Extracts of Physalis peruviana Protect Astrocytic Cells Under Oxidative Stress With Rotenone

Abstract

The use of medicinal plants to counteract the oxidative damage in neurodegenerative diseases has steadily increased over the last few years. However, the rationale for using these natural compounds and their therapeutic benefit are not well explored. In this study, we evaluated the effect of different Physalis peruviana extracts on astrocytic cells (T98G) subjected to oxidative damage induced by rotenone. Extracts of fresh and dehydrated fruits of the plant with different polarities were prepared and tested in vitro. Our results demonstrated that the ethanolic extract of fresh fruits (EF) and acetone-dehydrated fruit extract (AD) increased cell viability, reduced the formation of reactive oxygen species (ROS) and preserved mitochondrial membrane potential. In contrast, we observed a significant reduction in mitochondrial mass when rotenone-treated cells were co-treated with EF and AD. These effects were accompanied by a reduction in the percentage of cells with fragmented/condensed nuclei and increased expression of endogenous antioxidant defense survival proteins such as ERK1/2. In conclusion, our findings suggest that ethanolic and acetone extracts from P. peruviana are potential medicinal plant extracts to overcome oxidative damage induced by neurotoxic compounds.

Full text

ORIGINAL RESEARCH
published: 20 July 2018
doi: 10.3389/fchem.2018.00276
Frontiers in Chemistry | www.frontiersin.org 1July 2018 | Volume 6 | Article 276
Edited by:
Eduardo Dellacassa,
University of the Republic, Uruguay
Reviewed by:
Michael Erich Netzel,
The University of Queensland,
Australia
Alam Zeb,
University of Malakand, Pakistan
*Correspondence:
George E. Barreto
gesbarreto@gmail.com;
gsampaio@javeriana.edu.co
Specialty section:
This article was submitted to
Food Chemistry,
a section of the journal
Frontiers in Chemistry
Received: 27 August 2017
Accepted: 18 June 2018
Published: 20 July 2018
Citation:
Areiza-Mazo N, Robles J,
Zamudio-Rodriguez JA, Giraldez L,
Echeverria V, Barrera-Bailon B,
Aliev G, Sahebkar A, Ashraf GM and
Barreto GE (2018) Extracts of Physalis
peruviana Protect Astrocytic Cells
Under Oxidative Stress With
Rotenone. Front. Chem. 6:276.
doi: 10.3389/fchem.2018.00276
Extracts of Physalis peruviana
Protect Astrocytic Cells Under
Oxidative Stress With Rotenone
Natalia Areiza-Mazo 1, Jorge Robles 2, Jairo A. Zamudio-Rodriguez 1, Lisandro Giraldez 3,
Valentina Echeverria 4,5, Biviana Barrera-Bailon 1, Gjumrakch Aliev 6, 7,8 ,
Amirhossein Sahebkar 9,10,11 , Ghulam Md Ashraf 12 and George E. Barreto 1
*
1Departamento de Nutrición y Bioquímica, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá, Colombia,
2Departamento de Química, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá, Colombia, 3Departamento de
Química e Exatas, Universidade Estadual do Sudoeste da Bahia, Jequié, Brazil, 4Facultad de Ciencias de la Salud,
Universidad San Sebastián, Concepción, Chile, 5Bay Pines VA Healthcare System, Research and Development, Bay Pines,
FL, United States, 6Institute of Physiologically Active Compounds, Russian Academy of Sciences, Chernogolovka, Russia,
7GALLY International Biomedical Research Consulting LLC., San Antonio, TX, United States, 8School of Health Science and
Healthcare Administration, University of Atlanta, Johns Creek, GA, United States, 9Neurogenic Inflammation Research
Center, Mashhad University of Medical Sciences, Mashhad, Iran, 10 Biotechnology Research Center, Pharmaceutical
Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran, 11 School of Pharmacy, Mashhad University of
Medical Sciences, Mashhad, Iran, 12 King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia
The use of medicinal plants to counteract the oxidative damage in neurodegenerative
diseases has steadily increased over the last few years. However, the rationale for using
these natural compounds and their therapeutic benefit are not well explored. In this study,
we evaluated the effect of different Physalis peruviana extracts on astrocytic cells (T98G)
subjected to oxidative damage induced by rotenone. Extracts of fresh and dehydrated
fruits of the plant with different polarities were prepared and tested in vitro. Our results
demonstrated that the ethanolic extract of fresh fruits (EF) and acetone-dehydrated fruit
extract (AD) increased cell viability, reduced the formation of reactive oxygen species
(ROS) and preserved mitochondrial membrane potential. In contrast, we observed a
significant reduction in mitochondrial mass when rotenone-treated cells were co-treated
with EF and AD. These effects were accompanied by a reduction in the percentage
of cells with fragmented/condensed nuclei and increased expression of endogenous
antioxidant defense survival proteins such as ERK1/2. In conclusion, our findings suggest
that ethanolic and acetone extracts from P. peruviana are potential medicinal plant
extracts to overcome oxidative damage induced by neurotoxic compounds.
Keywords: Physalis peruviana, uchuva, phenolics, astrocytes, oxidative stress, rotenone
INTRODUCTION
Free radicals and reactive oxygen species (ROS) are common by-products of cellular metabolism
and xenobiotic exposure (Circu and Aw, 2010). These molecules play important roles in the defense
against pathogens and mediate intracellular communication and regulation (Schieber and Chandel,
2014). They also interfere with highly complex biological pathways that regulate cell growth, cell
death and senescence. This interference is exerted through various mechanisms such as acting on
phagolysosome formation and enzymatic degradation, autophagy, chemoattraction, inflammation,
and acting as redox messengers (Paiva and Bozza, 2014). However, excessive ROS generation

Areiza-Mazo et al. Uchuva Extracts Protect Cells
can lead to cell damage through destabilization of the
mitochondrial oxidative phosphorylation supercomplexes, which
in turn will stimulate further ROS production. This vicious
cycle in most cases can induce oxidative stress and subsequent
damage to vital biomolecules such as lipids, proteins, and
DNA (Halliwell, 2007; Chaban et al., 2014). Overproduction
of ROS has been associated with several chronic diseases such
as cancer, autoimmune disorders, cardiovascular disease, and
neurodegenerative diseases (Halliwell, 2007; Kim et al., 2010;
Blach-Olszewska et al., 2015; Gasiorowski et al., 2017).
Brain tissue is especially vulnerable to oxidative stress owing
to its high oxygen consumption and the oxidizing capacity of
monoamine oxidase on neurotransmitters such as dopamine that
results in H2O2overproduction (Barreto G. E. et al., 2011).
Other factors such as the presence of highly reactive copper
and iron ions, high concentrations of polyunsaturated fatty acids
and low concentrations of the antioxidant enzymes catalase and
glutathione peroxidase account for the suceptibility of the brain
tissue to oxidative damage (Cui et al., 2004; Albarracin et al.,
2012). Oxidative stress has been associated with mitochondrial
dysfunction, a pathological mechanism that occurs in most
neurodegenerative diseases including Alzheimer’s disease (AD)
and Parkinson’s disease (PD) (Cabezas et al., 2012, 2015; Denzer
et al., 2016). In this regard, defects in mitochondrial energy
metabolism may result in a decrease in high-energy phosphate
reserves, antioxidant defense impairment, deterioration of
membrane potential, and deregulation of calcium homeostasis,
thereby leading to excitotoxicity and neuronal death (Federico
et al., 2012; Chaturvedi and Flint Beal, 2013; Abeti and Abramov,
2015). Therapeutic efforts aiming at improving brain antioxidant
defense are essential to alleviate damage especially during
neurodegeneration (Barreto et al., 2017). To counteract neuronal
degeneration, astrocytes, the second most abundant cells in the
brain after neurons (Barreto G. E. et al., 2011; Barreto G. et al.,
2011; Garzon et al., 2016), play a vital role. This protective
role of astrocytes against oxidative damage is exerted through
releasing endogenous antioxidant species such as glutathione and
superoxide dismutase (SOD), or removing glutamate (Barreto G.
et al., 2011).
There is an increasing interest in studying the neuroprotective
effects of natural products and functional foods. Phytochemicals
are exogenous antioxidants that are widely present in fruits and
vegetables. For example, polyphenols from berries (blueberries
and grapes) have shown antioxidant and anti-aging effects (Kim
et al., 2010; Vuong et al., 2010; Xia et al., 2010; Bornsek et al.,
2012; Queiroz et al., 2017), suggesting potential neuroprotective
actions. Additionally, previous animal studies have shown
that consumption of flavonoid-rich foods improves cognitive
function, vascular function and synaptic plasticity in the brain
(Rendeiro et al., 2015).
In a continuing search for natural compounds to be
used for the prevention and treatment of PD, the present
study investigated the properties of different fractions isolated
from the fruits of Physalis peruviana, commonly known
as goldenberry. This plant possesses antioxidant and anti-
inflammatory properties associated with its polyphenol content
(Horn et al., 2015; Yildiz et al., 2015). Goldenberry has a high
content of vitamins A, C, E, D, and B complex, polyphenols,
withanolides, and carotenoids (Xu et al., 2017; Etzbach et al.,
2018). Some of these components of goldenberry act as
“scavengers” of free radicals and can prevent cell damage and
neuroinflammation induced by oxidative stress (Abdel Moneim,
2016; Sang-Ngern et al., 2016). The present work aimed to
perform a preliminary evaluation of the antioxidant properties
of goldenberry components in PD. We assessed the effect of
different fractionated extracts of P. peruviana on the response of
astrocytic cells to rotenone, an inhibitor of the complex I of the
mitochondrial electron transport chain that is used to generate a
cellular model of PD.
MATERIALS AND METHODS
Chemicals
The chemicals used to isolate the plant extracts, including
ethanol, dichloromethane, petroleum benzine, acetone, and
ethyl acetate, were purchased from Merck (Merck & Co., Inc.,
Kenilworth, NJ, USA).
Plant Material
The fruits of P. peruviana plant were obtained from an organic
farm located at 2,900 m above sea level in Cundinamarca,
Colombia. Before use, each fruit was selected based on the
absence of the signs of bacterial or fungal infection, or structural
damage. The vegetal material was obtained with the calyx present
to preserve the integrity of the fruit. Fruits were divided in two
parts of ∼1,000 g. One part was heat dehydrated at 45◦C for 4
days, macerated and stored until the day of use. Another part was
homogenized in a food chopper and used fresh.
Preparation of Crude Extracts
The preparation followed the protocol developed previously
by Domínguez (1979). Briefly, fresh fruit was submerged into
an ethanol solution at room temperature (RT) under mild
agitation. Then, ethanol was evaporated using a rotary evaporator
(BUCHI, RE 111. Flawil, Switzerland) at 40◦C until a pure
ethanolic fresh fruit (EF) fraction was obtained. About 80% of the
ethanolic extract was used for liquid-liquid fractionation, and the
remaining 20% was used to perform bioassays. The first fraction
was obtained with petroleum benzine, then dichloromethane
and finally with ethyl acetate. Each fraction was evaporated to
obtain the respective fractions (BF), (DF), and (AF). The final
material was lyophilized (FreeZone 2.5 Liter Benchtop Freeze
Dry System, Labconco©, Kansas City, MO, USA) to obtain the
lyophilized extract (L). On the other side, the dehydrated fruit
was submerged into a petroleum benzene solution at RT with
mild agitation, then evaporated to obtain the respective fraction
(Benzene Dehydrated; BD). Subsequently, the resulting residual
material was extracted first with dichloromethane, then with
acetone, and lastly with ethanol, and each solvent was evaporated
to obtain the pure fractions dichloromethane dehydrated (DD),
acetone dehydrated (AD), and ethanolic dehydrated (ED).
Materials were submerged, with mild agitation, in 2 L of each
solvent for a period of 2 days to obtain the individual extracts.
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Areiza-Mazo et al. Uchuva Extracts Protect Cells
The obtained fractions were weighed and diluted in 99.9% DMSO
and stored at 20◦C.
Determination of the Total Phenolic
Content
Folin-Ciocalteu reagent (F9252. Sigma-Aldrich R
, St. Louis, MO,
USA) assay was used for determining the content of phenols
(Mena et al., 2012). The testing mix consisted of 50 mg extracts
(100 µL), 800 µL of distilled water, and 100 µL of Folin-
Ciocalteau. The mix was incubated in the dark for 8 min.
Subsequently, 50 µL of 7.5% sodium carbonate was added and
the new mix solution incubated for 1 h. Finally, the phenolic
content was determined spectrophotometrically measuring the
absorbance of the mix at 760 nm and a standard curve made with
known concentrations of gallic acid.
Cell Culture
T98G [T98-G] Homo sapiens brain glioblastom (ATCC R

CRL-1690TM) cell line was maintained under exponential
growth in Eagle Modified by Dulbeco (DMEM) (12-
917F Lonza R
Walkersville, MD, USA) culture medium,
supplemented with 10% fetal bovine serum (FBS), antibiotics
(penicillin/streptomycin) and amphotericin at 37◦C. Cell
cultures were maintained in a humidified atmosphere containing
5% CO2(Ávila Rodriguez et al., 2014).
Drug Treatments
Cells were seeded in multi-well plates and allowed to grow for
24 h. Afterwards, the cultured cells were serum-deprived for
24 h prior to treatments. Then, cultured cells were exposed to
rotenone [50 µM] (R8875. Sigma-Aldrich R
, St. Louis, MO, USA)
for 24 h, as described by Cabezas et al. (2015).
Cell Viability
T98G cell viability was tested using MTT (5 mg/ml stock
solution) [3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyltetrazolium
bromide] assay (M2128. Sigma-Aldrich R
, St Louis, MO, USA)
(Swarnkar et al., 2012; Riss et al., 2013). Cells were seeded into
96-well plates in DMEM culture media containing 10% bovine
fetal serum at a seeding density of 10,000 cells per well and
allowed to grow for 24 h. Afterward, cells were serum deprived
for 24 h, and finally treated with golden berries extracts at 25, 50,
100 y 200 µg/ml for 12, 18, and 24 h. Cell viability was assessed
following the treatments by adding 0.45 mg/ml per well MTT
solution for 4 h at 37◦C in the dark. Afterwards, formazan crystals
were solubilized with dimethyl sulfoxide (DMSO; 276855.Sigma-
Aldrich R
, St Louis, MO, USA) and the absorbance at 490 nm
was determined. Each assay was performed with a minimum of
six replicate wells for each condition. The amount of released
formazan, which is directly proportional to the number of live
cells, was determined by optical density (OD) at 540 nm in a
spectrophotometer. The values were normalized to the value
of the control culture without extract added containing 0.01%
DMSO, which was considered 100% survival. Rotenone-treated
cells were used as the control for neurotoxicity.
Determination of Reactive Oxygen Species
(ROS)
To measure the potential neuroprotective effect of the
goldenberry extracts from superoxide (O2−) and oxygen
peroxide (H2O2) production induced by rotenone, ROS
production was evaluated by cytometry and fluorescence
microscopy as described (Torrente et al., 2014). Briefly, cells
were seeded at a density of 25,000 cells per well into 48-well
plates in a DMEM culture medium containing 10% FBS and
then subjected to 24 h of serum deprivation before treatments.
Then, cells were treated with 10 µM dihydroethidium (DHE;
37291. Sigma-Aldrich R
, St Louis, MO, USA) or 1 µM 20,70-
dichlorofluorescein diacetate (DCFDA; 35845. Sigma-Aldrich R
,
St Louis, MO, USA), in the dark at 37◦C for 30 min. Then cells
were washed in PBS, detached with trypsin (Trypsin/EDTA
500 mg/l:200 mg/L- BE02-034E. LONZA, Walkersville, MD,
USA) and analyzed for flow cytometry in a Guava EasyCyteTM
cytometer (Millipore, Billerica, MA, USA). Each assay was
performed with a minimum of three replicates per condition.
For DHE fluorescence imaging analysis, cells were seeded
at a density of 20,000 cells per well into 48-well plates in a
DMEM culture medium containing 10% FBS. On the next day,
cells were co-treated with 50 µM rotenone plus a plant extract.
After 24 h, cells were incubated with DHE for 30 min. Finally,
cells were washed with PBS and photographed in an Olympus
IX53 fluorescence microscope- ex/em 340/510 nm (OLYMPUS
CORPORATION, Shinjuku, Tokyo, Japan). The images were
processed with Image J software (NIH, Bethesda, MD, USA),
and the mean fluorescence intensity of randomly selected cells
was determined by fluorescence microscopy (Olympus IX53
microscope, ex/em 340/510 nm; OLYMPUS CORPORATION,
Shinjuku, Tokyo, Japan) using a 20X objective. The number
of fluorescent cells was determined in at least eight randomly
selected areas (0.03 mm2) from each experimental group. Each
experiment was performed in triplicate.
Cytoplasmic Nitric Oxide Concentration
(NO)
Nitric oxide production was evaluated by determining nitrite
levels with the Griess reagent (G4410; Sigma-Aldrich R
, St Louis,
MO, USA). Cells were seeded at a density of 25,000 cells per
well into 48-well plates in DMEM containing 10% FBS. Then,
cells were subjected to serum deprivation for 24 h and co-
treatment with 50 µM rotenone plus the respective extract. Once
treatments were completed, the culture medium was harvested
and maintained at −80◦C until used. Duplicates of 100 µL of
the culture medium were added to 96-well plates and mixed with
100 µL of Griess reagent. Quantification was performed using
spectrophotometry (Omega Star Fluorometer; BMG LabTech,
Ortenberg, Germany) measuring absorbance at 540 nm. Each
assay was performed in triplicates.
Lipid Peroxidation
Lipid peroxidation is an indicator of oxidative stress and the
reactive aldehydes generated, like 4-hydroxynonenal (HNE)
may well act as “second toxic messengers.” To visualize lipid
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Areiza-Mazo et al. Uchuva Extracts Protect Cells
peroxidation, fluorescence of HNE was measured. Cells were
washed twice with PBS after rotenone plus the respective extract
in co-treatment, and then fixed in 4% paraformaldehyde for
10 min. Cells were permeabilized with 1% Triton X-100, diluted
in PBS with 2.5% serum, for 20 min at RT, and stored at
−20◦C. Then, cells were immunostained with rabbit anti-HNE
antibodies (1:500; ab46545, Abcam, Cambridge, MA, USA) and
analyzed using fluorescence microscopy as described in section
Determination of Reactive Oxygen Species (ROS).
Nuclear Fragmentation
To visualize nuclear morphology after co-treatment with
rotenone and the respective extract, cells were washed twice with
PBS and then fixed in 4% paraformaldehyde for 20 min (Ávila
Rodriguez et al., 2014). Cells were then stained with 2.5 mg/mL
DNA dye Hoechst 33342 (14533. Sigma-Aldrich R
, St Louis, MO,
USA) in PBS for 15 min at RT and analyzed using fluorescence
microscopy as described in section Determination of Reactive
Oxygen Species (ROS). Data was expressed as the percentage of
cells presenting nuclear fragmentation.
Determination of Mitochondrial Membrane
Potential (19m)
Mitochondrial membrane potential was evaluated using
tetramethyl rhodamine methyl ester (TMRM). TMRM (T5428.
Sigma-Aldrich R
, St Louis, MO, USA) is a cell penetrating
cationic fluorescent dye sequestered by active mitochondria.
After 24 h of established treatments, cells were loaded in the dark
with 500 nM TMRM at 37◦C for 30 min. Thereafter, cells were
washed with PBS and quantified using flow cytometry in a Guava
R Easy CyteTM (Millipore) cytometer. As an experimental
control, we used the protonophore uncoupler carbonyl cyanide
m-chlorophenylhydrazine (CCCP; Sigma-Aldrich R
, St Louis,
MO, USA; 10 mM) to dissipate the membrane potential and
define the baseline for the analysis (Cabezas et al., 2015).
Determination of the Mitochondrial Volume
Mitochondrial mass was evaluated using Nonyl Acridine
Orange (NAO; A1372. Invitrogen, Waltham, MA, USA), a
cell penetrating cationic fluorescent dye sequestered by active
mitochondria (Cabezas et al., 2015). Cells were seeded at a
density of 20,000 cells per well into 48-well plates in a DMEM
culture medium containing 10% FBS and then were treated
according to the experimental paradigm. After 24 h of treatment,
cells were loaded with 5 µM NAO at 37◦C for 30 min in the
dark. Afterwards, cells were washed with PBS and mitochondrial
volume was evaluated using flow cytometry in a Guava R Easy
CyteTM (Millipore) cytometer.
Mitochondrial and Endoplasmic Reticulum
Calcium Concentration
Mitochondrial and endoplasmic reticulum calcium
concentrations were evaluated using the Rhod-2 (R1245MP,
Thermo Fisher Waltham, MA, USA) and Mag-fura-2 (M1292,
Thermo Fisher Waltham, MA, USA) according to the protocol
described by Avila et al. with minor modifications (Ávila
Rodriguez et al., 2014). Cells were seeded at a density of 20,000
cells per well into 48-well plates in a DMEM culture medium
containing 10% FBS and then were treated according to the
previous experimental paradigm of 24 h of serum deprivation.
After 24 h of treatments, cells were incubated with 5 µM Rhod-2
or 3 µM Mag-fura-2 in standard medium for 30 min in the
incubator. Single cell fluorescence was excited at 545 nm in an
Omega Star Fluorometer (BMG LabTech, Ortenberg, Germany).
Determination of Antioxidant Status
The expression of antioxidant molecules and the protein
concentration of superoxide dismutase (SOD, Mn-SOD)
(1:1,000; PA5-30604, Thermo Fisher, Waltham, MA, USA),
catalase (CAT) (1:1,000; PA5-18531, Thermo Fisher. Waltham,
MA, USA) and glutathione peroxidase (GPx) (1:1,500; PA5-
30593, Thermo Fisher, Waltham, MA, USA) and protein
kinases ERK1 (13-8600, Thermo Fisher, Waltham, MA, USA)
and ERK1/2 (13-6200, Thermo Fisher, Waltham, MA, USA)
were determined using Western blotting according to the
protocol described by Baez-Jurado et al. (2017).β-tubulin
immunoreactivity (1:100; 2128, Cell Signaling, Danvers, MA,
USA) was used for protein loading and transfer. T98G cells
were lysed on ice with RIPA Lysis, and Extraction Buffer
Thermo ScientificTM (89900, Thermo Fisher, Waltham, MA,
USA) supplemented with HaltTM Protease Inhibitor Cocktail,
EDTA-free (100X) (78430, Thermo Fisher, Waltham, MA,
USA). Protein content was estimated using the PierceTM BCA
Protein Assay Kit. Equal amounts of protein were dissolved in
sample buffer containing 5% β-mercaptoethanol and boiled.
Then, proteins were separated by electrophoresis in SDS–PAGE,
transferred onto a PVDF membrane and blocked in 5% skim
milk dissolved in Tris-buffered saline containing 0.05% Tween
20 (TBS-T) at RT for 1 h. The membranes were incubated at
4◦C overnight with antibodies against the desired protein. The
immunoreactivity was visualized by incubating the membrane
with the specific secondary antibody (IRDye R
Antibodies)
for 1 h and detected using Odyssey CLx Imaging System
Specifications (LI-COR Biosciences). The intensity of each band
was quantified using Image J software (National Institutes of
Health, Bethesda, MD, USA). All data are normalized to control
values on each gel.
Statistical Analysis
Data were tested for normal distribution using the Kolmogorov–
Smirnov test and homogeneity of variance by Levene’s test.
Then, data were examined using analysis of variance, followed
by Dunnet’s post-hoc test for comparisons between controls and
treatments and Tukey’s post-hoc test for multiple comparisons
between the means of treatments and time points. Data is
presented as mean ±SEM values. A statistically significant
difference was defined at p<0.05. The results were analyzed
using GraphPad Prism software version 5.00 for Windows
(GraphPad Software, San Diego, CA, USA).
RESULTS
Cytotoxic Effect of P. peruviana Extracts
on T98G Cells
The analysis of the cytotoxic effect of the fruit extracts from
P. peruviana on T98G cells allowed us to establish both the
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Areiza-Mazo et al. Uchuva Extracts Protect Cells
extract concentration with the least toxic effect and the optimal
treatment conditions. Our results showed a significant decrease
in cell viability (>50%, p<0.0001) in cells exposed to both
petroleum benzene and dichloromethane extracts of the fresh
fruit (BF, DF) as well as petroleum benzene and dichloromethane
extracts of dehydrated fruit (BD, DD) at various incubation
times (12, 18, and 24 h) and concentrations (25, 50, 100, and
200 µg/ml; Supplementary Figure 1). Reductions of 78.7 or
40% in cell viability were observed when cells were exposed
to 200 µg/mL of BD for 18 h (Supplementary Figure 1A) or
200 µg/Ml of DD extract for 12 h (Supplementary Figure 1B).
This cytotoxic effect was independent of the concentration
of the extract used and the duration of incubation with
BD, DD, and BF extracts (Supplementary Figures 1A–C). DF
extract showed a cytotoxic effect on T98G cells that was
dependent on both duration and concentration of exposure.
No reduction in cell viability was observed at 25 µg/mL DF
(p=0.53) for 12 h or at 25 µg/mL (p=0.95) and 50 µg/mL
(p=0.9262) for 18 h but a cytotoxic effect was obser ved in
all concentrations after 24 h (Supplementary Figure 1D). In
contrast, ethanolic extracts (EF from fresh fruit and ED from
dehydrated fruit), ethyl acetate extracts (AF), acetone extracts
(AD), and lyophilized samples (L) did not affect cell viability
at 25, 50, and 100 µg/mL concentrations in all three times
tested (Supplementary Figures 2A–E). Indeed, some extracts
such as AD (50 µg/mL for 12 h) increased cell viability by 53%
(Supplementary Figure 2B). The ED (200 µg/ml for 24 h) and
EF (for 12 and 18 h) significantly decreased cell viability by
49, 30, and 43.5%, respectively (p=0.0003) (Supplementary
Figures 2A,C). Similarly, AD extract at the higher concentration
(200 µg/mL for 18 and 24 h) had a toxic effect, decreasing
cell viability by 74.7 and 71.9%, respectively (p<0.0001)
(Supplementary Figure 2B).
EF Extract Increased Cell Survival Against
Rotenone in T98G Cells
Based on the toxicity assays, ED, EF, AD, AF, and L extracts at
25 µg/mL were selected to assess their cytoprotective effects. Cells
were co-treated with the plant extracts and 50 µM rotenone for
24 h. The results showed that rotenone reduced cell viability by
52% (p=0.013). However, EF extracts improved cell survival
by 51% (p=0.0073). Also, a non-significant increase (p<0.05)
in cell viability was attained with AD (33.4%) and ED (21.7%),
AF (11.9 %), while L extract diminished viability by 3%
(Figure 1A).
Correlation of Phenol Content of Fruit Extracts and
Protective Effect on T98G Cells
Quantification of phenols in extracts ED, EF, AD, AF, and L
was performed. The highest content of phenolic compounds was
present in the AD extract with 11.13 mg gallic acid equivalents/g
of dry mass, followed by the ED extract with 10.6 mg equivalents
(Table 1). The lowest concentrations were found in the EF extract
with 6.5 mg equivalents, while AF presented 5.9 mg equivalents
and the freeze-dried extract with 4.2 mg gallic acid equivalents/g
sample (Table 1). No correlation was found between quantity of
phenols and percentage of survival (Figure 1B).
EF and AD Extracts Reduced ROS Levels
in Cells Upon Oxidative Damage
The formation of superoxide radical and hydrogen peroxide
showed a significant increase by 160% (p<0.0001) for
superoxide and over 200% (p<0.0001) for hydrogen peroxide
in cells exposed to rotenone (Figures 2A,C,F). Similarly, DCFA
fluorescence increased from 10.7 to 60 in rotenone-treated cells
and decreased to 22 and 32.3 (p=0.0027 and p=0.0261)
in cultures co-treated with EF and AD (25 µg/mL for 24 h),
respectively (Figure 2F). The formation of DHE showed a
significant decrease of 82.9% (p=0.016) and 121.5% (p=0.0004)
in cells co-treated with the extracts EF and AD at 25 µg/mL
for 24 h, respectively (Figures 2A,D,E). Confirming the previous
results, the fluorescence analysis indicated a higher emission
of the fluorochrome DHE in cells treated with rotenone and
a decrease in the cells co-treated with the EF and AD extracts
(Figures 2B–E).
Analysis of the conditioned media of T98G cells co-treated
with rotenone, and EF or AD extracts using the Griess reagent did
not show statistically significant differences in nitrite production
between the groups (Figure 2G). The lowest mean nitrite
concentration was 14 ±2µM for AD treatment and the
maximum concentration was 17 ±5µM when cells were treated
with rotenone alone.
Extracts of P. peruviana Decreased Lipid
Peroxidation in Rotenone-Treated Cells
Peroxidation of membrane lipids was quantified as a measure
of oxidative damage. Our results demonstrated that rotenone
increased HNE fluorescence from 8 to 12.6. In contrast, there was
a significant decrease in peroxidation levels, expressed as lower
HNE fluorescence from 2.06 to 9 and 9.2 when cells were exposed
to AD and EF extracts (25 µg/mL) (p<0.0001), respectively
(Figures 2H–L).
EF and AD Extracts Preserved
Mitochondrial Functions
Mitochondrial membrane potential (19m) showed a significant
decrease of 70.98% (p<0.0001) in cells exposed to rotenone
(Figure 3A). In contrast, EF and AD improved mitochondrial
potential by 17.9% (p=0.0013) and 12.2% (p=0.0097)
compared to rotenone alone (Figure 3A). On the other hand,
mitochondrial mass increased significantly by 51% in rotenone-
insulted cells (p=0.0189) and decreased by 47.6% (p=0.03)
after treatment with AD (25 µg/mL). Moreover, EF (25 µ
g/ml) induced a 19.4% decrease in mitochondrial mass, though
this difference was not statistically significant (p=0.56;
Figure 3B).
Plant Extracts Modulate Calcium Levels in
Cells Stimulated With Rotenone
When calcium levels were analyzed, a significant decrease in
mitochondrial Ca2+levels (19%) (p=0.034) was found in
cells exposed to rotenone (Figure 3C). A similar decrease was
observed in cells co-treated with rotenone and EF (17.5%;
p=0.057). However, this decrease was lower in cells treated
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Areiza-Mazo et al. Uchuva Extracts Protect Cells
FIGURE 1 | Effect of extracts of Physalis peruviana on the viability of T98G cell. (A) Percentage of survival of T98G cells measured by MTT against the toxic effect of
50µM rotenone for 24 h and simultaneous treated with extracts EF, ED, AF, AD, and L (25µg/ml). A 52% decrease in cell survival (p=0.0126) was detected after
exposure to rotenone. In the co-treatment with EF extract, cellular survival was increased in 51% (p=0.073), followed by AD extract in 33% (p=0.1290), ED in
21.7% (p=0.4839), AF in 11.9% (p=0.8990). There was no significant effect of the lyophilized extract. (B) Correlation of the phenol content and percentage of cell
survival of the extracts EF, ED, AF, AD, and L before the insult with 50µM rotenone. The content of phenols in the extracts EF, ED, AF, AD, and L was determined by
the Folin-Ciocalteu method.
TABLE 1 | Quantification of phenols and relation of percentage of survival of T98g
cells in relation with Rotenone control treatment.
Extract mg gallic acid
equivalents/grams of
dry mass
% Cell survival
Dehydrated fruit ethanolic
extract (ED)
10.6 21.7
Fresh fruit ethanol extract
(EF)
6.5 51.4
Dehydrated fruit acetone
extract (AD)
11.1 33.4
Dehydrated fruit ethyl
acetate extract (AF)
5.9 11.9
Freeze- dried (L) 4.2 −3.31
with AD extracts (8.9%; p=0.48). In contrast, ER calcium
in T98G cells showed a slight, but non-significant, increase of
13% in cells stimulated with rotenone and cells co-treated with
rotenone and EF or AD (25 µg/mL) relative to the control
culture exposed to culture medium containing 0.05% DMSO
(Figure 3D). There were no difference in ER calcium between
the insulted cells and those treated with the extracts mentioned
above.
EF Extract Decreased Nuclear
Fragmentation Induced by Rotenone
The number of fragmented and condensed nuclei quantified by
fluorescence microscopy indicated an increase of 15.6% after
treatment with 50 µM rotenone (Figure 3E). In parallel, a non-
significant decrease in the condensed nuclei was observed in
the cells co-treated with the AD extracts (5%), and a significant
decrease (p=0.0118) in those co-treated with EF (12.9%;
Figures 3F–I).
EF and AD Extracts Stimulated the
Expression of Antioxidant Enzymes
To assess changes in the antioxidant protein levels in astrocytic
cells exposed to rotenone alone or rotenone plus extracts,
the expression of the antioxidant enzymes SOD, catalase and
glutathione peroxidase was examined. The results showed an
increase of 65% in SOD when the cells were exposed to
rotenone +AD extracts in comparison to control values
(Figure 4A). On the contrary, catalase expression level did
not change either in cells treated with rotenone alone,
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Areiza-Mazo et al. Uchuva Extracts Protect Cells
FIGURE 2 | Production of reactive oxygen and nitrogen species (ROS) lipid peroxidation in T98G cells. (A) Mean fluorescence of DHE from superoxide radical in T98G
cells. Quantification of superoxide radical in T98G cells after co-treatment with 50 µM rotenone plus EF or AD at 25 µg/ml by flow cytometry. Determination of DHE by
fluorescence microscopy. (B) Cells exposed to 0.5% DMSO; (C) Cells exposed to 50 µM rotenone (p<0.0001); (D) Cells exposed to 50 µM rotenone plus 25 µg/ml
EF (p=0.0027); (E) Cells exposed to 50 µM rotenone plus 25 µg/ml AD (p=0.0261). (F) Mean fluorescence of DCFA in T98G cells. Quantification by flow cytometry
of hydrogen peroxide in T98G cell cultures after co-treatment with 50 µM rotenone plus EF and AD (25 µg/ml). (G) Nitrite concentration in T98G cells determined by
Griess reagent after treatment with 50 µM rotenone and co-treatment with EF and AD at 25 µg/ml. (H) Mean fluorescence of HNE in cell cultures of T98G after
co-treatment with rotenone 50 µM plus EF and AD at 25 µg/ml. Determination of HNE by fluorescence microscopy. (I) Cells exposed to 0.5% DMSO; (J) Cells
treated with 50 µM rotenone (p<0.0001); (K) Cells co-treated with 50 µM rotenone plus EF (25 µg/ml; p<0.0001); (L) cells co-treated with 50 µM rotenone and
25 µg/ml AD (p<0.0001). Bar scales: 10 µm.
rotenone +EF, or rotenone +AD extracts (Figure 4A).
As for glutathione, an increase in the expression by 30.3%
was observed in rotenone-treated cells, and by 26% in the
rotenone +EF group compared to control. There were no
significant changes in the expression of antioxidant enzymes
in cells treated with AD extracts when compared to controls
(Figure 4B).
ERK1 and ERK2 expression levels were also assessed.
Increases by 93, 105, and 30% in ERK1 expression were
observed in cells treated with rotenone (50 µM) alone, co-treated
with rotenone and EF, and co-treated with rotenone and AD,
respectively (Figure 4C). As for the ERK1/ERK2 ratio, there
was a 29.6% increase in the rotenone-treated cells, while a 36%
increase was observed after co-treatment with EF. Finally, we
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Areiza-Mazo et al. Uchuva Extracts Protect Cells
FIGURE 3 | Determination of mitochondrial parameters, the content of cellular calcium and cell morphology. (A) Mean TMRM fluorescence in T98G cells.
Determination of mitochondrial membrane potential by flow cytometry in T98G cell cultures after co-treatment with 50 µM rotenone plus EF and AD at 25 µg/ml. (B)
Mean fluorescence of NAO in T98G cells by flow cytometry in T98G cell cultures after co-treatment with 50µM rotenone plus EF and AD at 25 µg/ml. (C)
Determination of mitochondrial calcium concentration in T98G cells. Mean fluorescence of RHOD 2, mitochondrial calcium indicator, in T98G cell cultures after
co-treatment with 50 µM rotenone plus EF and AD at 25 µg/ml. (D) Determination of endoplasmic calcium concentration in T98G cells. Mean fluorescence of
Mag-fura-2 fluorometer, calcium indicator in cytoplasmic reticulum in cell cultures of T98G after co-treatment with 50µM rotenone plus EF or AD at 25µg/ml. (E)
Percentage of cells with fragmented/condensed nuclei in T98G cells, measured by Hoescht staining after treatment with 50 µM rotenone and co-treatment with
extracts EF and AD at 25 µg/ml. Determination of Hoescht by fluorescence microscopy in (F) Cells exposed to 0.5% DMSO, (G) Cells damaged with 50 µM rotenone
(p=0.0059), (H) Cells co-treated with 50 µM rotenone plus EF 25 µg/ml, (I) Cells co-treated with 50 µM rotenone and 25 µg/ml AD (p=0.0247). Bar scales: 10 µm.
found a 15% reduction in ERK1/ERK2 when cells were co-treated
with AD.
DISCUSSION
The interest in studying natural polyphenols as neuroprotective
(Vauzour et al., 2008; Akinrinmade et al., 2017) and nootropic
compounds has grown in recent years. A major limitation
challenging the effectiveness of polyphenols for the mentioned
purposes is the ability of these compounds to cross the blood-
brain barrier (Subash et al., 2014; Queiroz et al., 2017). Different
types of berries have been reported to exert neuroprotective
effects via modulating enzymes with antioxidant activity,
improving cognitive function, decreasing lipid peroxidation and
ROS production and increasing SOD expression in an in vitro
model of glutamate-induced toxicity as well as in vivo models
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Areiza-Mazo et al. Uchuva Extracts Protect Cells
FIGURE 4 | Expression of antioxidant defense and ERK proteins in T98G cells. (A) Expression of Superoxide dismutase (SOD) and Catalase determined by Western
Blot in T98G cell cultures after co-treatment with 50 µM rotenone plus EF and AD at 25 µg/ml. (B) Expression of Glutathione determined by Western Blot in T98G cell
cultures after co-treatment with 50 µM rotenone plus EF and AD at 25 µg/ml. (C) Expression of ERK and MAPK proteins determined by Western blotting on T98G
cells after co-treatment with rotenone plus EF and AD at 25 µg/ml.
of neurodegenerative diseases and senescence-accelerated rats
(Kolosova et al., 2006; Forbes-Hernandez et al., 2016; Lee
et al., 2017). For instance, addition of pomegranate antioxidant
extracts to the diet of obese rats exerted antioxidant effects and
reversed hyperlipidemia and cerebral oxidative stress (Amri et al.,
2017; Oviedo-Solís et al., 2017). Also, phenols were shown to
exert neuroprotective effects in neuroblastoma cells and HT22
hippocampal mouse cells instigated with oxidative stress (Tavares
et al., 2013; Vepsäläinen et al., 2013).
In the present study, we explored whether different extracts
isolated from P. peruviana could counteract the oxidative damage
induced by rotenone in T98G cells. Our findings showed that
EF and AD extracts improved cell survival and preserved
mitochondrial functions in rotenone-treated cells.
P. peruviana has been reported to exert beneficial effects
in various chronic diseases. This plant contains peruvioses
and sucrose esters that are responsible for the reported
hypoglycemic and hypocholesterolemic effects and the observed
benefits reported in improving health conditions like cancer,
cardiovascular and neurodegenerative diseases (Ramdan, 2011;
Abdel Moneim et al., 2014; Al-Olayan et al., 2014; Bernal
et al., 2018). Other protective actions of P.peruviana rely on
its antioxidant, anti-inflammatory and anti-apoptotic properties
that are probably exerted by the bioactive compounds present in
leaves, stems, fruits and calyx of the plant (Martínez et al., 2010;
Franco et al., 2014; Toro et al., 2014). Indeed, previous studies
have shown a high content of polyphenols and carotenoids that
are strong ROS scavengers, in the plant fruits (Lan et al., 2009;
Yen et al., 2010; Ramdan, 2011; Dkhil et al., 2014; Toro et al.,
2014; Yildiz et al., 2015). It is quite known the presence of
these bioactive compounds in different parts of P.peruviana.
However, in our study, the analysis of phenols has been limited
to calculated equivalents of gallic acid with spectrophotometric
or HPLC methods (Hassanien, 2011; Zhang et al., 2013; Cortés
Díaza et al., 2015).
Exposure of astrocytic cells to rotenone is a validated method
to simulate the neuropathological conditions of PD (Cabezas
et al., 2015) and allows preliminary screening of neuroprotective
compounds with antioxidant properties. This experimental
approach allowed us to determine which fractions of the
P. peruviana whole extract have antioxidant or cytotoxic effects.
This information will be instrumental to identify beneficial
secondary metabolites from P. peruviana, which might be tested
in future in vivo models to validate any therapeutic effects.
To assess the beneficial actions of the phytochemicals from
P. peruviana, further analytical studies should be performed
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Areiza-Mazo et al. Uchuva Extracts Protect Cells
using mass spectrometry in order to better understand their
interference and modulatory effects on the intrinsic pathological
mechanisms of these diseases.
Many beneficial properties of P. peruviana pure extracts had
already been identified but our work is the first to evaluate the
effects of P. peruviana with different polarities in an in vitro
model of a neurodegenerative disease that is associated with
oxidative stress. Our results indicated that the effect of the
extracts on cell viability was dependent on the concentration
and incubation time applied on the T98G cells. Low and
medium polarity extracts, which are those obtained by petroleum
benzene and dichloromethane, showed a significant cytotoxic
effect that was dependent on the time and the concentration
applied (Supplementary Figure 1). On the other hand, extracts
of medium-high polarity and polar extracts showed lower
cytotoxicity (Supplementary Figure 2). In fact, when the AD,
AF, EF, and ED extracts were tested, minor increases in cell
viability were observed. For instance, cells exposed to AD
extract at 25 µg/ml for 12 h showed a 53% increase in cell
viability (Supplementary Figure 2). The cytotoxic effect of low
polarity extracts is consistent with the findings of previous
studies; for instance, benzene and dichloromethane extracts
from Ficus Sycomorus were found to be less toxic compared
with those obtained with ethyl acetate or ethanol (Al-Matani
et al., 2015; Tang et al., 2015). Consistent with previous studies,
extracts of goldenberry obtained with ethanol and polar solvents
(acetone and ethyl acetate) showed higher biological activities
and neuroprotective effects.
Previous studies reported an increase in cell survival
after exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MTPT)-treated cells to goldenberry extracts. This increase in
viability was accompanied by cytoplasmic membrane protection
from ROS damage, increased expression of the antioxidant
enzymes SOD and catalase, as well as improved cognitive
function and memory (Faria et al., 2005; Fortalezas et al., 2010;
Debnath et al., 2011; Jeong et al., 2013). Also, in in vitro and
in vivo studies performed by Kim et al. (2010), a decrease
in symptoms related to PD such as bradykinesia and loss of
dopaminergic neurons was noted after exposure to ethanolic
extract of blackberries. The authors postulated that the mulberry
extract and its polyphenols could serve as natural drugs for the
prevention and treatment of PD (Kim et al., 2010).
It should be noted the overall phenolic content of the most
polar fractions did not correlate with the percentage of cell
survival. Although the Folin-Ciocalteu reagent assay indicates
the total phenols of a sample, there are differences in the
methods used to obtain the extracts. The presence of water
changes the elution capacity of solvents. Thus, in the fresh
fruit extract, water facilitates the elution of some hydrosoluble
components of the fruit into ethanol. Ethanol was the first
fraction obtained, making it possible to elute the more significant
portion of the polar elements associated with water. On the
other side, with the dehydrated fruit, the solvent elution capacity
varied and the more polar components are extracted by acetone
and are eluted with higher selectivity than the less polar
components. Among the phenols, the most polar compounds
are the glycosylated flavonoids. These extraction differences and
variations in antioxidant compounds may somehow explain the
increase in the percentage of cell survival, mainly observed with
EF and AD extracts. Furthermore, the Folin-Ciocalteu reagent
assay is not specific for phenolic components, as it also recognizes
the phenolic structures of proteins and many other (reducing)
compounds (Huang et al., 2005; Prior et al., 2005). In our study,
to minimize the content of protein-related phenolic structures,
the cellular components were discarded by centrifugation.
Further studies should determine and characterize the phenolic
components of P. peruviana, an analysis that goes beyond the
goal of our present study.
Enhanced production of ROS in the brain is a phenomenon
that occurs in a large number of neurodegenerative diseases
(Vauzour et al., 2008), suggesting a role for oxidative stress
in triggering cell death. Decrease in superoxide radical and
hydrogen peroxide levels observed in cells treated simultaneously
with rotenone and EF or AD suggests that these extracts
possess antioxidant activity (Figures 3A,F). It is not known
whether the effect elicited by these extracts is due to free
radical scavenging or reduction of ROS formation. Our findings,
however, show that these extracts can directly reduce oxidative
stress (Figures 2C,D,E). This is supported by the reduction in
membrane lipid oxidation (3% decrease in HNE fluorescence),
which may be associated with a reduction of intracellular ROS
levels. In this regard, high concentrations of carotenoids, the
pigments that impart the yellow color of the fruit, may play
a fundamental role in the scavenging of oxidant compounds,
since these molecules have a high affinity for ROS (Rodriguez-
Amaya, 2010). Likewise, the expression of antioxidant enzymes
may be induced by an increase in ROS that activates the cellular
defense system, which can either inhibit oxidative damage or
trigger an apoptotic process in astrocytes (Barreto G. E. et al.,
2011; Barreto G. et al., 2011). An increase in ERK expression
could activate cell survival pathways and reduce cell death (Essa
et al., 2012). Nitric oxide is a reactive nitrogen species and can
further promote oxidative damage (Chaturvedi and Flint Beal,
2013). We found a slight increase in nitrite concentration in
cells treated with rotenone and a decrease when the cells were
treated simultaneously with the extracts but the variations were
not significant compared to the control.
The mitochondria play a fundamental role in the regulation
of cellular oxidative status as well as response to oxidative
stress (Federico et al., 2012). For this reason, mitochondrial
dysfunction is directly associated with increased ROS content
in the brain in most neurodegenerative diseases (Cabezas
et al., 2012; Chaturvedi and Flint Beal, 2013; Ruszkiewicz
and Albrecht, 2015). In fact, mitochondrial dysfunction has
been established as one of the early and vital features of
neurodegeneration (Federico et al., 2012). The results presented
in this study indicated a decrease in the mitochondrial membrane
potential in cells exposed to rotenone. The loss of mitochondrial
membrane potential is one of the initial steps that occur
in the process of mitochondrial dysfunction and subsequent
cell death (Federico et al., 2012). This phenomenon may also
explain our observation of decreased cell viability in rotenone-
treated cells. Nevertheless, the results showed that EF and AD
extracts preserved mitochondrial membrane potential against
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Areiza-Mazo et al. Uchuva Extracts Protect Cells
rotenone stimulation (Figure 3A), suggesting that these extracts
are mitochondrial protectors.
Another indicator of mitochondrial dysfunction is alteration
of mitochondrial mass (Ávila Rodriguez et al., 2014; Cabezas
et al., 2015). An increase in mitochondrial mass was detected
in cells treated with rotenone, with an opposite turnout when
cells were treated with either EF or AD. We postulate that the
slight increase in mitochondrial mass in the rotenone group
may represent a process of abnormal mitochondrial dynamics
due to the loss of fusion/fission mitochondrial homeostasis
(Parrado-Fernández et al., 2016). In fact, there are reports
in the literature indicating that cellular stress promotes the
mitochondrial fusion process (Westermann, 2010), which may
explain the increase of mitochondrial mass in the cells exposed
to rotenone. In this sense, it is reasonable to suggest that the
decrease in the mitochondrial mass induced by the exposure
of cells to the extracts was due to the ability of the extracts
in reducing oxidative stress and maintaining mitochondrial
membrane potential. However, future studies investigating the
mode of action of phytochemicals in modulating mitochondrial
dynamics are necessary to clarify the mechanisms underlying
the effect of these extracts on the mitochondrial mass. On the
other hand, a decrease in mitochondrial Ca2+in the rotenone-
treated cells was also observed in our study, yet these levels
slightly increased when the cells were co-treated with AD
(Figure 3C). These findings are in agreement with previous
studies showing that cranberries prevent deregulation of the
Ca2+in hippocampal cells in rats (Brewer et al., 2010). However,
continuous measurements to test the effects of extracts on
rapid variations of calcium ion in the RE and mitochondria of
astrocytes (Abeti and Abramov, 2015) are still required.
The antioxidant capacity of a compound can be extrapolated
to activities that go beyond capturing or preventing the formation
of ROS (Nones et al., 2010). For example, it has been stated
that the antioxidant capacity of a compound is greater when
it also stimulates antioxidant-related pathways such as ERK
signaling (Spencer, 2008). In this direction, it has been reported
that flavonoids have strong antioxidant activities owing to their
capacity in stimulating survival pathways involving enzymes
such as ERK1 and 2. In our study, increased expression of
ERK1 protein was observed when the cells were co-treated
with rotenone and EF. Indeed, cells treated with AD extract
showed higher expression of SOD and lower concentration
of superoxide radical. These findings suggest that the tested
extracts might enhance the endogenous antioxidant defense
system (Halliwell, 2007). Nonetheless, it would be necessary
to identify the active ingredients in these extracts. In future
experiments, more refined analysis of subfractions of the
tested extracts using HPLC and Mass spectrometry techniques
are needed to elucidate the identity and quantity of the
bioactive compounds. Finally, verification of the observed
antioxidant effects of P. peruviana extracts in animal models
of neurodegenerative diseases associated with oxidative stress
merits further investigation.
CONCLUSIONS
The present study suggested that the EF and AD extracts from
P. peruviana fruits may have cytoprotective and antioxidant
effects in brain cells exposed to neurotoxic stimuli. These effects
were expressed as reduced cell death, preserved mitochondrial
functions and attenuated nuclei damage. Moreover, our results
demonstrated that: (i) different effects of P. peruviana fruit
extracts depend on the polarity of the extraction solvent; (ii)
extracts of medium-high polarity (namely AD and EF) have
protective effects on the mitochondria; and (iii) AD and EF
extracts might induce cytoprotective actions in rotenone-treated
cells by enhancing the cellular antioxidant content which has
a positive impact on cell survival. More studies should be
performed with the aim of assessing the bioavailability and
bioactivity of P. peruviana extracts in vivo. Finally, our study
opens a new avenue of research aiming at the identification of
natural compounds as potential drugs to treat neurodegenerative
diseases.
AUTHOR CONTRIBUTIONS
NA-M, JR, JZ-R, LG, BB-B, VE, GA, AS, GMA, and GB designed
the experiments. NA-M, GB, GA, AS, GMA, VE, JR, BB-B
analyzed the results. NA-M, GA, AS, GMA, VE, BB-B, GMA and
GB wrote and revised the manuscript. All authors have approved
the final revised manuscript.
ACKNOWLEDGMENTS
This work was supported by the Pontificia Universidad Javeriana,
Bogotá, Colombia, and Colciencias by the Young Researchers
program.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fchem.
2018.00276/full#supplementary-material
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Frontiers in Chemistry | www.frontiersin.org 13 July 2018 | Volume 6 | Article 276