Behavioural Brain Research 219 (2011) 197204

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Behavioural Brain Research

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Memory enhancing effects of saffron in aged mice are correlated with antioxidant protection
Magdalini A. Papandreou a , Maria Tsachaki b , Spiros Efthimiopoulos b , Paul Cordopatis c , Fotini N. Lamari c , Marigoula Margarity a,
a b c

Laboratory of Human & Animal Physiology, Department of Biology, University of Patras, 26504 Patras, Greece Division of Animal & Human Physiology, Department of Biology, University of Athens, Greece Laboratory of Pharmacognosy & Chemistry of Natural Products, Department of Pharmacy, University of Patras, Greece

a r t i c l e

i n f o

a b s t r a c t
Brain aging is characterized by cognitive decline and memory decits that could be the result of oxidative stress and impaired cholinergic function. In this study, the effects of a daily, 7-day, intraperitoneal administration of saffron on cognitive functions were examined in both healthy adult (4 months old) and aged (20 months old), male Balb-c mice (n = 8/group), by passive avoidance test. Whole brain homogenates (minus cerebellum) were collected for examination of brain oxidative markers, caspase-3 and acetylcholinesterase (AChE) activity. Results showed that saffron-treated mice exhibited signicant improvement in learning and memory, accompanied by reduced lipid peroxidation products, higher total brain antioxidant activity and reduced caspase-3 activity in both age groups of mice. Furthermore, saltand detergent-soluble AChE activity was signicantly decreased only in adult mice. Thus, we showed, for the rst time, that the signicant cognitive enhancement conferred by saffron administration in mice, is more closely related to the antioxidant reinforcement. Next, we compared the effect of saffron (1250 g/mL), crocetin and safranal (1125 M) on H2 O2 -induced toxicity in human neuroblastoma SH-SY5Y cells. Both saffron and crocetin provided strong protection in rescuing cell viability (MTT assay), repressing ROS production (DCF assay) and decreasing caspase-3 activation. These data, together with earlier studies suggest that crocetin is a unique and potent antioxidant, capable of mediating the in vivo effects of saffron. 2011 Elsevier B.V. All rights reserved.

Article history: Received 29 September 2010 Received in revised form 31 December 2010 Accepted 7 January 2011 Available online 14 January 2011 Keywords: Crocetin Saffron Safranal Acetylcholinesterase Learning/memory SH-SY5Y Oxidative stress Free-radicals Aging

1. Introduction Normal aging is associated with a slow decline in brain functions such as sensory and motor performance, and at times, this decline is accompanied by progressive memory loss, dementia

Abbreviations: Ac-DEVD-pNA, Asp-Glu-Val-Asp p-nitroanilide; Acetyl-CoA, acetyl coenzyme A; ACh, acetylcholine; AChE, acetylcholinesterase; ATCC, American type culture collection; ATCI, acetylthiocholine iodide; BW, body weight; ChAT, choline acetyltransferase; DCF, 2 ,7 -dichlorouorescein; DCFH-DA, 2 ,7 dichlorouorescein diacetate; DMSO, dimethyl sulphoxide; DS, detergent soluble; EDTA, ethylenediaminetetraacetic acid; FeSO4 , ferrous sulphate; FRAP, ferric reducing antioxidant power; FRASC assay, ferrous reducing ascorbic acid assay; GSH, reduced glutathione; H2 O2 , hydrogen peroxide; i.c.v., intracerebroventricullar injections; IL, initial latency; i.p., intraperitoneal injections; MTT, 3,(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide; MDA, malondialdehyde; OH, hydroxyl radical; 6-OHDA, 6-hydroxydopamine hydrobromide; PBS, phosphate-buffered saline; p-NA, p-nitroanilide; ROS, reactive oxygen species; RT, room temperature; SH-SY5Y, human neuroblastoma cells; SS, Salt-soluble; STL, step-through latency; TPTZ, 2,4,6-Tris(2-pyridyl)-s-triazine. Corresponding author. Tel.: +30 2610 997430; fax: +30 2610 969273. E-mail address: margar@upatras.gr (M. Margarity). 0166-4328/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2011.01.007

and cognitive dysfunctions, ultimately resulting in limited functionality. In both aged humans and rodents, cognitive impairment has been correlated to the accumulation of oxidative damage to lipids, proteins, nucleic acids [8,16,44] and the vulnerability of various neurotransmitters/neurotrophin systems activity to oxidative stress [21,40,60]. In addition, an age-related decline in cholinergic function is thought to be partially responsible for the memory disorders occurring during senescence. One of the major markers of cholinergic function is the activity of the enzyme acetylcholinesterase (AChE) which is known to be decreased with aging in various cerebral areas [54] and synaptic plasma membranes [20]. AChE activity is also known to be decreased by free radicals and increased oxidative stress [41]. This data has led to the suggestion that various antioxidant supplements and phytochemical components might be benecial for preserving brain functions and forestalling the age-related decits [64]. Crocus sativus L. is cultivated in many countries for the culinary uses of its styles (saffron). For more than 3000 years saffron is in constant use as a drug. Particularly in traditional Indian medicine, saffron has been used for the treatment of cogni-

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M.A. Papandreou et al. / Behavioural Brain Research 219 (2011) 197204 into the dark compartment, before, and 24 h, after the delivery of a mild foot shock (25 V, 3 mA, 5 s) to their paws. All training and testing sessions were carried out during the light phase (08:00 to 14:00 h). 2.4. Tissue preparation Mice were killed on day 7 by transcardial perfusion with ice-cold 0.95% NaCl (10 mL/10 g BW) and the brain (minus the cerebellum) was weighed and homogenized (10%, w/v, 30 mM Na2 HPO4 , pH 7.6) with a glass homogenizer (Thomas, Philadelphia, USA, No. B 13957) at 9500 rpm, thrice. The homogenates were then centrifuged (23,113 g, 4 C, 2 h) to recover the salt-soluble fraction (SS) of AChE. The pellets were re-extracted with an equal volume of 30 mM Na2 HPO4 , pH 7.6, containing 1% Triton X-100 and the suspensions were centrifuged at 23,113 g at 4 C for 2 h to recover the detergent-soluble fraction (DS) [11]. Protein concentrations were determined by the Bradford assay. 2.5. Acetylcholinesterase (AChE) activity determination AChE activity was determined using the colorimetric assay of Ellman, as previously described [14,53], with acetylthiocholine iodide (ATCI) as a substrate. The AChE activity was expressed as mmol/min/g of tissue protein. 2.6. Measurement of caspase-3 activity in mice brain Tissue samples (100 mg/mL) were assayed with the caspase-3 colorimetric assay (CASP-3-C, SigmaAldrich) kit, according to the manufacturer protocol, using acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) as substrate. Caspase-3 was expressed as nmol/h/mg of protein. The assay showed good linearity (1500 M, y = 0.0022x 0.0104, R2 = 0.9971). 2.7. Assessment of antioxidant parameters Ferric-reducing antioxidant power (FRAP assay) measures the ability of antioxidants to reduce the [Fe(TPTZ)2 ]3+ complex to the blue-coloured [Fe (TPTZ)2 ]2+ [6] and was assayed as previously described [53]. Ferrous sulphate (FeSO4 ) was used as a standard and the assay showed good linearity (101000 M, y = 0.001x 0.0009, R2 = 0.9986) and sensitivity. The antioxidant capacity of the brain samples was expressed as mol FeSO4 /g of wet tissue. In the above mentioned set-up for the FRAP assay, incubation with ascorbate oxidase from Curcubita sps (SigmaAldrich) (4 U/mL), prior to the addition of the FRAP reagent, enables the determination of ascorbic acid (FRASC assay) [47]. The experiment was performed as previously described [6]. FRASC showed good linearity (5250 M, y = 0.0018x + 0.0017, R2 = 0.9993) and sensitivity with a detection limit of 5 M ascorbic acid. Malondialdehyde (MDA) was determined uorometrically after reaction with thiobarbituric acid, as previously described [22,26,53]. The assay showed good linearity (0.055 M, y = 6.7697x 0.29, R2 = 0.9907) and sensitivity. The level of MDA was expressed as mol/g tissue protein. The level of reduced glutathione (GSH) was estimated uorometrically after reaction with o-phthalaldehyde, as previously described [40,53]. A good linearity was obtained for GSH in the range of 0.5100 M (y = 1.492x 0.5693, R2 = 0.9972). The level of GSH was expressed as mol/g of protein. 2.8. Cell culture and drugs treatment

tive dysfunctions. Chemical analysis has revealed the presence of unusual carotenoids glycosides (crocins), which are mono-, or diglycosyl-esters of crocetin, (2E,4E,6E,8E,10E,12E,14E)-2,6,11,15tetramethyl-2,4,6,8,10,12,14-hexadecahepta-enedioic acid, and small amounts of monoterpene aldehydes, like picrocrocin and safranal [71]. Saffron is efcient in enhancing cognitive behavior in adult rodents which have previously been exposed to amnestic agents (e.g. scopolamine, ethanol or acetaldehyde) [57,58,69,70,79]. Saffron was effective in inhibiting TNF -induced apoptosis of PC12 cells [66] and protected neurons from the neurotoxic activity of 6-hydroxydopamine hydrobromide [2]. Crocin constituents also inhibited amyloid aggregation in vitro [54], while saffron was shown to be similarly effective with donepezil in patients with mild-to-moderate Alzheimers disease [3]. Previous in vitro studies [51,54] have shown that saffron has antioxidant properties, albeit moderate. However, despite the continuously increasing literature on saffron neuroprotective effects in vitro and in various disease-models, none has so far examined its effect either on healthy adult and aged mice, or on cholinergic neurotransmission. The aim of the present study was to investigate the effect of a 7-day intraperitoneal administration of saffron [60 mg/kg body weight (B.W.)] on learning and memory of healthy, adult and aged, male Balb-c mice, their brain AChE activity, caspase-3 activity and oxidative status. Results showed that the short-term supplementation of both healthy adult and aged mice signicantly enhanced cognitive performance in the passive avoidance test and attenuated brain oxidative stress markers, whereas AChE activity was attenuated signicantly only in adult mice. In order to further investigate the antioxidant properties of saffron in stress conditions and identify the bioactive components, human neuroblastoma SH-SY5Y cells were incubated with saffron extract, crocetin and safranal. Results showed that both saffron and crocetin provided strong protection and inhibited ROS production and caspase-3 activation.
2. Materials and methods 2.1. Plant material and extraction Greek saffron (red threads) was kindly provided by the Cooperative Association of Krokos in Kozani, in West Macedonia, Greece. The preparation of the extract was performed as previously described [54]. Crocetin (CRT) (purity >98%) was prepared by saponication of saffron extract, as previously described [28], while safranal was purchased from SigmaAldrich Corporation, St. Louis, MO, USA. 2.2. Animals The animals used in this study were adult (4-months-old) and aged (20-monthsold) male Balb-c mice. Gross examination of the animals at sacrice suggested aging related changes (i.e. hair loss) but not overt disease (e.g. tumors, dermatopathies etc). Mice were kept in polyacrylic cages (38 cm 23 cm 10 cm) with eight animals per cage and housed in a room under controlled temperature 2426 C, relative humidity 5060% and with 12 h lightdark cycles. Food in form of dry pellets and water were available ad libitum. The inbred rst generation male mice (n = 16, 4 months old; 35.8 1.3 g BW) were randomly divided into 2 groups: a vehicle group (Control) and a saffron (60 mg/kg BW)-treated group (n = 8/group). Respectively, aged animals (n = 16, 20 months old; 39.0 2.1 g BW) were accordingly divided to a Control and a saffron (60 mg/kg BW)-treated group (n = 8/group). All procedures were in accordance with Greek National Laws (Animal Act, PD 160/91). Saffron extract was administered i.p. daily at a nal volume 20 L, while control mice received 20 L of saline. Saffron solution was prepared fresh daily, right before administration, by dissolving the dry extract in saline and by ltering through membrane lters of 0.2 m internal diameter. 2.3. Behavioral testing: passive avoidance task Mice were subjected to a passive avoidance test [27,29] on day 6, after a double training and an initial acquisition trial on day 5. The test is based on negative reinforcement to examine long-term memory [43,50]. It was performed according to previously described procedures [53] using a two-compartment passive avoidance apparatus (white/dark, separated by a black wall with a guillotine door in the middle part). Results were expressed as the mean initial (IL, max. time 120 s) and step-through (STL, max. time 300 s) latency times recorded once animals had crossed

Human neuroblastoma SH-SY5Y cell line was obtained from the American Type Culture Collection (ATCC). Cultures were grown in DMEM containing 10% heat-inactivated fetal bovine serum, 4.5 g/L glucose, 2 mM l-glutamine, 100 g/mL penicillin and 100 g/mL streptomycin sulphate. Conuent cultures were subcultured using 0.5 g/L trypsin/0.2 g/L EDTA into 96-well plates at a density of 1.5 104 cells/well. All experiments were carried out 24 h after the cells were seeded. Prior to all treatments, cells were incubated in serum-free media, for 24 h. Finally, cells were co-incubated with 250, 500 and 750 M H2 O2 and the tested phytochemicals (1250 g/mL saffron and 1125 M CRT and safranal), or vehicle as control (medium containing max. 0.1%, v/v DMSO), for 18 h. For caspase-3 experiments, cells were incubated with 250 M H2 O2 in the presence or absence of the tested phytochemicals (1250 g/mL saffron and 1125 M CRT and safranal), or vehicle as control (medium containing max. 0.1%, v/v DMSO), for 12 h. 2.9. Assessment of cell viability and reactive oxygen species (ROS) levels Cell viability was estimated using 3,(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT reagent) [59]. Briey, on completion of the treatment, cells were washed with pre-warmed PBS and incubated with DMEM w/o containing MTT at the nal concentration of 2.5 mg/mL for 150 min. Supernatants were carefully aspirated and the resultant formazan product was dissolved in DMSO and detected by a UVvis spectrometer (Denley WellScan, West Sussex, UK) at 545 nm. The formation of ROS was evaluated by means of the probe 2 ,7 dichlorouorescein diacetate (DCF-DA) (SigmaAldrich Corporation, St. Louis, MO, USA) [modied by [76]]. Cells were washed with pre-warmed PBS and incubated

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Fig. 1. The effect of saffron on initial latency (IL) and step-through latency (STL) in the double trial passive avoidance test, in (A) adult and (B) aged Balb-c mice. Individual values are presented as circles (closed for IL and open for STL) for control animals and triangles (closed for IL and open for STL) for saffron-treated ones, whereas lines represent the mean values. IL: max. time allowed 120 s and STL: max. time allowed 300 s. Statistical analysis was performed with GraphPad Instat 3 software, using the non-parametric MannWhitney test (n = 8 animals/group). (a) p < 0.01 statistical difference in comparison to control. (b) p < 0.01 statistical difference between the two age groups of mice receiving saffron extract. with 50 M DCF-DA in PBS at 37 C for 30 min. Fluorescence was then quantied in a microplate-reader (Bio-Tek Instruments Inc., Ville St. Laurent, Que, Canada) at excitation wavelength 485 nm and emission wavelength 530 nm. 2.10. Measurement of caspase-3 activity in SH-SY5Y cells Caspase-3 activity was measured with acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) as the substrate, as per the manufacturers protocol (CASP-3-C, SigmaAldrich). Liberation of p-NA was measured at 405 nm with a UVvis spectrometer (Denley WellScan). The changes in absorbance were standardized using graded concentrations of free p-NA solutions (1200 M). The assay showed good linearity (y = 0.0022x + 0.0077, R2 = 0.998). Results were calculated as release of p-NA in pmol/min/mg protein. 2.11. Statistical analysis Data are presented as means S.E. Statistical analysis was performed with GraphPad Instat 3 software (GraphPad Instat Software, Inc. USA) using the nonparametric MannWhitney test (p < 0.05). In all tests, a criterion of p 0.05 (two-tailed) was considered necessary for statistical signicance.

3. Results 3.1. Effect of saffron on rodent passive avoidance test learning ability Passive avoidance behavior is based on negative reinforcement and is used to examine long-term memory. Step-through latency reected the long-term memory of animals. Signicant increase in step-through latency value shown improvement in memory. The effect of saffron injected i.p. to mice for 6 days on the stepthrough latency is shown in Fig. 1. No signs of writhing syndrome or changes in the emotional state of mice were observed after safTable 1 The effect of saffron on AChE activity in adult and aged mice braina . Animal groups Adult Control Saffron-treated Aged Control Saffron-treated Salt soluble (SS)-AChE (mmol/min/g of tissue protein) 0.211 0.008 0.102 0.003** 0.111 0.007 0.103 0.002

fron administration. As shown in Fig. 1, the performance of each mouse was largely concise, although the motor activity of aged mice appeared slightly reduced. The latter observation is reected by lower mean initial and step-through latencies of both groups of aged mice than those of adult mice. For instance, control aged mice showed a 6977% decrease in their latency times, in comparison to their control adult littermates. In the control and saffron-treated groups of adult mice, the mean initial latency was not signicantly different (32.75 9.02 and 23.63 5.62, respectively) indicating that both groups behaved the same in the training trial. The same behavior was observed in the control and the saffron-treated aged mice (7.64 1.56 and 10.99 1.91, respectively), suggesting no major age-related differences in latency time prior to training. Saffron-treated adult mice exhibited signicant increase (149%, p = 0.03) in stepthrough latency, as compared to the control group (246.25 35.95 vs. 94.25 34.74). Accordingly, saffron-treated aged mice exhibited signicantly higher latencies than their control littermates (68.38 12.60 vs. 29.62 3.76). Overall, saffron administration facilitated learning in both adult and aged mice, as is evident by the delay of transfer in the dark chamber (shock) 1 day after the acquisition trial. 3.2. Effect of saffron on brain AChE activity Given the implication of the cholinergic system in maintaining normal cognitive function, the activity levels of AChE were measured in mice brain, with the salt- and detergent-soluble fractions of brain homogenates as a marker of central cholinergic status. The molecular forms G4 and G1 of AChE are predominantly present in

Detergent soluble (DS)-AChE (mmol/min/g of tissue protein) 0.818 0.049 0.509 0.022** 0.667 0.033 0.754 0.053

a Animals were administered with an i.p. daily injection (20 L) of either saline (control group) or 60 mg/kg B.W saffron extract (saffron group) for 7 days (n = 8 animals/group). The mice brain AChE levels are expressed as mean S.E. of 8 values each analyzed in triplicate. Statistical analysis was performed with GraphPad Instat 3 software, using the non-parametric MannWhitney test. ** p < 0.0001 statistical difference in comparison to control. p < 0.05 statistical difference between the two age groups of control mice. p < 0.01 statistical difference between the two age groups of control mice.

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Table 2 Effect of saffron in biochemical parameters of mice brain oxidant statusa . Animal groups Adult Control Saffron-treated Aged Control Saffron-treated FRAP (mol FeSO4 /g wet tissue)b 7.11 0.13 8.02 0.24** 5.25 0.30 8.59 0.32** Ascorbic acid (g/g wet tissue)b 115.90 6.86 152.50 6.05** 89.19 11.75 219.88 11.51* , MDA (mol/g protein)c 2.18 0.16 1.20 0.11** 2.80 0.18 1.56 0.10** GSH (mol/g protein)c 25.83 1.08 29.94 1.21** 19.78 1.35 23.46 0.74* ,

Statistical analysis was performed with GraphPad Instat 3 software, using the non-parametric Mann-Whitney test. a Animals were administered with an i.p. daily injection (20 L) of either saline (control group) or 60 mg/kg B.W. saffron extract (saffron-treated group) for 7 days (8 animals/group). b Data are the mean S.E. of 8 values each analyzed in triplicate. c Data are the mean S.E. of 8 values each analyzed once.parametric MannWhitney test. * p < 0.05 statistical difference in comparison to control. ** p < 0.001 statistical difference in comparison to control. p < 0.05 statistical difference between the age groups. p < 0.001 statistical difference between the age groups.

detergent (DS) and salt (SS) soluble fraction of brain homogenate, respectively. AChE activity in the salt-soluble fractions of brain tissue of control aged mice was signicantly lower (p < 0.05) than that of control adult mice (Table 1). In adult mice, saffron administration resulted in a signicant decrease in AChE-specic activity in both fractions, with the percentage of inhibition ranging between 38 and 52%. However, saffron failed to affect AChE activity in whole brain of aged mice.

Thus, it appears that saffron administration conferred signicant antioxidant protection and attenuated caspase-3 activity in both age groups of mice. 3.4. H2 O2 -induced toxicity on human neuroblastoma SH-SY5Y cell line H2 O2 can freely penetrate the cell membrane and generates the highly reactive hydroxyl radical, OH, within the cell, ultimately inducing multiple pathways that will eventually result into an apoptotic cascade of events. To determine the effective concentrations of H2 O2 , SH-SY5Y cells were treated with various concentrations of H2 O2 (501000 M), for 18 h, and both the viability and freeradicals production of the cells were assessed by the MTT-reducing activity and DCF-assay. Treatment with 50 and 100 M H2 O2 for 18 h had virtually no great effect on the viability of the cell line (data not shown). Treatment with 250, 500 and 750 M H2 O2 decreased cell viability by 43 1%, 52 1% and 63 1%, respectively, compared to untreated cells. ROS accumulation was also signicantly increased in the SHSY5Y cell line after incubation with H2 O2 (250 M), as shown by the increase in relative uorescence units (73,026 4566 DCF/MTT values) compared to the untreated (control) cells (45,973 2535 DCF/MTT values). To determine whether the H2 O2 -induced cytotoxicity was due to apoptosis, activation of caspase-3 was determined, using Ac-DEVD-pNA as a substrate. As shown in Table 3, caspase-3 was signicantly activated (up to 52%) after incubation of the neuroblastoma SH-SY5Y cells with 250 M H2 O2 , for 12 h. 3.5. Effects of saffron against H2 O2 -induced toxicity on SH-SY5Y cell line As illustrated in Fig. 2 treatment of SH-SY5Y cells with saffron and crocetin for 18 h, signicantly prevented the cell death caused

3.3. Effect of saffron on brain oxidative status and caspase-3 activity Since oxidative stress can affect both the aging process and AChE activity [8], we examined the existence of a possible correlation between age-related alterations in AChE and brain oxidative status and caspase-3 activity. As shown in Table 2, the whole brain homogenates of control aged mice had statistically signicant lower total antioxidant activity (26%) and ascorbic acid (23%) levels than control adult mice. Of statistical importance were also the increases observed in the whole brain lipid peroxidation products (22%) in aged mice, which was accompanied by a 23% decrease in GSH levels (Table 2). Saffron administration signicantly increased the total brain antioxidant activity, ascorbic acid and GSH levels in both age groups of mice. Saffron administration also caused a marked decrease in MDA levels (4463%) (Table 2). Comparison of the control littermates of both age groups showed a statistically signicant increase (17%, p = 0.02) in the levels of caspase-3 in aged mice (52.9 2.1 nmol pNA/h/mg vs. 45.4 1.4 nmol pNA/h/mg protein), which is in agreement with the increased levels of MDA, previously mentioned. Administration of saffron attenuated caspase-3 activity by 13% in adult mice (39.9 1.5 nmol pNA/h/mg vs. 45.4 1.4 nmol pNA/h/mg protein of the control) and 25% in aged mice (39.5 1.3 nmol pNA/h/mg vs. 52.9 2.1 nmol pNA/h/mg protein of the control), respectively.

Table 3 Effect of saffron, crocetin and safranal against H2 O2 -induced activation of caspase-3 levels in SH-SY5Y cellsa . Treatment 80 min 120 min Control 2.80 0.10 1.84 0.07 250 M H2 O2 (12 h) 4.16 0.15 2.80 0.10 +10 g/mL saffron (12 h) 2.99 0.09 2.21 0.31 +10 M CRT (12 h) 2.84 0.17* 1.96 0.10* , +10 M safranal (12 h) 3.31 0.05 2.08 0.07* ,

a Absorbance of the hydrolysis of caspases-3 substrate was measured at 405 nm for 80 min and 120 min, respectively, after treatment of the SH-SY5Y cells with 250 M H2 O2 , in the presence or absence of saffron (10 g dry extract/mL) and its constituents (10 M CRT/or safranal), for 12 h. Results were calculated as release of p-NA in pmol/min/mg protein. Caspase-3 levels are expressed as mean S.E. of four independent experiments analyzed in duplicate. Statistical analysis was performed with GraphPad Instat 3 software, using the non-parametric MannWhitney test. * p < 0.05 statistical difference in comparison to H2 O2 . p < 0.05 statistical difference in comparison to control.

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Fig. 2. The effects of the tested phytochemicals on H2 O2 -induced cytotoxicity on SH-SY5Y human neuroblastoma. Cells were co-treated with varying concentrations of H2 O2 (250750 M) and the tested phytochemicals (1250 g dry extract/mL for saffron and 0.1125 M for CRT and safranal, respectively) for 18 h. Cell viability was determined by the MTT assay. Experimental values are the mean S.E. values of six determinations and each included quadruple sets. Statistical analysis was performed with GraphPad Instat 3 software, using the non-parametric MannWhitney test. * p < 0.0001 vs. H2 O2 . p < 0.05 vs. control. Percentages indicate the percentage of cell death in the presence of the H2 O2 .

by H2 O2 , in a non-concentration dependent manner, with the percentages of viability ranging from 69 to 98% in relation to the control group (100% viability). Although safranal co-incubation led to signicantly higher viability (5671%) compared to H2 O2 -treated, it failed to preserve viability levels equal to those in untreated cells. Moreover, co-incubation of SH-SY5Y cells with the tested phytochemicals and H2 O2 completely abolished H2 O2 -induced ROS accumulation, in a non-concentration-dependent manner

(Fig. 3AC). Saffron and safranal treatment preserved ROS levels equal to those in untreated cells, whereas crocetin reduced, further, ROS levels at all tested concentrations. Furthermore, both saffron and its constituents attenuated H2 O2 -induced caspase-3 activation, in the order of CRT (3038%) > safranal (2131%) > saffron (2138%) (Table 3), even up to 120 min of absorbance measurement, with the enzymes levels being near to those of the untreated cells. Thus, cell culture results showed that saffron and crocetin provide strong protection in

Fig. 3. Effect of saffron and its constituents on H2 O2 -induced ROS accumulation on wild type (SH-SY5Y) human neuroblastoma cells. Quantitative analysis of DCF uorescence intensity after treatment with 250 M H2 O2 in the presence/or absence of test compounds at various concentrations for 18 h. Values are mean S.E. calculated from six different experiments performed in sixtruple. Statistical analysis was performed with GraphPad Instat 3 software, using the non-parametric MannWhitney test. * p < 0.05 vs. H2 O2 alone. p < 0.05 vs. control. Percentages indicate the percentages of decrease observed after incubation with the tested phytochemicals in the presence of H2 O2 .

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rescuing cell viability, repressing ROS production and decreasing H2 O2 -induced caspase-3 activation.

4. Discussion Aging is characterized by a deterioration of cognitive function, including learning and memory. In the present investigation, in order to determine if Balb-c mice displayed age-related memory impairments, the retention of an aversive experience was measured. Passive avoidance paradigm is widely utilized for testing learning and memory in rats and mice [27,29]. In this procedure, the acquisition latency (initial latency) to enter the compartment on the rst day is an indicator of visual ability and motor activity and the retention latency (step-through latency) to enter the compartment on the second day indicates memory of the aversive experience. In the present investigation, the mean initial latency and step-through latency values for the control aged mice were signicantly lower (6977%) than those of the control adult mice. Our ndings are in agreement with numerous studies showing that aging is associated with decreases in fear conditioning in rats and mice [30,65]. A substantial part of age-related memory impairments have been attributed to alterations in various neurotransmitter systems [7,38], among which is the cholinergic; deteriorations in the integrity of basal forebrain cholinergic neurons have been shown to occur along with many other changes in limbic/cortical systems [13,15,17,35,45]. The cholinergic hypothesis of geriatric memory dysfunction suggests that the reduction of cholinergic markers, such as acetylcholine (ACh), choline acetyltransferase (ChAT) and AChE, is a critical component for the age- and dementia-associated memory decits [5,56,72]. Additional lines of mostly indirect evidence establishing a relationship between age-related changes in mnemonic function and the cholinergic system come from studies in both humans and rodents, after either brain lesions and/or administration of anti-cholinergic drugs [9,10,17,18]. The alterations occurring in the cholinergic system of mouse brain as a function of aging were examined in the present study by recording AChE activity, the main enzyme responsible for the metabolism of ACh to choline and acetyl-CoA. AChE exists into different molecular forms, each of which presents different localization in the neuron. The G1 form, for instance, is cytosolic, while the G4 form is membrane bound and requires detergent for solubilization. Therefore, the SS- and DS-fractions of AChE contain predominantly the G1 and G4 forms, respectively [36]. AChE is an important therapeutic target. Reversible inhibitors of this enzyme have been used as cognitive enhancers in the treatment of Alzheimers disease and other dementia disorders [31,33,73]. Total AChE activity is found to be lower in AD than in normal brains [9,30,50], while decreases in AChE activity have also been reported with aging in various cerebral areas [53,60,63] and synaptic plasma membranes [20]. Signicantly lower (p < 0.001) levels of AChE were observed in control aged (20-months-old) mice whole brains than in their adult littermates, mainly because of a decrease of the G1 form (47% vs. 18% of G4 ). The former observation could possibly be ascribed to a possible loss of post-synaptic enzyme activity [78], rather than a loss of cholinergic neurons during aging, as according to Lams et al. [32] the expression of AChE can be regulated independently of the survival of cholinergic neurons following an insult. Our results are in good agreement with previous reports on aged rodents [12,30,37,75]. For instance, in rats (<12-months-old), a progressive decrease in AChE concentration was observed with aging in the cerebral hemisphere [42] and more specically in the cerebral cortex [23]; minor or no alterations where observed in rat cerebellum or in the caudate nucleus [39], while similar age-dependant alteration in whole SS-brain

homogenates have also been reported for male Fischer rats [62]. Signicant age-related decreases in AChE have also been reported in whole brain of C57BL/10 mice (>16-months-old) [52], and in the hippocampus of C57BL/6J mice [75], but not in the cerebellum. In the light of these ndings, it seems that there are regional [68] and dynamic age changes in AChE, since the largest declines occurred in specic brain areas and before 1216-months of age, suggesting thus, that the reason behind AChE changes is far more complicated than initially thought of and might be more related to development than aging [63]. This assumption is interesting in view of the conicting reports from both human and animals on the validity of the cholinergic hypothesis, with the studies showing age-related changes in cholinergic markers being about equal in number to those that failed to demonstrate this loss. Reasons mentioned to account for these discrepancies are differences in species and strains of experimental animals, age and sex, tissue sampling and methods of assay. Although it is not possible, at present, to assign a denite factor to explain the pattern of decit observed in the enzyme activity in old mice, previous studies in both rodents and humans have shown that there is a close correlation between age-related alterations in AChE and increased oxidative stress [41], as well as between agerelated losses of cognitive function with oxidative protein damage in the brain [16,46]. Specically the current study has demonstrated higher caspase-3 activity and MDA levels, along with decreased antioxidant activity (GSH, total antioxidant power and ascorbic acid levels) in control aged mice (Table 2). Similar age-dependent declines have also been reported in the literature in most tissues in aged rodents [23,25,67]. Saffron administration forestalled/reversed those age-related decits. In particular, short-term administration of saffron resulted in enhanced cognitive function in both adult and aged mice, as evidenced by the signicantly high STL values (131149%) 1 day after the aversive experience. These results are in line with other reports on the long-term memory enhancing effects of saffron administration on chemically-induced amnesic rodents [1,48,57,58,69,70], with the protective effects been ascribed largely to the competition with the inhibitory actions of the amnestic agents (ethanol/or acetaldehyde) on binding to the NMDA receptors [1]. The exact mechanism(s) through which peripherally administered saffron exerts its cognitive enhancing effects remain yet to be elucidated, although our results showed to be only partially dependent on cholinergic markers functions, such as AChE (Table 1), since saffron extract failed to affect AChE activity in whole brain fractions of aged mice, in contrast to the marked decreases (3852%) observed in adult mice. The discrepancy observed between the enhanced cognitive behavior of saffron-treated aged mice with their unaltered brain AChE activity levels may be due to the implication of other neurotransmitter systems system in maintaining cognitive function. Short-term administration of saffron also resulted in signicant alterations in rodent brain oxidative stress parameters and antioxidant mechanisms, as evidenced by the decreased caspase-3 and MDA levels and increased total brain antioxidant activity, ascorbic acid and GSH levels in both adult and aged mice (Table 2). Similar protective effects of saffron by modulation of lipid peroxidation, antioxidants and detoxication systems have also been observed in genotoxins-induced oxidative stress in Swiss albino mice [61], in renal ischemiareperfusion (IR)-induced oxidative injury in rats [24] and in reperfusion-induced oxidative/nitrative injury to cerebral microvessels after global cerebral ischemia in mice [80]. Thus, our in vivo results indicate that the signicant cognitive enhancement conferred by saffron administration in aged mice, is more closely related to the antioxidant reinforcement, rather than to AChE inhibition, per se, not precluding other synergestic/pleiotrophic effects. Furthermore, it conrms the notion

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that memory is a complex process requiring the coordination of many neurotransmitter system, apart from the cholinergic [34]. Only in healthy adult mice were the memory-enhancing properties of saffron further correlated with cholinergic function as evidenced by the decreased AChE levels, indicating the need for an optimal balance/preservation level to exist between neuronal cholinergic transmission and cognitive performance. In order to further investigate the antioxidant properties of saffron in stress conditions and identify its bioactive components, the human neuroblastoma SH-SY5Y cell line was incubated with the tested phytochemicals in the presence of H2 O2 . Exposure of cultured cells to H2 O2 resulted in a dose-dependent viability loss and increase in ROS production and caspase-3 activity levels. These phenotypes induced by H2 O2 , were totally reversed by the tested phytochemicals, with crocetin being the most effective even at high H2 O2 concentrations (Figs. 2 and 3, Table 3). Similar protective effects afforded by the carotenoid constituents of saffron against cellular oxidative damage are also reported in the literature [47,48,55]. Overall, our results showed that crocetin, the aglycon crocin metabolite, is a unique and potent antioxidant capable of combating oxidative stress, even at the higher applied concentrations of H2 O2 . The latter is in agreement with other reports on PC12 [49] and hepatic cells derived from rats [19,74], showing protective effects of crocetin against ROS-induced hepatotoxicity and genotoxicity. In particular, in experiments performed in rats, crocetin showed protective effects in the striatum and substantia nigra of a hemiParkinsonian (6-hydroxydopamine, 6-OHDA) [2] model. Crocetin has been demonstrated to be one of the main metabolites of orally administered crocin in rodents, which when absorbed, is partially metabolized to mono- and di-glucocuronide conjugates [4,77] and could be mainly responsible for these pharmacological activities, although it still remains to be elucidated its ability to cross the blood-brain barrier. The latter is of particular importance since it will allow us to elucidate whether the central measures are a direct effect of saffron/or CRT, or whether this is secondary to a peripheral effect on other tissues. In conclusion, our ndings show, in vitro and in vivo, the antioxidant properties of saffron and its cognitive enhancing effect in both adult and aged mice. The latter seems to be more closely related to the higher brain antioxidant properties and only partially related to the inhibition of brain AChE activity. Protective effects were also afforded by saffron and crocetin against H2 O2 -induced toxicity in SH-SY5Y cells. Despite the fact that the mechanisms underlying these effects are still unknown and require more pharmacological and neurochemical research to establish any therapeutic advantage, saffron seems to be valuable in the prevention/treatment of age-related diseases and oxidative stress.

Acknowledgments The present study is co-nanced by the E. U. European Social Fund (80%) and the Greek Ministry of Development-GSRT (20%) and by GlaxoSmithKline. The authors would like to kindly acknowledge Prof. L. Stefanis from Biomedical Research Foundation of the Academy of Athens (BRFAA) for providing the human neuroblastoma SH-SY5Y cell lines.

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