Molecules 2010, 15, 8543-8552; doi:10.
3390/molecules15128543
OPEN ACCESS
molecules
ISSN 1420-3049
[Link]/journal/molecules
Article
Characterization and Quantification of Polyphenols in Amazon
Grape (Pourouma cecropiifolia Martius)
Daise Lopes-Lutz, Judith Dettmann, Chamila Nimalaratne and Andreas Schieber *
Department of Agricultural, Food and Nutritional Science, University of Alberta, 410
Agriculture/Forestry Centre, Edmonton, AB T6G 2P5, Canada
* Author to whom correspondence should be addressed; E-Mail: Schieber@[Link];
Tel.: +1-780-492-2912; Fax: +1-780-492-4265.
Received: 26 October 2010; in revise form: 18 November 2010 / Accepted: 23 November 2010 /
Published: 26 November 2010
Abstract: The phenolic profile of Amazon grape fruit (Pourouma cecropiifolia Martius)
was investigated by high-performance liquid chromatography-electrospray ionization mass
spectrometry (HPLC-ESI-MS/MS). For this purpose, suitable extraction and liquid
chromatographic methods were developed. Anthocyanins, flavonols and chlorogenic acids
were found mainly in the peel. Besides the main anthocyanins, i.e. delphinidin 3-glucoside,
cyanidin 3-glucoside and cyanidin 3-(6”-malonyl)glucoside, several minor anthocyanins
were identified in the peel. Among these, cyanidin 3,5-diglucoside, delphinidin 3-
galactoside, cyanidin 3-rutinoside, cyanidin 3-(3”-malonyl)glucoside, malvidin 3-
glucoside, pelargonidin 3-glucoside, peonidin 3-glucoside and petunidin 3-glucoside were
characterized on the basis of their fragmentation patterns in MS/MS experiments. The total
anthocyanin content in the peel was 420.26 ± 3.07 mg kg-1 fresh weight. The pulp
contained mainly 5-O-caffeoylquinic acid (210.39 ± 3.43 mg kg-1 fresh weight). Rutin was
the predominant flavonol found in Amazon grape (peel 155.45 ± 2.06 mg kg-1 fresh weight
and pulp 2.64 ± 1.21 mg kg-1 fresh weight). Total polyphenols content was higher in the
peel than in the pulp.
Keywords: Pourouma cecropiifolia Martius; Amazon grape; anthocyanins; polyphenols;
LC-MS
Molecules 2010, 15 8544
1. Introduction
Fruits are excellent sources of bioactive components, mainly secondary plant metabolites such as
carotenoids and polyphenols. Recently, anthocyanins, along with other phenolics, have attracted much
interest since they are major antioxidants in our diet and may impart health benefits linked to their
antimicrobial, anti-inflammatory, and anticarcinogenic activities, insulin secretion ability, and
neuroprotective effects [1]. Interest in the role of antioxidants in human health has prompted research
in the field of food science to assess new sources of fruit antioxidants.
Amazon grape (Pourouma cecropiifolia Martius), which belongs to the Moraceae family, is a
tropical fruit that has a sweet and juicy flesh with a characteristic flavor which resembles Muscat grape
with a mild wintergreen mint aroma. It is the only species of Pourouma that is cultivated for the
consumption of the fruits in several countries located in the western Amazon Basin since pre-Hispanic
times and is also referred to as mapati. The dark purple spherical drupes (2-4 cm) are eaten fresh or
processed into jams, jellies, marmalades and wine. It is a dioecious tree that grows to 12 m and starts
producing fruits in the third year [2,3]. Previous reports on the fruit composition revealed that the pulp
had low acidity and high contents of sugars and minerals such as potassium, calcium and phosphorous.
The flavor composition was also investigated and the principal volatile constituents were methyl
salicylate and the oxygenated monoterpenes: cis-linalool oxide (furan isomer), trans-linalool oxide
(furan isomer), linalool, p-menth-1-en-9-al, α-terpineol and geraniol. These constituents enabled a
correlation of the mapati flavor with that of white varieties of Vitis vinifera, since the characteristic
volatiles of the latter are linalool, furanoid linalool oxides, α-terpineol, β-ionone, geraniol, nerol and
citronelol [4]. Very recent in vitro studies suggest promising cytotoxic effects of Amazon grape
extracts on several cancer cell lines, which however need to be confirmed using in vivo experiments
[5]. To obtain a more comprehensive knowledge of the composition of this tropical fruit, the aim of the
present investigation was to identify and quantify individual phenolic compounds in the peel and pulp
of Amazon grape by HPLC-DAD-ESI-MS/MS.
2. Results and Discussion
Because of the presumed benefits to human health and the trend to natural food additives including
pigments, there is a constant search for novel sources of anthocyanins. In many cases, studies are not
limited to anthocyanins but comprise also other flavonoids and phenolic acids. Using LC-ESI-MS/MS
methods, the profile of phenolic compounds in Amazon grape was characterized. The major
compounds of each class of polyphenols present were quantified by external calibration using
LC-DAD methods.
The results of Folin-Ciocalteu assay showed that the peel had the highest polyphenols content. Total
polyphenols contents were 84.66 ± 1.22 mg gallic acid equivalent in 100 g fresh peel and
8.85 ± 3.74 mg gallic acid equivalent in 100 g fresh pulp. Although this assay is usually employed to
determine “total polyphenols” it should be noted that it actually measures the reducing capacity of a
sample and, therefore, can be considered another assay for the determination of the antioxidant
activity. As a result, the Folin-Ciocalteu assay tends to overestimate the “true” total phenols content
due to interference of reducing substances such as ascorbic acid.
Molecules 2010, 15 8545
The amount of anthocyanin pigments measured in the peel was 420.26 ± 3.07 mg kg-1 fresh weight.
The identification of individual anthocyanins was accomplished by HPLC-DAD-MS/MS analysis.
From Figure 1 it can be seen that a satisfactory separation of three major anthocyanins and several
minor compounds could be achieved within 20 min. The wavelength extracted chromatogram at
520 nm showed no interfering compounds, allowing the analysis of Amazon grape anthocyanins
directly using the extract without partitioning with ethyl acetate.
Figure 1. Separation of anthocyanins extracted from Amazon grape peels by HPLC with
diode array detection (520 nm).
mAU
3 4
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11
1800
1600
1400
1200
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800
600
400 8
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200 2 9
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7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5 min
The identification of major anthocyanins (Table 1) was based on comparison of their retention times
with those of reference compounds and their elution order on reversed-phase C18 columns [6,7]. Peak
assignment was confirmed by mass spectrometry. MS2 experiments yielded product ions at m/z 287
(cyanidin), 303 (delphinidin), 317 (petunidin), 271 (pelargonidin), 301 (peonidin) and 331 (malvidin),
in most cases formed after the loss of 162 a.m.u. attributed to a hexose. CID of the [M]+ ion at m/z 611
resulted in fragments at 449 and 287, corresponding to the aglycone cyanidin and the loss of two
hexose moieties. Compounds 8 and 11 had [M]+ ions at 535 Da. CID of the molecular ion of
compound 11 led to the formation of a predominant fragment at m/z 287 and another fragment of lower
intensity at m/z 449 formed by the loss of 86 a.m.u., which can be ascribed to release of the malonyl
moiety. This fragmentation behavior is in accordance with literature data for cyanidin 3-(6”-
malonyl)glucoside; the more stable cyanidin 3-(3”-malonyl)glucoside (compound 8) yielded only a
product ion at m/z 287, showing that the acyl linkage to the 6”-position of the sugar is more labile than
the corresponding linkage to the 3”-position [8]. Furthermore, the order of elution of compounds was
Molecules 2010, 15 8546
found to be in accordance with that described previously under reversed-phase conditions [6,7].
Delphinidin 3-glucoside, cyanidin 3-glucoside and cyanidin 3-(6”-malonyl)glucoside have recently
been identified in Amazon grape [5].
Table 1. Identification and contents of anthocyanins in Amazon grape peel.
Peak tR [M]+ MS/MS Content a
Identity
no. (min) (m/z) (m/z) (mg/kg fresh wt)
1 9.62 611 449/287 cyanidin 3,5-diglucoside 1.53 ± 0.82
2 9.90 465 303 delphinidin 3-galactoside 0.56 ± 0.74
3 10.82 465 303 delphinidin 3-glucoside 104.42 ± 2.45
4 11.75 449 287 cyanidin 3-glucoside 244.57 ± 2.13
5 12.26 595 449/287 cyanidin 3-rutinoside 3.03 ± 0.85
6 12.68 479 317 petunidin 3-glucoside 0.94 ± 0.56
7 13.35 433 271 pelargonidin 3-glucoside 0.69 ± 0.54
8 13.89 535 287 cyanidin 3-(3”-malonyl)glucoside 7.10 ± 0.87
9 14.45 463 301 peonidin 3-glucoside 3.02 ± 0.13
10 15.03 493 331 malvidin 3-glucoside 4.06 ± 0.52
11 16.01 535 449/287 cyanidin 3-(6”-malonyl)glucoside 50.36 ± 2.46
a
Content calculated as cyanidin-3-glucoside equivalent.
Ethyl acetate partition is particularly recommended if other phenolic compounds need to be
subsequently analyzed by mass spectrometry. This is due to the large amount of anthocyanins in the
peel extract that could make MS assignments difficult. Also, it was necessary to develop a separate
HPLC method for the analysis of hydroxycinnamic acid derivatives and flavonols. Hydroxycinnamic
acid derivatives were the most abundant group of polyphenols in Amazon grape peel and pulp.
Chlorogenic acid (5-O-caffeoylquinic acid) was the predominant hydroxycinnamate, with contents of
685.44 ± 5.31 and 210.39 ± 3.43 mg kg-1 fresh weight in the peel and pulp, respectively. The
LC-MS/MS data showed the presence of neochlorogenic (3-O-caffeoylquinic acid), chlorogenic, 4,5-
O-dicaffeoyl quinic and 5-O-feruloyl quinic acids in Amazon grape extracts (Table 2; Figure 2).
Chlorogenic acid was identified by comparison of the retention time and MS data with a commercial
standard. Neochlorogenic acid, 5-O-feruloyl quinic acid and 4,5-O-dicaffeoylquinic acid were
identified by their elution order under reversed-phase conditions and their MS fragmentation pattern.
3-O-Caffeoyl quinic acid gave the same base peak as 5-O-caffeoylquinic acid but could be
distinguished by a more intense fragment at m/z 179 derived from caffeic acid. For 5-O-feruloyl quinic
acid, the MS2 base peak m/z 191 was derived from the cinnamic acid moiety. The identification of 4,5-
O-dicaffeoylquinic acid was based on the similarity of MS2 and MS3 spectra with those reported by
Clifford et al. [9]. The ion m/z 173 is diagnostic for substitution at position 4.
Molecules 2010, 15 8547
Table 2. Identification of polyphenols in Amazon grape peel and pulp.
Peak [M-H]-
tR (min) MS/MS (m/z) Identity Peel Pulp
no. (m/z)
1 19.93 353 191/179 3-O-caffeoyl quinic acid (neochlorogenic + +
acid)
2 22.10 289 245/205/179 catechin +
3 22.45 353 191 5-O-caffeoyl quinic acid (chlorogenic acid) + +
4 23.13 577 425 procyanidin B + +
5 24.64 289 245/205/179 epicatechin + +
6 27.72 367 191 5-O-feruloyl quinic acid + +
7 29.46 609 301 rutin + +
8 31.12 463 301 quercetin 3-galactoside + +
9 32.49 463 301 quercetin 3-glucoside + +
10 34.4 433 301 quercetin 3-xyloside +
11 35.18 433 301 quercetin 3-arabinopyranoside +
12 43.21 515 353/179/173/135 4,5-O-dicaffeoyl quinic acid + +
Figure 2. Separation of phenolic acids and other flavonoids extracted from Amazon grape
peels by HPLC with diode array detection (320 nm).
mAU
3
2800
2600
2400
2200
2000
1800
1600
1400
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10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50
min
Flavonols, flavan-3-ols and a proanthocyanidin dimer were also identified in Amazon grape
extracts. Quercetin derivatives were the characteristic flavonols found, rutin being the most abundant.
Molecules 2010, 15 8548
The rutin content in the peel was 155.45 ± 2.06 mg kg-1 fresh weight and in the pulp 2.64 ± 1.21 mg
kg-1 fresh weight. The identification of quercetin derivatives in Amazon grape was based on
comparison of their retention times and mass spectrometric data with those of pure standards of rutin,
quercetin-3-galactoside and quercetin-3-glucoside, and mango peel extract which was characterized by
Schieber et al. [10]. Catechin and epicatechin were the only free flavan-3-ols found, and their retention
times and mass spectra were compared with those of reference compounds. A proanthocyanidin dimer
was tentatively identified based on MS fragments [11].
3. Experimental Section
3.1. Chemicals
Sodium hydroxide, water (HPLC grade), methanol (HPLC grade), acetonitrile (HLPC grade),
formic acid and acetic acid were purchased from Fisher Scientific (Ottawa, ON, Canada).
Folin-Ciocalteu reagent (2N), sodium carbonate, gallic acid and polyamide for column
chromatography were supplied by Sigma–Aldrich (Oakville, ON, Canada); cyanidin 3-glucoside
chloride, cyanidin 3-rutinoside chloride and cyanidin 3,5-diglucoside chloride were obtained from
Extrasynthèse (Genay Cedex, France). Chlorogenic acid, ()-catechin, (-)-epicatechin, rutin, quercetin-
3-glucoside, and quercetin-3-galactoside were purchased from ChromaDex (Santa Ana, CA, USA). A
C18 Sep-Pak cartridge (1 g) was obtained from Waters (Milford, MA, USA).
3.2. Plant material
Ripe fruits (3 kg) were harvested in December 2009 from trees cultivated in Manaus, Amazon,
Brazil. The most uniform fruits were separated and frozen at -70 °C until analysis. The average weight
of the fruits was 10.4 g (52.3% pulp, 20.1% seed, and 7.4% peel).
3.3. Sample preparation
Frozen fruits were peeled manually using a stainless steel knife. Polyphenols were extracted by
homogenizing frozen peel (12 g) and frozen pulp (22 g) in 80% aqueous methanol containing 0.1%
formic acid (70 mL) for 30 s. The mixture was sonicated for 20 min and centrifuged for 30 min
(13,000 g, 4 °C). The samples were extracted another three times using the same procedure. For the
spectrophotometric determination of total polyphenols, the supernatants were combined, adjusted to a
defined volume, and filtered through 0.45 µm filter. For high-performance liquid chromatography
analysis, the combined supernatants were evaporated to dryness at 40 °C under vacuum. The residue
was dissolved in water containing 0.1% formic acid (pH adjusted to 1.5 with formic acid) and
transferred to a polyamide column. The resin (10 g) was eluted with aqueous 0.1% formic acid
solution (60 mL) to remove sugars and then with 0.1% formic acid in methanol (200 mL) to recover
the polyphenols. The eluate was evaporated at 40 °C under vacuum to remove the organic solvent and
the residue dissolved in water containing 0.1% formic acid. A 10 µL aliquot was injected for HPLC
analysis of anthocyanins [7].
Molecules 2010, 15 8549
For the analysis of non-anthocyanin polyphenols, the extraction was performed as described above
and the residue obtained after evaporation of methanol was dissolved in water containing 0.1% formic
acid (pH adjusted to 1.5 with formic acid). The aqueous solution was extracted four times with 50 mL
of ethyl acetate and the organic phase was evaporated to dryness. The residue was dissolved in water
and the pH adjusted to 7 with 1 N NaOH solution. It was applied to a C18 Sep-Pak cartridge (1 g).
After washing with water containing 0.1% formic acid (10 mL), the polyphenol fraction 1 was
collected. Polyphenol fraction 2 was recovered after eluting the C18 Sep-Pak cartridge with ethyl
acetate. Both fractions were evaporated to dryness using a rotary evaporator. The residue was
dissolved in 80% aqueous methanol (0.1% formic acid) [8].
3.4. Determination of total phenols by the Folin-Ciocalteu assay
The total phenols content of the extracts was measured using a modified colorimetric Folin-
Ciocalteu assay as described by Singleton et al. [12]. One mL of diluted fruit extract and 1 mL of
Folin-Ciocalteu reagent were transferred to a 100 mL volumetric flask. After 3 min, a 20% aqueous
solution of sodium carbonate (Na2CO3, 10 mL) was added, and the flask was made up to volume with
distilled water. The absorbance at 765 nm was measured after 1 h, and the measurement was compared
to a standard curve of gallic acid. Concentrations were expressed as milligrams of gallic acid
equivalents per 100 g of fresh weight ± SD for triplicate fruit extracts.
3.5. HPLC analysis of anthocyanins
Anthocyanin analysis was conducted using a 1200 Series Agilent Technologies HPLC (Agilent,
Palo Alto, CA, USA) which was equipped with a model G1315D diode array detector (DAD), a model
G1379B degasser, a model G1312A binary gradient pump, a model G1329A thermoautosampler and a
model G1316A column oven. The reversed-phase separation was performed on a 250 mm × 4.6 mm
i.d. Symmetry C18 column, particle size 5 µm (Waters, MA, USA) with a Nova-Pak 4 µm C18 guard
column 3.9 × 20 mm operating at 35 °C and at a flow rate of 1.0 mL/min. The compounds were
separated with gradient elution of (A) 4.5% aqueous formic acid and (B) 80% acetonitrile in solution
A. The gradient program was: 0-15% B (9 min), 15-45% B (22 min), 45-100% B (6 min), 100-0% B
(1 min), 2 min 0% B. The injection volume was 10 uL. Monitoring was performed at 520 nm and the
diode array detector was set at an acquisition range from 200 nm to 700 nm at a spectral acquisition
rate of 1.25 scans s-1 (peak width 0.2 min). Quantification was conducted by LC-DAD with external
calibration using a set of seven standard dilutions of cyanidin-3-glucoside at 520 nm. A stock solution
of 1 mg/mL in 80% methanol in aqueous 0.1% formic acid was prepared and stored at -20 °C. The
calibration curves were linear over the range of 1 to 200 µg/mL with a correlation coefficient of
≥ 0.99. For quantification, peak areas were correlated with concentrations in accordance with the
calibration curves. Data are reported as means ± standard deviations of triplicate independent analyses.
3.6. HPLC analysis of phenolic acids and other flavonoids
The analysis of phenolic acids and flavonols was conducted with a 1200 Series Agilent
Technologies HPLC (Agilent, Palo Alto, CA, USA) equipped with a model G1315D diode array
Molecules 2010, 15 8550
detector (DAD), a model G1379B degasser, a model G1312A binary gradient pump, a model G1329A
thermoautosampler and a model G1316A column oven. The reversed-phase separation was performed
on a 250 mm × 4.6 mm i.d. Luna C18(2) column, particle size 5 µm (Phenomenex, Torrance, CA,
USA) with an AQ 4 × 20 mm C18 precolumn (Phenomenex) operating at 20 °C and at a flow rate of
0.5 mL/min. The compounds were separated with gradient elution of (A) 2% aqueous acetic acid
solution and (B) 0.5% aqueous acetic acid solution and acetonitrile (50:50, v/v). Samples were eluted
with the following gradient: 0% B (5 min), 0-40% B (10 min), 40-60% B (40 min), 60-80% B
(10 min), 80-100% B (10 min), 100% B (30 min), and 100-0% B (2 min). The injection volume was
20 uL. Monitoring was performed at 280 and 320 nm and the diode array detector was set at an
acquisition range from 200 nm to 700 nm at a spectral acquisition rate of 1.25 scans s-1 (peak width
0.2 min). Quantification was performed by LC-DAD using external calibration with a set of seven
standard dilutions of rutin at 320 nm and chlorogenic acid at 280 nm. Data acquisition, peak
integration, and calibrations were performed with Agilent Chemstation software. A stock solution of
1 mg/mL in 100% methanol was prepared and stored at -20 °C. The calibration curves were linear over
the range of 1 to 500 µg/mL with a correlation coefficient of ≥ 0.99. For quantification, peak areas
were correlated with concentrations in accordance with the calibration curves. Data are reported as
means ± standard deviations of triplicate independent analyses.
3.7. HPLC-ESI-MS/MS determination of individual polyphenols
The chromatographic system consisted of an Agilent 1200 Series HPLC unit comprising a degasser,
binary pump, autosampler, thermostated column compartment, and diode array detector (Agilent
Technologies, Palo Alto, CA, USA) connected to a linear ion trap mass spectrometer 4000 QTRAP
system (AB Sciex, ON, Canada) which was equipped with an ESI Turbo V™ source. The reversed-
phase separation of anthocyanins was performed on a 250 mm × 4.6 mm i.d. Symmetry C18 column,
particle size 5 µm (Waters, MA, USA) with a Nova-Pak C18 guard column 4 µm 3.9 × 20 mm using
the same conditions as described above.
For the HPLC-MS analysis of anthocyanins, the effluent from the column after the DAD detector
was directly introduced into the electrospray ion source (ESI). The tuning of MS instrument
parameters was performed by compound optimization using a 100 ng/mL solution of cyanidin 3-
glucoside dissolved in 80% methanol/20% formic acid (0.1%) solution. The optimization of ion source
parameters was conducted using a flow rate of 1.0 mL/min. High-purity nitrogen gas (99.995 %) was
used as the nebulizing (GS1) and heating gas (GS2). The values for optimum spray voltage, source
temperature, GS1, GS2 and curtain gases were +3.0 kV, 600 °C, 40, 40 and 20 psi, respectively.
An information-dependent acquisition (IDA) method, EMS → 4 EPI, was used to profile the
anthocyanins in positive mode. The spectra were obtained over a range from m/z 50 to 1300 in 1 s. LIT
fill time was set at 20 ms. The IDA threshold was set at 100 cps, above which enhanced product ion
(EPI) spectra were collected from the eight most intense peaks. The EPI scan rate was 4000 amu s-1.
Collision-induced dissociation (CID) spectra were acquired using nitrogen as the collision gas under a
collision energy of 20 eV (CES15). The other MS parameters used were as follows: declustering
potential (DP), 96 V; entrance potential (EP), 10V; and collision exit potential (CXP) 3V.
Molecules 2010, 15 8551
The separation of the non-anthocyanin phenolic compounds was performed on a 250 mm × 4.6 mm
i.d. Luna C18(2) column, particle size 5 µm (Phenomenex, Torrance, CA, USA), using the same
conditions described above. The mass spectra were recorded in negative mode; the flow rate was
maintained at 0.5 mL/min with the pneumatically assisted electrospray probe using high-purity
nitrogen gas (99.995%) as the nebulizing (GS1) and heating gas (GS2). The values for optimum spray
voltage, source temperature, GS1, GS2, and curtain gases were -4 kV, 600 °C, and 50, 30, and 25 psi,
respectively. An information-dependent acquisition (IDA) method, EMS → 4 EPI, was used to
identify phenolic compounds. Both Q1 and Q3 were operated at low and unit mass resolution. The
spectra were obtained over a range from m/z 50 to 1300 in 1 s. LIT fill time was set at 20 ms. The IDA
threshold was set at 100 cps, above which enhanced product ion (EPI) spectra were collected from the
eight most intense peaks. The EPI scan rate was 4000 amu s-1. Collision-induced dissociation (CID)
spectra were acquired using nitrogen as collision gas under a collision energy of -20 eV. The other MS
parameters were as follows: declustering potential (DP), -70 V; entrance potential (EP), -10V; and
collision exit potential (CXP), -7V. Data acquisition was interfaced to a computer workstation Analyst
1.5 (Applied Biosystems, CA, USA).
4. Conclusions
This study clearly shows that LC-ESI-MS/MS is a powerful technique enabling fast separation and
characterization of polyphenols reported herein for the first time in Amazon grape. The high sensitivity
of this hyphenated technique allowed expanding previous characterization of Amazon grape to
additional eight minor anthocyanins, four quercetin glycosides, catechin, epicatechin, procyanidin B
and four hydroxycinnamic acid derivatives. In this study, only peel and pulp samples were investigated
for their profile of phenolic compounds. It would be desirable to characterize also the polyphenols
present in the seeds, which constitute approximately 20% of the total fruit weight and are by-products
of processing, to assess their potential as a source of natural bioactive components.
Acknowledgements
A.S. acknowledges funding from the Research Chairs of Canada. This research was funded, in part,
by the Natural Sciences and Engineering Research Council of Canada (NSERC).
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Sample Availability: No Available.
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