Effects of Saffron (Crocus sativus L.) and its
Active Constituent, Crocin, on Recognition and
Spatial Memory after Chronic Cerebral
Hypoperfusion in Rats
Hossein Hosseinzadeh,1 Hamid Reza Sadeghnia,2 Fatemeh Abbasi Ghaeni,1
Vahideh Sadat Motamedshariaty1 and Seyed Ahmad Mohajeri1*
1Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
2Department of Pharmacology, Department of New Sciences and Technology, Neuroscience Research Center, School of Medicine,
Mashhad University of Medical Sciences, Mashhad, Iran
Cerebral ischemia produces brain damage and related behavioral deficits such as memory. In this study, a rat
model of chronic cerebral hypoperfusion was used to determine whether saffron extract and crocin, which are
potent antioxidants and free radical scavengers, can reduce vascular cognitive impairment. Male adult Wistar rats
were administered different doses of an aqueous solution of crocin or hydroalcohol extract of saffron intraperitoneally (i.p.) 5days after permanent occlusion of the common carotid arteries. Spatial learning and memory
were assessed in training trials, 7–11days after common carotid artery ligation using the Morris water maze.
The results showed that the escape latency time was significantly reduced from 24.64s in the control group to
8.77 and 10.47s by crocin (25mg/kg) and saffron extract (250mg/kg). The traveled distance to find the platform
was also changed from 772cm in the control group to 251 and 294cm in the crocin (25mg/kg) and saffron extract
(250mg/kg) groups. The percentages of time spent in the target quadrant, in comparison with the control group
(24.16%), increased to 34.25% in the crocin (25mg/kg) and 34.85% in the saffron extract (250mg/kg) group. This
study suggests that saffron extract and crocin improve spatial cognitive abilities following chronic cerebral
hypoperfusion and that these effects may be related to the antioxidant effects of these compounds. Copyright
# 2011 John Wiley & Sons, Ltd.
Keywords: Crocus sativus; crocin; memory; chronic cerebral hypoperfusion; antioxidant.
INTRODUCTION
Vascular dementia is widely considered one of the most
important and common forms of dementia after
Alzheimer’s disease (AD) and some studies indicate that
the incidence and prevalence of the disease increases exponentially after the age of 65 (Murray et al., 2007). Vascular dementia is caused by a cerebrovascular disease
that generally occurs in the elderly. People with vascular
dementia commonly experience a decline in thought
processes (cognitive impairment), caused by cerebrovascular diseases with pathological features of ubiquitous cerebral arteriosclerosis and infarction (He et al.,
2008). Due to the increasing number of elderly people
in the world, dementia, which is known by the progressive loss of memory and cortical functions, has given rise
to enormous socioeconomic problems (Liao et al., 2004).
It has been proved that vascular dementia is related to
ischemia, hypoxia or hemorrhagic damage to specific
corresponding regions involved in cognition and memory (Decarli, 2004).
Little is known about the role of antioxidants in the
pathogenesis of vascular dementia and its associated
neuronal damage. Increasing the free radical formation, together with reducing the antioxidant defense,
may cause neuronal injury. A low concentration of
antioxidants may influence the development of vascular
dementia. In a study by Ryglewicz et al., low levels of
plasma alpha-tocopherol were observed in patients with
vascular dementia indicating a reduced antioxidant
defense in these subjects (Ryglewicz et al., 2002).
Saffron is the dried stigmas of Crocus sativus and one of
the most expensive spices. It could be used as a drug,
textile dye and culinary adjunct (Mohajeri et al., 2010).
Chemical analysis of its stigmas has indicated the presence
of crocin (Fig. 1) as a water-soluble carotenoid, monoterpene aldehyde and its glucoside (safranal and picrocrocin)
and flavonoids (quercetin and kaempferol) (Pitsikas et al.,
2007). Saffron is commonly cultivated in Iran, Spain,
India, Switzerland, Italy and other countries (Hadizadeh
et al., 2010). The saffron and its colored carotenoid (crocin) have anticonvulsant (Hosseinzadeh and Talebzadeh,
2005; Hosseinzadeh and Sadeghnia, 2007), antioxidant
(Hosseinzadeh and Sadeghnia, 2005; Hosseinzadeh
et al., 2005b; Ochiai et al., 2007; Hosseinzadeh et al.,
2009; Goyal et al., 2010; Mousavi et al., 2010), antitumor
(Molnar et al., 2000) and antidepressant (Akhondzadeh
et al., 2004; Hosseinzadeh et al., 2004; Moshiri et al.,
2006) effects. It also has memory improving properties
(Pitsikas et al., 2007). Zhang et al. indicated that saffron
and its colored components reduced ethanol-induced
* Correspondence to: Assistant Professor Seyed Ahmad Mohajeri,
Pharmaceutical Research Center, School of Pharmacy, Mashhad University
of Medical Sciences, Mashhad, Iran.
E-mail: mohajeria@mums.ac.ir
PHYTOTHERAPY RESEARCH
Phytother. Res. 26: 381–386 (2012)
Published online 19 July 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/ptr.3566
Copyright # 2011 John Wiley & Sons, Ltd.
Received 15 March 2011
Revised 23 April 2011
Accepted 06 May 2011
memory impairment in the passive avoidance test in
mice (Zhang et al., 1994). Saffron extract counteracted
recognition memory deficits and antagonized scopolamine-induced performance impairments in the passive
avoidance task in the rat (Pitsikas and Sakellaridis,
2006). In a study by Pitsikas et al., the efficiency of
crocin to counteract scopolamine-induced detrimental effects on spatial memory was reported (Pitsikas
et al., 2007). In this study, the vascular dementia model
was produced by permanent bilateral ligation of the
common carotid arteries, which can induce hypoperfusion and spatial memory impairment (Guang and Du,
2006; He et al., 2008). Our aim was to determine
whether saffron or its main constituents, crocin, as antioxidants have beneficial effects in the cerebral ischemia
model in rats.
MATERIALS AND METHODS
Chemicals. The total saffron extract (Saharkhiz Saffron
Co.) and crocin were isolated from the red dried stigmas
(saffron) of Crocus sativus, as described previously
(Hadizadeh et al., 2010). Briefly, saffron stigma powder
(10g) was suspended in 25mL ethanol 80% at 0
C and
shaken by vortex for 2min. After centrifugation at
4000rpm for 10min, the supernatant was separated.
Then 25mL of 80% ethanol was added to the sediment
and the extraction was repeated again. This step was
repeated six more times. The total volume of solvent
used for 10g saffron stigmas in the extraction process
was 200mL (825mL). For preparation of the total
hydroalcohol extract of saffron, the resulting solution
was dried in a rotary evaporator system in darkness at
35
C. For preparation of crocin (Hadizadeh et al.,
2010), the resulting solution was kept in a thick walled
glass container at 5
C for 24days in darkness. The
container was sealed during this period. The obtained
crystals were separated from the solution and washed
with acetone to remove the remaining water. The purity
of the crocin crystals was tested with HPLC and was
more than 97%. The total amount of crocin in the saffron extract was determined as 10–15%. The saffron extract and crocin were dissolved in normal saline (0.9%)
before injection. Xylazine and ketamine were obtained
from Loughrea, Co. (Galway, Ireland) and Rotexmedica
GmbH (Germany), respectively.
Animals. Male Wistar rats weighing 200–230g were
obtained from the animal facilities of the Pharmaceutical Research Center, BuAli Research Institute of
Mashhad University of Medical Sciences. The animals
were housed five per cage with a 12/12h light/dark cycle
at 212
C and had free access to food and water.
About 70 rats were divided into eight groups: (1)
sham-operated animals underwent the same surgical
procedure without ligation of the common carotid arteries (n=14); (2) control group received 0.9% saline solution (n=14); (3), (4) and (5) saffron groups received
saffron extract (50, 100 and 250mg/kg/day, n=7); (6),
(7) and (8) crocin groups received crocin (5, 10, 25mg/
kg/day, n=7). Handling and experimental procedures
for all animals were in accordance with the Mashhad
University of Medical Sciences Ethics Committee Acts.
Surgery and experimental procedure. The rats were
anesthetized with a mixture of ketamine (60mg/kg)
and xylazine (6mg/kg) (i.p.). The surgical technique
for the induction of cerebral ischemia was adapted from
the earlier published method of Xu et al. (2010), with
some modifications. In the ischemic rats, the left common carotid artery was exposed through a midline neck
incision and double ligated with 4–0 type surgical silk.
After 3days, the right common carotid artery was
ligated in the same way. The sham-operated rats
received the same two-step operation procedure except
the ligatures. During the surgery, their body temperature
was monitored and maintained at 37.50.5
C by means
of a heating lamp. The animals received drugs or vehicle
intraperitoneally 1h after the second step of operation
and the same dosage every day for 5days.
Behavioral assessment. The spatial memory performances were evaluated using a Morris water maze 7
days after induction of hypoperfusion. The water maze
was a black circular tank 136cm in diameter and 60cm
in height. The tank was filled with water (201
C) to a
depthof 35cm.Themazewaslocatedin aroom containing
extra-maze cues. The walls of the pool and the platform
were dyed black to conceal the platform. The maze was
divided geographically into four quadrants (northeast
(NE), northwest (NW), southeast (SE), southwest
(SW)) and starting positions (north (N), south (S), east
(E), west (W)) that were equally spaced around the perimeter of the pool. A hidden platform (diameter: 10cm)
Figure 1. Chemical structure of crocin.
382 H. HOSSEINZADEH ET AL.
Copyright # 2011 John Wiley & Sons, Ltd. Phytother. Res. 26: 381–386 (2012)
was located in the SE quadrant (target quadrant), 1cm
below the surface of the water. A video camera was
mounted directly above the water maze to record the rats’
swim paths. A tracking system was used to measure the
escape latency time, traveled distance and swimming
speed of each rat, and also the percentage of time in the
target quadrant.
The rats were given four training trials each day on 5
consecutive days. For each training trial, the rats were
placed in the water facing the pool wall at one of the
four starting positions (north, south, east or west pole)
in a different order each day and allowed to swim until
they reached the platform located in target quadrant of
the maze in every trial. The latency to reach the platform was recorded for up to 60s. They remained on
the platform for 20s before being removed. The experimenter guided any rat that had failed to reach the platform within 60s to it for 20s and the maximum latency
was scored. One final test trial with the platform
removed was conducted 24h after the last training trial
to assess the memory of the correct platform location.
After the trials, the rat was dried with a towel and placed
in a holding cage under a heating lamp before it was
returned to the home cage (Nakagawa and Takashima,
1997; Hosseinzadeh et al., 2005a).
Statistical analysis. Data are expressed as meanSEM.
The traveled distance, swimming speed of each rat and
the percentage of the time in the target quadrant were
assessed by one-way analysis of variance (ANOVA)
followed by the Tukey’s post hoc test. Data obtained
from the latency time tests were analysed by two-way
ANOVA. A value of p<0.05 was considered statistically
significant.
RESULTS
The mean latency time in finding the hidden platform
decreased during the training period in all groups. The
control group took longer to find the platform than the
sham-operated rats. This prolongation of latency was
shortened by the total saffron extract and crocin. This
effect was dose dependent in the saffron extract and
crocin groups (Fig. 2a and b). In the final test trial, the
swimming time in the target quadrant was used to evaluate the spatial memory performance. The sham-operated group and the saffron and crocin-treated groups
swam longer in the target quadrant than the control
group (Fig. 3a and b). In the final test trial, the percentage of time traveled in the target quadrant in the shamoperated group was 35.66%, in the control group was
24.16%, in the saffron extract (50, 100 and 250mg/kg)
groups this was increased to 25.18%, 32.84% and
34.85% and the data for crocin (5, 10, 25mg/kg) groups
were 30.20%, 33.26% and 34.25%. But only the saffron
250mg/kg and crocin 10 and 25mg/kg groups were
statistically different from the control group (p<0.05).
Figure 4a and b represent the traveled distance to reach
the hidden platform on day 5. The data indicated that in
comparison with the control group, the traveled
distance to find the platform decreased in the saffron
(100 and 250mg/kg) and crocin groups (at all doses)
and these results were dose dependent in the treated
groups. Figure 5 shows the speed of the animals in each
group. The results indicated that no difference was
observed between the groups in 5days.
DISCUSSION
Permanent bilateral occlusion of the common carotid arteries of rats can reproduce cerebral hypoperfusion and
causes spatial memory dysfunction. Thus, this model is
suitable for studying the memory deficits associated with
cerebral circulation impairments (Farkas et al., 2004,
2005; Xu et al., 2010). In our experiment, the escape latency time during trials, the percentage of time spent in
the target quadrant, the traveled distance to reach the
hidden platform on day 5 and the speed of animals in
each group were used to assess acquisition of the water
maze task. The data showed that in comparison with the
control group, crocin (at all doses) and saffron extract
Figure 2. Escape latency time (meanSEM) to reach hidden
platform by crocin (a) and saffron extract (b) treated rats in the
Morris water maze. +++ p<0.001 compared with sham group,
*p<0.05, **p<0.01, ***p<0.001 compared with control group
(n=14, in sham and control group and n=7, in crocin and saffron
extract groups).
MEMORY ENHANCING EFFECT OF SAFFRON AND CROCIN 383
Copyright # 2011 John Wiley & Sons, Ltd. Phytother. Res. 26: 381–386 (2012)
(at 100 and 250mg/kg) could significantly reduce the escape latency time and the traveled distance in finding
the hidden platform. Also, in the final test trial, the percentage of time traveled in the target quadrant, at
higher doses of saffron extract (250mg/kg) and crocin
(10, 25mg/kg) groups, was significantly increased in
comparison with the control group. No significant difference was observed in the swim speed between groups
(Fig. 5). It meant that decreasing the escape latency
was not due to the effect of saffron extract or crocin
on swimming speed. These data indicated that the saffron extract and crocin did not have any effect on motor
ability. Therefore, the present study demonstrates that
saffron extract and crocin improved cognitive deficits
induced by chronic cerebral hypoperfusion in rats. This
neuroprotective effect may be due to the antioxidative
properties of saffron and crocin. Other workers have also
reported that antioxidants could protect against cognitive deficits induced by chronic cerebral hypoperfusion.
Xu et al. indicated that icariin has protective effects on
learning ability and memory in a rat model of chronic
cerebral hypoperfusion. Icariin is an antioxidant and a
major constituent of flavonoids derived from the Chinese
medicinal herb Epimedium brevicornum Maxim (Xu
et al., 2010). In another study, edaravone as a potent
antioxidant and free radical scavenger had protective
effects against white matter lesions and endothelial injury in a rat chronic hypoperfusion model and suggested
that edaravone is potentially useful for the treatment of
cognitive impairment (Ueno et al., 2009). As shown in a
study by Xu et al. (2010), green tea polyphenols, which
are potent antioxidants and free radical scavengers, can attenuate vascular cognitive impairment (Xu et al., 2010).
The ethanol root extract of Pongamia pinnata has also
shown a protective effect in ischemia-reperfusion injury
and long-term hypoperfusion in rats (Raghavendra
et al., 2007). These studies indicate that the antioxidant
effects of these compounds play an important role in
improving the spatial cognitive abilities after chronic
cerebral hypoperfusion.
It has been shown that saffron and crocin are potent
antioxidants (Asdaq and Inamdar, 2010; Ochiai et al.,
2004; Papandreou et al., 2006). They scavenge free
radicals, especially superoxide anions and, thus, may
protect cells from oxidative stress (Abe and Saito,
2000). In a study by Ochiai et al., crocin prevented the
death of rat pheochromocytoma (PC-12) cells by its
antioxidant effects being stronger than those of alphatocopherol (Ochiai et al., 2004). Crocin is likely to prevent ethanol-induced inhibition of hippocampal long
term potentiation (LTP) by antagonizing the inhibitory
effect of ethanol on NMDA receptors, though it is not
Figure 4. Distance swum (mean+SEM) to reach the platform on
day 5 by crocin (a) and saffron extract (b) treated rats in Morris
water maze. +++p<0.001 compared with sham group, *p<
0.05, **p<0.01, ***p<0.001 compared with control group
(n=14, in sham and control group and n=7, in crocin and saffron
extract groups).
Figure 3. Percentage of time spent (mean+SEM) in the target
quadrant of the pool in the final test trial by crocin (a) and saffron
extract (b) treated rats in the Morris water maze. +p<0.05 compared with sham group, *p<0.01, compared with control group
(n=14, in sham and control group and n=7, in crocin and saffron
extract groups).
384 H. HOSSEINZADEH ET AL.
Copyright # 2011 John Wiley & Sons, Ltd. Phytother. Res. 26: 381–386 (2012)
clear whether crocin acts directly on the NMDA receptor channel complex or indirectly modulates NMDA receptor functions (Abe et al., 1998; Abe and Saito, 2000).
Other researchers indicated that saffron extracts significantly antagonized the scopolamine-induced memory
performance deficits (Pitsikas and Sakellaridis, 2006).
Thus, saffron extract and its constituents, especially
crocin, improve the impairment of certain types of
learning and memory through different mechanisms.
Permanent cerebral hypoperfusion produces excess free
radicals (Liao et al., 2004; Liu et al., 2007; Xu et al., 2010)
and reactive oxygen species (ROS) that can damage the
weakened antioxidant defense system of the brain,
thereby inducing neuronal degeneration and death. After
chronic cerebral hypoperfusion, antioxidant capabilities
changed in the cortex and hippocampus (Xu et al.,
2010). Liu et al. (2007) showed elevated superoxide
dismutase (SOD) activity in the cortex and hippocampus
of animals underwent permanent bilateral occlusion of
the common carotid arteries and considered this a compensatory rise in antioxidant activity that indicated the
brain’s antioxidant machinery was activated when overwhelmed by oxidative stress. Huang et al. (2008) also
found reduced antioxidant activity after chronic
cerebral hypoperfusion. Saffron extract and crocin
scavenge free radicals, especially superoxide anions,
and thereby may protect cells from oxidative stress
(Abe and Saito, 2000). Accumulating evidence suggests that cellular stress induced by free radicals is
responsible for a variety of CNS neurodegenerative disorders, including Alzheimer’s disease and Parkinson’s
disease (Abe and Saito, 2000). It is demonstrated that
crocin significantly decreases the formation of peroxidized membrane lipids and restores SOD activity
compared with a-tocopherol activity (Ochiai et al.,
2004). The restoration of SOD activity suggests that
crocin has an important role in modulating antioxidative
effects. Crocin also suppressed the activation of caspase-
8 caused by serum/glucose deprivation (Ochiai et al.,
2004). Mousavi et al. found that saffron extract and
crocin decreased the toxicity of glucose, in PC12 cells,
by reducing the ROS production (Mousavi et al., 2010).
Crocin is the main active constituent and antioxidant
in saffron. Therefore, it may be responsible for the
memory enhancing effect of saffron extract. These
results suggest that saffron and crocin as potent
antioxidants may combat oxidative stress in neurons
and could be useful in the therapy of brain neurodegenerative disorders such as vascular dementia.
Acknowledgements
The authors gratefully acknowledge the Vice Chancellor of Research,
Mashhad University of Medical Sciences for financial support through
grant number 88748. The authors also thank Saharkhis Saffron Co. for
supplying saffron.
Conflict of Interest
The authors have declared no conflict of interest.
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