Journal of Functional Foods 46 (2018) 243–249

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Journal of Functional Foods

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

Sour cherry (Prunus cerasus L.) juice protects against hydrogen peroxide- T
induced neurotoxicity by modulating the antioxidant response
⁎ ⁎
Guillermo Cásedasa, Elena González-Burgosb, , Carine Smithc, Víctor Lópeza, ,
María Pilar Gómez-Serranillosb
a
Department of Pharmacy, Faculty of Health Sciences, Universidad San Jorge, 50.830 Villanueva de Gállego, Zaragoza, Spain
b
Department of Pharmacology, Pharmacognosy and Botany, Faculty of Pharmacy, Universidad Complutense de Madrid, 28040 Madrid, Spain
c
Department of Physiological Sciences, Science Faculty, Stellenbosch University, Stellenbosch, South Africa

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

Keywords: Sour cherry (Prunus cerasus L.) juice is a relevant source of polyphenols and antioxidants. The purpose of this
Sour cherry juice study was to evaluate the protective effects and mechanism of action of sour cherry juice under oxidative stress
Antioxidant enzymes conditions using hydrogen peroxide as neurotoxic agent in the SH-SY5Y cell line. Cells were pre-treated with the
ROS juice for 24 h followed by the exposure to 0.1 mM hydrogen peroxide for 30 min. Sour cherry juice prevented
Polyphenols
cells from hydrogen peroxide-induced toxicity and from the increase of reactive oxygen species and lipid per-
Neuroprotection
SH-SY5Y
oxidation. Moreover, sour cherry juice restored the ratio GSH/GSSG as well as the activity of antioxidant en-
zymes (catalase, superoxide dismutase, glutathione reductase and glutathione peroxidase). Furthermore, the in
vitro ORAC and FRAP assays confirmed the antiradical and reducing activity. These results demonstrate that sour
cherry juice could be of interest for the prevention of oxidative stress-related pathologies in the central nervous
system.

1. Introduction increase of thiobarbituric acid reactive substances (TBARS) in cortical

Reactive oxygen species (ROS) exist as products of normal cellular
physiology and play vital roles in the stimulation of signalling pathways
in plant and animal cells (Jabs, 1999). Among these ROS are included
hydroxyl radical, superoxide anion, hydrogen peroxide, singlet oxygen,
nitric oxide, hypochlorous acid and peroxynitrite (Rodríguez-Chávez,
Coballase-Urrutia, Nieto-Camacho, & Delgado-Lamas, 2015). When
ROS increase upon certain levels, they may give rise to a cell damage
(DNA base modifications, lipid peroxidation, protein oxidation) and
could contribute to disease pathogenesis of major neurodegenerative
disorders such as Parkinson’s disease (PD), Alzheimer’s disease (AD),
multiple sclerosis and amyotrophic lateral sclerosis (ALS) (Kelsey,
Hulick, Winter, Ross, & Linseman, 2011). However, under certain
conditions, cells recruit intrinsic antioxidant defence systems to uphold
intracellular redox homeostasis and protect themselves against oxida-
tive damage increasing levels of superoxide dismutase, catalase and
glutathione in its reduced form (GSH) (Ye, Han, Wang, & Yu, 2009). In
fact, previous clinical studies have demonstrated a reduced activity of
antioxidant enzymes (Zemlan, Thienhaus, & Bosmann, 1989), an
tissue in patients with Alzheimer’s disease (Subbarao, Richardson, &
Ang, 1990) and low GSH in the substantia nigra of patients with Par-
kinson’s disease (Andersen, 2004).
From an international perspective, the market of fruit juices re-
presents a great impact in the economy and nutrition of certain coun-
tries. For example, in the United States, revenue amounts to US$
9,943 m and an average per capita consumption of 17.1L in 2017
(statista).
Historically, fruit juices have been also recommended by paedia-
tricians as source of vitamins and water in infants and young children in
order to prevent constipation and ameliorate hydration but the current
recommendations from the American Academy of Pediatrics state that
fruit juice should not be introduced into the diet of infants before
12 months of age (Heyman, Abrams, 2017). In adults and adolescents,
fruit juice may represent a source of interesting bioactive molecules and
the European Association of the Study of Obesity has recently devel-
oped a Healthy Hydration Working Group, arguing for the urgency of
offering healthy drinks (Lafontan, 2014).
Previous research shows that sour cherry and related products may

⁎
Corresponding authors at: Facultad de Ciencias de la Salud, Universidad San Jorge, Campus Universitario Villanueva de Gállego, Autovía A-23 Zaragoza-Huesca Km. 299, 50.830
Villanueva de Gállego, Zaragoza, Spain (Víctor López). Departamento de Farmacología, Farmacognosia y Botánica, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza
Ramón y Cajal s/n, 28040 Madrid, Spain (Elena González-Burgos).
E-mail addresses: elenagon@ucm.es (E. González-Burgos), ilopez@usj.es (V. López).

https://doi.org/10.1016/j.jff.2018.04.055
Received 22 December 2017; Received in revised form 21 March 2018; Accepted 26 April 2018
Available online 26 May 2018
1756-4646/ © 2018 Elsevier Ltd. All rights reserved.
G. Cásedas et al.
be healthy foods due to the presence of certain bioactive molecules. A
study carried out in 34 human subjects concluded that a daily con-
sumption of sour cherries for 30 days showed a reduction in oxidative
stress suppressed the formation of reactive oxygen species by circu-
lating phagocytes due to its high content in polyphenols, specifically
anthocyanins (Bialasiewicz et al., 2017). The antioxidant activity of
sour cherry juice and its capacity for inhibiting enzymes present in the
central nervous system suggest that sour cherry juice may have also
certain neuroprotective activity (Cásedas, Les, Gómez-Serranillos,
Smith, & López, 2016). Animal studies have also demonstrated a neu-
roprotective effect for sour cherry products; for example, rats with age-
related deficits presented amelioration in behavioural and neuronal
function when they were fed with tart cherry-supplemented diet
(Thangthaeng et al., 2016). Another study run in animals concluded
that tart cherry treatment decreased behavioural and pathological
deficits in a mouse model of Alzheimer’s disease (Matchynski et al.,
2013). A maternal dietary supplementation study in mice with food rich
in polyphenols also showed a neuroprotective effect in neonatal mouse
brain (Loren, Seeram, Schulman, & Holtzman, 2005). Studies also re-
veal that anthocyanins, which are present in sour cherry products are
potential neuroprotective agents ((Kim, Ho, Young, Hyun, & Lee, 2005).
The aim of this study is to evaluate, for the first time, the protective
effects and mechanism of action of sour cherry juice under oxidative
stress conditions using hydrogen peroxide as neurotoxic agent in the
human neuroblastoma SH-SY5Y cell line.
2. Materials and methods
2.1. Reagents and chemicals
Fetal bovine serum (FBS), phosphate-buffered saline (PBS) and
gentamicin were purchased from Gibco (Invitrogen, Paisley, UK). 3-
(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT)
and dimethyl sulphoxide (DMSO), 2,2́ -azobis(2-methylpropionami-
dine)-di-hydrochloride (AAPH), 2,4,6- Tris (2-pyridyl)-1,3,5-triazine
(TPTZ), Dulbecco’s modified Eagle’s medium (DMEM), hydrogen per-
oxide solution (30% w/w), 6-hydroxy-2,5,7,8-tetramethylchromane-2-
carboxylic acid (TROLOX), 2,7-dichloro-dihydrofluorescein diacetate
(DCFH-DA) were purchased by Sigma-Aldrich (St. Louis, MO, USA).
2.2. Sour cherry juice material
Sour cherry juice (Rabenhorst®) was provided by a specialized in-
dustry. Cherries were harvested from different crops from Germany and
Austria. The juice was lyophilized using a Genesis VirTis 25 EL lyo-
philizer (Wizard 2.0 control system) over 7 days. The phytochemical
analysis showed a relevant content of polyphenols such as phenolic
acids and anthocyanins. Main compounds detected were ascorbic acid,
chlorogenic and neochlorogenic acid, gallic acid and cyanidin 3-glu-
cosyl-rutinoside (Table 1). The nutritional information per 100 mL of
juice is provided as supplementary material. All the information about
Table 1
Polyphenol content of sour cherry juice.
Compound µg/mg of lyophilized juice (S.E.)
Ascorbic acid 0.011 (0.003)
Gallic acid 0.047 (0.001)
Ellagic acid –
Catechin – reactive species of thiobarbituric acid (TBARS) (Mitsuru Uchiyama,
Chlorogenic acid 0.593 (0.001)
Neochlorogenic acid 1.580 (0.012)
Cyanidin 3-glucosil-rutinoside 0.081 (0.000)
Total polyphenols (Folin method) 9.835 ± 1.092
Total anthocyanins (HPLC method) 0.194 (0.003)
–: not detected; S.E. = standard error.
244
Journal of Functional Foods 46 (2018) 243–249
lyophilization and phytochemical analysis was previously published
(Cásedas et al., 2016).
2.3. SH-SY5Y cell line
2.3.1. Cell culture
Human neuroblastoma SH-SY5Y cells were cultured in DMEM sup-
plemented with 10% fetal bovine serum (FBS) and 1% gentamicin, and
maintained in a humidified atmosphere of 5% CO2 at 37 °C (González-
Burgos, Fernández-Moriano, Lozano, Iglesias, & Gómez-Serranillos,
2017). SH-SY5Y cells were seeded in different culture plates (96-well
plate, 12-well plate and Petri plate) at a fitting density. DMEM was
regularly replaced every two or three days and sub-cultured once they
reached 80–90% confluence.
2.3.2. Cell treatments
Cells were pre-treated with lyophilized sour cherry juice in a range
of concentrations from 25 µg/mL to 500 µg/mL for 24 h previous to
hydrogen peroxide exposure (100 µM) for 30 min. The lyophilized sour
cherry juice was first dissolved in DMSO and then serial dilutions in PBS
were made, being the final concentration of DMSO less than 0.1%. The
protective effects were performed at 25–50–100 µg/mL because these
concentrations were not toxic and represent an amount of anthocyanins
that could be physiologically relevant.
2.4. Cell viability: MTT assay
Cells were cultured in 96-well plates at a density of 5 × 104 cells per
well. After treatments, cells were incubated with MTT solution (2 mg/
mL) for 1 h at 37 °C. Successively, the medium was replaced with DMSO
in each well to dissolve formazan crystals. Finally, absorbance was
measured at 550 nm with a FLUOstar Omega microplate reader (BMG
Labtech, Ortenberg, Germany). All experiments were performed in tri-
plicate and results expressed as percentage of control (100%).
2.5. Production of intracellular reactive oxygen species (ROS)
DCFH-DA (2,7-dichloro-dihydrofluorescein diacetate) is a fluor-
escent reagent which is used to detect the production of ROS at in-
tracellular level (LeBel, Ischiropoulos, & Bondy, 1992). Experiment day
cells were incubated in PBS-glucose + 50 µL of DCFH-DA (0.01 M) at
37 °C and preserved from the light for 30 min. After this time, cells were
twice washed with PBS-glucose and then different concentration of
lyophilized sour cherry juice and H2O2 were added. Generation of ROS
was measured at several times (0′ 10′ 30′ 60′ 90′) in a microplate
fluorescence reader (FLx800, Bio-Tek Instrumentation) at λexcitation of
480 nm and λemission of 580 nm.
2.6. BCA assay: Protein content
Bicinchoninic acid (BCA) assay is a colorimetric method to de-
termine the total concentration of protein using bovine serum albumin
(BSA) as a reference standard protein. Absorbance was measured at
550 nm using a FLUOstar Omega microplate reader (BMG Labtech,
Ortenberg, Germany) (Smith et al., 1985).
2.7. Thiobarbituric acid reactive species (TBARS)
Lipid peroxidation was determined by measuring the formation of
1978). After treatments, cell pellets were frozen at −80 °C. Then, 50 μL
of cells were defrosted at room temperature, mixed with 100 μL of TBA-
TCA-HCl reaction mixture and boiled at 100 °C for 10 min. Finally, the
reaction was stopped on ice for 20 min and total cell lysates were
centrifuged for 10 min at 3000 rpm (4 °C). Samples were moved to a 96-
well plate and absorbance was read at 530 nm using a FLUOstar Omega
G. Cásedas et al. Journal of Functional Foods 46 (2018) 243–249

microplate reader (BMG Labtech, Ortenberg, Germany). (A)

2.8. Measurement of reduced/oxidized glutathione index ratio (GSH/ 100
GSSG)

% Cell viability
of control
To measure glutathione levels, a fluorometric method was com-
pleted using a fluorescent reagent (OPT) (Hissin & Hilf, 1976). Oxidized
50
levels (GSSG) were determined by adding 50 μL of lysed cells and 3 μL
of 7.5 mM of N-ethylmaleimide (NEM) to the well. After 5 min in the
dark at room temperature, 150 μL of 0.1 N NaOH buffer (pH 12) and
20 μL of 1 mg/mL ortho-phthalaldehyde (OPT) were added to the mix-
ture and incubated for 15 min. On the other hand, 50 μL of lysed cells 0
and 150 μL of 0.1 M phosphate buffer – 5 mM EDTA (pH 8) were mixed

ol

25

50

0
10

20

30

40

50
with 20 μL of 1 mg/mL OPT to calculate reduced glutathione (GSH)

tr
on
C
levels after 15 min of incubation. Standard curves were performed at
different concentrations of GSH and GSSG to determine final con- Sour cherry juice (μg/ml)
centrations of glutathione levels. Emission wavelength was set up at
(B)
350 nm and excitation at 420 nm to measure the absorbance in a mi-
100
croplate fluorescence reader (FLx800, Bio-Tek Instrumentation).

% Cell viability
2.9. Determination of antioxidant enzymatic activity

(of control)
**** ** **
2.9.1. Catalase (CAT) activity
50 #
A volume of 670 μL of a H2O2 (15 mM) buffer solution were mixed
with 30 μL of total cell lysates. The activity was measured in a quartz
cuvette for 1 min at 25 °C and the absorbance was measured at 240 nm
using Uvikon Spectrophotometer 930 (Kontron Instruments, UK). Molar
extinction coefficient of H2O2 was 43.6 mL nmol−1 cm−1 and results 0
were expressed as percentage of CAT activity of the control.

ol

25

50

0
O
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10
tr

2.9.2. Superoxide dismutase (SOD) activity
Following the procedure of (Marklund & Marklund, 1974), the re-
action mixture contained 10 μL of pyrogallol (0.15 mM) diluted in HCl
(10 mM), 25 μL of total cell lysates and 765 μL of Tris–DTPA buffer
(50 mM), pH 8.2. The absorbance was recorded at 420 nm for 1 min
using an Uvikon Spectrophotometer 930 (Kontron Instruments, UK).
Results were expressed as percentage of SOD activity of the control.
2.9.3. Glutathione peroxidase (GPx) activity
GPx activity was determined in a 96-well plate following the pro-
cedures written by (Rotruck et al., 1973) and (Wilson, Zucker, Huang, &
Spector, 1989). One hundred μL of 50 mM phosphate buffer −6.3 mM
EDTA (pH 7.4), 20 μL of total cell lysates, 2 μL of 0.048 U GR, 20 μL of
10 mM GSH were incubated for 5 min in dark conditions. After this
incubation time, 20 μL of 1.2 mM NADPH and 20 μL of 63.50 mM H2O2
were added to start the reaction. Absorbance was read at 340 nm every
minute for 10 min using a FLUOstar Omega microplate reader (BMG
Labtech, Ortenberg, Germany).
2.9.4. Glutathione reductase (GR) activity
The activity of GR was assessed using the protocol established by
(Staal, Helleman, Wael, & Veeger, 1969) and performed in a 96-well
plate. A volume of 116 μL of 50 mM phosphate buffer −6.3 mM EDTA
(pH 7.4), 10 μL of total cell lysates, 7 μL of 5 mM GSSG and 7 μL of
1.2 mM NADPH were added into the well. Absorbance was measured at
340 nm every minute for 10 min using a FLUOstar Omega microplate
reader (BMG Labtech, Ortenberg, Germany).
2.10. In vitro antioxidants assays
2.10.1. Oxygen radical antioxidant capacity (ORAC) method
ORAC assay was performed to measure the capacity of sour cherry
juice to scavenge peroxyl radicals. Trolox was used as antioxidant re-
ference compound. Sour cherry juice was dissolved at a concentration
of 1 mg/mL in methanol/PBS (50:50) and then diluted so that the
concentrations in the wells ranged from 1 and 500 µg/mL. Lyophilized
H
2
on
C
Sour cherry juice (μg/ml)
+ H2O2 100μM
Fig. 1. Effects of sour cherry juice on SH-SY5Y cell viability measured by MTT
assay. (A) Cytotoxicity of 24-h treatment with different concentrations of sour
cherry juice from 25 to 500 µg/mL (B) Protective effect of sour cherry juice on
hydrogen peroxide-induced neurotoxicity. SH-SY5Y cells were pretreated with
different concentrations of sour cherry juice for 24 h previous to H2O2 (100 µM,
30 min). Results are expressed as mean ± standard deviation of data from
three independent experiments. **p < 0.001 versus hydrogen peroxide;
****p < 0.0001 versus hydrogen peroxide; #p < 0.0001 versus control.
samples were incubated with fluorescein (70 nM) for 10 min at 37 °C in
96-well plates. After incubation, AAPH (12 mM) was added and fluor-
escence was recorded for 98 min at excitation and emission wave-
lengths of 485 nm and emission of 520 nm, respectively, in a FLUOstar
Optima fluorimeter (BMG Labtech, Ortenberg, Germany) (Dávalos,
Gómez-Cordovés, & Bartolomé, 2004). Results were expressed as TE/
mg extract.
2.10.2. Ferric reducing antioxidant potential (FRAP) method
FRAP assay was carried out in order to determine the ferric anti-
oxidant activity of sour cherry juice. The reaction measures reduction of
ferric 2,4,6-tripyridyl-s-triazine (TPTZ) to a coloured product using
ferric chloride and buffer solution (Fernández-Moriano, González-
Burgos, Divakar, Crespo, & Gómez-Serranillos, 2016). Sour cherry juice
(1 mg/mL) was mixed with FRAP reagent and incubated 30 min at
37 °C. Absorbance was read at 595 nm in a FLUOstar Omega microplate
reader (BMG Labtech, Ortenberg, Germany). Results were expressed as
Fe2+/g extract.
2.11. Statistical analysis
Each experiment was performed at least three times. Data represent
means ± SEM for the total number of experiments done. All data were

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G. Cásedas et al. Journal of Functional Foods 46 (2018) 243–249

### Fig. 2. Effects of sour cherry juice on in-

(A) #### #
Control tracellular reactive oxygen species (ROS)
measured by dichlorofluorescein assay and
production (% of control)

H2O2 (100 μM) lipid peroxidation. (A) SH-SY5Y cells were

150
Intracellular ROS

pretreated with different concentrations of

Sour cherry juice (25 μg/ml) sour cherry juice (25, 50 and 100 µg/mL)
**** Sour cherry juice (50 μg/ml) previous to hydrogen peroxide (100 µM) for
** measuring intracellular ROS production
Sour cherry juice (100 μg/ml) along 90 min. (B) Effects on lipid peroxida-
100
tion expressed as percentage of thiobarbituric
acid reactive substances (TBARS) compared
to control cells. Results are expressed as
mean ± standard deviation of data from
three independent experiments. **p < 0.01
50 versus hydrogen peroxide; ***p < 0.0005
0 20 40 60 80 100 versus hydrogen peroxide; ****p < 0.0001
Time (min) versus hydrogen peroxide; #p < 0.05 versus
control; ###p < 0.001 versus control;
(B) 300 #### ####p < 0.0001 versus control.
(% of control)

200 ***
TBARS

*** ***

100

0
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25

50

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10
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H
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on
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Sour cherry juice (μg/ml)

+ H2O2 100μM

Table 2 the cells.

Effects of 24-h pretreatments with different concentrations of sour cherry juice
previous to hydrogen peroxide (100 µM, 30 min) on glutathione index redox
(IR) and related enzymes in SH-SY5Y cells. Effects on GR activity, GPx activity
and glutathione levels expressed as index redox (reduced glutathione/oxidized
glutathione; GSH/GSSG). Results are expressed as mean ± standard error of
meaning deviation of data from three independent experiments. *p < 0.05
versus hydrogen peroxide; **p < 0.01 versus hydrogen peroxide; ***p < 0.001
versus hydrogen peroxide; ## p < 0.01 versus control; #### p < 0.0001
versus control.
Treatments GR activity (%) GPx activity (%)
The MTT method was also used to evaluate the protective effect of
sour cherry juice on H2O2-induced damage. As shown in Fig. 1B, ex-
posure to the cells to 100 μM H2O2 for 30 min caused a significant re-
duction of cell viability (more than 70%). However, the 24-h pre-
treatment with sour cherry juice previous to H2O2 exposure improved
the cell viability at the tested concentrations of 25, 50 and 100 μg/mL
(Fig. 1B). Since these three concentrations of 25, 50 and 100 μg/mL
exerted a cytoprotective effect on neuron-like cells, these were chosen
to perform the following experiments.
IR (GSH/GSSG)

Control 100 100 4.828 ± 0.43 3.2. Effect of sour cherry juice on intracellular reactive oxygen species
H2O2 7.65 ± 1.54b 7.463 ± 0.42#### 2.285 ± 0.38## (ROS) production in SH-SY5Y cells
25 25.40 ± 8.46* 35.28 ± 0.76** 4.843 ± 0.60*
50 31.20 ± 3.51** 42.85 ± 7.06** 5.110 ± 1.24
H2O2 increased intracellular ROS formation up to 150% compared
100 33.84 ± 3.71** 27.31 ± 3.92* 5.280 ± 0.85*
to control cells (100%) as evidenced in the dihydrofluorescein assay
(Fig. 2A). However, when SH-SY5Y cells were treated with sour cherry
analysed using Student t-test or one-way ANOVA with a post hoc juice (25, 50 and 100 μg/mL), the levels of intracellular ROS were
Tukey’s test. Significant differences were considered when p-value < significantly decreased in a time-concentration dependent manner
0.05. Statistical analyses and figures were performed in GraphPad compared to those cells treated only with H2O2, being the concentra-
Prism v.6. tions of 25 and 50 μg/mL of sour cherry juice the most active by de-
creasing ROS formation in 30% over control cells after 90 min treat-
ment (p < 0.001).
3. Results
3.3. Effect of sour cherry juice on redox biomarkers in SH-SY5Y cells
3.1. Effect of sour cherry juice on SH-SY5Y cell viability
We then measured the effect of sour cherry juice on the redox
Initially, the MTT assay was used to test whether sour cherry juice biomarkers: lipid peroxidation and index ratio of reduced glutathione
could affect SH-SY5Y cell viability. For this purpose, cells were treated and oxidized glutathione.
with a range of concentrations from 25 to 500 μg/mL for 24 h. As shown The level of thiobarbituric acid-reactive substances (TBARS) as in-
in Fig. 1A, none of the assayed concentrations affected the viability of dicative of lipid peroxidation degree in cells was higher than 200% in

246
G. Cásedas et al. Journal of Functional Foods 46 (2018) 243–249

(A) 6 compared to control cells. On the other hand, pretreatments with the
three assayed concentrations restored GSH and GSSG levels as evi-
denced in index ratio GSH/GSSG. The most active concentration was
** 100 μg/mL which significantly enhanced the antioxidant response
Catalase Activity

** **
(UI/mg protein)

4 GSH/GSSG to control levels (Table 2).

3.4. Effect of sour cherry juice on antioxidant enzymes activity in SH-SY5Y

#
cells
2

After assessing the effect of sour cherry juice on redox biomarkers,

we then evaluated the effect on antioxidant enzymes activity. As shown
in Fig. 3 and Table 2, exposure to 100 μM H2O2 for 30 min produced a
0
reduction in the enzymatic activity of CAT, SOD, GPx and GR by 1.8,
l

25

50

0
o

O
2

10
11.6, 7.5 and 7.7%, respectively, compared to control cells. Pretreat-
tr

H
2
on

ments with sour cherry juice for 24 h ameliorated the CAT, GR and GPx
C

Sour cherry juice (μg/ml) activities. Particularly, sour cherry juice significantly increased CAT
+ H2O2 100μM activity up to 3.03 U/mg protein compared to H2O2-treated cells at the
concentration of 25 μg/mL (Fig. 3). Moreover, the concentrations of 25
(B) 30 and 50 μg/mL of sour cherry juice notably increased the activity of GPx
by 50.6 and 60.1%, respectively (Table 2). Furthermore, the activity of
GR was also significantly increased up to 39.92 and 43.20% at 50 and
100 μg/mL, respectively (Table 2).
(UI/mg protein)
SOD activity

20

# 3.5. Evaluation of in vitro antioxidant activity

10 Finally, we evaluated the in vitro antioxidant activity of the sour

cherry juice using ORAC and FRAP assays which are indicative of an-
tiradical and reducing properties. As shown in Table 3, ORAC value was
0.60 TE/mg of extract and FRAP value was 3.71 Fe2+/g of extract.
0
4. Discussion
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25

50

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10
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H
2
on
C

Sour cherry juice (μg/ml)
+ H2O2 100μM
Fig. 3. Effects of 24-h pretreatments with different concentrations of sour
cherry juice previous to hydrogen peroxide (100 µM, 30 min) on antioxidant
enzymes activity in SH-SY5Y cells. (A) Effect on CAT activity. (B) Lack of effect
on SOD activity. Results are expressed as mean ± standard deviation of data
from three independent experiments. **p < 0.01 versus hydrogen peroxide;
#p < 0.05 versus control.
Table 3
Antioxidant activity of sour cherry juice (Prunus cerasus L.) measured by
Oxygen Radical Antioxidant Capacity (ORAC) and Ferric Reducing Antioxidant
Potential (FRAP) assays. Results are expressed as mean ± standard deviation.
*p < 0.05.
Antioxidant assays Sour cherry juice
ORAC 0.60 ± 0.21
(μmol Trolox Equivalents per mg of extract)
FRAP 3.71 ± 1.29
(µmol Fe2+g−1 extract)
those SH-SY5Y cells treated only with H2O2. On the other hand, 24-h
pretreatments with sour cherry juice considerably reduced cellular lipid
peroxidation, being the concentrations of 25 and 50 μg/mL the most
active by decreasing TBARS levels to 129.2 and 114.9%, respectively,
compared to H2O2 treated cells (Fig. 2B).
The content in reduced glutathione (GSH), which has potent anti-
oxidant properties, and the content in oxidized glutathione (GSSG)
were measured and expressed as an index ratio GSH/GSSG. In SH-SY5Y
control cells, GSH concentration was 4.8 times higher than GSSG con-
centration. However, when cells were exposure to 100 μM H2O2 for
30 min, glutathione redox status was altered, increasing GSSG level and
consequently, reducing significantly GSH/GSSG index ratio to half
Consistent evidence suggest that phytonutrient-rich foods con-
sumption may be linked to a reduced risk or progression of major
neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s
disease (Cilla, Alegría, Attanzio, Garcia-Llatas, & Tesoriere, 2017;
Denzer and Münch, 2016). The intake of antioxidant vitamins such as
vitamin E and C may be in relation with a reduction in the prevalence
and incidence of Alzheimer’s disease (Zandi et al., 2004). Most of the
observed therapeutic actions for plant-based food are related to their
antioxidant properties. Polyphenols are compounds with antioxidant
potential based on their capacity to scavenge free radicals and to en-
hance endogenous antioxidant defences (Pisoschi, Pop, Cimpeanu, &
Predoi, 2016; Wilson et al., 2017). These natural antioxidants are pre-
sented mostly in fruits and vegetables, being sour cherry juice a great
source of these bioactive compounds (Cásedas et al., 2016; Mayta-
Apaza, Marasini, & Carbonero, 2017). In recent years, there is a
growing interest to better understand not only the nutritional value of
food components but also the health-beneficial properties. The use of
antioxidant compounds to decrease oxidative stress situation derived
from an excessive ROS generation or from a decrease of the antioxidant
defence physiological system and could help to prevent certain neuro-
degenerative diseases (Hussain et al., 2016).
Although there may be certain controversial points about the ca-
pacity of anthocyanins and other flavonoids to reach the central ner-
vous system, it has been previously demonstrated that these compounds
can be metabolized in the digestive system, absorbed and cross the
blood-brain barrier (Faria et al., 2014; Fornasaro et al., 2016; Talavéra
et al., 2005). Once in the central nervous system, anthocyanins could be
responsible for the prevention of certain biochemical disorders such as
modulation of oxidative stress (Pogačnik et al., 2016).
To the best of our knowledge, the neuroprotective activity of cherry
polyphenols has only been tested with extracts from various cultivars of
fresh sweet and sour cherries on PC12 cells exposed to H2O2 (Kim et al.,
2005). However, none study has previously investigated and deepen on
the neuroprotective mechanisms of action of sour cherry juice.

247
G. Cásedas et al.
Therefore, we have examined for the first time how sour cherry juice
exert a potential neuroprotective action under oxidative stress condi-
tions through redox control mechanisms. For this purpose, H2O2 has
been chosen as neurotoxic agent and the human neuroblastoma SH-
SY5Y cells as neuronal cell line, which is considered one the best
models for neuroprotection studies (Xie, Hu, & Li, 2010). H2O2 is found
at high concentrations in the brain because this organ requires over
25% of body’s total oxygen consumption (Milton, 2004). The neuro-
pathological features of this ROS lie in its capacity to form the most
potent oxidant, hydroxyl free radical, in presence of ferrous iron.
Moreover, experimental findings involve amyloid β peptide’s redox
chemistry mechanisms in Alzheimer’s disease onset and progression;
amyloid β-containing plaques produce H2O2 which interact with metal
ions leading to a cascade of ROS generation (Milton, 2004). Further-
more, H2O2 is also involved in the aetiology of Parkinson’s disease;
exogenous H2O2 participate in the aggregation of α-Synuclein (α-Syn)
in Lewy bodies and in consequence, in the death of dopaminergic
neurons (Xu et al., 2015).
The colorimetric MTT assay, based on the capacity of mitochondrial
dehydrogenases enzymes of viable cells to covert the yellow tetra-
zolium salt MTT into a soluble dark blue formazan product, was used to
determine the most effective non-cytotoxic concentrations of freeze-
dried sour cherry juice under oxidative stress experimental conditions.
Our findings demonstrated that the lowest tested concentrations of sour
cherry juice exerted a significant protective effect against hydrogen
peroxide-induced cell damage. Since H2O2 across cell membranes and it
is able to produce free radicals metal ion-catalyzed (Halliwell, 2001),
we analyse the effect of sour cherry juice on intracellular ROS pro-
duction by fluorimetric methods. All the assayed concentrations of sour
cherry juice could protect against hydrogen peroxide-induced ROS
generation even exhibited better results than untreated cells. The lower
intracellular ROS production in treated cells may be attributed to the
anthocyanins present in sour cherry juice (Lim et al., 2005). Antho-
cyanins have the ability to form a protective barrier against ROS
through hydrogen bonds in the lipid-water interface of the cell mem-
brane (Oteiza, Erlejman, Verstraeten, Keen, & Fraga, 2005). In addition,
anthocyanins are able to inhibit the formation of ROS induced by H2O2
at membrane level with potential beneficial effects as neuroprotective
and antihyperglycemic agents (Tarozzi et al., 2007).
The level of lipid peroxidation and the content in GSH and GSSG are
biomarkers of oxidative stress for clinical use. Numerous previous stu-
dies have shown high concentrations of isoprostanes, neuroprostanes,
acrolein and 4-hydroxy-2-nonenal as lipid peroxidation by-products in
brain of patients with Alzheimer's disease (Montine et al., 2002).
Moreover, malondialdehyde (MDA) has been also found increased in
substantia nigra and in plasma of Parkinson’s disease patients (Dexter
et al., 1989; Sanyal et al., 2009). Harmful effects such as loss of fluidity,
disorder of cell membrane leading to release of cell organelles, in-
creases in permeability to ions, and finally inactivation of membrane
enzymes are linked to high levels of lipid peroxidation (Ye et al., 2009).
Results presented herein demonstrate that sour cherry juice could avoid
TBARS increasing in H2O2-induced cells and consequently, prevent
from lipid peroxidation. In a recent work, Howatson et al. (2010) re-
vealed that tart cherry juice has a protective role against oxidative
stress in runners through TBARS decrease and other parameters of
muscle inflammation. Regarding GSH and GSSG levels, the reduced
glutathione is the most important endogenous antioxidant compound to
deal with reactive oxygen species and re-balance redox status. Our
findings show that pretreatments with sour cherry juice restore GSH/
GSSG ratio alterations caused by H2O2, suggesting that GSH system is
involved in the cytoprotective mechanism of action of sour cherry juice.
The primary antioxidant enzymes CAT, SOD and GPx participate in
ROS detoxification. CAT transforms H2O2 into molecular oxygen and
water in the peroxisome (Zhang et al., 2016). SOD, located in the mi-
tochondria and cytosol, catalyses superoxide radical transformation
into H2O2 and O2 and, GPx participates in hydroperoxides reduction to
248
Journal of Functional Foods 46 (2018) 243–249
alcohols and oxygen by a coupled reaction with the secondary anti-
oxidant enzyme GR (Meng-Chang Tsai, 2015). Pretreatments with sour
cherry juice enhanced the antioxidant activity of these enzymes in the
human neuroblastoma SH-SY5Y cells which could explain its cytopro-
tective effect against oxidative stress. Although it is the first time that
sour cherry juice is reported to enhance the activity of the physiological
enzymes CAT and GR in neuronal cells, our work is in accordance with
a previous study in which sour cherry juice was able to enhance SOD
and GPx activities in the liver mice fed with this product (Šarić et al.,
2009).
Finally, and since one of the antioxidant mechanism of action pro-
posed for polyphenols compounds is their ability to scavenge free ra-
dicals and their reducing capacity, we have also performed two in vitro
antioxidant assays widely known as the ORAC and FRAP methods. The
antioxidant capacity of the juice was confirmed, being these results in
agreement with the previous literature (Wojdyło, Nowicka, Laskowski,
& Oszmiański, 2014).
In conclusion, these results demonstrate that sour cherry juice
protects neurons from oxidative stress by scavenging ROS and enhan-
cing the activity of physiological systems such as CAT, GPx, GR and
reduced glutathione.
Acknowledgements
Universidad San Jorge is acknowledged for financial support and
providing Guillermo Cásedas a PhD scholarship. The Department of
Pharmacology (Faculty of Pharmacy, Universidad Complutense de
Madrid) is thanked for hosting Guillermo Cásedas during the experi-
mental work. Natur Import is recognized for supplying the juice and
CITA Aragón for lyophilization.
Conflicts of interest
The authors declare that they do not have any conflict of interest.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.jff.2018.04.055.
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