Food and Chemical Toxicology 60 (2013) 45–51

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Food and Chemical Toxicology

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

Passiflora manicata (Juss.) aqueous leaf extract protects against reactive

oxygen species and protein glycation in vitro and ex vivo models
Maurilio da Silva Morrone a,⇑, Adriano Martimbianco de Assis a, Ricardo Fagundes da Rocha a,
Juciano Gasparotto a, Andressa Córneo Gazola b, Geison Modesti Costa b, Silvana Maria Zucolotto b,
Leonardo H. Castellanos c, Freddy A. Ramos c, Eloir Paulo Schenkel b, Flávio Henrique Reginatto b,
Daniel Pens Gelain a, José C.F. Moreira a
a
Centro de Estudos de Estresse Oxidativo, Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
b
Programa de Pós-graduação em Farmácia, Centro de Ciências da Saúde, Universidade Federal de Santa Catarina, Florianópolis, Brazil
c
Departamento de Química, Universidad Nacional de Colombia, Bogotá, Colombia

a r t i c l e i n f o a b s t r a c t

Article history: The leaf extracts of many species of genus Passiflora have been extensively investigated for their
Received 6 December 2012 biological activities on several rat tissues, but mainly in the central nervous system and liver. They posses
Accepted 11 July 2013 anxiolytic-like, sedative effects and antioxidant properties. Evidences suggest a key role of C-glycosylfl-
Available online 20 July 2013
avonoids in the biological activities of Passiflora extracts. Some species (such as P. manicata) of the genus
are still poorly investigated for their chemical and biological activity. In this work, we aim to investigate
Keywords: both antioxidant and antiglycation properties of aqueous extract of P. manicata leaves (PMLE) in vitro and
Antioxidant
ex vivo models. Crude extract showed the C-glycosylflavonoid isovitexin as the major compound. Isoori-
C-glycosylflavonoids
Oxidative stress
entin and vitexin were also identified. In TRAP/TAR assay, PMLE showed a significant antioxidant activity.
Passiflora manicata PMLE at concentrations of 10 and 100 lg mL 1 significantly decreasing LDH leakage in rat liver slices.
Protein glycation Antioxidant effect also was observed by decreased in oxidative damage markers in slices hence hydrogen
peroxide was added as oxidative stress inductor. PMLE inhibited protein glycation at all concentrations
tested. In summary, P. manicata aqueous leaf extract possess protective properties against reactive oxy-
gen species and also protein glycation, and could be considered a new source of natural antioxidants.
Ó 2013 Published by Elsevier Ltd.

1. Introduction 1996; Mors et al., 2000). Many of these species are cultivated
due to their use as edible fruits or in preparation of juices, but their
The genus Passiflora is the most important genus of the family leaves are also employed in folk medicine, mainly as sedative and
Passifloraceae, comprising about 500 species (Lewis and Lewis, tranquilizer (Pio Corrêa, 1978; Lorenzi and Matos, 2008).
1977; Pio Corrêa, 1978). Popularly known in Europe and North The leaf extracts of P. incarnata, P. edulis var. edulis, P. edulis var.
America as passionflower, in South America several species of flavicarpa and P. alata, have been extensively investigated for their
Passiflora are widely distributed, being popularly named as biological activities on several rat tissues, but mainly in the central
‘‘maracujá’’, ‘‘curuba’’ and ‘‘badea’’, among others (Arbelaez, nervous system (CNS) (Speroni et al., 1996; Soulimani et al., 1997;
Petry et al., 2001). In this context, P. alata and P. edulis extracts
present anxiolytic-like and sedative effects without affecting mem-
Abbreviations: AAPH, 2,2-azobis[2-amidinopropane]; ANOVA, analysis of vari-
ance; AUC, area under curve; BSA, bovine serum albumin; CAT, catalase; CDNB,
ory processes in Wistar rats (Barbosa et al., 2008). In liver, P. alata
chloro-dinitro benzene; CNS, central nervous system; DNPH, dinitrophenylhidr- and P. edulis leaves extracts exert protective activity against oxida-
azine; DTNB, 5,5-dithionitrobis 2-nitrobenzoic acid; GPx, glutathione peroxidase; tive damage to biomolecules, such as lipids and proteins (Rudnick
HPLC-UV DAD, high performance liquid chromatography equipped with Diode et al., 2007). Recent studies have shown that C-glycosylflavonoids
Array Detection; LDH, lactate dehydrogenase; PMLE, Passiflora manicata leaf
play an important role in the biological activities of Passiflora ex-
extract; TRAP, total reactive antioxidant potential; TAR, total antioxidant reactivity;
H2O2, hydrogen peroxide; TBARS, thiobarbituric reactive species; SOD, superoxide tracts (De Paris et al., 2002; Coleta et al., 2006; Santos et al.,
dismutase. 2006; Sena et al., 2009).
⇑ Corresponding author. Address: Rua Ramiro Barcelos 2600, Anexo Depto. The biological activity of many medicinal plants and other
Bioquímica, Lab 32, CEP 90035-003, Porto Alegre, RS, Brazil. Tel.: +55 51 33085577; natural products are directly related to their flavonoid content
fax: +55 51 33085535.
(Rice-Evans, 2004). Flavonoids (polyphenols) are known to possess
E-mail address: maurilio.bio@gmail.com (M. da Silva Morrone).

0278-6915/$ - see front matter Ó 2013 Published by Elsevier Ltd.

http://dx.doi.org/10.1016/j.fct.2013.07.028
46 M. da Silva Morrone et al. / Food and Chemical Toxicology 60 (2013) 45–51

antioxidant properties and might play a role in the prevention of 2.5. Total reactive antioxidant capacity (TRAP) and total antioxidant capacity (TAR)
various diseases associated with oxidative stress, such as cancer,
The in vitro antioxidant activity of the P. manicata leaf extracts was estimated by
diabetes, inflammatory and neurodegenerative diseases (Havsteen, the total reactive antioxidant potential (TRAP) assay, as previously described (Lissi
2002; Manach et al., 2004). These molecules act as antioxidants by et al., 1992). This assay is based on the peroxyl radical (generated by AAPH solution,
scavenging reactive oxygen species (ROS) and chelating redox- 2,2-azobis[2-amidino-propane], with luminol) quenching by sample compounds.
active transition metals (Rice-Evans et al., 1996). Under regular The measuring is performed by chemiluminescence detection. The results were
transformed in percentage and the area under curve (AUC) calculated as described
physiological conditions, ROS participate as intracellular messen-
by Dresch et al. (2009) with GraphPad Software (San Diego, CA, USA – version 5.00).
gers and regulatory molecules. Their concentrations are regulated Total antioxidant reactivity (TAR) was analyzed using the same samples utilized
by balancing systems comprised by different antioxidants, antiox- for TRAP readings. TAR results were calculated as the ratio of light intensity in ab-
idant enzymes, and proteins (Kowaltowski et al., 2009). Further- sence of samples (I0)/light intensity right after extract addition. Although TAR and
more, non-enzymatic antioxidants obtained from diet or other TRAP evaluations are obtained in the same experiment, they represent different
observations, since TAR is more related to the antioxidant quality (reactivity, the
processes regulate oxidative reactions in the body which prevent scavenging capacity in a short-term period) and TRAP is more related to the antiox-
oxidative stress by removing ROS (Halliwell, 2007). idant amount and kinetic behavior (Lissi et al., 1995).
In spite of the interest in studying some common species of the
genus Passiflora (P. incarnata, P. edulis var. edulis, and P. edulis var.
flavicarpa) other species are still poorly investigated for their 2.6. Animal procedure and tissue preparation
chemical and biological activities, although these are extensively
Male Wistar rats (Rattus novergicus) from our breeding colony were used in the
used in traditional folk medicine, in the diet and in the psychedelic present study. Ten rats were killed by decapitation in each experiment. The com-
ceremonies in South American countries. That is the case of P. man- plete cerebral cortex and liver were dissected, weighed, and cut into 0.3-mm slices
icata, which produces a fruit similar to the banana passion fruit using a McIlwain tissue chopper. The protocol used in this research followed the
(curuba) that are traditionally reported as both toxic and halluci- guidelines of the Committee on Care and Use of Experimental Animal Resources,
School of Veterinary Medicine and Animal Science of the University of São Paulo,
nogenic fruits, popularly named as ‘‘el diablito’’ in the northern an-
Brazil, and was approved by the Local Ethics Committee (Comitê de Ética no Uso
dean countries. Moreover, the leaves of this plant are reported as de Animais – UFRGS).
antimicrobial agent (Ramón, 2008) and have resistance to helmin-
tic attacks (Torres, 2007). Previous works have reported the pres-
ence of flavonoids as vitexin, isovitexin and orientin in leaf 2.7. Ex vivo assays – antioxidant activity
extracts of P. manicata (Abourashed et al., 2002; Zulcolotto et al.,
The antioxidant activity of the PMLE was first evaluated in assays using only the
2011). These compounds, when presented in extracts, are believed
extract diluted in incubation medium to measure the citotoxic effects of the extract
to act as antioxidants; nevertheless, the biological properties of and redox parameters in slices. To better clarify the antioxidant effect of the extract,
these leaf extract are still unknown. In this way, the aim of this a new incubation was performed, but using H2O2 as oxidative stress inducer diluted
study was to investigate both antioxidant and antiglycation prop- in the incubation medium. At first, the results were compared to both groups which
contained Trolox [200 nM] and the control system. In the second incubation the ef-
erties of aqueous extract of P. manicata leaves (PMLE) in vitro and
fects of the PMLE at slices were compared to control, induced and Trolox groups.
ex vivo models. CNS frontal cortex and rat liver slices were separated in five or six groups at second
incubation: control (only with medium); Trolox 200 nM, stress induced control
(H2O2 [0.5 M] in medium); and three groups of different concentrations of extract
2. Materials and methods
(1, 10, 100 lg mL 1) diluted in the medium in absence or presence of hydrogen per-
oxide. The incubations were as follows: in the first assay, the slices were incubated
2.1. Reagents
with extracts diluted in the medium for 1 h. The second procedure was similar but
H2O2 [0.5 M] was added at the end of the 60 min in the predetermined groups for
2.1.1. HPLC assay
more 30 min. Both CNS frontal cortex and rat liver slices (300 mM) were incubated
Acetonitrile and formic acid (HPLC-grade) were provided by TediaÒ (Brazil).
for 60 or 90 min at 37 °C under 95% of O2/5% of CO2 in a shaking water bath
Water was purified with a Milli-Q system (MilliporeÒ, Bedford, USA). All the solu-
(60 oscillations/min) in a medium of oxygen-equilibrated Krebs–Ringer phosphate
tions prepared for HPLC were filtered through a 0.45 mm membrane before use. Iso-
buffer – 10 mM glucose, pH 7.4. After incubations, the slices were removed and the
orientin, vitexin and isovitexin were purchased from Sigma–AldrichÒ Co. (St. Louis,
medium was centrifuged at 12,000g for 10 min. In the first incubation, the superna-
USA).
tant portion was used to quantify lactate dehydrogenase activity using a commer-
All other reagents utilized were purchased from Sigma–AldrichÒ Co. (St. Louis,
cial kit (LDH Liquiform™, Brazil).
USA).

2.2. Plant material 2.8. Redox assays

The leaves of P. manicata (Juss.) were collected in Villa de Leyva, Boyacá, Colom- 2.8.1. Protein quantification
bia. The plant material was identified by Prof. Luis Carlos Jimeénez and voucher All the results were normalized by the protein content using bovine albumin as
specimen was deposited at the Instituto Nacional de Ciencias, Universidad Nacional standard (Lowry et al., 1951).
de Colombia (COL 530663).

2.8.2. TBARS assay

2.3. Extract preparation
The formation of thiobarbituric acid reactive species (TBARS) was measured
during an acid-heating reaction as an index of lipid peroxidation. This is a widely
The leaves of P. manicata were air-dried at 40 °C, powdered and extracted by
adapted parameter for the measure of lipid redox state, as previously described
infusion with boiling water (95 °C, plant:solvent 1:10,w/v) for 10 min. Thereafter,
(Draper and Hadley, 1990). The samples were mixed with 0.6 mL of 10% trichloro-
the aqueous extract was filtered, lyophilized and stored at 20 °C until tested
acetic acid (TCA) and 0.5 mL of 0.67% thiobarbituric acid, then heated in a boiling
water bath for 25 min. TBARS were determined by the absorbance in a spectropho-
2.4. HPLC assay tometer at 532 nm.

The HPLC system consisted of a PerkinElmer Series 200 equipped with Diode
Array Detection (DAD), quaternary pump, on-line degasser and autosampler, and 2.8.3. Total protein thiol content
the separation was achieved on a Vertical VertSep C18 column (250  4.6 mm i.d.; After the incubation, tissue samples were analyzed for proteins thio content,
5 lm), using a linear gradient system of acetonitrile (solvent A) and formic acid which was used as an estimation of oxidative alterations in proteins. As previously
0.5% (solvent B), as follow: 15–35% A (0–15 min). The flow rate was kept constant described by Ellman (1959), an aliquot of the sample was diluted in SDS 0.1%. Then,
at 1.2 mL/min and the chromatograms were recorded at 340 nm, with spectra ac- 0.01 M 5,5-dithiobis-2-nitrobenzoic acid (DTNB) in ethanol was added. The intense
quired over a range of 200–450 nm. The injection volume was 20 lL and the data yellow color was developed and read in a spectrophotometer at 412 nm after
were processed using the ChromeraÒ software. 60 min.
 M. da Silva Morrone et al. / Food and Chemical Toxicology 60 (2013) 45–51 47

Fig. 1. HPLC–DAD fingerprint of crude aqueous extracts of P. manicata leaves at 340 nm. 1: Isoorientin; 2: Vitexin; and 3: Isovitexin.

2.8.4. Protein carbonylation Furthermore, isoorientin (rt = 6.7 min) and vitexin (rt = 8.2 min)
The formation of carbonyl groups in liver slices was used as a parameter for oxi- were also identified (Fig. 1).
dative damage to proteins, based on the reaction with dinitrophenylhidrazine
(DNPH), as previously described by Levine et al. (1990). Proteins were precipitated
Redox properties of PMLE were first evaluated using a method
by the addition of 20% TCA and re-solubilized in DNPH. Then, the absorbance was based on quenching of luminol-enhanced chemiluminescence
read in a spectrophotometer at 370 nm. (CL) of AAPH, the TRAP and TAR assays (Fig. 2). All concentrations
tested produced an antioxidant effect, observed in TRAP assay
2.8.5. Antioxidants enzymes activities (Fig. 2A). At the concentrations of 1, 10, 100 lg mL 1 and
The activities of three important antioxidant enzymes were analyzed in slices 1 mg mL 1 the extract also showed significant antioxidant capacity
after the incubations: catalase (CAT), glutathione peroxidase (GPx) and superoxide
according to TAR measurements (Fig. 2B).
dismutase (SOD). CAT activity was assayed by measuring the rate of decrease in
H2O2 absorbance in a spectrophotometer at 240 nm. To determine GPx activity,
In order to evaluate the potential biological the effects of PMLE,
the rate of NAD(P)H oxidation was measured in a spectrophotometer at 340 nm we selected the concentrations that were effective in TRAP/TAR as-
in the presence of reduced glutathione, tert-butyl hydroperoxide and glutathione says (1, 10 and 100 lg mL 1) for tests in an ex vivo experimental
reductase. SOD activity was assessed by quantifying the inhibition of superoxide- model of tissue slices. Rat brain and liver slices were incubated
dependent adrenaline auto-oxidation in a spectrophotometer at 480 nm (Aebi,
with the extract diluted in the incubation medium and the effect
1984; Flohé and Gunzler, 1984; Misra and Fridovich, 1972).
of the different concentrations of PMLE on lactate dehydrogenase
(LDH) release, a parameter of cytotoxicity, were compared with
2.9. Protein glycation
the effect of the standard antioxidant Trolox (200 nM), a synthetic
According to the method previously described (Vinson and Howard, 1996), bo- analogue of vitamin E (Fig. 3). Whereas the addition of Trolox in
vine serum albumin (BSA, 7 mg/mL) in phosphate buffer (50 mM, pH 7.4) contain- the incubation medium substantially decreased cell death as de-
ing 0.02% (w/v) sodium azide was pre-incubated with the PMLE at final tected by reduced LDH leakage in both CNS frontal cortex and rat
concentrations of 1, 10 and 100 lg mL 1 for 30 min at room temperature (25 °C).
liver slices, PMLE at concentrations of 10 and 100 lg mL 1 showed
Glucose (25 mM) and fructose (25 mM) solutions were added to the reaction mix-
ture. After incubating at 37 °C for 7 days, the fluorescent reaction products were as- a similar effect in rat liver slices, significantly decreasing LDH
sayed in a fluorescence reader with an excitation wavelength of 350 nm and an leakage.
emission wavelength of 450 nm. Isolated tannic acid (Sigma–Aldrich) was used as In addition, redox parameters were also quantified at tissue
a positive polyphenol control at two concentrations. Results were expressed as arbi-
slices in order to evaluate the effect of PMLE on oxidative stress.
trary units of fluorescence from the glycated protein.
In the CNS frontal cortex slices, the groups incubated with P. man-
icata extract did not show significant effects when compared to the
2.10. Statistical analysis
control group. There were no significant differences on oxidative
Data were expressed as means ± standard deviation (SD) of triplicates from damage markers and in antioxidant enzyme activities of slices
three independent experiments. Differences between treatment groups were com- incubated with PMLE when compared to control groups (data not
pared by two-way ANOVA, followed by Tukey’s test for approximately normally show).
distributed variables.
We next studied the protective effects of PMLE against the oxi-
dative stress inducer H2O2. This molecule was chosen due to its
3. Results role in the physiological and pathological stress in both CNS frontal
cortex and liver. Brain and liver slices were co-incubated with
The chemical analysis by HPLC-UV/DAD of crude extract of P. PMLE and hydrogen peroxide. In rat frontal cortex slices, PMLE
manicata showed isovitexin (rt = 8.8 min) as the major compound. inhibited the formation of TBARS induced by H2O2 (Fig. 4a); total
48 M. da Silva Morrone et al. / Food and Chemical Toxicology 60 (2013) 45–51

Fig. 2. TRAP TAR measurements. (A) The total reactive antioxidant potential (TRAP)
of P. manicata extract at different concentrations. A free radical source (AAPH)
generating system produces peroxyl radicals at a constant rate, and the effect of Fig. 3. The effect of the P. manicata aqueous leaf extract on LDH leakage in both
different concentrations of P. manicata extract on free radical-induced chemilumi- frontal cortex of central nervous system (A) and liver slices (B). Results are
nescence is measured as area under a curve for 60 min. (B) Total antioxidant expressed as leakage of the enzyme into the incubation medium compared to a
reactivity (TAR) values are calculated as the ratio of light intensity in absence of homogenized fresh slices ± SD of triplicates from three independent experiments.

samples (I0)/light intensity right after P. manicata extract addition (I) and expressed p < 0.05 compared to control. (Two-way ANOVA followed by Tukey’s post hoc test).
as percent of inhibition. The experiments were performed in triplicate, and bars
represent mean ± SEM of four different experiments. p < 0.0001; # concentrations
4. Discussion
used in ex vivo assays. (Two-way ANOVA followed by Tukey’s post hoc test).

Recent studies carried out over the past few years have shown
that flavonoids (polyphenols) from medicinal plants may play a
protein thiol oxidation was also reversed by the co-treatment with protective role against oxidative stress (Rice-Evans et al., 1996;
PMLE (Fig. 4b). Moreover, Trolox at 200 nM concentration was able Manach et al., 2004). Moreover, antioxidant and pro-oxidant bio-
to prevent both TBARS formation and total protein thiol oxidation chemical agents have been presenting important roles in therapeu-
when co-incubated with hydrogen peroxide at frontal cortex of tic strategies for prevention and/or management of oxidative
CNS slices. Protein carbonyl levels in the brain samples were not damage. In our work, isovitexin, vitexin and isoorientin were iden-
detectable in all groups (data not shown). In liver slices, PMLE tified in the crude extract by the HPLC–DAD analysis. Previous
did not affect thiol oxidation induced by H2O2 (data not shown). work on flavonoid composition of P. manicata leaves reported the
On the other hand, H2O2-induced TBARS formation was signifi- presence of isovitexin, vitexin, orientin and isoorientin (Aboura-
cantly inhibited by co-treatment with PMLE at all doses (Fig. 5a) shed et al., 2002). Since the ability of plant extract to scavenge
while the protein carbonylation was reversed at the higher concen- ROS and/or metal chelating seems to be related to the chemical
tration of PMLE, indicating an antioxidant activity in liver tissue structure of phenolic compounds (Halliwell et al., 1995; Rice-Evans
(Fig. 5b). Especially at liver, Trolox 200 nM was not capable to pre- et al., 1996), we presume that the aqueous leaf extract of P. mani-
vent both protein carbonylation and TBARS formation when com- cata may present some important antioxidant properties, probably
pared to control group. related to its phenolic content. In this regard, we decided to inves-
Finally, to evaluate the effect of PMLE on protein glycation, we tigate the possible interactions of leaf extract with different types
performed an in vitro assay using a glycation-inducing reaction of ROS.
system with purified bovine albumin, fructose and glucose. PMLE The potential of PMLE to scavenge peroxyl radicals in vitro, by
was added to this incubation system, and we observed a significant using TRAP/TAR assays, indicated a significant antioxidant capac-
inhibition of protein glycation at 1, 10 and 100 lg mL 1 concentra- ity. Antioxidant activity of leaf extracts from medicinal plants (Zai-
tions (Fig. 6). The classic polyphenolic compound tannic acid was nol et al., 2003) and fruits (Banerjee et al., 2005) show a direct
also tested and it exhibited a significant inhibition of protein glyca- linear relationship between the total phenolic content and the total
tion at concentration of 1 and 10 lg mL 1. These results indicate a antioxidant activity, indicating that phenolic compounds might be
protective action of the PMLE against glycation when applied in the major contributors to the antioxidant activities of these ex-
this in vitro model. tracts. In our study, we identified the polyphenolic compounds
 M. da Silva Morrone et al. / Food and Chemical Toxicology 60 (2013) 45–51 49

H2 O2 [0.5M] H 2 O2 [0.5M]
(A) (A)
5 * 3 *
*

nMol TBARS/mg ptn

nMol TBARS/mg ptn

4
2
3

2 1

0 0

-1

-1

-1

M
l

d
-1

-1

-1

ro
M
l

ce

L
ro

0n
ce

.m

.m

.m
nt
0n

du
.m

.m

.m
nt

20
co
du

µg

µg

µg
20
co

In
µg

µg

µg

ox
In

ox

10
1

0
10
1

10
0

ol
10

ol

Tr
Tr

(B) H2 O2 [0.5M]
H 2 O2 [0.5M]
(B) 0.3 25

µ mol carbonil/mg ptn

mmol SH / mg ptn

20 **
0.2 * ** *
15 *
10
0.1
5

0.0 0
-1

-1

-1

-1

-1

-1
l

M
d

M
l

d
ro

ro
ce

0n

ce

0n
.m

.m

.m
nt

.m

.m

.m
nt
du

du
20
co

20
µg

µg

µg

co

µg

µg

µg
In

In
ox

ox
10
1

10
1

0
10

ol

10

ol
Tr

Tr
Fig. 4. The effect of the P. manicata aqueous leaf extract upon redox parameters in Fig. 5. The effect of the P. manicata aqueous leaf extract upon redox parameters in
frontal cortex of CNS slices. (A) TBARS levels in presence of induced oxidative stress; liver slices. (A) TBARS levels in presence of induced oxidative stress; (B) Carbonyls
(B) total protein thiol content in presence of induced oxidative stress. Results are levels in presence of induced oxidative stress. Results are expressed as means ± SD
expressed as means ± SD of triplicates from three independent experiments. of triplicates from three independent experiments. p < 0.05 compared to control;

p < 0.05 compared to control. (Two-way ANOVA followed by Tukey’s post hoc test). 
p < 0.01 compared to control. (Two-way ANOVA followed by Tukey’s post hoc
test).

(Isoorientin, vitexin and isovitexin) in the extract by HPLC–DAD

Arbitrary units of fluorescence

analysis and also confirmed antioxidant activity of PMLE by scav- 200

enging peroxyl radicals by TRAP/TAR assays. These results lead
us to conclude that the presence of the compounds identified by 150 *
HPLC–DAD in our sample could be related with their in vitro anti-
oxidant activity (Halliwell et al., 1995; Rice-Evans et al., 1996). ***
100 ***
The unbalance of ROS production and antioxidant defense in *** ***
cells leads to various forms of reversible and irreversible oxidative
50
modifications of proteins (carbonylation), lipids (formation of
hydroperoxide lipid derivates) and DNA (formation of adducts
and strand breaks) that eventually result in loss of molecular func- 0
ed

id

id
LE

LE

LE

tions (Giorgio et al., 2007). Trolox, a standard antioxidant used is

ac

ac
at

PM

PM

PM
yc

several studies of antioxidant potential evaluation in vitro, in vivo

ic

ic
L -1

L -1

L -1

nn

nn
gl

.m

.m

.m

and ex vivo (Chung et al., 2006; Itoh et al., 2000; Perfeito et al.,
SA

ta

ta
L -1

L -1
µg

µg

µg
B

2007) was used here to evaluate and comparate the protective

.m

.m
l

10
1

0
ta

10

µg

µg
To

properties of PMLE in ex vivo CNS and liver models. The co-incuba-

10
1

tion with PMLE at concentrations 10 and 100 lg mL 1 provided

significant antioxidant protection to rat liver slices (similarly to Fig. 6. Inhibition of protein glycation by P. manicata aqueous leaf extract. Results
Trolox), as show by the decrease in LDH leakage. When we ana- are expressed as arbitrary units of fluorescence ± SD of from three independent
experiments performed in quadruplicate. p < 0.05 compared to total glycated BSA;
lyzed CNS frontal cortex slices, this parameter was not significantly 
p < 0.0001 compared to total glycated BSA. (Two-way ANOVA followed by
altered in relation to the untreated (control) group. In our second Tukey’s post hoc test).
ex vivo model, we used hydrogen peroxide as oxidative stress indu-
cer. Lipid peroxidation is one of the effects induced by oxidative A previous study of our group showed antioxidant properties
stress and occurs readily in tissues due to the presence of highly of P. alata and P. edulis leaf extracts in an ex vivo model: liver
oxidizable polyunsaturated fatty acids in cell membranes (Cini slices which were co-incubated with the extracts had lower levels
et al., 1994). In addition, other oxidized molecules (proteins) are of carbonyl groups and TBARS when oxidative stress was induced
often functionally inactivated by oxidative stress, which may affect by iron sulfide (Rudnick et al., 2007). Here, the groups of slices
the activity of enzymes, receptors, and membrane transporters treated with the extract and H2O2 showed lower levels of car-
(Stadman, 2001). bonyl and TBARS as well. In frontal cortex slices the groups
50 M. da Silva Morrone et al. / Food and Chemical Toxicology 60 (2013) 45–51

co-incubated with all doses of PMLE showed a significant de- Acknowledgements

crease of TBARS, indicating the potential of the extract as antiox-
idant agent (Fig. 4a). At the same slices, PMLE also prevent total The Brazilian research funding agencies FAPERGS (PqG
protein thiol oxidation as showed in Fig. 4b, since this parameter 1008860, PqG 1008857, ARD 11/1893-7, PRONEX 1000274) CAPES
could be considered a very early product of protein oxidation. On (PROCAD 066/2007), CNPq and PROPESQ UFRGS supported this
the other hand, the extract antioxidant activity in the liver slices study.
was effective into prevent TBARS formation in all doses and
inhibited protein carbonylation at higher concentration as
showed in Fig. 5. In this way, oxidative damage markers (lipid References
and proteins) presented a attenuation in liver slices co-incubated
Abourashed, E.A., Vanderplank, J.R., Khan, I., 2002. High-speed extraction and HPLC
with the extract in all concentrations. In addition, in rat liver fingerprinting of medicinal plants – I. Application to Passiflora flavonoids.
slices PMLE was more efficient into prevent oxidative stress when Pharm. Biol. 40, 81–91.
Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126.
compared to standard antioxidant Trolox since this last molecule
Arbelaez, E.P., 1996. Plantas utiles de Colombia, fifth ed. Jardim Botânico José
when present in incubation medium was not able to prevent the Celestino Nutis, Bogotá.
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