-nitrophenyl-α-D-glucopyranoside,

161

mercaptoethanol,

162

2,2′-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), 2,4,6-tris(2-pyridyl)-

163

s-triazine (TPTZ), fluorescein sodium salt (FL), and Folin-Ciocalteu reagent were

164

purchased from sigma-Aldrich (St. Louis, MO, USA). and ascorbic acid (VC) was

165

purchased from Fluka (Buchs, Switzerland). Dimethyl sulfoxide (DMSO) and

166

polyamide resin were purchased from Sinopharm Chemical Reagent Co., Ltd. Trolox

Natural

Product

dithiothreitol

R&D

Co.,

host).

Ltd.

PTP1B

p-nitrophenyl

(DTT),

Cyanidin-3-O-sambubioside,

(all standards with

(human,

phosphate

recombinant), (pNPP),

2,2-diphenyl-1-picrylhydrazyl


β-

(DPPH),

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and 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) were purchased from

168

Beijing Biotopped Science & Technology Co., Ltd. Acetonitrile, methanol and formic

169

acid (all HPLC grade) were purchased from Fisher (Fairlawn, NJ).

170

2.2 Sample preparation

171

Fresh berries were collected and carefully washed, preprocessed by vacuum

172

freeze-drying, and crushed into powder. Next, the freeze-dried powder (5 g) was

173

extracted using ethanol water (75:25, v/v) for 3 h at 35 °C with ultrasonic extraction

174

apparatus, until it became white to obtain crude polyphenols. The acquired crude

175

polyphenols were filtered through Büchner funnel. Next, filtrates were collected.

176

rotary evaporation apparatus was used to remove the solvent under vacuum, and

177

residues were further lyophilized for investigation.

A

178

The crude polyphenols were re-suspended in 15 mL of 10% ethanol containing

179

0.1% HCl and loaded onto a polyamide column (3 mm × 72 mm, 25 g) using a

180

gradient of 10%, 30%, 50%, 70%, and 90% ethanol (containing 1% formic acid) as

181

elution solvents to obtain five fractions (A-E) under darkness and pressureed

182

conditions. Sugar mainly remained in A, and the main polyphenolic profiles

183

(especially anthocyanins) were concentrated in fraction B. Eluents were dried under

184

vacuum and stored at - 20 °C before use.

185

2.3 Polyphenol, flavonoids and anthocyanin contents

186

Total phenolic content (TPC) of crude polyphenols and 30% elution fractions of

187

berries were examined following previously reported Folin-Ciocalteu method.22

188

Briefly, The total phenolic content were expressed as (mg GAE/g DW) from gallic

189

acid calibration curve. Total flavonoids content (TFC) of 30% elution fractions of

190

berries were determined colorimetrically as reported previously.23 The results were

191

expressed as mean of milligrams catechin equivalents (CE)/g dry weight (DW). And

192

each 30% elution fraction of berries were quantified for anthocyanin content (TMA)

193

using a pH differential method as described by Wrolstad.24 The results were calculated



194

as in mg of cyanidin-3-O-glucoside equivalents/g dry weight (DW).

195

2.4 Chromatographic analysis

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HPLC analytical procedures with a run time of 40 min were performed on an

197

Agilent 1260 HPLC system (Agilent Technologies, Waldbronn, Germany). The

198

system

199

thermostatically controlled column compartment and diode-array detector. The The

200

column applied in this work was an Agilent HC-C18 column (250×4.6 mm, 5 µm) at a

201

flow rate of 0.8 mL/min. The mobile phase 1% formic acid in acetonitrile (A) and 1%

202

formic acid in water (B) was employed. A linear gradient with 10% A in B for 7 min

203

followed by a linear gradient to 28% A in B at 40 min with a column regeneration

204

time of 10 min between injections, injection volume was 15 µL. The column

205

temperature was maintained at 25 °C, and the detection wavelength was at 280 nm,

206

and UV spectra from 190 to 400 nm were also recorded for peak characterization. In

207

the quality analysis, the standards was used at the following concentrations:

208

protocatechuic acid, 1.0300 mg/mL; chlorogenic acid, 1.6824 mg/mL; catechin,

209

1.1444 mg/mL; cyanidin-3-O-glucoside, 1.4290 mg/mL; caffeic acid, 1.55132 mg/mL;

210

and cyanidin-3-O-rutinoside 1.6147 mg/mL.

211

2.5 ESI FT-ICR MS analysis analysis

equipped

with

quadruple

pump,

online

degasser,

auto-sampler,

212

The instrument used for these studies were carried out on a FT-ICR MS and a 7.0

213

T superconducting magnet (Bruker Daltonics, Bremen, Germany) equipped

214

electrospray ionization (ESI) interface at a nebulizing gas pressure of 4.0 bar, a dry

215

gas flow rate of 8.0 L/min, with a capillary voltage of -3.0 kV, an end plate offset of

216

-500 V and a transfer capillary temperature of 200 °C. Full-scan MS data was

217

acquired over an m/z range of 50-3000 utilized the positive ion mode (m/z M+H+),

218

and the collision energy was initially set at 25 eV for the MS/MS experiments of the

219

preferred ions and then modified according to the fragments. Precursor ions were

220

subjected to collision-induced dissociation (CID) to generate the fragment ions, and


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the fragmentation patterns were proposed for the structural identification of

222

constituents. FT-MS control, Bruker Compass-Hystar and DataAnalysis Software

223

(Bruker, Germany) were used to control the equipment and for data acquisition and

224

analysis, respectively.

225

2.6 Antioxidant activities

226

Free radical scavenging capacity (DPPH and ABTS assays) and ferric reducing

227

antioxidant power (FRAP assays) were conducted to measure antioxidant capacity of

228

each 30% elution fraction of berries in vitro, ascorbic acid as the antioxidant standard

229

according to the method reported previously.21 ORAC assay was evaluated using a

230

previously methods described by Ehlenfeldt et al and carried out on a varioskan flash

231

analyzer (Thermo Scientific, USA). 25

232

2.7 α-Glycosidase inhibitory assay and PTP1B enzymatic assay

233

The α-glycosidase activity was assessed through a previously reported method.21

234

Enzymatic activity was quantified by measuring absorbance at 405 nm. The 30%

235

elution fractions of berries were performed with different concentrations dissolved in

236

1% DMSO and acarbose was used as positive control. The IC50 value (µg/mL) was

237

defined as the concentration that inhibited 50% of α-glycosidase activity.

238

The different concentrations (in 1% DMSO) 30% elution fractions were tested

239

against the PTP1B and NaVO4 was used as positive control. The method was

240

conducted as our previously report.21 Dephosphorylation of pNPP generated product

241

pNP can be monitored at 405 nm.

242

2.8 In silico molecular docking study

243

To investigate the relationship between anthocyanins and PTP1B, six kinds of

244

anthocyanidins (pelargonidin, cyanidin, delphinidin, petunidin, malvidin and peonidin)

245

glycoside in berries were carried out as ligands in silico molecular docking study26



246

The three-dimensional structure of PTP1B (PDB ID:2QBQ) was obtained from the

247

online Protein Data Bank (PDB). Ligand structures optimization were processed by

248

ChemBioDraw (PerkinElmer), and the molecular docking was implemented using

249

AutoDock Tools (version: 1.5.6), AutoDock 4.2 package (Autodock 4.2 and Autogrid

250

4.2), with the help of The PyMOL Molecular Graphics System (Version 1.7.4.5)

251

(PyMOL Molecular Graphics System, San Carlos, CA, USA), which can be used to

252

better visualize the interactions between ligands and receptors. X, Y, Z were set to

253

47.028, 11.528 and 0.228 Å in the cubic grid box as the center dimensions. water

254

molecule and persisting inhibitors were removed from the crystal structure of 2QBQ.

255

Blind docking over the whole receptor was carried out using Genetic Algorithm, and

256

the resultant complex structures were investigated by using the conformations of the

257

most favorable binding energy.

258

2.9 Statistical analysis

259

All the samples were replicated in triplicate. One-way analysis of variance

260

(ANOVA) was performed using SPSS 16.0 program for windows (SPSS Inc., Chicago,

261

IL, USA).

262

3 Results and discussion

263

3.1 Total phenolic, flavonoid, and monomeric anthocyanin content

264

Environmental factors, such as light intensity, nutrient availability, season,

265

degree of ripeness, and cultivars, may contribute to differences in phenolic, flavonoid

266

and anthocyanin contents.27,28 In addition, different purification and analysis methods

267

may lead to varied results.29 The purification of anthocyanins has been researched by

268

solid-phase extraction (SPE), high-speed counter-current chromatography (HSCCC),

269

column chromatography (CC), supercritical CO2, and preparative high-performance

270

liquid chromatography (HPLC).30,31

271

Based on our previous research, the polyamide resin with different

272

concentrations of ethanol elution is a useful purification method for anthocyanins. The


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total phenolic, flavonoid and monomeric anthocyanin were higher than the that found

274

in the literature.32 Therefore, the four berries were purified with polyamide and eluted

275

with 30% ethanol in order to enrich anthocyanins. This process was performed under

276

darkness and pressure conditions in to avoid oxidation. Total monomeric anthocyanin

277

content ranged from 1.4 ± 0.17 mg/g DW to 739.6 ± 17.14 mg/g DW, with blueberry

278

possessing the highest content, followed by bilberry (644.1 ± 17.34 mg/g DW),

279

mulberry (15.5 ± 2.27 mg/g DW) and cranberry. Delphidin, cyanidin, petunidin,

280

peonidin, and malvidin were detected in blueberry and bilberry. Cranberry mainly

281

contains cyanidin and malvidin, and mulberry mainly consisted of cyanidin. Similar to

282

anthocyanin, the total phenolic content was the highest in blueberry (804.0 ± 20.56

283

mg/g DW) followed by bilberry, mulberry, and cranberry. Gallic acid, protocatechuic

284

acid, chlorogenic acid, catechin, and caffeic acid were found in all berries. Bilberry

285

contained 641.2 ± 15.72 mg/g DW of total flavonoids, with blueberry and mulberry

286

having 598.0 ± 17.11 and 427.6 ± 15.94 mg/g DW, respectively. Cranberry contained

287

the lowest with 183.3 ± 12.13 mg/g DW. The derivatives of myricetin and quercetin

288

were found in cranberry and bilberry. (Table 1)

289

3.2 FT-ICR MS analysis

290

Positive ion mode MS was used to detect anthocyanins, and the results are

291

presented in Fig. 1, Fig. 2 and Table 2. Peaks 12, 13, and 15 were identified by spiking

292

experiments with authentic standards as cyanidin-3-O-sambubioside (m/z 581.15071,

293

1.06 ppm, m/z 287.05478), cyanidin-3-O-glucoside (m/z 449.10513, 0.71 ppm, m/z

294

287.05410), and cyanidin-3-O-rutinoside (m/z 595.16146, 0.23 ppm, m/z 287.05419),

295

respectively. Cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside are the main

296

anthocyanins in berries.

297

Peaks 3, 4, 20, and 22 showed [M +] ion with m/z value of 627.15131 (1.07 ppm),

298

597.14106 (0.46 ppm), 579.16687 (1.46 ppm), and 609.18055 (0.93 ppm),

299

respectively, which suggested that they are disaccharide derivatives of anthocyanins.

300

Precursor ion scan demonstrated that the ion with m/z of 627.15131 was the precursor



Page 12 of 35

301

of the ion with m/z of 303.04873, indicating that anthocyanin contained delphinidin

302

within its structure. The MS/MS product ion scan produced a fragment ion with m/z

303

of 303.04873, which corresponds to the loss of two glucose molecules

304

([M-glucose-glucose]+).

305

delphinidin-3,5-O-diglucoside. Peak 20 showing an ion with m/z of 579.16687 and

306

fragment ion with m/z of 271.06954 coincident with formulas C27H31O14 (1.46 ppm)

307

was identified as pelargonidin-3-O-rutinoside, which was rarely found in the berries.

308

In

309

delphinidin-3-O-hexose-pentoside and peonidin-3-O-rutinoside, respectively. Among

310

the four anthocyanins, delphinidin-3,5-O-diglucoside and peonidin-3-O-rutinoside

311

were only detected in blueberry, and pelargonidin-3-O-rutinoside was only detected in

312

mulberry.

a

similar

Therefore,

manner,

peaks

peak

4

and

3

was

22

were

identified

identified

as

as

313

Peaks 5, 7, 9, and 11 showed [M +] ion with m/z value of 465.10047 (0.77 ppm),

314

465.09996 (0.67 ppm), 435.08968 (0.66 ppm), and 435.08956 (0.59 ppm),

315

respectively. In MS/MS product ion scan, these anthocyanins contain common

316

sub-ion with m/z of 303.04, which indicated they contained basic parent structure

317

delphinidin. It is known that galactose, glucose and arabinose are common aglycones

318

linked to the parent structure, and the elution series (retention time from short to long)

319

was galactose, glucose, and arabinose.6 So Peaks 5, 7, 9, and 11 were identified as

320

delphinidin-3-O-galactoside,

321

delphinidin-3-O-pentoside. Following a similar method, the peaks 8, 17, and 18 were

322

monosaccharide derivatives of cyanidin, peaks 16, 19, and 23 were monosaccharide

323

derivatives of petunidin, peaks 25, 27, 28, and 29 were monosaccharide derivatives of

324

malvidin, and peaks 21, 24, and 26 were monosaccharide derivatives of peonidin.

325

Among the anthocyanins monosaccharide derivatives, there are 18 anthocyanins

326

monosaccharide were detected in blueberry, and 13 in bilberry.

delphinidin-3-O-glucoside,

and

two

327

Peaks 31 and 32 showed [M +] ion with m/z value of 507.11072 (0.68 ppm) and

328

595.16170 (0.34 ppm). The MS/MS product ion scan produced fragment ions with

329

m/z of 303.04906 and 287.05413, respectively, suggested they contained


Page 13 of 35


330

skeleton structure delphinidin and cyanidin, respectively. By high mass accuracy

331

measurements,

332

delphinidin-3-O-(6''-acetyl)-galactoside and cyanidin-3-O-(6''-coumaroyl)-glucoside.

333

The malonyl, acetyl and coumaroyl are common substituent groups to the

334

anthocyanins.

these

anthocyanins

can

be

identified

as

335

The non-anthocyanidin phenolic compounds were also investigated. This group

336

included gallic acid; protocatechuic acid; chlorogenic acid; caffeic acid; catechin; and

337

derivatives of myricetin, quercetin, and kaempferol. The phenolic acids and

338

flavonoids show low response in positive ionization, so gallic acid, protocatechuic

339

acid, chlorogenic acid, caffeic acid, and catechin were identified by comparing with

340

the standard substances.

341

Among the non-anthocyanidin phenolic compounds, derivatives of myricetin,

342

quercetin, and kaempferol were the most abundant flavonoids. Peaks 33, 34, 35, 36,

343

37, and 39 with precursor and fragment ions m/z values of 303.04 and 151.52,

344

respectively, suggested that these were derivatives of quercetin, and identified as

345

quercetin-3-O-rutinoside,

346

quercetin-3-O-arabinoside, quercetin-3-O-pentoside, and quercetin, respectively. Peak

347

38 showed a [M+H+] ion with m/z value of 449.10520 (1.07 ppm) and fragment ion

348

m/z value of 287.05409, which was identified as kaempferol-3-O-glucoside. Peak 30

349

with 481.09488 (1.02 ppm) was identified as myricetin-3-O-hexoside. The derivatives

350

of quercetin were the main compounds in bilberry. The myricetin and quercetin

351

derivatives are the main ingredients in cranberry. however, kaempferol derivatives

352

were only detected in blueberry.

quercetin-3-O-galactoside,

quercetin-3-O-glucoside,

353

In short, as high-resolution and high-throughput power was needed to detect

354

minor or trace compounds in complex mixtures, FT-ICR MS was used to identify the

355

anthocyanins, phenolic acid and flavonoids from the berries for the first time.

356

Thirty-nine polyphenols, including 26 anthocyanins, 9 flavonoids, and 4 phenolic

357

acids, were identified accurately. Among the six types of anthocyanins, 24 and 17

358

anthocyanins were detected in blueberry and bilberry, respectively. The elution order



Page 14 of 35

359

of the anthocyanins from shortest to longest were as follows: delphinidin, cyanidin,

360

petunidin, peonidin and malvidin. The reason may be the number of hydroxyls and

361

methoxyls attached to the skeleton. Cyanidin aglycone was common in the berries,

362

whereas malvidin aglycone was mainlyfound in bilberry and cranberry.

363

3.3 Quality analysis

364

Quantitation is vital for the quality control of functional food. The literature

365

about validation procedure for anthocyanins in plants and foods are rare, may be

366

because the scarcity of anthocyanin standards. In this study, the parameters for

367

validation, such as linearity, limits of detection (LOD), and limits of quantitation

368

(LOQ),

369

protocatechuic acid, chlorogenic acid, catechin and caffeic acid. (Table 3a)

were

tested

using

cyanidin-3-O-glucoside,

cyanidin-3-O-rutinoside,

370

Calibration curves were built by injection of six appropriate concentrations of

371

reference substances three times, and statistical data showed good linearity (R2 ＞

372

0.99). LOD and LOQ were calculated by peak height, which were 3 and 10 times of

373

instrument noise. LODs and LOQs varied from 0.0085µg/mL to 2.3859 µg/mL and

374

0.03824 µg/mL to 10.7366 µg/mL, respectively.

375

The anthocyanin, phenolic and flavonoid contents in the berries are presented in

376

Table 3b. Cyanidin-3-O-rutinoside was the highest in blueberry, bilberry, and

377

mulberry, accounting for 36.63%, 26.40%, and 66.56% of total anthocyanins,

378

respectively. In cranberry, the highest was malvidin-3-O-glucoside (52.42% of total

379

anthocyanins). As six kinds of anthocyanins have the same skeleton, and differ in

380

substituent, similar absorptivities at 280 nm were expected. Therefore, anthocyanidins

381

in tandem with monosaccharides were estimated based on cyanidin-3-O-glucoside as

382

calibrant, and anthocyanidins in tandem with disaccharides were estimated based on

383

cyanidin-3-O-rutinoside. The concentration of some anthocyanins (such as

384

pelargonidin-3-O-rutinoside,

385

delphinidin-3-O-(6''-acetyl)-galactoside) were below the the LOQ, so they also not

386

quantified in the samples

387

delphinidin-3-O-galactoside,

and

Discrepant results of phenolic, flavonoid, and anthocyanin contents between


Page 15 of 35


388

chemical reaction and HPLC are not unusual, because the principles are different. For

389

example, the Folin-Ciocalteu assay relies on the redox reaction of phenolic

390

compounds with a mixture of phosphotungstic and phosphomolybdic acids in an

391

alkaline medium to create a blue-colored complex that can be quantified at 750 nm. It

392

reacts strongly with compounds that easily donate electrons such as mono- and

393

dihydroxylated phenolics. The pH differential method for anthocyanin content is

394

based on the different structures of anthocyanin, which showed different color in pH

395

1.0 to 4.5.

396

3.4 Antioxidant activities

397

The berries exhibit strong antioxidant activities because of their high amounts of

398

anthocyanins, phenolic acids, and flavonoids. Moreover, evaluation of antioxidant

399

activity was analyzed by many methods. By using the ORAC method, Prior et al first

400

reported blueberries have higher antioxidant activities than other fruits and

401

vegetables.33 In this paper, DPPH, ABTS, FRAP, and ORAC assays were used to

402

evaluate antioxidant activity by scavenging the radicals generated in vitro. The results

403

are shown in Table 1, and agree with the research reported by Wu, X.34

404

In the DPPH free-radical scavenging activities, the EC50 for 30% fraction of

405

blueberry was the highest (

Identification of Anthocyanins from Four Kinds of Berries and Their

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