Pharmacological Reports 67 (2015) 115–122

Contents lists available at ScienceDirect

Pharmacological Reports
journal homepage: www.elsevier.com/locate/pharep

Original research article

Pinocembrin attenuates hippocampal inflammation, oxidative

perturbations and apoptosis in a rat model of global cerebral ischemia
reperfusion
Muhammed A. Saad *, Rania M. Abdel Salam, Sanaa A. Kenawy, Amina S. Attia
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Cairo University, Cairo, Egypt

A R T I C L E I N F O A B S T R A C T

Article history: Background: Pinocembrin is a major flavonoid molecule isolated from honey and propolis. It has versatile
Received 24 June 2014 pharmacological and biological activities including antimicrobial, anti-inflammatory, antioxidant, and
Received in revised form 13 August 2014 anticancer activities as well as neuroprotective effects against cerebral ischemic injury. The purpose of
Accepted 14 August 2014
the current study was to determine the possible mechanisms of neuroprotection elicited by pinocembrin
Available online 27 August 2014
with specific emphasis on chronic prophylactic use before the induction of global cerebral ischemia
reperfusion.
Keywords:
Methods: Global cerebral ischemia–reperfusion (I/R) was induced by bilateral carotid artery occlusion
Pinocembrin
Ischemia/reperfusion
for 15 min followed by 60 min reperfusion period. Animals were randomly allocated into 3 groups
Oxidative stress (n = 28): Sham operated, I/R control and rats treated with pinocembrin (10 mg/kg, po) daily for 7 days
Apoptosis then I/R was induced 1 h after the last dose of pinocembrin. After reperfusion rats were killed by
Inflammation decapitation, brains were removed and both hippocampi separated and the following biochemical
parameters were estimated; lactate dehydrogenase activity, oxidative stress markers (lipid peroxides,
nitric oxide and reduced glutathione), inflammatory markers (myeloperoxidase, tumor necrosis factor-
alpha, nuclear factor kappa-B, interleukin-6 and interleukin-10), apoptotic biomarkers (caspase 3 and
cytochrome C), neurotransmitters (glutamate, gamma aminobutyric acid) and infarct size were assessed.
Results: Pinocembrin ameliorated damage induced by I/R through suppressing oxidative stress,
inflammatory and apoptotic markers as well as mitigating glutamate and lactate dehydrogenase activity.
One of the more significant findings to emerge from this study is that pinocembrin normalized the infarct
size elevated by I/R.
Conclusions: Pinocembrin showed a neuroprotective effects through antioxidant, anti-inflammatory and
antiapoptotic mechanisms.
ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp.
z o.o. All rights reserved.

and antioxidant activities [1–3]. The extensive research on

Introduction
Pinocembrin, one of the most important flavonoids isolated from
honey and propolis. It possesses anti-inflammatory, antimicrobial,
Abbreviations: EB, Evan’s blue; ELISA, enzyme-linked immunosorbent assay; GSH,
reduced glutathione; HPLC, high performance liquid chromatography; I/R,
ischemia/reperfusion; IFN, interferon; IL-6, interleukin-6; IL-10, interleukin-10;
LDH, lactate dehydrogenase; MPO, myeloperoxidase; NF-kB, nuclear factor kappa
B; NOx, nitric oxide; SO, Sham operated.; TBARs, thiobarbituric acid reactive
substances; TNF-a, tumor necrosis factor-alpha..
* Corresponding author.
E-mail address: muhammed.abdullatif@gmail.com (M.A. Saad).
pinocembrin indicated that it has potential biological activities,
which have made a further interest in studying its activities in
many other disorder of global concern. Stroke is one of these
disorders as it is considered the leading cause of morbidity and
mortality world-wide [4]. Acute brain ischemia–reperfusion (I/R)
injury is the major pathophysiological sign of ischemic stroke [5].
Pinocembrin showed antiapoptotic effects by attenuating
endoplasmic reticulum stress in a model of focal and global (I/
R) [6,7]. Furthermore, it decreased neurological score, brain edema
and concentrations of Evan’s blue (EB) and fluorescein sodium in
brain tissue suggesting an alleviation in blood–brain barrier injury
induced by global cerebral I/R [8,9]. Additionally, pinocembrin has
proven to be a possible neuroprotective agent against global

http://dx.doi.org/10.1016/j.pharep.2014.08.014
1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
116 M.A. Saad et al. / Pharmacological Reports 67 (2015) 115–122
cerebral I/R injury through its antioxidant, anti-excitotoxic and
anti-inflammatory effects [6,10]. Wang et al. [11] suggested that
inhibiting soluble epoxide hydrolase and then increasing the
potency of epoxyeicosatrienoic acids may be one of the mecha-
nisms through which pinocembrin provides cerebral protection.
Another possible mechanism for neuroprotection may be through
anti-inflammatory actions as evidenced by the reduced expres-
sions of interleukin-1beta (IL-1b), tumor necrosis factor-alpha
(TNF-a), and inducible NO synthase shown by pinocembrin [12].
So far, there has been considerable research on the curative
effect of pinocembrin if given on acute basis, however, there has
been little discussion about the prophylactic effect of pinocembrin
if given chronically before induction of I/R. Therefore, it deemed of
importance to study the prophylactic effect of pinocembrin if given
on chronic basis. Although extensive research has been carried out
on pinocembrin, no single study exists which adequately covers its
effect on infarct size, neurotransmitter as GABA and glutamate and
nuclear factor kappa-B (NF-kB). Furthermore, few writers have
been able to draw on any systematic research into the full
mechanism of neuroprotection induced by pinocembrin against
damage induced by I/R. Hence the current investigation were
aimed to study the prophylactic effect of pinocembrin against
injury induced by global cerebral I/R if given chronically for seven
days before the induction of I/R. The central question in this
dissertation asks how pinocembrin exerts its neuroprotective
effect, and was answered through making a full investigation of the
possible mechanisms of its neuroprotection by studying the
changes in oxidative stress, inflammatory and apoptotic biomark-
ers as well as neurotransmitters and infarct size.
Materials and methods
Animals
Male Wistar rats weighing; 250–300 g were obtained from the
National Scientific Research Center (Giza, Egypt). Animals were
housed for at least one week in the laboratory room prior to testing.
They were kept under controlled environmental conditions; room
temperature (24–27 8C), constant humidity (60  10%), with
alternating 12 h light and dark cycles. Food (standard pellet diet) &
water were allowed ad libitum. The Ethics Committee of Faculty of
Pharmacy Cairo University approved this study. All animals’
procedures were performed in accordance to the institutional Ethics
Committee and in accordance with the recommendations for the
proper care and use of laboratory animals. Unnecessary disturbance
of animals was avoided. Animals were treated gently; squeezing,
pressure and tough maneuver were avoided.
Global cerebral ischemia and experimental material preparation
Animals were randomly allocated into 4 groups (n = 28 rats per
group) as follows:
Group I: Sham operated (SO).
Group II: ischemia–reperfusion group (I/R).
Group III: pinocembrin pretreatment; rats were given pino-
cembrin (10 mg/kg, po) daily for 7 days [13], then I/R was
induced 1 h after the last dose of pinocembrin.
Each group was subdivided into 2 subsets. The first subset
(n = 24 rats) was used for biochemical estimations, while the
second subset (n = 4 rats) served for measurement of infarction
size.
In all groups rats were anaesthetized with thiopental (50 mg/
kg, ip) and midline ventral incision was made in the neck. Bilateral
carotid artery occlusion using small artery clips was done to
induce global cerebral ischemia for 15 min followed by 60 min
reperfusion period except for the SO group in which the artries
were exposed for 75 min without occlusion. After reperfusion rats
were euthanized by cervical dislocation. Brains were removed
immediately and both hippocampi were dissected on ice cold
plates. The first subset (n = 24 rats) were subdivided into 2 sets, in
one set of animals (n = 16 rats), the hippocampi were homoge-
nized in isotonic ice cold saline using a glass homogenizer
(HeidolphDiax 900, Germany). This homogenate was used for the
determination of NOx, TBARs, GSH, IL-10, IL-6, NFkB and TNF-a
contents as well as Cytochrome C and Caspase 3 activities. An
aliquot of 60 ml of this homogenate was centrifuged at
105,000  g for 45 min at 4 8C using cooling ultra-centrifuge
(Sorvall-Compiplus T-880, Dupont, USA) to separate the cytosolic
hippocampal fraction, which was used for determination of the
activity of LDH. In another subset (n = 8 rats), the hippocampi
were divided into two portions: one hippocampus was homoge-
nized in hexadecyltrimethylammonium bromide (0.5%) in potas-
sium phosphate buffer (100 mM, pH 6) for the determination of
myeloperoxidase (MPO) activity, while the other hippocampus
was homogenized in 70% high performance liquid chromatogra-
phy (HPLC) methanol (1/10, weight/volume) for the determina-
tion of neurotransmitter (Glutamate and GABA) contents. All
parameters were normalized to protein content, measured
according to Lowry et al. [14].
Determination of lactate dehydrogenase (LDH) activity in rat
hippocampus
The activity of LDH in rat hippocampus was determined by
colorimetric kinetic determination using kit supplied by Biosys-
tems (Biosystems, S.A. Costa Brava, 30 08030 Barcelona, Spain)
according to the method of Lorentz et al. [15]. LDH specifically
catalyzes the oxidation of lactate to pyruvate with the subsequent
reduction of NAD to NADH. The rate at which NADH forms is
proportional to LDH activity. The method determines the increase
in NADH absorbance per minute using a double beam spectropho-
tometer at wavelength 340 nm.
Determination of lipid peroxides content in rat hippocampus
The lipid peroxidation products are determined by the
estimation of the level of thiobarbituric acid reactive substances
(TBARs) that are measured as MDA according to the method of
Mihara and Uchiyama [16]. MDA is the decomposition product of
the process of lipid peroxidation and is used as an indicator of this
process.
The principle of the assay depends on a colorimetric determi-
nation of a pink pigment product, which results from the reaction
of TBARS with thiobarbituric acid in acidic medium at high
temperature. To increase the sensitivity of the method, the
resultant colored product was extracted in n-butanol and
measured at two wavelengths, namely 520 and 535 nm using
Shimadzu double beam spectrophotometer (UV-150-02, Japan), to
exclude interfering substances. The difference in absorbance at
both wavelengths is used to calculate the content of TBARS in the
sample.
Determination of reduced glutathione (GSH) content in rat
hippocampus
Determination of GSH content in rat hippocampus was
performed according to the method of Beutler et al. [17]. The
method depends on the fact that both protein and non-protein SH-
groups (mainly GSH) react with Ellman’s reagent [5,50 -dithiobis-
(2-nitrobenzoic acid)] (DTNB) to form a stable yellow color of 5-
mercapto-2-nitrobenzoic acid, which can be measured at 412 nm.
In order to determine the GSH content in tissue, precipitation of
 M.A. Saad et al. / Pharmacological Reports 67 (2015) 115–122
protein SH-groups before the addition of Ellman’s reagent is
necessary.
Determination of nitric oxide (NOx) content in rat hippocampus
NOx was determined in rat hippocampus according to the
method described by Miranda et al. [18]. Nitric oxide is relatively
unstable in the presence of molecular oxygen, with an apparent
half life of approximately 3–5 s and is rapidly oxidized to nitrate
(NO3) and nitrite (NO2) totally designated as NOx. A high
correlation between endogenous nitric oxide production and
nitrite/nitrate (NOx) levels has been established, hence providing a
reliable and quantitative estimate of nitric oxide output in vivo. The
assay determines total nitrite/nitrate level based on the reduction
of any nitrate to nitrite by vanadium followed by the detection of
total nitrite (intrinsic + nitrite obtained from reduction of nitrate)
by Griess reagent. The Griess reaction entails formation of a
chromophore from the diazotization of sulfanilamide by acidic
nitrite followed by coupling with bicyclic amines such as N-(1-
naphthyl) ethylenediamine. The chromophoric azo derivative can
be measured colorimetrically at 540 nm.
Determination of myeloperoxidase (MPO) activity in rat hippocampus
MPO enzyme, being a plentiful constituent of neutrophils,
serves as a marker for tissue neutrophil content. Since MPO is
located within the primary granules of neutrophils, extraction of
MPO depends upon procedures to disrupt the granules which
render MPO soluble in aqueous solution. This could be achieved by
sonication in potassium phosphate buffer (50 mM, pH 6) contain-
ing 0.5% hexadecyltrimethylammonium bromide (HTAB) [19],
where HTAB is a detergent that releases MPO from the primary
granules of the neutrophil [20].
Determination of caspase-3 activity in rat hippocampus
The hippocampal activity of caspase-3 enzyme was measured
by Enzyme-linked immunosorbent assay (ELISA) using kit supplied
by R&D Systems (Quantikine Active Caspase-3 ELISA, Catalog #
KM300, R&D Systems, Inc., Minneapolis, MN, USA).
Determination of nuclear factor-kappa B (NF-kB) in rat hippocampus
The hippocampal content of NF-kB was measured by Enzyme-
linked immunosorbent assay (ELISA) using kit supplied by ALAab
(E1824r, EIAab Science Co., Wuhan, China).
Determination of cytochrome-c in rat hippocampus
The hippocampal content of cytochrome-c was measured by
Enzyme-linked immunosorbent assay (ELISA) using kit supplied by
ALAab (E0594r, EIAab Science Co., Wuhan, China).
Determination of tumor necrosis factor alpha (TNF-a) in rat
hippocampus
The hippocampal content of TNF-a was measured by Enzyme-
linked immunosorbent assay (ELISA) using kit supplied by R&D
Systems (Quantikine Rat TNF-a ELISA, Catalog # RTA00, R&D
Systems, Inc., Minneapolis, MN, USA).
Determination of interleukin-6 (IL-6) content in rat hippocampus
The hippocampal content of IL-6 was measured by Enzyme-
linked immunosorbent assay (ELISA) using kit supplied by R&D
Systems (Quantikine1 ELISA, Rat IL-6 Immunoassay, Catalog
Number R6000B, R&D Systems, Inc., Minneapolis, MN, USA).
Determination of interleukin-10 (IL-10) content in rat hippocampus
The hippocampal content of IL-10 was measured by Enzyme-
linked immunosorbent assay (ELISA) using kit supplied by R&D
Systems (Quantikine1 ELISA, Rat IL-10 Immunoassay, Catalog
Number R1000, R&D Systems, Inc., Minneapolis, MN, USA).
117
Determination of protein content in rat hippocampus
The hippocampal protein content of tissue supernatant was
determined using the method of Lowry et al. [14]. The method is
characterized by a great sensitivity and is described for measuring
protein in solution which is present in very low concentration as
little as 0.2 mg of protein. The final color is produced from the
reduction of phosphomolybdic-phosphotungstic reagent in Folin
reagent by copper-treated protein in alkaline medium at room
temperature.
Determination of GABA and glutamate contents in rat hippocampus
Hippocampus was homogenized in 70% high performance
liquid chromatography (HPLC) methanol (1/10, weight/volume)
and was used for the estimation of glutamate and GABA using a
fully automated high-pressure liquid chromatography system
(HPLC; Perkin-Elmer, MA, USA). Brain amino acids were deter-
mined by the phenylisothiocyanate derivatization technique
described by Heinrikson and Meredith [21].
Brain infarct size
At the end of 60 min reperfusion period, animals (n = 4) were
intracardiacally perfused with isotonic saline and sacrificed by
spinal dislocation. Brains were then sliced into 2 mm coronal
sections and incubated with 1% triphenyltetrazolium chloride
(TTC) at 37 8C in 0.2 M Tris buffer (pH 7.4) for 20 min. While viable
cells stain bright red when TTC is converted to red formazone
pigment by NAD and lactate dehydrogenase, infracted cells lose the
enzyme as well as cofactor and thus remain unstained or stain dull
yellow. The brain slices were placed over glass plate and the
infarcted areas were traced by a 100 squares in 1 cm2 transparent
plastic grid. In each brain slice, the average infarcted areas of both
sides as well as the non infarcted area were determined. Infarcted
area was expressed as a percentage of total brain area [22,23].
Statistical analysis
Values were expressed as mean  SEM using a computer
software program Statistical Package for the Social Sciences ‘‘SPSS’’
(Version 16.0.). One-Way Analysis of Variance followed by Tukey-
Kramer multiple comparisons post-hoc test was used for comparing
the means of the different groups. The criterion for statistical
significance was set at the p < 0.05 level. Graphical representation
was conducted using GraphPad Prism (version 5).
Results
Effect of pinocembrin (10 mg/kg, po) on cerebral infarct size
Global cerebral I/R resulted in a significant increase in infarct
size to 260% the SO group. Pinocembrin decreased the elevated
infarct size to 145% of that in SO group but it still was higher than
the normal level (Fig. 1).
Effect of pinocembrin (10 mg/kg, po) on lactate dehydrogenase (LDH),
Myeloperoxidase (MPO) activities, and inflammatory biomarkers in
hippocampal tissue
Global cerebral I/R was accompanied with elevated hippocam-
pal LDH activity to about 263% of the SO group. Pinocembrin
decreased and normalized LDH activity to about 104% of the SO
group. Global cerebral I/R was associated with neutophil infiltera-
tion observed as an elevation in hippocampal MPO activity to 149%
of the SO group. This effect was offset by pinocembrin in which
MPO activity decreased to about 88% the SO group returning back
to the normal level.
[(Fig._1)TD$IG]
118 M.A. Saad et al. / Pharmacological Reports 67 (2015) 115–122

Fig. 1. Effect of pinocembrin (10 mg/kg, po) on brain coronal sections. (I) Coronal sections showing the infarct areas (in white) in (A) Sham operated control, (B) ischemia/
reperfusion brain, (C) and pinocembrin (10 mg/kg, po). (II) Summary of the quantitative analysis of infarct areas. Values were expressed as mean  SEM of 4 rats. *Significantly
different from Sham operated control at p < 0.05. @Significantly different from Ischemia reperfusion at p < 0.05. Statistical analysis was performed by ANOVA followed by Tukey’s
post-hoc test.

Global cerebral I/R showed a significant increase in hippocam-
pal NF-kB, TNF-a, IL-6 contents together with reduction in IL-10
content. NF-kB content reached 331% of that in the SO group
whereas TNF-a and IL-6 were increased to 491% and 149% of the SO
group, respectively. On the other hand IL-10 content reached 29%
of the SO group.
Pinocembrin reduced and normalized NF-kB, TNF-a and IL-6
contents to be 144%, 117% and 78% of that in SO group,
respectively. However, pinocembrin failed to change the reduced
IL-10 content (Table 1).
Effect of pinocembrin (10 mg/kg, po) on oxidative stress and apoptotic
biomarkers contents in hippocampal tissue
Global cerebral I/R resulted in a significant increase in
hippocampal TBARs content to 209% accompanied with a
reduction in GSH content to 28% compared to SO group.
Pinocembrin showed a potential antioxidant power through a
reduction in TBARs (Fig. 2a) and NOx contents (Fig. 2c) to about 83%
and 23% of that in SO group, respectively. Furthermore, pinocem-
brin elevated the declined hippocampal reduced GSH content to
90% of the SO group (Fig. 2b). Pinocembrin normalized TBARs and
GSH contents and reduced NOx less than normal level.
Global cerebral I/R resulted in obvious increase in hippocampal
caspase-3 activity to 254% of the SO group. Additionally,
hippocampal Cyt-c content was elevated to about 488% following
I/R compared to the SO group. Treatment with pinocembrin
significantly normalized and reduced both caspase-3 activity and
cytochrome c content to about 113% and 98% compared to SO
group (Fig. 2d and e).
Effect of pinocembrin (10 mg/kg, po) on neurotransmitters contents in
hippocampal tissue
Global cerebral I/R showed a significant increase in both
glutamate and GABA contents in the hippocampus. Glutamate
content reached to about 609% that measured in the SO group
while GABA content increased to 368% the SO group. Pinocembrin

Table 1
Effect of pinocembrin (10 mg/kg, po) on hippocampal: cytosolic lactate dehydrogenase (LDH) activity, myeloperoxidase (MPO) activity, nuclear factor kappa-B (NF-kB), tumor
necrosis factor-alpha (TNF-a), interleukin-10 (IL-10) and interleukin-6 (IL-6) contents in rats subjected to global cerebral ischemia reperfusion.

Groups Parameter

LDH content MPO activity NFK-B content TNF-a content IL-10 content IL-6 content
(U/g protein) (mU/mg protein) (ng/mg protein) (Pg/mg protein) (Pg/mg protein) (Pg/mg protein)
Mean  SE Mean  SE Mean  SE Mean  SE Mean  SE Mean  SE

Sham operated control 30.57  1.49 26.83  4.24 0.11  0.01 3.02  0.39 16.71  2.34 27.11  1.74
Ischemia reperfusion 80.43*  16.41 42.53*  2.42 0.35*  0.02 14.85*  0.96 4.96*  1.22 40.41*  5.38
pinocembrin (10 mg/kg, po) 32.01@  7.02 23.84@  2.01 0.15@  0.02 3.55@  0.44 12.43  1.88 21.17@  1.33

Values were expressed as mean  SEM of 6 rats. Statistical analysis was performed by ANOVA followed by Tukey’s post-hoc test.
*
Significantly different from Sham operated control at p < 0.05.
@
Significantly different from Ischemia reperfusion at p < 0.05.
[(Fig._2)TD$IG] M.A. Saad et al. / Pharmacological Reports 67 (2015) 115–122 119

Fig. 2. Effect of pinocembrin (10 mg/kg, po) on hippocampal oxidative stress biomarkers (a) thiobarbituric acid reactive substances (TBARs), (B) reduced glutathione (GSH)
and (c) nitric oxide (NOx) contents; apoptotic markers (d) Caspase-3 avtivity and (e) cytochrome-c (Cyt-c) content in rats subjected to global cerebral ischemia reperfusion.
Values were expressed as mean  SEM of 6 rats. *Significantly different from Sham operated control at p < 0.05. @Significantly different from Ischemia reperfusion at
p < 0.05. Statistical analysis was performed by ANOVA followed by Tukey’s post-hoc test.

failed to significantly change the elevated GABA content as it is still
higher than normal level attaining 478% of the SO group (Fig. 3b),
but reduced the glutamate content to about 206% of the SO group
returning back to normal level (Fig. 3a).
Discussion
Oxidative stress is a well recognized factor of ischemia
reperfusion injury. It induces damage of lipids and proteins, and
consequently might cause changes in activity of transport systems
[24]. This study produced results which corroborate the findings of
a great deal of the previous work in this field. Global 15 min
ischemia by two-vessel occlusion followed by 1 h reperfusion was
induced in the present work. Following the induction of I/R, a
marked elevation in TBARs and NOx contents were observed in rats’
hippocampi indicating a higher than normal peroxynitrite level.
Furthermore, the content of reduced glutathione showed an
obvious decline compared to the Sham operated group. The
increase in the prooxidant parameters and the decrease in
antioxidant parameters shown in our results clearly confirm that
the induction of oxidative stress is an inevitable result of I/R. Attack
of proteins and lipids by free radicals leads to lipid peroxidation as
well as protein modifications that will finally participate in the
injury induced by I/R.
Treatment with pinocembrin in the current investigation
showed a protective effect against oxidative stress induced by I/
R. Pinocembrin reduced the elevated TBARs and NOx contents and
ameliorated the declined level of reduced GSH. TBARs can react
with NO to produce peroxynitrite (ONOO), which is a strong
oxidative radical that causes protein nitration and dysfunction
[25]. NO generated from neuronal NO synthase nitrosylates
protein, which leads to cell dysfunction. The reduction in TBARs
and NOx contents shown in our experiment after treatment with
pinocembrin suggests a reduction in ONOO content and
amelioration of protein nitration and nirosylation. Pervious report
supports our findings as pinocembrin administrated at the start of
reperfusion showed a reduction of NO, both neuronal and
inducible NO syntheses, reactive oxygen species and an increase
in GSH [10].
[(Fig._3)TD$IG]
120 M.A. Saad et al. / Pharmacological Reports 67 (2015) 115–122
Fig. 3. Effect of pinocembrin (10 mg/kg, po) on hippocampal neurotransmitters (a) glutamate and (b) gamma amino butyric acid (GABA) contents in rats subjected to global
cerebral ischemia reperfusion. Values were expressed as mean  SEM of 6 rats. *Significantly different from Sham operated control at p < 0.05. @Significantly different from
Ischemia reperfusion at p < 0.05. Statistical analysis was performed by ANOVA followed by Tukey’s post-hoc test.
Oxygen derived free radicals generated during I/R can induce
TNF-a [26]. TNF-a starts a number of intracellular adaptive
metabolic responses among them an increase in the intracellular
Ca++-concentration with generation of calcium pyrophosphate
complexes which can bind to intracellular protein complexes so
called inflammasomes [27]. The inflammasomes activates NF-kB
which plays a central role in the generation of an inflammatory
response as it causes an activation and formation of other pro-
inflammatory factors such as IL-1b, IL-6, tumor necrosis factor
(TNF)-a, and interferon (IFN)-g potentiating the inflammatory
response [28].
There are similarities between the attitudes expressed by I/R in
this study and those described by Pan et al. [28] as induction of
global cerebral ischemia reperfusion in the present work showed
an obvious elevation in proinflammatory mediators as MPO, NF-
kB, TNF-a and IL-6. Additionally the anti-inflammatory mediator
IL-10 was significantly reduced after I/R injury.
In the current investigation pinocembrin reversed all the
previously mentioned inflammatory reactions except IL-10 con-
tent. The reduction in the activity of MPO by pinocembrin in the
current work confirms the reduction of the cytotoxic MPO
metabolites as hypochlorous acid and tyrosyl radical. Furthermore,
TNF-a which is an inflammatory mediator of neuronal death ‘a
member of the death-inducing ligand (DIL) family’ is reduced after
exposure to pinocembrin. This inhibitory effect shown with
pinocembrin for TNF-a acts upstream on the caspases and
suggests a suppressive effect on the extrinsic pathway of apoptosis.
Additionally, pinocembrin reduced NF-kB, suggesting a reduction
in the transcription of all genes regulated by such transcription
factor as inducible nitric oxide synthase (iNOS), cyclooxygenase II
(COX-2), manganese-conjugated superoxide dismutase (MnSOD),
antiapoptotic proteins such as Bcl2 and inhibitor of apoptosis
factors (IAFs). Also, pinocembrin diminished IL-6 content which is
a pro-inflammatory cytokine secreted by T cells and macrophages
to stimulate immune response during infection and after trauma.
From all the previous knowledge, pinocembrin had proven to
possess a potent anti-inflammatory activity and suggests a
possible use of pinocembrin in many other inflammatory
disorders.
This finding corroborates the ideas of Soromou et al. [29] who
demonstrated that in the mouse model of LPS-induced acute lung
injury, pinocembrin reduced the development of pulmonary
edema, ameliorated histological severities, as well as infiltration
of neutrophil, lymphocyte and macrophage, which were elevated
by LPS administration.
Prolonged oxygen deprivation during a period of ischemia
results in an ATP-depletion causing a swelling of mitochondria and
eventually results in the release of cytochrome c. Cytochrome c
activates an apoptotic signaling cascade and participate in the
induction of an inflammatory response via generation of IL-1b
[30]. Apart from the formation of calcium phosphate complexes,
the elevation of the intracellular calcium concentration also
enhances the activation of phospholipases as well as proteases
‘‘caspases’’ which execute programmed cell death (apoptosis). The
two major well-studied apoptotic processes are the extrinsic and
intrinsic pathways [31,32]. The extrinsic pathway is mediated by
tumor necrosis factor receptors, while the intrinsic pathway is
largely centered around and/or regulated by the mitochondria
through the release of cytochrome c [33].
The findings observed in this study mirror those of the previous
studies as rats subjected to I/R in the present experiment showed
an elevated contents of TNF-a, cytochrome c as well as caspase
3 activities. Therefore, it could be suggested that I/R can induce
apoptosis through both the intrinsic and the extrinsic pathways.
In the intrinsic mechanisms of apoptosis Cytc binds with
apoptotic protein-activating factor-1 (Apaf-1) and activates
caspase-9 which subsequently activates caspase-3. Activated
caspase-3 cleaves DNA repair enzymes, which leads to DNA
damage and apoptosis [34]. In the extrinsic mechanisms of
apoptosis the engagement of death receptors located on the
plasma membrane that belongs to the tumor necrosis factor
receptor superfamily activates caspase-8 which initiates down-
stream activation of caspase-3, thus paving the way for the
execution phase of apoptosis, as discussed above [35]. Treatment
with pinocembrin before the induction of I/R reduced TNF-a,
cytochrome c contents as well as caspase 3 activity. These results
show clearly that pinocembrin possesses antiapoptotic actions by
inhibiting both the extrinsic and intrinsic pathways.
Previous reports have shown that pinocembrin treatment
attenuated endoplasmic reticulum stress induced apoptosis as
shown by reduced scores of neurological deficit, infarct size, and
neuron apoptosis in the ischemia/reperfusion rats [7]. Further-
more, pinocembrin reduced the expressions of TNF-alpha and IL-
1b after induction of focal cerebral ischemia by occlusion of middle
cerebral artery [12].
Brain and other aerobic tissues starts a series of biochemical
reactions called the ischemic cascade after seconds to minutes of
ischemia secondary to injury, stroke, or cardiac arrest following
heart attack [36]. This cascade starts by failing of the neuron’s
normal process for making ATP for energy due to lack of oxygen.
This leads to switching of the cell to anaerobic metabolism,
producing lactic acid. The failure of the ATP-dependant ion
transport pumps, force the cell to become depolarized, allowing
ions, including calcium (Ca++), to flow into the cell and intracellular
 M.A. Saad et al. / Pharmacological Reports 67 (2015) 115–122
calcium levels get too high. The Ca++ loading into the cell triggers
the release of the excitatory amino acid neurotransmitter
glutamate which in turn stimulates AMPA receptors and Ca++-
permeable NMDA receptors, allowing more calcium to flow into
the cells. The excess Ca++ entry overexcites cells and causes the
generation of harmful chemicals like reactive oxygen species, free
radicals and calcium-dependent enzymes such as phospholipases
in a process called excitotoxicity. Calcium can also cause the
release of more glutamate [37]. It is therefore likely that such
connections exist between glutamate level and Ca++ which plays a
major role in the damage induced by I/R. Although the role of
glutamate was clearly justified, debate continues about the role of
GABA in I/R. For instance, the GABA(B) receptor was found to plays
a significant role in the inflammatory response and neutrophil-
dependent ischemia reperfusion injury such as stroke [38]. On the
other hand, GABA receptor agonists have been shown to have a
neuroprotectant effect in reducing infarct size and improving
functional outcome in animal models of cerebral ischemia
[39]. The elevation in the contents of both glutamate and GABA
contents following global cerebral I/R in the present investigation
clearly assists in our understanding of the role of neurotransmit-
ters in the damage induced by I/R.
To our knowledge this is the first work to study the effect of
pinocembrin on the neurotransmitters contents. Pinocembrin
normalized the elevated glutamate content, but failed to reduce
the elevated GABA content as a result of I/R. The former effect could
be added as a possible mechanism for neuroprotection elicited by
pinocembrin.
Although less than the I/R group, the observed brain damage in
the Sham operated group may be due to the exposure of rats to
75 min of anesthesia under the effect of thiopental. Duan et al. [40]
showed that thiopental caused a prolonged duration of uncon-
sciousness with a high rate of mortality, and exaggerated the
neurological deficits after brain ischemia. Furthermore, the infarct
volume was increased in thiopental-anesthetized rats suggesting
that thiopental is toxic to the brain cells. One of the key findings of
the present investigation is that pinocembrin alleviated the
increase in infarct size and LDH activity after induction of I/R.
Our results are in accordance with the previously published results
of [10] who demonstrated that neuronal survival rates were
increased, LDH release was decreased and apoptosis were
alleviated when pinocembrin was present during reoxygenation.
Conclusions
This paper has given an account of and the reasons for the
widespread use of pinocembrin the master constituent of propolis
and honey. Returning to the question posed at the beginning of this
study, it is now possible to state that pinocembrin has an obvious
prophylactic effect against the injury induced by I/R. Additionally,
the study has gone some way toward enhancing our understanding
of the mechanism of neuroprotective effect of pinocembrin which
was thought to be through suppressing oxidative stress, inflam-
matory reactions, excitotoxicity and apoptotic markers as well as
mitigating infarct size elevated by I/R. It is recommended that
further research be undertaken to assess the efficacy of pinocem-
brin when combined with other neuroprotective strategies.
Funding
The authors have no support or funding to report.
Conflict of interest
The authors declare that there are no conflicts of interest.
121
References
[1] Estevinho L, Pereira AP, Moreira L, Dias LG, Pereira E. Antioxidant and antimi-
crobial effects of phenolic compounds extracts of Northeast Portugal honey.
Food Chem Toxicol 2008;46(12):3774–9.
[2] Feng R, Guo ZK, Yan CM, Li EG, Tan RX, Ge HM. Anti-inflammatory flavonoids
from Cryptocarya chingii. Phytochemistry 2012;76(10):98–105.
[3] Kumar MA, Nair M, Hema PS, Mohan J, Santhoshkumar TR. Pinocembrin
triggers Bax-dependent mitochondrial apoptosis in colon cancer cells. Mol
Carcinog 2007;46(3):231–41.
[4] Wardlaw JM, von KR, Farrall AJ, Chappell FM, Hill M, Perry D. A large web-
based observer reliability study of early ischaemic signs on computed tomog-
raphy. The Acute Cerebral CT Evaluation of Stroke Study (ACCESS). PLoS ONE
2010;5(12):e15757.
[5] Sinanovic O. Neuropsychology of acute stroke. Psychiatr Danub 2010;22(2):
278–81.
[6] Shi LL, Chen BN, Gao M, Zhang HA, Li YJ, Wang L, et al. The characteristics of
therapeutic effect of pinocembrin in transient global brain ischemia/reperfu-
sion rats. Life Sci 2011;88(11–12):521–8.
[7] Wu CX, Liu R, Gao M, Zhao G, Wu S, Wu CF, et al. Pinocembrin protects brain
against ischemia/reperfusion injury by attenuating endoplasmic reticulum
stress induced apoptosis. Neurosci Lett 2013;546(5):57–62.
[8] Gao M, Liu R, Zhu SY, Du GH. Acute neurovascular unit protective action of
pinocembrin against permanent cerebral ischemia in rats. J Asian Nat Prod Res
2008;10(5–6):551–8.
[9] Meng F, Liu R, Gao M, Wang Y, Yu X, Xuan Z, et al. Pinocembrin attenuates
blood–brain barrier injury induced by global cerebral ischemia–reperfusion in
rats. Brain Res 2011;1391:93–101.
[10] Liu R, Gao M, Yang ZH, Du GH. Pinocembrin protects rat brain against oxidation
and apoptosis induced by ischemia–reperfusion both in vivo and in vitro. Brain
Res 2008;1216:104–15.
[11] Wang SB, Pang XB, Gao M, Fang LH, Du GH. Pinocembrin protects rats against
cerebral ischemic damage through soluble epoxide hydrolase and epoxyei-
cosatrienoic acids. Chin J Nat Med 2013;11(3):207–13.
[12] Gao M, Zhu SY, Tan CB, Xu B, Zhang WC, Du GH. Pinocembrin protects the
neurovascular unit by reducing inflammation and extracellular proteolysis in
MCAO rats. J Asian Nat Prod Res 2010;12(5):407–18.
[13] Punvittayagul C, Pompimon W, Wanibuchi H, Fukushima S, Wongpoomchai R.
Effects of pinocembrin on the initiation and promotion stages of rat hepato-
carcinogenesis. Asian Pac J Cancer Prev 2012;13(5):2257–61.
[14] Lowry OH, Rosebrough NJ, Farr AL. Protein measurement with the Folin phenol
reagent. J Biol Chem 1951;193(1):265–75.
[15] Lorentz K, Klauke R, Schmidt E. Recommendation for the determination of the
catalytic concentration of lactate dehydrogenase at 37 degrees C. Standardi-
zation Committee of the German Society for Clinical Chemistry, Enzyme
Working Group of the German Society for Clinical Chemistry. Eur J Clin Chem
Clin Biochem 1993;31(12):897–9.
[16] Mihara M, Uchiyama M. Determination of malonaldehyde precursor in tissues
by thiobarbituric acid test. Anal Biochem 1978;86(1):271–8.
[17] Beutler E, Duron O, Kelly BM. Improved method for the determination of blood
glutathione. J Lab Clin Med 1963;61:882–8.
[18] Miranda KM, Espey MG, Wink DA. A rapid, simple spectrophotometric method
for simultaneous detection of nitrate and nitrite. Nitric Oxide 2001;5(1):62–71.
[19] Bradley PP, Christensen RD, Rothstein G. Cellular and extracellular myeloper-
oxidase in pyogenic inflammation. Blood 1982;60(3):618–22.
[20] Schultz J, Kaminker K. Myeloperoxidase of the leucocyte of normal human
blood. I. Content and localization. Arch Biochem Biophys 1962;96:465–7.
[21] Heinrikson RL, Meredith SC. Amino acid analysis by reverse-phase high-
performance liquid chromatography: precolumn derivatization with pheny-
lisothiocyanate. Anal Biochem 1984;136(1):65–74.
[22] Malik ZA, Singh M, Sharma PL. Neuroprotective effect of Momordica charantia
in global cerebral ischemia and reperfusion induced neuronal damage in
diabetic mice. J Ethnopharmacol 2011;133(2):729–34.
[23] Rehni AK, Bhateja P, Singh N, Jaggi AS. Implication of mast cell degranulation in
ischemic preconditioning-induced prevention of cerebral injury. Fundam Clin
Pharmacol 2008;22(2):179–88.
[24] Murin R, Drgova A, Kaplan P, Dobrota D, Lehotsky J. Ischemia/reperfusion-
induced oxidative stress causes structural changes of brain membrane pro-
teins and lipids. Gen Physiol Biophys 2001;20(4):431–8.
[25] Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA. Apparent hy-
droxyl radical production by peroxynitrite: implications for endothelial injury
from nitric oxide and superoxide. Proc Natl Acad Sci U S A 1990;87(4):1620–4.
[26] Meldrum DR, Dinarello CA, Cleveland Jr JC, Cain BS, Shames BD, Meng X, et al.
Hydrogen peroxide induces tumor necrosis factor alpha-mediated cardiac
injury by a P38 mitogen-activated protein kinase-dependent mechanism.
Surgery 1998;124(2):291–6.
[27] Jamilloux Y, Seve P, Henry T. Inflammasomes in human diseases. Rev Med
Interne 2014 [Epub ahead of print].
[28] Pan Y, Chen XY, Zhang QY, Kong LD. Microglial NLRP3 inflammasome activa-
tion mediates IL-1beta-related inflammation in prefrontal cortex of depressive
rats. Brain Behav Immun 2014 [Epub ahead of print].
[29] Soromou LW, Chu X, Jiang L, Wei M, Huo M, Chen N, et al. In vitro and in vivo
protection provided by pinocembrin against lipopolysaccharide-induced in-
flammatory responses. Int Immunopharmacol 2012;14(1):66–74.
[30] Kosieradzki M, Pratschke J, Kupiec-Weglinski J, Rowinski W. Ischemia/reperfu-
sion injury, its mechanisms, and prevention. J Transplant 2012;2012:610370.
122 M.A. Saad et al. / Pharmacological Reports 67 (2015) 115–122
[31] Fulda S, Gorman AM, Hori O, Samali A. Cellular stress responses: cell survival
and cell death. Int J Cell Biol 2010;2010:214074.
[32] Hotchkiss RS, Strasser A, McDunn JE, Swanson PE. Cell death. N Engl J Med
2009;361(16):1570–83.
[33] Gupta S, Kass GE, Szegezdi E, Joseph B. The mitochondrial death pathway: a
promising therapeutic target in diseases. J Cell Mol Med 2009;13(6):1004–33.
[34] Broughton BR, Reutens DC, Sobey CG. Apoptotic mechanisms after cerebral
ischemia. Stroke 2009;40(5):e331–9.
[35] Love S. Apoptosis and brain ischaemia. Prog Neuropsychopharmacol Biol
Psychiatry 2003;27(2):267–82.
[36] Kernagis DN, Laskowitz DT. Evolving role of biomarkers in acute cerebrovas-
cular disease. Ann Neurol 2012;71(3):289–303.
[37] Mehta A, Prabhakar M, Kumar P, Deshmukh R, Sharma PL. Excitotoxicity:
bridge to various triggers in neurodegenerative disorders. Eur J Pharmacol
2013;698(1–3):6–18.
[38] Rane MJ, Gozal D, Butt W, Gozal E, Pierce Jr WM, Guo SZ, et al. Gamma-amino
butyric acid Type B receptors stimulate neutrophil chemotaxis during ische-
mia–reperfusion. J Immunol 2005;174(11):7242–9.
[39] Liu J, Wang LN. Gamma aminobutyric acid (GABA) receptor agonists for acute
stroke. Cochrane Database Syst Rev 2013;2:CD009622.
[40] Duan YF, Liu C, Zhao YF, Duan WM, Zhao LR. Thiopental exaggerates
ischemic brain damage and neurological deficits after experimental
stroke in spontaneously hypertensive rats. Brain Res 2009;1294:
176–82.