Uterotonic Effects of Aqueous and Methanolic Extracts of Lannea acida in Wistar Rats: An In Vitro Study

Lannea acida (Anacardiaceae), commonly called Kikié in the Noun division (West-Cameroon), is a tree whose bark is used locally to facilitate delivery. This study was aimed at evaluating the in vitro uterotonic effects of aqueous and methanol extracts of L. acida in Wistar rats. Uterine strips isolated from rats pretreated with 5 μg estradiol (48 h) were mounted in a single-organ bath containing aerated and thermostated De Jalon solution (37 °C). After equilibration, non-cumulative effects of L. acida extracts were recorded after application. The effect of the methanol extract (the most active extract) was monitored in the presence of atosiban (a competitive antagonist of oxytocin receptors), atropine (a specific type 3 muscarinic receptor antagonist), nifedipine (an L-type calcium channel antagonist), and 2-Aminoethoxydiphenyl borate (2-ADB, a specific antagonist of inositol 1,4,5-triphosphate receptors type 1), and in calcium-free medium containing EGTA to elucidate its mechanism of action. L. acida induced uterine contraction in a concentration-dependent manner with the methanol extract (1.506 ± 0.032 gf) being the most effective. Administration of atosiban (2 μmol/L) and atropine (1 μmol/L) reduced the contractile effect of L. acida. Complete inhibition was observed with nifedipine, 2-APB, and calcium-free medium containing EGTA. These results suggest that L. acida possesses uterotonic effects mediated through oxytocin receptors with mobilization of extracellular calcium.


Background
Parturition is the delivery of a fully grown fetus after completion of the normal pregnancy period. This biological process is characterized by an increase in myometrial contractility and the dilatation of the uterine cervix [1]. In fact, as term approaches, the quiescent uterus becomes activated by estrogens. This leads to increased expression of various contraction-associated proteins (CAPs) such as prostaglandins and oxytocin receptors [2]. Also, an increase in gap junction formation between adjacent myometrial cells allows electrical synchrony within the myometrium and ensures effective coordination of contractions [3]. During labor process, the frequency and intensity of myometrial smooth muscle contractions are essential contributing factors to normal delivery [4]. In women with slow and/or weak uterine activity during labor, uterotonic molecules (oxytocin and prostaglandins) are regularly used [5,6]. In extreme cases, caesarian sections can be required [7]. However, these modern technics bear several side effects such as postpartum bleedings commonly associated with uterotonic compounds, esthetic consequences (presence of scar) of the surgical solutions [8]. Furthermore, in developing countries where the accessibility of health services is limited, people largely rely on medicinal plant for primary health problems [9].
Many medicinal plants with uterotonic properties have been reported. These include among other Newbouldia laevis [10], Ananas comosus [11], and Foeniculum vulgare [12]. Lannea acida (Anacardiaceae), commonly called Kikié in the Noun Division (West-Cameroon), is a small deciduous tree of about 8-to 12-m height. The decoction of its stem barks is traditionally used as fertility enhancer and at late gestational stage to facilitate parturition. Scientific evidences showed that this plant possesses androgenic [13], estrogenic [14], and contractile properties in isolated rat vas deferens and seminal vesicles [15]. Phytochemical screening of this plant revealed the presence of alkaloids, glycosides, and tannins compounds [16,17]. However, less is known on the effects of this plant on uterine contractility in rats. This study was therefore undertaken to determine the effect of L. acida on uterine activity of healthy non-pregnant rat and its mechanism of action.

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 methanol extract preparation.

Extracts Preparation
To obtain the aqueous extract, 500 g of the plant powder was mix in 3 L of distilled water and boiled for 10 min. 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 13.5 g of the methanol extract, giving an extraction yield of 5.4%. For bioactivity investigations, the aqueous and methanol extracts were dissolved in distilled water.

Animals
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 Directive 86/609/EEC, of the 24th November 1986 [18].

Isolated Rat Uterus Preparation
The preparation of estrogenized uterus was performed according to the procedure described by Watcho et al. [19]. Briefly, 48 h before the experiment, virgin female rats were subcutaneously injected with 17-β-estradiol benzoate (5 μg 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.

Drug Challenges
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 −6 mol/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 [15]. 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), or nifedipine (5 μmol) (an L-type calcium channel antagonist) before administration of oxytocin, acetylcholine, or KCl, respectively. The experiment was repeated with the plant extract in the presence of each antagonist. Furthermore, the effects of the plant extract were tested in the presence of 2-amino-ethoxyphenylborate (100 μmol) (2-ADB, a specific antagonist of inositol 1,4,5triphosphate 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: The calcium-free De Jalon solution was prepared by substitution of CaCl 2 with EGTA as describe by Aziba [20].

Statistical Analysis
The data is expressed as means ± SEM. One-way analysis of variance (ANOVA) was used to compare the means between groups and Tukey HSD post hoc was then followed to separate statistical differences. The results were significantly different when p < 0.05. Data analysis was performed using Statistica Software (version 8.0).

Effects of the Aqueous and Methanol Extracts of L. acida on Uterine Contractility
Contrary to methanol extract (Fig. 1b), a dose-dependent contractile effect on the rat myometrial strips was recorded after application of aqueous extract of L. acida (Fig. 1a). As shown in Fig. 1c, the contraction force increased gradually after the application of the aqueous extract of L. acida. In the uterus samples treated with the methanol extract of L. acida, the contraction force increased at low (1.09 mg/mL) and moderate (2.18 mg/ mL) concentrations but decreased at high concentrations (3.27 and 4.36 mg/mL). The methanol extract was more effective at low concentrations while the aqueous extract produced the highest effect at high concentration (Fig. 1a, b, c).

Relative Potency of the Aqueous and Methanol Extracts of L. acida and Other Uterotonics
The effect of L. acida extracts and agonists on the relative potency of rat uterus is shown in Table 1. The tension recorded after application of oxytocin was increased by 24.17% and 15.41% compared to aqueous and methanol extracts of L. acida, respectively. Acetylcholine also increased the uterus tension by 28.29% and 20% compared to aqueous and methanol extracts of L. acida, respectively. The uterus tension increased by 50.49% and 44.77% in the samples treated with potassium chloride, compared to aqueous and methanol extracts of L. acida, respectively (Table 1). Potassium chloride was the most effective drug.

Effects of some Antagonists on Uterine Contraction Induced by Plant Extracts and Agonists
Effect of Atosiban and Atropine on Uterine Contraction Induced by L. acida Oxytocin, acetylcholine, and L. acida induced uterine contraction after application. The contractile effects of oxytocin were totally inhibited by atosiban (Fig. 2a). Atosiban also inhibited the contractile effect of the methanol extract of L. acida. However, the second administration of the plant extract (before washout period) caused moderated contractions of the uteri strips (Fig. 2b). The contraction force increased significantly (p < 0.05) after oxytocin and L. acida applications, compared to control (Fig. 2c).
The contractile effect of acetylcholine was totally inhibited after atropine (a specific type 3 muscarinic receptor antagonist) application (Fig. 2d). Additionally, atropine partially inhibited the contractile effect of the methanol extract of L. acida (Fig.  2e). The contraction force recorded after atropine injection was significantly (p < 0.05) elevated compared to control (Fig. 2f).

Effect of Nifedipine and Ca 2+ -Free Medium with EGTA on Uterine Contraction Induced by L. acida
The contractile effects of KCl and methanol extract of L. acida were completely abolished in the presence of Fig. 1 Effects of aqueous (a) and methanol (b) extracts of L. acida on isometric uterine contractions. c Mean force contraction generated from five isolated uterine horns obtained from different estrogenized rats, which were exposed to various concentrations of L acida (0; 1.09; 2.18; 3.27; and 4.36 mg/ mL); W O, washout period; AE, aqueous extract; ME, methanol extract 3.042 ± 0.041 nifedipine (5 μmol, an L-type calcium receptor antagonist) ( Fig. 3a and b). The maximum contraction forces of KCl and L. acida were significantly (p < 0.05) reduced compared to control (Fig. 3c). Compare to the effect in De Jalon solution, acetylcholine or L. acida had no contractile effect in calcium free medium with 2 mMol/L of EGTA (Fig. 4a-c). The contractile effect of oxytocin and L. acida on uterine smooth muscle was significantly (p < 0.05) inhibited by 2-APB (a specific antagonist of inositol 1,4,5-triphosphate receptors type 1) with the Emax values of 14.82 ± 3.64% and 9.53 ± 1.66%, respectively (Fig. 5a-c).

Discussions
This study demonstrated the in vitro uterotonic effect of aqueous and methanol extracts of L. acida, which may justify its traditional usage to facilitate parturition. The uterine strips isolated from rats pretreated (subcutaneously) with17-βestradiol were used in this work because of their high sensitivity to uterotonic agents [2]. L. acida extracts (1.09 to 4.36 mg/mL) stimulated uterine contraction with a maximal effect obtained at 2.18 mg/mL with the methanol extract and 4.36 mg/mL with the aqueous extract. Comparatively, the methanol extract of L. acida was more effective at low concentrations (1.09 and 2.18 mg/mL) while the aqueous extract had its highest effect at high concentrations (3.27 and 4.36 mg/mL). Over 2.18 mg/mL of methanol extract in the medium, the contraction force decreased with increasing concentration. This adverse effect observed in vitro could be attributed to high concentrations of active molecules such as alkaloids, saponins, and flavonoids in the methanol extract, leading to uterine receptors internalization or desensitization [17]. It has been demonstrated that alkaloids and saponins induce contractile activities on smooth muscle [22,23]. The alkaloids (Imperialine-3β-D-glucoside) are known for their ability to contract uterine smooth muscle in contrast to the flavonoids (spinosine) that relax it [24]. Apart from L. acida (Anarcadiceae family), other Anacardiaceae species including Spondias mombin [24] were also reported to stimulate uterine contraction (0.75 mg/mL), suggesting that the uterotonic effect is common to the Anacardiaceae family. Similar studies using rat [12,25] and mouse [26] uterine strip have led to conclude that bioactive compounds found in L. acida are responsible of the contractile effects. In the current study, the concentration (2.18 mg/mL) which gave the maximal effect was higher than the concentration (1.6 mg/ mL and 2 mg/mL) used by Watcho [19] and salleh and Ahmad [27], respectively. Like oxytocin, acetylcholine, and potassium chloride which are standard uterotonic agents, L. acida induced a concentration-dependent contractile effect. To apprehend the contractile effects of L. acida on uterine strips, antagonization assays were made using atosiban (a competitive antagonist of oxytocin receptors) and atropine (a non-competitive antagonist of muscarinic channels) to elucidate the mechanism of action of the most active extract (methanol). The contractile effect of the methanol extract of L. acida was inhibited after atosiban (2 μmol) or atropine (1 μmol) application. Our findings suggest that L. acida-induced uterine contraction was mediated mainly via oxytocin receptors as evidenced by the highest degree of inhibition registered with atosiban.
Moreover, low inhibition by atropine indicates that the muscarinic receptors are partially involved in the uterine contractile response to L. acida. These results are similar to the action observed by Watcho et al. [19] and Salleh and Ahmad   receptors. The mechanism of action of stimulants depends mainly on pharmaco-mechanical coupling since membrane depolarization receptors appear to belong to the G protein family. Activation of the receptor which is coupled to G protein alpha stimulates uterine contraction by activating the phospholipase C/Ca 2+ -dependent pathway [28].G protein-coupled membrane receptors mobilize extracellular calcium via L-type calcium channels activated by DAG/ PKC and/or intracellular calcium via IP3 receptors [29]. In addition, acetylcholine opens ion channels without depolarization of membrane while KCl induced contraction by depolarizing membrane which causes the influx of Ca 2+ [30]. Ca 2+ then binds to calmodulin, which activates the myosin light chain kinase leading to phosphorylation of myosin light chains, triggering contraction [31].
In order to verify the involvement of L-type calcium channels and in turn the involvement of extracellular calcium in the mechanism of action of L. acida, a test with nifedipine and in free-Ca 2+ De Jalon with 2 mM EGTA was performed using potassium chloride and acetylcholine. Nifedipine (1 μMol), an L-type calcium channel antagonist, suppressed the stimulatory effects of L. acida. Also, contractions of L. acida or acetylcholine were abolished in free-Ca 2+ De Jalon with 2 mM EGTA after 30 min. In myometrial smooth muscle, calcium is sequestered in caveolae and recycled through L-type Ca 2+ channels. The system can recycle Ca 2+ efficiently between the caveolae and the reticulum using L-type Ca 2+ channels and IP 3 mediated Ca 2+ release from the reticulum (by acetylcholine) [32]. These results suggest that L. acida could mobilize extracellular calcium by stimulating L-type calcium channel receptors.
Given the intracellular origin of calcium in the uterine smooth muscle contraction process, an additive experiment was performed using 2-APB, a non-specific type 1 IP3 receptor [33]. 2-APB inhibited the contractile effects of L. acida and oxytocin, suggesting that these drugs act through IP3 pathway. Moreover, the inhibitory effect of atosiban suggests that L. acida act through oxytocin receptors and mostly depends of extracellular calcium. Nevertheless, the contraction produced does not depend solely on extracellular Ca 2+ as evident from the total inhibition on Emax by 2-APB.
Oxytocin binds to its G protein-coupled receptor and activates phospholipase C (PLC), which in turn increases inositol trisphosphate (IP3) and diacylglycerol (DAG) levels. DAG induced extracellular Ca 2+ influx through voltage-operate channels such as L-type calcium channel. IP3 activates the IP3 receptor at the sarcoplasmic reticulum membrane which release Ca 2+ into the cytosol and amplify contractions [34]. Since 2-APB inhibits the contractile effect of L. acida, 2-APB may interact with TRPC and IP3 receptors [35] leading to increased extracellular Ca 2+ influx in smooth muscle cells [34]. These results suggest that bioactive compounds present in the methanol extract of L. acida could induce (via the myometrial membrane receptors) the release of intracellular calcium. This moderate Ca 2+ release induces the opening of the calcium channels thus causing an increase in the calcium flow at the origin of the contractions. These results are similar to those of Sharma et al. [36] who showed that histamine acts by mobilizing first calcium reserves and extracellular calcium.

Conclusion
Based on these findings, it appears that L. acida triggered uterine smooth muscle contraction in vitro. These uterotonic effects of L. acida are mediated through oxytocin receptors with mobilization of extracellular and intracellular calcium. This result justifies the use of L. acida in traditional medicine to facilitate childbirth.