Aerial parts (leaves, stem, flowers and seeds) of SA were collected and their specimen was submitted at Herbarium of Pakistan (Quaid-i-Azam University Islamabad, Pakistan). They were shade dried at room temperature, chopped and ground mechanically to a mesh size of 1 mm.
Preparation of plant extracts
One kg dried sample of SA was extracted twice with 4 l of absolute methanol at 25°C. The extracts were filtrated through Whatman No. 1 fiter paper and concentrated using a rotary evaporator (Panchun Scientific Co., Kaohsiung, Taiwan) under reduced pressure at 40°C. The dried extract was stored at 4°C for in-vivo investigations.
In-vitro acetylcholinesterase inhibition assay
The assay for AChE activity was conducted using the method of Ellman et al. , having acetylthiocholine iodide (ATCI) as a substrate. The rate of production of thiocholine is determined by the continuous reaction of the thiol with 5,5-dithiobis-2-nitrobenzoate (DTNB) ion to produce the yellow anion of 5-thio-2- nitro-benzoic acid. Briefly, in the 96well plates, 25 μl of 15 mM ATCI, 75 μl of 3 mM DTNB and 50 μl of 50 mM Tris HCl, pH 8.0, containing 0.1% bovine serum albumin (BSA), and 25 μl of SA (5–150 μg/ml) was added and the absorbance was measured at 405 nm after 5 min of incubation at room temperature. After 25 μl of 0.22 U/ml of AChE from electric eel (Sigma–Aldrich Corporation, St. Louis, MO, USA) was added, the absorbance was measured again after 5 min of incubation at room temperature. Percentage of inhibition was calculated by comparing the rate of enzymatic hydrolysis of ATCI for the samples to that of the blank (50% aqueous methanol in the buffer). Galanthamine (1–32 μM) was used as a reference standard and was supplied by Sigma–Aldrich. All determinations were carried out at least five times, and in triplicate, at each concentration of the standard and samples.
30 Male, albino rats (180–190 g, b.w.), were provided by National Institute of Health Islamabad and were kept in ordinary cages at room temperature of 25 ± 3°C with a 12 h dark/light cycle. They were allowed free access to food in form of dry pellets and water and randomly divided into three groups: a vehicle group (n = 10) (Control), SA-treated group (n = 10) with SA 50 mg/kg b.w. (SA 50) and SA-treated group (n = 10) with 100 mg SA/kg b.w., (SA100). The SA extract was administered orally daily to SA50 and SA100 groups at a final volume 200 μl, while rats in the C group received 200 μl of saline. The study protocol was approved by Ethical Committee of Quaid-I-Azam University Islamabad for laboratory animal feed and care.
2.5. Behavioral testing: step-through a passive avoidance task
Rats were subjected to a step-through test on day 6, after a double training and an initial acquisition trial on day 5. It was performed according to previously described procedures, using a two-compartment passive avoidance apparatus (white/dark, separated by a black wall with a guillotine door in the middle part), with minor modifications of the time intervals. In detail, on day 5, the animals were allowed to habituate in the experimental room for 1 h prior to experiments. One hour later, each rat was placed in the illuminated chamber for the acquisition trial and was left to habituate to the apparatus. One hundred seconds later, the guillotine door was opened, and the animal was allowed to enter the dark compartment. The latency with which the animal crossed into the dark compartment was recorded. Animals that waited more than 100 s to enter the dark compartment were eliminated from the experiments. Once the animal crossed with all four paws to the next compartment, the guillotine door was closed, and the rat was taken into its home cage. The trial was repeated after 30 min as in the acquisition trial, where after 5 s the guillotine door was opened and as soon as the animal crossed to the dark compartment the door was closed and a foot shock (25 V, 3 mA, 5 s) was immediately delivered to the grid floor of the dark room. Thereafter, the rat was immediately removed from the apparatus and returned to its home cage. In this trial, the initial latency (IL) of entrance into the dark chamber was recorded (maximum time allowed was 120 s). Twenty-four hours after training, a retention test was performed to determine long-term memory. Each animal was placed in the light compartment for 20 s. The door was opened, and the step-through latency (STL) was measured for entering into the dark compartment. The test session ended when the animal entered the dark compartment or remained in the light compartment for 300 s (criterion for retrieval). During these sessions, no electric shock was applied. All training and testing sessions were carried out during the light phase between 08:00 and 14:00 h.
Ex vivo assessment of antioxidant enzymes
After completions of experiment rats were killed, and whole intact brain was carefully removed and incubated on an ice chilled for cleaning. The cerebellum was separated immediately while rest of brain tissue was homogenized in a phosphate buffer (pH 7.6), centrifuged at 20,000 × g, 4◦C for 2 h to obtain a soluble salt part (SS). Re-extraction of the pellets was carried out to get a soluble detergent part (DS) . The supernatant was collected and stored at −20◦C. Protein concentrations were determined by the Bradford assay with Bovine serum albumin as standard (0.05–1.00 mg/ml).
AChE activity was determined using the colorimetric assay of Ellman et al. , as previously described. Briefly, in the 96 well plates, 25 μl of 15 mM ATCI, 75 μl of 3 mM DTNB and 75 μl of 50 mM Tris–HCl, pH 8.0, containing 0.1% BSA, were added and the absorbance was read at 405 nm after five min incubation at room temperature. Any increase in absorbance due to the spontaneous hydrolysis of the substrate was corrected by subtracting the rate of the reaction before adding the enzyme. Then, 25 μl of sample (SS and DS fraction of brain homogenates) was added, and the absorbance was read again after 5 min of incubation at room temperature. The AChE activity is expressed as mol/min/g of tissue protein. All determinations were carried out twice and in triplicate.
Catalase assay (CAT)
CAT activities were determined with reaction solution contained: 2.5 ml of 50 mmol phosphate buffers (pH 5.0), 0.4 ml of 5.9 mmol H2O2 and 0.1 ml tissue homogenate. Changes in absorbance of the reaction solution at 240 nm were determined after one minute. One unit of catalase activity was defined as an absorbance change of 0.01 as units/min .
Superoxide dismutase assay (SOD)
SOD activity was estimated by the method of Kakar et al. . Reaction mixture of this method contained: 0.1 ml of phenazine methosulphate (186 μmol), 1.2 ml of sodium pyrophosphate buffer (0.052 mmol; pH 7.0), 0.3 ml of the supernatant after centrifugation (1500 × g for 10 min followed by 10,000 × g for 15 min) of homogenate was added to the reaction mixture. Enzyme reaction was initiated by adding 0.2 ml of NADH (780 μmol) and stopped after 1 min by adding 1 ml of glacial acetic acid. Amount of chromogen formed was measured by recording color intensity at 560 nm. Results are expressed in units/mg protein.
Glutathione-S-transferase assay (GST)
The glutathione-S-transferase activity mixture consisted of 1.475 ml phosphate buffer (0.1 mol, pH 6.5), 0.2 ml reduced glutathione (1 mmol), 0.025 ml 1-Chloro-2,4-dinitrobenzene (CDNB) (1 mmol) and 0.3 ml of homogenate in a total volume of 2.0 ml. The changes in the absorbance were recorded at 340 nm and enzymes activity was calculated as nmol CDNB conjugate formed/min/mg protein using a molar extinction coefficient of 9.6 × 103 M-1 cm-1.
Glutathione reductase assay (GSR)
Glutathione reductase activity was determined by method of Carlberg and Mannervik . The reaction mixture consisted of 1.65 ml phosphate buffer: (0.1 mol; pH 7.6), 0.1 ml ethylenediaminetetraacetic acid (EDTA) (0.5 mmol), 0.05 ml oxidized glutathione (1 mmol), 0.1 ml nicotinamide adenine dinucleotide phosphate (NADPH) (0.1 mmol) and 0.1 ml of homogenate in a total volume of 2 ml. Enzyme activity were quantitated at 25°C by measuring disappearance of NADPH at 340 nm and was calculated as nmol NADPH oxidized/min/mg protein using a molar extinction coefficient of 6.22 × 103 M-1 cm-1.
Glutathione peroxidase assay (GSH-Px)
Glutathione peroxidase activity was assayed by the method of Mohandas et al.  (1984). The reaction mixture consisted of 1.49 ml phosphate buffer (0.1 mol; pH 7.4), 0.1 ml EDTA (1 mmol), 0.1 ml sodium azide (1 mmol), 0.05 ml glutathione reductase (1 IU/ml), 0.05 ml GSH (1 mmol), 0.1 ml NADPH (0.2 mmol), 0.01 ml H2O2 (0.25 mmol) and 0.1 ml of homogenate in a total volume of 2 ml. The disappearance of NADPH at 340 nm was recorded at 25°C. Enzyme activity was calculated as nmol NADPH oxidized/min/mg protein using a molar extinction coefficient of 6.22 × 103 M-1 cm-1.
Reduced glutathione assay (GSH)
Reduced glutathione was estimated by the method of Jollow et al. , using DTNB as a substrate. The yellow color developed was read immediately at 412 nm and expressed as μmol GSH/g tissue.
Estimation of a lipid peroxidation assay (TBARS)
The assay for lipid peroxidation was carried out following the modified method of Iqbal et al. . The reaction mixture in a total volume of 1.0 ml contained 0.58 ml phosphate buffer (0.1 mol; pH 7.4), 0.2 ml homogenate sample, 0.2 ml ascorbic acid (100 mmol), and 0.02 ml ferric chloride (100 mmol). The reaction mixture was incubated at 37°C in a shaking water bath for 1 h. The reaction was stopped by addition of 1.0 ml 10% trichloroacetic acid. Following addition of 1.0 ml 0.67% thiobarbituric acid, all the tubes were placed in boiling-water bath for 20 min and then shifted to crushed ice-bath before centrifuging at 2500 × g for 10 min. The amount of TBARS formed in each of the samples was assessed by measuring optical density of the supernatant at 535 nm using a spectrophotometer against a reagent blank. The results were expressed as nmol TBARS/min/mg tissue at 37°C using a molar extinction coefficient of 1.56 × 105 M-1 cm-1.
Data were expressed as standard error mean (Mean ± SEM) and one-way analysis of variance (ANOVA). The least significant difference (LSD) was determined using post hoc testing for inter group comparisons at a probability level of 0.05% and 0.01%. SPSS ver 14.0 (Chicago, IL, USA) and Microsoft Excel 2003 (Roselle, IL, USA) were used for the statistical and graphical evaluations.