Collection of LP
Whole plant of Launaea procumbens at maturity was collected from District Bannu during Dec 2010, after identification the plant by Prof. Dr. Mir Azab Khan, Dean Faculty of Biological Sciences and submitted their voucher specimen at the Herbarium of Biotechnology, UST Bannu, KPK, Pakistan for future reference. After shade drying and chopping, plant was ground mechanically.
Plants extract preparation
1 kg dry powder of Launaea procumbens was socked in 4 liter aqueous methanol (80% methanol: 20% water) for 7 days. After one week of socking extract was filtrated using whatman filter 45. Filtrate was dried using rotary evaporator at 40 °C temperature and low pressure. The crude methanolic extract was stored at 4 ºC for in vivo investigations.
In vitro acetylcholinesterase inhibition assay
Method of Ellman et al. was used for assessment of AChE activity. Briefly, reaction mixture composed of 25 μl of ATCI (15mM), 75 μl of DTNB (3mM) and 50 μl of Tris- HCl, pH 8.0, (50mM), BSA (0.1%), and 25 μl of LPME was mixed and took OD at 405 nm after incubation for 5 min at room temperature. Inhibition of AChE was measured using blank in percentage. Experiments were repeated in triplicate.
Ethical approval of the study protocol
The study protocol was approved by an Ethics Committee of Quaid-i-Azam University for the proposed study as well as Feeding and Care of Laboratory Animals.
30 rats (180–190 g, b.w.), were provided by NIH Islamabad, Pakistan. The entire rats were placed at 25±3ºC with a half day light and dark cycle. Food and water was supplied timingly. Rats were divided randomly into three groups as;
Group 1 (Control)
Group II 100 mg /kg b.w. LPME
Group III 200 mg /kg b.w. LPME
Experiment was conducted for 7 days. At the end of the experiment all animals were sacrificed; blood was drawn prior to the excision of brain, then treated with liquid nitrogen and stored at -80°C for further enzymatic analysis.
Behaviors study (step-through passive avoidance task) was carried out using modified protocol as used by Kameyama et al. . During this procedure, briefly after first training and acquisition test at 5th day rats were allowed into two chamber (light/dark) equipment with passive avoidance. At 5th day rats were allowed for the attainment test in the light compartment. After hundred seconds, animals were allowed to enter the dark chamber by opening door and recorded the latency with removing rats that reach after 100 seconds using electric shock. After 30 min the trial was repeated. Initial latency (IL) was recorded for entrance into the dark chamber. After 24 hours, rats were tested for step-through latency (measuring time into dark section) for 5 min. Experiment was repeated during 09:00am and 15:00pm.
Estimation of oxidative status
Homogenization of brain tissue was carried out in phosphate buffer (pH 7.6), centrifuged at 20,000rpm×g at 4°C for 2 hour, to obtain a soluble salt part (SS). Re-extraction of the pellets was carried out to get a soluble detergent part (DS) . Supernatant of both parts were stored at −20°C. BSA was used for estimation of protein with different concentrations. Activities of various antioxidant enzymes are in brain tissue homogenate were measured as.
Catalase activity (CAT)
CAT activities were determined by the method of Chance and Maehly  with some modification. The reaction solution of CAT activities contained 2.5 ml of 50 mM phosphate buffer (pH 5.0), 0.4 ml of 5.9 mM H2O2 and 0.1 ml enzyme extract. Changes in absorbance of the reaction solution at 240 nm were determined after one minute. One unit of CAT activity was defined as an absorbance change of 0.01as units/min.
Super oxide dismutase assay (SOD)
SOD activity was estimated by the method of Kakkar et al. . Reaction mixture of this method contained 0.1 ml of phenazine methosulphate (186 μM), 1.2 ml of sodium pyrophosphate buffer (0.052 mM, pH 7.0), 0.3 ml of supernatant after centrifugation (1500 xg, 10 min followed by 10,000 × g, 15 min) of 10% homogenate was added to the reaction mixture. Enzyme reaction was initiated by adding 0.2 ml of NADH (780 μM) 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.
Reduced glutathione assay (GSH)
Reduced glutathione was estimated by the method of Jollow et al. . 1.0 ml sample of 10% homogenate was precipitated with 1.0 ml of (4%) sulfosalicylic acid. The samples were kept at 4ºC for 1 hr and then centrifuged at 1200 × g for 20 min at 4ºC. The total volume of 3.0 ml assay mixture composed of 0.1 ml filtered aliquot, 2.7 ml phosphate buffer (0.1 M, pH 7.4) and 0.2 ml DTNB (5,5-dithiobis-2-nitrobenzoic acid), (100 mM). The yellow color of the mixture was developed, read immediately at 412 nm on a Smart SpecTM plus Spectrophotometer and expressed as μM GSH/g tissue.
Glutathione-S-transferase assay (GST)
Glutathione-S-transferase activity was assayed by the method of Habig et al. . The reaction mixture consisted of 1.475 ml phosphate buffer (0.1 M, pH 6.5), 0.025 ml (CDNB) (1 mM), 0.2 ml reduced glutathione (1 mM), and 0.3 ml of 10% 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 nM CDNB conjugate formed/min/mg protein using a molar extinction coefficient of 9.6 × 103/M cm.
Glutathione reductase assay (GSR)
Glutathione reductase activity was determined by method of Carlberg and Mannervik . The reaction solution composed of 1.65 ml phosphate buffer: (0.1 M, pH 7.6), 0.1 ml EDTA (0.5 mM), 0.1 ml NADPH (0.1 mM) 0.05 ml oxidized glutathione (1 mM), and 0.1 ml 10% homogenate in a total volume of 2 ml. Enzyme activity was quantitated at 25 ºC by measuring disappearance of NADPH at 340 nm and was calculated as nM NADPH oxidized/min/mg protein using molar extinction coefficient of 6.22 ×103/M cm.
Glutathione peroxidase assay (GSH-Px)
Glutathione peroxidase activity was assayed by the method of Mohandas et al. . The reaction mixture consisted of 1.49 ml phosphate buffer (0.1 M, pH 7.4), 0.1 ml sodium azide (1 mM), 0.05 ml glutathione reductase (1 IU/ml), 0.05 ml GSH (1 mM) 0.1 ml EDTA (1 mM), 0.1 ml NADPH (0.2 mM), 0.01 ml H2O2 (0.25 mM) and 0.1 ml 10% homogenate in a total volume of 2 ml. The disappearance of NADPH at 340 nm was recorded at 25ºC. Enzyme activity was calculated as nM NADPH oxidized/min/mg protein using molar extinction coefficient of 6.22 × 103/M cm.
Estimation of lipid peroxidation assay (TBARS)
The assay for lipid peroxidation was carried out following the method of Iqbal et al. . The reaction mixture in a total volume of 1.0 ml contained 0.58 ml phosphate buffer (0.1 M, pH 7.4), 0.2 ml homogenate sample, 0.2 ml ascorbic acid (100 mM), and 0.02 ml ferric chloride (100 mM). The reaction mixture was incubated at 37ºC for 1 h in a shaking water bath. The reaction was stopped by addition of 1.0 ml 10% trichloroacetic acid. After addition of 1.0 ml 0.67% thiobarbituric acid, all the tubes were boiled in a 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 spectrophotometer against a reagent blank. The results were expressed as nM TBARS/min/mg tissue at 37ºC using molar extinction coefficient of 1.56 × 105/M cm.
In Vivo AChE assessment
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 DTNB and 75 μl of 50 mM Tris–HCl, pH 8.0, containing 0.1% BSA, were added and absorbance was read at 405 nm after five min incubation at room temperature. Any increase in absorbance due to the pontaneous 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 ofincubation at room temperature. The AChE activity is expressed as mol/min/g of tissue protein. All determinations were carried out twice and in triplicate.
Computer software SPSS 13.0 was used to determine the level of probability at LSD 0.05%.