Phenolics of Selected Cranberry Genotypes (Vaccinium macrocarpon

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Phenolics of selected cranberry genotypes (Vaccinium macrocarpon Ait.) and their antioxidant efficacy Gihan Abeywickrama, Samir C. Debnath, Priyatharini Ambigaipalan, and Fereidoon Shahidi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04291 • Publication Date (Web): 22 Nov 2016 Downloaded from http://pubs.acs.org on November 26, 2016

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Journal of Agricultural and Food Chemistry

Phenolics of selected cranberry genotypes (Vaccinium macrocarpon Ait.) and their antioxidant efficacy Gihan Abeywickrama1, Samir C. Debnath1,2 Priyatharini Ambigaipalan3 and Fereidoon Shahidi1, 3*

Departments of Biology1 and Biochemistry, 3Memorial University of Newfoundland, St. John’s, NL, Canada A1B 3X9 2

St John’s Research and Development Centre, Agriculture and Agri-Food Canada, St. John's,

NL, Canada A1E 0B2

*Corresponding author E-mail: [email protected] Tel: 709 864 8552 Fax: 709 864 2422 1

ACS Paragon Plus Environment


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ABSTRACT

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Free, esterified and bound phenolic fractions of berries from five different cranberry genotypes

3

and two market samples were evaluated for their total phenolic, flavonoid and monomeric

4

anthocyanin contents as well as their antioxidant efficacy using TEAC, ORAC, DPPH radical,

5

reducing power and ferrous ion chelation capacity assays. HPLC-MS/MS analysis was

6

performed for two of the rich sources (Pilgrim and wild clone NL2) of phenolics and high

7

antioxidant activity. Among the genotypes, Pilgrim showed the highest phenolic and flavonoid

8

contents and wild clone NL3 and NL2 showed the highest monomeric anthocyanin and

9

proanthocyanidin content, respectively. Protocatechuic and syringic acids were only detected in

10

Pilgrim, while luteolin 7-O glucoside, quercetin 3-O rhamnoside, quercetin 3-O galactoside,

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proanthocyanidin B-type and myricetin 3-O galactoside were found in wild clone NL3 genotype.

12

Moreover, proanthocyanin trimer A-type and dimer B-type predominated in the wild clone NL2,

13

while proanthocyanidin dimer B and trimer A were predominant in Pilgrim.

14 15

Keywords: Cranberry, genotypes, phenolic compounds, proanthocyanidns, insoluble-bound

16

phenolics, antioxidant efficacy

17 18 19 20 21 22 23

2


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INTRODUCTION

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Berries are small fruits that are highly recognized for their pleasing colour, fine texture and unique

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flavour. Among the twenty most commonly consumed fruits in the North American diet, cranberry is

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ranked high among fruits in both antioxidant quality and quantity because of its considerable amount

28

of flavonoids, phenolic acids and other phytochemicals.1 These phytochemicals include a variety of

29

beneficial compounds, including phenolic compounds (especially proanthocyanidins type A),

30

essential minerals, fatty acids, dietary fiber, provitamin A, as well as vitamins C and B-complex. The

31

variety and concentration of antioxidants are highly dependent on the species and genotype. Pre-

32

harvest practices, environmental conditions, maturity stage at harvest, post-harvest storage and

33

processing operations also play important roles in the phytochemical profiles of cranberries.2

34

Phenolic compounds can be considered as the most abundant antioxidants in the human diet,

35

especially in the western world. The compounds that can delay or inhibit the effects of oxidation

36

have been considered as antioxidants, including compounds that either inhibit specific oxidizing

37

enzymes or react with oxidants before they damage critical biological molecules. As antioxidants,

38

phenolic compounds prevent the formation of free radicals, which have deleterious health effects, or

39

neutralize them once formed, and are therefore important in disease risk reduction.3 Several studies

40

in the literature have revealed the presence of the original forms of food phenolic compounds. For

41

example, unmodified proanthocyanidin, in human plasma in addition to its metabolites (methylated,

42

glucuronidated, and sulfated proanthocyanidins) as exemplified for the proanthocyanidin-rich diet4.

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Thus, these findings suggest the importance of phenolic compounds in their original form against

44

oxidative damage in a human trial.

45

Phenolic constituents in cranberry exist as free, soluble ester and insoluble-bound forms and not

46

equally distributed in the whole fruit. Phenolic acids are capable of forming ester and ether linkages

47

due to their carboxylic and hydroxyl groups, respectively5. These linkages facilitate cross-linking of

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phenolics with cell wall macromolecules and generally referred to as insoluble-bound phenolics that

49

could be released by alkali, acid or enzymatic pre-treatment of samples5. In our study, phenolic 3



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constituents of cranberries were determined for these forms using alkali hydrolysis prior to analysis.

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The insoluble-bound phenolics escape digestion in the small intestine and undergo fermentation in

52

the large intestine by microbes in the gut6. Insoluble-bound phenolics consist mainly of phenolic

53

acids, which exert health benefits such as anticancer, anti-inflammation and cardioprotective effects6.

54

This would fill the apparent gap in the existing knowledge with respect to phenolic profile of free,

55

esterified and insoluble-bound forms in cranberries and would provide information about the

56

contribution of each fraction of cranberry phenolics on antioxidant potential in food systems.

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Specifically, this study compares ripe berries from five different cranberry genotypes along with two

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market samples, mature/ripe (burgundy colour) and immature (red colour) fruits. Recognition of

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variation in phenolic profile and antioxidant properties among genotypes could be useful for plant

60

breeders, who may wish to develop or select potentially health-promoting genotypes.

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MATERIALS AND METHODS

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Materials

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Ripe berries from Pilgrim and wild clones NL1, NL2, NL3, and PE1 genotypes were obtained from

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the Research and Development Centre of the Agriculture and Agri-Food Canada, St. John's, NL,

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Canada. All plants were grown in a greenhouse in plastic pots (25 × 18 cm, 6 L equivalents; East-

66

Chem Inc, Mount Pearl, NL, Canada) containing 2 peat: 1 perlite (v/v), under natural light conditions

67

at a maximum PPF of 90 µmole m-2 s-1 at 20 ± 2 °C, and 85% relative humidity. While Pilgrim

68

(‘Prolific' × 'McFarlin') is a hybrid developed by a cooperative cranberry breeding program in the

69

USA, each Canadian wild clone represented a single plant selected from the wild based on vigour,

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berry colour, size and yield per plant and apparent freedom from disease and insects. The distance

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between two selected plants within a community was more than 10 meters. Irrigation and artificial

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fertilization [100 mg L-1 N from Peters Azalea Neutral Fertilizer 20N - 8P - 20K (Plant Products Co.

73

Ltd, Brampton, ON, Canada)] were applied when necessary. Each winter chilling and dormancy

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requirements were met by maintaining the plants at or below 6° C for 12 weeks.7 Two market

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samples (mature and immature fruits) were purchased from Costco in St. John’s, NL, Canada in 4


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November 2013; these were produced in Nova Scotia, Canada. These samples were selected based

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on their morphological characteristics. All chemicals and reagents were purchased from Sigma-

78

Aldrich Canada Ltd (Oakville,ON, Canada) or Fisher Scientific Ltd. (Ottawa, ON, Canada). Trolox

79

(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was purchased from Acros Organics (Fair

80

Lawn, NJ, USA)

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Preparation of crude extracts

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Berries were homogenized in a Waring blender (model 33BL73, Waring Products Division

83

Dynamics Co. of America, New Hartford, CT, USA) and kept frozen at -20ºC until used within one

84

week for the extraction of phenolics.

85

Extraction of phenolics

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Phenolic compounds (free, esterified, and insoluble-bound) were extracted and fractionated as

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described by Naczk and Shahidi.8 Homogenized samples (100g) were sonicated in an ultrasonic bath

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(300 Ultrasonik, Whittemore Enterprises, Inc., Rancho Cucamonga, CA, USA) for 25 min at 30 °C

89

under reflux conditions with 100 mL of a mixture of acetone-water-acetic acid (70:29.5:0.5, v/v/v).

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Following centrifugation at 4000 ×g, the supernatants were collected and the procedure was carried

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out twice. Combined supernatants were evaporated under vacuum at 40 °C and then freeze dried.

92

Residues of cranberry samples were used for the extraction of insoluble bound phenolics.

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Lyophilized crude phenolic extract (250 mg in 10 mL) was acidified with 6 M HCl (pH 2), and free

94

phenolics were extracted with diethyl ether / ethyl acetate (1:1, v/v) five times followed by vacuum

95

evaporation. . Neutralized water phase was lyophilized and hydrolyzed with 2M NaOH for 4 h under

96

nitrogen atmosphere, followed by solvent extraction and vacuum evaporation. A similar procedure,

97

as described above for esterified phenolic compounds, was carried out for extraction of insoluble

98

bound phenolics from the resultant residues.

99

Determination of total phenolic content

100

Total phenol content was determined as described by Singleton and Rossi9 using Folin Ciocalteu’s

101

phenol reagent (0.5 mL) and results were expressed as gallic acid equivalents (GAE; mg gallic acid 5



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eq/g of dried fruit weight). The absorbance values read after 3 min at 725 nm were used in the

103

determinations

104

Determination of total flavonoid content

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Total flavonoid content of cranberry samples was determined by the colourimetric method described

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by Chandrasekara et al.10. The extract was mixed with appropriate amount of reagents and the

107

absorbance was read at 510 nm.. Total flavonoid content was calculated as micromoles of catechin

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equivalents (CE) per gram of dried cranberry

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Determination of total monomeric anthocyanin content

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The total monomeric anthocyanin content was determined by the pH differential method.11 The

111

extract was diluted in 0.025 M potassium chloride and 0.4M acetate buffer to adjust the pH to 1 and

112

4.5, respectively. After completion of the reaction, the absorbance of each dilution was read at 520

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and 700 nm, respectively. The monomeric anthocyanin content was calculated using the equation

114

given below.

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Monomeric anthocyanins content as cyanidin-3-glucoside equivalents (mg / g of dry matter) =A ×

116

MW × DF/ (€ × W)

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Where A = absorbance (A520nm - A700nm) pH 1.0 – (A520nm - A700nm) pH 4.5, MW = molecular weight

118

of cyanidin-3- glucoside (C15 H11 O6, 449.2), DF =dilution factor, € = molar absorptivity (26,900),

119

and W= sample weight (g).

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Measurement of total antioxidant capacity using trolox equivalent antioxidant capacity

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(TEAC)

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The TEAC determination was carried out using 2, 2’-azinobis-(3-ethylbenzothiazoline-6-sulfonate)

123

radical cation (ABTS•+)12. The sample solution was mixed with 1.96 mL of ABTS•+ solution

124

(prepared in 2.5 mM phosphate buffer, pH 7.4 containing 0.15M NaCl) and the absorbance was

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monitored at 734 nm over a six min period. A standard curve was prepared using trolox and TEAC

126

values calculated using following equation.

6


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TEAC values were determined as follows: ∆A

128

∆A

129

absorbance at a given time, m= slope of the standard curve, [trolox] = concentration of trolox, and d=

130

dilution factor.

131

DPPH radical scavenging capacity (DRSC) using electron paramagenetic resonance (EPR)

132

spectrometry

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The DRSC assay was carried out according to the method explained by Madhujith and Shahidi13 with

134

slight modifications. Solutions of DPPH in methanol (0.3mM, 2 mL) were added to cranberry

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extracts (500 µL) and kept in the dark for 10 min. The spectrum was recorded using a Bruker e-scan

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EPR spectrophotometer (Bruker Biospin Co., Billercia, MA, USA). The parameters were set as

137

reported before and DRSC was calculated using the equation below.

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DRSC % =100 – [EPR signal intensity of extract / EPR signal intensity of control] x 100. The results

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were expressed as micromoles of trolox equivalents per gram of dried cranberry sample.

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Determination of oxygen radical absorbance capacity (ORAC)

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The ORACFL determination was carried out using fluorescein and AAPH as the radical generator in a

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Fluostar Optima plate reader (BMG Labtech, Durham, NC, USA)10. Fluorescence was recorded

143

every minute for 60 min and the ORAC of the extracts was calculated as trolox equivalents using a

144

standard curve (1-10 µM). Filters with an excitation wavelength of 485 nm and an emission

145

wavelength of 520 nm were used. 13

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Reducing power activity

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The reducing power of extracts was determined by the method of Amarowicz et al.14 Phenolic extract

148

(0.3–1.0 mg) was mixed with 2.5 mL of a 0.2 M phosphate buffer (pH 6.6) solution and 2.5 mL of a

149

1% (w/v) solution of potassium ferricyanide [K3Fe(CN)6]; the mixture was then incubated in a water

150

bath at 50 °C for 20 min. A 10% (w/v) solution (2.5 mL) of trichloroacetic acid (TCA) was added

151

and centrifuged at 3010 xg for 10 min. After that, the supernatant (2.5 mL) was added to distilled

trolox

= m x [trolox], TEAC =

{∆A

trolox

extract/

= {A

t=0 trolox

-A

t=6 min trolox}

- ∆A

solvent (0-6 min),

m} x d, Where, ∆A= reduction in absorbance, A=

7



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water (2.5 mL) and 0.5 mL of a 0.1% (w/v) solution of ferric chloride were added. Absorbance was

153

read spectrophotometrically at 700 nm and results were expressed as micromoles of trolox

154

equivalents (TE) per gram of dried sample.

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Measurement of iron (II) chelation capacity

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The ability of the phenolic extract to chelate iron (II) was assessed as described by Liyana-Pathirana

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et al.15 Each extract (0.3–1.0 mg, 0.2 mL) was mixed with 0.02 mL of 2 mM FeCl2 and 5 mM

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Ferrozine (0.2 mL), followed by vigorous shaking and then standing for 10 min. The absorbance was

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read spectrophotometrically at 562 nm. The results were expressed as micromoles of EDTA

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(ethylenediaminetetraacetic acid) equivalents per gram of dried sample.

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Hydroxyl radical scavenging assay using EPR Spectroscopy

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The hydroxyl radical scavenging capacity was determined according to Chandrasekara and Shahidi.10

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The extracts (0.2-3.0 mg/ml, 0.1 mL) were mixed with 0.1 mL of H2O2, (10 mM) and 0.2 mL of

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DMPO (17.6 mM) and 0.1 mL of FeSO4 (0.1 mM). After 3 min, the mixture was injected to a Bruker

165

e-scan EPR spectrophotometer. The results were expressed as micromoles of catechin equivalents

166

(CE) per gram of dried sample.

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Determination of 2-thiobarbituric acid reactive substances (TBARS) in cooked ground pork

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model system

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The effect of cranberry extracts on the inhibition of production of TBA reactive substances (TBARS)

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in cooked pork was determined.16,17 Phenolic extracts and BHT were added separately to ground pork

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(20 g) and cooked in a thermostated water bath at 80 ± 2ºC for 40 min with intermittent stirring.

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After the usual work out, the samples were analyzed for TBARS on days 0, 4, 7 and 14, using the

173

absorbance values at 532 nm. The TBARS were then calculated and expressed as milligrams MDA

174

equivalents per kg of sample.

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Determination of major phenolic compounds by HPLC/ESI-MS/MS

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Phenolic compounds present in the cranberry samples were determined by using an Agilent 1100

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HPLC unit (Agilent Technologies, Palo Alto, CA, USA) with a UV-diode array detector (UV-DAD). 8


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RP-HPLC Agilent 1100 system had a quaternary pump (G1311A), a degasser (G1379A) and a ALS

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automatic sampler (G1329A), a ALS Therm (G1130B), a Colcom column compartment (G1316), A

180

diode array detector (DAD, G1315B) and a system controller linked to Chem Station Data Handling

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System (Agilent Technologies). A slightly modified version of the method outlined by Lin and

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Hanly18 was used. Genotypes of wild clone NL2 and Pilgrim extracts were passed through a syringe

183

filter (0.45-µm) before injection into a reverse phase C18 column (250 mm length, 4.6 mm i.d., 5 µm

184

particle size, Sigma-Aldrich Canada Ltd.) with a guard column and the column temperature was 25

185

°C. The mobile phase consisted of a combination of B (0.1% formic acid in acetonitrile) and D (0.1%

186

formic acid in water). The gradient was as follows: 0 min, 100% D; 5 min, 10% B; 35 min, 15% B;

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45 min, 40% B; 55 min, 100% B; 65 min, 100% D and then held for 10 min before returning to the

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initial conditions. The flow rate was 0.5 mL/min and the phenolic acids and flavonoids were detected

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at 280, 306, 350 nm, whilst anthocyanins were identified at 520 nm. HPLC of cranberry extracts was

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analyzed online by using a mass selective detector (LC-MSD-Trap-SL, Agilent). In mass spectral

191

analysis, ESI (electrospray ionization) at negative mode for phenolic acids and flavonoids as well as

192

positive mode for anthocyanin detection was used to provide detailed structural information through

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collision induced dissociation. The scan range set was from m/z 50 to 2000, using smart parameter

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setting, drying nitrogen gas at 350 °C, flow rate of12 L/min, and nebulizer gas pressure of 70 psi.

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Phenolic acids [protocatechuic, p-coumaric, gallic, caffeic, ferulic, syringic and sinapic acids] and

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flavonoids [(+)-catechin, (−)-epicatechin, kaemferol and quercetin] were identified by comparison

197

with their authentic standards’ retention times and fragment ions. Other compounds were tentatively

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identified using tandem mass spectrometry (MSn), UV spectral and literature data.

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Statistical analysis

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All experiments were triplicated and the results were expressed as mean ± standard deviation. The

201

significant differences were determined at p < 0.05 using analysis of variance (ANOVA) followed by

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Turkey’s tests using SPSS 16.0 for Windows.

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RESULTS AND DISCUSSION

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Total phenolic content (TPC)

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Polyphenolic compounds from cranberry extracts reduced the Folin-Ciocalteu reagent and formed a

206

blue complex that was read at 725 nm. Pilgrim showed the highest free phenolic content followed by

207

NL2, NL3, PE1, NL1, and mature and immature market samples (Table 1). The highest amount of

208

free (1.56±0.01 mg GAE/g dw), esterified (1.29±0.04 mg GAE/ g dw) and bound (0.16±0.03 mg

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GAE/g dw) phenolics were found in Pilgrim. Total phenolic content that is the sum of free, esterified

210

and bound phenolics of cranberry genotypes followed the decreasing order of Pilgrim (~3.01 mg

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GAE/g dw) > Wild clone NL2 (~2.38 mg GAE/g dw) > NL3 (~1.69 mg GAE/g dw) > PE1 (~1.52

212

mg GAE/g dw) > market-mature (~61 mg GAE/g dw) > market-immature (~0.41 mg GAE/g dw) >

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NL1 (~0.18 mg GAE/g dw). Furthermore, the results indicated that the TPC in the free phenolic

214

fraction of all genotypes was the highest compared to the bound and esterified extracts, except in the

215

market samples. In agreement with this finding, Sun et al.19 reported that phenolics in the free form

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were higher than that of bound form in cranberry, strawberry, apple, red grape, peach, pear,

217

grapefruit, banana and lemons. In general, the phenolic contents in all tested cranberry genotypes

218

were higher than those in the market samples and those given in the existing literature.4,19

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Furthermore, based on Kahkonen et al.20, the phenolic content of their cranberry sample was similar

220

to all five genotypes tested in this work. Costantino et al.21 quantified TPC to be 2.56 mg/g in fresh

221

bilberry, and 2.21 mg/g in bilberry press residues. Zielinski et al.22 reported that compounds such as

222

simple carbohydrates and/or amino acids may be present in the crude extracts and could interfere

223

with determinations of TPC by the Folin-Ciocalteu assay, leading to discrepancies. It is also of

224

interest to indicate that TPC in all tested cranberry genotypes were higher compared to the European

225

cranberry genotypes.23 These also lend support to the fact that the total amount of phenolics in

226

cranberry varies among different genotypes, and depends on the cultivar/clone, preharvest practices,

227

environmental conditions, and maturity stage at harvest, all of which control the accumulation of

228

phenolic compounds by synthesizing different quantities and types of phenolics.2 10


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Total flavonoid content (TFC)

230

Flavonoids exhibit a variety of biological activities both in vitro and in vivo.24 In this study, Pilgrim

231

exhibited the highest total flavonoid content (2.00 mg CE/g dw), followed by clones NL2 (1.00 mg

232

CE /g dw), NL3, PE1 and NL1. It was also noted that the free phenolic fractions contained the

233

highest amounts of flavonoids in all tested genotype varieties compared to the esterified and the

234

insoluble-bound fractions (Table 1), except in the immature market sample. In agreement with this

235

finding, Meyers et al.25 reported that bound fractions contributed the least in strawberry compared to

236

the corresponding free and esterified fractions. In general, in all extracts the TFC in the insoluble-

237

bound fraction were lower than their corresponding free and esterified fractions. The TFC in bound

238

phenolic fraction of pilgrim had the highest level (0.14 mg of CE/g of dried fruit weight), whereas

239

the wild clone PE1 showed the lowest (0.02 mg of CE/g of dried fruit weight). In contrast to the

240

results obtained in this study, Tuloi et al.26 reported that TFC (0.46 mg CE/g of fresh fruit) in

241

cranberry were less than the genotypes tested here. The TFC reported in this study for different

242

cranberry genotypes were lower than those reported for other berries, namely strawberry, blueberry

243

and raspberry18 and four different raspberry genotypes examined by Liu et al.27 The variety of berries

244

may account for differences in the results obtained as well as possible variations in the extraction

245

conditions, genotypes considered, environmental conditions, and maturity stage at harvest.2

246

Total monomeric anthocyanin contents

247

In the present study, soluble extracts of cranberry cultivars and market samples were evaluated for

248

their total monomeric anthocyanin content, and presented as mg cyanidin-3-O-glucoside equivalents

249

(Table 1). The highest content of monomeric anthocyanins was in wild clone NL3 (2.22±0.10 mg/g

250

dw), while monomeric anthocyanin content in Pilgrim genotype was 1.15±0.07 mg/ g dw. This result

251

is in agreement with that of Viskelis et al.28, who reported that the total monomeric anthocyanin in

252

overripe Pilgrim was 1.37 mg/g of sample. There was a significant difference (p NL2 > NL3 > NL1 > market mature > PE1 > market

264

immature. Esterified phenolic fraction of Pilgrim showed the highest RP (1.12±0.02 mmoles trolox

265

equivalents (TE) /g dw), followed by clones NL2 (0.72±0.09 mmoles TE/g dw), NL3, PE1, and

266

mature and immature market samples, respectively, except in NL1, where free phenolic fraction

267

showed the highest RP compared to that of its insoluble-bound and soluble ester forms. The RP of

268

the insoluble-bound phenolic was comparable to those of the free and esterified phenolics that

269

showed the lowest activity. Meantime, the bound phenolic extract of Pilgrim had the highest RP

270

(0.18 ± 0.01 mmoles TE /g dw), whereas immature market sample showed the lowest (0.04 ± 0.00

271

mmoles TE /g dw). According to RP activity of cranberry, esterified phenolic extracts that are the

272

dominant form of phenolics, contributed most to the antioxidant activity of cranberry followed by

273

free and bound phenolic fractions. In general, the RP in all tested cranberry genotypes was higher

274

than those given in the existing literature for cranberry, wild and cultivated red raspberry, blueberry,

275

blackberry, red current, gooseberry and cornelian cherry. 31,32

276

Ferrous ions chelating activity

277

Chelating agents, which bind prooxidant metals, are effective as secondary antioxidants.16 The

278

present study demonstrated significant chelating activity for free, esterified and bound phenolic

279

extracts of cranberries against Fe2+ in all tested genotypes (Table 2; Figure 1A). The results of this

280

study indicate that phenolic extracts of Pilgrim may serve well as a potential source of chelating 12


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agents, where bound phenolics extract had chelating activity of 3.88 ±0.19 µmol EDTA /g of dw,

282

while free and esterified phenolic extract exhibited 1.27 ±0.06 µmol of EDTA /g dw and 4.27 ±0.71

283

µmol of EDTA /g dw metal chelating activity, respectively. In this study, the total ferrous ion

284

chelating activity of cranberry genotype extracts decreased in the order of Pilgrim > NL2 > NL3 >

285

PE1 > NL1> immature market > mature market (Figure 1A). No significant difference (p>0.05)

286

existed between esterified phenolics of NL2, NL1, Pilgrim, and NL3 (Table 2). In general, lower

287

metal chelations were observed compared to those of blueberry33. In addition to that, a similar trend

288

was observed between metal ion chelation and TPC and TFC in all tested cranberry genotypes. This

289

indicates that phenolic and flavonoid compounds are the major constituents in the fruits that

290

contribute to their metal chelation activity33.

291

Trolox equivalent antioxidant capacity (TEAC)

292

TEAC assays measure the ability of cranberry phenolic extracts to reduce the in vitro formed radicals

293

(Table 3; Figure 1B). In this study, cranberry wild clone NL3 exhibited the highest total TEAC

294

(979.66 µmoles TE equivalents /g dw). The results indicated that esterified phenolic extract of NL3

295

had 5.4 times higher TEAC than that of its free phenolic fraction. According to Tulio et al.26, TEAC

296

of cranberry extract ranged from 13.04 to 13.78 µmoles TE/100g of fresh weight and a study by

297

Seeram et al.34 showed that TEAC of cranberry juice ranged from 6.7 to 14.8 µmoles TE/mL.

298

Furthermore, TEAC of bilberry seeds ranged from 5.8 to 84.4 µmoles of TE/g of berry seed press

299

residues and those for black currant were 67.2 to 74.7 µmoles of TE/g of berry seed press residues,

300

respectively.35 The total TEAC values of all tested cranberry genotypes did not show any correlation

301

with TPC and TFC of samples. In agreement with this finding, some studies have reported a lower

302

TPC for berries exhibiting a higher total antioxidant capacity.35 This demonstrates that the contents of

303

total phenolics and flavonoids may not sufficiently explain the observed antioxidant activity of fruit

304

and plant phenolic extracts which are mixtures of different compounds with varying activities in the

305

test samples. Furthermore, the TEAC value of an extract represents the sum of antioxidant

306

compounds, which depend on the solvent used to extract them from their source matrix. 13



Page 14 of 40

307

Analysis of oxygen radical absorbance capacity (ORAC)

308

In this study, ORAC values of esterified, free and bound phenolics in all tested cranberry genotypes

309

were in the range of 10.05-3.69, 8.14-4.38 and 4.44-0.93 mmoles of TE/g dw, respectively (Table 3).

310

On a dried fruit weight basis, Pilgrim had a significantly higher ORAC value than the other

311

genotypes. The contribution of esterified phenolics of cranberry extracts towards the total ORAC was

312

higher than the free and insoluble-bound phenolic counterparts. Esterified phenolics of wild clone

313

NL3 exhibited the highest ORAC followed by NL2, Pilgrim, mature market sample, immature

314

market sample, PE1 and NL1. There were no significant differences (p˃0.05) among esterified

315

phenolic fractions of NL2, Pilgrim, NL3 and immature market samples. The total ORAC for

316

cranberry genotypes were in the order of Pilgrim > mature market > NL2 > immature market > NL3

317

> PE1 > NL1 (Figure 1C). This trend is very similar to those of TPC and TFC of the samples, except

318

for the market samples. Thus, samples with higher total phenolic contents exhibited higher

319

antioxidant activity. These results suggest that the antioxidant activity of fruits is derived mainly

320

from the contribution of their phenolic compounds. In agreement with this trend, previous research

321

has shown a linear relationship between total phenolic content and ORAC in various berry crops36,

322

suggesting that the phenolic compounds contribute to their oxygen radical absorbance capacities and

323

the total phenolic content is a better indicator for oxygen radical absorbing components in berries.

324

However, little information is available on the contribution of individual phenolic compounds to total

325

antioxidant activity in berry crops37 and also ORAC value of all tested cranberry genotypes had the

326

highest activity compared to the reported values for cranberry, blackberry, raspberry and

327

strawberry.36,37

328

DPPH radical scavenging capacity (DRSC) using EPR spectroscopy

329

The DRSC for cranberry genotypes were in the order of Pilgrim > NL2 > NL3 > immature market >

330

PE1 > mature market > NL1 (Table 4). DRSC of all genotypes were mainly contributed by their

331

esterified phenolics, except in cranberry wild clone NL1, where the free phenolic extracts displayed

332

the highest activity. There were no significant differences (p>0.05) among esterified phenolic extract 14


Page 15 of 40


333

of NL2, Pilgrim, NL3, PE1, mature market and immature market samples. The insoluble-bound

334

phenolic extracts in all genotypes had the lowest DPPH radical scavenging activity as compared to

335

their free and esterified counterparts, except in immature market sample, where the free phenolic

336

forms had a lower activity. According to Tulio et al.26, the DRSC activity of cranberry phenolic

337

extracts ranged from 6.13 to 6.28 µmoles TE/100g of fresh weight. This trend is also very similar to

338

the TPC and TFC of samples, indicating that samples with higher phenolic content exhibit higher

339

antioxidant activity. However, wild clone NL1 which had a higher total phenolic content than market

340

samples (immature and mature) showed a lower DRSC, because of the existing differences in the

341

chemical composition and structural features contributing to scavenging activity. Shahidi and

342

Naczk38 reported that the antioxidant activity of a given food or food product depends on the

343

chemical nature of its constituents and, not always their quantities, as the efficacy of compounds

344

present varies considerably.

345

Hydroxyl radical scavenging capacity using EPR spectroscopy

346

Hydroxyl radical is a biologically relevant radical species that can cause severe damage to

347

biomolecules. Hydroxyl radical scavenging activity of free, esterified and bound phenolics were

348

75.82-251.50, 24.96-251.89 and 16.42-183.24 µmoles CE/g dw, respectively (Table 4). The main

349

contribution for all genotypes was from their free phenolics, except in NL2 and Pilgrim, where the

350

esterified phenolic extracts showed the highest values. This trend is fairly different from that for

351

DPPH radical scavenging activity, possibly due to the existing differences in their scavenging power

352

for the two radicals. In addition, the bound phenolics in all genotypes showed the lowest hydroxyl

353

radical scavenging activity as compared to their free and esterified forms, except for the immature

354

market sample. As shown in Table 4, clone NL3 had the highest total hydroxyl radical scavenging

355

activity (604.85 µmoles CE/g dried fruit weight) whereas immature market sample had the lowest.

356

Ou et al.39 have evaluated a number of phenolic compounds for their hydroxyl radical scavenging

357

capacity and ORAC and revealed that the former is consistently lower than ORAC among the tested

358

compounds. This pattern was true for most foods tested by Ou et al.39 A similar trend was observed 15



Page 16 of 40

359

for all tested cranberry genotypes as well. Ou et al.39 observed that phenolic compounds with metal

360

chelation potential showed higher hydroxyl radical scavenging values, whereas the compounds with

361

poor metal chelation activity displayed lower values. Phenolics act as metal chelators, thereby

362

blocking the reaction sites for H2O2, which leads to reduce concentration of Fe (II) and hydroxyl

363

radicals.

364

Determination of TBARS in cooked meat model system

365

The 2-thiobarbituric acid (TBARS) method is widely used for measuring lipid peroxidation in muscle

366

foods due to its simplicity and rapid nature. According to Figure 1D, the percentage inhibition of

367

TBARS formation in the BHT treated sample was 53.84% on day 7 of storage. The samples arranged

368

in the order of their effectiveness in inhibiting the formation of TBARS (%) and reported as MDA

369

equivalents inhibition (%) were in the order of Pilgrim (48.67%) > NL3 (42.55%) > PE1 (32.60%) >

370

mature market (20.96%) > NL2 (20.54%) > NL1 (12.44%) > immature market (11.58%). Pilgrim

371

was highly effective in inhibiting oxidation in a cooked pork model system and immature market

372

extracts showed lowest protective effect against lipid oxidation (Figure 1D). The iron released from

373

porphyrin ring could act as catalyst of lipid oxidation in comminuted pork model system and the rate

374

of releasing iron depends on time, intensity and temperature of cooking.40 It indicated that phenolic

375

extract of all five tested cranberry genotypes may act as effective iron chelators as well as peroxyl

376

radicals scavengers . Phenolics of Pilgrim displayed effective ferrous ion chelating activities whereas

377

immature market sample showed a weak activity. The inhibition of formation of TBARS for all

378

tested cranberry genotypes was similar to that of TPC and TFC of the samples except cranberry wild

379

clone NL2 genotype. Thus, samples with higher phenolic contents better inhibited lipid oxidation and

380

rancidity development. In agreement with this trend, extracts enriched in phenolic acids,

381

anthocyanins, flavonols and proanthocyanidins showed the greatest inhibitory effect on lipid

382

oxidation in cooked pork.41

383

Identification and quantification of major phenolic compounds of cranberry genotypes by

384

using HPLC/ESI-MS/MS 16


Page 17 of 40


385

The predominant phenolic acids and flavonoids present in cranberry samples were identified and

386

quantified using HPLC/ESI-MS/MS (Table 5 and 7; Supplementary material, Figure 1). The results

387

showed that the content and type of phenolic compounds varied depending on the cranberry genotype

388

as well as phenolic fraction considered. However, as expected, the cranberry genotypes tested

389

showed essentially the same phenolic compounds in each fraction though the contents differed

390

between the two tested genotypes, which confirm the fact that phenolic content is influenced by the

391

variety. The highest flavonoid level was detected in the free phenolic fractions of the tested

392

cranberry genotypes (Table 6). As observed in previous studies, and confirmed in this study, soluble

393

phenolic fraction, composed of free and esterified fractions, of two tested cranberry genotypes

394

showed higher TPC and antioxidant activity than that of insoluble-bound phenolics in several in

395

vitro systems.19

396

Phenolic acids

397

This family of compounds includes derivatives of hydroxycinnamic acids (HCA; also known as

398

phenylpropanids) and hydroxybenzoic acids (HBA)42,48. These compounds rarely occur in the free

399

form; they are instead associated with other types of compounds such as arabinoxylans or cellulose in

400

the cell walls or may be glycosylated to simple sugars or occur as soluble esters. In this work, in

401

agreement with previous research findings, the soluble phenolics that included both the free and

402

esterified fractions, was present at a higher concentration, as reflected in TPC and antioxidant

403

activity, than that of the insoluble-bound phenolics for the two tested cultivars, as shown in several in

404

vitro systems. The concentration of HBA derivatives is generally low in food of plant origin, the

405

exception being for the majority of berries, whose content of protocatechuic, ellagic, and gallic acids

406

is very high.42 Among HCA and HBA, p-coumaric acid (from 2 ± 0.16 to 245 ± 1.16 µg of p-

407

coumaric acid/ g dw) was the most abundant compound followed by caffeic acid (from 5 ± 0.91 to

408

123 ± 4.98 µg/g dw), ferulic acid (from 4 ± 0.09 to 39 ± 2.22 µg/g dw) and chlorogenic acid (from 6

409

± 0.90 to 47 ± 1.76 µg/g dw) in cranberry wild clone NL2 and cultivar Pilgrim (Table 6). HBA and

410

HCA were quantified using the calibration curve of their corresponding standards at 280nm. These 17



Page 18 of 40

411

results are in accordance with previous studies that found gallic, chlorogenic, p-hydroxybenzoic,

412

ferulic and p-coumaric acids as being predominant in cranberry pomace.43 According to Velioglu et

413

al.44, the European cranberry bush fruits contained 2037 mg kg-1 chlorogenic acid. This is more than

414

5 times higher than our results and those of other researchers.45 Chlorogenic acid concentration may

415

be varied among cranberry genotypes, stage of maturity, extraction method and climate factors (light,

416

temperature).46

417

Flavonols

418

Flavonols are found in abundance in Ericaceae fruits such as cranberry, blueberry, and bilberry. In

419

this study, we were able to identify and quantify four major flavonols in wild clone NL2 and cultivar

420

Pilgrim; these were quercetin 3-O-rhamnoside (343 ± 7.18 µg of CE/ g dw), myricetin 3-O-

421

arabinoside, quercetin 3-O-galactoside and myricetin 3-O-galactoside (Table 6). Flavonols were

422

quantified with the calibration curve of catechin at 280 nm, except for epicatechin, which was

423

quantified with its own calibration curve at 280 nm. Myricetin 3-O-arabinoside was found only in the

424

free fraction of cultivar Pilgrim, whereas it was detected in all fractions of Wild clone NL2.

425

Similarly, kaempherol hexoside and myricetin 5-O-galactoside were detected in different fractions in

426

both cultivars. Thus the distributions of phenolics vary between these two cultivars, possibly due to

427

genetic variation or other factors that require further clarification. In agreement with this study, Chen

428

and Zuo47, also identified two major flavonol glycosides, quercetin galactoside and quercetin

429

arabinoside, in American cranberry fruits.

430

Anthocyanins

431

Anthocyanins are generally found in red, purple, and blue fruits of berry crops. Their concentrations

432

in

433

temperature)50.Anthocyanins were quantified with the calibration curve of catechin at 280 nm (Table

434

6). The major anthocyanins detected in cranberry wild clone NL2 and Pilgrim were cyanidin-3-O-

435

arabinoside and cyanidin-3-O-galactoside. Cyanidin-3-O-arabinoside and cyanidin-3-O-galactoside

436

were predominant in cranberry wild clone NL2 and Pilgrim, respectively. This agrees with the results

food

tend

to

increase

as

fruits

ripen

in

response

to

climatic

factors

(light,

18


Page 19 of 40


437

reported by Grace et al.51 Commercial cranberry contained six anthocyanins, which were

438

galactosides, glucosides, and arabinosides of both cyanidin and peonidin and wild Alaskan lowbush

439

cranberry displayed only cyanidin glycosides as the dominant anthocyanin, with non-detectable

440

levels of peonidins. However, Neto29 reported that the major anthocyanins in cranberry are

441

galactosides and arabinosides of cyanidin and peonidin. In the HPLC analysis, anthocyanins were

442

quantified mainly in free extracts of wild clone NL2 and Pilgrim. Anthocyanins in bound and

443

esterified extracts were found in trace amounts and hence could not be quantified. In agreement with

444

this, Brown et al.30 did not report peonidin-3-O-glucoside in their samples, which might be due to

445

their presence at a lower level than the detection limit of this method. In this study, during the

446

fractionation of the insoluble-bound phenolics, the residue of the cranberry sample obtained after

447

extraction of soluble phenolics was hydrolyzed with NaOH and the water phase was neutralized to

448

pH 7 with NaOH. According to da Costa et al.52, anthocyanins can be found in different chemical

449

forms depending on the pH of the solution. At pH 1, the red coloured flavylium cation is the

450

predominant species. At pH between 2 and 4, the quinoidal blue coloured species dominate while at

451

pH between 5 and 6 only two colourless species can be observed, which are carbinol pseudobase. At

452

pH values higher than 7, anthocyanins are degraded to simple phenolic acids53 hence absence of

453

anthocyanins in the bound phenolics examined in this work.

454

Flavanols and Proanthocyanidins

455

Catechin and epicatechin are important constituents of fruits, and their presence has been reported in

456

cranberry.48 Quantification of catechin and epicatechin is shown in Table 6 and the latter was

457

predominant in cranberry wild clone NL2 and cultivar Pilgrim, mainly in the free phenolic form, at

458

874 ± 42.11 and 1798 ± 56.29 µg / g dw. Meanwhile, catechin was present at corresponding values

459

of 66 ± 0.44 and 34 ± 2.64 µg/ g, respectively. Velioglu et al.44 identified catechin and epicatechin in

460

European cranberry bush with a catechin content of 290 mgL-1. According to Maatta-Riihinen et

461

al.49, catechin and epicatechin contents in cranberry were 417 and 447 µg/ mg dw, respectively.

462

These values were lower than those in our study. The concentrations of catechin and epicatechin may 19



Page 20 of 40

463

vary depending on the cultivar, stage of maturity, extraction method, climatic factors (light,

464

temperature), as well as the storage time and presence of other substances.38 Flavan-3-ols in

465

cranberry occur as monomers, or in the oligomeric and polymeric forms (called proanthocyanidins;

466

PACs). The contents of PACs in cranberry vary according to the nature of the inter-flavan linkage,

467

constitutive units, and degree of polymerization (DP). Proanthocyanidins were quantified with the

468

calibration curve of catechin at 280 nm. HPLC quantification revealed that the highest amount of

469

proanthocyanidins existed as free phenolics followed by esterified and insoluble-bound fractions in

470

both NL2 and Pilgrim. Wild clone NL2 had slightly higher proanthocyanidin content (~1175 (µg/g)

471

in comparison with Pilgrim cranberries (~1047 (µg/g). Both A- and B-type PACs were found in

472

cranberry. B-type PACs are those in which monomeric units are linked through the C4 position of the

473

upper unit and the C6 or C8 positions of the lower unit, whereas A-type PACs contain an additional

474

ether-type bond between the C2 position of the upper unit and the hydroxyl group at C7 or C5 of the

475

lower unit (C2−O−C7 or C2−O−C5). It has been estimated that A-type PACs account for 65% of total

476

PACs in cranberry.53 In agreement with this study, A-type PACs dimers and trimers were more

477

abundant than B-type PACs dimers and trimers in wild clone NL2 and Pilgrim (Table 6). Grace et

478

al.51 showed a similar trend, whereas commercial cranberry had higher percentages of A-type dimers

479

and trimers (23.2 and 12.1%, respectively) than B-type analogues (9.2 and 3.6%, respectively).

480

In this study, the phenolic constituents of cranberry were fractionated into their respective free,

481

esterified and insoluble-bound forms to provide a complete picture of their compositional

482

characteristics. Phenolics were predominantly present in the free and esterified forms in all the

483

examined cranberry genotypes, except the market samples. Thus, cranberries, rich in a number of

484

phytochemicals, serve as promising candidates for further development, as reflected in the two

485

varieties (wild clone NL2 and Pilgrim) that exhibited excellent characteristics in terms of their

486

antioxidant potential compared to those in the market and other tested samples. Moreover, use of

487

various methods, such as RP, ORAC, TEAC, and DRSC for the determination of antioxidant activity

488

revealed the mechanism of cranberry polyphenols as a source of potential antioxidative compound. 20


Page 21 of 40


489

Abbreviations used: Trolox equivalent antioxidant capacity (TEAC), oxygen radical absorbance

490

capacity (ORAC), DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging capacity (DRSC), 2-

491

thiobarbituric acid reactive substances (TBARS), Gallic acid equivalents (GAE), dried fruit weight

492

(dw).

493

Supporting Information. Representative HPLC chromatogram of esterified phenolic fraction of

494

cranberry wild clone NL2 with selected phenolic compounds (Figure).

21



495

Page 22 of 40

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607 (46) Bohm, B. A. Introduction to flavonoids, Department of Botany, University of British Columbia, 608 609 610

Vancouver, BC, Canada. 1998; pp. 503-514. (47) Chen, H.Y.; Zuo, Y. Identification of flavonol glycosides in American cranberry fruit. Food Chem. 2007, 101, 1357-1364.

611 (48) Macheix, J.J.; Fleuriet, A. Phenolic acids in fruits. In: Flavonoids in Health and Disease. Rice 612

Evans, C.A. and Packer, L., Eds., Marcel Dekker Inc, New York, Basel, 1998, pp 35-60.

613 (49) Maatta-Riihinen, R.K.; Kahkonen, P.M.; torronen, R. A.; heinonen, M.I. Catechins and 614

Procyanidins in Berries of Vaccinium Species and Their Antioxidant Activity. J. Agric. Food

615

Chem. 2005, 53, 8485-8491.

616 (50) Brouillard, R.; Chassaing, S.; Fougerousse, A. Why are grape/fresh wine anthocyanins so simple 617

and why is it that red wine colour lasts so long. Phytochemistry. 1997, 64, 1179-1186.

618 (51) Grace, M.H.; Massey, A. R.; Mbeunkui, F.; Yousef, G. G.; Lila, M. A. Comparison of Health619

Relevant Flavonoids in Commonly Consumed Cranberry Products. J. Food Sci. 2012, 77, 176-

620

183. 27



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621 (52) da Costa, C.T.; Horton, D.; Margolis, S. A. Analysis of anthocyanins in foods by liquid 622

chromatography, liquid chromatography–mass spectrometry and capillary electrophoresis.

623

J. Chromatogr. A. 2006, 881 (1-2), pp. 403-410.

624

(53) Sanchez-Patan, F.; Bartolome, B.; Martin-Alvarez, P.J.; Anderson, M.; Amy Howell. A.;

625

Monagas, M. Comprehensive Assessment of the Quality of Commercial Cranberry Products.

626

Phenolic Characterization and in Vitro Bioactivity. J. Agric. Food Chem. 2012, 60, 3396-

627

3408.

628

Acknowledgement

629

We are grateful to the Natural Science and Engineering Research Council (NSERC) of Canada for

630

financial support in the form of a discovery grant to FS

631 632 633 634 635 636 637 638 639 640 641

28


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642

Figure Captions

643

Figure 1. (A) Total Ferrous Iron Chelating Activity; (B) Total Trolox Equivalent Antioxidant

644

Capacities (TEAC); (C) Total Oxygen Radical Absorbance Capacity (ORAC) of Cranberry Fruit

645

Samples on a Dry Weight Basis; and (D) Thiobarbituric Acid Reactive Substances (TBARS) in

646

Cooked Pork Model System of Dried Cranberry on a Dry Weight Basis After 7 days of Storage at

647

Refrigerated Temperature

648 649 650 651 652 653 654 655 656 657

29



Page 30 of 40

658

Table 1. Total Content of Phenolics, Flavonoids and Monomeric Anthocyanins of Cranberry Fruit Samples on a Dry Weight (dw)

659

Basis a

Cranberry genotype

Total Phenolics

Total Flavonoids

Total Monomeric

(mg GAE equivalents/g dw )

(mg CE equivalents/g dw)

Anthocyanins (mg /g dw)

Free

Esterified

Bound

Free

Esterified

Bound

Wild clone NL2

1.38±0.07b

0.86±0.04b

0.14±0.01a

0.73±0.03b

0.15±0.03c

0.12±0.03a

1.25±0.11b

Pilgrim

1.56±0.10a

1.29±0.04a

0.16±0.09a

0.94±0.02a

0.92±0.03a

0.14±0.04a

1.15±0.07b

Wild clone NL3

1.11±0.08c

0.47±0.04c

0.11±0.01 b

0.68±0.04c

0.22±0.03b

0.06±0.00b

2.22±0.10a

Wild clone PE1

1.04±0.04c

0.40±0.01c

0.08±0.01b

0.76±0.04b

0.07±0.00d

0.02±0.00c

0.54±0.05e

Wild clone NL1

0.90±0.04d

0.10±0.02e

0.08±0.00b

0.68±0.25c

0.04±0.00d

0.06±0.00b

0.71±0.04d

Market-immature

0.15±0.01e

0.22±0.00d

0.04±0.00c

0.03±0.00d

0.04±0.00d

0.04±0.00c

0.41±0.02e

Market-mature

0.27±0.02e

0.27±0.01d

0.07±0.00b

0.06±0.00d

0.05±0.00d

0.03±0.00c

0.91±0.04c

a

Data are expressed as means ± SD (n=3). Values with the same letter in the same column are not significantly different (p>0.05). GAE, gallic acid

equivalents; and CE, catechin equivalents.

30


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Table 2. Antioxidant Capacity (Reducing Power and Iron Chelation) of Cranberry Fruit Extracts on a Dry Weight (dw) Basis a

Cranberry Sample/ Genotype

Reducing Power (mmoles TE equivalents/g dw) Free Esterified Bound

Iron Chelating Activity (µmoles EDTA equivalents/ g dw) Free Esterified Bound

Wild clone NL2 Pilgrim

0.39±0.00c 0.66±0.07a

0.72±0.09b 1.12±0.02a

0.12±0.00b 0.18±0.01a

1.13±0.02a 1.27±0.06a

2.12±0.07c 4.27±0.71a

5.16±0.34a 3.88±0.19b

Wild clone NL3

0.34±0.09c

0.66±0.00c

0.12±0.01b

1.2±0.12a

4.48±0.11a

2.51±0.00c

Wild clone PE1

0.22±0.03d

0.26±0.04d

0.09±0.00c

0.31±0.03b

3.23±0.05b

2.02±0.02d

Wild clone NL1

0.49±0.00b

0.15±0.01e

0.11±0.00b

0.03±0.00c

1.42±0.01c

2.53±0.04c

Market-immature

0.14±0.00e

0.19±0.02e

0.04±0.00d

0.41±0.15b

0.51±0.01d

0.46±0.01e

Market-mature

0.21±0.00d

0.23±0.03d

0.11±0.01b

0.32±0.11b

0.31±0.00d

0.32±0.00e

a

Data are expressed as means ± SD (n=3). Values with the same letter in the same column are not significantly different (p>0.05). TE, trolox; and EDTA, Ethylenediaminetetraacetic acid.

31



Page 32 of 40

Table 3. Antioxidant Capacity (TEAC and ORAC) of Cranberry Fruit Extracts on a Dry Weight (dw) Basis a

Cranberry Genotype

TEAC (µmoles TE equivalents/g dw )

ORAC (mmoles TE equivalents/g dw)

Free

Esterified

Bound

Free

Esterified

Bound

Wild clone NL2

243.46±3.01d

368.08±3.90c

43.91±1.21b

6.36±0.77b

9.94±0.21a

1.02±0.09c

Pilgrim

364.33±1.39b

361.45±2.75c

36.17±2.01c

6.63±0.23b

9.62±0.79a

4.44±0.18a

Wild clone NL3

147.11±2.09e

801.69±7.03a

30.80±0.92d

4.38±0.52d

10.05±0.5a

2.02±0.16b

Wild clone PE1

322.54±2.05c

221.71±1.92d

30.54±0.89d

8.14±0.59a

4.87±0.49c

0.93±0.04c

Wild clone NL1

176.41±1.99f

62.88±2.94f

31.45±0.75d

6.73±0.29b

3.69±0.53c

2.62±0.17b

Market-immature

95.48±3.01g

157.67±2.01e

41.85±2.01b

5.72±0.46c

8.72±0.36a

2.71±0.26b

Market-mature

391.94±2.33a

421.72±4.23b

59.52±1.05a

6.91±0.39b

8.93±0.06a

2.23±0.36b

a

Data are expressed as means ± SD (n=3).Values with the same letter in the same column are not significantly different (p>0.05). TEAC, trolox equivalents antioxidant capacity; ORAC, oxygen radical absorbance capacity; and TE, trolox equivalents.

32


Page 33 of 40


Table 4. DPPH Radical Scavenging Activity and Hydroxyl Radical Scavenging Capacity of Phenolics in Cranberry Fruit Extracts on a Dry Weight (dw) Basis a

Cranberry Genotype

DPPH Radical Scavenging Activity (µmoles TE equivalents /g dw)

Hydroxyl Radical Scavenging Capacity (µmoles CE equivalents /g dw)

Free

Esterified

Bound

Free

Esterified

Bound

Wild clone NL2

165.39±3.31a

949.54±7.15a

98.58±2.15a

197±0.34b

251.89±3.82a

29.74±1.15d

Pilgrim

154.21±2.95a

1095.91±6.11a

82.41±1.55b

183.85±0.51b

198.91±1.75c

183.24±3.03a

Wild clone NL3

161.99±0.71a

948.97±1.15a

68.79±4.11c

251.50±2.47a

229.17±1.99b

124.18±2.2b

Wild clone PE1

164.05±4.86a

861.94±8.78a

53.82±2.93c

249.90±4.05a

205.84±0.15c

72.87±1.17c

Wild clone NL1

163.75±5.83a

93.56±1.16b

62.95±2.73c

200.38±1.15b

155.84±3.11d

80.98±0.93c

Market-immature

94.64±1.03b

916.57±6.35a

109.32±3.32a

75.82±3.15c

24.96±0.35f

64.81±1.05c

Market-mature

84.43±1.52b

743.47±4.19a

80.44±1.01b

80.40±1.45c

79.88±0.77e

16.42±0.55e

a

Data are expressed as means ± SD (n=3). Values with the same letter in the same column are not significantly different (p>0.05)

33



Page 34 of 40

Table 5. Individual Phenolic Compounds Identified in Cranberrya Phenolic Compounds Peak No

Molecular Weight (g/moles)

[MH]¯/[MH]+ (m/z)a

ESI Fragments (m/z)

Hydrobenzoic and Hydrocinnamic Acids 1

Gallic

170

169

125

2

Protocatechuic

154

153

109

3

Sinapic

224

223

179

4

Syringic

198

197

153

5

Caffeic

180

179

135,167

6

p-coumaric

164

163

119,139

7

Ferulic

194

193

135

8

Chlorogenic

354

353

191

9

(+)-Catechin

290

289

245

10

(-)-Epicatechin

290

289

245

11

Quercetin

302

301

121,179

12

Kaempherol hexoside

448

447

257,285,327,401

13

Luteolin 7-O-glucoside

448

447

121,177,285,313,381

Flavonoids

34


Page 35 of 40


14

Cyanidin 3-O-arabinoside

419

417

15

Proanthocyanidin dimer B- type

578

577

287,289,407,425

16

Proanthocyanidin dimer A- type

576

575

285,289,407,423

17

Quercetin 3-O-rhamnoside

449

448

303

18

Proanthocyanin trimer A-type

864

863

449,559,693,711,737

19

Cyanidin 3-O-galactoside

449

448

285,287

20

Myricetin 3-O-arabinoside

451

450

317

21

Myricetin 5-O-galactoside

479

477

257,262,298,317,355

22

Proanthocyanidin A- type

592

591

285,303,421,451,465, 573

23

Procyanidin tetramer A-type

1152

1151

449,693,737,863,981

24

Procyanidin trimer

866

865

739,713,577,575,451,407,289,287

25

Quercetin 3-O-galactoside

464

463

301, 271, 255, 151

26

Proanthocyanidin B- type

594

593

556,456,449,423,303,289, 285

27

Myricetin 3-O-galactoside

479

478

257,262,298,355

28

Proanthocyanidin dimer

574

575

283,289,323,421,529, 555

29

Proanthocyanidin trimer

866

865

739,713,577,575,451,407,289, 287

285,287

a

Phenolic acids and flavonoids were detected at negative mode and anthocyanins were identified at positive mode

35



Page 36 of 40

Table 6. Content of Prominent Compound (µg/g Dried Cranberry Sample) in the Extracts of Wild Clone NL2 and Pilgrim Quantified Using HPLCESI-MS/MS

Peak No

Phenolic Compounds

Wild Clone NL2

Pilgrim

InsolubleBound

Free

Esterified

InsolubleBound

Free

Esterified

4±0.10a

-

-

3±0.90a

-

-

2 Protocatechuic

-

-

-

24±0.91a

-

24±2.19a

3 Sinapic

-

-

59±1.67a

-

-

0.05±0.00b

4 Syringic

-

-

-

3±0.87a

-

-

5 Caffeic

7±0.02c

5±0.91c

79±3.12b

-

123±4.98a

6 p-coumaric

26±0.10b

2±0.16c

245±1.66a

30±0.23b

-

5±1.16c

7 Ferulic

4±0.09b

-

39±2.12a

-

-

39±2.22a

-

31±1.41b

-

-

47±1.76a

6±0.90c

9 (+)-Catechin

5±0.24d

66±0.44b

119±5.33a

2±0.61d

34±2.64c

73±3.15b

10 (-)-Epicatechin

5±0.34e

874±42.11b

76±1.65d

30±3.23e

1798±56.29a

171±5.41c

-

112±4.92b

8±0.11d

-

157±13.46a

37±2.98c

5±0.10c

-

23±1.12b

7±0.51c

121±9.78a

-

-

-

3±0.60a

-

-

-

14 Cyanidin 3-O-arabinoside

3±0.09c

43±0.19b

4±0.72c

2±0.10c

84±9.34a

-

15 Proanthocyanidin dimer B- type

2±0.00c

336±16a

7±0.40c

-

338±11.12a

24±1.87b

Hydroxybenzoic and Hydroxycinnamic Acids 1 Gallic

8 Chlorogenic Flavonoids

11 Quercetin 12 Kaempherol hexoside 13 Luteolin 7-O-glucoside

36


Page 37 of 40


2±0.00d

74±2.30b

35±1.98c

4±0.87d

134±2.54a

-

17 Quercetin 3-O-rhamnoside

-

-

343±7.18a

-

-

-

18 Proanthocyanin trimer A-type

-

442±7.32a

196±8.23b

-

459±32.31a

22±6.75c

19 Cyanidin 3-O-galactoside

-

-

7±0.18b

-

82±4.15a

-

20 Myricetin 3-O-arabinoside

8±0.08c

150±6.75b

273±6.53a

-

166±2.79b

-

21 Myricetin 5-O-galactoside

-

-

316±3.75a

-

51±1.79b

-

22 Proanthocyanidin A- type

-

5±0.09a

6±0.92a

-

3±0.19b

-

23 Procyanidin tetramer A-type

-

15±1.31a

-

-

16±1.11a

-

24 Procyanidin trimer

-

3±0.67b

-

-

16±2.44a

-

25 Quercetin 3-O-galactoside

54±2.12 b

126±7.12 a

-

-

-

-

26 Proanthocyanidin B- type

10±0.87b

18±1.12 a

-

-

-

-

27 Myricetin 3-O-galactoside

2±0.11a

-

-

-

-

-

28 Proanthocyanidin dimer

6±0.34b

-

-

16±0.37a

-

-

29 Proanthocyanidin trimer

-

-

18±1.12a

-

16 Proanthocyanidin dimer A- type

15±1.89a

*HBA and HCA were quantified with the calibration curve of corresponding standard at 280nm, Flavonoids were quantified with the calibration curve of catechin at 280nm, Epicatechin was quantified with the calibration curve of epicatechin at 280nm

37



Page 38 of 40

a 10 b

b

8 c 6 d 4 e

2

e

0

25 20

a b

b

c d

15 10 5 0

d

b

C

1200 TEAC(µmoles of Trolox E.q./g dw)

A

12

Inhibition of TBARS formation (%)

ORAC activity (mmoles T.E /g)

Metal Chelation (µmoles of EDTA/g dw)

Figures B

a 1000

b c

800

d e

600 f

400

f

200 0

60

a

50

D

b

40 30

a

c d

d

20

e

e

10 0

Figure 1

38


Page 39 of 40


TOC Graphic

TEAC(µmoles of Trolox E.q./g of dried cranberry)

Bioactives 1200

a

1000

600

b

c

800

d

e

400 200

f

f

0

39



Page 40 of 40

40


Phenolics of Selected Cranberry Genotypes (Vaccinium macrocarpon

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