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European Journal of Pharmaceutics and Biopharmaceutics 85 (2013) 339345

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European Journal of Pharmaceutics and Biopharmaceutics


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

Research paper

Curcumin loaded solid lipid nanoparticles: An efcient formulation


approach for cerebral ischemic reperfusion injury in rats
Vandita Kakkar a, Sravan Kumar Muppu b, Kanwaljit Chopra b, Indu Pal Kaur a,
a
b

Department of Pharmaceutics, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India
Department of Pharmacology, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India

a r t i c l e

i n f o

Article history:
Received 24 December 2012
Accepted in revised form 11 February 2013
Available online 27 February 2013
Keywords:
Bioavailability
Cerebral ischemia
Curcumin
Oxidative/nitosative stress
Solid lipid nanoparticles

a b s t r a c t
Scope: To evaluate curcumin loaded solid lipid nanoparticles (C-SLNs) in the experimental paradigm of
cerebral ischemia (BCCAO model) in rats.
Methods and results: Oral administration of free curcumin and C-SLNs (25 and 50 mg/kg) was started
5 days prior and continued for 3 days after BCCAO. Alleviation in behavioral, oxidative and nitrosative
stress, acetylcholinesterase, mitochondrial enzyme complexes, and physiological parameters were
assessed. Conrmation of effective brain delivery of C-SLNs (p.o) was done using biodistribution studies
in mice and confocal microscopy of rat brain section.
There was an improvement of 90% in cognition and 52% inhibition of acetylcholinesterase versus cerebral
ischemic group (I/R). Neurological scoring improved by 79%. Levels of superoxide dismutase, catalase,
glutathione, and mitochondrial complex enzyme activities were signicantly increased, while lipid peroxidation, nitrite, and acetylcholinesterase levels decreased (p < 0.05) after C-SLNs administration. It is
noteworthy to report the restoration of SOD, GSH, catalase, and mitochondrial complex enzyme levels
equivalent to sham control values. Gamma-scintigraphic studies show 16.4 and 30 times improvement
in brain bioavailability (AUC) upon oral and i.v administration of C-SLNs versus solubilized curcumin
(C-S).
Conclusions: Study indicates protective role of curcumin against cerebral ischemic insult; provided it is
packaged suitably for improved brain delivery.
2013 Elsevier B.V. All rights reserved.

1. Introduction
Several components of reactive oxygen species (ROS) are
involved in ischemic reperfusion (I/R) injury and associated
neuronal loss. Presence of polyunsaturated rich fatty acid sites
which are highly prone to free radical induced peroxidation and
a low content of protective antioxidant enzyme content make the

Abbreviations: AChE, acetylcholinesterase; BCCAO, bilateral common carotid


artery occlusion; BA, bioavailability; BSLN-I/R, blank SLNs; BBB, bloodbrainbarrier; C-S, Curcumin solubilized in Tween 80; C-SLNs, curcumin loaded solid lipid
nanoparticles; free curcumin, curcumin suspended in 1% sodium carboxy methyl
cellulose; EPM, elevalted plus maze; free curcumin, 25 mg/kg and 50 mg/kg, CUR
25-I/R and CUR 50-I/R, respectively; GCI, global cerebral ischemia; iNOS, inducible
nitric oxide synthase; ITL, initial transfer latency; I/R, ischemic reperfusion; LPO,
lipid peroxidation; MDA, malondialdehyde; MWM, Morris water maze; NO, nitric
oxide; ROS, reactive oxygen species; RTL, retention transfer latency; GSH, reduced
glutathione; SOD, cytosolic superoxide dismutase; CAT, catalase activity; NO, nitric
oxide; SLNs, solid lipid nanoparticles.
Corresponding author. Department of Pharmaceutics, University Institute of
Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India. Tel.: +91
172 2534191; fax: +91 172 2543101.
E-mail address: indupalkaur@yahoo.com (I.P. Kaur).
0939-6411/$ - see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejpb.2013.02.005

brain tissue highly prone to the oxidative damage [1]. Inducible nitric oxide synthase (iNOS) generated after I/R injury, results in
excessive nitric oxide (NO) production leading to neuronal loss.
Ischemia is caused by nitric oxide release from vascular endothelium in order to limit the induced damage by improved local blood
ow [2]. Restoration of the blood ow triggers a chain of negative
consequences post-free oxygen radical generation. This includes
reaction of superoxide and hydroxyl radical with unsaturated lipids of biomembranes, resulting in the generation of lipid peroxide
radical and fragmentation products such as malondialdehyde
(MDA) [3]. Ozkul et al. [4] report on the negative correlation of
these oxidative stress parameters (MDA & NO) with clinical outcomes in stroke patients using Canadian Neurological scale scores.
The enzyme, acetylcholinesterase (AChE), is one of the major components of the cholinergic system in the central and peripheral
nervous system. AChE hydrolyzes acetylcholine, and this plays a vital role in terminating neurotransmission at cholinergic synapses.
Interestingly, the neuronal injury associated with ischemia is
accompanied by changes in the cholinergic system [5,6]. AChE
and pro-inammatory cytokines are increased during cerebral
ischemia in rat brains. Many AChE inhibitors have been reported

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to show anti-inammatory and neuroprotective effects [7,8]. In


rats, hippocampal damage associated with cerebral ischemia is
also accompanied by impairment in learning and memory [911].
Antioxidants (tempol, LY231617, LY178002, and U-101033E)
are reported to reduce various effects associated with I/R injury
[12]. Natural polyphenolics like curcumin show potent antioxidant
and anti-inammatory effect and thus may have a role in cerebral
ischemia [13,14]. Although the exact mechanism of such activities
needs complete elucidation, the antioxidative property seems to
underlie pleiotropic biological activity of curcumin.
Effectiveness of curcumin has been established in a wide variety
of human diseases [15] but is yet to be approved as a therapeutic
agent, due to its poor bioavailability (BA). Later is attributed to its
poor absorption and stability at physiological pH, rapid metabolism,
and rapid systemic elimination. To overcome its poor pharmacokinetics and develop it for neuronal disorders, we propose the use of
curcumin loaded solid lipid nanoparticles (C-SLNs) [1618]. SLNs
are lipidic nanoparticles with an ability to penetrate the blood
brain-barrier (BBB) and a potential for CNS disorders [19]. We have
earlier reported on 32155 times enhancement of systemic bioavailability of curcumin from developed C-SLNs in our lab [20,21] along
with its anticancer activity in in vitro cancer cell lines [22].
In the present study, we evaluated the neuroprotective potential of C-SLNs in bilateral common carotid artery occlusion
(BCCAO) induced global cerebral ischemia (GCI) in rats by achieving an improved systemic and cerebral BA of curcumin. Curcumin
is reported to protect rat myocardium against ischemic insult, and
the protective effect is attributed to its antioxidant properties and
inhibition of xanthine dehydrogenase/oxidase conversion and
resultant superoxide anion production [23].
2. Materials and methods
2.1. Animals
Male Wistar rats (250300 g) bred at Central Animal House
(CAH) of Panjab University, Chandigarh, India, were used for the
study. Animals were housed under standard (25 2 C, 6070%
humidity) laboratory conditions on 12 h dark and light cycle. Standard rat chow pellets and water were allowed ad libitum. Animals
were acclimatized to laboratory conditions before the test. All
behavioral experiments were carried out between 10.00 and
17.00 h. The experimental protocols were approved by the Institutional Animal Ethical Committee (IAEC) and conducted according
to the guidelines of Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA).
2.2. Drugs
Curcumin (95% curcumin and 5% of methoxycurcumin and bis
methoxycurcumin as the other two curcuminoids) was a gift from
Sanat Products Limited, Delhi, India. Soy Lecithin (Hi Media, India);
Tween 80 (S.D. Fine Chemicals Ltd., India); Compritol 888 ATO
(Glyceryl Behenate, gift sample from Gattefosse, USA) were also
used in the study.
All other chemicals and reagents were of analytical grade and
were used without further purication.
2.3. Preparation of C-SLNs
Polysorbate 80 (45.45%), soy lecithin (0.58%), and water were
placed together in a beaker and heated to the lipid melt temperature. Lipid (7.27%) on the other hand was also melted (8285 C).
Curcumin was added to the aqueous phase containing polysorbate
80, following which the hot aqueous emulsier mix was dropped

at once into the lipid melt, under magnetic stirring to obtain a clear
microemulsion. The hot microemulsion thus formed was transferred into an equivalent amount of cold water (2 C) under continuous mechanical stirring (5000 rpm) for 1.5 h. In the aqueous
medium, SLNs are formed by crystallization of the oil droplets
present in the microemulsions [21]. The prepared SLNs were stored
in refrigerator until further analysis.
All other chemicals were of analytical grade or reagent grade
purchased from commercial suppliers.
2.4. Radiolabelling and biodistribution study
The formulations [C-SLNs and C-S (Curcumin solubilized in
Tween 80)] were radiolabelled with 99mTc as per standard procedure [24] and administered orally using a dosing cannula to mice.
Animals were sacriced 4 h post-administration, after collecting
blood by cardiac puncture and their brains harvested. The radioactivity was expressed as% administered dose/gram organ.
2.5. Confocal microscopy
Envisaging the uorescent nature of curcumin, brain of animals
administered, C-S, and C-SLNs (50 mg/kg) were harvested, 1 h
post-administration and stored at 80 C for 3 days, before obtaining cryosections, which were observed under confocal laser scanning microscopy CLSM (A1R-Objective type CFI PLAN APO VC,
Nikon, Japan at 400 magnication).
2.6. Induction of global cerebral ischemia
Global cerebral ischemia/reperfusion was induced as modied
method of Jingtao [25] with slight modications. Briey, the rats
were anaesthetized using sodium thiopental (45 mg/kg, i.p.). Both
common carotid arteries were exposed over a midline incision
and dissection on ventral side of the neck between the sternocleidomastoid and the stern hyoid muscles parallel to the trachea. Trachea was exposed followed by the left and right common carotid
artery. The left and right common carotid arteries were freed from
all muscles, ligaments, its adventitial sheath, and vagus nerve was
carefully separated and maintained. The induction of ischemia was
performed by occluding bilateral common carotid arteries (BCCAO)
with clamps for 10 min followed by 72 h reperfusion, and the skin
was closed with stitches using waxed silk suture. During the
BCCAO, animals were observed for, maintenance of dilated pupils,
absence of a cornea reex on exposure to light and maintenance of
rectal temperature at 37 0.5 C. Development of hypothermia
was prevented using a heating lamp on the surgery table. Animals
which did not match these criteria and showed seizures were excluded from study. Sham control animals underwent surgery,
without BCCAO. After 4 and 12 h of BCCAO, 1 ml of sterile 5% dextrose solution was administered to all the rats. After the completion of reperfusion period, animals were assessed for neurological
outcome after 24 h. Unless otherwise stated, for all the parameters,
rats were killed and the brains were isolated after 72 h of
reperfusion.
2.7. Treatment groups
A total of 96 rats were used in the study. Curcumin suspended
in 1% sodium carboxy methyl cellulose (free curcumin) and C-SLNs
administered by oral gavage were investigated for their protective
potential against BCCAO. Group I sham operated rats (Sham control); Group II rats subjected to ischemia and reperfusion (I/R Control); Groups III and IV comprised of I/R rats receiving free
curcumin (25 mg/kg and 50 mg/kg; CUR 25-I/R and CUR 50-I/R,
respectively); Group V received blank SLNs (BSLN/SLN 0-I/R),

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Groups VI and VII constituted I/R rats receiving C-SLNs (25 mg/kg
and 50 mg/kg; SLN 25-I/R and SLN 50-I/R, respectively). Rats were
pretreated for 5 days before BCCAO, and the treatment was continued for another 3 days. BSLN/SLN0 were evaluated for any inherent
effects of constituents of SLNs. All behavioral, biochemical, and
mitochondrial studies were conducted for all the groups to which
animals were assigned randomly.
2.8. Physiological parameters
Daily recordings of body weight and rectal temperature using
Telethermometer (Century Pvt. Ltd., India) were done throughout
the study. Change in body weight was calculated in comparison
with the pretreatment body weight.
2.9. Behavioral and biochemical assessment
Neurological evaluation was carried out to verify successful
BCCAO on an eight point behavioral rating scale [26], and onset
of cerebral ischemia was established. After this animals were
tested in a spatial version of Morris water maze (MWM) test
[27]. A probe trial was performed on the eighth day [28] where
in the extent of memory consolidation was assessed. Memory
acquisition and retention were also tested using elevated plus
maze (EPM) test on days 7 and 8 [29]. The retention trial latency
was expressed as percentage of initial trial latency.
The locomotor activity of BCCAO rats was monitored using
Actophotometer (IMCORP, India). It was placed in a darkened, light,
and sound attenuated and ventilated testing room and performed
as per the literature [30]. Memory evaluation was complemented
by assessment of acetylcholinesterase activity according to the
method of Ellman and Courtney [31].
2.10. Estimation of lipid peroxidation, reduced glutathione, superoxide
dismutase and catalase
Oxidativenitrosative stress parameters were determined in the
brain post-mitochondrial supernatant, prepared by centrifuging rat
brain homogenates in chilled phosphate buffer; pH 7.4 at 10,500g
for 20 min at 4 C. The malondialdehyde content (MDA), a measure
of lipid peroxidation, was assayed in the form of thiobarbituric
acid-reactive substances by the method of Wills [32]. Reduced glutathione (GSH) was assayed by the method of Jollow et al. [33]
while cytosolic superoxide dismutase (SOD) and catalase activity
(CAT) was assayed by the method of Kono [34] and Claiborne
[35], respectively.
2.11. Nitrite estimation
Nitrite was estimated using the Greiss reagent and served as an
indicator of nitric oxide (NO) production [36]. Nitrite concentration
was calculated using a standard curve for sodium nitrite. Nitrite
levels were expressed as percentage of control.
2.12. Mitochondrial complex estimation
Rat brain mitochondria were isolated by the method of Berman and Hastings [37]. Complex I (NADH dehydrogenase activity)
was measured spectrophotometrically by the method of King and
Howard [38]. The method involves catalytic oxidation of NADH to
NAD+ with subsequent reduction in cytochrome c. The reaction
mixture contained 0.2 M glycylglycine buffer pH 8.5, 6 mM NADH
in 2 mM glycylglycine buffer, and 10.5 mM cytochrome C. The
reaction was initiated by addition of requisite amount of solubilized mitochondrial sample and followed absorbance change at
550 nm for 2 min.

Complex II (Succinate dehydrogenase (SDH) activity) was measured spectrophotometrically according to King and Howard [39].
The method involves oxidation of succinate by an articial electron
acceptor, potassium ferricyanide. Complex III was estimated using
MTT assay which is an indirect method to measure its activity.
MTT, a pale yellow substrate, produces a purple product when
incubated with living cells, and the number of viable cells/well is
directly proportional to the intensity of the colored product
formed, which is measured using ELISA [39]. Cytochrome oxidase
activity was assayed in brain mitochondria according to the method of Sottocasa et al. [40].
2.13. Statistical analysis
All data were analyzed via one-way analysis of variance (ANOVA) using SigmaStat 2.0 software (Jandel Scientic); Data presented are mean SEM (n = 6). A value of p < 0.05 was considered
signicant.
3. Results
Total drug content and entrapment efciency of C-SLNs with an
average particle size of 134.6 15.4 nm (Mastersizer 2000, Malvern Instruments, UK) were 92.33 1.63% and 81.92 2.91%
(n = 6), respectively. The in vitro release was predominantly by diffusion phenomenon and was prolonged up to 7 days [21]. Ratio of
area under the curves (AUC) after 4 h of oral administration of CSLNs/C-S was 8.135 in blood and 16.4 in brain. On i.v. administration, the AUCbrain for C-SLNs was 30.82 times that of C-S (Table 1).
Direct conrmation of the targeting of C-SLNs to brain was further
obtained by CLSM. Cryosections of brain of animals administered
C-SLNs revealed the presence of uorescent particles (Fig. 1).
I/R group animals underwent weight loss and hyperthermia.
C-SLNs treatment signicantly increased the body weight and restored body temperature to normal, while free curcumin did not
show any effect. I/R injury resulted in a signicant increase in neurological decit and a decrease in both ambulatory and rearing
activity coupled with decreased muscle grip strength in comparison with sham control. High neurological score of 5.0 in I/R group
(Table 2) conrms onset of BCCAO related changes. Free curcumin
did not show any effect, while C-SLNs signicantly restored all the
activities (Table 2). A signicant increase in initial transfer latency
(ITL) and retention transfer latency (RTL) in the EPM was observed
in I/R rats. C-SLNs treatment signicantly attenuated, while free
curcumin did not show any effect (Table 2). Animals treated with
C-SLNs showed a signicant improvement in memory consolidation in MWM when compared to I/R and curcumin treated rats.
The time spent in the target quadrant was signicantly lower in
I/R animals as compared to the sham control group (Table 2). This
was complemented with increased acetylcholinesterase activity in
I/R rats. Later did not change on free curcumin treatment, while CSLNs signicantly reversed the rise (Fig. 2A).
I/R injury caused signicant oxidative damage as indicated by
increase in LPO and nitrite concentration, as well as depletion of
GSH, CAT, and SOD activity in brain homogenates. Curcumin

Table 1
Biodistribution of radiolabelled C-SLNs and C-S expressed in terms of ratios of their
area under curves (AUC).
AUC

Ratio (AUCC-SLNs/AUCC-S)

Blood

8.135
16.4
0.686
30.82

AUCOral
Brain
AUCOral
Blood
AUCI.V
Brain
AUCI.V

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Fig. 1. Confocal micrograph of rat brain cryosection 1 h after p.o administration of


C-SLNs at 400. Adapted from own research work Kakkar et al., 2011 [43]. (For
interpretation of the references to color in this gure legend, the reader is referred
to the web version of this article.)

treatment did not show any signicant effect on any of these oxidativenitrosative stress parameters, while C-SLNs signicantly restored the later (Table 3) to sham control values.
Furthermore, I/R injury was also found to signicantly impair
mitochondrial enzyme complex I, II and IV activity in rat brains.
CUR did not show any effect, while C-SLNs signicantly ameliorated their activity. A small insignicant decrease in complex III
activity in I/R rats was not affected by CUR or C-SLNs treatment
(Fig. 2B).
4. Discussion
Pluripharmacology of curcumin makes it a leading phytochemical being looked up by researchers for its potential in prevention
of human diseases. However, a proof for its in vivo clinical effectiveness is still lacking. Presently, we prepared and evaluated CSLNs in I/R injury in rats. BloodAUCoral values for C-SLNs were
8.135 times greater than that for C-S, conrming a prolonged circulation of former. The ratio of Blood AUCi.v. C-SLN/C-S in blood is <1
while the ratio in brain very promisingly indicates 30 times higher
and preferential distribution of curcumin from C-SLNs into brain.
Similar observations were also made for oral administration. To
our knowledge, this is the rst study reporting an enhanced distribution of curcumin by 30 times to brain by using a suitable delivery system. Improved levels in the brain may be accredited to the
small average particle size of C-SLNs which bypass the liver rst

pass metabolism a major site of curcumin degradation. Free curcumin undergoes signicant metabolic transformation resulting in a
poor oral bioavailability of <1%. Use of surfactants such as Tween
80 and lecithin, in the preparation of SLNs contribute toward their
increased permeability across the intestinal membrane, a high
afnity between lipid particles and intestinal membrane, may also
improve bioadhesion to the GI tract wall. Curcumin embedded
within solid lipid matrix of SLNs is thus not only protected against
enzymatic degradation during absorption, but also attains a long
circulation time and a reduced clearance in vivo following absorption. Furthermore, a passive uptake of these small SLNs by pinocytosis and endocytosis and an active uptake by brain endothelial
cells are purported. Later is attributable to the adsorption of circulating plasma apolipoprotein E onto the surface of the SLNs (due to
the presence of hydrophilic polysorbate 80 in the outer coat) which
are then selectively up taken by APO E receptors present at the
BBB. Although studies by other researchers [41,42] working on
the BA enhancement of curcumin indicate a 23 times increase
in its plasma concentration, we report an increase of 32155 times
[20,21].
Conrmation of delivery of C-SLNs to brain was obtained by
CLSM of brain cryosection which revealed the presence of uorescent nanoparticles [43] (Fig. 1).
A decrease in body weight and increase in rectal temperature
has been reported after ischemia reperfusion. Gracia and Liu [44]
reported that such effects are due to the change in feeding
behavior and injury to the anterior hypothalamus. C-SLNs treatment restored normal body temperatures and a signicant increase in BW indicating protection against I/R-induced damage to
hypothalamus.
In the present study, memory consolidation task in the MWM
task demonstrates cognitive function. In probe trial, the time
spent in target quadrant was signicantly decreased in I/R injury
rats as compared to sham control. Administration of C-SLNs signicantly increased the time spent in target quadrant indicating
intact memory in treated rats, while no such effects were observed in CUR group. The results from EPM further substantiated
the ndings of MWM test as C-SLNs reduced the increased% ITL of
I/R injury rats. Acetylcholine, a neurotransmitter associated with
learning and memory, is degraded by the enzyme acetylcholinesterase, terminating the physiological action of the neurotransmitter. In addition to their role in cholinergic transmission,
cholinesterases may also play a role in morphogenesis and neurodegenerative diseases. In the present study, the acetylcholinesterase activity was signicantly increased in I/R injury rats as
compared to sham control. This increase in acetylcholinesterase
activity leads to a diminished cholinergic transmission leading
to cognitive decits. C-SLNs treatment signicantly inhibited
AChE activity in I/R injury rats.

Table 2
Effect of free curcumin and C-SLNs on neurological scoring, elevated plus maze (EPM); locomotor activity; rota rod (RR) activity; and Morris water maze (MWM) in I/R injury rats
in seconds.
Groups

Neurological scoring (17)

EPM [RTL (s)]

Mean locomotor activity (s)

RR [ITL (s)]

MWM (TSTQ)

Sham control
I/R control
CUR 25
CUR 50
B SLN
SLN 25
SLN 50

1.2 0.12
5.4 0.75
5.2 0.68
5.1 0.75
5.2 0.43
2.4 0.37
2.2 0.25

42.42 1.37
96.32 6.42a
87.09 2.89
74.65 2.08
89.69 0.48a
57.61 2.41b
48.41 2.69b

392.67 10.93
176.20 9.30a
198.75 10.75
239 5.96
221.00 5.71a
290.67 15.57b
323.5 10.97b

85.00 1.37
23.00 6.42a
27.00 2.89
29.00 2.08
25.00 0.48a
67.00 2.41b
71.00 2.69b

68.33 0.88
17.20 1.32
20.75 1.18
23.67 0.88
24.67 0.88
60.00 6.24
63 0.88

Note: In I/R all the values were signicantly different from those of the sham control at ap < 0.05. All the values of the treatment groups were compared with I/R group and
those found to be signicantly different at p < 0.05 are marked as.b
RTL retention transfer latency (s).
ITL Initial transfer latency (s).
TSTQ time spent in target quadrant (s).

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Fig. 2. Effect of free curcumin (25 and 50 mg/kg), B SLNs and C-SLNs (25 and 50 mg/kg) on (A) Acetylcholinesterase levels (B) mitochondrial complex activity in I/R injury rats.

Note section Treatment Groups. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)

Table 3
Effect of free curcumin and C-SLNs on LPO, GSH, Nitrite, SOD and CAT levels.
GROUPS

LPO (nmoles/mg protein)

GSH (nmoles/mg protein)

Nitrite (lg/ml)

SOD (units /mg protein)

Catalase (lM of H2+/min/mgprotein)

Sham control
I/R control
CUR 25
CUR 50
B SLN
SLN 25
SLN 50

1.02 0.07
3.47 0.03a
2.95 0.22
2.57 0.13
3.41 0.34a
1.74 0.06b
1.39 0.10 b

20.10 0.0005
7.70 0.0006a
8.80 0.0008
1.06 0.0008
6.9 0.0012a
12.1 0.0007b
15.6 0.0027b

134.17 5.21
278.83 15.77a
280.83 2.45
237.08 11.47
256.39 26.62a
165.42 7.92b
149.72 4.00b

2.736 0.20
0.511 0.04a
0.607 0.03
0.988 0.25
0.688 0.31a
1.929 0.27b
2.150 0.27 b

16.68 1.58
7.70 0.54a
9.20 1.01
10.95 0.31
7.80 1.14a
15.06 2.27b
16.41 0.88 b

Data is expressed as mean S.E.M.


Note: In I/R all the values were signicantly different from those of the sham control at ap < 0.05. All the values of the treatment groups were compared with I/R group and
those found to be signicantly different at p < 0.05 are marked as.b

Oxidative damage and free radicals generation are documented


to increase in the animal brain after cerebral ischemia/reperfusion
injury [45]. The cytoprotective activity of curcumin is reported in
in vitro and in vivo model of cytotoxicity by various research
groups. Curcumin showed protective effect in isoproterenol induced myocardial ischemia in rats [46]. It reduces renal damage
and inammation in renal ischemia reperfusion injury [47]. Luo
and co-workers [48] showed the inhibitory activity of curcumin
over oxidative stress induced activation of AP1 and NF-kB, upregulation of c-fos, c-jun, and phosphorylation of c-jun proteins. Curcumin has been found to inhibit JNK, PKC, iNOS, and COX-2
enzymes [49] and is reported to show a membrane stabilizing
activity [46]. In the present study, ischemic reperfusion signicantly caused oxidative damage as indicated by increase in MDA
levels and nitrite concentration along with marked decrease in
the activity of catalase, SOD, and reduced glutathione in ischemic
brain. Curcumin treatment as solid lipid nanoparticles (C-SLNs)
at the doses of 25 and 50 mg/kg (but not the free curcumin;
CUR) signicantly ameliorated I/R-induced oxidative and nitrosative stress.

Peroxidation of lipid bilayer is reported after cerebral IR injury


[50]. In the present study, 10 min of ischemia and 72 h of reperfusion resulted in elevated levels of MDA in brain. The interaction of
curcumin with the membrane impacts its ability to protect the
body from harmful free radicals in several ways. First, the exact
localization of curcumin in the membrane will affect the availability of curcumin to oxidants and other antioxidants. Experiments
have shown an increased lipid peroxidation in contralateral cortex
(in addition to ipsilateral cortex) in the ischemic rat [51].
Second, the rate of lipid peroxidation is inuenced to a strong
degree by the physical state of the membrane in a way that is
not completely understood. Treatment with C-SLNs (25 and
50 mg/kg) reduced the extent of MDA production. This may be
attributed to a higher permeability of curcumin achieved by CSLNs resulting in a better quenching of oxidants and also by its
membrane stabilizing effects. Curcumin is proposed to modify
the energetic penalty, that is caused by deformation of the membrane, and there by altering the physical properties of the membrane such as the hydrophobic thickness of the membrane, the
elastic modulus, the lateral pressure prole, or other physical

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parameters leading to energetically favorable bilayers and can


therefore have a strong inuence on the conformational equilibria
of membrane proteins [52]. C-SLNs with a polysorbate 80phospholipid coat are expected to intermingle with these membrane lipids resulting in physiologically signicant effects as witnessed in the study.
Substantial evidence in the literature proves the potential of
curcumin and its metabolites to reduce oxidative stress induced lipid peroxidation reaction [53]. It scavenges the components of
reactive oxygen species-superoxide, hydroxyl, singlet oxygen, nitric oxide, and peroxynitrite [46]. The decrease in brain MDA level
in the present study on C-SLNs treatment may be due to antioxidant property of curcumin. During cerebral ischemia, xanthine
dehydrogenase, an enzyme, which is involved in purine metabolism is irreversibly converted to xanthine oxidase by sulfhydryl
cleavage, and this conversion is found to be inhibited by curcumin
[54]. During reperfusion, sudden supply of molecular oxygen
(which serves as a substrate for xanthine oxidase for nucleotide
metabolism) results in the production of heavy burst of superoxide. Xanthine oxidase derived superoxide formation accounts for
major contribution of total superoxide produced during IR injury.
Once superoxide initiates the oxidative burst, hydrogen peroxide,
hydroxyl radical, and peroxynitrite are formed during the catabolism of superoxide resulting in the neurotoxicity of superoxide.
As curcumin is found to have the xanthine oxidase inhibitory activity [53], it can directly prevent the production of superoxide (and
hence the other components of ROS) during cerebral ischemia
reperfusion.
Normally, there is a balance between the ROS production and
the endogenous scavenging system which detoxies the ROS.
During IR, sudden burst of ROS cannot be handled efciently by
endogenous system which protects neurons in the normal conditions. As a result, the highly reactive ROS damages the components (cellular membranes, proteins, and DNA) of neuron.
Accumulation of hydrogen peroxide was reported to impair the
mitochondrial function [54]. Superoxide along with hydroxyl radical produces modication in the primary, secondary, and tertiary
structure and aggregation and/or fragmentation of cellular proteins including SOD and peroxidase [54]. Dysfunction of SOD
and GPx may result in loss of protective activity exerted by these
enzymes, and this may be the reason why we observed a reduction in SOD activity and GSH levels after IR injury. If so, an antioxidant treatment should prevent the loss of SOD activity and
GSH levels by effectively scavenging the excess ROS. In the present study, the reduction in the SOD activity was prevented by CSLNs administration. The increased SOD activity may also be due
to the increased expression of SOD gene product by curcumin
treatment. It has been reported that curcumin up-regulates the
SOD gene expression in rat kidney after ischemia/reperfusion injury [55]. Curcumin treatment also reversed the reduction in the
GSH levels. It is reported that curcumin and its metabolite tetrahydrocurcumin induce glutathione peroxidase [56]. Our results
are in agreement with [55]; they have shown that the chronic
treatment of rats with curcumin increased the activity of SOD
and GPx in liver homogenate.
Superoxide anion produced as a result of IR injury reacts with
nitric oxide to form peroxynitrite. In addition to superoxide, it also
has hydroxyl radical scavenging activity. This would have resulted
in the decreased inactivation of GPx and SOD functionality as well
as decreased membrane destruction by lipid peroxidation. All
these effects put together result in the neuroprotection achieved
by C-SLNs treated ischemic animals. In conclusion, curcumin offered signicant neuroprotection in BCCAO model of global cerebral ischemia. Neuroprotection shown by C-SLNs may be
attributed to inhibition of lipid peroxidation and increase in endogenous antioxidant defense enzymes (Fig. 3).

Fig. 3. Formation of reactive oxygen species as a consequence of cerebral ischemic


reperfusion injury and mechanisms by which curcumin rescues the neurons. (For
interpretation of the references to color in this gure legend, the reader is referred
to the web version of this article.)

Mitochondria are considered to be a major source of ROS in the


cells, which include superoxide anion, hydrogen peroxide and hydroxyl free radical [54]. Decrease in mitochondrial enzyme complex activity is another hallmark of cerebral ischemia and
reperfusion injury which is a major cause of brain cell death. In line
with these ndings, signicant impairment in mitochondrial enzyme complex I (NADH dehydrogenase), complex II (succinate
dehydrogenase) and signicant reduction in cytochrome c oxidase
activity and slight decrease in number of viable cells (MTT assay)
was observed by us in ischemia reperfusion injured animal group.
C-SLNs treatment signicantly restored these mitochondrial enzyme complex activities, except the complex III (MTT assay) suggesting its role in restoration of mitochondrial enzyme complex
activities [57].
This study revealed that C-SLNs with their improved bioavailability are an effective armament against cerebral ischemic insult,
and this effect could be an attribute to enhanced antioxidant potential and/or inhibitory effects on XD/XO conversion and resultant
superoxide anion production, achieved by packaging curcumin into
a suitable carrier system.
Acknowledgments
The facilities provided by Dr. Anil K. Mishra and Technical help
provided by Ms. Krishna Chuttani from Institute of Nuclear Medicine and Allied Sciences, Brig. S.K. Mazumdar Marg, Delhi,
110054, India is highly acknowledged.
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