Uterotonic Effects of Aqueous and Methanolic Extracts of Lannea Acida in Wistar Rats: An in Vitro Study.

Uterotonic Effects of Aqueous and Methanolic Extracts of Lannea Acida in Wistar Rats: An in Vitro Study. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Uterotonic Effects of Aqueous and Methanolic Extracts of Lannea Acida in Wistar Rats: An in Vitro Study. esther simo ngadjui, Jibril Yves Kouam, Georges Romeo Fozin Bonsou, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-52315/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Jan, 2021 Read the published version in Reproductive Sciences → Version 1 posted You are reading this latest preprint version Abstract Background: Lannea acida (Anacardiaceae), commonly called Kikié in the Noun division (West-Cameroon), is a tree whose bark is used locally to solve difficult childbirth. This study aimed to evaluate the in vitro uterotonic effects of aqueous and methanolic extracts of L. acida in female Wistar rats. Uterine strips isolated from female rats pretreated (48h) with oestradiol (5μg) were mounted in a single-organ bath containing a well aerated and thermostated De Jalon solution (37°C). The effects of L. acida extracts were recorded in a non-cumulative manner after application. The effect of the methanolic 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. Results: L. acida induced uterine contraction in a concentration-dependent manner with the methanolic extract (1.506 ± 0.032 gf) being the most effective. Administration of atosiban (2 μmol/l), atropine (1 μmol/l), nifedipine (5 μmol), 2-APB (100 μmol), and calcium free medium containing EGTA (2 mmol) reduced the contractile effect of L. acida . Complete inhibition was observed with nifedipine, 2-APB, and calcium free medium containing EGTA. Conclusions: These results suggest that L. acida possesses an uterotonic effect mediated through oxytocin receptors with mobilization of extracellular calcium. Sexual & Reproductive Medicine Cancer Biology Lannea acida calcium uterine contraction in vitro rats Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Parturition is the process ofdelivery of a fully-grown fetus on the 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 byestrogens.This leads to increased expression of various contraction-associated proteins (CAPs) such as prostaglandin 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 are limited due to postpartum bleeding commonly associated to uterotonic compoundsand financial and esthetic consequences of the surgical solution[ 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 family), commonly called Kikié in the Noun Division (West-Cameroon) is a small deciduous tree of about 8 to 12 meters height. The decoction ofitsstem 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 in healthy non-pregnant rat and its mechanism of action. Results Effects of the aqueous and methanolic extracts of L. acida on uterine contractility A dose-dependent contractile effect on the rat myometrial strips was recorded after application of aqueous extract of L. acida (Fig. 1 A). On the contrary, the effect of methanolic extract was not dose-dependent (Fig. 1 B). Indeed, the maximum contractile effect was observed at moderate concentration (2.18 mg) while the highest concentration (4.36 mg) was less active. As shown in Fig. 1 C, the contraction force increased gradually after the application of the aqueous extract of L. acida . In the uterus samples treated with the methanolic 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 methanolic extract was more effective at low concentrations while the aqueous extract produced the highest effect at high concentration (Fig. 1 A, B, C). Relative potency of the aqueous and methanolic extracts of L. acida and other uterotonincs 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 methanolic extracts of L. acida respectively. Acetylcholine also increased the uterus tension by 28.29% and 20% compared to aqueous and methanolic 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 methanolic extracts of L. acida respectively (Table 1 ). Potassium chloride was the most effective drug. Table 1 Relative potency of L. acida extracts compared to other uterotonics Treatments Tension (gf) Methanolic extract of L. acida 1.506 ± 0.032 Aqueous extract of L. acida 1.680 ± 0.230 Oxytocin 1.986 ± 0.063 Acetylcholine 2.100 ± 0.087 Potassium chloride 3.042 ± 0.041 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. 2 A). Atosiban also inhibited the contractile effect of the methanolic extract of L. acida . However, the second administration of the plant extract (before washout period) caused moderated contractions of the uteri strips (Fig. 2 B). The force of contraction increased significantly (p < 0.05) after oxytocin and L. acida applications compared to control (Fig. 2 C). The contractile effect of acetylcholine was totally inhibited after atropine (a specific type 3 muscarinic receptor antagonist) application (Fig. 2 D). Additionally, atropine partially inhibited the contractile effect of the methanolic extract of L. acida (Fig. 2 E). The force of contraction recorded after atropine injection was significantly (p < 0.05) elevated compared to control (Fig. 2 F). Effect of nifedipine and Ca 2+ free medium with EGTA on uterine contraction induced by L. acida The contractile effects of KCl and methanolic extract of L . acida were completely abolished in the presence of nifedipine (5 µmol, an L-type calcium receptor antagonist) (Fig. 3 A and B). The maximum contraction forces of KCl and L . acida were reduced significantly (p < 0.05), compared to control (Fig. 3 C). 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. 4 A-C). Effect of 2-aminoethoxydiphenyl borate (2-APB) on uterine contractions induced by oxytocin and L. acida 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 14.82 ± 3.64% and 9.53 ± 1.66% respectively (Fig. 5 A-C). Discussions This study demonstrated the in vitro uterotonic effect of aqueous and methanolic extracts of L. acida , which may justified its traditional used to facilitate parturition. The uterine strips isolated from rats pretreated (subcutaneously) with17-β-estradiol were used in this work because of its 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 a concentration of 2.18 mg/ml with the methanolic extract and 4.36 mg/ml with the aqueous extract. Comparatively, the methanolic 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 methanolic extract in the medium, the contraction force decreased with increasing concentration. This adverse effect carried out in vitro could be attributed to the high concentration of the active principle such as alkaloids, saponins and flavonoids [ 17 ] in methanolic extract than the aqueous extract that can trigger an internalization or desensitization of the uterine receptors. Recently, 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) 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 for the contractile effects. In the current study, the maximal concentration (2.18 mg/ml) which gives the maximal effect is highest than the maximal concentration (1.6 mg/ml and 2 mg/ml) used respectively by Watcho [ 19 ] and salleh and Ahmad [ 27 ]. Like oxytocin, acetylcholine and potassium chloride which are good standard uterotonic agents, L. acida induced a concentration-dependent contractile effect. Despite several pharmacological and molecular studies on the elucidation of signaling pathways including metabotropic receptors [ 28 ], the mechanism of action with the most active extract (methanol) was carried out using atosiban (a competitive antagonist of oxytocin receptors) and atropine (a non-competitive antagonist of muscarinic channels) which are the major influence of extracellular calcium in the uterus strips.The contractile effect of the methanolic 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 the oxytocin receptor as evidenced by the highest degree of inhibition of the atosiban while the lowest inhibition by atropine suggests that L. acida binding to the muscarinic receptors produced the least degree of contraction. These results are similar to the action observed by Watcho et al. [ 19 ] and Salleh and Ahmad [ 27 ], which showed that F. asperifolia and F. deltoidea respectively increase the contractile activity of rat uterine smooth muscle via multiple membrane receptors. The mechanism of action of stimulants depends mainly on pharmaco-mechanical coupling since membrane depolarization receptors appear to belong tothe 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 [ 29 ]. G protein-coupled membrane receptors mobilize extracellular calcium via L-type calcium channels activated by DAG/PKC and/or intracellular calcium via IP3 receptors [ 30 ]. In addition, acetylcholine opens ion channels without depolarization of membrane while KCl induced contraction by depolarizing membrane which causes the influx of Ca 2+ [ 31 ]. Ca 2+ then binds to calmodulin, which activates the myosin light chain kinase leading to phosphorylation of myosin light chains, triggering contraction [ 32 ]. 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 were performed using potassium chloride and acetylcholine. Nifedipine (1 µMol), an L-type calcium channel antagonist, suppressed the stimulatory effects of L. acida . Though all contractions of L. acida or acetylcholine were abolished in free-Ca 2+ De Jalon with 2 mM EGTA after 30 minutes. In myometrial smooth muscle, calcium is sequestered in caveolae and recycles through L-type Ca 2+ channels. The system can recycle Ca 2+ efficiently between the caveolae and the SR using L-type Ca 2+ channels and IP 3 mediated Ca 2+ release from the SR (by acetylcholine) [ 33 ]. These results suggest that L. acida could mobilize extracellular calcium by stimulating L-type calcium channels 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 [ 34 ]. Because 2-APB inhibited the contractile effects of L. acida and oxytocin, we can therefore suggest that these drugs act through IP3 pathway. Moreover, the inhibitory effect of atosiban suggests that L. acida act through oxytocin receptors and mostly depend 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 [ 35 ]. Since 2-APB inhibits the contractile effect of L. acida , 2-APB may interact with TRPC and IP3 receptors [ 36 ] because Inositol 1,4,5-triphosphate activates TRPC3 channels and increases extracellular Ca 2+ influx in smooth muscle cells [ 35 ]. These results suggest that bioactive compounds present in the methanolic 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. [ 37 ] who showed that histamine acts by first mobilizing 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 calcium. This result justifies the use of L. acida in traditional medicine to facilitate childbirth. Methods 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. 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. 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. 2010 Council Directive 2010/63/EU of 22 November 2010 [18]. Experimental design Isolated rat uterus preparation The preparation of estrogenized uterus was performed according to the procedure described by Watcho et al. [19]. 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. Drugs 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. 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 CaCl 2 with EGTA as describe by Aziba[20]. 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) [21]. Statistical analysis 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. Abbreviations 2-ADB 2-Aminoethoxydiphenyl borate EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid) CAPs contraction-associated proteins DAG diacylglycerol TRPC3 Transient Receptor Potential Cation Channel Subfamily C Member 3 PKC Protein kinase C IP3 inositotrisphosphate Declarations Acknowledgement The authors are grateful to the University of Dschang Cameroon, for the research facilities. Authors’ Contributions Esther Ngadjui, Pierre Watcho,Jibril Yves Kouam and Georges Romeo Fozin Bonsou participated in the study design. Aimé Césaire Tetsatsi Momo, Jibril Yves Kouam and Patrick Brice Defo Deeh collected the data and carried out the statistical analysis. Pierre Watcho, Modeste Wankeu-Nya, Patrick Brice Defo Deeh and Telesphore Benoit Nguelefack drafted the manuscript. All authors read and approved the final manuscript. Funding The present work was carried out with authors’ personal funds. Availability of data and materials The data used to support the findings of this study are available from the corresponding author upon request. Ethics approval and consent to participate 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 [18]. Consent for publication Not applicable Competing interests The authors declare that they have no competing interest. References Kuijsters NPM, Methorst WG, Kortenhorst MSQ, Rabotti C, Mischi M, Schoot BC. 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Extra and intracellular calcium signaling pathways differentially regulate Histamine induced myometral contraction during early and mid-pregnancy stage in buffaloes ( Bubalus bibalis ). Anim Reprod Sci. 2017b;179:10–9. Cite Share Download PDF Status: Published Journal Publication published 21 Jan, 2021 Read the published version in Reproductive Sciences → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-52315","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":1230224,"identity":"8bd45cdc-1513-42c6-8f98-9d17cc3a8f2f","order_by":0,"name":"esther simo ngadjui","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIie3QMUvDUBDA8SuBZClkTQnYr/CyaEHafJULD3TpB+ggUgjoUuiaQfoZMgmFDhcO0uVB1wgOcXmzbh0cTEKFLokZhb7/cu/gfssDMJn+Z/ZpIgIuAET9XnQK65yoE1E9CcDgqQdx3UNefu3eZ+tn1N7Hhsc3TkyQ7drJKJFOkGgtE1XeedErB9tVjkC6nYjCsv0hsYQCGzJIi7mwPqmdhAe2/e+KjBvywmFNgDqIAGn7QDwTDVly9CfxCnk9WhFjoEo5wfxepipH6iLuOtPekTi82s+jt+PD7TTdx1nZRX6LljDEasbN1gNU3wDg1IePfY5NJpPpwvoBbPFiMNI+hF8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-2971-7537","institution":"Universite de Dschang Faculte des Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"esther","middleName":"simo","lastName":"ngadjui","suffix":""},{"id":1230225,"identity":"30a2b0f9-372f-4e14-b289-2b29f85f3735","order_by":1,"name":"Jibril Yves Kouam","email":"","orcid":"","institution":"Universite de Dschang","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jibril","middleName":"Yves","lastName":"Kouam","suffix":""},{"id":1230226,"identity":"5fe52966-292e-42b5-b637-22ac4f82f7c0","order_by":2,"name":"Georges Romeo Fozin Bonsou","email":"","orcid":"","institution":"Universite de Dschang","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Georges","middleName":"Romeo Fozin","lastName":"Bonsou","suffix":""},{"id":1230227,"identity":"e35e4963-7c63-43a1-bf3b-d35af3b67866","order_by":3,"name":"Aimé Césaire Tetsatsi Momo","email":"","orcid":"","institution":"Universite de Dschang","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Aimé","middleName":"Césaire Tetsatsi","lastName":"Momo","suffix":""},{"id":1230228,"identity":"d224f93d-b4b4-455b-a094-cc91686cd977","order_by":4,"name":"Patrick Brice Defo Deeh","email":"","orcid":"","institution":"Universite de Dschang","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Patrick","middleName":"Brice Defo","lastName":"Deeh","suffix":""},{"id":1230229,"identity":"a7d269e7-7d14-455c-80d5-f5200fb55b5a","order_by":5,"name":"Modeste Wankeu-Nya","email":"","orcid":"","institution":"Universite de Dschang","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Modeste","middleName":"","lastName":"Wankeu-Nya","suffix":""},{"id":1230230,"identity":"6d5e07ea-152d-4f99-8e05-e01fc3da71fa","order_by":6,"name":"Telesphore Benoit Nguelefack","email":"","orcid":"","institution":"Universite de Dschang","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Telesphore","middleName":"Benoit","lastName":"Nguelefack","suffix":""},{"id":1230231,"identity":"4d66ac94-d48c-4aa0-9bcd-7d061cef5817","order_by":7,"name":"Pierre Watcho","email":"","orcid":"","institution":"Universite de Dschang","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Pierre","middleName":"","lastName":"Watcho","suffix":""}],"badges":[],"createdAt":"2020-08-01 11:46:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-52315/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-52315/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s43032-021-00465-x","type":"published","date":"2021-01-21T19:49:27+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":1827341,"identity":"47e218d2-3b13-409c-8e66-5f019a30557d","added_by":"auto","created_at":"2020-08-06 21:53:12","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":604938,"visible":true,"origin":"","legend":"Effects of aqueous (A) and methanolic (B) extracts of L. acida on isometric uterine contractions. C: mean force contraction generated from five isolated uterine horns obtained from different oestrogenized 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= methanolic extract.","description":"","filename":"figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-52315/v1/figure1.jpg"},{"id":1827342,"identity":"fd6f0484-6d62-4c19-95aa-368c3095a39f","added_by":"auto","created_at":"2020-08-06 21:53:12","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1137477,"visible":true,"origin":"","legend":"Effect of methanolic extract of L. acida and agonists in the presence of selected antagonists. (a,b) Representative tracings of isometric uterine contraction following agonists (a) and L. acida (b) administration in the presence of various antagonists and (c) mean Emax following administration of agonist sand L. acida. n= 5 rats per group, ME= methanolic extract; OT= oxytocin; Ach= acetylcholine; W O= washout period.*: p\u003c0.05 compared to control.","description":"","filename":"figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-52315/v1/figure2.jpg"},{"id":1827343,"identity":"1b8f25a9-e4b7-4eb7-b093-4b2909d7f565","added_by":"auto","created_at":"2020-08-06 21:53:12","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":628974,"visible":true,"origin":"","legend":"Effect of potassium chloride and methanolic extract of L. acida in the presence of nifedipine. (A,B) Representative tracings of isometric uterine contraction following potassium chloride and L. acida administration in the presence of antagonists and (C) mean Emax following administration of L. acida at 2.18 mg/ml and agonists alone and in the presence of nifedipine. n= 5 rats per group, ME= methanolic extract; KCl= Potassium chloride. W O= washout period.","description":"","filename":"figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-52315/v1/figure3.jpg"},{"id":1827344,"identity":"77567c5a-4548-4ef8-b608-6645fac550f6","added_by":"auto","created_at":"2020-08-06 21:53:13","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":604053,"visible":true,"origin":"","legend":"Representative physiographic recording of acetylcholine (A) and L. acida (B) De Jalon and in free-Ca2+ solution with EGTA on myometrial rat strips. Chart (C) shows the Emax of acetylcholine and L. acida drugs administrated in De Jalon and in free Ca2+ solution. L. acida have any effect without Ca2+. ","description":"","filename":"figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-52315/v1/figure4.jpg"},{"id":1827345,"identity":"504a3b83-1011-49a2-ad89-56ee358cfba4","added_by":"auto","created_at":"2020-08-06 21:53:13","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":574364,"visible":true,"origin":"","legend":"Effect of IP3 receptors blocker on uterine contraction induced by oxytocin (A) and methanolic extracts of L. acida (B). (C) Show the chart of oxytocin and L. acida drugs administrated. OT= ocytocine and M E = methanolic extract. W O= washout period.","description":"","filename":"figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-52315/v1/figure5.jpg"},{"id":13569461,"identity":"2e162cae-6dd9-4b01-be2b-656d3d418b34","added_by":"auto","created_at":"2021-09-17 03:40:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1045478,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-52315/v1/30b0c6c6-fe0c-4998-8c58-f7e170705f28.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eUterotonic Effects of Aqueous and Methanolic Extracts of Lannea Acida in Wistar Rats: An in Vitro Study.\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003eParturition is the process ofdelivery of a fully-grown fetus on the completion of the normal pregnancy period. This biological process is characterized by an increase in myometrial contractility and the dilatation of the uterine cervix [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In fact, as term approaches, the quiescent uterus becomes activated byestrogens.This leads to increased expression of various contraction-associated proteins (CAPs) such as prostaglandin and oxytocin receptors [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Also, an increase in gap junction formation between adjacent myometrial cells allows electrical synchrony within the myometrium and ensures effective coordination of contractions [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. During labor process, the frequency and intensity of myometrial smooth muscle contractions are essential contributing factors to normal delivery [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In women with slow and/or weak uterine activity during labor, uterotonic molecules (oxytocin and prostaglandins) are regularly used [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In extreme cases, caesarian sections can be required [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, these modern technics are limited due to postpartum bleeding commonly associated to uterotonic compoundsand financial and esthetic consequences of the surgical solution[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Furthermore, in developing countries where the accessibility of health services is limited, people largely rely on medicinal plant for primary health problems [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMany medicinal plants with uterotonic properties have been reported. These include among other \u003cem\u003eNewbouldia laevis\u003c/em\u003e [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], \u003cem\u003eAnanas comosus\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and \u003cem\u003eFoeniculum vulgare\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. \u003cem\u003eLannea acida\u003c/em\u003e (Anacardiaceae family), commonly called \u003cem\u003eKiki\u0026eacute;\u003c/em\u003e in the Noun Division (West-Cameroon) is a small deciduous tree of about 8 to 12 meters height. The decoction ofitsstem barks is traditionally used as fertility enhancer and at late gestational stage to facilitate parturition. Scientific evidences showed that this plant possesses androgenic [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], estrogenic [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and contractile properties in isolated rat vas deferens and seminal vesicles [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Phytochemical screening of this plant revealed the presence of alkaloids, glycosides and tannins compounds [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. 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 \u003cem\u003eL. acida\u003c/em\u003e on uterine activity in healthy non-pregnant rat and its mechanism of action.\u003c/p\u003e "},{"header":"Results","content":" \u003cp\u003e \u003ch2\u003eEffects of the aqueous and methanolic extracts of L. acida on uterine contractility\u003c/h2\u003e\u003c/p\u003e \u003cp\u003eA dose-dependent contractile effect on the rat myometrial strips was recorded after application of aqueous extract of \u003cem\u003eL. acida\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). On the contrary, the effect of methanolic extract was not dose-dependent (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Indeed, the maximum contractile effect was observed at moderate concentration (2.18\u0026nbsp;mg) while the highest concentration (4.36\u0026nbsp;mg) was less active. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, the contraction force increased gradually after the application of the aqueous extract of \u003cem\u003eL. acida\u003c/em\u003e. In the uterus samples treated with the methanolic extract of \u003cem\u003eL. acida\u003c/em\u003e, the contraction force increased at low (1.09\u0026nbsp;mg/ml) and moderate (2.18\u0026nbsp;mg/ml) concentrations, but decreased at high concentrations (3.27 and 4.36\u0026nbsp;mg/ml). The methanolic extract was more effective at low concentrations while the aqueous extract produced the highest effect at high concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B, C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003ch2\u003eRelative potency of the aqueous and methanolic extracts of L. acida and other uterotonincs\u003c/h2\u003e\u003c/p\u003e \u003cp\u003eThe effect of \u003cem\u003eL. acida\u003c/em\u003e extracts and agonists on the relative potency of rat uterus is shown in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The tension recorded after application of oxytocin was increased by 24.17% and 15.41% compared to aqueous and methanolic extracts of \u003cem\u003eL. acida\u003c/em\u003e respectively. Acetylcholine also increased the uterus tension by 28.29% and 20% compared to aqueous and methanolic extracts of \u003cem\u003eL. acida\u003c/em\u003e respectively. The uterus tension increased by 50.49% and 44.77% in the samples treated with potassium chloride, compared to aqueous and methanolic extracts of \u003cem\u003eL. acida\u003c/em\u003e respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Potassium chloride was the most effective drug.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRelative potency of \u003cem\u003eL. acida\u003c/em\u003e extracts compared to other uterotonics\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTension (gf)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethanolic extract of \u003cem\u003eL. acida\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.506\u0026thinsp;\u0026plusmn;\u0026thinsp;0.032\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAqueous extract of \u003cem\u003eL. acida\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.680\u0026thinsp;\u0026plusmn;\u0026thinsp;0.230\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOxytocin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.986\u0026thinsp;\u0026plusmn;\u0026thinsp;0.063\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcetylcholine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.100\u0026thinsp;\u0026plusmn;\u0026thinsp;0.087\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePotassium chloride\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.042\u0026thinsp;\u0026plusmn;\u0026thinsp;0.041\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEffects of some antagonists on uterine contraction induced by plant extracts and agonists\u003c/h2\u003e \u003cp\u003e \u003ch2\u003eEffect of atosiban and atropine on uterine contraction induced by L. acida \u003c/h2\u003e \u003cp\u003eOxytocin, acetylcholine and \u003cem\u003eL. acida\u003c/em\u003e induced uterine contraction after application. The contractile effects of oxytocin were totally inhibited by atosiban (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Atosiban also inhibited the contractile effect of the methanolic extract of \u003cem\u003eL. acida\u003c/em\u003e. However, the second administration of the plant extract (before washout period) caused moderated contractions of the uteri strips (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The force of contraction increased significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) after oxytocin and \u003cem\u003eL. acida\u003c/em\u003e applications compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe contractile effect of acetylcholine was totally inhibited after atropine (a specific type 3 muscarinic receptor antagonist) application (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Additionally, atropine partially inhibited the contractile effect of the methanolic extract of \u003cem\u003eL. acida\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The force of contraction recorded after atropine injection was significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) elevated compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003ch2\u003eEffect of nifedipine and Ca\u003c/b\u003e \u003csup\u003e \u003cb\u003e2+\u003c/b\u003e \u003c/sup\u003e free medium with EGTA on uterine contraction induced by L. acida\u003c/h2\u003e \u003cp\u003eThe contractile effects of KCl and methanolic extract of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eacida\u003c/em\u003e were completely abolished in the presence of nifedipine (5\u0026nbsp;\u0026micro;mol, an L-type calcium receptor antagonist) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). The maximum contraction forces of KCl and \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eacida\u003c/em\u003e were reduced significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Compare to the effect in De Jalon solution, acetylcholine or \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eacida\u003c/em\u003e had no contractile effect in calcium free medium with 2 mMol/l of EGTA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003ch2\u003eEffect of 2-aminoethoxydiphenyl borate (2-APB) on uterine contractions induced by oxytocin and L. acida\u003c/h2\u003e\u003c/p\u003e \u003cp\u003eThe contractile effect of oxytocin and \u003cem\u003eL. acida\u003c/em\u003e on uterine smooth muscle was significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) inhibited by 2-APB (a specific antagonist of inositol 1,4,5-triphosphate receptors type 1) with the Emax values 14.82\u0026thinsp;\u0026plusmn;\u0026thinsp;3.64% and 9.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66% respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e "},{"header":"Discussions","content":" \u003cp\u003eThis study demonstrated the \u003cem\u003ein vitro\u003c/em\u003e uterotonic effect of aqueous and methanolic extracts of \u003cem\u003eL. acida\u003c/em\u003e, which may justified its traditional used to facilitate parturition. The uterine strips isolated from rats pretreated (subcutaneously) with17-β-estradiol were used in this work because of its high sensitivity to uterotonic agents [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. \u003cem\u003eL. acida\u003c/em\u003e extracts (1.09 to 4.36\u0026nbsp;mg/ml) stimulated uterine contraction with a maximal effect obtained at a concentration of 2.18\u0026nbsp;mg/ml with the methanolic extract and 4.36\u0026nbsp;mg/ml with the aqueous extract. Comparatively, the methanolic extract of \u003cem\u003eL. acida\u003c/em\u003e was more effective at low concentrations (1.09 and 2.18\u0026nbsp;mg/ml) while the aqueous extract had its highest effect at high concentrations (3.27 and 4.36\u0026nbsp;mg/ml). Over 2.18\u0026nbsp;mg/ml of methanolic extract in the medium, the contraction force decreased with increasing concentration. This adverse effect carried out \u003cem\u003ein vitro\u003c/em\u003e could be attributed to the high concentration of the active principle such as alkaloids, saponins and flavonoids [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] in methanolic extract than the aqueous extract that can trigger an internalization or desensitization of the uterine receptors. Recently, it has been demonstrated that alkaloids and saponins induce contractile activities on smooth muscle [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Apart from \u003cem\u003eL. acida\u003c/em\u003e (Anarcadiceae) other Anacardiaceae species including \u003cem\u003eSpondias mombin\u003c/em\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] were also reported to stimulate uterine contraction (0.75\u0026nbsp;mg/ml), suggesting that the uterotonic effect is common to the Anacardiaceae family. Similar studies using rat [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and mouse [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] uterine strip have led to conclude that bioactive compounds found in \u003cem\u003eL. acida\u003c/em\u003e are responsible for the contractile effects. In the current study, the maximal concentration (2.18\u0026nbsp;mg/ml) which gives the maximal effect is highest than the maximal concentration (1.6\u0026nbsp;mg/ml and 2\u0026nbsp;mg/ml) used respectively by Watcho [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and salleh and Ahmad [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLike oxytocin, acetylcholine and potassium chloride which are good standard uterotonic agents, \u003cem\u003eL. acida\u003c/em\u003e induced a concentration-dependent contractile effect. Despite several pharmacological and molecular studies on the elucidation of signaling pathways including metabotropic receptors [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], the mechanism of action with the most active extract (methanol) was carried out using atosiban (a competitive antagonist of oxytocin receptors) and atropine (a non-competitive antagonist of muscarinic channels) which are the major influence of extracellular calcium in the uterus strips.The contractile effect of the methanolic extract of \u003cem\u003eL. acida\u003c/em\u003e was inhibited after atosiban (2\u0026nbsp;\u0026micro;mol) or atropine (1\u0026nbsp;\u0026micro;mol) application. Our findings suggest that \u003cem\u003eL. acida\u003c/em\u003e-induced uterine contraction was mediated mainly via the oxytocin receptor as evidenced by the highest degree of inhibition of the atosiban while the lowest inhibition by atropine suggests that \u003cem\u003eL. acida\u003c/em\u003e binding to the muscarinic receptors produced the least degree of contraction. These results are similar to the action observed by Watcho \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and Salleh and Ahmad [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], which showed that \u003cem\u003eF. asperifolia\u003c/em\u003e and \u003cem\u003eF. deltoidea\u003c/em\u003e respectively increase the contractile activity of rat uterine smooth muscle via multiple membrane receptors. The mechanism of action of stimulants depends mainly on pharmaco-mechanical coupling since membrane depolarization receptors appear to belong tothe G-protein family. Activation of the receptor which is coupled to G protein alpha stimulates uterine contraction by activating the phospholipase C/Ca\u003csup\u003e2+\u003c/sup\u003e dependent pathway [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. G protein-coupled membrane receptors mobilize extracellular calcium via L-type calcium channels activated by DAG/PKC and/or intracellular calcium via IP3 receptors [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In addition, acetylcholine opens ion channels without depolarization of membrane while KCl induced contraction by depolarizing membrane which causes the influx of Ca\u003csup\u003e2+\u003c/sup\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Ca\u003csup\u003e2+\u003c/sup\u003e then binds to calmodulin, which activates the myosin light chain kinase leading to phosphorylation of myosin light chains, triggering contraction [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn order to verify the involvement of L-type calcium channels and in turn the involvement of extracellular calcium in the mechanism of action of \u003cem\u003eL. acida\u003c/em\u003e, a test with nifedipine and in free-Ca\u003csup\u003e2+\u003c/sup\u003e De Jalon with 2\u0026nbsp;mM EGTA were performed using potassium chloride and acetylcholine. Nifedipine (1 \u0026micro;Mol), an L-type calcium channel antagonist, suppressed the stimulatory effects of \u003cem\u003eL. acida\u003c/em\u003e. Though all contractions of \u003cem\u003eL. acida\u003c/em\u003e or acetylcholine were abolished in free-Ca\u003csup\u003e2+\u003c/sup\u003e De Jalon with 2\u0026nbsp;mM EGTA after 30 minutes. In myometrial smooth muscle, calcium is sequestered in caveolae and recycles through L-type Ca\u003csup\u003e2+\u003c/sup\u003e channels. The system can recycle Ca\u003csup\u003e2+\u003c/sup\u003e efficiently between the caveolae and the SR using L-type Ca\u003csup\u003e2+\u003c/sup\u003e channels and IP\u003csub\u003e3\u003c/sub\u003e mediated Ca\u003csup\u003e2+\u003c/sup\u003e release from the SR (by acetylcholine) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. These results suggest that \u003cem\u003eL. acida\u003c/em\u003e could mobilize extracellular calcium by stimulating L-type calcium channels receptors.\u003c/p\u003e \u003cp\u003eGiven 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 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Because 2-APB inhibited the contractile effects of \u003cem\u003eL. acida\u003c/em\u003e and oxytocin, we can therefore suggest that these drugs act through IP3 pathway. Moreover, the inhibitory effect of atosiban suggests that \u003cem\u003eL. acida\u003c/em\u003e act through oxytocin receptors and mostly depend of extracellular calcium. Nevertheless, the contraction produced does not depend solely on extracellular Ca\u003csup\u003e2+\u003c/sup\u003e as evident from the total inhibition on Emax by 2-APB.\u003c/p\u003e \u003cp\u003eOxytocin 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\u003csup\u003e2+\u003c/sup\u003e influx through voltage-operate channels such as L-type calcium channel. IP3 activates the IP3 receptor at the sarcoplasmic reticulum membrane which release Ca\u003csup\u003e2+\u003c/sup\u003e into the cytosol and amplify contractions [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Since 2-APB inhibits the contractile effect of \u003cem\u003eL. acida\u003c/em\u003e, 2-APB may interact with TRPC and IP3 receptors [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] because Inositol 1,4,5-triphosphate activates TRPC3 channels and increases extracellular Ca\u003csup\u003e2+\u003c/sup\u003e influx in smooth muscle cells [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. These results suggest that bioactive compounds present in the methanolic extract of \u003cem\u003eL. acida\u003c/em\u003e could induce (via the myometrial membrane receptors) the release of intracellular calcium. This moderate Ca\u003csup\u003e2+\u003c/sup\u003e 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. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] who showed that histamine acts by first mobilizing calcium reserves and extracellular calcium.\u003c/p\u003e "},{"header":"Conclusion","content":" \u003cp\u003eBased on these findings, it appears that \u003cem\u003eL. acida\u003c/em\u003e triggered uterine smooth muscle contraction \u003cem\u003ein vitro\u003c/em\u003e. These uterotonic effects of \u003cem\u003eL. acida\u003c/em\u003e are mediated through oxytocin receptors with mobilization of extracellular calcium. This result justifies the use of \u003cem\u003eL. acida\u003c/em\u003e in traditional medicine to facilitate childbirth.\u003c/p\u003e "},{"header":"Methods","content":"\u003ch2\u003eCollection of plant material and preparation of extracts\u003c/h2\u003e\n\u003cp\u003eFresh stem barks of \u003cem\u003eL. acida\u003c/em\u003e (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.\u003c/p\u003e\n\u003ch2\u003eExtracts preparation\u003c/h2\u003e\n\u003cp\u003eTo 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 \u003cem\u003eL. acida\u003c/em\u003e 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.\u003c/p\u003e\n\u003ch2\u003eAnimals\u003c/h2\u003e\n\u003cp\u003eHealthy 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 \u003cem\u003ead libitum\u003c/em\u003e. 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 [18].\u003c/p\u003e\n\u003ch2\u003eExperimental design\u003c/h2\u003e\n\u003ch3\u003eIsolated rat uterus preparation \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eThe preparation of estrogenized uterus was performed according to the procedure described by Watcho et al. [19]. Briefly, 24h before the experiment, virgin female rats were subcutaneously injected with 17-\u003cem\u003e\u0026beta;\u003c/em\u003e-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\u0026deg;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.\u003c/p\u003e\n\u003ch2\u003eDrugs challenges\u003c/h2\u003e\n\u003cp\u003eAfter the equilibration period during which spontaneous contractions were registered, non-cumulative concentration-response curves to oxytocin (0.054 - 3 \u0026times; 10\u003csup\u003e-10 \u003c/sup\u003emol/l), acetylcholine (2.13 - 17 \u0026times; 10\u003csup\u003e-6\u003c/sup\u003emol/l), potassium chloride (5.3 - 42.1 mmol/l) and \u003cem\u003eL. acida \u003c/em\u003eextract (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. \u003cstrong\u003eDetermination of the mechanism of action of \u003cem\u003eL. acida\u003c/em\u003e\u003c/strong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; To determine the mechanism of action of \u003cem\u003eL. acida\u003c/em\u003e, the tissue was pre-incubated for 30 min with atosiban (2 \u0026mu;mol) (an oxytocin receptor inhibitor), atropine (1 \u0026mu;mol) (a specific type 3 muscarinic receptor antagonist) and nifedipine (5 \u0026mu;mol) (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 \u0026mu;mol) (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) \u0026times; 100CF = contraction force.The calcium-free De Jalon solution was prepared by substitution of CaCl\u003csub\u003e2\u003c/sub\u003e with EGTA as describe by Aziba[20].\u003cstrong\u003eDrugs\u003c/strong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Estradiol benzoate (17-\u0026beta;-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 [\u0026alpha;-(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) [21].\u003c/p\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eThe data was expressed as means \u0026plusmn; 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\u0026lt; 0.05.\u003c/p\u003e"},{"header":"Abbreviations","content":" \u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e2-ADB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2-Aminoethoxydiphenyl borate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEGTA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e(ethylene glycol-bis(β-aminoethyl ether)-N,N,N\u0026prime;,N\u0026prime;-tetraacetic acid)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCAPs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003econtraction-associated proteins\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDAG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ediacylglycerol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTRPC3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTransient Receptor Potential Cation Channel Subfamily C Member 3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePKC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProtein kinase C\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIP3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einositotrisphosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe authors are grateful to the University of Dschang Cameroon, for the research facilities.\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026rsquo; Contributions\u003c/h2\u003e\n\u003cp\u003eEsther Ngadjui, Pierre Watcho,Jibril Yves Kouam and Georges Romeo Fozin Bonsou participated in the study design. Aim\u0026eacute; C\u0026eacute;saire Tetsatsi Momo, Jibril Yves Kouam and Patrick Brice Defo Deeh collected the data and carried out the statistical analysis. Pierre Watcho, Modeste Wankeu-Nya, Patrick Brice Defo Deeh and Telesphore Benoit Nguelefack drafted the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThe present work was carried out with authors\u0026rsquo; personal funds.\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eThe data used to support the findings of this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eAll 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 [18].\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e \u003cspan\u003eKuijsters NPM, Methorst WG, Kortenhorst MSQ, Rabotti C, Mischi M, Schoot BC. 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Phytochemical screening contractile activity of \u003cem\u003eFoeniculum vulgare\u003c/em\u003e seeds on rat intestine and uterine muscles. J Pharmacogn Phytochem. 2017;6:346\u0026ndash;50.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eSalleh N, Ahmad VN. \u003cem\u003eIn vitro\u003c/em\u003e effect of \u003cem\u003eFicus deltoidea\u003c/em\u003e on the contraction of isolated rat\u0026rsquo;s uteri is mediated via multiple receptors binding and is dependent on extracellular calcium. BMC Complement Altern Med. 2013;13:359.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eSharma A, Nakade P, Choudhury S, Garg SK. Functional involvement of proteins kinase C, Rho-kinase and TRPC3 decreases while PLC increases with advancement of pregnancy in mediating oxytocin-induced myometral contractions in water buffaloes (\u003cem\u003eBubalus bubalis\u003c/em\u003e). 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Calcium channels, rho-kinase, protein kinase-C, and phospholipase-C pathways mediate mercury chloride-induced myometrial contractions in rats. Biol Trace Elem Res. 2018;187:418\u0026ndash;24.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eDaniel EE, El-Yazbi A, Cho WJ. Caveolae and calcium handling, a review and a hypothesis. J Cell Mol Med. 2006;10:529\u0026ndash;44.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eSaleem H, Tovey SC, Molinski TF, Taylor CW. Interactions of antagonists with subtypes of inositol 1,4,5-trisphosphate (IP3) receptor. Br J Pharmacol. 2014;171:3298\u0026ndash;312.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eSong T, Hao Q, Zheng Y, Liu QH, Wang XY. Inositol 1,4,5-trisphosphate activates TRPC3 channels to cause extracellular Ca\u003csup\u003e2+\u003c/sup\u003einflux in airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol. 2015;309:1455\u0026ndash;66.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eBencze M, Behuliaka M, Vavř\u0026iacute;nov\u0026aacute; A, Zicha J. Broad-range TRP channel inhibitors (2-APB, flufenamic acid, SKF-96365) affect differently contraction of resistance and conduit femoral arteries of rat. Eur J Pharmacol. 2015;765:533\u0026ndash;40.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eSharma A, Nakade P, Choudhury S, Yadav R, Garg K. Extra and intracellular calcium signaling pathways differentially regulate Histamine induced myometral contraction during early and mid-pregnancy stage in buffaloes (\u003cem\u003eBubalus bibalis\u003c/em\u003e). Anim Reprod Sci. 2017b;179:10\u0026ndash;9.\u003c/span\u003e \u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lannea acida, calcium, uterine contraction, in vitro, rats","lastPublishedDoi":"10.21203/rs.3.rs-52315/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-52315/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBackground: \u003c/em\u003e\u003c/strong\u003e\u003cem\u003eLannea acida\u003c/em\u003e (Anacardiaceae), commonly called Kikié in the Noun division (West-Cameroon), is a tree whose bark is used locally to solve difficult childbirth. This study aimed to evaluate the \u003cem\u003ein vitro \u003c/em\u003euterotonic effects of aqueous and methanolic extracts of \u003cem\u003eL. acida\u003c/em\u003e in female Wistar rats. Uterine strips isolated from female rats pretreated (48h) with oestradiol (5μg) were mounted in a single-organ bath containing a well aerated and thermostated De Jalon solution (37°C). The effects of \u003cem\u003eL. acida\u003c/em\u003e extracts were recorded in a non-cumulative manner after application. The effect of the methanolic 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. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003e\u003cem\u003eResults: \u003c/em\u003e\u003c/strong\u003e\u003cem\u003eL. acida\u003c/em\u003einduced uterine contraction in a concentration-dependent manner with the methanolic extract (1.506 ± 0.032 gf) being the most effective. Administration of atosiban (2 μmol/l), atropine (1 μmol/l), nifedipine (5 μmol), 2-APB (100 μmol), and calcium free medium containing EGTA (2 mmol) reduced the contractile effect of \u003cem\u003eL. acida\u003c/em\u003e. Complete inhibition was observed with nifedipine, 2-APB, and calcium free medium containing EGTA.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConclusions: \u003c/em\u003e\u003c/strong\u003eThese results suggest that \u003cem\u003eL. acida \u003c/em\u003epossesses an uterotonic effect mediated through oxytocin receptors with mobilization of extracellular calcium.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","manuscriptTitle":"Uterotonic Effects of Aqueous and Methanolic Extracts of Lannea Acida in Wistar Rats: An in Vitro Study.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-08-06 21:53:10","doi":"10.21203/rs.3.rs-52315/v1","editorialEvents":[{"type":"communityComments","content":1}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"739fd3d4-3265-4f2d-b040-e758ccd8e8d7","owner":[],"postedDate":"August 6th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":250165,"name":"Sexual \u0026 Reproductive Medicine"},{"id":250166,"name":"Cancer Biology"}],"tags":[],"updatedAt":"2021-08-06T19:49:27+00:00","versionOfRecord":{"articleIdentity":"rs-52315","link":"https://doi.org/10.1007/s43032-021-00465-x","journal":{"identity":"reproductive-sciences","isVorOnly":false,"title":"Reproductive Sciences"},"publishedOn":"2021-01-21 19:49:27","publishedOnDateReadable":"January 21st, 2021"},"versionCreatedAt":"2020-08-06 21:53:10","video":"","vorDoi":"10.1007/s43032-021-00465-x","vorDoiUrl":"https://doi.org/10.1007/s43032-021-00465-x","workflowStages":[]},"version":"v1","identity":"rs-52315","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-52315","identity":"rs-52315","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}