Research report
Memory enhancing effects of saffron in aged mice are correlated with antioxidant protection

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Abstract

Brain aging is characterized by cognitive decline and memory deficits 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 significant 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, salt- and detergent-soluble AChE activity was significantly decreased only in adult mice. Thus, we showed, for the first time, that the significant cognitive enhancement conferred by saffron administration in mice, is more closely related to the antioxidant reinforcement. Next, we compared the effect of saffron (1–250 μg/mL), crocetin and safranal (1–125 μM) on H2O2-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.

Highlights

► Aging is accompanied by cognitive impairment and oxidative stress. ► Saffron administration enhances rodent performance in passive avoidance test. ► Cognitive enhancement is closely related to the cerebral antioxidant reinforcement. ► Cerebral acetylcholinesterase is decreased in adult mice after saffron administration. ► Saffron and crocetin protect SH-SY5Y cells against H2O2-induced cytotoxicity.

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 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 beneficial for preserving brain functions and forestalling the age-related deficits [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 cognitive dysfunctions. Chemical analysis has revealed the presence of unusual carotenoids glycosides (crocins), which are mono-, or di-glycosyl-esters of crocetin, (2E,4E,6E,8E,10E,12E,14E)-2,6,11,15-tetramethyl-2,4,6,8,10,12,14-hexadecahepta-enedioic acid, and small amounts of monoterpene aldehydes, like picrocrocin and safranal [71]. Saffron is efficient 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 TNFα-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 Alzheimer's 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 significantly enhanced cognitive performance in the passive avoidance test and attenuated brain oxidative stress markers, whereas AChE activity was attenuated significantly 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.

Section snippets

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 saponification of saffron extract, as previously described [28], while safranal was purchased from Sigma–Aldrich Corporation, St. Louis, MO, USA.

Animals

The animals used in this study were adult (4-months-old) and aged (20-months-old) male Balb-c mice. Gross

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 reflected the long-term memory of animals. Significant increase in step-through latency value shown improvement in memory. The effect of saffron injected i.p. to mice for 6 days on the step-through latency is shown in Fig. 1. No signs of writhing syndrome or changes in the emotional state of mice were observed after saffron administration. As shown in Fig. 1, the

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 first day is an indicator of visual

Acknowledgments

The present study is co-financed 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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