Saturation binding (8 point) curves are measured by incubating either crude extracts of HEK 293 cells stably expressing 5-HT3 receptors, or Guinea pig membrane preparations, in 0.5 mL incubations containing 10 mM HEPES buffer (pH 7.4) and 0.1-1 nM [³H]granisetron or 1-10 nM [³H]N-methylScopolamine. Competition binding (10 point) is determined by incubating the same receptors preparations in 0.5 mL HEPES buffer containing either 0.6 nM [³H]granisetron or 0.6 nM [³H]N-methylScopolamine, and differing concentrations of competing ligands. Non-specific binding is determined with 1 mM quipazine or 10 μM Scopolamine respectively. Incubations are terminated by filtration onto Whatman GF/B filters wetted with HEPES buffer+0.3% polyethyleneimine, followed by two rapid washes with ice-cold HEPES buffer. Protein concentration is calculated using a Lowry protein assay with bovine serum albumin standards. Radioactivity is measured using a Tri-Carb 2100 TR scintillation counter^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[2]
The mice are weighed, labeled and grouped into seven groups of 5 animals each after which all animals are pre-injected intraperitoneally with 3 mg/kg Scopolamine. Groups 1-3 are administered 0.2 mL equivalent doses of 4 mg/kg, 6 mg/kg and 8 mg/kg of the extract of Morinda lucida while groups 4-6 are given same doses of Peltophorum pterocarpum extract and group 7 is given 0.2 mL of distilled water (negative control) for 3 consecutive days.
Rats^[3]
Healthy male Wistar rats (12 months old) weighing 180–200 g are used in this study. Rats are divided into five groups (n=6/group); Group I-normal control, Group II-disease control (Scopolamine hydrobromide 3 mg/kg, i.p.), Group III-Scopolamine+Quercetin (25 mg/kg, p.o.), Group IV-standard treatment (Scopolamine+Donepezil hydrochloride 3 mg/kg, p.o.), and Group V-Scopolamine+Quercetin (25 mg/kg, p.o.)+Donepezil (3 mg/kg, p.o.). Group III, IV, and V rats are dosed every 24 h interval with respective drugs for 14 consecutive days. The acquisition trail for Morris water maze, elevated plus maze, and passive avoidance paradigm is carried out on the 14^th day, and Scopolamine (3 mg/kg, i.p.) is administered on the 14^th day after the acquisition trail to all groups except normal control group, which provoke the cognitive impairment in rats. Retention of memory is tested on the 15^th day, and on the same day, rats are sacrificed and brain tissues are isolated to estimate acetylcholinesterase enzyme (AchE) and brain oxidative stress markers such as lipid peroxidase (LPO), glutathione (GSH) (reduced). ELISA kit is used to estimate β amyloid (Aβ_1-42) level. The hippocampus of rat brains is dissected out and studied for histopathological changes.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Lochner M, et al. The muscarinic antagonists Scopolamine and atropine are competitive antagonists at 5-HT3 receptors. Neuropharmacology. 2016 Sep;108:220-8.  [Content Brief]

  [2]. O ET, et al. COGNITIVE-ENHANCING PROPERTIES OF MORINDA LUCIDA (RUBIACEAE) AND PELTOPHORUM PTEROCARPUM (FABACEAE) IN SCOPOLAMINE-INDUCED AMNESIC MICE. Afr J Tradit Complement Altern Med. 2017 Mar 1;14(3):136-141.  [Content Brief]

  [3]. Pattanashetti LA, et al. Evaluation of neuroprotective effect of Quercetin with Donepezil in Scopolamine-induced amnesia in rats. Indian J Pharmacol. 2017 Jan-Feb;49(1):60-64.  [Content Brief]