Various pharmacological agents have been used to treat pseudopregnancy, including anti-prolactins, progestogens, serotonin agonists, and dopamine agonists. Progestins are not fully effective in the treatment of pseudopregnancy and cause recurrence of the syndrome. Furthermore, they have a wide range of serious side effects. Therefore, progestogens have not been used for the treatment of pseudopregnancy. In recent years, medical therapy used to treat pseudopregnancy include dopamine agonists and serotonin antagonists. Dopamine agonists are preferred more frequently in the treatment of pseudopregnancy because they cause the least side effects and longer duration of action. Prolactin secretion is regulated by multiple neuro-transmitters and hormones. The major control mechanism of prolactin secretion is the activation of prolactin-inhibiting dopaminergic neurons in the hypothalamus. Ergot derivatives such as bromocriptine or cabergoline reduce prolactin secretion since they have a strong dopamine D2-receptor agonist activity. The serotonin antagonists indirectly inhibit prolactin secretion by stimulating the secretion of endogenous dopamine. Although dopamine agonist drugs that are selective prolactin inhibitors have the ability to suppress prolactin, they are expensive drugs (Gobello et al. 2001; Gobello 2006; Ramsey 2017; Root et al. 2018). Therefore, novel drug candidates have been investigated for the treatment of pseudopregnancy, among them natural products are promising ones. According to the previous studies, Pulsatilla alpina subsp. apiifolia (Scop.) Nyman (Martin et al. 1988); Ferula asafoetida H.Karst. (Bharath Kumar et al. 2017); Urtica dioica L. (Bisht et al. 2017); Thuja orientalis L. (Park et al. 2014) and Matricaria chamomilla L. (Kabiri et al. 2019) used for the treatment of pseudopregnancy due to their dopaminergic effect. The dopaminergic effect of Artemisia absinthium L. has been revealed by previous studies (Kharoubi et al. 2010; Zeraati et al. 2014). Therefore, in our previous study, we investigated the therapeutic effects of the different extracts of A. absinthium on pseudopregnant rats (Demirel et al. 2018). The petroleum ether, dichloromethane and methanol extracts were administered to the rats at 100 mg/kg dose (per os) for ten days. According to the results, petroleum ether extract of the plant exhibited modulatory effect on hormonal and histological changes against pseudopregnancy. This active extract was rich in nonpolar and volatile compounds and over 70% of the compounds were found to be hydrocarbons. In the present study, we aimed to assess the potential activity of A. absinthium essential oil, which also contains nonpolar ingredients, in the treatment of pseudopregnancy in rats.
The results have shown that A. absinthium essential oil at 25 mg/kg dose displayed beneficial effects in the pseudopregnancy model in rats by considering the reduction in volume of uterine and the weights of uterine, ovary and mammary tissues when compared to the control group. Moreover, A. absinthium essential oil showed histological changes of ovarian and mammary tissues in the treatment groups.
The normal estrous cycle in rats is 4 to 5 days. The estrous cycle consists of 4 stages: proestrus (12 h), estrus (12 h), metestrus (21 h), and diestrus (57 h) (Paccola et al. 2013; Cora et al. 2015). In pseudopregnancy, after ovulation, the diestrus period is prolonged by 13–18 days, the corpora lutea become permanent and progesterone continues to be secreted (Anderson and Musah 2013; Abd-Elkareem 2017). In the first days of pseudopregnancy, the progestative process is similar to the gestational period and the structure of the CL is regulated by the production of prolactin. The nocturnal surge of prolactin stimulates the neuroendocrine pathway in pseudopregnant rats (Terkel 1988). These changes in the estrous cycle can be revealed by vaginal cytology (Cora et al. 2015). Irregular estrous cycle is observed in pseudopregnant rats. The nucleated epithelial cells of the diestrus stage continue to be seen in the extended period. In the present study, vaginal cytology was assessed on the 0, 5th, and 10th day of treatment protocol in pseudopregnant rats. The nucleated epithelial cells of the diestrus stage were seen in all the rats of the C Group during the experimental procedure. In the treatment groups, irregular-shaped cornified squamous epithelial cells were found at the end of the experiment. It was thought that the estrous cycle started in these rats. These findings in treatment groups were similar. Thus, it was concluded that vaginal cytology was not a determinative factor on the effect of A. absinthium essential oil on pseudopregnancy.
Although there is no embryo implantation in the uterus, in pseudopregnancy cases, the histomorphological changes are detected in the tissue (Abd-Elkareem 2017). These changes are nonspecific trauma-related decidual cell differentiation and hyperemic and swelling featured uterine tissue which are observed at the middle stage of pseudopregnancy (Peel et al. 1979; Anderson and Musah 2013). Owing to the enhancement of plasma progesterone level, mucosal plica, glandular formation, epithelial proliferation and thickening of the uterine wall are seen in this syndrome. Epithelial proliferation, crypt formation, increased mucosal plica and glands are characteristic findings of the last stage of pseudopregnancy (Albers et al. 2015; Abd-Elkareem 2017). The numbers of CL were significantly increased in the C group when compared to the treatment groups. Thus, similar to other studies, increased mucosal plica and glandular hypertrophy in endometrium were distinct in C and T-1 groups. Mucosal plica was determined in some cases in the other groups. The previous studies have shown that the mammary epithelial cells are increased at the stage of metestrus as seen in pseudopregnant females (Hvid et al. 2012). In the current study, while mammary gland, alveolar and ductal developments were found to be much enhanced in C and T-1 groups, the mammary tissue was recovered in T-2 and R groups. In overall assessment, 25 mg/kg of A.absinthium essential oil was determined as the effective dose on uterine and mammary tissue in pseudopregnant rats.
In our previous study, GC-MS analysis revealed that, among the volatile compounds, cis-chrysantenyl acetate and β-thujone were the major ones in the most active petroleum ether extract (Demirel et al. 2018). According to the phytochemical findings of the present study, cis-chrysanthenyl acetate was the main compound with a percentage of 17.8. Moreover, sabinyl acetate, terpinen-4-ol, caryophyllene oxide and (E)-nuciferol were determined as other main compounds. These findings were in accordance with the outcome of the previous studies in which cis-epoxyocimene, cis-chrysanthenyl acetate, bornyl acetate, eucalyptol, myrcene, linalool, neryl butanoate, sabinene, trans-sabinyl acetate, trans-thujone, α- and β-thujone were detected in the essential oil of the plant (Bailen et al. 2013; Batiha et al. 2020; Dhen et al. 2014; Jiang et al. 2021; Judzentiene and Budiene 2010; Julio et al. 2015; Orav et al. 2006; Pino et al. 1997; Rezaeinodehi and Khangholi 2008). In agreement with our results, cis-chrysanthenyl acetate was reported as the major component in the essential oil of A. absinthium. Sharopov et al. (2012) investigated three A. absinthium samples from two different localities and cis-chrysanthenyl acetate (17.9%) was the main component in one of the samples. In addition, high concentration of cis-chrysanthenyl acetate was reported by Bailen et al. (2013) in the essential oils obtained from A. absinthium grown under various environmental conditions. Similar results were also reported by Judzentiene and Budiene (2010), Julio et al. (2015) and Obistioiu et al. (2014). However, chamazulene (Msaada et al. 2015), bornyl acetate (Pino et al. 1997), neryl butanoate (Orav et al. 2006), sabinene (Orav et al. 2006) were found to be the main compounds in the essential oils of A. absinthium from different countries. The observed differences could be explained by climatic (rainfall, temperature, sunlight etc.), geographical conditions (altitude, soil structure etc.) and harvest time.
Thujone, a psychoactive compound, causes hallucinations and enhances the activity of dopamine. Medicinal plants that have thujone have been reported to be used as an abortifacient, contraceptive and uterotonic and for the treatment of amenorrhea and uterine carcinomas (Dhiman et al. 2012). However, long-term use of A. absinthium essential oil can be toxic and cause mental disorders such as insomnia, convulsions, and hallucinations. Judzentiene et al. (2012) investigated wormwood essential oil toxicity depending on oil composition. The most toxic essential oils were found to be those containing significant amounts of trans-sabinyl acetate and thujon, while other samples containing an equivalent amount of sabinyl acetate but not thujon were found to be significantly less toxic. α- and β-thujone were listed as potentially dangerous compounds in A. absinthium by The European Food Safety Authority (EFSA). However, α- and β-thujone content in the oil between 0% and 70.6% does not cause any side effects (Szopa et al. 2020). In our study, β-thujone content was determined as 0.1% in the oil.