Identification and Quantification of Flavonoids from Two Southern

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

Identification and quantification of flavonoids from two Southern Italy cultivars of Allium cepa L. Var. Tropea (red onion) and Montoro (copper onion) and their capacity to protect human erythrocytes from oxidative stress Idolo Tedesco, Virginia Carbone, Carmela Spagnuolo, Paola Minasi, and Gian Luigi Russo J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 12 May 2015 Downloaded from http://pubs.acs.org on May 13, 2015

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

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

Page 1 of 35

Journal of Agricultural and Food Chemistry

1

Identification and quantification of flavonoids from two Southern Italy cultivars of Allium

2

cepa L. Var. Tropea (red onion) and Montoro (copper onion) and their capacity to protect

3

human erythrocytes from oxidative stress

4 5

Idolo Tedesco*, Virginia Carbone*, Carmela Spagnuolo*, Paola Minasi and Gian Luigi Russo§

6 7

Institute of Food Sciences, National Research Council, 83100, Avellino, Italy

8 9

*Equal contribution

10 11

§

12

Dr. Gian Luigi Russo

13

Istituto Scienze dell’Alimentazione – Consiglio Nazionale delle Ricerche

14

Via Roma 64

15

83100 – Avellino

16

Italy

17

Phone: +39 0825 299331

18

Fax: + 39 0825 781585

19

E-mail: [email protected]

To whom correspondence should be addressed:

20

1 ACS Paragon Plus Environment


Page 2 of 35

21

ABSTRACT

22

Onions (Allium cepa) are consumed worldwide and represent an important source of dietary

23

phytochemicals with proven antioxidant properties, such as phenolic acids, flavonoids,

24

thiosulfinates and anthocyanins. Epidemiological and experimental data suggest that a regular

25

consumption of onions is associated with a reduced risk of degenerative disorders. Therefore, it is

26

of interest to investigate the biological properties of different varieties of onions. Here, we

27

characterized for the first time a variety of onion, called “Ramata di Montoro” (coppery onion from

28

Montoro) grown in a niche area in Southern Italy and compared its phenolic profile and antioxidant

29

properties to a commercial ecotype of red onion, namely Tropea, also present in Southern Italy. An

30

analytical method based on high performance liquid chromatography coupled with UV detector and

31

mass spectrometry was used to separate and characterize the phenolic fraction (anthocyanins and

32

flavonols) extracted from both coppery and red types. The main compounds detected in the two

33

ecotypes were: quercetin and quercetin glucosides, isorhamnetin glucosides, kaempferol glucoside

34

and, among anthocyanins, cyanidin glucosides. Tropea ecotype onion showed a higher content of

35

flavonols (632.82 mg/kg fresh weight) than the Montoro type onion (252.91 mg/kg fresh weight).

36

Accordingly, the antioxidant activity of the former was 2.8-fold higher compared to the latter. More

37

pronounced were the differences existing between the 4 anthocyanins detected in the two ecotypes,

38

with those in the Tropea ecotype onion present at concentrations between 20 and 230-fold higher

39

than in the Montoro type onion. Both extracts reduced LDL oxidation of about 6-fold and protected

40

human erythrocytes from oxidative damage induced by HClO of about 40%. In addition, as a

41

consequence of HClO treatment, GSH concentration in erythrocytes was reduced of about 50% and

42

pre-treatment with onion extracts induced a recovery of GSH level of about 15-22%. Qualitative

43

differences highlighted in the chemical composition of the two phenolic extracts, especially the total

44

content of anthocyanins which was 30-fols higher in Montoro type onion compared to Tropea

45

ecotype can be associated to the protective effects measured against oxidative damage induced in

46

human erythrocytes. 2 ACS Paragon Plus Environment

Page 3 of 35

47


ABBREVIATIONS

48 49

ROS, reactive oxygen species; HOCl, hypochlorous acid; RNS, reactive nitrogen species; COM,

50

Coppery Onion from Montoro; RTO, Red Tropea onion; RBCs, red blood cells; LDL, low density

51

lipoprotein; GSH, glutathione; HPLC, High Performance Liquid Chromatography; QE, equivalent

52

of quercetin; FW, fresh weight;

53 54



55

Page 4 of 35

INTRODUCTION

56 57

Onions are the second most produced vegetable crop after tomatoes with about 3,642,000 ha grown

58

annually worldwide and a production of 53.6 Mt 1, 2. Their large distribution is probably due to the

59

versatile culinary uses as raw food or different modes of cooking (baked, boiled, braised, grilled,

60

fried, roasted, etc.). In addition, onions are considered among the healthier vegetables due to the

61

high levels of non-nutrient compounds, such as phenolic acids, flavonoids, thiosulfinates and

62

anthocyanins. For these reasons, it is of interest to study and characterize the chemical and

63

biological properties of new varieties, including those grown in niche geographical areas, which are

64

produced in limited amount, but may represent a potential source of new biological compounds.

65

Onions are among the richest sources of dietary flavonoids. In bulbs, only compounds belonging to

66

the flavonols, the anthocyanins, and the dihydroflavonols have been detected. As reviewed 3, yellow

67

onions contain 270–1187 mg/kg FW of flavonols, whereas red onions contain 415–1917 mg/kg FW

68

of flavonols. Quercetin derivatives (quercetin 4′-glucoside and quercetin 3,4′-diglucoside) are the

69

predominant flavonols in all onion cultivars. The anthocyanins of red onions (approximately 10% of

70

the total flavonoid content) are mainly cyanidin glucosides acylated with malonic acid or

71

nonacylated 3. Storage of onions for 6 weeks in different conditions resulted in a decrease of 64-

72

73% of total anthocyanins which paralleled with a reduction in the total antioxidant activity of 29-

73

36% 4. To this regards, it is worthwhile to mention that onions are considered among the major

74

sources of antioxidant compounds 5 whose level of activity is related to the different cultivars and

75

colors 6-10.

76

The scientific literature of the last twenty years or so has been influenced by the syllogism that

77

since free radicals damage cellular structures, their scavenging by antioxidants is health protective.

78

As a consequence, an increased uptake of antioxidants, such as those deriving from a fruits and

79

vegetables-rich diet, may increase resistance to reactive species induced pathologies. Although this

80

concept probably overestimates the antioxidant capacity of phytochemicals and, possibly, generated 4 ACS Paragon Plus Environment

Page 5 of 35


81

misinterpretations in the field 11, 12, biochemical and genetic studies on cellular and animal models

82

on the mechanism(s) of action of phytochemicals provide a functional explanation of how and why

83

a diet rich in fruits and vegetables can protect against degenerative diseases 13, 14.

84

Therefore, scientists in this field are constantly in search of novel agronomic species and/or local

85

varieties possessing enhanced protective effects against oxidative damage in cellular and animal

86

models. In the present work, we focused our attention on a local variety of onion, so-called “Ramata

87

di Montoro” (coppery onion from Montoro area; COM), whose cultivation was born and passed

88

down through generations in the area of Montoro Inferiore, a village located in the province of

89

Avellino (Southern Italy). We compared the antioxidant properties of polyphenols isolated from

90

COM to a better characterized, commercial ecotype, namely red Tropea onion (RTO), a variety of

91

red onion grown in a specific area of Calabria, a region in Southern Italy 15.

92

In the present study, an analytical method based on high performance liquid chromatography

93

(HPLC) coupled with UV detector and mass spectrometry was used to separate and characterize the

94

phenolic fraction (anthocyanins and flavonols) extracted from cultivated bulbs of COM compared

95

to RTO ecotype. Polyphenolic extracts from the two onions were evaluated for their antioxidant

96

activity. Subsequently, we investigated the protective role of phenolic extracts from RTO and COM

97

against low density lipoprotein (LDL) oxidation and hypochlorous acid-induced oxidative damage

98

in normal red blood cells (RBCs) and their interference with the antioxidant defense systems active

99

in erythrocytes. Oxidized LDL contain a diverse set of toxic species, such as aldehydes, oxysterols

100

and lipid peroxides 16. They are considered as an important initial step in the development of

101

atherosclerosis; therefore, lowering LDL levels by independent mechanisms is considered

102

protective against the likelihood of atherosclerotic events 17-19. Previous studies indicated that

103

phytochemicals, such as phenolic acids, flavonoids and anthocyanins, inhibited LDL oxidation 20, 21

104

22, 23

105

exposed to continuous oxidative stress deriving from endogenous24, or exogenous sources 25. For

106

these reasons, RBCs have developed efficient enzymatic and not-enzymatic antioxidant defenses to

. RBCs have been selected as the study model since, for their physiological role, they are



Page 6 of 35

107

preserve themselves from oxidative damage. In addition, due to their mobility, erythrocytes can be

108

considered as ideal antioxidant scavengers acting throughout the circulation and reducing the

109

damaging mass effect of elevated levels of ROS on different tissues 24-26. Therefore, we established

110

a novel in vitro model using RBCs isolated from whole blood, treated with HClO. Millimolar

111

concentrations of HClO generates osmotic fragility and formation of transient membrane pores with

112

the consequent hemolysis of erythrocytes 27. Treatment with HClO cannot be seen as an

113

experimental adaptation to allow the functionality of our assay on RBCs, but it is very close to

114

physiological processes; in fact, following inflammatory tissue injury and microbial killing,

115

neutrophils and monocytes generate hyper-production of HClO in the millimolar range 28.

116

Based on these observations, we firstly chemically characterized the polyphenolic contents of RTO

117

and COM ecotypes, the latter representing a new variety, while the former was used as a reference,

118

being already described and characterized. Subsequently, we compared their protective effect

119

against oxidative damage taking advantage of two different assays: capacity to reduce LDL

120

oxidation and protection of human RBCs from healthy subjects from oxidative insult caused by

121

HClO treatment.

122


Page 7 of 35

123


MATERIAL AND METHODS

124 125 126 127

Chemicals.

128

Ciocalteu’s reagent; 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ); glutathione (GSH); phtaldialdehyde,

129

trichloroacetic acid (TCA); hydrogen peroxide (H2O2); lipoprotein low density from human plasma

130

(LDL); cupric sulfate anhydrous; xylenol orange; ferrous ammonium sulfate; butylated

131

hydroxytoluene (BHT) were purchased from Sigma-Aldrich (Milan, Italy). Isoquercitrin (quercetin-

132

3-O-glucoside) was obtained from Fluka (Buchs SG, Switzerlandand). Cyanidin-3-O-glucoside

133

chloride (Kuromanin chloride); delphinidin-3-O-glucoside chloride (myrtillin chloride); quercetin-

134

3,4'-di-O-glucoside; quercetin-4'-O-glucoside (Spiraeoside); kaempferol-3-O-glucoside were

135

purchased from Extrasynthese (Genay, France). Ferric chloride, sodium carbonate, sodium

136

hypochlorite were from Carlo Erba (Milan, Italy). Phosphate-buffered saline (PBS) tablets were

137

purchased from Invitrogen (S. Giuliano Milanese, Milan, Italy). HPLC grade water (18.2 mΩ) was

138

prepared using a Millipore Milli-Q purification system (Millipore Corp., Bedford, MA, USA). All

139

other chemicals used were of research highest purity grade.

Methanol and formic acid were obtained from Merck (Darmstadt, Germany). Quercetin; Folin

140 141

Onion collection and sample treatment. Cultivated bulbs of onion, Allium cepa L. Tropea

142

ecotype, were collected in Tropea (Calabria, Italy), while cultivated bulbs of brown onion, Allium

143

cepa L. Montoro type, were kindly provided by a local Committee promoters of Cipolla Ramata di

144

Montoro (www.cipollaramatadimontoro.it) located in Montoro (Campania, Italy). The name Cipolla

145

Ramata (coppery onion) derives from the color of the external skin of the bulb. This variety is

146

cultivated in a niche geographical area in Southern Italy (17 areas within Avellino and Salerno

147

districts).

148

For each variety two bulbs were finely chopped and 10 g were extracted with 20 mL of 80%

149

aqueous methanol, at room temperature, on an horizontal shaker. After 10 min samples were placed 7 ACS Paragon Plus Environment


150

in an ultrasonic bath and sonicated for 15 min and then filtered through filter paper. Extracts were

151

then dried in rotary evaporator (LaboRota 4000 /HB Efficient, Heidolph) and stored at -20°C until

152

being used.

Page 8 of 35

153 154

Analysis of total phenolic content and antioxidant capacity. Dry extracts were suspended in PBS

155

to obtain a stock solution of 50 mg/mL (w/v) and kept at 4°C and in the dark. This stock solution

156

was used in all biological assays performed in the present study 29 and for the analysis of total

157

phenolic content and antioxidant capacity. The amount of total phenols was determined according

158

to the Folin-Ciocalteu’s procedure 30, 31 using quercetin as a reference standard. Briefly, 50 L of

159

Folin–Ciocalteu’s reagent and 500 L of distilled water were added to 10 L of suitable aqueous

160

dilution of the stock solution (50 mg/mL) (w/v). The reaction mixture was mixed and allowed to

161

stand for 2 min. Finally, 0.1 mL of sodium carbonate and 0.34 mL of distilled water were added and

162

the solution was incubated in the dark for 120 min. The samples absorbance was measured at 760

163

nm. The results were expressed as micromolar equivalent of quercetin (QE) in 1 mg/mL of onion

164

extracts. All measurements were carried out in triplicates.

165

The antioxidant capacity of different onion extracts were determined by a ferric reducing

166

antioxidant power (FRAP) assay, as reported 32 and results calculated as micromolar QE in 1

167

mg/mL of the two onion extracts.

168

Ascorbic acid (AA) content in onion extracts was determined by FRASC method 33. Samples were

169

treated with 10 IU ascorbate oxidase/mL, diluted in FRAP solution (20 mM FeCl3 and 10 mM

170

TPTZ in acetate buffer, pH 3.5) and finally incubated for 5 min at 37°C. The absorbance was

171

measured at 595 nm and micromolar concentrations of AA were calculated using a standard curve.

172 173

HPLC-UV analyses. Extracts from cultivated bulbs of the two different onion types were

174

reconstituted in 1% formic acid and analysed by HPLC-UV/Vis using a HP 1110 Series HPLC

175

(Agilent, Palo Alto, CA, USA) equipped with a binary pump (G-1312A) and an UV detector (G8 ACS Paragon Plus Environment

Page 9 of 35


176

1314A). Individual phenols were separated on a Hypersil BDS C18 column (250 mm x 4.6mm, 5

177

µm) (Thermo, Bellefonte, PA, USA) at a flow rate of 1 mL/min; solvent A was 1% formic acid and

178

solvent B was 1% formic acid in methanol and water (50:50, v/v). After a 5 min hold at 20%

179

solvent B, elution was performed according to the following conditions: from 20% (B) to 80% (B)

180

in 22 min, isocratic elution (80% B) for the next 13 min, from 80% (B) to 95% (B) in 10 min,

181

followed by 15 min of maintenance. Flavonols were monitored at 340 nm while anthocyanins at

182

520 nm. Standard curves for each flavonol standard were prepared over a concentration range of 1–

183

180 µg/mL with six different concentration levels and triplicate injections at each level.

184

Quantification of anthocyanins was performed with external calibration curves generated by

185

repeated injections of a fixed volume of standard solutions of cyanidin-3-O-glucoside over a

186

concentration range of 0.5–20 µg/mL with five different concentration levels and duplicate

187

injections at each level. All samples were prepared and analysed in duplicate. Results were

188

expressed as mg/kg of fresh weight (FW).

189 190

HPLC-ESI-ITMSn analysis. Identification of phenolic compounds in the extracts from cultivated

191

bulbs of the two different onion ecotypes was effectuated by HPLC–ESI/MS and MSn

192

fragmentation analyses using a SURVEYOR MS micro HPLC (Thermo Finnigan, San Josè, CA,

193

USA) coupled with a Finnigan LCQ DECA XP Max ion trap mass spectrometer (Thermo Finnigan,

194

San Josè, CA, USA), equipped with Xcalibur® system manager data acquisition software (Thermo

195

Finnigan, San José, CA, USA). Individual phenols were separated on a Hypersil BDS C18 column

196

(250 mm x 2.1 mm, 5 µm) (Thermo, Bellefonte, PA, USA) at a flow rate of 200 µL/min. The HPLC

197

conditions were as described for the HPLC-UV system. Mass spectra were recorded from mass-to-

198

charge ratio (m/z) 50 to 1200 both in negative and in positive ionization mode. The capillary voltage

199

was set at -13 V, the spray voltage was at 4.5 kV and the tube lens offset was at -15 V in negative

200

ion mode while, in positive ion mode, the capillary voltage was set at 38 V, the spray voltage was at



201

3 kV and the tube lens offset was at 50 V. The capillary temperature was 275°C. Data were

202

acquired in MS, MS/MS and MSn scanning mode.

Page 10 of 35

203 204

LDL oxidation. The assay was performed incubating 0.1 mL of LDL (200 g protein) with or

205

without onion extracts (0.25 mg/mL; w/v) for 15 min, then oxidation was induced by adding 50 M

206

CuSO4 at 37°C for 15 h. Subsequently, 0.9 mL of FOX reagent (1 mM xylenol orange, 2.5 mM

207

ferrous ammonium sulfate and 4.4 mM BHT in methanol) were added to the samples. After 30 min,

208

samples were centrifuged in a microfuge at maximal speed for 5 min, and absorbance of the

209

supernatants was detected spectrophotometrically at 560 nm. Values were expressed as hydrogen

210

peroxide equivalents 30, 34.

211 212

Blood samples. Whole blood samples were obtained from 16 healthy donors, collected in EDTA-

213

treated tubes and immediately used. All blood samples were obtained after informed consent.

214

Erythrocytes were isolated by centrifugation, 2000xg for 15 min, to remove plasma, platelets and

215

buffy coat and washed twice in PBS. Erythrocyte pellets were suspended in PBS and aliquots of 1.2

216

x105 cells/L were used in all experiments presented.

217 218

Reaction of human erythrocytes with HOCl and hemolysis assay. NaOCl was diluted with PBS,

219

and the pH of the solution was adjusted to 7.4 immediately before use. At this pH, the solution

220

contains approximately equimolar amounts of HClO (hypochlorous acid) and NaOCl, and is

221

referred to hereafter as HClO 35.

222

Diluted erythrocytes in PBS (1.2 x105 cells/L) were pre-treated with onion extracts at the indicate

223

concentrations for 45 min and then added with 0.3 mM of HClO for 15 min at 37°C. After

224

incubation with HClO, samples were centrifuged at 2000x g for 2 min and the percent of hemolysis

225

determined spectrophotometrically at 540 nm 24. 10 ACS Paragon Plus Environment

Page 11 of 35


226 227

GSH measurement and catalase assay. In parallel with hemolysis assay, GSH concentration and

228

catalase enzymatic activity were measured. GSH determination was evaluated by a

229

spectrofluorimetric method. Following treatments, erythrocyte pellets were washed with PBS and

230

then proteins were precipitated using TCA (5.0% v/v final concentration in 0.1 M HCl, 10 mM

231

EDTA). Fluorescence of the supernatants was measured at 340 nm (excitation wavelength) and 460

232

nm (emission wavelength). GSH concentration was calculated from a standard curve and refereed

233

as percentage compared to controls (untreated samples) 24.

234

To determine catalase activity, after treatment, erythrocytes were diluted with cold distilled water,

235

ethanol and chloroform (1.5:1:1, v/v) to precipitate the hemoglobin. Samples were shaken

236

vigorously and centrifuged at 13000xg for 3 min and the water-ethanol layer was used. Catalase

237

activity was evaluated as the first-order kinetic constant of the rate of disappearance of H2O2,

238

measured by absorbance at 240 nm, as reported previously 36. Values were expressed as specific

239

activity of the samples and referee as percentage of the control (untreated sample).

240 241

Statistical Analysis. Data are presented as mean values ± standard deviation (s.d.). For LDL

242

oxidation, hemolysis assay, GSH measurement, catalase assay, concentration of individual and total

243

polyphenolics determined by HPLC significance was measured using Student’s t-test. Number of

244

determinations are as reported in figure legends.

245



246

Page 12 of 35

RESULTS AND DISCUSSION

247 248

Total polyphenol content and antioxidant activity. Total phenolic content of onion extracts

249

indicate that the highest content of total polyphenols was found in Tropea onion ecotype (29.35 M

250

QE). They were 2.3-fold higher than those present in Montoro onion (12.68 M QE) (Table 1). As

251

expected, considering the result of the Folin-Ciocalteu assay, the antioxidant capacity was

252

proportional to the polyphenol content, with RTO showing the highest values which resulted about

253

3-fold higher than COM extract (Table 1).

254

While for COM no previous data were reported in the literature on its total polyphenol content and

255

antioxidant activity to allow a comparison with our results, the information on red onion ecotypes

256

are largely available. Although the use of different protocols of extractions complicates the

257

comparison among data from different articles, our determinations are comparable to those reported

258

by others. As an example, Marotti and Piccaglia 37 measured a total amount of flavonoids in the

259

ecotype Tropea rossa tonda of 762.9 mg/kg, not far from 632.82 mg/kg of total flavonols reported

260

in Table 3. In three red ecotypes from Spain, total flavonols ranged between 211.7-304.3 mg/kg,

261

lower than Tropea onion, but in the same order of magnitude 2. The comparative evaluation of the

262

antioxidant capacity appears more complicated, since the different methods and units used to

263

measure it. However, in general, red onion ecotypes present higher values of antioxidant capacity

264

independently from the scale of color. A good example in this sense is reported by a Turkish group

265

who demonstrated that, in 14 different cultivars differing in color, the red onions had higher

266

antioxidant activities than yellow and white onions although the formers had the richest phenolic

267

contents 15.

268 269

Identification and quantification of polyphenols in the two different onion types extracts.

270

HPLC-UV/Vis chromatograms of extract from cultivated bulbs of Allium cepa L. Montoro type are

271

shown in Figure 1. Nine flavonols and four anthocyanins were identified and quantified (Table 2 12 ACS Paragon Plus Environment

Page 13 of 35


272

and 3). The main flavonols found in this onion type were quercetin-3,4'-di-O-glucoside and

273

quercetin-4'-O-glucoside (spiraeoside), which accounted for ∼85% of the total flavonol content.

274

Results obtained from the HPLC-UV/Vis and ESI–ITMSn analyses of Tropea ecotype onion and the

275

total phenolic content measured by HPLC-UV/Vis (expressed as mg/kg of FW) were also reported

276

in Table 3. RTO showed a higher content of flavonols (632.82 mg/kg FW) than COM (252.91

277

mg/kg FW). Only kaempferol-3-O-glucoside was present in the COM and absent in RTO. More

278

pronounced were the differences existing between the 4 anthocyanins detected in the two ecotypes,

279

with those in RTO present at concentrations between 20 (compound 12 in Table 3) and 230

280

(compound 10 in Table 3)-fold higher than in COM. All these differences, including the significant

281

difference in the anthocyanin content, may justify the high level of antioxidant capacity measured in

282

RTO (Table 1).

283

As to anthocyanins, both RTO and COM contained cyanidin glucosides, acylated or not with

284

malonic acid (Table 2 and 3), which are known to be present in various cultivars of red onions 3, 38,

285

39

286

our analyses did not confirm these results. In fact, the HPLC analysis at 340 nm of RTO,

287

specifically used for monitoring flavonols, showed the presence of two very intense peaks at tR

288

25.35 min and tR 32.51 min and minor peaks at tR 26.72 min and tR 36.01 min (data not shown).

289

Compounds present in these fractions produced molecular ions at m/z 627, 465, 641 and 479

290

respectively, in ESI–ITMSn analysis carried out in positive ion mode, and were identified as

291

flavonols quercetin-3,4'-diglucoside, quercetin-4-O-glucoside, isorhamnetin-3,4'-diglucoside and

292

isorhamnetin-4'-glucoside. In addition, the ESI–ITMSn analysis of these fractions carried out in

293

negative ion mode further confirmed the presence of these flavonols (Table 3). In contrast,

294

compounds originating the same m/z values (m/z 627, 465, 641 and 479) were previously identified

295

as delphinidin and petunidin derivatives3, 4. It is important to note that quercetin and delphinidin as

296

well as isorhamnetin and petunidin displayed the same m/z value in the mass spectrum in positive

297

ESI mode (m/z 303 pseudomolecular ion [M+H]+ for quercetin and protonated molecular ion M+

. It has been reported the presence of delphinidin and petunidin derivatives in RTO 3, 4. However,



Page 14 of 35

298

for delphinidin; m/z 317 pseudomolecular ion [M+H]+ for isorhamnetin and protonated molecular

299

ion M+ for petunidin). Moreover quercetin-3,4'-diglucoside, quercetin-4-O-glucoside, isorhamnetin-

300

3,4'-diglucoside and isorhamnetin-4'-glucoside originated MS/MS spectra in positive ion mode very

301

similar to those of delphinidin (glucosyl-glucoside), delphinidin-3-glucoside, petunidin (glucosyl-

302

glucoside) and petunidin glucoside, respectively, so that, in this case, tandem mass spectrometry

303

does not lead to a definite discrimination of these compounds and accurate HPLC data are crucial to

304

achieve a conclusive result. In order to rule out the presence of at least delphinidin-3-glucoside, the

305

corresponding standard was analyzed by HPLC-UV/Vis under the reported experimental condition.

306

In agreement with the elution order reported by Wu and Prior (2005)40, the retention time of this

307

compound (tR 22.64 min) was lower than that of cyanidin 3-glucoside (tR 25.88) and of any peak in

308

our HPLC chromatogram (Figure 2), thus, definitively ruling out the presence of delphinidin-3-

309

glucoside in RTO.

310 311

Effect of onion extracts on oxidized-LDL. To confirm the antioxidant role of onion extracts and to

312

highlight potential differences between them, we evaluated their ability to reduce the oxidative state

313

of LDL (Figure 3). We incubated commercial LDL with 50 M Cu2+ for 15 h to obtain the

314

oxidized-LDL. Pre-treating isolated LDL for 30 min with 0.25 mg/mL (w/v) of onion extracts

315

resulted in a strong and significant reduction in oxidized LDL (Figure 3). The extracts deriving

316

from COM and RTO were tested at the same final concentration of 0.25 mg/mL, corresponding to

317

3.1 M QE and 7.2 M QE, respectively. Therefore, although the relative polyphenolic amounts of

318

COM was 2.3-fold lower than RTO, they exerted a similar and significant protective effect when

319

compared to oxidized-LDL (Cu2+ bar in Figure 3), while the difference between RTO + Cu2+ and

320

COM+ Cu2+ time points was not significant. These data suggest that the higher antioxidant capacity

321

of RTO, emerging from Table 1, does not reflect an increased protective activity against LDL

322

oxidation compared to COM.

323 14 ACS Paragon Plus Environment

Page 15 of 35


324

Protective effect of onion extract against erythrocyte oxidative damage. Based on the previous

325

results, we hypothesized that the antioxidant activity of the two onion extracts (Table 1 and Figure

326

3) could be protective in maintaining redox homeostasis in human erythrocytes. Based on this

327

model, we stimulated RBCs isolated from 16 healthy donors, with 0.3 mM HClO to obtain an

328

hemolysis of about 25% (Figure 4). Pre-treating with 0.25 mg/mL (w/v; corresponding to 3.1 M

329

QE COM and 7.2 M QE RTO) of the two onion extracts for 45 min resulted in a significant

330

protective effect against HClO-induced hemolysis of 15 and 17% for RTO and COM, respectively

331

(Figure 4A). The selected concentration applied (e.g., 0.25 mg/mL) was not hemolytic per se;

332

moreover, from previous data we deduct that the total concentration of polyphenols applied in our

333

assay was in the same order of magnitude as that reachable in vivo following the ingestion of an

334

amount of onion enough to bring the blood concentration of quercetin near to 1 M 41-43.

335

It is worthwhile to note that also in this case, as above for LDL oxidation, the two onion types

336

induced a similar protective effect, even though the 2.31- and 2.86-fold higher amount of

337

polyphenolic content and antioxidant capacity in RTO compared to COM, respectively (Table 1).

338

To better clarify this point, we treated RBCs with equal concentrations of polyphenols (final

339

concentration 2.7 M QE). In this case, the total amount of polyphenols expressed as QE in our

340

assays was of 0.963 g for RTO and 0.978 g for COM. As reported in Figure 4B, we observed

341

that COM protected significantly RBCs against HClO-induced hemolysis, while the limited

342

reduction observed for RTO was not significant compared to the control (HClO bar). Mathematical

343

elaboration of data presented in Figure 4A and 4B (data not shown) indicates that, to increase by

344

1% the protective effect of RTO against HClO, 0.208 µg QE of extract are necessary. In the case of

345

COM, only 0.081 µg QE (2.5-fold less) are sufficient. This calculation further demonstrates the

346

higher efficacy of COM extract compared to RTO.

347

In addition, we can exclude that the presence of AA could contribute to the protection from HClO

348

damage shown in Figure 4. In fact, measuring by FRASC assay the AA amount present in 0.25 15 ACS Paragon Plus Environment


Page 16 of 35

349

mg/mL of COM and RTO extracts, we observed that AA was null in RTO and present at a

350

concentration of 0.277 M in COM. When we repeated the assay in the presence AA in a wide

351

range (0.11-0.55 M), no reduction of HClO dependent hemolysis was observed (data not shown).

352 353

Effects of onion extracts on antioxidant defenses in human erythrocytes. RBCs excellently

354

regulate intracellular oxidative stress; in fact, through the combined activities of GSH peroxidase,

355

catalase, SOD and shunt of the hexose monophosphate they are able to protect themselves against

356

ROS44. We measured GSH level in RBCs isolated from 16 healthy donors, pretreated with 0.25

357

mg/mL of COM and RTO extracts (corresponding to 3.1 M and 7.2 M QE, respectively) for 45

358

min before HClO addition. As a consequence of HClO treatment, we observed an about 50%

359

reduction in GSH concentration present in RBCs. Pre-treatment with onion extracts induced a

360

recovery of GSH level of about 22 and 15% for RTO and COM, respectively (Figure 5A). Data

361

were significant for the former and the trend of increased production of GSH was confirmed in

362

COM. Similarly to diseases associated with increased production of ROS, the presence of a strong

363

electrophile, as HClO, causes a rapid consumption of GSH in RBCs 24, 45 and restoration of the

364

normal erythrocyte GSH concentration has been shown to have positive therapeutic effects45, 46. The

365

protective role of onion extracts against oxidative stress was also confirmed by their effect on

366

catalase activity which increases of 2.3-fold compared to basal level after HClO treatment and

367

decreases after pretreatment with the two onion extracts (Figure 5B).

368

We hypothesized that treatment of whole human blood with a HClO concentration not far from that

369

reachable in vivo following an inflammatory process, induces large hemolysis in RBCs caused by a

370

burst of intracellular ROS. Pre-treatment of whole blood with onion extracts generates a protective

371

effect against HClO-induced hemolysis. We postulated that the bioactive compounds in RTO and

372

COM extracts trigger downstream events leading to increased GSH concentration which, in turns,

373

protects erythrocytes from the subsequent oxidative damage caused by HClO (Figure 5 A and B). 16 ACS Paragon Plus Environment

Page 17 of 35


374

How are the molecules present in the extracts responsible for the de novo GSH synthesis is actually

375

under investigation in our laboratory. A model for GSH synthesis and turnover in the human

376

erythrocyte has been recently published and represents the starting point for future studies 47.

377

We described for the first time to our knowledge the flavonoid composition of a new ecotype of red

378

onion, the coppery onion, cultivated in a well limited area of about 40 ha within the districts of

379

Avellino and Salerno in Southern Italy. We also characterize the protective effects of COM against

380

oxidative stress experimentally induced on LDL and RBCs. COM was compared to a red onion

381

variety, i.e., RTO selected for its close geographic origin (Southern Italy), its high concentration of

382

polyphenols 15, 37 and larger commercial distribution. From our study an important observation

383

emerged: although total polyphenol concentration and single flavonoid compounds were both

384

significantly higher in RTO compared to COM, the biological activity was comparable, or even

385

more pronounced in COM. This result led us to formulate the following hypotheses: 1. different

386

qualitative composition in the flavonol and anthocyanin families in the two ecotypes; 2. synergistic

387

associations within the pool of flavonoids in COM resulting in an enhanced antioxidant capacity

388

compared to RTO; 3. polyphenols in red and coppery onions are not directly responsible for the

389

protective, antioxidant effect which, in turns, can be attributed to compounds of unknown chemical

390

structure present in relatively comparable amounts in the two extracts; 4. it may be possible that, at

391

least for RBCs, the protective effect of onion extracts resulting in the reconstitution of depleted

392

intracellular GSH pools, occurs towards mechanisms independent from the antioxidant activity of

393

the extracts and involving specific binding to intracellular targets. Hypotheses 3-4 require intense

394

experimental efforts, currently in progress, to be verified.

395

We focused our attention on the different polyphenolic composition between the two ecotypes. Data

396

reported in Tables 2 and 3 indicate that in COM anthocyanins are almost absent (about 0.46% of the

397

total), compared to those present in RTO (4.54%), a difference of about one order of magnitude.

398

This may suggest that in Figure 4B, when we assayed comparable amount of QE, the relative

399

abundance of anthocyanins in RTO could counteract the antioxidant effect of the flavonols present. 17 ACS Paragon Plus Environment


Page 18 of 35

400

However, initial attempts to identify a cause-effect relationship between the ratio

401

flavonols/anthocyanins and protection from oxidative damage in erythrocytes did not provide

402

conclusive answers.

403

Perhaps, we are facing a situation not totally novel regarding the mechanisms of action of phenolic

404

compounds. In fact, several cases exist in the literature demonstrating that flavonoids may exert

405

their biological activities independently from the capacity to scavenge ROS. Quercetin represents a

406

perfect example in this context, as we largely demonstrated in leukemic cell lines resistant to

407

apoptotic stimuli 48-50.

408

In summary, the present work reports for the first time the chemical characterization of polyphenols

409

present in a new variety of onion from Southern Italy and suggests that the phenolic-enriched

410

extract prepared from this ecotype exerts a protective effect against oxidative damage in LDL and

411

human erythrocytes. We also highlighted the possibility that synergistic combination of specific

412

phenolic compounds and/or the contribution of uncharacterized molecules may explain the

413

quantitative differences in biological activities measured when extracts deriving from different

414

onion varieties are compared.

415


Page 19 of 35

416


AKNOWLEDGEMENTS

417 418

We gratefully thank the Committee promoters of Montoro coppery onion (Comitato dei Promotori

419

della IGP Cipolla Ramata di Montoro; www.cipollaramatadimontoro.it) for providing us with

420

Montoro type onions. This work was partially supported by a grant from the Italian Ministry of

421

Economy and Finance to the National Research Council for the project "Innovazione e Sviluppo del

422

Mezzogiorno - Conoscenze Integrate per Sostenibilità ed Innovazione del Made in Italy

423

Agroalimentare - Legge n. 191/2009” and by Regione Campania in the framework of the “Rete di

424

Spettrometria di Massa della Campania” (RESMAC).

425



426

Page 20 of 35

FIGURE LEGENDS

427 428

Figure 1. HPLC chromatograms of extract from cultivated bulbs of Allium cepa L. Montoro type

429

recorded at 340 nm (A) and 520 nm (B). Peaks are labelled according to Table 2.

430 431

Figure 2. HPLC chromatograms of extract from cultivated bulbs of Allium cepa L. Tropea ecotype

432

recorded at 520 nm: a, Cyanidin 3-glucoside; b, Cyanidin-3-laminaribioside (Cyanidin 3-

433

glucosylglucoside); c, Cyanidin 3-(6’’-malonylglucoside); d, Cyanidin-3-malonyl-laminaribioside

434

(Cyanidin3-malonylglucosylglucoside) (Panel A) and of delphinidin-3-O-glucoside standard (de-3-

435

O-glu) (Panel B).

436 437

Figure 3. Effect of onion extracts on LDL oxidation. LDL were incubated for 30 min in the

438

presence of 0.25 mg/mL (w/v) of the two different onion extracts before addition of 50 M of Cu2+

439

for 15 h. At the end of treatment, FOX assay was performed to determinate the level of H2O2

440

generated following LDL oxidation. Bar graphs represent the mean ± s.d. Experiments were carried

441

out in duplicates. Symbols indicate significance: p

Identification and Quantification of Flavonoids from Two Southern

Recommend Documents