Article

pubs.acs.org/JAFC

Characterization of Phenolic Compounds and Antioxidant Activity of

Solanum scabrum and Solanum burbankii Berries
J. Oszmiański, J. Kolniak-Ostek, and A. Wojdyło*
Department of Fruit, Vegetable and Grain Technology, Wroclaw University of Environmental and Life Sciences, 37/41
Chełmońskiego Street, 51 630 Wroclaw, Poland

ABSTRACT: The purpose of this research was to quantify and characterize phenolic compounds and to measure the
antioxidant activity of Solanum scabrum and Solanum burbankii berries. The antioxidant activity of Solanum berry extracts was
assayed by electrochemical and spectrophotometric methods, whereas liquid chromatography (LC)/quadrupole time-of-flight
mass spectrometry and ultra-performance LC-photodiode detector were used for identification and quantification of their
polyphenols. Eighteen phenolic compounds were identified in these fruits. The presence of seven phenolic acid derivatives and
two flavonols was reported for the first time. In both cultivars, the major compound was found to be anthocyanin petunidin-3-(p-
coumaroyl-rutinoside)-5-O-glucoside. Additional anthocyanins in S. burbankii and S. scrabum berries were characterized as
petunidin, delphinidin, and malvidin with the same glycosidic substitution pattern and acylation with p-coumaric and ferulic acids.
S. scabrum was richer in phenolic compounds, especially anthocyanins, and was characterized by more powerful antioxidant
activity than S. burbankii.
KEYWORDS: UPLC-PDA−Q/TOF-MS, identification, Solanaceae, phenolic acid, flavonols, acylated antocyanins,
antioxidant activity, electrochemical detector

■ INTRODUCTION
Solanaceae are a family that includes a number of toxic and
edible plants. The name of the family originates from the Latin
Solanum, “the nightshade plant”.1 Most likely, the name comes
from the perceived resemblance that some of the flowers bear
to the sun and its rays, and in fact, a species of Solanum
(Solanum nigrum) is known as the “sunberry”. Solanaceae
species are often rich in alkaloids the toxicity of which to
humans and animals ranges from mildly irritating to fatal in
small quantities.1 In most parts of the world, particularly in
Europe and North America, these species are considered to be
troublesome weeds of agriculture, but in many developing
countries, they constitute a minor food crop, with the shoots
and berries not only being used as vegetables and fruits, but
also for various medicinal and local uses. Some of species,
however, are often considered to be edible forms, and
consumption of their leaves and fruits in man’s diet is
widespread, particularly in Africa and SE Asia.3 In Poland and
other countries of Eastern Europe two Solanum species are
well-known and cultivated, namely, Solanum scabrum and
Solanum burbankii, which are consumed fresh and as home-
made products in the form of juices, sauces, and jams.
S. scabrum is relatively easy recognizable by its large-sized
leaves, strong stem with distinct dented wings, and many-
seeded fruits 15−17 mm in diameter that do not drop at
maturity. It is additionally characterized by vigorous growth and
easy cultivation.1,2 The berry is green when immature and
purple to purplish black when ripe, with a high concentration of
anthocyanins. Therefore, these anthocyanin pigments are used
as colorants in fruit juices and apple sauce. Additionally, S.
scabrum is a widely cultivated species and is used as a leafy
vegetable, as a medicinal plant, and as a source of ink dye.3
The second investigated berry is S. burbankii, being an
interspecies nightshade hybrid developed by Luther Burbank.
Its berries are tiny, smaller (approximately 0.5 cm in diameter)
than those of S. scabrum, with a different flavor and sweet taste.
The low-growing plant hides its berries well under the foliage
and is a bit more difficult to harvest than other edible
nightshades. They are good fresh but even better when
sweetened and cooked. Short plants produce good yields rather
quickly. Similarly to S. scabrum, S. burbankii is very rich in
anthocyanin. Its fruits are widely used for the preparation of
pies, jams, and sauces, with an intense purple color.4,5
Some of the anthocyanin composition of the S. scabrum and
S. burbankii berries has already been established;2 however,
these authors did not identify other phenolic compounds
presented in these fruits, i.e., quercetin and hydroxycinnamic
acid derivatives. The major pigments in the berries of this
species were anthocyanidins, especially petunidin-3-(p-cou-
maroyl-rutinoside)-5-O-glucoside (93%). Seven other pigments
included delphinidin-3-O-rutinoside-5-O-glucosides acylated
with p-coumaric, ferulic, and an unidentified acid; malvidin-3-
O-rutinoside-5-O-glucosides acylated with p-coumaric and
ferulic acid; and petunidin-3-O-rutinoside-5-O-glucoside acy-
lated with two p-coumaric acid moieties. Over 97% of the
anthocyanins present in S. scabrum are acylated with cinnamic
acids. In other fruits belonging to the family of Solanum, i.e., in
Solanum quineese, Francis et al.1 identified also petunidin-3-(p-
coumaroyl-rutinoside)-5-O-glucoside, as the major pigment,
and 3-O-rutinoside-5-O-glucosides of petunidin or malvidin.
Received: July 18, 2013
Revised: January 27, 2014
Accepted: January 29, 2014
Published: February 7, 2014

© 2014 American Chemical Society 1512 dx.doi.org/10.1021/jf4045233 | J. Agric. Food Chem. 2014, 62, 1512−1519
Journal of Agricultural and Food Chemistry
Spectral inspection indicates that there is a considerable
number of interfering compounds that absorb in the UV−vis
region. These compounds make it difficult to confirm the
identical nature of the individual pigment peaks.
Anthocyanins are able to capture free radicals by donation of
phenolic hydrogen atoms.10,11 Acylation of the anthocyanin
molecule improves their stability through intramolecular and/
or intermolecular copigmentation and self-association reactions.
Therefore, sources of acylated anthocyanins of the S. scabrum
and S. burbankii berries may ensure the desirable stability for
food applications. The anthocyanins with acylating substituents
are more stable during processing and storage than the
unacylated pigments.
The antioxidative activity of phenolic compounds, especially
anthocyanin, was studied by different chemical methods. The
Folin−Ciocalteu method, the oxygen radical absorbance
capacity (ORAC) assay, and the Trolox equivalent antioxidant
capacity (TEAC) assay are the most considered methods.
Kilmartin et al.13 developed an electrochemical method for the
characterization of the antioxidant properties of wine and wine
phenolics. Recently, Janeiro et al.14 studied the redox behavior
of anthocyanins by voltammetric techniques and found that all
the phenolic hydroxyl groups can be oxidized electrochemically.
Also differences in electroactive substituents on analogous
structures can lead to characteristic differences in their
voltammetric behavior.15 Pereira at al.16 studied the effects of
substitution for the hydroxyl group at position C4′ in the B-ring
of the flavylium cation, which indicated that the para-position of
the ring B is important for the increase in the delocalization of
π-electrons in the chromophore. Electrochemical studies reveal
general trends in the electron-donating abilities of flavonoids.
Cren-Olivé at al.17 shown the importance the catechol group in
the B-ring, which is more easily oxidizable than the resorcinol
group in the A-ring. They indicated that the 4′-OH is the group
that deprotonates preferentially in the flavylium cation of
cyanidin and cyanidin-3-O-glucoside.17
The purpose of this research was to tentatively identify and
quantify phenolic compounds, main flavonoids, phenolic acids,
and anthocyanins for better characterization of S. scabrum and
S. burbankii berries. Additionally, the study was aimed at
monitoring the changes in the antioxidant activity by
spectrometric methods (ABTS and FRAP assay) and an
electrochemical technique, cyclic voltammetry. These rapid
analytical techniques are very useful in determining the
antioxidant activity, which is a consequence of the ability of
an antioxidant to donate electrons around the potential of the
anodic wave.
■
MATERIALS AND METHODS
Reagents and Standard. Acetonitrile and methanol for UPLC
and LC/MS purity, formic acid, leucine enkephalin, 2,2-azinobis(3-
ethylbenzothiazoline-6-sulfonic acid (ABTS), 6-hydroxy-2,5,7,8-tetra-
methylchroman-2-carboxylic acid (Trolox), 2,4,6-tri(2-pyridyl)-s-tria-
zine (TPTZ), ammonium formate, and methanol were purchased from
Sigma-Aldrich (Steinheim, Germany). Quercetin-3-O-glucoside and
-3-O-rutinoside; peonidin, delphinidin and petunidin aglycon; p-
coumaric acid; and sinapic acid were purchased from Extrasynthese
(Lyon, France). Chlorogenic acid, neochlorogenic acid, and
cryptochlorogenic acid were purchased from TRANS MIT GmbH
(Giessen, Germany).
UPLC-grade water, prepared by using an HLP SMART 1000s
system (Hydrolab, Gdańsk, Poland), was additionally filtered through
a 0.22 μm membrane filter immediately before use.
1513
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Plant Material. S. scabrum and S. burbankii berries were collected
in 2011 from the Garden of Medicinal Plants herbarium at the Medical
University in Wroclaw, Poland, by cultivation in the University’s
experimental field. The fruits were harvest successively during 10 days
and after harvest, the berries (100 g per day, finally 1 kg) were
immediately frozen and stored at −20 °C until used for analysis.
Extraction Procedure. The extraction procedure of polyphenols
was carried out as described previously by Oszmiański et al.18 The
frozen berries were quick thawed in a microwave oven and heated only
to 20 °C and then directly crushed in a laboratory mill (IKA A11).
This extraction procedure was applied because use of the microwave
oven was previously found to be the best extraction method when
berries were exposed for 1 min. Polyphenols were isolated from berries
by maceration with water containing 200 mg/L SO2 and subsequently
extracted in a ultrasonic bath at 20 °C for 15 min. The ratio of this
solvent to berries was 10:1 (v/v). The homogenized mixture was then
centrifuged for 5 min (4000 rpm; MPW 360R; Warsaw, Poland), and
the extraction was repeated twice. The experiment were performed in
triplicate.
Four milliliters of the extract was passed through a C18 Sep-Pak
cartridge (360 mg/0.7 mL; Waters) preconditioned with 5 mL of ethyl
acetate, 5 mL of methanol, and 5 mL of 0.01 N HCl. The loaded
cartridge was washed with 3 mL of 0.01% HCl and then the cartridge
was dried with a flow of nitrogen gas for 3 min. Phenolic compounds
other than anthocyanins were eluted with 20 mL of ethyl acetate. The
solvent was removed using a rotary vacuum evaporator at 20 °C, and
the phenolic residue was dissolved in 20% acetonitrile for UPLC-
PDA−MS analysis. Anthocyanins adsorbed in the Sep-Pak C18 were
eluted with methanol containing 0.5% HCOOH, and the anthocyanin
fraction was analyzed by the UPLC-PDA−MS method.
Absorption Spectra of Berry Extracts. Berry extract was used
for measurement of absorption spectra, which were recorded in the
visible wavelength range from 450 to 600 nm. These spectra were
measured using a PC 2401 UV−vis spectrophotometer (Shimadzu;
Tokyo, Japan) in a 10-mm path length cell, with buffer solutions as a
reference. Buffer solutions of two different pH values (pH 2.5 and 7.5)
were prepared by mixing 50 mM ammonium formate with acetonitrile
(50/50; v/v). pH was adjusted with ammonium hydroxide. All
experiments were repeated in triplicate.
Identification of Polyphenols by the UPLC-PDA−Q/TOF-MS
Method. Identification of polyphenol of S. scabrum and S. burbankii
berry extracts was carried out using an ACQUITY ultra-performance
LC system (UPLC) with binary solvent manager (Waters Corp.;
Milford, MA) and a Micromass Q-Tof Micro mass spectrometer
(Waters; Manchester, UK) equipped with an electrospray ionization
(ESI) source operating in negative and positive mode. Separations of
individual polyphenols were carried out using a UPLC BEH C18
column (1.7 μm, 2.1 × 100 mm, Waters Corp.; Milford, MA) at 30 °C.
Samples (10 μL) were injected, and elution was completed in 15 min
with a sequence of linear gradients and isocratic flow rates of 0.45 mL
min−1. The mobile phase was composed of solvent A (4.5% formic
acid, v/v) and solvent B (100% acetonitrile). The program began with
isocratic elution with 99% A (0−1 min), and then a linear gradient was
used until 12 min, lowering A to 0%; from 12.5 to 13.5 min, the initial
composition (99% A) was used and then held constant to re-
equilibrate the column. Analysis was carried out using full scan, data-
dependent MS scanning from m/z 100 to 1500. Leucine enkephalin
was used as the internal reference compound during ESI-MS accurate
mass experiments and was permanently introduced via the LockSpray
channel using a Hamilton pump. The lock mass correction was ±1.000
for the mass window. All quadrupole time-of-flight mass spectrometry
(Q/TOF-MS) chromatograms are displayed as base peak intensity
(BPI) chromatograms and scaled to 12 400 counts per second (cps)
(=100%). The effluent was led directly to an electrospray source with a
source block temperature of 130 °C, desolvation temperature of 350
°C, capillary voltage of 2.5 kV, and cone voltage of 30 V. Nitrogen was
used as desolvation gas at flow rate 300 L h−1.
The characterization of the single components was carried out via
the retention time and the accurate molecular masses. Each compound
was optimized to its estimated molecular mass [M − H]− in the
dx.doi.org/10.1021/jf4045233 | J. Agric. Food Chem. 2014, 62, 1512−1519
 Table 1. Qualitative and Quantitative (mg/100 g dm) Characterization of S. scabrum and S. burbankii Phenolic Compound Profiles with UPLC-PDA−MS/MSa
peak compd tR (min) λmax (nm) [M + H]+/[M − H]− (m/z) and MS/MS fragments in ESI-MS-Q-TOF S. burbankii S. scabrum
−
1 3-caffeoylquinic acid 2.27 323 [M − H] 353.0870 (100%) 0.83 ± 0.03 0.82 ± 0.04
191.0555 (100%)/173.0446 (43%)/135.0452 (62%)
2 delphinidin-3-O-rutinoside-5-O-glucoside 3.33 525 [M + H]+ 773.2146 (100%) 0.24 ± 0.01 1.37 ± 0.08
611.1609 (26%)/465.1017 (21%)/303.0496 (100%)
3 5-caffeoylquinic acid 3.61 324 [M − H]− 707.1823 (100%) 47.64 ± 2.47 7.81 ± 1.50
353.0875 (88%)/191.0558 (100%)
4 4-caffeoylquinic acid 4.16 324 [M − H]− 353.0879 (100%) 0.29 ± 0.10 0.20 ± 0.04
191.0555 (84%)/173.0449 (100%)
5 petunidin-3-O-rutinoside-5-O-glucoside 4.43 529 [M + H]+ 787.2300 (100%) 2.11 ± 0.26 7.80 ± 1.07
625.1765 (35%)/479.1188 (25%)/317.0671 (100%)
6 malonyl-caffeoylquinic acid 4.66 325 [M − H]− 439.0872 (100%) 15.95 ± 0.85 14.24 ± 1.51
353.0879 (20%)/233.0660 (100%)/191.0555 (32%)/173.0449 (20%)
7 malvidin-3-O-rutinoside-5-O-glucoside 5.53 526 [M + H]+ 801.2440 (100%) 2.56 ± 0.41 ndb
639.1934 (42%)/493.1325 (30%)/331.0814 (100%)
Journal of Agricultural and Food Chemistry

8 malonyl-caffeoylquinic acid 5.76 325 [M − H]− 439.0872 (100%) 6.10 ± 1.20 5.67 ± 1.01
395.0979 (29%)/353.0879 (20%)/233.0665 (97%)/191.0555 (25%)/173.0453 (100%)
9 acetylo-p-coumaroylquinic acid 6.24 310 [M − H]− 379.1031 (100%) 0.44 ± 0.04 0.38 ± 0.05
319.0818 (31%)/163.0386 (245%)/145.0296 (46%)
10 quercetin-3-O-rutinoside 8.30 352 [M − H]− 609.1432 (100%) 4.96 ± 0.87 1.62 ± 0.11
301.0277 (86%)

1514
11 delphinidin-3-O-p-coumaroyl-rutinoside-5-O-glucoside 8.37 528 [M + H]+ 919.2509 (100%) 2.37 ± 0.12 0.24 ± 0.07
757.1982 (70%)/465.1034 (46%)/303.0496 (100%)
12 quercetin-3-O-glucoside 8.44 351 [M − H]− 463.0887 (100%) 1.69 ± 0.15 1.68 ± 0.13
301.0277 (100%)/173.0447 (30%)
13 delphinidin-3-O-feruoyl-rutinoside-5-O-glucoside 8.89 529 [M + H]+ 949.2607 (100%) 7.71 ± 0.64 21.08 ± 0.68
787.2077 (85%)/465.1032 (55%)/303.0496 (100%)
14 sinapoyl malic acid 9.10 328 [M − H]− 339.0716 (100%) 0.98 ± 0.06 0.84 ± 0.07
223.0606 (100%)/149.0241 (57%)
15 petunidin-3-O-p-coumaroyl-hexoside-5-O-hexose 9.30 531 [M + H]+ 933.2642 (100%) 63.26 ± 5.75 156.87 ± 3.87
771.2128 (99%)/479.1187 (59%)/317.0671 (100%)
16 petunidin-3-O-feruoyl-hexoside-5-O-hexose 9.82 532 [M + H]+ 963.2780 (100%) 0.91 ± 0.17 13.67 ± 1.17
801.2242 (100%)/479.1100 (87%)/317.0671 (86%)
17 malvidin-3-O-p-coumaroyl-hexoside-5-O-hexose 10.28 532 [M + H]+ 947.2829 (100%) 2.66 ± 0.11 3.82 ± 0.66
785.2296 (99)/493.1341 (55%)/331.0814 (100%)
18 malvidin-3-O-feruoyl-hexoside-5-O-hexose 10.75 532 [M + H]+ 977.2892 (100%) 0.24 ± 0.04 1.37 ± 0.11
815.2397 (100%)/613.3407 (86%)/493.1344 (54%)/331.0814 (97%)
total phenolics (mg/100 g) 160.95*** 239.35***
a
Results represent mean values of triplicated determinations ± standard deviation (p < 0.001). bnd: not detected.
Article

dx.doi.org/10.1021/jf4045233 | J. Agric. Food Chem. 2014, 62, 1512−1519

Journal of Agricultural and Food Chemistry Article

Figure 1. UPLC profile at 320 nm (phenolic acid and quercetin derivative) and 520 nm (anthocyanins) of Solanum berry extracts. For abbreviations
of the peak labels, see Table 1

1515 dx.doi.org/10.1021/jf4045233 | J. Agric. Food Chem. 2014, 62, 1512−1519

Journal of Agricultural and Food Chemistry
negative and positive mode before and after fragmentation. The data
obtained from UPLC/MS were subsequently entered into the
MassLynx 4.0 ChromaLynx Application Manager software. On the
basis of these data, the software is able to scan different samples for the
characterized substances.
The runs were monitored at the following wavelengths:
hydroxycinnamates at 320 nm, flavonol glycosides at 360 nm, and
anthocyanins at 520 nm. Photodiode detector (PDA) spectra were
measured over the wavelength range 200−600 nm in steps of 2 nm.
Retention times and spectra were compared with those of pure
standards within 200−600 nm. Calibration curves were run for the
external standards 3-, 4-, and 5-O-caffeoylquinic acids, p-coumaric acid,
quercetin-3-O-rutinoside and -3-O-glucoside, peonidin-3-O-rutinoside,
delphinidin-3-O-rutinoside, and petunidin-3-O-rutinoside at concen-
trations ranging from 0.05 to 5 mg/mL. Amounts of 3-, 4-, and 5-O-
caffeoylquinic acids were calculated for the quantification of the
neochlorogenic, cryptochlorogenic, and chlorogenic acids and their
isomers. The concentrations of p-coumaric and sinapic acid containing
compounds were calculated by using p-coumaric and sinapic acid,
respectively. The concentrations of delphinidin-, petunidin-, and
malvidin-containing compounds were calculated by using delphinidin,
petunidin, and malvidin aglycons, respectively. Obtained results was
express as milligrams per 100 g of dry matter (dm).
Analysis of Antioxidant Activity. The protocols for analysis were
prepared as described previously by Wojdyło et al.19 The ABTS•+
activity of the sample was used for measurement of antioxidant
activity. The total antioxidant potential of the sample was determined
using a ferric reducing ability of plasma (FRAP) assay. For all analyses,
a standard curve was prepared using different concentrations of
Trolox. All determinations were performed in triplicate using a
Shimadzu UV-2401 PC spectrophotometer (Kyoto, Japan). The
results were corrected for dilution and are expressed in millimole of
Trolox per 100 g dm.
Cyclic Voltammograms. The mixture of 50 mM ammonium
formate (pH 7.4) and acetonitrile (50/50, v/v) was prepared as the
supporting electrolyte. All experiments were performed on a ROXY
EC system (Antec; Zoeterwoude, The Netherlands). Electrochemical
conversions were accomplished in an electrochemical thin-layer cell
(ReactorCell, Antec). The reactor cell consisted of a three-electrode
arrangement including a working electrode, an auxiliary electrode, and
a reference electrode. Glassy carbon was used as working electrode
material. The accessible area of the working electrode was 15.1 mm2.
The inlet block of the cell was employed as auxiliary electrode and the
HyREF (Antec) electrode was used as reference electrode. The
working electrode and the auxiliary electrode inlet block were
separated by a 50-μm spacer, giving a cell volume of approximately
750 nL. Potentials (0−3000 mV) were applied using a purposive
potentiostat (ROXY potentiostat, Antec). Shredded S. scabrum and S.
burbankii berry extracts (∼2.5 g) were mixed with 3 mL of ammonium
formate and then centrifuged for 10 min at 15 000 rpm (20.878g;
MPW-360R; Warsaw, Poland). Prior to each run, the surface of the
glassy carbon electrode was freshly abraded with 12 μm flattening plate
and rinsed with redistilled water and methanol. The scan was taken in
the potential range between 0 and 3000 mV with a scan rate 50 mVs−1.
Samples were pumped through the cell at a flow rate of 5 μL/min by a
syringe pump model 74900 (Cole Parmer; Vernon Hills, IL). Cyclic
voltammograms were also recorded for 0.25 mM cyanidin, malvidin,
and pelargonidin aglycon standards solutions.
■
RESULTS AND DISCUSSION
Characterization and Quantification of Phenolic
Compounds. The data for retention times (tR), wavelengths
of maximum absorbance (λmax), protonated/deprotonated
molecules ([M + H]+/[M − H]−), diagnostic fragments, and
identification for each peak of the phenolic compound detected
in the S. scabrum and S. burbankii berry extracts are listed in
Table 1. The compounds were characterized according to their
retention time at 320 nm for phenolic acid derivatives, 360 nm
1516
Article
for quercetin derivatives, and at 520 nm for anthocyanins and
are shown in Figure 1, respectively.
Eighteen phenolic acid derivatives and flavonoids were
detected in the S. scabrum and S. burbankii berry extracts.
Among them, there were three nonacylated anthocyanin
hexosides and six acylated anthocyanin hexosides.
Nine compounds other than antocyanin phenolic ones were
tentatively identified in the S. scabrum and S. burbankii berry
extracts for the first time. Among them, there were seven
phenolic acid derivatives and two flavonol hexosides.
Chlorogenic acid (5-caffeoylquinic acid, compound 1) and its
isomers were found at different tR, showing the deprotonated
molecule [M − H]− (m/z 353) and the ion corresponding to
the deprotonated quinic acid (m/z 191) in the full scan mode.
Reference substances neochlorogenic acid (3-caffeoylquinic
acid, compound 3) and cryptochlorogenic (4-caffeoylquinic
acid, compounds 4) were confirmed by injection of standards
and the subfractions in neutral loss of m/z 162, which also
corresponds to the loss of a caffeic acid unit. Several isomers of
chlorogenic acid have been found in other plant sources.20,21
Compounds 6 and 8 possessing two malonyl-caffeoylquinic
acid derivatives were detected in full-scan experiments showing
m/z 439, 191, and 173 at the tR of 4.66 and 5.76 min in
decreasing concentrations. The mass spectra of the peaks in full
scan mode showed the typical fragmentation of the malonyl
acid and quinic acid of chlorogenic acid derivatives at m/z 86
and 191, respectively.
Acetylo-p-coumaroylquinic acid (9) and sinapoyl-malic acid
(14) were found in the analyzed berries as well. The compound
9 (tR 6.24 min, λmax 310 nm) produced the parent ion at m/z
163 and compound 14 (tR 9.10 min, λmax 328 nm) produced an
ion at m/z 233, corresponding to p-coumaric acid and sinapic
acid, respectively. The chemical nature of the nonphenolic acid
side chains was established using high resolution in source
fragmentation experiments in a negative ion mode showing
fragment ions with the neutral losses of 60 amu (m/z 379 − 60
= 319) and 116 amu (m/z 339 − 116 = 223) for the acetyl and
maloyl residues, respectively (Table 1).
Two glucosides of quercetin, namely, quercetin-3-O-rutino-
side (10, rutin) and quercetin-3-O-glucoside (12, isoquerci-
trin), were identified. Quercetin-3-O-rutinoside was detected at
the tR of 8.30 min, whereas quercetin-3-O-glucoside was found
at the tR of 8.44 min, both of them exhibiting a mass spectrum
in full-scan mode with m/z 609 and 463, respectively, and a
precursor ion scan m/z 301 as quercetin aglycon. Comparison
of the mass spectra and tR with those of the respective
standards confirmed the occurrence of these glycosylated
flavonols in the S. scabrum and S. burbankii berry extracts. The
fragmentation pathway of O-glycosylated flavonoids starts with
the cleavage of the glycosydic bonds and elimination of the
sugar moieties with charge retention on the aglycon. In
compounds containing two or more sugars at the same aglycon
carbon, ions arising from the cleavage of the glycosidic bonds
between sugar units are weak. Compounds such as quercetin-3-
O-glucosyl-rutinoside and quercetin-3-O-rutinoside were typical
for the Solanaceae family and have been previously reported in
potato.22 Quercetin, a major representative of the flavonol
subclass found at high concentrations in fruit and vegetables,
has recently received considerable attention. This flavonoid
displays the ability to prevent the oxidation of LDL by
scavenging free radicals and chelating transition metal ions. As a
result, quercetin may aid in the prevention of certain diseases,
such as cancer, atherosclerosis, and chronic inflammation by
dx.doi.org/10.1021/jf4045233 | J. Agric. Food Chem. 2014, 62, 1512−1519
Journal of Agricultural and Food Chemistry Article

retarding oxidative degradation and inducing enzymes that The concentrations of low molecular weight flavonols
detoxify carcinogens and also by blocking cancer formation by (quercetin) and hydroxycinnamic acid conjugates (caffeic, p-
deactivating at least 30 types of potentially carcinogenic coumaric and sinapic acid) were higher in the S. burbankii
agents.23,24 berries (78.89 mg/100 g) than in the S. scabrum berries (33.13
The UPLC method was applied for short run times mg/100 g) (Table 1). In both berries, 3-caffeoylquinic acid was
combined with a photodiode array detector (PDA), and a dominant, and the concentration of 5-caffeoylquinic acid was
quadrupole/time-of-flight mass spectrometer (Q/TOF-MS) higher than that of 4-caffeoylquinic acid. The content of
enables better characterization of individual compounds, acylated derivatives of anthocyanins showed the largest
especially with high molecular mass, i.e., acetylated anthocya- differences between the investigated berries. S. scabrum berries
nins. Three nonacylated anthocyanins were presented as were much more richer sources of anthocyanins (206.22 mg/
delphinidin-O-3-rutinoside-O-5-glucoside (2; m/z 773, [M + 100 g) than S. burbankii berries (82.06 mg/100 g). Price et al.2
H]+), petunidin-3-O-rutinoside-5-O-glucoside (5; m/z 787, [M found more anthocyanin compounds in S. scabrum berries (350
+ H]+), and malvidin-3-O-rutinoside-5-O-glucoside (7; m/z mg/100 g). The concentration of acylated antocyanins was
801, [M + H]+). Malvidin-3-O-rutinoside-5-O-glucoside was higher than that of the nonacylated antocyanins in Solanum
detected only in S. burbankii berry extract. The ESI mass berries. Petunidin-3-O-p-coumaroyl-rutinoside-5-O-glucoside
spectrum of pigments showed fragment ions at [M − 162 was the dominant acylated antocyanin in the investigated
(glucose)]+ corresponding m/z 611, 625, and 639, respectively, berries.
and the next fragment ions at [M − 146 (hexoside, probably Figure 2 shows the spectral character of S. scabrum (A) and S.
rhamnoside)]+ m/z 465, 479, and 493, respectively. The last burbankii (B) berry extracts at pH 2.5 and 7.5, indicating that
fragment ions at [M − 162 (hexoside, probably glucoside)]+
m/z 303 (dephinidin), 317 (petunidin) and 331 (malvidin)
were aglycons, as listed in Table 1. Seven other compounds 11
and 13−18 (tR 8.37−10.78 min; molecular weights m/z 918−
976 amu) were also clearly detected in Solanum berry extracts
(Table 1). Acylated anthocyanin glycosides were easily
identified on the basis of the increase in the mass of the
parent ions and the wavelength maxima (330−336 nm) of their
UV−vis spectra. Thus, peaks with the tR at 8.37, 8.89, 9.30,
9.82, 10.28, and 10.75 min were identified as 3-O-p-coumaroyl-
hexoside-5-O-hexoside (11) and 3-O-feruoyl-hexoside-5-O-
hexoside (13) of delphinidin, 3-O-p-coumaroyl-hexoside-5-O-
hexoside (15) and 3-O-feruoyl-hexoside-5-O-hexoside (16) of
petunidin, and 3-O-p-coumaroyl-hexoside-5-O-hexoside (17)
and 3-O-feruoyl-hexoside-5-O-hexoside (18) of malvidin,
respectively. The ESI mass spectrum of the acylated pigment
compound 11 showed a flavylium cation at m/z 919 [M + H]+
together with a fragment ion at m/z 757 [M − 162 (glucose)]+.
On the basis of observations of two additional fragment ions at
m/z 465 [M − 292 (the presence of hexoside, probably
rhamnose, and p-coumaric acid)], and m/z 303 derived from
successive losses of 162 amu, the presence of glucose moiety
was confirmed, for structural elucidation. Similar fragmentation
ions were observed for the remaining acetylated anthocyanins
(Table 1). Although these compounds have already been
presented in earlier works,1,2 to enable their complete
identification, their structures should be fully elucidated by
means of NMR or by comparison with authentic compounds, if
existing.
In addition, nonacylated anthocyanins were shown to be the Figure 2. Spectral character of S. scabrum (A) and S. burbankii (B)
most abundant compounds in wild species of berry and other berry extracts at pH 2.5 and 7.5.
fruits.25,26 The presence of acylated anthocyanins is typical of
grapes, blueberry, blackberry, and some vegetables, i.e., red these berries contain acylated anthocyanin. Changing the pH
cabbage, radish, and red-fleshed potato.27 from 2.5 to 7.5 did not result in any significant reduction in
Recent research has shown that anthocyanins with acylating absorbance at a wavelength of 530 nm, as is observed in the
substituents are more stable during processing and storage than case of nonacylated anthocyanins. The variation of pH values
other natural pigments.6,9,27 Brouillard et al.28 explain that the influences anthocyanin properties, such as color, stability,
effect of the improved stabilization of the acylated anthocyanins spectral shape, and absorption peak.29 In our research, it could
has been attributed to the stacking of the acyl groups with the be observed from Figure 2 that the anthocyanin extracts from S.
pyrylium ring of the flavylium cation, thereby reducing the scabrum (A) and S. burbankii (B) berries had the maximum
susceptibility of a nucleophile attack of water and subsequent absorption around 530 nm at pH 2.5 and 7.5. Anthocyanin
formation of intramolecular copigmentation of a pseudobase or solutions, at both pH values, exhibited bright red-violet color,
a chalcone. and their absorbances at 530 nm varied slightly. Acylated
1517 dx.doi.org/10.1021/jf4045233 | J. Agric. Food Chem. 2014, 62, 1512−1519
Journal of Agricultural and Food Chemistry
anthocyanins are also more resistant to color fading with
increased pH than their unacylated analogs. Unacylated
anthocyanins are only stable at pH values where the flavylium
cation dominates. Cevallos-Casals et al.6 showed that vegetables
such as sweet potato and purple carrot, which contain acylated
anthocyanins, were more resistant to the solution pH than red
grape rich, which is in unacylated anthocyanins. Other
researchers confirmed the unusual stability of acylated
anthocyanins at pH >5.0.7,8
Antioxidant Activity and Cyclic Voltammetry of
Solanum Berries. The antioxidant activity of S. scabrum (a)
and S. burbankii (b) berries was tested by measuring their
activity to scavenge radical (Table 2). The ABTS radical
Table 2. Antioxidant Activity (mmol Trolox/100 g dm) of
Solanum Fruits
S. scabrum S. burbankii
ABTS 2.89 ± 0.37 2.07 ± 0.42
FRAP 1.65 ± 0.09 0.71 ± 0.06
Mean values with standard deviation of n = 3.
scavenging activity was higher in S. scabrum berry extract [2.89
mmol Trolox equiv (TE)/100 g dm] than in S. burbankii berry
extract (2.07 mmol TE/100 g dm). After comparing the
ABTS•+ activity of the Solanum berries with that of known food
products we found that they exhibited a lower antioxidant
activity than that of blueberries (4.59 mmol TE/100g dm),
which constitute one of the richest reported sources of
antioxidants.30 But, still, S. scabrum berry extract had a higher
antioxidant activity than Rubus species (0−2.53 mmol TE/100
g dm), which have been recommended as improving the
nutritive value due to its high antioxidant activities.31 In regard
to the FRAP activity, S. scabrum had 1.65 mmol TE/100 g dm
and S. burbankii had 0.71 mmol TE/100g dm, respectively. The
differences in polyphenols between Solanum varieties could be
preliminarily attributed to their different contents of antioxidant
activity. S. scabrum berry extract contained much more phenolic
compounds than S. burbankii berry extract.
The cyclic voltammetric technique (CV) was employed to
study the electrochemical behavior of S. scabrum and S.
burbankii berry polyphenol extracts, and results are presented in
Figure 3. The major current peak appears at approximately 2.4
V for S. scabrum and at 2.1 V for S. burbankii. According to
literature data, this peak may be attributed to powerful, low
formal potential phenolics characterized by an o-hydroxyphenol
or di- or trihydroxyphenol group. Petuninidin and delphinidin
anthocyanin derivatives were the main compounds of Solanum
berry, but higher contents of these compounds were
determined in S. scabrum. These anthocyanins possess three
and two catechol groups on the B-ring. Cevallos-Casals at al.6
explained that oxidation of phenolic compounds involved the
formation of a stable quinone, which can be reduced in the
reverse scan, this process appearing as a cathodic peak of the
current response. In the return scan, the electrochemical
behavior of S. burbankii extract is irreversible. Yakovleva et al.32
present the irreversible oxidation of some phenolic acids and
flavones in plants. However Tamura et al.12 found that
anthocyanins acylated by p-coumaric acid are much better
antioxidants than α-tocopherol or (+)-catechin. Besides the
color and antioxidant activity, anthocyanins have free radical
scavenging activity. The anthocyanin radical is more stable than
1518
Article
Figure 3. Cyclic voltammograms of S. burbankii and S. scabrum.
other radicals generated in the human body; hence the duration
of this radical is longer.12
In both cultivars, the major compound was found to be the
anthocyanin petunidin-3-(p-coumaroyl-rutinoside)-5-glucoside.
Eight additional anthocyanins in S. burbankii berries and 11 in
S. scabrum berries were identified as petunidin, delphinidin, and
malvidin with the same glycosidic substitution pattern as the
major pigment and varying degrees of acylation with p-
coumaric and ferulic acids. The presence of seven phenolic acid
mainly caffeoylquinic derivatives and two quercetin derivatives
was reported for the first time. S. scabrum extracts are richer in
anthocyanins but have less phenolic acid than S. burbankii. This
study demonstrated the usability of fast and effective electro-
chemical measurements and a spectrophotometric technique
for determination of the phenolic content as well as antioxidant
activity of Solanum berry extracts. S. scabrum exhibited the
highest TEAC values according to both CV and ABTS and
FRAP assay.
From the consumers’ point of view, the analyzed fruits are
good sources of bioactive compounds and colorants to be used
in the food industry.
■
AUTHOR INFORMATION
Corresponding Author
*Tel./fax: +48 71 320 7706. E-mail: aneta.wojdylo@up.wroc.pl.
Notes
The authors declare no competing financial interest.
■ REFERENCES
(1) Francis, F. J.; Harborne, J. B. Anthocyanins of the garden
huckleberry Solanum guineense. J. Food Sci. 1966, 31, 524−528.
(2) Price, C. L.; Wrolstad, R. E. Anthocyanin pigments of royal
Okanogan huckleberry juice. J. Food Sci. 1995, 60, 369−374.
(3) Manoko, M. L. K.; Van den Berg, R. G.; Feron, R. M. C.; Van der
Weerden, G. M.; Mariani, C. Genetic diversity of the African hexaploid
species Solanum scabrum Mill. and Solanum nigrum L. (Solanaceae).
Genet. Resour. Crop Evol. 2008, 55 (3), 409−41.
(4) D’Arcy, W. G. The Solanaceae since 1976 with a review of its
biogeography in Solanaceae III: Taxonomy chemistry and evolution
(Hawkes, J. G., Lester, R. N., Nee, M., Estrada, N. R. , Eds.; Academic
Press: London, 1991; pp 75−137.
dx.doi.org/10.1021/jf4045233 | J. Agric. Food Chem. 2014, 62, 1512−1519
Journal of Agricultural and Food Chemistry Article

(5) Rao, G. R. Role of fruit pigments in understanding the inter- (25) Liang, Z.; Yang, Y.; Cheng, L.; Zhong, G. Polyphenolic
relationships and mechanism of evolution of higher chromosomal composition and content in the ripe berries of wild Vitis species. Food
forms of the species of the Solanum nigrum L complex. Acta Bot. Indica Chem. 2012, 132, 730−738.
1978, 6 (Suppl), 41−47. (26) Wu, X.; Prior, R. L. Systematic identification and character-
(6) Cevallos-Casals, B. A.; Cisneros-Zevallos, L. Stability of ization of anthocyanins by HPLC−ESI-MS/MS in common foods in
anthocyanin-based aqueous extracts of Andean purple corn and red- the United States: Fruits and berries. J. Agric. Food Chem. 2005, 53,
fleshed sweet potato compared to synthetic and natural colorants. 2589−2599.
Food Chem. 2004, 86, 69−77. (27) Giusti, M. M.; Rodriguez-Saona, L. E.; Griffin, D.; Wrolstad, R.
(7) IdakaE. (Suntory Ltd.) Acylated anthocyanin and process for E. Electrospray and tandem mass spectroscopy as tools for
producing the same as well as pigment composition containing the anthocyanin characterization. J. Agric. Food Chem. 1990, 47, 4657−
same. Patent US 4999423 A, 1991. 4664.
(8) Delgado-Vargas, F.; Jimenez, A. R.; Paredes-Lopez, O. Natural (28) Brouillard, R.; Chassaing, S.; Fougerousse, A. Why are grape/
pigments: Carotenoids, anthocyanins and betalains-characteristics, fresh wine anthocyanins so simple and why is it that red wine color
biosynthesis, processing and stability. Crit. Rev. Food Sci. Nutr. 2000, lasts so long? Phytochemistry 2003, 64, 1179−1186.
40 (3), 173−289. (29) Dao, L. T.; Takeoka, G. R.; Edwards, R. H.; Berrios, J.; De, J.
(9) Fossen, T.; Ř vstedal, D. O.; Slimestad, R.; Andersen, Ř . M. Improved method for stabilization of anthocyanidins. J. Agric. Food
Anthocyanins from a Norwegian potato cultivar. Food Chem. 2003, 81, Chem. 1996, 46, 3569−3654.
433−437. (30) Kaur, C. K.; Kapoor, H. C. Antioxidants in fruits and
(10) Chen, Z. Y.; Chan, P. T.; Ho, K. Y.; Fung, K. P.; Wang, J. vegetablesThe millennium’s health. Inter. J. Food Sci. Technol.
2001, 36, 703−725.
Antioxidant activity of natural flavonoids is governed by number and
(31) Deighton, N.; Brennan, R.; Finn, C.; Davies, H. V. Antioxidant
location of their aromatic hydroxyl groups. Chem. Phys. Lipids 1996, 79
properties of domesticated and wild Rubus species. J. Agric. Food Chem.
(2), 157−163.
2000, 80, 1307−1313.
(11) Rice-Evans, C. A.; Miller, N. J.; Paganga, G. Structure−
(32) Yakovleva, L. E.; Kurzeev, S. A.; Stepanova, E. V.; Fedorova, T.
antioxidant activity relationships of flavonoids and phenolic acids. Free V.; Kuznetsov, B. A.; Koroleva, O. V. Characterization of plant
Radical Biol. Med. 1996, 20 (7), 933−956. phenolic compounds by cyclic voltammetry. Applied Biochem.
(12) Tamura, H.; Yamagami, A. Antioxidative activity of mono- Microbiol 2007, 43, 661−668.
acylated anthocyanins isolated from Muscat Bailey grape. J. Agric. Food
Chem. 1994, 42, 1612−1615.
(13) Kilmartin, P. A.; Hsu, C. F. A cyclic voltammetry method
suitable for characterizing antioxidant properties of wine and wine
phenolics. J. Agric. Food Chem. 2001, 49, 1957−1965.
(14) Janeiro, P.; Oliveira, B. A. M. Redox behaviour of anthocyanins
present in Vitis vinifera L. Electroanalysis 2007, 19, 1779−1786.
(15) Mazza, G.; Brouillard, R. Recent developments in the
stabilization of anthocyanins in food products. Food Chem. 1987, 25,
207−225.
(16) Pereira, G. K.; Donate, P. M.; Galembeck, S. E. Effects of
substitution for hydroxyl in the B-ring of the flavylium cation. J. Mol.
Struct. 1997, 392, 169−179.
(17) Cren-Olivé, C.; Hapiot, P.; Pinson, J.; Rolando, C. Free radical
chemistry of flavan-3-ols: Determination of thermodynamic parame-
ters and of kinetic reactivity from short (ns) to long (ms) time scale. J.
Am. Chem. Soc. 2002, 124, 14027−14038.
(18) Oszmiański, J.; Lee, C. Y. Isolation and HPLC determination of
phenolic compounds in red grapes. Am. J. Enol. Vitic. 1990, 41, 204−
206.
(19) Wojdyło, A.; Oszmiański, J.; Laskowski, P. The polyphenolic
compounds and antioxidant activity of new and old apple varieties. J.
Agric. Food Chem. 2008, 56, 6520−6530.
(20) Narváez-Cuenca, C.-E.; Vincken, J.-P.; Gruppen, H. Identi-
fication and quantification of (dihydro) hydroxycinnamic acids and
their conjugates in potato by UHPLC−DAD−ESI-MSn. Food Chem.
2012, 130, 730−738.
(21) Schmidt, S.; Zietz, M.; Schreiner, M.; Rohn, S.; Kroh, L. W.;
Krumbein, A. Identification of complex naturally occurring flavonoid
glycosides in kale (Brassica oleracea var. sabellica) by high-performance
liquid chromatography diode-array detection/electrospray ionization
multi-stage mass spectrometry. Rapid Commun. Mass Spectrom. 2010,
24, 2009−2022.
(22) Shakya, R.; Navarre, D. A. Rapid screening of ascorbic acid
glycoalkaloids and phenolics in potato using high-performance liquid
chromatography. J. Agric. Food Chem. 2006, 54, 5253−5260.
(23) Ackland, M. L.; Van de Waarsenburg, S.; Jones, R. Synergistic
antiproliferative action of the flavonols quercetin and kaempferol in
cultured human cancer cell lines. In Vivo 2005, 19, 69−76.
(24) Fresco, P.; Borges, F.; Marques, M. P. M.; Diniz, C. The
anticancer properties of dietary polyphenols and its relation with
apoptosis. Curr. Pharm. Des. 2010, 16, 114−134.

1519 dx.doi.org/10.1021/jf4045233 | J. Agric. Food Chem. 2014, 62, 1512−1519