Journal of Parkinsons Disease 3 (2013) 7783

DOI 10.3233/JPD-130173
IOS Press
77
Research Report
Saffron Pre-Treatment Offers
Neuroprotection to Nigral and Retinal
Dopaminergic Cells of MPTP-Treated mice
Sivaraman Purushothuman
a
, Charith Nandasena
a
, Cassandra L. Peoples
b
, Nabil El Massri
b
,
Daniel M. Johnstone
a
, John Mitrofanis
b,
and Jonathan Stone
a
a
Discipline of Physiology, University of Sydney, Australia
b
Discipline of Anatomy & Histology, University of Sydney, Australia
Abstract.
Background: There is growing evidence that the spice saffron, which contains powerful anti-oxidants, offers protection against
neurodegenerative disorders, including age-related macular degeneration and Alzheimers disease.
Objective: We examined whether saffron pre-treatment protects dopaminergic cells of the substantia nigra pars compacta (SNc)
and retina in an acute MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model of Parkinsons disease.
Methods: BALB/c mice received MPTP or saline injections over a 30 hour period, followed by six days survival. For ve days
prior to injections, the drinking water of the saffron groups was supplemented with saffron (0.01% w/v), while non-saffron
groups received normal tap water. After the survival period was complete, brains were processed for tyrosine hydroxylase (TH)
immunochemistry and the number of TH
+
cells was analysed using the optical fractionator method.
Results: In both the SNc and retina, non-conditioned MPTP-injected mice had a reduced number of TH
+
cells (3035%)
compared to the saline-injected controls. Saffron pre-conditioning mitigated the reduction, with pre-conditioned MPTP-injected
mice having SNc and retinal TH
+
cell numbers close to control levels, signicantly (2535%) higher than in non-conditioned
MPTP-injected mice.
Conclusions: Our results indicated that saffron pre-treatment of mice saved many dopaminergic cells of the SNc and retina from
parkinsonian (MPTP) insult.
Keywords: Tyrosine hydroxylase, substantia nigra, amacrine cell, neuroprotection, Parkinsons disease
ABBREVIATIONS
6OHDA 6 hydroxydopamine
ATP adenosine-5

-triphosphate
MPTP 1-methyl-4-phenyl-1,2,3,
6-tetrahydropyridine
NIr near-infrared light
SAF saffron
SNc substantia nigra pars compacta

Correspondence to: Professor John Mitrofanis, Department of

Anatomy & Histology F13, University of Sydney 2006, Australia.
E-mail: john.mitrofanis@sydney.edu.au.
SNr substantia nigra pars reticulata
TH tyrosine hydroxylase
INTRODUCTION
Parkinsons disease is a severe, progressive break-
down of body movement, its pathology characterised
by a loss of dopaminergic cells within the substantia
nigra pars compacta (SNc) of the midbrain. This loss
triggers a cascade of abnormal basal ganglia circuitries
that manifest in the clinical signs of Parkinsons
disease, namely lead-pipe rigidity, akinesia and tremor.
ISSN 1877-7171/13/$27.50 2013 IOS Press and the authors. All rights reserved
78 S. Purushothuman et al. / Saffron Saves Dopaminergic Cells in MPTP-Treated Mice
The most common treatment, dopamine replacement
therapy, relieves signs but has no major effect against
disease progression, leaving a need for treatments able
to slow progression [1].
The cause of SNc cell loss in Parkinsons disease
is not clear but mitochondrial dysfunction is thought
to be central, as this is the primary mode of action
of Parkinsons disease-related toxins (eg. 1-methyl-
4-phenyl-1,2,3,6-tetrahydropyridine; MPTP). Further,
studies in animal models have reported that various
substances (eg, anti-oxidants) [1] or treatments (eg,
photobiomodulation or deep brain stimulation) [2, 3,
4] that prevent mitochondrial dysfunction are effective
in protecting dopaminergic pathways.
In this context, the neuroprotective effects of dietary
saffron (stigmata of Crocus sativus) have generated
interest recently. In animal models, saffron given prior
to stressful light is protective to retinal photoreceptor
morphology and function [5]; it has been shown also to
regulate many ncRNAs and genes related to membrane
integrity and repair [6]. In clinical trials of age-related
macular degeneration, saffron has been reported to
restore partially macular function and to stabilise the
macula against further degeneration [7]. In addition to
these neuroprotective roles, saffron has been shown to
have anti-inammatory actions, including for exam-
ple the inhibition of tissue necrosis factor [8], and its
bioactive components (crocin, crocetin) bind directly
to genomic DNA and protect against toxin-induced
mutations [9].
In this study, we examined whether dietary saffron
offers neuroprotection to two groups of dopaminergic
cells, namely those in the SNc and retina (amacrine
cells), of MPTP-treated mice (well-known animal
model of Parkinsons disease). We examined two
groups as to determine whether saffron treatment or
MPTP had a differential effect on distinct dopaminer-
gic cell groups in the mouse central nervous system.
MATERIALS AND METHODS
Subjects
Male BALB/c (n =40; 10 wks old) mice were
housed on a 12 h light/dark cycle with unlimited access
to food and water. All experiments were approved by
the Animal Ethics Committee of University of Sydney.
Experimental design
Four groups, each of ten mice, received intraperi-
toneal injections of either MPTP or saline, combined
with either saffron (SAF) pre-treatment (ingestion) or
not. We will refer to the different groups as:
(1) Saline: saline injections with no saffron
(2) Saline-SAF: saline injections with saffron
(3) MPTP: MPTP injections with no saffron
(4) MPTP-SAF: MPTP injections with saffron.
MPTP and saffron treatments
Following our previous work, we used an acute
MPTP mouse model, in which intraperitoneal injec-
tions of the MPTP toxin induced a depletion of
dopaminergic cells in the SNc and in the retina [3,
4]. Briey, we made two MPTP (25 mg/kg injections;
total of 50 mg/kg per mouse) or saline injections over
a 24 hour period. Thereafter, mice were allowed to sur-
vive for six days. Five days prior to the rst MPTP or
saline injections, saffron was introduced into the drink-
ing water of the saffron groups. 10 mg of saffron was
put into 100 ml water overnight, to allow the water
soluble components (including crocin and safranal)
to dissolve. Effectively, the mice drank an aqueous
extract of saffron, each mouse drinking an estimated
2 ml of the extract each day, ingesting the water-soluble
components of 110 mg saffron each day. On the day
of rst injections, the aqueous extract of saffron was
replaced with normal drinking water. The non-saffron
groups drank normal water throughout the experimen-
tal period. This saffron treatment regime was adapted
from that used by previous studies, in particular, those
reporting neuroprotective effects in the central nervous
system [5, 6], in which a measured volume of the
aqueous extract of saffron was delivered to animals
individually, injected into a vegetable matrix, which
the rat ate. Delivery by drinking water relies on the
animals natural thirst, and requires much less handling
of the individual animal. Studies of dose-response rela-
tionships for the neuroprotective action of saffron (Di
Marco et al., manuscript in preparation) indicate the
importance of extending the daily dose of saffron over
510 days, which is achieved easily via drinking water.
Immunocytochemistry
Following the survival period, mice were anaes-
thetised with an intraperitoneal injection of sodium
pentobarbital (60 mg/ml). They were then perfused
transcardially with 0.1 M phosphate-buffered saline
(pH 7.4), followed by 4% buffered paraformaldehyde.
The brains were removed and post-xed overnight
in the same solution. Next, brains were placed in
S. Purushothuman et al. / Saffron Saves Dopaminergic Cells in MPTP-Treated Mice 79
phosphate-buffered saline with the addition of 30%
sucrose until the tissue sank. The midbrain was then
sectionedcoronallyandseriallyusinga freezingmicro-
tome at 50m thickness. All sections were collected
in phosphate-buffered saline and then immersed in a
solution of 1% Triton (Sigma) and 10% normal goat
serum (Sigma) at room temperature for 30 minutes
each. Sections were then incubated in anti-tyrosine
hydroxylase (Sigma; 1:1000) for 48 hours (at 4

C),
followed by biotinylated anti-rabbit IgG (Bioscien-
tic; 1:200) for three hours (at room temperature)
and then streptavidin-peroxidase complex (Bioscien-
tic; 1:200) for two hours (at room temperature). To
visualise the bound antibody, sections were reacted in
a 3,3

- diaminobenzidine tetrahydrochloride (Sigma)

- phosphate-buffered saline solution. Sections were
mounted onto gelatinised slides, air-dried overnight,
dehydrated in ascending alcohols, cleared in His-
toclear and coverslipped using DPX. Most of our
immunostained sections were counterstained lightly
with neutral red. For the retinae, globes were removed
and post-xed for twenty minutes in 4% buffered
paraformaldehyde. Next, retinae were dissected free
from other structures in the globe as wholemounts.
Retinae were processed in same way as described
above, except that the streptavidin-peroxidase complex
was replaced by Extravidin-FITC complex (Sigma;
1:200). Retinae were then mounted onto glass slides,
coverslipped using Fluoromount (Sigma) and viewed
under a uorescence microscope. We found the FITC
method far more sensitive than the peroxidase (and
diaminobenzidine) method [4]. Negative control sec-
tions of midbrain and retinae were processed as
described above, except that no primary antibody was
used. These control sections were immunonegative.
Cell analysis
Following procedures outlined by previous stud-
ies [24], the number of TH
+
cells within the SNc
was estimated using the optical fractionator method
(StereoInvestigator, MBF Science). Briey, systematic
random sampling of sites within dened boundaries of
the SNc was undertaken. All TH
+
cells that came into
focus within the frame were counted. For the retinae,
they were scanned systematically under a uores-
cence microscope and every TH
+
cell was plotted and
total number was recorded. For comparisons between
groups, a one-way ANOVA test (F and p values) was
performed, in conjunction with a Tukey-Kramer mul-
tiple comparison test (q and p values) (using GraphPad
Prism programme).
Fig. 1. Graphs showing TH
+
cell number in the SNc (A) and
retina (B) in the four groups (n =10 per group). Columns show the
mean standarderror of the total number (of one side) ineachgroup.
The symbols in the MPTPgroup represent levels of signicant differ-
ence in number fromthe Saline groups in each series, while symbols
in the MPTP-SAF group represent levels of signicant difference in
number from the MPTP group; represents p <0.001, represents
p <0.01 and * represents p <0.05. Note the reduction in cell num-
ber in the MPTP groups of both SNc and retina. This reduction was
signicant. Note also that there were signicantly more cells in the
MPTP-SAF than MPTP groups of both regions.
RESULTS
In the section that follows, the number, distribution
and morphology of TH
+
cells in the SNc and retina
will be considered.
Number of SNc and retinal TH
+
cells
Figure 1 shows the estimated number of TH
+
cells
in the SNc (Fig. 1A) and retina (Fig. 1B) of the
four experimental groups. Overall, the variations in
number were signicant in the SNc (ANOVA test:
F=15.5; p <0.0001) and the retina (ANOVA test:
F=7.3; p <0.001). For the Saline and Saline-SAF
groups in SNc (Fig. 1A) and retina (Fig. 1B), the
numbers of TH
+
cells were similar; no signicant
differences were evident between these groups (Tukey-
Kramer test; p >0.05). For the MPTP group, TH
+
80 S. Purushothuman et al. / Saffron Saves Dopaminergic Cells in MPTP-Treated Mice
cell number in both regions was reduced compared
to the saline control groups (3035%). This reduc-
tion was signicant for both SNc (Tukey-Kramer test;
p <0.001) and retina (Tukey-Kramer test; p <0.01). In
the MPTP-SAF groups, TH
+
cell number was higher
than in the MPTP group of the SNc (25%) and retina
(35%) and these differences were signicant (Tukey-
Kramer test; p <0.01 for SNc and p <0.05 for retina).
The number of TH
+
cells in SNc or retina did not dif-
fer signicantly between the MPTP-SAFgroup and the
saline control groups (Tukey-Kramer test; p >0.05).
Distribution and morphology of SNc TH
+
cells
These patterns in the SNc are illustrated in the pho-
tomicrographs of Fig 2. The gures show TH
+
cells in
the SNc in each of the groups studied, namely Saline
(Fig. 2A), Saline-SAF (Fig. 2B), MPTP (Fig. 2C) and
MPTP-SAF(Fig. 2D). Althoughthere were fewer TH
+
cells in the MPTP group (Fig. 2C), those remain-
ing were similar in overall appearance to those seen
in the Saline (Fig. 2A), Saline-SAF (Fig. 2B) and
MPTP-SAF (Fig. 2D) groups. They had round or oval-
shaped somata with one to two labelled dendrites. The
schematic diagrams in Fig. 2 show the topographical
distribution of the TH
+
cells along the rostrocaudal
axis of the SNc (lateral region mapped) in the differ-
ent groups (Fig. 2A

). Note the reduction in TH

+
cell number across the SNc in the MPTP group com-
pared to the other groups, most notably the MPTP-SAF
group.
Distribution and morphology of retinal TH
+
cells
Figure 3 shows photomicrographs of TH
+
cells in
the retinae of Saline (Fig. 3A), Saline-SAF (Fig. 3B),
MPTP (Fig. 3C) and MPTP-SAF (Fig. 3D) groups. In
all groups, THimmunoreactivity was seen in amacrine
cells with large oval-shaped somata located mainly in
the inner nuclear layer and dendrites extending into
the inner plexiformlayer, often forming dendritic rings
(arrows Fig. 3). As with the SNc, although there were
fewer cells in the MPTP group, the morphology of
the retinal TH
+
cells was very similar in the different
groups (Fig. 3).
In summary, our results indicated that there were
fewer TH
+
cells in the SNc and retina of the MPTP
group (3035%) compared to the others. In particular,
there was a distinctly (2535%) higher number of TH
+
cells in saffron-conditioned, MPTP-stressed SNc and
retina, than in unconditioned, MPTP-stressed tissue.
DISCUSSION
The tissue-protective properties of saffron have been
studied in models of genotoxicity, inammation and,
especially, cancer. Its neuroprotective action has been
studied in clinical trials of macular degeneration [7, 11]
and dementia [10]. Neuroprotection at the cellular level
has been shown in models of retinal degeneration [4,
6] and in clinical trials in age-related macular degen-
eration [7, 11]. Our report is the rst, as far as we are
aware, toshowa direct neuroprotective effect of dietary
saffron at the cellular level in the brain. Our central
nding is that saffron conditioning reduces the death
of dopaminergic cells of the SNc in an acute toxicity
model; this is complemented by evidence that saffron
conditioning also reduced the loss of dopaminergic
amacrine cells in the retina. While it is not surpris-
ing conceptually that saffron protects brain as well as
retina, the nding gives encouragement to the testing
of saffron in models of other cerebral degenerations;
and to extend the clinical trial of saffron in age-related
macular degeneration to cerebral degenerations in
humans.
Comparison with previous studies on Parkinsons
and other neurodegenerative diseases
Ahmad and colleagues [12] have reported previ-
ously that pre-treatment with one of the bioactive
molecules found in saffron (crocetin, delivered
by intraperitoneal injection), improves locomotive
behaviour and increases anti-oxidant activity in 6
hydroxydopamine (6OHDA)-treated rats; they also
showed, from non-stereological analysis, a saving
of SNc cells. The present study extends this nd-
ing to MPTP toxicity, to orally ingested saffron, to
a detailed stereological analysis of SNc cell num-
bers and to the dopaminergic amacrine cells of the
retina. Treatment with saffron, or its key components,
crocetin and crocin, has been shown to have bene-
cial outcomes, both cellular and clinical, in other
neurological diseases. For instance, in a recent double-
blind, placebo-controlled study, saffron was reported
to improve cognitive function in patients with mild
to moderate Alzheimers disease [10]. Further, saffron
and crocetin have been shown to enhance substantially
memory function in aged mice [13]. A clinical trial
of saffron in early, dry age-related macular degen-
eration has shown signicant restoration of macular
function in patients [7], which was maintained over the
following 15 months [11]. Finally, several in vitro stud-
ies have reported the protective effects of carotenoids
S. Purushothuman et al. / Saffron Saves Dopaminergic Cells in MPTP-Treated Mice 81
A
SNc
A

Saline
B
SNc
SNr
B

VTA
Saline-SAF
C
SNc
SNr
C

MPTP
D
SNr
D

MPTP-SAF
SNc
SNr

A
Fig. 2. Photomicrographs (AD) and schematic diagrams (A

) of TH
+
cells in the SNc of the Saline (AA

), Saline-SAF (BB

), MPTP
(CC

) and MPTP-SAF (DD

) groups. Photomicrographs and schematics were from lateral SNc regions. The inset (A

) shows the sections,

across the rostrocaudal axis (from left to right), from where the SNc were mapped. In the schematic maps of the SNc, one black circle represents
one cell. These schematics provide an overall view of the patterns of TH
+
cell distribution across the SNc in one case of each different group.
The photomicrographs represent only a small region of the SNc, in fact a portion of just one of the schematic diagrams. They serve as examples
of the patterns of TH immunolabelling in one case of each group. Although there were fewer TH
+
cells in the MPTP group, those remaining
were similar in overall appearance to those seen in the other groups. All gures are of coronal sections; dorsal to top and lateral to right. Scale
bar =100m.
(crocin, picrocrocin and crocetin) from saffron against
neuronal injury [14] and of crocin fromsaffron against
reactive oxygen species induced toxicity in dopamin-
ergic PC12 cells [15]. Taken together, all these results
indicate that saffron treatment enhances the survival of
neurones in disease states. Our use of dietary saffron,
encompassing a mixture of components such as cretin
and crocin, is similar to that used by clinical trials in
humans and hence important for future therapeutic use
in Parkinsons disease patients.
Identifying the dopaminergic cells
Many studies have reported neuroprotection in
animal models of Parkinsons disease using TH
immunocytochemistry to identify dopaminergic cells
in the brain, specically in midbrain centres known to
contain concentrations of these cells [24]. A change
in TH
+
cell number after experimental manipulation
has generally been interpreted as an index of dopamin-
ergic cell loss or survival. It remains possible however,
that some changes in number are due to the loss of TH
in cells that are surviving. For two reasons, we sug-
gest that the bulk of our change in TH
+
cell number
reected overall cell survival. First, previous studies
have reported that a loss of TH by dopaminergic cells
generally leads to a death of the cells themselves [16]
and second, many experimental studies of MPTP tox-
icity have also shown changes in Nissl-stained cell
numbers in the SNc [2]. Nevertheless, whether a tran-
sient loss of TH or death, a key aspect of our study
was that saffron pre-treatment saved TH
+
cells during
82 S. Purushothuman et al. / Saffron Saves Dopaminergic Cells in MPTP-Treated Mice
Fig. 3. Photomicrographs of TH
+
amacrine cells in the retinae of Saline (A) Saline-SAF (B) MPTP (C) and MPTP-SAF (D) groups. Images are
of a mid-region of superior temporal retina in each case. Arrows indicate small dendritic rings formed by the TH
+
bres. These rings surround
other, non-dopaminergic amacrine cells. Although there were fewer TH
+
cells in the MPTP group, those remaining were similar in overall
appearance to those seen in the other groups. Scale bar =100m.
a period when MPTP treatment alone would have abol-
ished it [3, 4].
Mechanism how does saffron provide
neuroprotection?
Although our experiments did not address the mech-
anism that provided the neuroprotection, it is tempting
to speculate. One possibility is that saffron acts as a
direct anti-oxidant; it is, weight for weight, the most
powerfully anti-oxidant plant tested [17]. MPTP is
thought to impart its toxicity of dopaminergic cells
through complex mechanisms. Briey, once in the
brain, MPTP is converted into the active toxin, MPP
+
,
which concentrates within the mitochondria. There, it
inhibits the activity of complex 1 of the electron trans-
port chain, leading ultimately to ATP depletion and the
production of oxygen free radicals; this process causes
much damage to key proteins, including TH [18]. Saf-
fron may protect dopaminergic cells through its ability
to repress radical oxygen species production, decrease
caspase-3 activation, reduce lipid peroxidation prod-
ucts, and increase total brain anti-oxidant activity [13,
19]. Second, there is evidence that saffron regulates
cellular gene expression. Natoli and colleagues [6]
noted the large number of genes and ncRNAs regulated
by saffron preconditioning in their retinal light damage
model. Finally, there is the possibility that the neuro-
protective action of saffron in intact animals involves
the stimulation of more global systems, such as cir-
culating immune-like cells [20]. The effectiveness of
whole-saffron extract is reproduced by molecules that
are enriched in saffron, particularly crocin and cro-
cetin [2123]. Still unknown is whether saffron acts
locally, at the site of tissue damage (eg, regulating gene
expression and/or as an anti-oxidant), or by mobilising
circulating cells or cytokines (see above). The ability
of saffron to mitigate toxin-induced damage in clin-
ically signicant sites such as the SNc warrants the
further investigation of these mechanisms. We suggest
that these mechanisms are similar, if not the same [6],
as those used by near-infrared light (NIr) treatment in
the neuroprotection of dopaminergic cells in the SNc
[3] and retina [4] of an MPTP-treated mouse model.
CONCLUSIONS
These results on an animal model of Parkinsons dis-
ease provide encouragement that saffron pre-treatment
may be used to help save or prevent the death of SNc
S. Purushothuman et al. / Saffron Saves Dopaminergic Cells in MPTP-Treated Mice 83
and retinal dopaminergic cells in Parkinsons disease
patients, something that current drug therapy does not
do. Our results strengthen the ever growing template of
basic science needed for the establishment of a clinical
trial.
ACKNOWLEDGMENTS
We are forever grateful to Tenix corp, Salteri family
and Sir Zelman Cowen Universities Fund for sup-
porting our work. Sharon Spana provided rst class
technical help.
CONFLICT OF INTEREST
There was no conict of interest for any of the
authors.
REFERENCES
[1] Schapira AH (2009) Molecular and clinical pathways to neu-
roprotection of dopaminergic drugs in Parkinson disease.
Neurology, 17, S44-S50.
[2] Wallace BA, Ashkan K, Heise CE, Foote KD, Torres N,
Mitrofanis J, & Benabid AL (2007) Survival of midbrain
dopaminergic cells after lesion or deep brain stimulation of the
subthalamic nucleus in MPTP-treated monkeys. Brain 130,
2129-45.
[3] Shaw VE, Spana S, Ashkan K, Benabid AL, Stone J, Baker
GE, & Mitrofanis J (2010) Neuroprotection of midbrain
dopaminergic cells in MPTP-treated mice after near-infrared
light treatment. J Comp Neurol 1518, 25-40.
[4] Peoples C, Shaw VE, Jeffery G, Stone J, Baker GE, & Mitro-
fanis J (2012) Survival of dopaminergic amacrine cells after
near-infrared light (NIr) treatment in MPTP-treated mice.
ISRN Neurology, 850150
[5] Maccarone R, Di Marco S, & Bisti S (2008) Saffron supple-
ment maintains morphology and function after exposure to
damaging light in mammalian retina. Invest Ophthalmol Vis
Sci, 49, 1254-1261.
[6] Natoli R, Zhu Y, Valter K, Bisti S, Eells J, & Stone J (2010)
Gene and noncoding RNA regulation underlying photorecep-
tor protection: Microarray study of dietary antioxidant saffron
and photobiomodulation in rat retina. Mol Vis 16, 1801-1822.
[7] Falsini B, Piccardi M, Minnella A, Savastano C, Capolu-
ongo E, Fadda A, Balestrazzi E, Maccarone R, & Bisti S
(2010) Inuence of saffron supplementation on retinal icker
sensitivity in early age-related macular degeneration. Invest
Ophthalmol Vis Sci, 51, 6118-6124.
[8] NamKN, Park YM, Jung HJ, Lee JY, Min BD, Park SU, Jung
WS, Cho KH, Park JH, Kang I, Hong JW, & Lee EH (2010)
Anti-inammatory effects of crocin and crocetin in rat brain
microglial cells. Eur J Pharmacol, 648, 110-116.
[9] Hosseinzadeh H, Abootorabi A, &Sadeghnia HR(2008) Pro-
tective effect of Crocus sativus stigma extract and crocin
(trans-crocin 4) on methyl methanesulfonate-induced DNA
damage in mice organs. DNA Cell Biol, 27, 657-664.
[10] Akhondzadeh S, Sabet MS, Harirchian MH, Togha M,
Cheraghmakani H, Razeghi S, Hejazi SSh, Youse MH, Ali-
mardani R, Jamshidi A, Zare F, &Moradi A(2010) Saffron in
the treatment of patients with mild to moderate Alzheimers
disease: A16-week, randomized and placebo-controlled trial.
J Clinical Pharm Therap, 35, 581-588.
[11] Piccardi M, Marangoni D, Minnella AM, Savastano MC,
Valentini P, Ambrosio L, Capoluongo E, Maccarone R, Bisti
S, & Falsini B (2012) A longitudinal follow-up study of
saffron supplementation in early age-related macular degen-
eration: Sustained benets to central retinal function. Evid
Based Complement Alternat Med, 429124
[12] Ahmad AS, Ansari MA, Ahmad M, SaleemS, Yousuf S, Hoda
N, & Islam F (2005) Neuroprotection by crocetin in a hemi-
parkinsonian rat model. Pharm Biochem Behav, 81, 805-813.
[13] Papandreou MA, Tsachaki M, Efthimiopoulos S, Cordopatis
P, Lamari FN, & Margarity M (2011) Memory enhancing
effects of saffron in aged mice are correlated with antioxidant
protection. Behav Brain Res, 219, 197-204.
[14] Ochiai T, Shimeno H, Mishima K, Iwasaki K, Fujiwara M,
Tanaka H, Shoyama Y, Toda A, Eyanagi R, & Soeda S (2007)
Protective effects of carotenoids from saffron on neuronal
injury in vitro and in vivo. Biochim Biophys Acta, 1770, 578-
584.
[15] Mousavi SH, Tayarani NZ, & Parsaee H (2010) Protective
effect of saffron extract and crocin on reactive oxygen species-
mediated high glucose-induced toxicity in PC12 cells. Cell
Mol Neurobiol, 30, 185-191.
[16] Bj orklund A, Rosenblad C, Winkler C, & Kirik D (1997)
Studies on neuroprotective and regenerative effects of GDNF
in a partial lesion model of Parkinsons disease. Neurobiol
Dis 4, 186-200.
[17] Pellegrini N, Serani M, Salvatore S, Del Rio D, Bianchi
M, & Brighenti F (2006) Total antioxidant capacity of spices,
dried fruits, nuts, pulses, cereals and sweets consumed in Italy
assessed by three different in vitro assays. Mol Nutr Food Res,
50, 1030-1038.
[18] Przedborski S, Jackson-Lewis V, Djaldetti R, Liberatore G,
Vila M, Vukosavic S, & Almer G (2000) The parkinsonian
toxin MPTP: Action and mechanism. Restor Neurol Neurosci,
16, 135-142.
[19] Ordoudi SA, Befani CD, Nenadis N, Koliakos GG, & Tsimi-
dou MZ (2009) Further examination of antiradical properties
of Crocus sativus stigmas extract rich in crocins. J Ag Food
Chem, 57, 3080-3086.
[20] Stone J, Johnstone D, & Mitrofanis J (2012) The helmet
experiment in Parkinsons disease: An observation of the
mechanism of neuroprotection by near infra-red light. Pro-
ceedings of WALT P928C0072.
[21] Laabich A, Vissvesvaran G, Lieu K, Murata K, McGinn T,
Manmoto C, Sinclair J, Karliga I, Leung D, Fawzi A, &
Kubota R (2006) Protective Effect of Crocin against Blue
Light and White LightMediated Photoreceptor Cell Death
in Bovine and Primate Retinal Primary Cell Culture. Invest
Ophthalmol Vis Sci 47, 3156-3163.
[22] Wang Y, Han T, Zhu Y, Zheng C, Ming J, Rahman QL, & Qin
K, LP (2009) Antidepressant properties of bioactive fractions
from the extract of Crocus sativus. J Nat Med, 64, 24-30.
[23] Papandreou MA, Kanakis CD, Polissiou MG, Efthimiopou-
los S, Cordopatis P, Margarity M, & Lamari FN (2006)
Inhibitory activity on amyloid-beta aggregation and antioxi-
dant properties of Crocus sativus stigmas extract and its crocin
constituents. J Ag Food Chem, 54, 8762-8768.