Food Research International 122 (2019) 627–634

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Food Research International

journal homepage: www.elsevier.com/locate/foodres

Blackberry (Rubus ulmifolius Schott): Chemical composition, phenolic T

compounds and antioxidant capacity in two edible stages
⁎
Mayara Schulza, , Siluana Katia Tischer Seraglioa, Fabiana Della Bettaa, Priscila Nehringa,
Andressa Camargo Valeseb, Heitor Daguerb, Luciano Valdemiro Gonzagaa,
⁎
Ana Carolina Oliveira Costaa, Roseane Fetta,
a
Department of Food Science and Technology, Federal University of Santa Catarina, 88034-001, Florianópolis, SC, Brazil
b
National Agricultural Laboratory (LANAGRO-RS), Ministry of Agriculture, Livestock and Food Supply, 88102-600, São José, SC, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: The berries of the genus Rubus has been highlighted as important source of bioactive and health promoting
Fruits constituents, however, information about chemical composition and antioxidant potential of the specie Rubus
Ripening ulmifolius are still scarce. In this regard, this study aimed to assess the physicochemical characteristics, total
Physicochemical composition monomeric anthocyanins (TMA), individual phenolics, minerals, sugars, and antioxidant properties of mature
LC-ESI-MS/MS
and fully mature R. ulmifolius. With the advance of maturation, changes in the physicochemical composition
Capillary electrophoresis
Carbohydrate
suggest pleasant characteristics for consumption especially in the fully mature stage. High levels of TMA and
sugars (fructose and glucose) were also verified in the fully mature stage, as well as, expressive antioxidant
potential, with values of 241.06 μM Fe+2 g−1 for ferric reducing antioxidant power and 28.22 mg gallic acid
equivalent g−1 for Folin-Ciocalteu reducing capacity (all expressed in dry matter, DM). In contrast, minerals
(potassium, calcium, sodium) and most of the studied phenolic compounds showed the highest concentrations in
mature fruits. Among the phenolics investigated, 26 compounds were identified and quercetin and isoquercitrin
were the predominant phenolic compounds in the fruit. The results reinforce the nutritive and antioxidant
potential of Rubus ulmifolius in both maturation stages studied.

1. Introduction
The Rubus genus includes several berry species and is the largest
genus of the Rosaceae family. The species of this genus have been cul-
tivated for centuries because of their fruits; however, there are only few
domesticated species. These fruits are consumed fresh or as processed
products such as jam, wine, tea, ice cream, desserts, and bakery pro-
ducts (Bowen-Forbes, Zhang, & Nair, 2010; Zia-Ul-Haq, Riaz, De Feo,
Jaafar, & Moga, 2014).
Rubus ulmifolius Schott is a perennial shrub native to Europe and
North America and grows in many areas worldwide, including in South
American countries such as Brazil (Martins et al., 2014). This species
produces edible blackberry fruits, which are of the aggregate type,
globose, acidulated flavor and with black coloration at the end of ma-
turation (D'Agostino et al., 2015; Lorenzi, Bacher, Lacerda, & Sartori,
2006).
Due to increasing awareness about the valuable attributes of fruits,
the global consumption of berries and berries-based products has in-
creased considerably (Nile & Park, 2014; Padmanabhan, Correa-
Betanzo, & Paliyath, 2016). Berries present indispensable components
of a healthy diet, providing a wide spectrum of substances such as
dietary fiber, minerals, vitamins, other essential nutrients, and phyto-
chemicals, especially polyphenols (de Souza et al., 2014; Nile & Park,
2014). Considering that berries are a rich source of polyphenolics
compounds, these fruits have been recognized with potential human
health benefits due, mainly, to antioxidant and anti-inflammatory
properties of these compounds (Ahmad et al., 2015; Van de Velde,
Grace, Esposito, Pirovani, & Lila, 2016).
The chemical composition, antioxidant and health promoting con-
stituents of Rubus berries are usually well recognized (Ahmad et al.,

Abbreviations: CE-DAD, Capillary electrophoresis equipped with diode array detector; DM, Dry matter; FC, Folin-Ciocalteu reducing capacity; FRAP, Ferric reducing
antioxidant power; GAE, Gallic acid equivalent; LC-ESI-MS/MS, Liquid chromatography - electrospray ionization - tandem mass spectrometry; TMA, Total mono-
meric anthocyanins; TSS, Total soluble solids; TTA, Total titratable acidity
⁎
Corresponding authors at: Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Departamento de Ciência e Tecnologia de Alimentos, Rodovia
Admar Gonzaga, 1346, Itacorubi, CEP: 88034-001 Florianopolis, SC, Brazil.
E-mail addresses: schulzmay@gmail.com (M. Schulz), roseane.fett@gmail.com (R. Fett).

https://doi.org/10.1016/j.foodres.2019.01.034
Received 4 June 2018; Received in revised form 9 December 2018; Accepted 14 January 2019
Available online 15 January 2019
0963-9969/ © 2019 Elsevier Ltd. All rights reserved.
M. Schulz et al.
2015; Badhani, Rawat, Bhatt, & Rawal, 2015; de Souza et al., 2014;
Türkben, Sariburun, Demir, & Uylaşer, 2010; Yang & Choi, 2017). In R.
ulmifolius leaves, Tabarki et al. (2017) found kaempferol 3-O-rutinoside
and naringenine as dominant phenolic compounds, while Ali, Shaoib,
Shah, Shah, and Shuaib (2017) related flavonoids, tannins, alkaloids
and steroids in R. ulmifolius aerial parts. In contrast, there are few
published studies about the nutritional attributes, polyphenolics con-
stituents and antioxidant capacity of the fruits of R. ulmifolius (Ahmad
et al., 2015; Hajaji et al., 2017; Ruiz-Rodriguez et al., 2014), and none
of them were carried out with fruits grown in the American continent.
Therefore, the knowledge of the chemical and bioactive composition of
this fruit can be valuable to incentive its cultivation, especially in this
continent, and assess the differences between this species and other
blackberry species previously reported in the literature.
Previous studies have reported the influence of ripeness on physi-
cochemical characteristics, nutritional and bioactive components, and
antioxidant properties in berry fruits (Acosta-Montoya et al., 2010; Fu
et al., 2015; Gordon et al., 2012; Schulz et al., 2015; Seraglio et al.,
2018; Tosun, Ustun, & Tekguler, 2008). Results showed that during
ripening, anthocyanin pigments, antioxidant capacity, total soluble
solids, and sugars tend to increase. In contrast, titratable acidity and pH
tend to decrease, while non-anthocyanin phenolics and minerals are
claimed to not display a specific trend. These changes during matura-
tion are caused by a series of combined biochemical and physiological
processes. Therefore, understanding these changes can contribute to
determine the best ripening stage for consumption of fruits with better
quality and nutritional content (Glew et al., 2005; Schulz et al., 2015).
An analysis of chemical and bioactive components during ripening of
the R. ulmifolis blackberry has not been yet performed.
In this regard, the aim of this research was to determine the phy-
sicochemical characteristics, minerals, sugars, phenolics and anti-
oxidant properties of R. ulmifolius fruits cultivated in southern Brazil in
two edible maturation stages in order to provide data on the best stage
for nutritional and bioactive use.
2. Material and methods
2.1. Reagents and solutions
Analytical grade reagents and ultra-pure water (Milli-Q, Millipore,
Bedford, MA, USA) were used to prepare solutions. Ferric chloride,
ascorbic acid, acetone, potassium chloride, anhydrous sodium sulfate,
and gallic acid were purchased from Vetec (Cabo Frio, RJ, Brazil).
Sorbic acid, imidazole, cetyltrimethylammonium bromide (CTAB),
lactic acid, sodium hydroxide, acetic acid, sodium carbonate, 2,4,6-tris
(2-pyridil)-s-triazine (TPTZ), Folin-Ciocalteu reagent, citric acid, hy-
drochloric acid, methanol, ethyl acetate, sodium acetate, ultra-pure
standards of individual phenolics, sugars, and minerals were acquired
from Sigma-Aldrich (St. Louis, MO, USA).
2.2. Samples
The botanical identification of Rubus ulmifolius Schott was con-
firmed by Pedro Fiaschi, and the sample was deposited in the
Herbarium of the Department of Botany of the Federal University of
Santa Catarina, under identification numbers FLOR 61388 and FLOR
61389. The identification of the samples was carried out using the
traditional taxonomic methodology (Stuessy, 2009).
Fresh blackberries (R. ulmifolius) were harvest from an agricultural
property in Santa Catarina State, Brazil (latitude 26o85’02”S, longitude
52o98’17”W, altitude 515 m), in January 2015. The fruits were har-
vested in a single collection and classified into two edible maturation
stages, named as mature and fully mature stages. For each maturation
stage, the fruits (300 g) were handpicked from three randomly selected
plants. The classification of the maturation stages was performed ac-
cording to the external colour and physicochemical parameters, such as
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Food Research International 122 (2019) 627–634
Table 1
Physicochemical characteristics of blackberry (Rubus ulmifolius Schott) fruits in
two edible maturation stages.
Characteristics Blackberry
Mature Fully mature
Moisture content (g 100 g−1) 88.09 ± 0.24a 87.84 ± 0.28a
Total soluble solids (oBrix) 6.67 ± 0.58b 8.00 ± 0.00a
Total titratable acidity (g citric acid 100 g−1) 0.14 ± 0.01a 0.09 ± 0.01b
pH 3.48 ± 0.03b 3.71 ± 0.04a
TSS/TTA ratio 46.51 ± 3.57b 85.93 ± 5.13a
Weight of the fruit (g)* 5.05 ± 0.57a 5.17 ± 0.41a
Size of the fruit (cm)* 2.45 ± 0.39a 2.64 ± 0.34a
Results expressed as mean ± standard deviation. * Data presented are the
average of 35 fruits. a-b Mean values in the same row followed by different
letters indicate significant differences (p < .05) using Student's t-test. TSS/TTA
ratio - Total soluble solids/ Total titratable acidity.
total soluble solids, pH and total titratable acidity (Table 1). The fruits
without any damage were selected, packed in polyethylene bags with
the atmosphere modified with gaseous nitrogen, hermetically sealed
and immediately frozen at −18 ± 2 °C until the analysis (within
30 days) to avoid chemical and enzymatic reactions. For individual
phenolics, total monomeric anthocyanin, antioxidant capacity, minerals
and sugar analysis, the frozen fruits were triturated using a domestic
food processor (Britânia, Curitiba, PR, Brazil) and weighed soon after
reaching room temperature (25 ± 2 °C).
All the experimental analysis were performed in triplicate.
2.3. Physicochemical characterization
Blackberry samples were homogenized using a domestic food pro-
cessor prior to analyses. Moisture was determined using 5 g of triturated
sample in an oven at 105 ± 2 °C (Greenhouse Labor SP-400/1, Sao
Paulo, SP, Brazil) until constant weight according to the AOAC
International (2005) reference method 925.09. The moisture content
was expressed as g 100 g−1 and the dry matter was obtained by the
value of the moisture content subtracted from the value of 100; pH was
determined using a digital pH meter (DM-22, Digimed, Sao Paulo, SP,
Brazil) after dilution of 10 g of homogenized sample with 100 mL of
deionized water; titratable acidity was expressed as g citric acid
100 g−1, and determined by titration with NaOH 0.1 mol L−1 until
pH 8.2–8.4 according to reference method 942.15 (AOAC, 2005) after
dilution of 10 g of homogenized sample with 100 mL of deionized
water; total soluble solids were expressed as °Brix, and determined in
the homogenized sample at 20 °C using a refractometer (Tropenmodell
I, Carl Zeiss Jena, Germany) according to reference method 932.14
(AOAC, 2005).
The size and weight of the blackberry were measured with the
whole fruit. The size was expressed as cm, and measured with a caliper
(Digimess, Sao Paulo, SP, Brazil), while the weight was determined
using an analytical balance model AB204-SRS (Mettler Toledo, Zürich,
Switzerland) and expressed as g.
2.4. Analysis of phenolic compounds by LC-ESI-MS/MS
The extraction procedure of phenolic compounds was performed
according to the method proposed by Schulz et al. (2015), with adap-
tations (Seraglio et al., 2018). Initially, 1.0 ± 0.1 g of the sample was
manually stirred for 1 min with 9 mL of ultra-pure water and 20 mg of
ascorbic acid. A volume of 5 mL of 6 mol L−1 HCl was added to the
system and incubated at 85 °C for 30 min in an oven. The extract cooled
was sequentially extracted for 1 min with 5 mL of ethyl acetate for three
times. After centrifugation (5 min/ 1338 x g) (Fanem, model 280R, Sao
Paulo, SP, Brazil), the supernatants were combined, dried with 3 g of
anhydrous sodium sulfate, vaporized using a rotary evaporator
M. Schulz et al.
(Fisatom 802, Sao Paulo, SP, Brazil), and the extract reconstitute in
1 mL of ultrapure grade methanol. The reconstitute extract was diluted
1:9 (v/v) with mobile phase and injected in the liquid chromatography
system.
The identification and quantification of the individual phenolic
compounds were performed according to Seraglio et al. (2018) in a
liquid chromatography – electrospray ionization – tandem mass spec-
trometry (LC-ESI-MS/MS). An Agilent 1290 series chromatographic
system (Agilent Technologies, Wilmington, DE, USA) coupled with a
hybrid quadrupole linear ion trap mass spectrometer QTRAP® 5500 (AB
Sciex, Foster City, CA, USA) equipped with an electrospray ionization
source (ESI) was used. Identification and quantification of the com-
pounds was performed according to the retention time, precursor ion,
major fragment (quantification ion) and secondary fragment (con-
firmation ion) by comparison with commercial analytical standards.
Mass spectrometry parameters for multiple reaction monitoring (MRM)
transitions mode, calibration curves, and detection and quantification
limits for the 33 standards of phenolic compounds tested are listed in
Supplementary material 1. The results were expressed in μg 100 g−1 of
dry matter (DM).
2.5. Total monomeric anthocyanin (TMA)
The extraction of TMA was performed according to the method
described by Borges, Vieira, Copetti, Gonzaga, and Fett (2011), with
some adaptations (Seraglio et al., 2018). Firstly, 1.25 ± 0.10 g of the
sample was extracted with acidified methanol (1.5 mol L−1 HCl) in a
10 mL volumetric flask, for 24 h in the dark and with temperature
controlled (4 ± 2 °C). The samples were filtered through paper filters
(Whatman n° 1) and TMA content was immediately determined ac-
cording to the spectrophotometric pH differential method (Giusti &
Wrolstad, 2001). The anthocyanin content was expressed as mg cya-
nidin 3-glucoside (cy-3-glu) g−1 of DM.
2.6. Antioxidant capacity
To determine the antioxidant capacity, samples was sequentially
extracted according to Rufino et al. (2010), with adaptations (Seraglio
et al., 2018). Firstly, 2.0 ± 0.1 g of the sample were first extracted with
4 mL of ultrapure grade methanol/water (1:1, v/v) for 30 min in an
ultrasonic bath (Unique, model USC-1400, Sao Paulo, SP, Brazil). After
being centrifuged by 10 min/ 1338 x g, the supernatant was transferred
to a 10 mL volumetric flask. The residue was sequentially extracted
with 4 mL of acetone/water 7:3, v/v in the same conditions cited above.
The supernatant was transferred to the same 10 mL volumetric flask
and the volume adjusted with ultra-pure water.
The antioxidant capacity was determined through the evaluation of
the ferric reducing antioxidant power (FRAP) assay and Folin-Ciocalteu
reducing capacity (FC).
The FRAP assay was performed according to the method described
by Benzie and Strain (1996), with some modifications (Arnous, Makris,
& Kefalas, 2002). An aliquot of 200 μL of blackberry extracts was mixed
with 200 μL of FeCl3 (3 mmol L−1 in 5 mol L−1 citric acid) in a poly-
propylene tube and kept for 30 min at 37 ± 2 °C in a water bath (Fi-
satom, model 550, São Paulo, SP, Brazil). Then, an aliquot of 3.6 mL of
TPTZ solution (0.312 mg mL−1 in 0.05 mol L−1 HCl) was added to the
mixture. After 10 min, the absorbance was measured using a UV–Vis
spectrophotometer (Spectro Vision SB 1810–60 S, Beijing, China) at
620 nm. The reducing capacity was expressed as μmol Fe+2 equivalents
g−1 of DM.
The FC assay was determined using the Folin-Ciocalteu method (V.
L. Singleton & Rossi, 1965), and the results were expressed as mg gallic
acid equivalent (GAE) g−1 of DM.
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Food Research International 122 (2019) 627–634
2.7. Determination of sugars and minerals by CE-DAD
For sugars determination, 2.0 ± 0.1 g of sample was transferred to
a 50 mL volumetric flask and the volume made up with ultra-pure
water. The resultant solution was stirred for 1 min using a domestic
food processor (Britânia, Curitiba, PR, Brazil) and centrifuged for 5 min
at 1338 x g (Seraglio et al., 2018). The supernatant collected was in-
jected into a capillary electrophoresis (CE) system (Agilent Technolo-
gies, model 7100, Palo Alto, CA, USA) equipped with a diode array
detector (DAD), for the determination of fructose, glucose, and sucrose,
according to the method proposed by Rizelio et al. (2012b). The results
were expressed as g 100 g−1 of DM.
For minerals determination, 5.0 ± 0.1 g of sample was weighed in
the crucibles pretreated (washed with nitric acid, washed with water
and dried in an oven at 105 °C), carbonized and submitted to a muffle
furnace (Quimis, EQ 318D24, São Paulo, SP, Brazil) at 525 °C for 6 h.
The ashes were dissolved under heating with HCl 12 mol L−1 (100 μL),
transferred to a volumetric flask (5 mL), and the volume made up with
ultra-pure water (Seraglio et al., 2018). The extract was diluted 9:1 (v/
v) with Barium (internal standard) and injected into the CE system to
the determination of sodium, calcium, potassium, magnesium, and
manganese using the method previously described by Rizelio et al.
(2012a). The quantification results were expressed as mg g−1 of DM.
2.8. Statistical analysis
Data was expressed as mean ± standard deviation and analyzed
using Statistica 13.0 software (Statsoft Inc., Tulsa, OK, USA). The
Student's t-test was performed to identify significant differences
(p < .05) between the ripening stages.
3. Results and discussion
3.1. Physicochemical composition
In Table 1 are shown the variation of some physicochemical para-
meters obtained from the blackberry R. ulmifolius in two edible ma-
turation stages. The moisture content of blackberry fruits stayed prac-
tically constant (p > .05) in the two studied stages, similar behavior
observed for jabuticaba (Myrciaria cauliflora), and guabiju (Myrcianthes
pungens) (Seraglio et al., 2018). The blackberry moisture values were in
general similar to red raspberry (Rubus idaeus L.), mulberry (Morus nigra
L., Morus alba L., and Morus alba var. tatarica L.) and blackberry (Rubus
adenotrichus Schltdl), ranging from 80.79 to 89.00 g 100 g−1 (Acosta-
Montoya et al., 2010; Jiang & Nie, 2015; Mazur, Nes, Wold, Remberg, &
Aaby, 2014).
With the advance of the maturation, the total soluble solids (TSS)
increased (p < .05) in the blackberry fruits (6.67 and 8.00 oBrix, for
mature and fully mature stage, respectively). The increase in the TSS
during maturation was also observed for other berries, such as R. ade-
notrichus blackberry, guabiju, jambolan, and jabuticaba (Acosta-
Montoya et al., 2010; Seraglio et al., 2018), and can be related to the
hydrolysis of starch into sugars (Kulkarni & Aradhya, 2005; Patel &
Rao, 2014). These values were in accordance to those reported for
blackberry (R. adenotrichus) (5.03 to 7.70 oBrix) (Acosta-Montoya et al.,
2010) and different blackberry genotypes (Rubus subgenus Rubus
Watson) (8.07 to 10.97 oBrix) (Threlfall et al., 2016).
High TSS generally results in sweeter fruit especially when accom-
panied by a decrease in acidity (Fawole & Opara, 2013), fact observed
in this study. The total titratable acidity (TTA) was significantly
(p < .05) higher in the mature stage of blackberry, with mean value of
0.14 g citric acid 100 g−1, in accordance with the information reported
by Threlfall et al. (2016) that the TTA of blackberry fruits are nor-
mally < 1 g citric acid 100 g−1. As consequence of the decrease in
acidity, the pH increased significant (p < .05) with the maturation,
same tendency observed for other berries fruits (Acosta-Montoya et al.,
M. Schulz et al. Food Research International 122 (2019) 627–634

2010; Seraglio et al., 2018). The pH values for blackberry were similar Table 2
to those reported for raspberry Rubus hirsutus Thunb. and different Phenolic content (μg 100 g−1 of dry matter) in blackberry (Rubus ulmifolius
blackberry genotypes (Rubus subgenus Rubus Watson) fruits (Fu et al., Schott) fruits in two edible maturation stages.
2015; Threlfall et al., 2016). Phenolic compound Blackberry
The decrease in the TTA and the increase in the TSS is a process
generally observed during maturation, which may influence the flavor Mature Fully mature
characteristics of the fruits (Kulkarni & Aradhya, 2005). In this regard,
Coumarins
the TSS/TTA ratio can assist in this evaluation, suggesting the equili- 4-methylumbelliferone < LOD < LOD
brium between sugars and acidity (Dalla Nora et al., 2014), and has
Flavonoids
been reported as an important indicator of the fruit maturity, influen- Apigenin < LOQ < LOQ
cing the fruit taste and contributing as a quality criterion for processing (+)-Catechin 378.71 ± 24.37 a 156.61 ± 12.12 b
in the food industry (Acosta-Montoya et al., 2010; Fawole & Opara, Chrysin < LOQ 10.52 ± 0.11
2013). The highest TSS/TTA ratio in the fully mature blackberry (−)-Epigallocatechin gallate < LOD < LOD
(+)-Epicatechin 448.40 ± 7.53 a 250.82 ± 11.86 b
(p < .05) indicate that this stage has higher agreeable flavor and in- Galangin < LOD < LOQ
creasing the consumer acceptance, same behavior observed for other Hesperidin < LOD < LOD
berry and pomegranate fruits along maturation (Acosta-Montoya et al., Isoquercitrin 1526.86 ± 60.70b 1888.51 ± 2.76a
2010; Fawole & Opara, 2013; Seraglio et al., 2018). In addition, the Isorhamnetin 62.42 ± 0.17a 55.70 ± 7.00a
Kaempferol 513.44 ± 15.36a 399.48 ± 5.03b
TSS/TTA ratio values found in our study were higher than those re-
Luteolin < LOD 5.97 ± 0.21
ported for red guava, cambuci, raspberry, and different blackberry Naringenin 36.14 ± 1.97a 28.34 ± 2.04a
genotypes (Rubus subgenus Rubus Watson) (2.25 to 31.31) (Azevedo, Naringin < LOD < LOD
Silva, Jacomino, & Genovese, 2017; Dalla Nora et al., 2014; Fu et al., Quercetin 9149.75 ± 150.31a 5509.61 ± 135.05b
2015; Mazur et al., 2014; Threlfall et al., 2016), suggesting that the Pinobanksin 41.57 ± 2.25a 32.04 ± 0.66b
Pinocembrin 32.13 ± 1.95b 110.67 ± 1.10a
blackberry R. ulmifolius, especially in the fully mature stage, has plea- Rutin < LOD < LOD
sant characteristics for consumption.
Lignin-derived aldehydes
No significant difference was found among the size and weight in
Coniferaldehyde 72.54 ± 2.93a 14.11 ± 1.17b
the two ripening stages, possibly because both stages are already sui- Sinapaldehyde 16.13 ± 0.85a 16.22 ± 0.34a
table for consumption and at the end of the maturation cycle. This trend Syringaldehyde 56.22 ± 0.11a 16.24 ± 0.94b
was also observed for other fruits, such as pomegranate, cambuci, and Phenolic acids
blackberry (R. adenotrichus) fruits during maturation progress (Acosta- p-Aminobenzoic acid < LOQ < LOQ
Montoya et al., 2010; Azevedo et al., 2017; Fawole & Opara, 2013). Benzoic acid < LOQ < LOQ
Differences in the blackberry's composition have been reported in the Caffeic acid 399.91 ± 5.49a 75.52 ± 1.21b
Chlorogenic acid < LOD < LOD
literature and are considered strongly dependent on the genotype p-Coumaric acid 233.75 ± 18.48a 39.65 ± 1.66b
(Threlfall et al., 2016), as well as influenced by geographical and cli- 3,4-Dihydroxybenzoic acid 481.71 ± 0.81b 1288.81 ± 7.58b
mate conditions (Acosta-Montoya et al., 2010). Ferulic acid < LOQ 388.59 ± 22.19
Gallic acid 1510.97 ± 80.36a 750.26 ± 87.64b
Salicylic acid 203.39 ± 8.43b 296.62 ± 13.79a
3.2. Phenolic compounds
Sinapic acid 446.41 ± 2.78a 228.69 ± 17.25b
Syringic acid 50.48 ± 4.95a 40.84 ± 5.02a
In plants, phenolic compounds occur as soluble (conjugated glyco- Vanillic acid < LOD < LOD
sides and aglycones) and insoluble forms (bonded to structural plant Sum of phenolic compounds 15,745.59 11,603.82
components such as cellulose, pectin, proteins) (Acosta-Estrada,
Results expressed as mean ± standard deviation. a-b Mean values in the same
Gutiérrez-Uribe, & Serna-Saldívar, 2014). To evaluate the conjugated
row followed by different letters indicate significant differences (p < .05)
phenolic compounds, an acid hydrolysis associated with high tem-
using Student's t-test. LOD – Limit of detection. LOQ – Limit of quantification.
perature was applied to break the glycosidic bonds and released the
phenolic compounds. Acid hydrolysis can cause losses of phenolic
Medvidović-Kosanović (2009), however, in our study the value was up
compounds by degradation, a fact that can be minimized by the addi-
to six times higher. Ruiz-Rodriguez et al. (2014) evaluated seven phe-
tion of ascorbic acid in the hydrolysis system (Acosta-Estrada et al.,
nolic compounds in blackberry R. ulmifolius of the Spain and found
2014; Shahidi & Yeo, 2016).
gallic acid as the most abundant, while in blackberry R. fruticosus,
Of thirty-three non-anthocyanins phenolic compounds studied,
Radovanović, Andelković, Radovanović, and Andelković (2013) ob-
which are commonly investigated in fruits, ten phenolic acids were
served that gallic acid and rutin were the predominant phenolic com-
identified in blackberry R. ulmifolius (Table 2 and Fig. 1). Gallic acid
pounds.
was the major phenolic acid found (750.26 to 1510.97 μg 100 g−1 DM),
In addition to phenolic acids and flavonoids, one coumarin (4-me-
followed by 3,4-dihydroxybenzoic acid (481.71 to 1288.81 μg 100 g−1
thylumbelliferone) and three lignin-derived aldehydes (con-
DM). Benzoic and p-aminobenzoic acids were found under the limit of
iferaldehyde, sinapaldehyde, syringaldehyde) were also studied
quantification, and chlorogenic and vanillic acids were under the limit
(Table 2 and Fig. 1). 4-methylumbelliferone was under the limit of the
of the detection of the method. Gallic acid was also reported for
detection of the method and coniferaldehyde was the major lignin-de-
blackberries R. adenotrichus and R. glaucus and the values were 460 and
rived aldehydes, with contents of up to 72.54 μg 100 g−1 DM.
1800 μg 100 g−1 DM, respectively (Mertz, Cheynier, Günata, & Brat,
The present study represent a great advancement to elucidate the
2007).
polyphenolic composition of the blackberry R. ulmifolius, considering
Regarding to the flavonoids, thirteen compounds were identified
that there is only one study that evaluated non-anthocyanins phenolics
(Table 2 and Fig. 1). Quercetin and isoquercitrin were the flavonoids
in this species, which found quercetin and gallic acid (Ruiz-Rodriguez
quantified at highest concentrations (5509.61 to 9149.75 and 1526.86
et al., 2014).
to 1888.51 μg 100 g−1 DM, respectively), and the predominant phe-
The maturation process influenced the phenolic compounds con-
nolic compounds in the sample. Epigallocatechin gallate, hesperidin,
tents in blackberry fruits, with significant statistical difference
naringin, and rutin were under the limit of detection of the method.
(p < .05) between the two edible maturation stages (Table 2). The
Quercetin was also one of the main compounds found in the blackberry
highest concentrations of individual phenolics were found in the
R. fruticosus studied by Jakobek, Šeruga, Šeruga, Novak, and

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18

A
Intensity (cps)

10,11
8,9
12
1 16
5,6,7 19, 20,21,22,23
4
2,3
24,25

15
13,14

6.5

Time (min)

17,18

B
Intensity (cps)

2,3 10,11
16
8,9
13,14
1 19,20,21,22,23
4 5,6,7
24,25,26

12 15

6.5
Time (min)

Fig. 1. Total ion chromatogram (TIC) of mature (A) and fully mature (B) blackberry (Rubus ulmifolius Schott).
1 – gallic acid (2.32 min); 2 – p-aminobenzoic acid (3.05 min); 3 – 3,4-dihydroxybenzoic acid (3.18 min); 4 – (+)-catechin (3.82 min); 5 – caffeic acid (4.25 min); 6 –
(-)-epicatechin (4.27 min); 7 – syringic acid (4.30 min); 8 – p-coumaric acid (4.99 min); 9 – isoquercetin (5.01 min); 10 – syringaldehyde (5.07 min); 11 – sinapic acid
(5.13 min); 12 – ferulic acid (5.20 min); 13 – sinapaldehyde (5.64 min); 14 – coniferaldehyde (5.71 min); 15 – benzoic acid (5.84 min); 16 – salicylic acid (6.10 min);
17 – luteolin (6.24 min); 18 – quercetin (6.29 min); 19 – apigenin (6.73 min); 20 – naringenin (6.80 min); 21 – pinobanksin (6.83 min); 22 – kaempferol (6.85 min);
23 – isorhamnetin (6.92 min); 24 – chrysin (7.96 min); 25 – pinocembrin (8.10 min); 26 – galangin (8.13 min)

mature stage for most of the studied compounds, as well as for the sum (Schulz et al., 2015), jabuticaba, guabiju, and jambolan (Seraglio et al.,
of phenolic compounds. This behavior was also observed in other ber- 2018). These changes during maturation are related to the polyphenols
ries such as blackberry R. adenotrichus (Acosta-Montoya et al., 2010), synthesis, which occurs in the phenylpropanoid metabolic pathway
açaí (Euterpe oleracea) (Gordon et al., 2012), juçara (Euterpe edulis) under enzymatic control. The synthesis of these compounds changes

631
M. Schulz et al. Food Research International 122 (2019) 627–634

according to the maturation stage of the fruits, since the enzyme ac- observed for blackberry R. adenotrichus (29.00 to 35.00 22 mg GAE g−1
tivity also change during the different phases of growth and develop- DM) (Acosta-Montoya et al., 2010) and for raspberry R. hirsutus (Fu
ment of the fruit (Elmastaş, Demir, Genç, Dölek, & Güneş, 2017; Schulz et al., 2015) for FC assay. In the FC assay, sugars are potential inter-
et al., 2015). The high concentration of quercetin in the immature ferents in concentration higher than 0.1 g mL−1 and elevated tem-
fruits, for example, may be related to the protective function of the perature (Singleton, Orthofer, & Lamuela-Raventós, 1999). However, in
photosynthetic apparatus of the plant. Quercetin occurs in the chlor- this study, the assay was performed in room temperature and the sugar
oplast envelope membrane, presenting photoprotection action, espe- concentration in the extract was about 0.01 g mL−1, minimizing this
cially against lipid peroxidation (Dobrikova & Apostolova, 2015). interference.
Therefore, quercetin levels can decrease with the advancement of ma- The increase of the antioxidant capacity with the advancement of
turation due to the decrease in the chlorophyll content (Ghasemnezhad, the maturation was contrary to the behavior of the investigated non-
Sherafati, & Payvast, 2011). The lower concentrations of the flavonoids anthocyanin phenolics, fact already reported for other berries such as
catechin and epicatechin in the more mature fruits may be related to blackberry R. adenotrichus (Acosta-Montoya et al., 2010), raspberry R.
the synthesis of blackberry proanthocyanidins. The proanthocyanidins hirsutus (Fu et al., 2015), juçara (Schulz et al., 2015) and açaí (Gordon
biosynthesis evolve the reduction of leucocyanidin to catechin by the et al., 2012). These findings suggest that the compounds investigated
leucoanthocyanidin reductase and the reduction of cyanidin to epica- are not primarily responsible for the antioxidant potential of this fruit.
techin by the anthocyanidin reductase. Both flavonoids can form the However, the increase in TMA content with the advancement of ma-
terminal unit of the proanthocyanidin polymer (Bogs et al., 2005; Hong turation can be related to the increase of the antioxidant capacity of the
et al., 2014). R. ulmifolius, since these compounds present reducing properties at-
For TMA, the levels were significantly higher (p < .05) in the fully tributed to the hydroxyl groups attached to ring structures (Wang & Lin,
mature stage than the mature stage (11.72 and 2.61 mg cy-3-glu g−1 2000). The contribution of the anthocyanin fraction to the antioxidant
DM, respectively) (Table 3). These values were higher than those found capacity assessed by the FRAP method was confirmed by the study of
for blackberry R. adenotrichus, which ranged of 0.5 to 5.2 mg cy-3-glu Coklar and Akbulut (2017) in Mahonia aquifolium berries. Vitamin C is
g−1 DM during ripening (Acosta-Montoya et al., 2010). This trend of other possible compound related to the potential antioxidant observed,
increasing in total monomeric anthocyanin concentration along ma- since the increase in its content with the advancement of maturation
turation was also found for strawberry (Ornelas-Paz et al., 2013), was observed in strawberry, cherry, and mulberry fruits (Mahmood,
cherry (Mozetič, Trebše, Simčič, & Hribar, 2004; Serrano, Guillén, Anwar, Abbas, Boyce, & Saari, 2012). Contents of up to 27 mg ascorbic
Martínez-Romero, Castillo, & Valero, 2005), jabuticaba, guabiju, jam- acid 100 g FW were found by Ruiz-Rodriguez et al. (2014) in R. ulmi-
bolan (Seraglio et al., 2018), and mulberry (Lee & Hwang, 2017). It has folius.
been shown that anthocyanins are present in the fleshy parts of the fruit
and increase during maturation (González, Vogel, Razmilic, & Wolfram, 3.4. Sugars and minerals
2015), presenting a range of colors progressing from green to black
(Gould, Davies, & Winefield, 2009). The results for sugar determination in the two edible maturations
stages are given in Table 3. The concentrations of fructose, glucose and
total sugar significantly increased (p < .05) during maturation, pre-
3.3. Antioxidant capacity senting the highest concentrations on the fully mature stage. Fructose
and glucose were the major sugars found, and in all maturation stages
The results for antioxidant capacity are show in Table 3. During the of blackberry, sucrose was lower than the limit of quantification. The
maturation of blackberry, the fully mature fruit presented significantly increase in fructose, glucose and total sugar along maturation may be
(p < .05) higher values of antioxidant capacity in all tests, with values attributed to hydrolysis of starch into simple sugars (Kulkarni &
of 241.06 μM Fe+2 g−1 DM for FRAP assay and 28.22 mg GAE g−1 DM Aradhya, 2005). This trend has also been reported for other varieties of
for FC assay. This increase in the antioxidant capacity was also blackberry, such as Rubus L. (Tosun et al., 2008), R. adenotrichus
(Acosta-Montoya et al., 2010).
Table 3 Acosta-Montoya et al. (2010) determined sugars in three maturation
Antioxidant capacity, total monomeric anthocyanins content, sugars and mi- stages of blackberry R. adenotrichus and reported lower values than
nerals in blackberry (Rubus ulmifolius Schott) in two edible maturation stages.
those found in our study for fructose (2.5, 8.1 and 10.4 mg 100 g−1
Parameters Blackberry DM), glucose (2.9, 9.2 and 11.7 mg 100 g−1 DM) and total sugar (5.5,
13.1 and 22.1 mg 100 g−1 DM). Hassimotto, Da Mota, Cordenunsi, and
Mature Fully mature
Lajolo (2008) assessed sugars in five cultivars of fully mature black-
Antioxidant capacity berry (Rubus sp.) and found similar results for total sugar, but in con-
FRAP assay (μmol Fe+2 g−1 DM) 201.05 ± 4.69b 241.06 ± 4.44a trast to our findings they found glucose concentrations higher than
Folin-Ciocalteu assay (mg GAE g−1 DM) 19.18 ± 0.40b 28.22 ± 0.28a fructose. These differences among our data and those reported by the
Total monomeric anthocyanins content (mg 2.61 ± 0.06b 11.72 ± 0.24a
authors could be justified by differences in the cultivars, plant nutrition,
cy-3-glu g−1 DM)
Fructose 22.52 ± 0.74b 26.00 ± 0.47a soil, and climatic conditions (Fawole & Opara, 2013).
Glucose 17.38 ± 0.48b 19.12 ± 0.01a The changes in mineral composition in the two studied stages of
Sucrose < LOD < LOD blackberry are presented in Table 3. The concentrations of potassium,
Sum of quantified sugars (g 100 g−1 DM) 39.90 ± 1.22b 45.12 ± 0.46a sodium, calcium, and the sum of quantified minerals were significantly
K 24.09 ± 1.63a 15.40 ± 0.47b
Na 5.05 ± 0.22a 1.68 ± 0.03b
higher (p < .05) in the mature stage than in the fully mature stage,
Ca 6.39 ± 0.05a 4.33 ± 0.18b with exception of magnesium that was higher in the fully mature fruits.
Mg 3.35 ± 0.06b 7.07 ± 0.08a In both maturation stages, manganese was under the limit of detection.
Mn < LOD < LOD These variations in the mineral content at different stages may be re-
Sum of quantified minerals (mg g−1 of DM) 38.88 ± 1.43a 28.49 ± 0.18b
lated to biochemical changes that occur in the maturation process. For
Results expressed as mean ± standard deviation. a-b Mean values in the same example, the higher calcium levels in the mature stage may be due to
row followed by different letters indicate significant differences (p < .05) the reduction of this mineral in the cell wall during ripening, since
using Student's t-test. GAE – gallic acid equivalent. Cy-3-glu – cyanidin 3-glu- protopectin solubilization could have occurred resulting in calcium
coside. DM – dry matter. LOD – Limit of detection. Sucrose limit of release (Costa, de Baeta, Saraiva, Verissimo, & Ramos, 2011). Changes
detection = 0.022 g L−1. Mn limit of detection = 0.27 mg L−1. in the potassium levels may be due to its great mobility in plants, since

632
M. Schulz et al.
is an important component in the synthesis of compounds such as
proteins and starch, as well as is linked to the metabolism of carbo-
hydrates and enzymatic processes (Santos et al., 2015; Zhou et al.,
2018).
Potassium was the major mineral found in both stages in the studied
fruits, followed by calcium, magnesium and sodium. Ahmad et al.
(2015) also found potassium, calcium, sodium, and magnesium as the
major minerals in three blackberry species (R. ellipticus, R. niveus, R.
ulmifolius) grown in Himalayan. In general, our results for potassium,
calcium, and magnesium in both edible stages were higher than those
reported by Tosun et al. (2008) for three maturation stages of Rubus L.
(14.58, 15.39 and 13.75 mg g−1 for potassium; 0.96, 0.67, 0.81 mg g−1
for calcium; 4.51, 4.49 and 2.90 mg g−1 for magnesium, all of them
expressed in DM), by Ahmad et al. (2015) for R. ulmifolius of Hyma-
layan (8.60 mg g−1 DM for potassium, 6.20 mg g−1 DM for calcium,
1.48 mg g−1 DM for magnesium), and by De Souza et al. (2014) for
Rubus spp. (6.6 mg g−1 DM for potassium, 0.6 mg g−1 DM for calcium,
1.30 mg g−1 DM for magnesium).
4. Conclusion
The results obtained in our study show that blackberry Rubus ul-
mifolius is a high-value source of nutritional and bioactive compounds,
since presented a large number of phenolics (especially flavonoids and
phenolic acids), high antioxidant capacity, high mineral and mono-
saccharides. According to our findings, we can also conclude that the
maturation significant influenced the chemical composition of black-
berry, however, both edible maturation stages present relevant con-
centrations for nutritional and bioactive use.
The research on the nutritional and bioactive compounds aims to
encourage the cultivation and the consumption of fruits less explored
commercially as blackberry, which can contribute to new sources of
food with potential health benefits, income for agricultures, basic
knowledge to optimize harvests and valorization of biodiversity.
Acknowledgments
This study was financed in part by the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) –
Finance Code 001. The authors also wish to thank the Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and
Fundação de Amparo à Pesquisa do Estado de Santa Catarina (FAPESC)
for fellowships and financial support, and the Cabanha Seraglio for the
fruit samples.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodres.2019.01.034.
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