Collection of plant material and preparation of extracts
Fresh stem barks of L. acida (Anacardiaceae) were collected in January 2018 in the Noun Division (West-Cameroun). A sample was authenticated at the Cameroon National Herbarium (HNC-IRA) by Mr. Victor Nana, by comparison to the specimen deposited under the voucher number 40942 HNC. The barks were shade-dried and grinded into powder prior to aqueous and methanolic extracts preparation.
To obtain the aqueous extract, 500 g of the plant powder were mix in 3 L of distilled water and boiled for 10 minutes. The solution was allowed to cool at room temperature and filtered using Whatman paper No 4. The filtrate was oven-dried to obtain 20.4 g of the aqueous extract (extraction yield: 4.08%). The methanol extract was prepared by maceration of 250 g of the powder of L. acida stem barks in 1 L of methanol for 72 h at room temperature. The filtrate was evaporated under reduced pressure and oven-dried to obtain 13.5g of the methanol extract, giving an extraction yield of 5.4%. For bioactivity investigations, the aqueous and methanol extracts were dissolved in distilled water.
Healthy non-pregnant adult female Wistar rats weighing 150-170 g were obtained from the animal house of the Department of Animal Biology, Faculty of Science of the University of Dschang-Cameroon. They were housed in plastic cages and had access to water and standard rat chow ad libitum. All procedures were validated by the scientific committee of the Department of Animal Biology, University of Dschang, which follows the internationally accepted standard ethical guidelines for laboratory animal use and care as described in the European Economic Community guidelines; EEC. 2010 Council Directive 2010/63/EU of 22 November 2010 .
Isolated rat uterus preparation
The preparation of estrogenized uterus was performed according to the procedure described by Watcho et al. . Briefly, 24h before the experiment, virgin female rats were subcutaneously injected with 17-β-estradiol benzoate (13.28 nM per animal). To collect the uteri, animals were sacrificed by cervical dislocation under anesthesia and the uteri were promptly removed, cleaned of the connective tissue and cut into strips of about 1 cm of length. Each uterine strip was vertically mounted in an organ bath of 20 mL capacity containing fresh De Jalon solution of the following composition (mM): NaCl 153.85, KCl 5.64, CaCl2 0.55, MgSO4 0.08, NaOH 12.5 and glucose 2.78, and thermostated at 37°C. Strip tension was adjusted to 0.71 g and allowed to equilibrate for 45 min during which the physiological solution was changed every 15 min. Spontaneous and drug-induced myometrial contractions were recorded using an isometric force transducer (SS12LA, BSL: Variable Force Transducer) connected to an MP36 amplifier (Biopac student lab pro version 3.7.3) and displayed on a monitor.
After the equilibration period during which spontaneous contractions were registered, non-cumulative concentration-response curves to oxytocin (0.054 - 3 × 10-10 mol/l), acetylcholine (2.13 - 17 × 10-6mol/l), potassium chloride (5.3 - 42.1 mmol/l) and L. acida extract (1.09 - 4.23 mg/ml) were recorded during 5 min. The tissue was then washed by changing the bathing solution and allowed to rest for 15 min before the next stimulation. The experiment was repeated 5 times for each drug/extract concentration. At the end of this phase, the most active extract (methanol extract at lowest concentration) was chosen to investigate the mechanism of action of the plant uterotonic activity. Determination of the mechanism of action of L. acida To determine the mechanism of action of L. acida, the tissue was pre-incubated for 30 min with atosiban (2 μmol) (an oxytocin receptor inhibitor), atropine (1 μmol) (a specific type 3 muscarinic receptor antagonist) and nifedipine (5 μmol) (an L-type calcium channel antagonist) before administration of oxytocin, acetylcholine and KCl. The experiment was repeated with the plant extract in the presence of each antagonist. Furthermore, the effects of the plant extract was tested in the presence of 2-amino-ethoxyphenylborate (100 μmol) (2-ADB, a specific antagonist of inositol 1,4,5-triphosphate receptors type 1) and in free calcium medium containing EGTA (2 mmol) to investigate the involvement of the intracellular and extracellular calcium in the plant activity. The results were expressed as inhibition percentage and calculated as follows:Inhibition% = (CF without antagonist-CF with in the presence of the antagonist) / (CF without antagonist) × 100CF = contraction force.The calcium-free De Jalon solution was prepared by substitution of CaCl2 with EGTA as describe by Aziba.Drugs Estradiol benzoate (17-β-diol 3-benzoate), acetylcholine hydrochloride [ethanaminium, 2-(acetyloxy)-N,N,N-trimethyl-, chloride], Potassium Chloride, EGTA(Ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid) and Atosiban were purchased from Sigma Chemical (St Louis, MO, USA). Atropine sulfate [α-(hydromethyl) benzene acetic 8-methyl-8-azabicyclo (3.2.1) oct-3yl-ester], oxytocin and nifedipine were purchased from local suppliers. All chemicals were dissolved in distilled water except 2-APB (DMSO) .
The data was expressed as means ± SEM. One-way analysis of variance (ANOVA) followed by Tukey HSD post hoc were used to assess statistical difference among groups using Statistica Software (version 8.0). The results were significantly different when p< 0.05.