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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
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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,
11
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
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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
26
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.
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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
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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.
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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
58
market samples, mature/ripe (burgundy colour) and immature (red colour) fruits. Recognition of
59
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
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(‘Prolific' × 'McFarlin') is a hybrid developed by a cooperative cranberry breeding program in the
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USA, each Canadian wild clone represented a single plant selected from the wild based on vigour,
70
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-
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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
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Dynamics Co. of America, New Hartford, CT, USA) and kept frozen at -20ºC until used within one
84
week for the extraction of phenolics.
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Extraction of phenolics
86
Phenolic compounds (free, esterified, and insoluble-bound) were extracted and fractionated as
87
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).
90
Following centrifugation at 4000 ×g, the supernatants were collected and the procedure was carried
91
out twice. Combined supernatants were evaporated under vacuum at 40 °C and then freeze dried.
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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
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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.
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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
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determinations
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Determination of total flavonoid content
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Total flavonoid content of cranberry samples was determined by the colourimetric method described
106
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
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extract was diluted in 0.025 M potassium chloride and 0.4M acetate buffer to adjust the pH to 1 and
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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)
117
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)
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radical cation (ABTS•+)12. The sample solution was mixed with 1.96 mL of ABTS•+ solution
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(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
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values calculated using following equation.
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TEAC values were determined as follows: ∆A
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∆A
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absorbance at a given time, m= slope of the standard curve, [trolox] = concentration of trolox, and d=
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dilution factor.
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DPPH radical scavenging capacity (DRSC) using electron paramagenetic resonance (EPR)
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spectrometry
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The DRSC assay was carried out according to the method explained by Madhujith and Shahidi13 with
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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
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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
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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
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(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
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1% (w/v) solution of potassium ferricyanide [K3Fe(CN)6]; the mixture was then incubated in a water
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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=
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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
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read spectrophotometrically at 700 nm and results were expressed as micromoles of trolox
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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
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e-scan EPR spectrophotometer. The results were expressed as micromoles of catechin equivalents
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(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
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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
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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
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filter (0.45-µm) before injection into a reverse phase C18 column (250 mm length, 4.6 mm i.d., 5 µm
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particle size, Sigma-Aldrich Canada Ltd.) with a guard column and the column temperature was 25
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°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
193
collision induced dissociation. The scan range set was from m/z 50 to 2000, using smart parameter
194
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
198
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
202
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
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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
211
GAE/g dw) > Wild clone NL2 (~2.38 mg GAE/g dw) > NL3 (~1.69 mg GAE/g dw) > PE1 (~1.52
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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
216
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
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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
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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
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phenolic compounds by synthesizing different quantities and types of phenolics.2 10
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Total flavonoid content (TFC)
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Flavonoids exhibit a variety of biological activities both in vitro and in vivo.24 In this study, Pilgrim
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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
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highest amounts of flavonoids in all tested genotype varieties compared to the esterified and the
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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
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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
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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
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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
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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
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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
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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
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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
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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
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Page 22 of 40
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597 (42) Shahidi, F.; Naczk, M. Food Phenolics: Sources, Chemistry, Effects, Applications. Technomic 598
Publishing Company, Lancaster, PA, 1995, pp 1-331.
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599 (43) Zheng, Z.; Shetty, K. Solid-state bioconversion of phenolics from cranberry pomace and role of 600
Lentinus edodes ß-glucosidase. J. Agric. Food Chem. 2000, 48, 895-900.
601
(44) Velioglu,Y. S.; Ekici, L.; Poyrazoglu, E. S. Phenolic composition of European cranberrybush
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(Viburnum opulus L.) berries and astringency removal of its commercial juice. Int J Food Sci
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Tech. 2006, 41, 1011-1015.
604 (45) Chen, H.; Yuegang Zuo, Y.; Deng, Y. Separation and determination of flavonoids and other 605
phenolic compounds in cranberry juice by high-performance liquid chromatography. J.
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Chromatogr. A. 2001, 913, 387-395.
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
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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
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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.
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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.
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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.
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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)
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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
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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
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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
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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
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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
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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
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