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antioxidants
Article
A Flavonoid-Rich Extract of Mandarin Juice Counteracts
6-OHDA-Induced Oxidative Stress in SH-SY5Y Cells and
Modulates Parkinson-Related Genes
Santa Cirmi 1,2 , Alessandro Maugeri 2, Giovanni Enrico Lombardo 2, *, Caterina Russo 2,3, Laura Musumeci 2,
Sebastiano Gangemi 4, Gioacchino Calapai 5, Davide Barreca 2and Michele Navarra 2, *
Citation: Cirmi, S.; Maugeri, A.;
Lombardo, G.E.; Russo, C.;
Musumeci, L.; Gangemi, S.; Calapai,
G.; Barreca, D.; Navarra, M. A
Flavonoid-Rich Extract of Mandarin
Juice Counteracts 6-OHDA-Induced
Oxidative Stress in SH-SY5Y Cells
and Modulates Parkinson-Related
Genes. Antioxidants 2021,10, 539.
https://doi.org/10.3390/
antiox10040539
Academic Editor: Domenico Nuzzo
Received: 24 February 2021
Accepted: 26 March 2021
Published: 30 March 2021
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4.0/).
1Department of Pharmacy-Drug Sciences, University of Bari “Aldo Moro”, 70125 Bari, Italy;
santa.cirmi@uniba.it
2Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina,
98168 Messina, Italy; amaugeri@unime.it (A.M.); carusso@unime.it (C.R.); laura.musumeci@unime.it (L.M.);
davide.barreca@unime.it (D.B.)
3Fondazione “Prof. Antonio Imbesi”, 98123 Messina, Italy
4Department of Clinical and Experimental Medicine, University of Messina, 98125 Messina, Italy;
sebastiano.gangemi@unime.it
5Department of Biomedical and Dental Sciences and Morphofunctional Imaging, University of Messina,
98125 Messina, Italy; gioacchino.calapai@unime.it
*Correspondence: gelombardo@unime.it (G.E.L.); mnavarra@unime.it (M.N.)
Abstract:
Parkinson’s disease (PD) is a degenerative disorder of the nervous system due to unceasing
impairment of dopaminergic neurons situated in the substantia nigra. At present, anti-PD drugs
acting on dopamine receptors are mainly symptomatic and have only very limited neuroprotective
effects, whereas drugs slowing down neurodegeneration of dopaminergic neurons and deterioration
of clinical symptoms are not yet available. Given that, the development of more valuable pharmaco-
logical strategies is highly demanded. Comprehensive research on innovative neuroprotective drugs
has proven that anti-inflammatory and antioxidant molecules from food sources may prevent and/or
counteract neurodegenerative diseases, such as PD. The present study was aimed at the evaluation
the protective effect of mandarin juice extract (MJe) against 6-hydroxydopamine (6-OHDA)-induced
SH-SY5Y human neuroblastoma cell death. Treatment of differentiated SH-SY5Y cells with 6-OHDA
brought cell death, and specifically, apoptosis, which was significantly inhibited by the preincubation
with MJe through caspase 3 blockage and the modulation of p53, Bax, and Bcl-2 genes. In addition, it
showed antioxidant properties in abiotic models as well as
in vitro
, where it reduced both reactive
oxygen and nitrogen species induced by 6-OHDA, along with restored mitochondrial membrane
potential, and prevented the oxidative DNA damage evoked by 6-OHDA. Furthermore, MJe restored
the impaired balance of SNCA, LRRK2, PINK1, parkin, and DJ-1 gene levels, PD-related factors,
caused by 6-OHDA oxidative stress. Overall, these results indicate that MJe exerts neuroprotective
effects against 6-OHDA-induced cell death in SH-SY5Y cells by mechanisms involving both the
specific interaction with intracellular pathways and its antioxidant capability. Our study suggests a
novel possible strategy to prevent and/or ameliorate neurodegenerative diseases, such as PD.
Keywords:
neurodegenerative diseases; Parkinson’s disease; mandarin juice; Citrus reticulata; 6-
OHDA; neuroprotection; SH-SY5Y; oxidative stress; natural products
1. Introduction
Parkinson’s disease (PD) is an aging-related neurodegenerative disease (ND) whose
characteristics are progressive aggregation of
α
-synuclein in surviving neurons and se-
lective death of dopaminergic neurons in the substantia nigra, particularly its pars com-
pacta [
1
]. PD shows tremor at rest, rigidity, bradykinesia, slowed movements, and postural
instability. The Global Burden of Diseases 2016 has gathered the evidence on neurological
Antioxidants 2021,10, 539. https://doi.org/10.3390/antiox10040539 https://www.mdpi.com/journal/antioxidants
Antioxidants 2021,10, 539 2 of 16
disorders, showing that PD had an incidence of 1 million cases worldwide, of which more
than 150,000 in the EU, and PD-related deaths were more than 340,000 globally, with 60,000
just in the EU [
2
]. Although the actual triggers of PD remain undefined, the link between
oxidative stress, mitochondrial impairment, protein misprocessing, and genetic variation is
pivotal in the pathogenesis of the disease [3].
In the unceasing search of innovative neuroprotective drugs, anti-inflammatory and
antioxidant molecules from dietary sources have been suggested to prevent and/or coun-
teract NDs, such as PD [
4
–
6
]. In this context, nutraceuticals along with food supplements
have been proven to provide neuroprotective effects in several experimental models, and
their use as substitute to synthetic drugs or in combination with them is supported by
their ability to hinder oxidative stress as well as to interact with intracellular pathways
involved in NDs [
4
,
7
,
8
]. These properties might be ascribed to the presence of polyphenolic
compounds, abundantly present in fruits, vegetables, cereals, and beverages. The main
class of polyphenols is flavonoids, whose major dietary sources are fruits, especially citrus,
vegetables, tea, coffee, and red wine. They possess a remarkable spectrum of biological
activities, such as antioxidant, free radical scavenging, metal ion chelating, vasoprotective,
hepatoprotective, anti-cancer, anti-infective and anti-inflammatory [9–13].
Based on this, we investigated whether a flavonoid-rich extract from Citrus reticulata
Blanco (mandarin; MJe) protects differentiated SH-SY5Y cells from 6-hydroxydopamine
(6-OHDA)-induced neurotoxicity and explored its mechanism of action.
2. Materials and Methods
2.1. Drug
Citrus reticulata Blanco fruits were harvested from crops located in Sicily (Italy). The
flavonoid fraction of mandarin juice (MJe) was provided by the company “Agrumaria
Corleone” (Palermo, Italy). The extract was obtained by passing the mandarin juice through
columns equipped with flavonoid-adsorbent resins, which were then eluted. The extract
was centrifuged for 15 min at 6000 rpm and then spray-dried. One hundred milliliters of
MJe yielded 109 mg of dry extract. Aliquots of MJe were stored at
−
20
◦
C and defrosted
just before use.
2.2. Chemical Characterization of MJe
2.2.1. Reagents and Standard Solutions
HPLC-grade acetonitrile and DMSO were obtained by Sigma-Aldrich (St. Louis, MO,
USA). Vicenin-2, hesperidin, eriocitrin, narirutin, sinensetin, nobiletin, and tangeretin
were supplied from Extrasynthèse (Genay, France). Lucenin-2 4
0
-methylether and orientin
4
0
-methyl ether were separated from Citrus limetta and Citrus bergamia [
14
,
15
] and used
as standards. The Iso-Disc P-34, 3 mm diameter PTFE membrane (0.45
µ
m pore size) was
from Supelco (Bellefonte, PA, USA). All the other reagents and chemicals employed in this
study were of analytical grade and were purchased from Sigma (St. Louis, MO, USA).
2.2.2. Sample Preparation
A solution of DMSO/H
2
O (1:1) was added to the lyophilized powder to obtain
a final concentration of (10.0 mg/mL), and the mixture was centrifuged for 5 min at
3200 rpm. The supernatant liquid was filtered through an Iso-Disc P-34, 3 mm diameter
PTFE membrane with a 0.45
µ
m pore size (Supelco, Bellefonte, PA, USA) and utilized for
HPLC-DAD separation.
2.2.3. RP-DAD-HPLC Separation and Identification
Reverse phase-diode array detector-high performance liquid chromatography (RP-
DAD-HPLC) was performed using a Shimadzu system (Shimadzu Ltd., Canby, OR, USA),
consisting of a LC-10AD pump system, a vacuum degasser, a quaternary solvent mixing,
an SPD-M10A diode array detector, and a Rheodyne 7725i injector. The separation of
each compound was performed on a 250
×
4.6 mm i.d., 5 mm Discovery C18 column,
Antioxidants 2021,10, 539 3 of 16
supplied by Supelco (Bellefonte, PA, USA), equipped with a 20
×
4.0 mm guard column.
The column was placed in a column oven set at 30
◦
C. The injection loop was 20
µ
L,
and the flow rate was 1.0 mL/min. The mobile phase consisted of a linear gradient of
acetonitrile in H
2
O as follows: 5–20% (0–15 min), 20–30% (15–20 min), 30–50% (20–30 min),
50–100% (30–35 min), 100% (35–40 min), 100–5% (40–50 min), and 5% (50–60 min). UV
spectra were recorded between 200 and 600 nm, and simultaneous detection by diode array
was performed at 278, 310, and 325 nm. Each sample was tested three times and gave
superimposable chromatograms. Peak identification was performed by matching retention
time and UV spectra against reference compounds and spiking the samples with pure
reference compounds. The calibration lines were obtained using known concentrations
of pure compounds and selected to match the concentration present in the tested sample.
Quantitative analysis was carried out by integration of the areas of the peaks from the
chromatogram at 280 and 325 nm for flavanone and flavone derivatives, respectively. The
calibration curves were constructed, and linear regression equations were obtained by
plotting the ratios of compounds’ peak areas to the peak areas of the external standard,
against the known concentrations of pure compounds.
2.2.4. Acid Hydrolysis
Hydrolysis was performed on MJe according to already published procedure [
16
].
Briefly, 10 mL of HCl (6 M) in a methanol (25 mL)/water (10 mL) solution was added to
5 mL of MJe to obtain a solution of 1.2 M HCl in 50% aqueous methanol. As an antioxidant,
we added ascorbic acid (50 mg). After refluxing at 90
◦
C for 20 h under constant stirring, the
solution was cooled at room temperature, the solvents evaporated under reduced pressure,
and the residue suspended in 10 mL water/DMF (1:1). The mixture was filtered through
an Iso-Disc P-34 membrane and analyzed by HPLC.
2.3. Cell Culture and Treatment
Experiments were carried out using the SH-SY5Y human neuroblastoma cell line orig-
inally from ATCC (Rockville, MD, USA). Their differentiation was performed with 10
µ
M
retinoic acid (RA; Sigma–Aldrich, Milan, Italy) in MEM/Ham’s F12 medium supplemented
for 5 days [
17
]. All reagents were from Gibco (Life Technologies, Monza, Italy). The stock
solution of MJe 400 mg/mL was prepared in DMSO that was employed, upon further
dilution in culture medium, to obtain the working concentrations. The same percentages
of DMSO present in these dilutions served as vehicle controls that were tested in each
of the following experiments to confirm that no effect was induced by the solvent (data
not shown).
2.4. Cytotoxicity Assays
Cell viability was evaluated by the 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan
(MTT) test as reported by Morisi and collaborators [
18
]. Cells were seeded into 96-well
plates at density of 5
×
10
4
cells/well and left to attach overnight. Then, cells were treated
with MJe at various concentrations (from 0.001 to 1 mg/mL) for 1 h. As stressor, 6-OHDA
50
µ
M (Sigma–Aldrich, Milan, Italy) was added for additional 24 h [
17
]. Afterwards, the
plates were centrifuged, supernatants were removed, and fresh media without phenol red
containing 0.5 mg/mL of MTT (Sigma-Aldrich) was added to each well. Plates were put
in the incubator for an additional 4 h. Then, formazan crystals were solubilized in 100
µ
L
HCl/isopropanol 0.1 N. The absorbance was spectrophotometrically quantified through
iMark
™
microplate reader (Bio-Rad Laboratories, Milan, Italy) at a wavelength of 570 nm.
The viability was determined as percentage of viable cells in treated cultures compared to
those in untreated ones.
Cell death was assessed by the trypan blue (0.4% w/v; TB) exclusion test. Cells were
seeded onto 6-well plates at a density of 1
×
10
4
cells/well for 24 h and then treated
with MJe (0.001–1 mg/mL) for 1 h before exposure to 6-OHDA (50
µ
M) for another 24 h.
Then, cells were detached and stained with trypan blue dye before proceeding with cell
Antioxidants 2021,10, 539 4 of 16
count [
19
,
20
]. Dead cells were reported as percentage of stained (nonviable) vs. total
cells counted.
2.5. Detection of Apoptotic Cell Death and Caspase-3 Enzymatic Activity
The protective effect of MJe against 6-OHDA-induced cell death was analyzed by
fluorescence-activated cell sorting (FACS), using the Annexin V-fluorescein isothiocyanate
(FITC)/propidium iodide (PI) staining [
21
], a method employed to discriminate the living
status of cells.
SH-SY5Y were seeded similarly to TB assay, but pretreated for 1 h with MJe (0.1
and 0.5 mg/mL) and then exposed to 50
µ
M 6-OHDA for another 24 h. Next, cells were
collected and processed as already reported [
21
]. Finally, samples were run on a Novocyte
2000 cytofluorimeter (ACEA Biosciences Inc., San Diego, CA, USA).
Caspase 3 enzymatic activity was measured using a commercial kit (AbCam, Cam-
bridge, UK). SH-SY5Y cells were differentiated in 100 mm petri dishes (1.5
×
10
6
cells) and
then treated for 6 h, as explained above. Then, according to the manufacturer’s instructions,
the analysis was carried out on cell lysates [
17
]. Absorbance was measured at 405 nm by a
microplate spectrophotometer.
2.6. Real-Time PCR Analysis
For the evaluation of MJe treatment on the expression of genes encoding for regulatory
proteins of apoptosis, SH-SY5Y cells were seeded and treated as for the apoptosis evaluation.
Afterwards, total RNA of treated or untreated SH-SY5Y cells was extracted and reverse
transcribed following procedures of Curròand collaborators [
22
]. Messenger RNA levels
were analyzed by quantitative real-time PCR, using SYBR green as a fluorescent probe.
The analysis was carried out on a 7300 Real-Time PCR System (Applied Biosystems).
β
-
Actin was used as housekeeping control, and a standard dissociation stage was included
to assess primer specificity. Data were collected and analysed using the 2
−∆∆CT
relative
quantification method [
22
]. Values are presented as fold change relative to untreated cells.
The primer sequences used for real-time PCR are listed in Table 1.
Table 1. Oligonucleotide primers used for real-time PCR.
Gene Product NCBI Reference Sequence Primer Sequence
p53 NM_000546.6 Forward: 50-GTGTGGAGTATTTGGATGAC-30
Reverse: 50-ATGTAGTTGTAGTGGATGGT-30
Bax NM_138764.5 Forward: 50-GGACGAACTGGACAGTAACATGG-30
Reverse: 50-GCAAAGTAGAAAAGGGCGACAAC-30
Bcl-2 NM_000657.3 Forward: 50-ATCGCCCTGTGGATGACTGAG-30
Reverse: 50-CAGCCAGGAGAAATCAAACAGAGG-30
SNCA NM_000345.4 Forward: 5’-TGACAAATGTTGGAGGAGCA-3’
Reverse: 5’-TGTCAGGATCCACAGGCATA-3’
LRRK2 NM_198578.4 Forward: 5’-TCAGCTTGTTGTTGGACAGC-3’
Reverse: 5’-ACTGCGTGAGGAAGCTCATT-3’
PINK1 NM_032409.3 Forward: 5’-ACGTTCAGTTACGGGAGTGG-3’
Reverse: 5’-GGCTAGTCAGGAGGGAAACC-3’
DJ-1 NM_007262.5 Forward: 5’-GGGTGCAGGCTTGTAAACAT-3’
Reverse: 5’-GGACAAATGACCACATCACG-3’
PARK2 NM_004562.3 Forward: 5’-CTGACACCAGCATCTTCCAG-3’
Reverse: 5’-CCAGTCATTCCTCAGCTCCT-3’
β-Actin NM_001101.5 Forward: 50-TTGTTACAGGAAGTCCCTTGCC-30
Reverse: 50-ATGCTATCACCTCCCCTGTGTG-30
Antioxidants 2021,10, 539 5 of 16
2.7. Evaluation of MJe Anti-Oxidant Activity through Abiotic Assays
The total phenolic content of MJe was evaluated through the Folin–Ciocalteu assay and
its total antioxidant activity through the oxygen radical absorbance capacity (ORAC) assay,
following Ferlazzo and co-workers [
23
]. The 2,2-Diphenyl-1-picrylhydrazyl (DPPH
•
) was
employed to test the radical scavenging activity of our extract, in accordance to Lombardo
and co-workers [
24
]. The reducing power of MJe was determined according to Ferlazzo
and collaborators through the potassium ferricyanide reducing power assay [
25
]. All tests
were repeated three times, and the results are expressed as means
±
SEM. As standards,
we employed gallic acid, Trolox, and ascorbic acid, in relation to the assay performed.
2.8. Measurement of Glutathione, Catalase and Superoxide Dismutase Activity
Catalase (CAT) and superoxide dismutase (SOD) activities and glutathione (GSH)
content were measured using commercial assay kits (AbCam). SH-SY5Y cells were plated
at density of 5
×
10
5
cells/well in 6-well plates and were treated with MJe 24 h later
at a concentration 0.1 and 0.5 mg/mL for 1 h. Then, 50
µ
M of 6-OHDA was added for
additional 24 h. Then, the assays were conducted according to the manufacturer’s protocols
on cell lysates. The absorbance was recorded by an iMark
™
microplate reader (Bio-Rad
Laboratories) at 450 nm for SOD, 570 nm for CAT, and 405 nm for GSH [25,26].
2.9. Determination of Reactive Oxygen Species (ROS) and Mitochondrial Membrane
Potential (∆Ψm)
Reactive oxygen species (ROS) and mitochondrial membrane potential (
∆Ψ
m) were
measured fluorometrically, as oxidative stress-related biomarkers. In both assays, cells
were seeded on 96-well plates (5
×
10
4
cells/well) and treated as explained for caspase 3
activity (see Section 2.5).
ROS were quantified using probe 2
0
,7
0
-dichlorodihydrofluorescein diacetate (DCFH-
DA) 25
µ
M (Sigma-Aldrich) [
23
] while variation of
∆Ψ
m were estimated by measuring
Rhodamine 123 incorporation (R123; Sigma-Aldrich), as reported by [
25
]. The fluorescence
was recorded by a microplate reader (POLARstar Omega, BMG Labtech, Ortenberg, Ger-
many) at 485 nm excitation and 535 nm emission for DCFH-DA and 488 nm excitation and
525 nm emission for R123.
2.10. Cytofluorimetric Evaluation of 8-oxo-dG
Oxidative DNA damage was assessed as levels of 8-Oxo-2’-deoxyguanosine (8-oxo-
dG) employing the FITC-labelled avidin probe, which is highly affine to 8-hydroxyguanine
(8-OH-Gua), being structurally similar to biotin. Briefly, cells were permeabilized with
methanol at
−
20
◦
C for 15 min and incubated with avidin-FITC conjugate (0.2
µ
M) at
37
◦
C for 1 h. The fluorescence was recorded cytofluorimetrically with Novocyte 2000
cytofluorimeter at 495 nm excitation and 520 nm emission (ACEA Biosciences Inc.) [23].
2.11. Determination of NO Accumulation in SH-SY5Y Culture Supernatant
The production of nitric oxide (NO) was assayed by a colorimetric commercial kit
(Sigma-Aldrich). In a 6-well plate, 5
×
10
5
cells/well were preincubated with MJe for 1 h,
and then treated with 50
µ
M 6-OHDA for 24 h. Supernatants were collected and processed
as described by Ferlazzo and colleagues [
17
]. Absorbance was recorded at a wavelength of
540 nm by an iMark™ microplate reader (Bio-Rad Laboratories).
2.12. Statistical Analyses
One-way analysis of variance (ANOVA) was used to interpret the data. Multiple
comparisons of the means of the groups were performed using the Tukey–Kramer test
(SigmaPlot Version 12.0, Systat Software, San Jose, CA, USA).
Antioxidants 2021,10, 539 6 of 16
3. Results
3.1. Chromatographic Analysis of MJe
The identification of the compounds present in the lyophilized powder was performed
by RP-DAD-HPLC. The preliminary inspection of chromatographic separation at 280 and
325 nm let us easily identify and discriminate the presence of flavanone and flavone basic
skeletons in the lyophilized powder. Both classes of flavonoids showed maxima of absorp-
tion (called band II) in the 240–280 nm range, but only flavones had a further well-defined
absorption maximum in the 300–380 nm range (called band I). The comparison, together
with inspection of UV spectra recorded in correspondence to each chromatographic peak,
showed that compounds 1–3 and 7–9 possessed a flavone skeleton, whereas 4–6 belonged
to the flavanone class (Figure 1). Moreover, the inspection of chromatogram recorded after
treatment with aqueous HCl (data not shown) indicated that compounds 1–3 were resistant
to acidic hydrolysis, whereas the remaining compounds were not resistant, suggesting the
presence of C-linked saccharide moieties in the former, while the latter were O-glycoside
compounds. Taking into consideration the retention time, UV spectra and spiking the
samples with pure reference compounds, the main peaks of the chromatogram have been
identified as vicenin-2 (1), lucenin-2 4
0
-methyl ether (2), orientin4
0
methylether (3), erioc-
itrin (4), narirutin (5), hesperidin (6), sinensetin (7), tangeretin (8), and nobiletin (9). The
flavonoid profile identified after the separation of compounds present in the lyophilized
powder was dominated by the presence of the flavanone hesperidin, which was by far the
most abundant component (about 353.6 mg/g), followed by a significant amount of di-
C-glucosyl flavone (6,8-di-C-glucosyl-apigenin, about 55.2 mg/g) and another rutinoside
(narirutin), although in smaller amount (about 47.8 mg/g). The quantitative analysis of all
the identified flavonoids is depicted in Table 2.
Antioxidants 2021, 10, x FOR PEER REVIEW 6 of 16
3. Results
3.1. Chromatographic Analysis of MJe
The identification of the compounds present in the lyophilized powder was per-
formed by RP-DAD-HPLC. The preliminary inspection of chromatographic separation at
280 and 325 nm let us easily identify and discriminate the presence of flavanone and fla-
vone basic skeletons in the lyophilized powder. Both classes of flavonoids showed max-
ima of absorption (called band II) in the 240–280 nm range, but only flavones had a further
well-defined absorption maximum in the 300–380 nm range (called band I). The compar-
ison, together with inspection of UV spectra recorded in correspondence to each chroma-
tographic peak, showed that compounds 1–3 and 7–9 possessed a flavone skeleton,
whereas 4–6 belonged to the flavanone class (Figure 1). Moreover, the inspection of chro-
matogram recorded after treatment with aqueous HCl (data not shown) indicated that
compounds 1–3 were resistant to acidic hydrolysis, whereas the remaining compounds
were not resistant, suggesting the presence of C-linked saccharide moieties in the former,
while the latter were O-glycoside compounds. Taking into consideration the retention
time, UV spectra and spiking the samples with pure reference compounds, the main peaks
of the chromatogram have been identified as vicenin-2 (1), lucenin-2 4′-methyl ether (2),
orientin4′methylether (3), eriocitrin (4), narirutin (5), hesperidin (6), sinensetin (7), tange-
retin (8), and nobiletin (9). The flavonoid profile identified after the separation of com-
pounds present in the lyophilized powder was dominated by the presence of the fla-
vanone hesperidin, which was by far the most abundant component (about 353.6 mg/g),
followed by a significant amount of di-C-glucosyl flavone (6,8-di-C-glucosyl-apigenin,
about 55.2 mg/g) and another rutinoside (narirutin), although in smaller amount (about
47.8 mg/g). The quantitative analysis of all the identified flavonoids is depicted in Table
2.
Figure 1. Reverse phase-diode array detector-high performance liquid chromatography (RP-
HPLC-DAD) separation of the compounds present in the lyophilized powder registered at 280 (A)
Figure 1.
Reverse phase-diode array detector-high performance liquid chromatography (RP-HPLC-
DAD) separation of the compounds present in the lyophilized powder registered at 280 (
A
) and
325 nm (
B
). Vicenin-2 (1), lucenin-2 4
0
-methyl ether (2), orientin4
0
methylether (3), eriocitrin (4),
narirutin (5), hesperidin (6), sinensetin (7), tangeretin (8), nobiletin (9). Peak identification was
performed by matching retention time and UV spectra against commercially available reference
compounds and spiking the samples with pure reference compounds.
Antioxidants 2021,10, 539 7 of 16
Table 2. Quantitative determination of the identified compounds.
mg/g (Dried Extract)
Peak Compounds Mean SD
1 Vicenin-2 55.2 2.5
2 Lucenin-2 40-methyl ether 21.5 1.7
3 Orientin40methylether 1.4 0.21
4 Eriocitrin 8.2 1.6
5 Narirutin 47.8 1.9
6 Hesperidin 353.6 20.5
7 Sinensetin 2.0 0.3
8 Tangeretin 6.4 0.5
9 Nobiletin 17.3 1.4
3.2. MJe Prevents 6-OHDA Induced SH-SY5Y Cell Death
To evaluate the potential neuroprotective effect of MJe, SH-SY5Y cells were preincu-
bated with MJe and then exposed to 6-OHDA before the assessment of cell viability. The
incubation of SH-SY5Y cells with 6-OHDA reduced cell viability by 37
±
5.1% compared to
that of control cells (p< 0.001; Figure 2A), whereas pretreatment with MJe at concentration
of 0.1, 0.5 and 1 mg/mL restored cell viability up to 77
±
6.1, 83
±
6.9, and
85 ±7.9%
,
respectively (p< 0.05 and p< 0.01 vs. 6-OHDA-injured cells; Figure 2A). Data of TB
assay followed the same pattern of those of MTT test (Figure 2B). In particular, 6-OHDA
increased cell death by 28
±
2.5%, whereas 0.01, 0.1, 0.5, and 1 mg/mL MJe maintained it
to 23.1 ±1.3, 19.4 ±1.2, 15.9 ±2.6, and 14.4 ±2.0%, respectively (p< 0.05 and p< 0.01 vs.
6-OHDA-exposed cells; Figure 2B).
Antioxidants 2021, 10, x FOR PEER REVIEW 7 of 16
and 325 nm (B). Vicenin-2 (1), lucenin-2 4′-methyl ether (2), orientin4′methylether (3), eriocitrin (4),
narirutin (5), hesperidin (6), sinensetin (7), tangeretin (8), nobiletin (9). Peak identification was
performed by matching retention time and UV spectra against commercially available reference
compounds and spiking the samples with pure reference compounds.
Table 2. Quantitative determination of the identified compounds.
mg/g (Dried Extract)
Peak Compounds Mean SD
1 Vicenin-2 55.2 2.5
2 Lucenin-2 4′-methyl ether 21.5 1.7
3 Orientin4′methylether 1.4 0.21
4 Eriocitrin 8.2 1.6
5 Narirutin 47.8 1.9
6 Hesperidin 353.6 20.5
7 Sinensetin 2.0 0.3
8 Tangeretin 6.4 0.5
9 Nobiletin 17.3 1.4
3.2. MJe Prevents 6-OHDA Induced SH-SY5Y Cell Death
To evaluate the potential neuroprotective effect of MJe, SH-SY5Y cells were preincu-
bated with MJe and then exposed to 6-OHDA before the assessment of cell viability. The
incubation of SH-SY5Y cells with 6-OHDA reduced cell viability by 37 ± 5.1% compared
to that of control cells (p < 0.001; Figure 2A), whereas pretreatment with MJe at concentra-
tion of 0.1, 0.5 and 1 mg/mL restored cell viability up to 77 ± 6.1, 83 ± 6.9, and 85 ± 7.9%,
respectively (p < 0.05 and p < 0.01 vs. 6-OHDA-injured cells; Figure 2A). Data of TB assay
followed the same pattern of those of MTT test (Figure 2B). In particular, 6-OHDA in-
creased cell death by 28 ± 2.5%, whereas 0.01, 0.1, 0.5, and 1 mg/mL MJe maintained it to
23.1 ± 1.3, 19.4 ± 1.2, 15.9 ± 2.6, and 14.4 ± 2.0%, respectively (p < 0.05 and p < 0.01 vs. 6-
OHDA-exposed cells; Figure 2B).
Figure 2. Protective effect of mandarin juice extract (MJe) against 6-hydroxydopamine (6-OHDA)-
induced cytotoxicity. Cell viability was detected with the 1-(4,5-dimethylthiazol-2-yl)-3,5-diphe-
nylformazan (MTT) assay (A). The viability variation was calculated as percentage of viable cells
in treated cultures relative to those in untreated ones. Cell death was estimated by the trypan blue
(TB) assay and expressed as percentage of nonviable (blue stained) cells relative to total counted
cells (B). Results are displayed as mean ± standard error of the means (SEM) from three independ-
ent experiments in eight replicates (MTT; N = 24) or in triplicate (TB; N = 9). *** p < 0.001 vs. ctrl; ° p
< 0.05, °° p < 0.01 and °°° p < 0.001 vs. 6-OHDA 50 μM.
3.3. MJe Reduces the Apoptotic Cell Death Induced by 6-OHDA
Figure 2.
Protective effect of mandarin juice extract (MJe) against 6-hydroxydopamine (6-OHDA)-induced cytotoxicity. Cell
viability was detected with the 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay (
A
). The viability variation
was calculated as percentage of viable cells in treated cultures relative to those in untreated ones. Cell death was estimated
by the trypan blue (TB) assay and expressed as percentage of nonviable (blue stained) cells relative to total counted cells (
B
).
Results are displayed as mean
±
standard error of the means (SEM) from three independent experiments in eight replicates
(MTT; N = 24) or in triplicate (TB; N = 9). *** p< 0.001 vs. ctrl; ◦p< 0.05, ◦◦ p< 0.01 and ◦ ◦ ◦ p< 0.001 vs. 6-OHDA 50 µM.
3.3. MJe Reduces the Apoptotic Cell Death Induced by 6-OHDA
The cytoprotecting activity elicited by MJe was also evaluated by flow cytometry
through an Annexin V-FITC/PI assay. As shown in Figure 3A,B, the incubation of SH-
SY5Y cells with 6-OHDA for 24 h augmented the percentage of cells in early (8
±
0.3%,
Annexin V+/PI
−
) and late (28
±
2.0%, Annexin V+/PI+) apoptosis, as well as in necrosis
(
16 ±1.1%,
Annexin V
−
/PI+). The pretreatment with 0.1 mg/mL MJe for 1 h reduced the
number of cells undergoing apoptosis (4
±
0.3% and 13
±
0.7% of early and late apoptosis,
respectively), likewise, in presence of MJe 0.5 mg/mL, it was further reduced to 5
±
0.2%
and 8
±
0.1%, respectively (Figure 3A,B). The incidence of apoptosis in 6-OHDA-treated
Antioxidants 2021,10, 539 8 of 16
cells was confirmed by the results of the caspase 3 activity assay. The exposure to 6-
OHDA for 6 h increased caspase 3 activity of SH-SY5Y cells compared to that of unexposed
ones by 250
±
15% (p< 0.001; Figure 3C). Pretreatment with MJe (0.1 or 0.5 mg/mL) for
1 h inhibited the activity of caspase 3 induced by 6-OHDA to 140
±
9 and 123
±
10%,
respectively (
p< 0.001
), while MJe alone, at both tested concentrations, had no effect on
this enzymatic activity (Figure 3C).
Antioxidants 2021, 10, x FOR PEER REVIEW 8 of 16
The cytoprotecting activity elicited by MJe was also evaluated by flow cytometry
through an Annexin V-FITC/PI assay. As shown in Figure 3A,B, the incubation of SH-
SY5Y cells with 6-OHDA for 24 h augmented the percentage of cells in early (8 ± 0.3%,
Annexin V+/PI−) and late (28 ± 2.0%, Annexin V+/PI+) apoptosis, as well as in necrosis (16
± 1.1%, Annexin V−/PI+). The pretreatment with 0.1 mg/mL MJe for 1 h reduced the num-
ber of cells undergoing apoptosis (4 ± 0.3% and 13 ± 0.7% of early and late apoptosis, re-
spectively), likewise, in presence of MJe 0.5 mg/mL, it was further reduced to 5 ± 0.2% and
8 ± 0.1%, respectively (Figure 3A,B). The incidence of apoptosis in 6-OHDA-treated cells
was confirmed by the results of the caspase 3 activity assay. The exposure to 6-OHDA for
6 h increased caspase 3 activity of SH-SY5Y cells compared to that of unexposed ones by
250 ± 15% (p < 0.001; Figure 3C). Pretreatment with MJe (0.1 or 0.5 mg/mL) for 1 h inhibited
the activity of caspase 3 induced by 6-OHDA to 140 ± 9 and 123 ± 10%, respectively (p <
0.001), while MJe alone, at both tested concentrations, had no effect on this enzymatic ac-
tivity (Figure 3C).
Figure 3. MJe mitigated the apoptosis induced by 6-OHDA in SH-SY5Y cells. (A) Evaluation of
apoptosis was performed cytofluorimetrically by the Annexin V/PI test. Representative Annexin V
vs. PI dot plots are shown, where necrotic, late apoptosis, viable cells, and early apoptosis cells are
in Q1, Q2, Q3 and Q4, respectively. (B) The histogram shows the percentage of cells in each quad-
rant, representing the mean ± SEM of three different experiments in triplicate (N = 9). (C) Data of
caspase 3 activity are presented as the mean of three experiments ± SEM in triplicate (N = 9). °°° p <
0.001 vs. control; *** p < 0.001 vs. 6-OHDA 50 μM.
3.4. Protective Effect of MJe on mRNA Levels of Apoptosis-Related Genes Modulated by 6-
OHDA
As shown in Figure 4, the incubation of SH-SY5Y cells with 6-OHDA for 24 h signif-
icantly enhanced the mRNA levels of the proapoptotic B-cell lymphoma 2 (Bcl-2)-associ-
ated X protein (Bax) and p53 genes up to 1.8 ± 0.2- and 2.2 ± 0.3-fold, respectively (p < 0.01
and p < 0.001), along with decreasing those of the antiapoptotic Bcl-2 up to 3 ± 0.9-fold (p
< 0.01). These effects were significantly hampered by pre-exposure to MJe at both 0.1 (1.35
± 0.2-fold and 1.55 ± 0.1-fold down, p < 0.05, for Bax and Bcl-2, and 1.2 ± 0.1-fold, p < 0.01,
for p53) and 0.5 mg/mL concentrations (1.25 ± 0.1-fold, p < 0.05, for Bax and 1.25 ± 0.1-fold
down and 1.4 ± 0.2-fold, p < 0.01 for Bcl-2 and p53) (Figure 4).
Figure 3.
MJe mitigated the apoptosis induced by 6-OHDA in SH-SY5Y cells. (
A
) Evaluation of apoptosis was performed
cytofluorimetrically by the Annexin V/PI test. Representative Annexin V vs. PI dot plots are shown, where necrotic, late
apoptosis, viable cells, and early apoptosis cells are in Q1, Q2, Q3 and Q4, respectively. (
B
) The histogram shows the
percentage of cells in each quadrant, representing the mean
±
SEM of three different experiments in triplicate (N = 9). (
C
)
Data of caspase 3 activity are presented as the mean of three experiments
±
SEM in triplicate (N = 9).
◦◦ ◦
p< 0.001 vs.
control; *** p< 0.001 vs. 6-OHDA 50 µM.
3.4. Protective Effect of MJe on mRNA Levels of Apoptosis-Related Genes Modulated by 6-OHDA
As shown in Figure 4, the incubation of SH-SY5Y cells with 6-OHDA for 24 h signifi-
cantly enhanced the mRNA levels of the proapoptotic B-cell lymphoma 2 (Bcl-2)-associated
X protein (Bax) and p53 genes up to 1.8
±
0.2- and 2.2
±
0.3-fold, respectively (p< 0.01
and p< 0.001), along with decreasing those of the antiapoptotic Bcl-2 up to 3
±
0.9-fold
(p< 0.01). These effects were significantly hampered by pre-exposure to MJe at both 0.1
(1.35
±
0.2-fold and 1.55
±
0.1-fold down, p< 0.05, for Bax and Bcl-2, and 1.2
±
0.1-fold,
p< 0.01, for p53) and 0.5 mg/mL concentrations (1.25
±
0.1-fold, p< 0.05, for Bax and
1.25 ±0.1-fold down and 1.4 ±0.2-fold, p< 0.01 for Bcl-2 and p53) (Figure 4).
Antioxidants 2021,10, 539 9 of 16
Antioxidants 2021, 10, x FOR PEER REVIEW 9 of 16
Figure 4. Protective effect of MJe on mRNA levels of apoptosis-related genes modulated by 6-
OHDA. SH-SY5Y cells were pretreated with MJe for 1 h and then exposed to 6-OHDA 50 μM for
additional 24 h and then assessed with real-time PCR. The 2
–ΔΔCT
method was employed to calcu-
late the relative quantities of mRNA. Results are expressed as fold change relative to untreated
cells. Data, expressed as mean ± SEM, represent the values obtained in three different sets of ex-
periments in triplicate (N = 9). ** p < 0.01, *** p < 0.001 vs. control; ° p < 0.05, °° p < 0.01 vs. 6-OHDA
50 μM.
3.5. Antioxidant Activity of MJe
The antioxidant and radical scavenging properties of MJe were demonstrated using
a range of tests as described in the Methods section. As shown in Table 3, the total phenolic
content expressed as milligrams of gallic acid equivalents (GAE) per gram of MJe evalu-
ated by Folin–Ciocalteau method was 117.76 ± 4.8. The valuable antiradical activity of MJe
shown in the DPPH test and expressed as milligrams of Trolox equivalents (TE) per gram
of extract (mgTE/g) was 60.07 ± 4.2, while in the Reducing Power test it was 53.6 ± 2.3
expressed as milligrams of ascorbic acid equivalent (AAE) per gram of MJe (mg AAE/g).
Moreover, the obtained values of 3753.7 ± 221.5 μmol TE/g MJe demonstrated high anti-
oxidant capacity of MJe against peroxyl radicals (Table 3).
Table 3. Antioxidant activity of MJe evaluated by abiotic assays. Results are reported as mean ±
SEM of three experiments performed in triplicate and expressed in standard equivalent/g of dried
extract (N = 9).
Folin-Ciocalteau (mg GAE/g) 117.76 ± 4.8
DPPH (mg TE/g) 60.07 ± 4.2
Reducing Power (mg AAE/g) 53.6 ± 2.3
ORAC (μmol TE/g) 3753.72 ± 221.5
3.6. Effects of MJe on 6-OHDA-Induced SOD and CAT Activities and GSH Content
We evaluated the levels of catalase and superoxide dismutase activities as well as
glutathione content to determine the extent of oxidative stress in the cells. As shown in
Figure 5, the treatment of SH-SY5Y cells with 6-OHDA caused a significant decrease in
the levels of these biomarkers in comparison to those of control cells (62 ± 2.3, 41 ± 1.7, and
53 ± 2.1% for SOD, CAT, and GSH, respectively, p < 0.001). Pretreatment with MJe at both
concentrations of 0.1 (62 ± 2.3, 41 ± 1.7, and 53 ± 2.1% for SOD, CAT, and GSH, respectively,
p < 0.01) and 0.5 mg/mL (88 ± 2.8, 65 ± 2.2, and 79 ± 2.0% for SOD, CAT, and GSH, respec-
tively, p < 0.001) for 1 h significantly augmented the activity of both CAT and SOD, along
with levels of GSH, indicative of a reduction of oxidative stress (Figure 5).
Figure 4.
Protective effect of MJe on mRNA levels of apoptosis-related genes modulated by 6-OHDA.
SH-SY5Y cells were pretreated with MJe for 1 h and then exposed to 6-OHDA 50
µ
M for additional
24 h and then assessed with real-time PCR. The 2
–∆∆CT
method was employed to calculate the relative
quantities of mRNA. Results are expressed as fold change relative to untreated cells. Data, expressed
as mean
±
SEM, represent the values obtained in three different sets of experiments in triplicate
(N = 9). ** p< 0.01, *** p< 0.001 vs. control; ◦p< 0.05, ◦ ◦ p< 0.01 vs. 6-OHDA 50 µM.
3.5. Antioxidant Activity of MJe
The antioxidant and radical scavenging properties of MJe were demonstrated using a
range of tests as described in the Methods section. As shown in Table 3, the total phenolic
content expressed as milligrams of gallic acid equivalents (GAE) per gram of MJe evaluated
by Folin–Ciocalteau method was 117.76
±
4.8. The valuable antiradical activity of MJe
shown in the DPPH test and expressed as milligrams of Trolox equivalents (TE) per gram
of extract (mgTE/g) was 60.07
±
4.2, while in the Reducing Power test it was 53.6
±
2.3
expressed as milligrams of ascorbic acid equivalent (AAE) per gram of MJe (mg AAE/g).
Moreover, the obtained values of 3753.7
±
221.5
µ
mol TE/g MJe demonstrated high
antioxidant capacity of MJe against peroxyl radicals (Table 3).
Table 3.
Antioxidant activity of MJe evaluated by abiotic assays. Results are reported as
mean ±SEM
of three experiments performed in triplicate and expressed in standard equivalent/g of dried extract
(N = 9).
Folin-Ciocalteau (mg GAE/g) 117.76 ±4.8
DPPH (mg TE/g) 60.07 ±4.2
Reducing Power (mg AAE/g) 53.6 ±2.3
ORAC (µmol TE/g) 3753.72 ±221.5
3.6. Effects of MJe on 6-OHDA-Induced SOD and CAT Activities and GSH Content
We evaluated the levels of catalase and superoxide dismutase activities as well as
glutathione content to determine the extent of oxidative stress in the cells. As shown in
Figure 5, the treatment of SH-SY5Y cells with 6-OHDA caused a significant decrease in
the levels of these biomarkers in comparison to those of control cells (62
±
2.3, 41
±
1.7,
and 53
±
2.1% for SOD, CAT, and GSH, respectively, p< 0.001). Pretreatment with MJe
at both concentrations of 0.1 (62
±
2.3, 41
±
1.7, and 53
±
2.1% for SOD, CAT, and GSH,
respectively, p< 0.01) and 0.5 mg/mL (88
±
2.8, 65
±
2.2, and 79
±
2.0% for SOD, CAT, and
GSH, respectively, p< 0.001) for 1 h significantly augmented the activity of both CAT and
SOD, along with levels of GSH, indicative of a reduction of oxidative stress (Figure 5).
Antioxidants 2021,10, 539 10 of 16
Antioxidants 2021, 10, x FOR PEER REVIEW 10 of 16
Figure 5. Effect of MJe on biomarkers of 6-OHDA-induced oxidative stress in SH-SY5Y cells. (A)
Effect of MJe on the activity of SOD in 6-OHDA-treated SH-SY5Y cells; (B) Effect of MJe on the
activity of CAT in 6-OHDA-treated SH-SY5Y cells; (C) Effect of MJe on the GSH levels in 6-
OHDA-treated SH-SY5Y cells. Data are showed as the mean ± SEM of three experiments in tripli-
cate (N = 9). *** p < 0.001 vs. ctrl; °° p < 0.01 and °°° p < 0.001 vs. 6-OHDA 50 μM.
3.7. MJe Reduces Oxidative Stress Induced by 6-OHDA
The increase of ROS is an acknowledged characteristic of several NDs, including PD.
Therefore, we measured the intracellular ROS amount through DCFH-DA. As shown in
Figure 6A, the exposure of SH-SY5Y cells to 50 μM 6-OHDA for 6 h caused a significant 2
± 0.15-fold intracellular ROS accumulation compared to that in the controls (p < 0.001;
Figure 6A). Pretreatment with MJe at both 0.1 and 0.5 mg/mL concentrations significantly
counteracted the increase in ROS caused by 6-OHDA, by 51 ± 0.2 and 63 ± 0.1%, respec-
tively (p < 0.001; Figure 6A). Moreover, the exposure of SH-SY5Y cells to 50 μM 6-OHDA
for 6 h significantly influenced the ΔΨm that, in comparison to that in control cells, de-
creased by 39 ± 0.6% (p < 0.01). The reduction of ΔΨm evoked by 6-OHDA was prevented
by MJe 0.1 and 0.5 mg/mL (81.3 ± 8 and 86.7 ± 7% p < 0.05, respectively; Figure 6B).
Figure 6. MJe diminished both generation of reactive oxygen species (ROS) and fall of mitochon-
drial membrane potential (ΔΨm) induced by 6-OHDA. (A) ROS accumulation was measured us-
ing the fluorescent probe DCFH-DA. (B) ΔΨm was assessed using the cationic fluorochrome R123.
Results are reported as percentage of the levels detected in untreated cells. Data are displayed as
the mean ± SEM of three experiments in triplicate (N = 9). °° p < 0.01 and °°° p < 0.001 vs. ctrl; * p <
0.05 and *** p < 0.001 vs. 6-OHDA 50 μM.
3.8. Protective Effects of MJe on 6-OHDA DNA Damage
Levels of 8-oxo-dG were measured to study the efficacy of MJe in containing DNA-
oxidative damage using a FITC-conjugated avidin probe (Figure 7). The exposure of SH-
SY5Y cells to 6-OHDA 50 μM for 24 h induced DNA oxidation of about 50 ± 2% in com-
parison to that in control cells. Pretreatment with 0.1 and 0.5 mg/mL of MJe for 1 h before
the exposure to 6-OHDA 50 μM (24 h) decreased oxidative DNA damage by 27 ± 1 and 33
Figure 5.
Effect of MJe on biomarkers of 6-OHDA-induced oxidative stress in SH-SY5Y cells. (
A
) Effect of MJe on the
activity of SOD in 6-OHDA-treated SH-SY5Y cells; (
B
) Effect of MJe on the activity of CAT in 6-OHDA-treated SH-SY5Y
cells; (
C
) Effect of MJe on the GSH levels in 6-OHDA-treated SH-SY5Y cells. Data are showed as the mean
±
SEM of three
experiments in triplicate (N = 9). *** p< 0.001 vs. ctrl; ◦ ◦ p< 0.01 and ◦◦ ◦ p< 0.001 vs. 6-OHDA 50 µM.
3.7. MJe Reduces Oxidative Stress Induced by 6-OHDA
The increase of ROS is an acknowledged characteristic of several NDs, including PD.
Therefore, we measured the intracellular ROS amount through DCFH-DA. As shown in
Figure 6A, the exposure of SH-SY5Y cells to 50
µ
M 6-OHDA for 6 h caused a significant
2±0.15-fold
intracellular ROS accumulation compared to that in the controls (p< 0.001;
Figure 6A). Pretreatment with MJe at both 0.1 and 0.5 mg/mL concentrations significantly
counteracted the increase in ROS caused by 6-OHDA, by 51
±
0.2 and 63
±
0.1%, respec-
tively (p< 0.001; Figure 6A). Moreover, the exposure of SH-SY5Y cells to 50
µ
M 6-OHDA for
6 h significantly influenced the
∆Ψ
m that, in comparison to that in control cells, decreased
by 39
±
0.6% (p< 0.01). The reduction of
∆Ψ
m evoked by 6-OHDA was prevented by MJe
0.1 and 0.5 mg/mL (81.3 ±8 and 86.7 ±7% p< 0.05, respectively; Figure 6B).
Antioxidants 2021, 10, x FOR PEER REVIEW 10 of 16
Figure 5. Effect of MJe on biomarkers of 6-OHDA-induced oxidative stress in SH-SY5Y cells. (A)
Effect of MJe on the activity of SOD in 6-OHDA-treated SH-SY5Y cells; (B) Effect of MJe on the
activity of CAT in 6-OHDA-treated SH-SY5Y cells; (C) Effect of MJe on the GSH levels in 6-
OHDA-treated SH-SY5Y cells. Data are showed as the mean ± SEM of three experiments in tripli-
cate (N = 9). *** p < 0.001 vs. ctrl; °° p < 0.01 and °°° p < 0.001 vs. 6-OHDA 50 μM.
3.7. MJe Reduces Oxidative Stress Induced by 6-OHDA
The increase of ROS is an acknowledged characteristic of several NDs, including PD.
Therefore, we measured the intracellular ROS amount through DCFH-DA. As shown in
Figure 6A, the exposure of SH-SY5Y cells to 50 μM 6-OHDA for 6 h caused a significant 2
± 0.15-fold intracellular ROS accumulation compared to that in the controls (p < 0.001;
Figure 6A). Pretreatment with MJe at both 0.1 and 0.5 mg/mL concentrations significantly
counteracted the increase in ROS caused by 6-OHDA, by 51 ± 0.2 and 63 ± 0.1%, respec-
tively (p < 0.001; Figure 6A). Moreover, the exposure of SH-SY5Y cells to 50 μM 6-OHDA
for 6 h significantly influenced the ΔΨm that, in comparison to that in control cells, de-
creased by 39 ± 0.6% (p < 0.01). The reduction of ΔΨm evoked by 6-OHDA was prevented
by MJe 0.1 and 0.5 mg/mL (81.3 ± 8 and 86.7 ± 7% p < 0.05, respectively; Figure 6B).
Figure 6. MJe diminished both generation of reactive oxygen species (ROS) and fall of mitochon-
drial membrane potential (ΔΨm) induced by 6-OHDA. (A) ROS accumulation was measured us-
ing the fluorescent probe DCFH-DA. (B) ΔΨm was assessed using the cationic fluorochrome R123.
Results are reported as percentage of the levels detected in untreated cells. Data are displayed as
the mean ± SEM of three experiments in triplicate (N = 9). °° p < 0.01 and °°° p < 0.001 vs. ctrl; * p <
0.05 and *** p < 0.001 vs. 6-OHDA 50 μM.
3.8. Protective Effects of MJe on 6-OHDA DNA Damage
Levels of 8-oxo-dG were measured to study the efficacy of MJe in containing DNA-
oxidative damage using a FITC-conjugated avidin probe (Figure 7). The exposure of SH-
SY5Y cells to 6-OHDA 50 μM for 24 h induced DNA oxidation of about 50 ± 2% in com-
parison to that in control cells. Pretreatment with 0.1 and 0.5 mg/mL of MJe for 1 h before
the exposure to 6-OHDA 50 μM (24 h) decreased oxidative DNA damage by 27 ± 1 and 33
Figure 6. MJe diminished both generation of reactive oxygen species (ROS) and fall of mitochondrial membrane potential
(
∆Ψ
m) induced by 6-OHDA. (
A
) ROS accumulation was measured using the fluorescent probe DCFH-DA. (
B) ∆Ψ
m was
assessed using the cationic fluorochrome R123. Results are reported as percentage of the levels detected in untreated cells.
Data are displayed as the mean
±
SEM of three experiments in triplicate (N = 9).
◦◦
p< 0.01 and
◦◦ ◦
p< 0.001 vs. ctrl;
*p< 0.05 and *** p< 0.001 vs. 6-OHDA 50 µM.
3.8. Protective Effects of MJe on 6-OHDA DNA Damage
Levels of 8-oxo-dG were measured to study the efficacy of MJe in containing DNA-
oxidative damage using a FITC-conjugated avidin probe (Figure 7). The exposure of
SH-SY5Y cells to 6-OHDA 50
µ
M for 24 h induced DNA oxidation of about 50
±
2% in
comparison to that in control cells. Pretreatment with 0.1 and 0.5 mg/mL of MJe for 1 h
before the exposure to 6-OHDA 50
µ
M (24 h) decreased oxidative DNA damage by 27
±
1
and 33
±
1% relative to stressed cells, respectively (Figure 7). The treatment with MJe alone
Antioxidants 2021,10, 539 11 of 16
at both concentrations tested did not induce DNA oxidation, since the emission values
approximately overlapped those recorded in control cells (data not shown).
Antioxidants 2021, 10, x FOR PEER REVIEW 11 of 16
± 1% relative to stressed cells, respectively (Figure 7). The treatment with MJe alone at
both concentrations tested did not induce DNA oxidation, since the emission values ap-
proximately overlapped those recorded in control cells (data not shown).
Figure 7. Protective effects of MJe on DNA oxidative damage induced by 6-OHDA. Levels of 8-
oxo-dG are measured as emission signals of fluorochrome FITC-labelled avidin. The plots are rep-
resentative of three independent experiments. The histograms show the percentage ± SEM of
healthy cells (non-fluorescent, M1) and the damaged ones (fluorescent, M2) of three separate ex-
periments in triplicate (N = 9).
3.9. MJe Reduces the Production of NO in SH-SY5Y Cells
The exposure of differentiated SH-SY5Y cells for 24 h to 6-OHDA brought a 79 ± 5%
increase of NO production (p < 0.01; Figure 8) that was hindered by pretreatment with
MJe for 1 h at both concentrations tested (47 ± 2.5 and 59 ± 2% lower, p < 0.01, compared
to that in 6-OHDA-treated cells; Figure 8), while MJe alone had no effect on NO produc-
tion (data not shown).
Figure 8. MJe prevented the release of NO induced by 6-OHDA. The levels of NO were measured
by a colorimetric assay. Differences in NO production are reported as percentage of NO value
detected in treated cells compared to those found in untreated ones. Results are expressed as
means ± SEM from three independent experiments in triplicate (N = 9). °° p < 0.01 vs. ctrl; ** p <
0.01 vs. 6-OHDA 50 μM.
Figure 7.
Protective effects of MJe on DNA oxidative damage induced by 6-OHDA. Levels of 8-oxo-dG are measured as
emission signals of fluorochrome FITC-labelled avidin. The plots are representative of three independent experiments. The
histograms show the percentage
±
SEM of healthy cells (non-fluorescent, M1) and the damaged ones (fluorescent, M2) of
three separate experiments in triplicate (N = 9).
3.9. MJe Reduces the Production of NO in SH-SY5Y Cells
The exposure of differentiated SH-SY5Y cells for 24 h to 6-OHDA brought a 79
±
5%
increase of NO production (p< 0.01; Figure 8) that was hindered by pretreatment with MJe
for 1 h at both concentrations tested (47
±
2.5 and 59
±
2% lower, p< 0.01, compared to
that in 6-OHDA-treated cells; Figure 8), while MJe alone had no effect on NO production
(data not shown).
Antioxidants 2021, 10, x FOR PEER REVIEW 11 of 16
± 1% relative to stressed cells, respectively (Figure 7). The treatment with MJe alone at
both concentrations tested did not induce DNA oxidation, since the emission values ap-
proximately overlapped those recorded in control cells (data not shown).
Figure 7. Protective effects of MJe on DNA oxidative damage induced by 6-OHDA. Levels of 8-
oxo-dG are measured as emission signals of fluorochrome FITC-labelled avidin. The plots are rep-
resentative of three independent experiments. The histograms show the percentage ± SEM of
healthy cells (non-fluorescent, M1) and the damaged ones (fluorescent, M2) of three separate ex-
periments in triplicate (N = 9).
3.9. MJe Reduces the Production of NO in SH-SY5Y Cells
The exposure of differentiated SH-SY5Y cells for 24 h to 6-OHDA brought a 79 ± 5%
increase of NO production (p < 0.01; Figure 8) that was hindered by pretreatment with
MJe for 1 h at both concentrations tested (47 ± 2.5 and 59 ± 2% lower, p < 0.01, compared
to that in 6-OHDA-treated cells; Figure 8), while MJe alone had no effect on NO produc-
tion (data not shown).
Figure 8. MJe prevented the release of NO induced by 6-OHDA. The levels of NO were measured
by a colorimetric assay. Differences in NO production are reported as percentage of NO value
detected in treated cells compared to those found in untreated ones. Results are expressed as
means ± SEM from three independent experiments in triplicate (N = 9). °° p < 0.01 vs. ctrl; ** p <
0.01 vs. 6-OHDA 50 μM.
Figure 8.
MJe prevented the release of NO induced by 6-OHDA. The levels of NO were measured by
a colorimetric assay. Differences in NO production are reported as percentage of NO value detected
in treated cells compared to those found in untreated ones. Results are expressed as means
±
SEM
from three independent experiments in triplicate (N = 9).
◦◦
p< 0.01 vs. ctrl; ** p< 0.01 vs. 6-OHDA
50 µM.
Antioxidants 2021,10, 539 12 of 16
3.10. Protective Effect of MJe on mRNA Levels of Parkinson-Related Genes Modulated
by 6-OHDA
As shown in Figure 9, the exposure of SH-SY5Y cells to 50
µ
M 6-OHDA for 24 h
significantly enhanced the levels of
α
-synuclein (SNCA) and leucine-rich repeat kinase
2 (LRRK2) genes up to 2.9
±
0.2- and 2.3
±
0.1-fold, respectively (p< 0.001), as well as
decreased those of phosphatase and tensin homolog (PTEN)-induced putative kinase 1
(PINK1), DJ-1, and parkin (PARK2) up to 2.2
±
0.15, 1.6
±
0.1 and 3
±
0.04-fold down,
respectively (p< 0.01). SNCA, LRRK2, and PARK2 were significantly modulated by the pre-
exposure to MJe at both 0.1 (1.3
±
0.15-fold down p< 0.01 for SNCA, 1.3
±
0.1-fold down,
p< 0.001 for LRRK2 and 2.7
±
0.1-fold p< 0.05 for PARK2, relative to 6-OHDA-stressed
cells) and 0.5 mg/mL concentrations (1.8
±
0.1-fold down for both SNCA and LRRK2,
and 3.7
±
0.3-fold for PARK2, p< 0.001, respect to 6-OHDA-stressed cells)
(Figure 9)
.
Concerning PINK-1 and DJ-1 genes, instead, only the highest concentration of MJe was
able to counteract the effect of 6-OHDA (2.0
±
0.1-fold p< 0.05 for PINK1, 1.9
±
0.2-fold
p< 0.01 for DJ-1, relative to 6-OHDA-stressed cells).
Antioxidants 2021, 10, x FOR PEER REVIEW 12 of 16
3.10. Protective Effect of MJe on mRNA Levels of Parkinson-Related Genes Modulated by 6-
OHDA
As shown in Figure 9, the exposure of SH-SY5Y cells to 50 μM 6-OHDA for 24 h
significantly enhanced the levels of α-synuclein (SNCA) and leucine-rich repeat kinase 2
(LRRK2) genes up to 2.9 ± 0.2- and 2.3 ± 0.1-fold, respectively (p < 0.001), as well as de-
creased those of phosphatase and tensin homolog (PTEN)-induced putative kinase 1
(PINK1), DJ-1, and parkin (PARK2) up to 2.2 ± 0.15, 1.6 ± 0.1 and 3 ± 0.04-fold down, re-
spectively (p < 0.01). SNCA, LRRK2, and PARK2 were significantly modulated by the pre-
exposure to MJe at both 0.1 (1.3 ± 0.15-fold down p < 0.01 for SNCA, 1.3 ± 0.1-fold down,
p < 0.001 for LRRK2 and 2.7 ± 0.1-fold p < 0.05 for PARK2, relative to 6-OHDA-stressed
cells) and 0.5 mg/mL concentrations (1.8 ± 0.1-fold down for both SNCA and LRRK2, and
3.7 ± 0.3-fold for PARK2, p < 0.001, respect to 6-OHDA-stressed cells) (Figure 9). Concern-
ing PINK-1 and DJ-1 genes, instead, only the highest concentration of MJe was able to
counteract the effect of 6-OHDA (2.0 ± 0.1-fold p < 0.05 for PINK1, 1.9 ± 0.2-fold p < 0.01
for DJ-1, relative to 6-OHDA-stressed cells).
Figure 9. Modulatory effects of MJe on mRNA levels of Parkinson-related genes modulated by 6-
OHDA. SH-SY5Y cells were pretreated with MJe for 1 h and then exposed to 6-OHDA 50 μM for
additional 24 h. Messenger RNA levels were quantified with real-time PCR, and their relative
quantities were calculated through the 2
–ΔΔCT
method. Results are expressed as fold change relative
to untreated cells. Data are expressed as mean ± SEM of three separate experiments in triplicate (N
= 9). * p < 0.05, *** p < 0.001 vs. control; ° p < 0.05, °° p < 0.01 and °°° p < 0.001 vs. 6-OHDA 50 μM.
4. Discussion
Citrus reticulata (mandarin), originated from Southeast China, is present in Europe
under a multitude of varieties and has been studied mainly for its anticancer properties
in different in vitro and in vivo models [10]. In these regards, we showed that MJe induces
antiproliferative activity in three different anaplastic thyroid carcinoma cell lines, block-
ing cell cycle in G2/M phase and inducing autophagy, as well as reducing cell migration
and affecting metalloproteinase activity [27].
To the best of our knowledge, this study is the first to assess the neuroprotective ef-
fect of MJe in 6-OHDA-stressed SH-SY5Y cells, a widely employed cell line to mimic the
cellular PD’s environment [28]. Among the most recognized PD models, we chose the 6-
OHDA, a neurotoxin inducing depletion of dopaminergic neurons in the substantia nigra
pars compacta, given the scientific evidence, both in vitro [29] ad in vivo [30,31], support-
ing its use.
First, we showed that MJe was able to defend cell viability from damage induced by
6-OHDA, as shown by MTT and trypan blue assays. Moreover, we found that our stressor
Figure 9.
Modulatory effects of MJe on mRNA levels of Parkinson-related genes modulated by
6-OHDA. SH-SY5Y cells were pretreated with MJe for 1 h and then exposed to 6-OHDA 50
µ
M
for additional 24 h. Messenger RNA levels were quantified with real-time PCR, and their relative
quantities were calculated through the 2
–∆∆CT
method. Results are expressed as fold change relative
to untreated cells. Data are expressed as mean
±
SEM of three separate experiments in triplicate
(N = 9)
. * p< 0.05, *** p< 0.001 vs. control;
◦
p< 0.05,
◦◦
p< 0.01 and
◦◦ ◦
p< 0.001 vs. 6-OHDA 50
µ
M.
4. Discussion
Citrus reticulata (mandarin), originated from Southeast China, is present in Europe
under a multitude of varieties and has been studied mainly for its anticancer properties in
different
in vitro
and
in vivo
models [
10
]. In these regards, we showed that MJe induces
antiproliferative activity in three different anaplastic thyroid carcinoma cell lines, blocking
cell cycle in G2/M phase and inducing autophagy, as well as reducing cell migration and
affecting metalloproteinase activity [27].
To the best of our knowledge, this study is the first to assess the neuroprotective effect
of MJe in 6-OHDA-stressed SH-SY5Y cells, a widely employed cell line to mimic the cellular
PD’s environment [
28
]. Among the most recognized PD models, we chose the 6-OHDA,
a neurotoxin inducing depletion of dopaminergic neurons in the substantia nigra pars
compacta, given the scientific evidence, both
in vitro
[
29
] ad
in vivo
[
30
,
31
], supporting
its use.
First, we showed that MJe was able to defend cell viability from damage induced
by 6-OHDA, as shown by MTT and trypan blue assays. Moreover, we found that our
stressor dramatically increased apoptotic events, as clearly demonstrated by Annexin V/PI
staining, an effect that was counteracted by MJe treatment both at 0.1 and 0.5 mg/mL,
though to different extent. Apoptosis is regulated by several factors that can push cells
Antioxidants 2021,10, 539 13 of 16
towards survival or programmed death. It is also acknowledged that increased ROS
levels unleash mitochondrial damage and hence the release of apoptosis inducers [
32
].
Proapoptotic proteins (i.e., Bax and Bad) and antiapoptotic ones (i.e., Bcl-2 and Bcl-XL) are
finely balanced to regulate the fate of each cell in the organism, a stability that comes to
an end in cellular degeneration [
33
]. Moreover, tumor suppressor p53 is sensitive to stress
such as DNA damage and hypoxia, being activated by phosphorylation and acetylation,
hence inducing cell-cycle arrest and apoptosis [
34
]. Accordingly, we evaluated the gene
expression of the abovementioned factors, finding that Bax and p53 were downregulated
by MJe treatment, whereas Bcl-2 was upregulated, in a completely opposite manner of our
stressor 6-OHDA. These clearly suggested that their regulation was crucial in the effect of
MJe in SH-SY5Y cells. Noteworthy, ROS are known to be able to directly activate the cascade
of caspases and hence the whole apoptotic machinery, directly aiming at caspase 3 [
35
].
Therefore, we also evaluated the involvement of this caspase in the protective effect of
MJe, and we witnessed a sharp hindering of its activation compared to the levels observed
in control cells, suggesting the role of caspase 3 as a keystone in the overall protective
mechanism brought by MJe. As is widely known, 6-OHDA increases ROS levels as a direct
result of mitochondrial impairment [
36
], being one of the possible cellular causes of PD,
as well as other neurodegenerative diseases. Consequently, we aimed at evaluating the
ROS levels in SH-SY5Y cells after 6-OHDA stress, finding a robust increase of the species,
which was blocked by MJe treatment. Moreover, our extract ameliorated
∆ψ
m, impaired
by 6-OHDA injury that, together with the reduction of ROS levels, are clear signals of
mitochondrial protection elicited by our extract. Unbalanced ROS production brings its
effects at the nucleus level, where oxidative damage on DNA gives birth to oxidized base
adducts, among which 8-oxo-dG is a relevant marker of early clinical manifestations of
cognitive impairment [
37
]. Therefore, we evaluated its presence in SH-SY5Y cells after
6-OHDA stress, detecting an increase of the portion of fluorescent cells, and hence carrying
this DNA oxidative adduct, whereas MJe was able to lower this effect.
The antioxidant capacity of MJe was also appreciated through the evaluation of typical
cellular biomarkers involved in this process, namely SOD, CAT, and GSH. The exposure
of SH-SY5Y cells to 6-OHDA decreases cellular antioxidant defense, and flavonoids are
acknowledged for hampering it [
38
]. Here, we found that MJe was able to restore both
SOD and CAT activity, two fundamental enzymes that act together to quench oxygen
radicals, as well as GSH levels. Interestingly, MJe proved to also be a great antioxidant in
abiotic models, where it was able to quench both oxygen (ORAC) and nitrogen (DPPH)
radicals along with reducing ferric ions into ferrous ones, effects likely ascribed to the high
polyphenol content, as assessed by Folin–Ciocalteu assay and in line with previous reports
on Citrus extracts [39,40].
Nitric oxide (NO) is another central signaling molecule whose overproduction is
acknowledged to be the cause of neuronal impairment, typical of neurodegenerative
diseases like PD [
41
]. Notably, MJe decreased the cellular levels of NO that were drastically
increased by 6-OHDA. Furthermore, our extract acted as both antioxidant and reducing
agent in different cell-free models, given its high polyphenolic content, reinforcing the
results we obtained
in vitro
. The etiology of PD is not fully understood yet, but scientific
evidence shows that mutations in SNCA, PINK1, parkin, DJ-1, and LRRK2 genes are at the
basis of familial cases of PD [
42
]. These encode for proteins that are tightly intertwined
in the process of mitophagy and hence in the regulation of neuron viability. After an
oxidative stimulus,
α
-synuclein increases in neurons, forming the so-called Lewy bodies,
agglomerates typical of PD. Overexpression of this protein induces an increase of ROS
at the cellular level, starting a vicious cycle where
α
-synuclein induce ROS and vice
versa. Gain-of-function mutation of LRRK2 gene brings to an increased susceptibility of
neurons to oxidative stress, as widely demonstrated. On the other hand, parkin and its
regulator PINK1 are known to be involved in mitochondrial survival and protection against
ROS, together with DJ-1 that homodimerizes, becoming another antioxidant neuronal
defense [
43
]. In our study, the oxidative stress induced by 6-OHDA brought an expected
Antioxidants 2021,10, 539 14 of 16
sharp increase of both SNCA and LRRK2, an effect that was hampered by MJe treatment.
Conversely, the mitochondrial antioxidant machinery, consisting of PINK1/parkin and
DJ-1, was negatively affected by the stressor. However, MJe was able to restore the levels
of genes encoding the abovementioned proteins and hence improving response against
oxidative stress. Our results are in line with previous reports in which natural products
were able to hamper neuronal oxidative stress elicited by 6-OHDA, lowering SNCA and
LRRK2 expression levels along with increasing those of PINK1/parkin and DJ-1 [44,45].
5. Conclusions
Overall, MJe hampered the oxidative stress induced by 6-OHDA treatment in our
cellular model, contemporarily targeting the mitochondria, nucleus, and cytoplasm, pro-
tecting these compartments by ROS overproduction and increasing the survival rate, along
with blocking apoptotic machinery. From a molecular point of view, MJe restored the gene
expression of factors linked to mitochondrial functionality, acknowledged to be crucial in
PD clinical outcomes, whose balance was impaired by 6-OHDA. Therefore, we suggest the
great validity of MJe in facing oxidative-based diseases, such as PD, that needs to be also
proven in more complex models to corroborate our statements.
Author Contributions:
S.C. performed the experiments, analyzed the data, and drafted the manuscript;
A.M. performed the experiments and drafted the manuscript; G.E.L., C.R. and L.M. supported the ex-
perimental procedures, S.G. and G.C. critically revised the manuscript, D.B. performed the chemical
analysis and critically revised the manuscript; M.N. conceived and designed the experiments as well
as critically revised the manuscript. All authors have read and agreed to the published version of
the manuscript.
Funding:
Research was supported by Grant from Sicily Region (PO FESR Sicilia 2007/2013, CUP
G73F11000050004 to MN, project “MEPRA”, n 133 of Linea d’Intervento 4.1.1.1).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article.
Conflicts of Interest: The authors declare no conflict of interests.
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