The rats were sacrificed 24 h after the last administration of PEAC after an overnight fast. The animals were sacrificed through cervical dislocation after the collection of blood samples from the retro-orbital plexus and the brain was excised and rinsed in cold Tris-KCl buffer. The brain, liver, heart, kidneys and spleen were harvested and weighed. The blood was centrifuged at 4000 rpm for 15 min at room temperature, serum was obtained and aliquots for determination of lipid profile and oxidative stress parameters. The whole brain was homogenized in 0.1 M cold sodium phosphate buffer (pH 7.4) using the Teflon homogenizer. The homogenate was centrifuged at 4 °C for 10 min at 10,000 rpm. The supernatant were kept in aliquots for later determination of oxidative stress parameters, acetylcholinesterase and interleukin-6.

Serum total cholesterol, triglyceride and HDL were estimated using Randox diagnostic kits (Randox Laboratories Limited, Antrim, United Kingdom). Low density lipoprotein (LDL) was calculated as LDL = (TC-HDL)-(TG/5) [39].

The risk of atherogenicity was determined by calculating the atherogenic index [40], HDL/LDL ratio [42] and coronary risk index [41].

The LPO end product malondialdehyde (MDA) concentration was measured in serum and brain supernatant using the thiobarbituric reacting substance (TBARS) assay [43]. Reduced glutathione (GSH) concentration in the serum and brain supernatant was determined using the Ellman’s reagent [44]. Assay for catalase enzyme activity in serum and brain supernatants of was determined using the colorimetric assay based on the yellow complex formation with H2O2 and molybdate [45].

The procedure described by Ellman et al. [46] was used to estimate AChE activity in the supernatant of the brain tissues. Briefly, 50 μL aliquots of brain supernatant was diluted 50 μL of phosphate buffer (0.1 M, pH 7.4) followed by addition of 50 μL of DTNB (0.0001 M) in a 96-well plate. The initial absorbance was first measured after 5 min of incubation with DTNB. Thereafter, 50 μL of acetylthiocholine iodide (0.028 M) was added to the mixture for 3 min and the absorbance again measured at 405 nm in a microplate reader (Micro READ 1000, Belgium). The rate of acetyl-cholinesterase activity (μmol/min/mg tissue) was calculated as described below [46]:


R Rate in moles of substrate hydrolyzed/min/g tissue.

A Change in absorbance/min.

Co = Original concentration of the tissue.

The concentration of IL-6 (BioLegend, USA) in the brain supernatant was determined according to the manufacturer’s instruction. IL-6 ELISA (BioLegend ELISA MAX™ Deluxe kit, USA) with sensitivity limit of 4 pg/mL. All the measurements were done at room temperature in accordance with BioLegend instructions using microplate reader with 450 nm filter(Micro READ 1000, Belgium). The concentration of IL-6 from the tissue was extrapolated from the standard curve of IL-6 standards included in the assay kits. The level of IL-6 in the brain was expressed as pg/mg protein.

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