Alzheimer's disease (AD) is a progressive neurodegenerative disorder associated with loss of neurons in distinct brain areas resulting in dementia.[1,2] The central cholinergic pathways play a prominent role in memory processes.[3] The allopathic system of medicine is yet to provide a radical cure for AD which principally relies on nootropic agents such as piracetam, aniracetam, fosracetam, and anticholinesterases such as donepezil, metrifonate, and rivastigmine.[4–10] One of the most important approaches for the treatment of this disease involves the enhancement of acetylcholine (Ach) levels in brain using AChE inhibitors.[11,12] Several studies have reported anticholinesterase activity of the plant extracts and drugs.[13–16] Certain reports have claimed that a few herbal extracts can act on the central nervous system, thereby enhancing the faculties of learning and memory. In our earlier report, cold and hot aqueous extracts of Ficus racemosa stem bark showed dose-dependent inhibition of rat brain acetylcholinesterase with IC50 values of 1813 and 1331 mg/mL, respectively.[17]

It is postulated that anti-inflammatory and antioxidant drugs are useful in controlling the progression and inflammatory damage to brain tissue as immunohistochemical studies have shown chronic inflammatory changes in AD.[18,19] In view of the above, F. racemosa bark having anti-inflammatory, antioxidant, and anticholinesterase activity[17,20,21] was evaluated for its ability to enhance ACh in rat brain and its effect on cognitive function using elevated plus-maze as the exteroceptive behavioral model to evaluate memory in rats.

Chemicals and plant material :
All the reagents and chemicals used in the study were of extra pure analytical grade. F. racemosa stem bark was identified by Dr. Shivprasad Hudeda, JSS Ayurvedic Medical College, Mysore, and the voucher specimen (BOT-001/2008) was deposited at the herbarium of Department of Studies in Botany, University of Mysore, Mysore, India. The bark was cut into small pieces, dried (50°C) and powdered, passed through 60 mesh sieve (BS) and stored in an air tight container at 4°C till further use.

Preparation of the extracts
Aqueous extract (FR) was prepared by extracting F. racemosa bark powder with distilled water (1:8 w/v) at 70°C in a temperature controlled mechanical shaker for 24 h, filtered and freeze dried (yield: 12% w/v).

Acetylcholine enhancing activity
Healthy male Wistar rats of 6 weeks were divided into the following three groups (n = 6).
Group I: Served as control and received 1 mL normal saline p.o.

Group II: FR group received F. racemosa extract dissolved in 1 mL distilled water (250 mg/kg p.o.).

Group III: FR group received F. racemosa extract dissolved in 1 mL distilled water (500 mg/kg p.o.).
The rats were housed in polyacrylic cages, maintained at 27 ± 2 °C, 45-60% RH, and 12-h photoperiod. They were provided with a standard pellet diet (Amrut feeds, Pune, India) and water ad libitum. The animals were maintained with the above treatment for a period of 4 weeks. After 4 weeks, the rats were euthanized by decapitation, brains were rapidly excised, hippocampi were dissected out on ice and placed in chilled NaCl solution (0.9%).[22] The hippocampi were then homogenized using a Teflon–glass homogenizer in freshly prepared 10% trichloroacetic acid (1:5 w/v) in ice cold conditions. The homogenates were centrifuged at 4°C (8000 × g; 10 min) and the supernatant was collected and immediately used for the estimation of Ach by the fluorimetric method.[23]

Elevated plus-maze
Elevated plus-maze served as the exteroceptive behavioral model to evaluate memory in rats. The apparatus for rats consisted of a central platform (10 × 10 cm2) connected to two open arms (50 × 10 cm2) and two covered (enclosed) arms (50 × 40 × 10 cm3), and the maze was elevated to a height of 50 cm from the floor (Parle and Singh, 2004). On the first day (i.e., seventh day of drug treatment), each rat was placed at the end of an open arm, facing away from the central platform. Transfer latency (TL) was defined as the time (in seconds) taken by the animal to move from the open arm into any one of the covered arms with all its four legs. TL was recorded on the first day (training session) for each animal. The rat was allowed to explore the maze for another 2 min and then returned to its home cage. Retention of this learned-task (memory) was examined 24 h after the first day trial. Significant reduction in the TL value of retention indicated improvement in memory.[24]

Statistical analysis
All values are expressed as mean ± SD. Data were analyzed by ANOVA followed by Tukey's multiple comparison tests for significant differences using SPSS 16.0 software. The values were considered significant at P ≤ 0.05.

Acetylcholine plays a central role in basic nerve transmission, concentration, memory, and learning. The leading pharmaceutical drugs used for senility are, in fact, aimed at elevating Ach levels in the brain.[25] Age-related neurodegenerative disease like AD often associated with decreased level of neurotransmitter, primarily Ach in the hippocampal region, the area which performs the major memory task. Defectiveness of the Ach in the cholinergic forebrain ultimately leads to dementia.[26] The reduction of cholinergic activity in the CNS of AD patients correlates with their deterioration in scores on dementia rating scales.[27]

This study reports the Ach and memory enhancing activity of aqueous extract of F. racemosa bark in rats. Administration of the extract at two levels namely 250 and 500 mg/kg significantly enhanced (P ≤ 0.05) Ach levels in hippocampi of rats compared to control [Figure 1]. ACh levels were increased from 42 nM/g in control to 53.9 and 67.7 nM/g by 250 and 500 mg/kg of the extracts, respectively. The percentage raise in Ach levels were found to be 22% and 38% at 250 and 500 mg/kg dose of the F. racemosa extract, respectively. These findings are in good agreement with an earlier study, wherein the leaf extract of Ficus hispida at 200 and 400 mg/kg increased Ach concentration by 21% and 31% in neonatal rats and by 48% and 55%, respectively, in adult rats.[28] The elevated levels of ACh in the hippocampal region could be attributed to the inhibition of acetylcholinesterase by F. racemosa bark extract.[17]

Figure 1:
Acetylcholine concentration in the hippocampi of various groups. Values are mean ± SD (n = 6), bars with different letters a, b, and c differ significantly (P ≤ 0.05) Memory refers to the storage, retention, and recollection of information including past experiences, knowledge, and thoughts.[24,29] Drugs that enhance acquisition and recall of associative memory represent important goals in the therapy of cognitive disorders. The changes in TL with the administration of F. racemosa extracts (250 and 500 mg/kg) are presented in Figure 2. The extract at both dosage levels showed significant reduction (P ≤ 0.05) in TL. The extract at 500 mg/kg showed significantly a higher reduction in TL compared to a 250 mg/kg dosage level. It is opinioned that anti-inflammatory, antioxidant, and cholesterol lowering drugs are useful in controlling the progression of AD[18] as chronic inflammation and abnormal accumulation of cholesterol increases β-amyloid (Aβ) plaques resulting in dementia.[30] Therefore, the memory enhancing activity of F. racemosa extract could be attributed to its anti-inflammatory, antioxidant, and hypocholesterolemic activity.[20,21,31]

AD is a complex neurodegenerative disorder, leading to accelerated cognitive decline and dementia. The clinical treatment strategy of AD mainly involves elevation of cholinergic hypofunction and administration of anti-inflammatory drugs, antioxidants, and life style management. The plant extract selected for investigation shown to have antioxidant and anti-inflammatory activity from previous studies. It is evident from our study that administration of this extract elevated Ach levels and improved memory in rats. The collective pharmacological actions attributed by F. racemosa extract may serve as beneficial and supporting agent in the treatment of AD

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