Gum Arabic

The gum of Acacia etbaica was collected from Klte Awulaelo district, Eastern Zone of Tigray region; paracetamol (batch no. 1706003, HEDEI JIHENG, Pharmaceuticals, Germany), PVP K-30 (BOAINKY, Pharmaceutical Ltd., India), and Acacia BP (batch no. 242, BOAINKY, Pharmaceutical Ltd., India) were kindly donated from Addis Pharmaceutical Factory Sh. Co. Lactose, magnesium stearate, potassium dihydrogen phosphate, sodium hydroxide, sodium chloride, sodium hydrogen phosphate dehydrate, acetone, HCl (LOBA CHEMIE PVT. LTD, England), maize starch (DBEBATS, India), talc (SISCO LAB, India), ethanol (Batch no. 026193, Fine Chemical General Trading, Ethiopia), and chloroform (CHEMIE PVT. LTD., England) were purchased from local market. All the chemicals were analytical grade and used as received.

Animal husbandry and experimental procedures were in accordance with the EU Directive 2010/63/EU for animal experiments and approved by the national ethical committee for care and use of animals (approval ID A16320). Every effort was made to minimize animal suffering and the number of animals used. Seven-week-old male C57Bl/6J mice from Janvier Labs (Le Genest-Saint-Isle, France) were housed in polypropylene cages and maintained in a temperature- and humidity-controlled pathogen-free facility with a 12 h light-dark cycle abd ad libitum access to food and water. Mice were handled daily for 1 week before experiment onset to minimize stress reactions due to manipulation. Mice were fed with A04 diet (Safe, Augy, France). They were supplemented by gavage (feeding probe V0105040, ECIMED, Boissy-Saint-Leger, France) with S. lavandulaefolia essential oil encapsulated with acacia gum (83.3 mg/kg/day), or S. officinalis aqueous leaf extract (166.7 mg/kg/day) or a mix of both vegetal extracts (Cognivia™; 250 mg/kg/day) or water for the control group, each morning for two weeks. S. lavandulaefolia essential oil was characterized for its content in terpenoids (as eucalyptol, camphor, α-, β-pinene and others) and S. officinalis aqueous leaf extract was characterized for its content in polyphenols (as rosmarinic acid, apigenin glucosides, luteolin glucosides and others). The encapsulation of essential oil was performed using acacia gum in solution with a specific shear stress in order to create dispersed and stabilized small oil droplets of essential oil (1 to 5 µm). Finally, the combination of both extracts was done using exactly the same materials. After the first day of gavage, mice were subjected to a Y-maze test to evaluate the impact of an acute dose on long-term memory. After two weeks of gavage, mice were submitted to a Morris water maze to evaluate the impact of a chronic dose on learning and long-term memory. At the end of the behavioral protocol, mice were sacrificed, and the brain structures implicated in memory (hippocampus, prefrontal cortex) and red blood cells were collected and frozen at −80 °C.

Two NRT products were tested, Nicorette Peppermint 2 mg lozenge, and Nicotinell Licorice 2 mg gum. The products weigh 0.6 g and 1.2 g per unit of use, respectively. The lozenge contains nicotine in the form of resinate, fillers (mannitol, xanthan gum, gum arabic, magnesium stearate, hypromellose, titanium dioxide, microcrystalline cellulose, potassium silicate, polysorbate 80), pH adjuster (sodium carbonate), sweetener (sucralose, acesulfame K) and flavorings. The gum contains nicotine polacrilex, chewing gum base, sweetener (acesulfame K, saccharin, sodium saccharin, sorbitol, xylitol, mannitol), pH adjusters (calcium carbonate, sodium carbonate, sodium bicarbonate), flavoring, glycerol, gelatine, titanium dioxide, canauba wax and talcum powder.

To measure the activity of PsEst3 towards bulky aromatic alcohol moieties and aliphatic chains, fluorescein dibutyrate (C4), fluorescein dioctanoate (C8) and fluorescein dilaurate (C12) were used as substrates. The fluorescein substrates were prepared in 2-ethoxyethanol at a concentration of 100 µM. The 50 µl reaction mixture consisted of 0.1 µM fluorescein derivative, PsEst3 (1.25 µg was used to measure activity) in 20 mM Tris–HCl buffer pH 8.0, 150 mM NaCl, 10% 2-ethoxyethanol and 0.005%(w/v) CHAPS and was directly prepared in a qPCR microtube. Fluorescein signal (SYBR Green filter; λ_ex = 492 nm, λ_em = 512 nm) was measured at 30°C for 10 min using a Qiagen Rotor-Gene Q 2plex qPCR cycler (Qiagen, Hilden, Germany). The fluorescein standard curve was obtained in the 0.01–0.1 µM range using the same buffer as used for enzymatic measurements (slope = 176.68, intercept = −0.403, R² = 0.9971). All measurements were conducted in triplicate and were corrected for non-enzymatic hydrolysis.
Substrate specificity was investigated using the following commercial p-nitrophenyl (pNP) esters: acetate, butyrate, hexanoate, octanoate and decanoate. All pNP derivatives were dissolved in dimethyl sulfoxide to 2 mM. The 80 µl reaction mixture consisted of 50 µg PsEst3 and 250 µMpNP ester in 0.1 M Tris–HCl pH 8.0 buffer which contained 0.1%(w/v) gum arabic and 1%(w/v) CHAPS to minimize autohydrolysis. The PsEst3 was pre-incubated at 30°C for 2 min before the pNP ester was added. The reaction was evaluated at 405 nm for 20 min (every 10 s) in a Multiskan GO microplate reader (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Assays were performed in triplicate and the values were normalized for non-enzymatic hydrolysis. The standard curve of the pNP derivatives was obtained in the 0–0.5 mM range using the same buffer as used for the enzymatic assay (slope = 4.568, intercept = 0.006109, R² = 0.9993).