Food and Chemical Toxicology 52 (2013) 163–170

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Food and Chemical Toxicology

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Investigation of the neuroprotective action of saffron (Crocus sativus L.)in aluminum-exposed adult mice through behavioral and neurobiochemicalassessment

Zacharoula I. Linardaki a, Malvina G. Orkoula b, Alexandros G. Kokkosis a, Fotini N. Lamari c,Marigoula Margarity a,⇑a Laboratory of Human and Animal Physiology, Department of Biology, University of Patras, 26504 Patras, Greeceb Laboratory of Instrumental Pharmaceutical Analysis, Department of Pharmacy, University of Patras, 26504 Patras, Greecec Laboratory of Pharmacognosy & Chemistry of Natural Products, Department of Pharmacy, University of Patras, 26504 Patras, Greece

a r t i c l e i n f o

Article history:Received 21 September 2012Accepted 8 November 2012Available online 17 November 2012

Keywords:AluminumSaffronMemoryCholinesterasesMonoamine oxidaseOxidative stress

0278-6915/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.fct.2012.11.016

Abbreviations: AChE, acetylcholinesterase; ATCBuChE, butyrylcholinesterase; BuTCh, butyrylthiochoChE, cholinesterase; DS, detergent-soluble; GSH, redperitoneal injections; IL, initial latency; MAO, monoamaldehyde; SS, salt-soluble; STL, step-through latency.⇑ Corresponding author. Tel.: +30 2610 997430; fax

E-mail addresses: linardaki@upatras.gr (Z.I. Li(M.G. Orkoula), magnusalex11@hotmail.com (A.G.(F.N. Lamari), margar@upatras.gr (M. Margarity).

a b s t r a c t

In the present study, the possible reversal effects of saffron against established aluminum (Al)-toxicity inadult mice, were investigated. Control, Al-treated (50 mg AlCl3/kg/day diluted in the drinking water for5 weeks) and Al + saffron (Al-treatment as previously plus 60 mg saffron extract/kg/day intraperitoneallyfor the last 6 days), groups of male Balb-c mice were used. We assessed learning/memory, the activity ofacetylcholinesterase [AChE, salt-(SS)/detergent-soluble(DS) isoforms], butyrylcholinesterase (BuChE, SS/DS isoforms), monoamine oxidase (MAO-A, MAO-B), the levels of lipid peroxidation (MDA) and reducedglutathione (GSH), in whole brain and cerebellum. Brain Al was determined by atomic absorption spec-trometry, while, for the first time, crocetin, the main active metabolite of saffron, was determined in brainafter intraperitoneal saffron administration by HPLC. Al intake caused memory impairment, significantdecrease of AChE and BuChE activity, activation of brain MAO isoforms but inhibition of cerebellarMAO-B, significant elevation of brain MDA and significant reduction of GSH content. Although saffronextract co-administration had no effect on cognitive performance of mice, it reversed significantly theAl-induced changes in MAO activity and the levels of MDA and GSH. AChE activity was further signifi-cantly decreased in cerebral tissues of Al + saffron group. The biochemical changes support the neuropro-tective potential of saffron under toxicity.

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1. Introduction

Aluminum (Al) is the third most abundant element in nature,making human exposure unavoidable (Verstraeten et al., 2008).The main entry sites of Al into the body are the gastrointestinaland respiratory tract and the skin (Verstraeten et al., 2008), en-abling distribution and accumulation in several tissues, like spleen,lungs, liver, kidneys, heart, bone and brain (Kumar and Gill, 2009),through systemic circulation. It has been demonstrated that Alcrosses the blood–brain barrier, implicating metal binding to

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h, acetylthiocholine iodide;line iodide; ce, cerebellum;uced glutathione; i.p., intra-ine oxidase; MDA, malondi-

: +30 2610 969273.nardaki), malbie@upatras.gr

Kokkosis), flam@upatras.gr

transferrin in the blood and a subsequent transferrin receptor-mediated mechanism of brain Al influx (Yokel et al., 1999).

High brain levels of Al induce cognitive deficiency and dementiaand, thus, Al is a widely accepted neurotoxin (Kawahara andKato-Negishi, 2011). Its implication in the pathogenesis ofneurodegenerative diseases has been suggested decades ago, butis seriously debated till now (Kawahara and Kato-Negishi, 2011;Tomljenovic, 2011). However, some researchers consider that themodel of chronic Al-induced neurotoxicity best describesAlzheimer’s disease, since it manifests many of the pathobiologicalhallmarks (Walton and Wang, 2009; Yokel, 2006; Zhang et al.,2003).

Al neurotoxicity is manifested through several behavioral andneurochemical alterations, which display diversity depending onthe animal species in question, the administration route and thechemical form of Al administered (Erasmus et al., 1993; Kumarand Gill, 2009). It has been demonstrated that Al promotesoxidative stress in the cerebral cortex and hippocampus of youngand aged rats, and damage of lipids, membrane-associated proteins

164 Z.I. Linardaki et al. / Food and Chemical Toxicology 52 (2013) 163–170

(Na+-K+ ATPase and protein kinase C) and endogenous antioxidantenzymes (Sethi et al., 2008; Sharma et al., 2009; Tripathi et al.,2009). Negative impacts of Al administration have been also ob-served in neurotransmitter systems of rodent brain regions, includ-ing serotoninergic (Kumar, 2002) and cholinergic systems (Julkaet al., 1995). The effects of Al on AChE activity remain controversialas both inhibition and activation have been reported (Julka et al.,1995; Sharma et al., 2009), whereas the respective data concerningBuChE activity are limited, though BuChE enzyme is considered toplay a supportive functional role in acetylcholine hydrolysis (Laneet al., 2006). Finally, behavioral deficits assessed with differentbehavioral tasks, have been observed in rats following Al exposure(Julka et al., 1995; Sethi et al., 2008; Tripathi et al., 2009).

Crocus sativus L. (Iridaceae) is a cultivated plant species in manycountries including Greece, since its styles (saffron) constitute anexquisite spice. Interestingly, saffron has been used as a medicinalagent for millenia (Schmidt et al., 2007). In recent years beneficialeffects of saffron have been demonstrated in models of neuronal,cardiovascular, respiratory and other disorders (Bathaie andMousavi, 2010), and are related to its unique composition; it is richin crocins, which are mono- and di-glycosides of crocetin. Previ-ously, we have reported the inhibitory activity on amyloid beta-peptide fibrillogenesis and protective action against H2O2-inducedtoxicity in human neuroblastoma cells of saffron and its crocinconstituents in vitro (Papandreou et al., 2006, 2011). Furthermore,i.p. administration of saffron (60 mg/kg body weight) to normaland aged mice for 7 days significantly improved learning andmemory as assessed by step-through passive avoidance test andthis was correlated with the significant cerebral antioxidant pro-tection conferred (Papandreou et al., 2011). Other studies have alsodemonstrated neuroprotective effects of saffron and its constitu-ents in vitro and in rodent models of brain disorders (amnesicand ischemic) (Hosseinzadeh and Sadeghnia, 2005; Ochiai et al.,2007; Pitsikas and Sakellaridis, 2006). The first small-scale clinicaltrials of saffron against depression and mild Alzheimer’s diseasehave brought forward promising results, although many issues re-main unresolved (Akhondzadeh et al., 2005, 2010; Noorbala et al.,2005). Yoshino et al. (2011) demonstrated the distribution ofcrocetin in brain of stroke-prone spontaneously hypertensive rats90 min after a single oral administration of pure crocetin, but ithas not been proven yet whether crocetin, the main crocin metab-olite, crosses the blood–brain barrier after saffron administration.

Taking into consideration the increasing evidence about saffronneuroprotective role we further investigated the possibleneuroprotective action of saffron extract in a model of chronicAl-induced neurotoxicity. Shati et al. (2011) demonstrated theameliorative effects of aqueous saffron extract administrationagainst Al-induced neurotoxicity, by presenting changes of brainantioxidant enzymes, serum tumor markers and brain expressionof genes (especially BCL-W, R-spondin and inositol polyphosphate4-phosphatase). However, the potential of saffron extract to re-verse the changes accompanying Al neurotoxicity has not beeninvestigated. Therefore, the present study based on a differentexperimental design (i.p. administration of saffron extract onlyfor the last 6 days of the Al treatment period) starts frombehavioral level, including learning and memory process, and isextended to subcellular level, focusing on determination of braincholinergic, monoaminergic and oxidant/antioxidant indices andcerebral Al and crocetin levels.

2. Material and methods

2.1. Plant material and extraction

Greek saffron (pure red styles of C. sativus) was kindly provided by the Cooper-ative Association of Krokos in Kozani, in West Macedonia, Greece and a sample hasbeen deposited at the herbarium of the Department of Biology. Dried styles of C.

sativus were extracted with methanol:water (50:50, v/v, 1 g in 60 mL) for 4 h inthe dark, under magnetic stirring, as previously described (Papandreou et al.,2006). The extract was centrifuged and evaporated to dryness in a Speed Vac sys-tem. The composition was screened with HPLC on a Supelcosil C-18 column as pre-viously described (Papandreou et al., 2006; Tarantilis et al., 1995). The dry residuewas stored at �30 �C until use.

2.2. Animals

Male adult (4 months-old) Balb-c mice were used in this study. The animalswere housed in groups in standard laboratory cages (5 mice per cage) in a temper-ature controlled room (20 ± 2 �C) with a 12 h light/dark cycle. Food in form of drypellets (feed composition: grain and grain by-products, oil seed products, minerals,vitamins and trace elements from Altromin Spezialfutter GmbH & Co. KG, Lage, Ger-many) was available ad libitum. Animals were randomly divided into three groups(n = 10/group). Control group: mice had access to normal drinking water over theexperimental period. Al treated group: mice received orally AlCl3 (aluminum chlo-ride anhydrous, purity >99%, Sigma–Aldrich, St. Louis, USA) (50 mg/kg body weight/day) dissolved in normal drinking water for a period of 5 weeks. Body weight andliquid intake were measured regularly to adjust the dose and achieve a constant in-take of Al. Al + saffron treated group: mice received orally AlCl3 (50 mg/kg bodyweight/day) dissolved in normal drinking water for 5 weeks and saffron extract(60 mg/kg body weight/day, 30 lL injection volume) intraperitoneally for the last6 days of the 5-week Al treatment.

AlCl3 solution was freshly prepared every 4 days over the experimental period,while saffron solution was prepared fresh daily, just before administration, by dis-solving the dry extract in saline. During the 6-day saffron treatment, control and Al-treated mice received intraperitoneally 30 lL of saline. Al and saffron dosage andschedule of exposure were based on the literature (Jyoti and Sharma, 2006) andon our previous study (Papandreou et al., 2011), respectively. All procedures werein accordance with Greek National Laws (Animal Act, PD 160/91).

2.3. Behavioral testing: step-through passive avoidance task

This test is based on negative reinforcement to examine long-term memory(Kaneto, 1997). A two-compartment passive avoidance apparatus (white/dark, sep-arated by a guillotine door) was used. Mice were subjected to single trial passiveavoidance task according to previously described procedures (Kaneto, 1997; Otanoet al., 1999; Papandreou et al., 2009). Briefly, on day 5 of saffron treatment an acqui-sition trial was performed 1 h after a 100 s-long habituation trial, while a retentiontrial was performed 24 h after the training session. Results were expressed as themean initial (IL, max. time 120 s) and step-through (STL, max. time 300 s) latencytime values recorded once mice crossed into the dark compartment, before and24 h after the delivery of a mild foot shock to their paws. All training and testingsessions were carried out during the light phase (08:00–14:00 h), one hour afterintraperitoneal administration of saffron.

2.4. Preparation of tissue homogenates

On the completion of the 5-week treatment period, all animals were sacrificedby cervical dislocation. Brain was excised immediately, and cerebellum was sepa-rated from the remaining whole brain. Liver was also removed and used as a refer-ence peripheral tissue. All tissues were rinsed with saline immediately. From thetissue samples isolated, only six tissues per group were used for the biochemicalassessments. The others were assayed for Al content by atomic absorption spec-trometry. The tissues for biochemical measurements and crocetin determinationwere weighed and homogenized (10%, w/v) with a glass-Teflon homogenizer inice-cold 30 mM Na2HPO4, pH 7.6. The homogenates were then centrifuged at15,000g for 20 min at 4 �C. Supernatants constituted the salt-soluble (SS) fractionsand were stored at �75 �C. The pellets were re-suspended in an equal volume of 1%(w/v) Triton X-100 (in 30 mM Na2HPO4, pH 7.6) and then centrifuged at 15,000g for20 min at 4 �C. Supernatants, the detergent-soluble (DS) fractions, were collectedand stored at �75 �C. This double-extraction method was performed in order to re-cover the cytosolic (SS fraction) and membrane-bound (DS fraction) isoforms ofcholinesterases (Das et al., 2005). Protein concentrations were determined by theBradford assay (Bradford, 1976).

2.5. Aluminum determination by atomic absorption spectrometry

2.5.1. Digestion of tissuesThe whole brain (-ce), cerebellum and liver samples from control, Al-treated

and Al + saffron treated mice were analyzed for Al concentration. Glassware wasnot used during sample preparation in order to avoid the possible contamination.Eppendorf tubes were used after immersing in a solution of nitric acid (HNO3): eth-anol (1:9, v/v) for 48 h and washing with ultra-pure water (Missel et al., 2005).Sample preparation was performed as previously described (Gulya et al., 1995) withmodifications. The dry weight of the tissues was measured after heating at 100 �Cfor 20 h. Then, the samples were digested with 1 mL/100 mg wet tissue (for wholebrain) and 1 mL/25 mg wet tissue (for cerebellum and liver) of nitric acid: sulphuric

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acid (HNO3:H2SO4, 1:1 v/v) at room temperature overnight. Mixtures were centri-fuged at 10,000g for 10 min to remove insoluble material. Al levels were deter-mined in the supernatants.

2.5.2. Atomic absorption spectrometryAl content in the supernatants was measured employing Atomic Absorption

Spectrometry (Perkin Elmer, AAnalyst 300 equipped with a HGA 700 Graphite Fur-nace and an AS-70 autosampler). The temperature of the furnace was programmedas described elsewhere (Johnson and Treble, 1992). Absorbance was measured at309.3 nm.

Preparation of samples for Al analysis: All samples were prepared in eppendorftubes and analyzed immediately after preparation. A 5% v/v HNO3 and 0.1% v/vMg solution prepared by proper dilutions from HNO3 solution (65%, Carlo Erba)and Mg stock standard solution (1000 lg/mL Mg2+ ±2 lg to 5% HNO3, Chem-Lab),was used as a matrix modifier in order to stabilize Al in the furnace.

The standard addition method was employed for the analysis. Four sampleswere prepared for each aliquot, adding known amounts (spikes) of the analyte(Al) from a stock standard solution (1000 ppm in 1 M HNO3 Fisher Scientific, UK).The spiked concentration ranged from 0 (unspiked sample) to 60 ppb. The volumewas kept equal for all samples by means of addition of the matrix modifier. Preli-minary tests assured that the overall concentration of Al was within the linearrange of the technique.

Analysis: Absorbance for each sample was measured three times. A calibrationcurve for each supernatant aliquot was constructed (OriginPro 8). The results areexpressed as lg Al/g wet tissue.

2.6. HPLC determination of crocetin in brain

Crocetin determination in whole brain (-ce) of Al + saffron treated mice wasperformed by an isocratic reversed-phase liquid chromatographic method devel-oped by Chryssanthi et al. (2011) for plasma analysis, with slight modifications.In detail, a high volume (3.5 mL) of the whole brain homogenate (100 mg/mL, SSfraction) was submitted to solid phase extraction on reversed phase Strata-X car-tridges (200 mg/3 mL) consisting of a surface modified with styrene–divinylben-zene polymer that were obtained from Phenomenex (Torrance, CA, USA). Thecrocetin eluate was evaporated to dryness in a speed Vac system and the dry resi-due was redissolved in 30 lL of the HPLC mobile phase. The samples were chro-matographed on a Luna C-18 column (4.6 � 250 mm, 5 lm) with a mobile phaseconsisting of methanol–water–trifluoroacetic acid (75.0:24.5:0.5, v/v/v) at a flowrate of 1.0 mL min�1. Crocetin was quantified using spiked brain standards at theconcentrations of 0.5, 1.0, 1.5, 2.0 and 3.0 lM of total crocetin (Chembiotin, purity>98%). Results are expressed as nmol of total crocetin/g wet tissue.

2.7. Biochemical assays

2.7.1. Determination of cholinesterase activityThe activity of SS and DS isoforms of AChE and BuChE in brain and liver samples,

was determined spectrophotometrically based on Ellman’s assay and previously de-scribed protocols (Ellman et al., 1961; Lassiter et al., 1998; Papandreou et al., 2009).Total ChE activity was measured by acetylthiocholine iodide (ATCh, Sigma–Aldrich,UK) as substrate, whereas BuChE activity was determined by S-butyrylthiocholineiodide (BuTCh, Sigma–Aldrich, Switzerland) as substrate, since BuTCh is hydrolyzedonly by BuChE (Lassiter et al., 1998). AChE activity was then calculated by subtract-ing BuChE activity from total ChE activity. Enzyme activity is expressed as lmol ofsubstrate hydrolyzed/min/g of tissue.

Fig. 1. Effect of saffron administration on initial (IL) and step-through (STL) latencytime of Al-exposed adult mice in step-through passive avoidance task. Al treatedmice displayed lower learning/memory capacity during the testing session of thetask, compared to the controls, whereas saffron co-treatment was ineffective. Eachhorizontal line represents the mean latency time of ten animals in each group(n = 10). Individual values are presented as squares for control mice, triangles for Altreated and circles for Al + saffron treated ones. ap < 0.05 vs. STL time of the controls(non-parametric Mann–Whitney test).

2.7.2. Determination of monoamine oxidase activityThe activity of both isoforms of MAO, MAO-A and MAO-B, was assessed in

mouse whole brain (-ce) and cerebellum by a fluorimetric assay based on previ-ously described protocols (Mahmood et al., 1994; Xu et al., 2010). Since MAO is amitochondrial membrane-bound enzyme, the DS fractions of brain tissues homog-enates were used. Briefly, 50 lL of brain sample was preincubated at 37 �C for20 min with 100 lL of either 1 lM R-(�)-Deprenyl hydrochloride (Deprenyl,MAO-B inhibitor, Sigma–Aldrich, USA) or 1 lM N-Methyl-N-propargyl-3-(2,4-dichlorophenoxy)propylamine hydrochloride (Clorgyline, MAO-A inhibitor, Sig-ma–Aldrich, USA) in 100 lL 30 mM Na2HPO4, pH 7.6. Then 10 lL of 3.07 mM kynur-amine dihydrobromide (Sigma–Aldrich, USA) was added to the reaction mixture assubstrate. The mixture was then incubated at 37 �C for 15 min again. After incuba-tion, the reaction was terminated by adding 100 lL of 0.6 M perchloric acid andcentrifuging the mixture at 1500g for 10 min to remove precipitated proteins. Analiquot of 0.3 mL of the supernatant was added to 2 mL of 1 N NaOH. The fluores-cence intensity of the produced 4-hydroxyquinoline was detected at an excitationwavelength of 315 nm and an emission wavelength of 380 nm, using a fluorescencespectrometer. The concentration of product was estimated from a correspondingstandard fluorescence curve of 4-hydroxyquinoline (1–100 lM) (Sigma–Aldrich,USA). MAO activity is expressed as nmol of 4-hydroxyquinoline formed/g tissue/min.

2.7.3. Estimation of lipid peroxidation and reduced glutathione levelsMalondialdehyde (MDA) levels, an index of tissue lipid peroxidation, were mea-

sured in cerebral and liver samples (SS fractions of tissue homogenates) following apreviously described fluorimetric assay (Papandreou et al., 2009). Lipid peroxida-tion levels are expressed as lmol MDA/g of tissue protein.

Reduced glutathione (GSH) content of brain and liver samples (SS fractions oftissue homogenates) was determined fluorometrically according to the procedureof Mokrasch and Teschke (1984). GSH levels are expressed as lmol/g of tissueprotein.

2.8. Statistical analysis

Data are presented as mean ± S.E.M. Statistical analysis was performed withGraphPad Instat 3 software using the nonparametric Mann–Whitney test for eval-uating statistically significant differences (p < 0.05) between the experimentalgroups.

3. Results

3.1. General observations

HPLC analysis showed that saffron extract contains a variety ofcrocins: trans-crocin-4 constitutes 46% of total crocin content ofthe crude extract, trans-crocin-3, 26%; cis-crocin-4, 12%; andtrans-crocin-2, 7%, as previously described (Papandreou et al.,2006; Tarantilis et al., 1995).

All animals developed normally during the experimental periodand no mortality was recorded during Al treatment. Also, therewere no differences on body weight gain and liquid intake, be-tween the experimental groups.

3.2. Effect of saffron on learning and memory ability of Al-exposedmice

Passive avoidance task was performed to examine long-termmemory. The initial latency (IL) time, measured at training trial,is an indicator of motor activity, and step-through latency (STL)time, measured at testing trial, indicates memory of the aversiveexperience. Al intake resulted in impaired long-term memory ofmice, as evidenced by the significantly lower (59%) mean STL timeof Al-treated animals compared to mean STL time of the controls(Fig. 1). The lack of difference of mean STL time between Al +saffron- and Al-treated groups (Fig. 1), shows that saffron extractco-administered with Al during the last 6 days of the treatmentperiod was ineffective in reversing Al-induced memory

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impairment. The mean IL time values were not significantly differ-ent among all experimental groups (Fig. 1), implying no differencein motor activity of mice in training session.

3.3. Al and crocetin levels in cerebral tissues

The levels of Al in whole brain (-ce), cerebellum and liver ofcontrol, Al-treated and Al + saffron treated mice are presented inTable 1. Al was not detected in whole brain (-ce) of control mice,whereas it was determined in high amounts in cerebellum. Inall animal groups Al levels in cerebellum were significantly(p < 0.05) higher than those of the remaining brain. Also, Al con-centrations were significantly (p < 0.05) increased in both cerebraltissues and liver of Al-treated mice compared to the controls anddid not differ significantly (p < 0.05) in the cerebral and liver tissueof Al + saffron treated mice, compared to Al-treated group.

Pooled whole brain (-ce) homogenates of Al + saffron treatedand control mice were analyzed by HPLC. HPLC chromatogram(Supplementary data) of Al + saffron treated brain (chromatogramB) shows a peak at 12.4 min, which is not detected in control brain(chromatogram A), and corresponds to trans-crocetin. UV–visibleabsorption spectrum of the peak presents a maximum double peakat 420–450 nm, characteristic of the carotenoids. Crocetin concen-tration, was 2.0 ± 0.1 nmol total crocetin/g tissue (n = 2) in wholebrain of mice treated with saffron extract and not detected in brainof control mice.

3.4. Effect on brain and liver ChE activity

The impact of Al or saffron treatment on the activities of bothisoforms (SS, DS) of AChE and BuChE in whole brain, cerebellumand liver of adult mouse is presented in Table 2. Al intake de-creased significantly (p < 0.05) the activity of both SS and DS iso-forms of AChE and BuChE in mouse whole brain (-ce) (26%, 17%

Table 1Al concentrationsa in cerebral tissues and liver of control, Al-treated and Al + saffrontreated adult mice.

Mice groups Whole brain (-ce) Cerebellum Liver

Control ND 7.77 ± 0.62 3.97 ± 0.15**

Al treated 1.30 ± 0.45** 11.67 ± 0.63* 7.62 ± 0.53*,**

Al + saffron treated 0.97 ± 0.12** 10.17 ± 1.47 10.22 ± 0.98*

ND, not detected.a Al concentrations are expressed as lg/g wet tissue. Data are the mean ± SEM

(n = 4 animals/group).* p < 0.05 vs. the control group.** p < 0.05 vs. cerebellum of the respective group (non-parametric Mann–Whitneytest).

Table 2Activity of SS- (salt soluble) and DS- (detergent soluble) isoforms of acetylcholinesterase (Al + saffron treated adult mice.

Mice groups Whole brain (-ce) Cereb

SS-ChE DS-ChE SS-Ch

AChE activity (lmol/min/g tissue)Control 11.17 ± 0.42 76.72 ± 0.99 6.32 ±Al treated 8.26 ± 0.20* 63.47 ± 3.57* 4.88 ±Al + saffron treated 6.99 ± 0.28*,# 45.43 ± 1.32*,# 3.69 ±

BuChE activity (lmol/min/g tissue)Control 1.78 ± 0.08 2.49 ± 0.07 2.47 ±Al treated 1.40 ± 0.07* 2.50 ± 0.05 1.93 ±Al + saffron treated 1.50 ± 0.07* 2.40 ± 0.06 1.84 ±

Data are the mean ± SEM (n = 6 animals/group).* p < 0.05 vs. the control group.# p < 0.05 vs. Al treated group (non-parametric Mann–Whitney test).

and 21%) and cerebellum (23%, 15% and 22%, 19%) compared tothe controls, except of the activity of DS-BuChE of whole brain(-ce) that remained unchanged. Further significant (p < 0.05)reduction of the SS- and DS-AChE activity in whole brain (-ce)(15%, 28%) and cerebellum (24%, 27%) was observed in Al + saffrontreated mice in comparison with Al treated group, whereas saffrontreatment did not further affect the activity of cerebral BuChE.Also, Al-treated mice displayed significantly (p < 0.05) decreasedliver BuChE (11% SS, 21% DS) and SS-AChE (25%) activitiescompared to the controls, while Al + saffron treated mice displayedfurther inhibition of liver BuChE (27% SS, 19% DS) and SS-AChE(31%) compared to Al-treated mice.

3.5. Effect on brain MAO activity

The effect of administration of either Al alone or in the presenceof saffron extract at the end of the treatment period, on MAO iso-forms activity of mouse whole brain (-ce) and cerebellum, is pre-sented in Fig. 2. Al-treated mice displayed significantly (p < 0.05)higher activities of MAO-A (19%) and MAO-B (10%) in their wholebrain (-ce) and significantly lower MAO-B activity (14%) in cerebel-lum, compared to the controls. Short-term co-administration ofsaffron restored MAO-A and MAO-B activity of whole brain (-ce)(decrease by 14% and 10%, respectively) and MAO-B activity (17%increase) of cerebellum, to normal levels.

3.6. Effect on brain and liver oxidant/antioxidant indices

MDA and GSH levels, as indicators of lipid peroxidation and cel-lular antioxidant defense, respectively, were measured in mousebrain tissues and liver of all experimental groups. As shown inFig. 3a, Al intake increased (18%) significantly (p < 0.05) MDA levelsonly in whole brain (-ce) in comparison with controls, while thesubsequent short-term co-administration of saffron restored(decrease by 22%) MDA levels to normal. Short-term co-administration of saffron also improved the antioxidant status ofcerebellum and liver by significantly lowering the levels of MDA,even though these tissues remained unaffected by Al treatment(Fig. 3a). Additionally, Al-treated mice displayed remarkablereduction in GSH content of whole brain (-ce) (44%), cerebellum(16%) and liver (29%), compared to the control animals, whereasAl + saffron treated mice exhibited elevated levels of GSH (29%,14% and 22%, respectively) compared to Al-exposed group (Fig. 3b).

4. Discussion

The present study investigates the neuroprotective potential ofa 6-day i.p. administration of saffron extract to adult mice that

AChE) and butyrylcholinesterase (BuChE) in brain and liver of control, Al treated and

ellum Liver

E DS-ChE SS-ChE DS-ChE

0.37 16.29 ± 0.61 5.93 ± 0.14 1.07 ± 0.310.14* 13.87 ± 0.22* 4.47 ± 0.36* 0.90 ± 0.150.18*,# 10.06 ± 0.17*,# 3.10 ± 0.34*,# 0.68 ± 0.17

0.09 1.53 ± 0.06 26.95 ± 0.68 9.64 ± 0.290.12* 1.24 ± 0.04* 23.86 ± 0.80* 7.60 ± 0.26*

0.07* 1.13 ± 0.07* 17.42 ± 0.42*,# 6.17 ± 0.25*,#

Fig. 2. Effect of saffron administration on monoamine oxidase activity (MAO-A andMAO-B) of Al-exposed adult mouse brain. Al-induced activation of both MAOisoforms in whole brain (-ce), inhibition of MAO-B in cerebellum and reversaleffects of saffron are evident. Data are the mean ± SEM (n = 6 animals/group).⁄p < 0.05 vs. the control group, #p < 0.05 vs. Al treated group (non-parametricMann–Whitney test).

Z.I. Linardaki et al. / Food and Chemical Toxicology 52 (2013) 163–170 167

were previously exposed to high Al intake through their drinkingwater for a 5-week period. To this end, cognitive behavior andbrain cholinergic, monoaminergic and oxidative indices were as-sessed. Our findings showed that long-term Al intake inducedlearning/memory decline in adult mice. Impaired performance of

Fig. 3. Effect of saffron administration on (a) lipid peroxidation (MDA) and (b) reduceincrease in MDA levels of whole brain (-ce), decrease in GSH levels of whole brain (-cmean ± SEM (n = 6 animals/group). ⁄p < 0.05 vs. the control group, #p < 0.05 vs. Al treate

rats on passive avoidance task has been shown by Bhalla et al.(2010) after long-term administration of a double dose of AlCl3

in drinking water, while Sethi et al. (2008) showed declined spatiallearning abilities of rats in Morris-water maze test after adminis-tration of the same dose of AlCl3 (50 mg/kg/day) in drinking waterfor 6 months. Saffron co-administration at the end of the treatmentperiod failed to reverse Al-induced cognitive deficits. In our previ-ous study, short-term saffron administration enhanced learning/memory in healthy adult and aged mice in passive avoidance task(Papandreou et al., 2011). However, in the current report theadministered dosage of saffron extract and/or the duration of expo-sure (which are the same as in the study of Papandreou et al., 2011)were insufficient to counteract Al-induced memory deficit.

Al levels were determined in whole brain (-ce), cerebellum andliver of all animal groups prior to biochemical evaluation. The val-ues of Al found in cerebral tissues, are comparable with those ofother studies that use Al as neurotoxic agent, although differentAl forms, doses and routes of administration are investigated(Esparza et al., 2005; Kaizer et al., 2008; Sánchez-Iglesias et al.,2007). Our findings showed high concentrations of Al in cerebel-lum of control mice (higher than liver), but none or below detec-tion limits in whole brain (-ce), in accordance with Bellés et al.(1998). In the current study, long-term AlCl3 intake through drink-ing water resulted in significantly increased Al concentrations inwhole brain, cerebellum and liver of adult mice. Our results are

d glutathione (GSH) levels of Al-exposed adult mouse brain and liver. Al-inducede), cerebellum and liver, and reversal effects of saffron are evident. Data are the

d group (non-parametric Mann–Whitney test).

168 Z.I. Linardaki et al. / Food and Chemical Toxicology 52 (2013) 163–170

in accordance with previous studies where AlCl3 is administered torodents (Bhalla and Dhawan, 2009; El-Maraghy et al., 2001). Theentry of Al into the brain through the blood–brain barrier has longbeen established (Yokel et al., 1999) and is also confirmed by ourresults. The accumulation of Al in brain is consistent with the ob-served impaired learning/memory ability of mice in passive avoid-ance test. However, cerebral Al levels did not differ significantlybetween Al- and Al + saffron treated mice, showing that short-termsaffron administration in the end of the treatment period did notaffect Al bioavailability.

Additionally, for the first time, we determined crocetin, the ma-jor metabolite (aglycon) of the bioactive crocin glycosides of saf-fron, in whole brain of Al + saffron treated mice, which wasabsent from the brain of control mice. The detection of crocetinin mouse brain demonstrates for the first time that this compoundcrosses the blood–brain barrier when saffron extract is adminis-tered for a short term through intraperitoneal route. In recent re-port (Chryssanthi et al., 2011), the presence of crocetin in humanplasma was shown after 2 and 24 h of saffron tea consumption,while only Yoshino et al. (2011) have demonstrated the distribu-tion of crocetin in rat brain after a single oral administration ofpure crocetin, up to now.

The fact that saffron short-term co-administration failed to re-verse the cognitive decline raises questions on the dosage, the per-iod and the route of administration, before definite conclusions canbe drawn about its effectiveness. However, our study presentssome remarkable brain biochemical alterations that followed saf-fron co-treatment.

Determination of ChE activity showed that the DS-AChE waspredominant in cerebral tissues, while approximately equal valuesof the activity of the two BuChE isoforms were recorded. Theseobservations are consistent with previous findings (Das et al.,2005; Fernández-Gómez et al., 2008). In accordance with otherstudies (Lassiter et al., 1998; Li et al., 2000), AChE activity consti-tutes the major fraction of total ChE activity in whole brain (-ce)and cerebellum, whereas BuChE activity predominates in liver. Inthe current work, Al intake resulted in significant decrease of theactivity of AChE and BuChE isoforms in mouse whole brain and cer-ebellum. Region-specific inhibition of the activity of rat cerebralmembrane-bound AChE and BuChE after Al intoxication, has beenshown by others (Julka et al., 1995). Previous studies have shownnecrotic rat hippocampal ultrastructural changes following thesame Al administration protocol (Jyoti and Sharma, 2006), provid-ing an explanation for our cholinotoxic and memory impairmentresults. However, Al has been suggested to interfere with the ac-tion of metabotropic glutamate receptors, enhancing excitotoxicityand neuronal injury (Blaylock, 2012). Also, Al impairs hippocampallong-term potentiation (LTP) in rats (Platt et al., 1995). Thus, be-sides the cholinotoxic effects of Al in the present study, theinvolvement of possible Al-induced glutamatergic neurotransmis-sion disturbances in the observed memory decline, should alsobe considered. In liver, both BuChE isoforms’ activity (the main li-ver ChE fraction) was significantly decreased following Al adminis-tration, while only SS-AChE activity was decreased significantly.However, increased activity of soluble and membrane-boundforms of rat liver BuChE after Al intake through diet for 100–115 days (Dave et al., 2002), has been previously demonstrated,but the Al dose and period of exposure are different.

Saffron short-term co-administration caused further significantreduction of cerebral AChE and liver BuChE isoforms activity. Cere-bral BuChE activity was not further affected and only SS-AChEactivity of liver was further significantly decreased after saffronco-treatment. The inhibitory effect of saffron on the activity ofbrain SS- and DS-AChE in adult healthy mice has been demon-strated in our previous study (Papandreou et al., 2011). However,the impact of saffron administration on brain BuChE and liver

ChE activities, has not been studied before. The considerable higherpercentage of inhibition of DS-AChE in whole brain of Al + saffrontreated mice compared to SS-AChE, suggests greater susceptibilityof the enzyme’s DS isoform to saffron treatment. Geromichaloset al. (2012) have recently demonstrated, for the first time, thatcrocetin inhibits AChE by binding at two different loci, the catalyticcenter and the peripheral anionic sites, as assessed by in vitro enzy-matic and molecular docking studies. Thus, the observed signifi-cant cerebral AChE inhibitory activity conferred by saffron short-term co-administration, may be attributed to a direct interactionof crocetin with the enzyme, since crocetin was detected in brainof Al + saffron treated mice and was absent in controls’ brain; how-ever, it remains to be investigated whether the crocetin concentra-tion is sufficient for such an effect. Furthermore, other indirectmechanisms cannot be excluded.

Monoamine oxidase (MAO, types A and B) is a mitochondrialmembrane-bound enzyme which catalyzes the oxidative deamina-tion of monoamines, thus regulating monoaminergic neurotrans-mission. Increased brain MAO activity has been recorded inneurodegenerative process (Hanish Singh et al., 2011; Mallajosyulaet al., 2008). Our results showed significant increase of whole brainMAO isoforms activity after Al intoxication, and an opposite effecton cerebellum MAO-B activity. Bhalla et al. (2010) have also pre-sented increased and decreased MAO activity in rat cerebrumand cerebellum, respectively, after long-term oral administrationof AlCl3. Activation of MAO isotypes in rat brain by prolonged in-take of Al has been also previously reported (Huh et al., 2005),but the underlying mechanisms have not yet been clarified.

Saffron co-treatment completely reversed Al-induced activationof brain MAO isoforms and inhibition of cerebellar MAO-B. The ef-fect of saffron administration on cerebral MAO activity has notbeen studied before. MAO inhibitors have long been well charac-terized for their antidepressant properties (Bortolato et al., 2008).The first small-scale clinical trials of saffron in the treatment ofmild to moderate depression, have shown significant benefits inthe mood of patients after a 6-week treatment (Akhondzadehet al., 2005; Noorbala et al., 2005). The antidepressant efficacy ofsaffron could, at least in part, correlated with brain MAO inhibitoryactivity of saffron extract and the presence of crocetin in brain thatare presented in our current study.

Oxidative stress has long been implicated in the initial and laterstages of neuronal degeneration (Melo et al., 2011). In the presentstudy, Al intake elevated significantly lipid peroxidation in wholebrain and decreased GSH content in both cerebral tissues and liver.Other reports have also demonstrated increased lipid peroxidationlevels and decreased GSH content in rat brain regions after long-term oral administration of AlCl3 (Jyoti and Sharma, 2006; Nehruand Bhalla, 2006). The resistance of liver to Al-induced lipid perox-idation, indicated by us and other studies (Kaneko et al., 2004), isexpected, as it constitutes the major site of detoxification in livingorganism. Although Al is not a redox-active metal, there is exten-sive experimental evidence on oxidative stress-mediated Al neuro-toxicity (Kumar and Gill, 2009). It has been demonstrated that Alinduces oxidative stress in cultured rat hippocampal neurons bypotentiating iron-mediated oxidative injury (Xie et al., 1996). Also,in vitro studies (Oteiza, 1994) have shown that Al stimulates iron-induced lipid peroxidation in isolated membrane fractions throughbinding to the membrane and promotion of changes in thearrangement of membrane lipids. Considering the byproduct(hydrogen peroxide) of MAO-mediated reactions, Al-induced acti-vation of brain MAO isoforms provided by our results could, atleast in part, contribute to the observed increased brain oxidativestress.

Reversal effects of saffron short-term co-administration wererecorded against Al-induced brain lipid peroxidation and GSH con-tent reduction in cerebral tissues and liver. In our previous study

Z.I. Linardaki et al. / Food and Chemical Toxicology 52 (2013) 163–170 169

(Papandreou et al., 2011), saffron administration significantlydecreased MDA levels and increased GSH content in the brain ofhealthy adult and aged mice. Shati et al. (2011) also showed thatintraperitoneal co-administration of saffron aqueous extract withAlCl3 ameliorated the disturbances in mouse brain lipid peroxida-tion and antioxidant enzymes’ activity induced by the metal wheninjected alone. Additionally, in our previous in vitro work (Papand-reou et al., 2011) both saffron extract and its crocetin componentprovided strong protection against H2O2-induced toxicity inhuman neuroblastoma SH-SY5Y cells, by repressing reactiveoxygen species production. It has been reported that crocin, thedi-gentiobiosyl ester of crocetin, promotes mRNA expression ofc-glutamylcysteinyl synthase, the rate-limiting enzyme of GSHsynthesis, in PC12 cells under hypoxic conditions (Ochiai et al.,2007). Accordingly, crocetin may mediate the observed brain anti-oxidant protection of saffron extract under Al neurotoxicity.

5. Conclusions

Our findings show that long-term intake of a relative high doseof AlCl3 through drinking water resulted in metal accumulation inadult mouse brain tissues and exerted neurotoxic effects, as evi-denced by the declined learning and memory capacity, metal-induced inhibition of ChEs, changes of MAO and the productionof oxidative damage. On the other hand, short-term co-administra-tion of saffron extract at the end of the treatment period benefi-cially affected mouse brain oxidative stress and antioxidantstatus markers and MAO activity that were disturbed by Al. Thebioavailability of crocetin in mouse brain after saffron extractadministration through intraperitoneal route demonstrated in thepresent study, supports the implication of this bioactive compo-nent in the observed neurochemical alterations. However, the par-ticular saffron treatment scheme was ineffective in reversingcognitive defects induced by the metal, although it had beneficialaction on biochemical markers of brain function. The findingssupport further investigation of the potential of saffron and itscrocin constituents as neuroprotective agents.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was partially supported by BIOFLORA network ofUniversity of Patras. Also, partial support from the University of Pa-tras Research Committee is greatly acknowledged.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.fct.2012.11.016.

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