Tocolytic action of essential oil from Annona leptopetala R. E. Fries is mediated by oxytocin receptors and potassium channels

Tocolytic action of essential oil from Annona leptopetala R. E. Fries is mediated by oxytocin receptors and potassium channels Paula Benvindo Ferreira, Italo Rossi Roseno Martins, Joedna Cavalcante Pereira, Ana Carolina de Carvalho Correia, Renata de Souza Sampaio, Maria da Conceição Correia Silva, Vicente Carlos de Oliveira Costa, Marcelo Sobral da Silva, Fabiana de Andrade Cavalcante and Bagnólia Araújo da Silva* a Programa de Pós-graduação em Produtos Naturais e Sintéticos Bioativos (PPgPNSB), Centro de Ciências da Saúde, Universidade Federal da Paraíba (UFPB), João Pessoa, Paraíba, Brazil. b Campus Senador Helvídio Nunes de Barros, Universidade Federal do Piauí (UFPI), Picos, Piauí, Brazil. c Unidade Descentralizada do Iguatu, Universidade Regional do Cariri (URCA), Iguatu, Ceará, Brazil. d Campus Garanhuns, Universidade de Pernambuco (UPE), Garanhuns, Pernambuco, Brazil. e Departamento de Ciências Farmacêuticas, Centro de Ciências da Saúde, Universidade Federal da Paraíba (UFPB), João Pessoa, Paraíba, Brazil. f Departamento de Fisiologia e Patologia, Centro de Ciências da Saúde, Universidade Federal da Paraíba (UFPB), João Pessoa, Paraíba, Brazil.

The development of new drugs is a long process associated with a powerful advance in experimental pharmacology. The use of isolated organs, various animal models and contemporary in vitro methods [6] are applicable tools to be used as an initial step to discover potential medicinal drugs. Experimental animal models are known to be useful and effective in this process. [6,7,8]. In addition, there are several articles that use in vitro models to investigate the medicinal properties of essential oils [9,10,11].
Thus, in the search for new agents to combat the uterine disorders, we decided to investigate a possible tocolytic effect of AL-EO on isolated rat uterus and thus assign this natural product as a tool to improve the pharmacotherapy associated to these conditions.

Materials and Methods
Virgin female Wistar rats (150-250 g) (n = 5) were pretreated with diethylstilbestrol 1.0 mg/kg (s.c.) 24 h prior to the estrus induction experiment. They were euthanized using a guillotine and uterus was immediately extracted, drenched in Locke-Ringer solution [12] and bubbled with a carbogenic mixture (95% O2 and 5% CO2). The organ was cut longitudinally and corns were suspended by a cotton thread in organ baths at 32 °C and the contractions were registered using a force transducer.
After the stabilization period, two similar concentration-response curves were obtained with carbachol (CCh) (10 −6 M) or oxytocin (10 -2 IU/mL). Then, AL-EO was pre-incubated with the uterus corns for 15 min with a single concentration of the essential oil in independent experiments before adding CCh or oxytocin [13]. Additionally, similar concentration-response curves or two similar cumulative concentration-response curves for OXY (10 -6 -10 -1 IU/mL) were obtained.
All trial procedures were done going along with the guidelines for the ethical use of animals in applied etiology studies and released by the Animal Use Ethics Committee of UFPB (protocol nº 0104/2014). The values were expressed as the mean and standard error of the mean (S.E.M.) and statistically analyzed by the Student's t-test or one-way variance analysis (ANOVA) followed by Tukey's test. The null hypothesis was rejected when p < 0.05. IC50 or EC50 were calculated by nonlinear regression [20] and Emax values were used as a measure of effectiveness. The data were analyzed by GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA). AL-EO (9, 27 and 81 µg/mL, n = 5) inhibited the cumulative concentration-response curves to OXY. These curves were shifted to the right in a non-parallel manner, with decreasing Emax from 100% to 96.9 ± 2.4; 72.7 ± 3.3; 55.4 ± 1.6 and 0%. EC50 values of OXY were attenuated from 6.7 ± 0.1 x 10 -5 IU/mL (control) to 9.2 ± 0.4 x 10 -5 , 3.8 ± 0.3 x 10 -5 and 3.8 ± 0.6 x 10 -4 IU/mL in the presence of 3, 9 and 27 µg/mL of AL-EO, respectively

Conclusions
Thus, this study shows that the essential oil from Annona leptopetala R. E. Fries exerts its tocolytic activity on rat uterus through a non-competitive pseudo-irreversible antagonism of OT receptors and a positive activation of K + channels, primarily the KV channels, which indirectly blockade the CaV, leading to a reduction in Ca 2+ influx and uterine smooth muscle relaxation.