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Protective Effect of a-Mangostin on Cardiac Reperfusion Damage

by Attenuation of Oxidative Stress


Mabel Buelna-Chontal,
1,2
Francisco Correa,
2
Sauri Hernandez-Resendiz,
2
Cecilia Zazueta,
2
and Jose Pedraza-Chaverri
1
1
Department of Biology, Faculty of Chemistry, National Autonomous University of Mexico, Mexico City, Mexico.
2
Department of Biochemistry, Ignacio Chavez National Institute of Cardiology, Mexico City, Mexico.
ABSTRACT This study was designed to investigate if a-mangostin (a-M), a xanthone present in the pericarp of Garcinia
mangostana L., was able to protect against reperfusion injury in Langendorff-reperfused hearts. It was observed that a-M
maintains the cardiac mechanical work, diminishes the area of infarct, and prevents the decrease in cardiac ATP and
phosphocreatine levels in the reperfused myocardium. The protective effect of this xanthone was associated with reduction of
oxidative stress. a-M treatment prevented reperfusion injury-induced protein oxidation (protein carbonyl content), lipid
peroxidation (malondialdehyde and 4-hydroxynonenal content), and diminution of glutathione content. In fact, after a-M
treatment, the values in these parameters were comparable to those obtained in nonreperfused hearts. In summary, a-M
induces a protective effect in postischemic heart associated to the prevention of oxidative stress secondary to reperfusion
injury.
KEY WORDS: a-mangostin oxidative stress reperfusion injury
INTRODUCTION
M
angostan (Garcinia mangostana L.) belongs to
the Guttiferae family and is called the queen of
fruits because many people agree that it possesses one of
the best avors in the world.
1
The pericarp of G. mangostana
has been used for many years in traditional medicine in
Southeast Asia, for the treatment of several diseases like
abdominal pain, diarrhea, dysentery, infections, suppura-
tions, and chronic ulcers.
2
Many benecial effects have been
attributed to the xanthones presents not only in the pericarp
but also in the edible portion of G. mangostana. The aqueous
extracts of G. mangostana pericarp have reactive oxygen
species (ROS) scavenging properties that have been associ-
ated with its neuroprotective effects against 3-nitropropionic
acid in primary cultures of cerebellar granule neurons.
3
The antioxidant a-mangostin (a-M) is the major xanthone
present in mangostan.
4
It has been reported that a-M is a
scavenger of peroxynitrite anion (ONOO),
5,6
singlet oxy-
gen (
1
O
2
), and superoxide anion (O
2

).
6
The neuroprotec-
tive effect of this xanthone against the toxicity induced by
3-nitropropionic acid was associated with its ability to at-
tenuate ROS production.
6
The renoprotective effect of this
xanthone against toxicity induced by cisplatin was related
to the attenuation of oxidative stress activity.
7,8
ROS are
involved in several myocardial pathologies like cardiac is-
chemia and reperfusion (I/R) injury. There is also substan-
tial evidence that ROS are generated during I/R in the heart.
The main ROS sources are xanthine/xanthine oxidase,
9
NADPH oxidase,
10
and complex I and complex III of the
mitochondrial electron transport chain.
11
Based in the above
data, in the present study it was explored if the antioxidant
a-M was able to attenuate the injury and oxidative stress
secondary to I/R.
MATERIALS AND METHODS
Isolation of a-M
The dry powder of mangosteen pericarp used for the
isolation of a-M as previously described
7,12
was obtained
from DNP International Co., Inc. (Whittier, CA, USA).
Animal treatment
All animal experiments were conducted in accordance with
the guidelines for the care and use of laboratory animals of the
U.S. National Institutes of Health. Male Wistar rats (weighing
400450g) were anesthetized with sodium pentobarbital
(60mg/kg) and anticoagulated with sodium heparin (1,000U/
kg). Five minutes after the heparin injection, a midstream
thoracotomy was performed, and the heart was rapidly
Manuscript received 10 September 2010. Revision accepted 14 February 2011.
Address correspondence to: Cecilia Zazueta, Departamento de Bioqumica, Instituto
Nacional de Cardiologa Ignacio Chavez, Juan Badiano No. 1, Colonia Seccion XVI,
Mexico 14080, D.F., Mexico, E-mail: azazuetam@yahoo.com or Jose Pedraza-Chaverri,
Facultad de Qumica, Edicio F, Universidad Nacional Autonoma de Mexico, Mexico
City, 04510, Mexico, E-mail: pedraza@unam.mx
JOURNAL OF MEDICINAL FOOD
J Med Food 14 (11) 2011, 13701374
# Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2010.0238
1370
excised and placed in ice-cold KrebsHenseleit buffer solu-
tion, consisting of 118mM NaCl, 4.75 mM KCl, 1.18mM
KH
2
PO
4
, 1.18 mM MgSO
4
$7H
2
O, 2.5 mM CaCl
2
, 25mM
NaHCO
3
, 5 mM glucose, and 100 lM sodium octanoate, pH
7.4. The heart was quickly xed onto a Langendorff heart
perfusion system and perfused retrogradely via the aorta at a
constant ux of 12 mL/minute with KrebsHenseleit solution
that was continuously bubbled with 95% O
2
and 5% CO
2
, at
37C. Mechanical work was measured at a left ventricular end
diastolic pressure of 10 mm Hg, using a latex balloon inserted
into the left ventricle and connected to a pressure transducer.
All variables were recorded using a computer acquisition data
system designed by the Instrumentation and Technical De-
velopment Department of the National Institute of Cardiology
(Mexico City, Mexico).
13
Experimental protocols
KrebsHenseleit buffer was perfused for 20 minutes to
stabilize the hearts. The hearts were subjected to global is-
chemia for 30 minutes, by turning off the pumping system,
and then to reperfusion for an additional 60 minutes (I/
R
60
).
13
Control hearts were continuously perfused as long as
I/R
60
hearts. In addition, some hearts were reperfused during
60 minutes with 2.5 lM a-M, after 30 minutes of ischemia.
The vehicle of a-M had no effect on cardiac function. Hearts
that developed arrhythmias before the ischemia were dis-
carded and replaced.
Measurement of infarct size
At the end of the experiment the hearts to be used for
infarct size calculations (n = 3 for each group) were frozen
at -20C for 24 hours. Later, the hearts were cut into ap-
proximately 3-mm slices simply by eye. Then, slices were
immersed in 1% triphenyltetrazolium chloride solution in
phosphate buffer (88 mM Na
2
HPO
4
and 1.8 mM NaH
2
PO
4
)
(pH 7.4) for 10 minutes at 37C,
14
incubated in a solution of
formalin for 5 minutes, and scanned on a Hewlett-Packard
Scanjet 3800 scanner (Hewlett-Packard, Palo Alto, CA,
USA). With the use of the U.S. National Institutes of Health
software Image J, each digitized image was subjected to
determination of the infarct area. The risk and the infarct
zones were traced, and the resulting areas were calculated in
terms of pixels.
Measurement of ATP and phosphocreatine content
Cardiac tissue was extracted with 3% perchloric acid
(nal concentration) and neutralized with 3 M Tris-KOH to
pH 7.3. ATP content was measured uorometrically as
NAD
+
reduction in a medium of pH 7.4, containing 50 mM
Tris, 10 mM MgCl
2
, 5 mM EDTA, 50 U/mL hexokinase,
50 U/mL glucose 6-phosphatase, and 0.5 mL of neutralized
extracts (at 340 nm excitation and 460 nm emission).
13
Phosphocreatine content was also determined in neutralized
acid extracts from cardiac tissue by using 19 U/mL creatine
kinase, 10 lM ADP, and 2.4 lM N-acetylcysteine according
to the procedure of Bergmeyer.
15
Markers of oxidative stress
At the end of the I/R protocol, the hearts were dismounted
from the Langendorff system and frozen in liquid nitrogen
until the determination. The hearts were homogenized for 15
seconds in a Brinkmann Polytron

(Kinematica, Lucerne,
Switzerland) in 50 mM phosphate buffer (pH 7.4) (1:3 ratio)
and then centrifuged at 5,000 g for 10 minutes at 4C.
Protein content in the supernatants was determined as
previously described.
16
Reduced glutathione (GSH) was
quantied using monochlorobimane, malondialdehyde and
4-hydroxynonenal levels were measured as markers of lipid
peroxidation, and protein carbonyls were determined as a
marker of oxidized proteins following derivatization with
dinitrophenylhydrazine with methods previously described.
7
Statistical analysis
Each experiment was performed in triplicate and repeated
at least three times. Data are presented as mean SEM
values for each experimental protocol. Signicance (P .05)
was determined for discrete variables by analysis of variance,
using the data analysis and technical program Microcal
Origin from Microcal Software, Inc. (Northampton, MA,
USA) (1999).
RESULTS
To evaluate the potential protective effect of a-M in
postischemic hearts, different concentrations of this xan-
thone were evaluated. First of all, it was observed that the
mechanical work in the I/R
60
group diminished drastically
since the rst minutes of reperfusion (Fig. 1). A partial,
FIG. 1. Protective effect of a-mangostin in the mechanical work of
postischemic hearts. After 20 minutes of stabilization the hearts were
subjected to 30 minutes of ischemia, then the pump system was re-
stored, and each heart was reperfused for 60 minutes: control (-),
ischemiareperfusion for 60 minutes (,), and (o) ischemia
reperfusion for 60 minutes + 2.5 lM a-mangostin. Data are mean
SEM of six experiments. *P< .001 versus control,
#
P<.01 versus
ischemiareperfusion for 60 minutes.
CARDIOPROTECTIVE EFFECT OF a-MANGOSTIN 1371
although nonsignicant, cardioprotective effect was found
in the I/R
60
groups treated with 1 lM, 2 lM, and 3 lM a-M,
compared with the I/R
60
hearts (data not shown). It is re-
markable that the mechanical work of the I/R
60
hearts
treated with 2.5 lM a-M was preserved (Fig. 1). This group
was not statistically different from the control group but was
different from the I/R
60
group.
Hearts subjected to 30 minutes of ischemia followed by
60 minutes of reperfusion with and without treatment were
used to measure the infarct size. A signicant reduction in
myocardial infarct size was observed in the I/R
60
+2.5 lM a-
M group compared with the I/R
60
group (5% vs. 70%) (Fig.
2). The infarct size in the control group was approximately
1%. In addition, a-M treatment was able to prevent the de-
crease in ATP and phosphocreatine content after 60 minutes
of reperfusion (Fig. 3). Furthermore, different oxidative
damage markers were evaluated. It was found that 2.5 lM a-
M was able to attenuate the decrease in GSH and the in-
crease in malondialdehyde, 4-hydroxynonenal, and oxidized
proteins induced by I/R (Fig. 4).
DISCUSSION
Cardiac damage induced by reperfusion interrupts bio-
chemical and physiological processes in the heart, an in-
terruption that compromises ATP production. The increase
in ROS during reperfusion has been pointed out as a main
factor in such injury.
17
Electronic paramagnetic resonance
studies have demonstrated a rapid increase in ROS pro-
duction after reperfusion of ischemic myocardium. Besides,
it has been demonstrated that O
2

is the predominant spe-


cies produced and that endothelial cells represent an im-
portant source of ROS.
18
On the other hand, the inhibition of
xanthine oxidase by allopurinol and tungsten is capable of
preventing O
2

generation, favoring cardioprotection in


guinea pig hearts.
19
Among the main targets of ROS attack
are polyunsaturated fatty acids of the lipid membrane, which
undergo lipid peroxidation and, thereby, alteration in
structure and in cellular function. Malondialdehyde and 4-
hydroxynonenal are decomposition products of the chain
reaction that leads to oxidation of polyunsaturated fatty
acids; therefore both are reliable markers of oxidative stress
produced by I/R. GSH has a critical role in cellular defense
against agents that cause oxidative stress; thus low levels of
GSH imply cellular alteration produced by oxidative stress.
In this situation, stable products like carbonyl groups are
FIG. 2. Infarct size in postischemic hearts treated with a-mangos-
tin. Heart slices were incubated with triphenyltetrazolium chloride. In
the viable tissue, the stain is reduced by active dehydrogenases,
producing 1,3,5-triphenylformazan (shown by intensity of the red
color). Infarcted tissue remains uncolored. Data are mean SEM
values of three experiments. *P< .005 versus control,
#
P<.05 versus
ischemiareperfusion for 60 minutes (I/R60).
FIG. 3. Cardioprotective effect of a-mangostin on (A) ATP and (B)
phosphocreatine content in isolated rat hearts subjected to ischemia
reperfusion. Data are mean SEM values of three experiments per-
formed in duplicate. *P< .05 versus control,
#
P< .05 versus I/R60.
1372 BUELNA-CHONTAL ET AL.
produced by ROS action on proteins, also making protein
carbonylation a reliable marker of oxidative stress.
In normal conditions the large amount of ATP necessary
to maintain contractile function and basal metabolism in the
heart is generated primarily by mitochondrial oxidative
metabolism. During ischemia, the ability of mitochondria to
sustain ATP synthesis is highly compromised; under such
conditions, cardiac cells maintain ATP levels by activating
the glycolytic pathway until oxidative metabolism is re-
stored during reperfusion.
20
However, if ischemia is pro-
longed, the cells accumulate glycolytic by-products, like
lactate and H
+
, which cause irreversible damage to cardiac
cells.
21
Paradoxically, re-introduction of oxygen during re-
perfusion enhances damage to ischemic cells, typically by
membrane rupture, followed by cell death. ROS overpro-
duction during reperfusion has been pointed out as a major
contributor to irreversible damage of mitochondrial function
and impaired recovery of physiological function in the
heart.
22
a-M is a component extracted from the pericarp of
mangosteen; this xanthone has antioxidant and anti-
inammatory properties, among others.
2
In vitro, a-M is a
potent scavenger of ONOO
-
,
1
O
2
, and O
2

.
5,6
Recently, Devi Sampath and Vijayaraghavan
23
demon-
strated that a-M provides protection against myocardial in-
farct induced by isoproterenol in rats; such an effect was
related to the activity of the intracellular defense system that
avoided lipid peroxidation. However, there are few studies
about the role of xanthones against myocardial I/R injury.
Therefore, we decided to investigate the potential protective
effect of a-M against the damage caused by reperfusion in
postischemic hearts. We found that 2.5 lM a-M diminished
the infarct size and improved the mechanical work in I/R
60
hearts during the rst minutes of reperfusion. We also
FIG. 4. Markers of oxidative stress in postischemic hearts treated with a-mangostin: (A) reduced glutathione (GSH), (B) malondialdehyde
(MDA), (C) 4-hydroxynonenal (4-HNE), and (D) oxidized proteins. DNPH, dinitrophenylhydrazine. Data are mean SEM values of six ex-
periments. *P<.05 versus control,
#
P<.01 versus I/R60.
CARDIOPROTECTIVE EFFECT OF a-MANGOSTIN 1373
observed that ATP and phosphocreatine levels were main-
tained in reperfused hearts treated with a-M. These results
were related to lowlevels of lipid peroxidation, a decrease in
protein carbonylation, and the preservation of high GSH
content, demonstrating that a-M signicantly protected
hearts against I/R cardiac dysfunction through its antioxi-
dant action, maintaining the function of the oxidative
phosphorylation machinery.
ACKNOWLEDGMENTS
This work was supported by grants DGAPA IN201910
and CONACYT 129838 to J.P.-C. and by grant CONACYT
80791 to C.Z.
AUTHOR DISCLOSURE STATEMENT
No competing nancial interests exist.
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