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Pimpinella anisum extract attenuates spontaneous and agonist-induced uterine contraction in term-pregnant rats
Journal of Ethnopharmacology 254 (2020) 112730
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Pimpinella anisum extract attenuates spontaneous and agonist-induced uterine contraction in term-pregnant rats
T
Mohammed F. Alotaibi Department of Physiology, College of Medicine, King Saud University, P.O Box 2925, Riyadh, 11461, Saudi Arabia
A R T I C LE I N FO
A B S T R A C T
Keywords: Anise Uterus Contraction Term-pregnant Rats Oxytocin
Ethnopharmacology relevance: Pimpinella anisum is a well-known traditional medicinal herb which has been used in folk medicine as an antiulcer, anticancer, antibacterial and as a muscle relaxant. Aim of the study: This study was performed to explore the modulatory effects of Pimpinella anisum on termpregnant rat uterine contractility and to investigate its possible underlying mechanisms. Material and methods: Intact uterine strips without endometrial layer were isolated from female term-pregnant Wistar rats (22 days of gestation) and mounted in a tissue bath apparatus for in vitro isometric force recording. The effects of different concentrations of Pimpinella anisum extract (PAE) (1, 3, 5, and 7 mg/mL) were examined on uterine contractions generated spontaneously or induced with oxytocin (5 nmol/L), Bay K8644 (1 μmol/L), and carbachol (10 μmol/L). In some experiments, PAE was applied on depolarized myometrium in the presence of high-KCl solution (60 mmol/L). The effect on Ca2+ release was also examined. Results: Application of PAE significantly reduced uterine contractions generated spontaneously or induced with oxytocin, Bay K8644, and carbachol in a concentration-dependent manner (n = 7; P < 0.01). In depolarized myometrium, PAE significantly reduced the tonic force induced by high-KCl solution (n = 7; P < 0.01). PAE prevented oxytocin-induced transient contraction in the entire absence of external calcium (n = 7; P < 0.01). Conclusion: The present findings demonstrate the potentials of PAE to relax pregnant uterine contractions possibly by blocking Ca2+ entry via L-type calcium channels and inhibiting Ca2+ release from the internal store. The tocolytic effects of PAE may be a potential adjuvant against strong premature uterine contractions which threaten early pregnancy although clinical studies are required.
1. Introduction The uterus is a relatively quiescent organ throughout pregnancy which transforms to a very excitable tissue at the onset of term or preterm labours. Preterm birth (delivery of a baby before 37 gestational weeks) is the most likely contributing factor to neonatal morbidity and mortality (Stanton et al., 2006). The underlying mechanisms of premature uterine stimulation during preterm labour are not fully understood but it involves a premature activation of contractile associated proteins including connexin-43, oxytocin and prostaglandin F2α receptors (Cook et al., 2000). Available medications to restrain forceful uterine activity during preterm labor are still questioned regarding their effectiveness, maternal/neonatal side effects, and long-term safety (Kim and Shim, 2006). Due to these considerations and due to socio-economic problems in different populations, several studies have been
performed in reproductive medicine to better find safe and cost-effective tocolytic medicinal plants to control uterine activity (Pereira-DeMorais et al., 2019; Monji et al., 2018). One of the oldest traditional plant that has been used worldwide in folk medicine is Pimpinella anisum L. (anise) that belongs to the Apiaceae family. Pimpinella anisum possesses antibacterial (Gülçın et al., 2003), antiviral (Lee et al., 2011), anticonvulsant (Karimzadeh et al., 2012), antitoxic (Jamshidzadeh et al., 2015), antioxidant (Gülçın et al., 2003), antiulcer (Al Mofleh et al., 2007), and muscle relaxant (Tirapelli et al., 2007) effects. Pimpinella anisum is grown for its seeds (aniseeds) and their beneficial properties are derived mainly from its essential oil which contains transanethole, cis-anethole, estragole, γ-himachalene, eugenol, α-cuparene, β-bisabolene, p-anisaldehyde, and terpenic compounds (Özcan and Chalchat, 2006; Orav et al., 2008; Gülçın et al., 2003). A full description of chemical compositions of Pimpinella anisum has been reported in
Abbreviations: PAE, Pimpinella anisum extract; IACC, Institutional Animal Care Committee; CCh, Carbachol; KCl, Potassium chloride; VGCCs, voltage gated calcium channels; OTRs, oxytocin receptors; DART-ToF-MS, Direct Analysis in Real Time – Time of Flight – Mass Spectrometry; AUC, area under the curve; ANOVA, analysis of variance E-mail address: [email protected]. https://doi.org/10.1016/j.jep.2020.112730 Received 13 September 2019; Received in revised form 13 February 2020; Accepted 29 February 2020 Available online 04 March 2020 0378-8741/ © 2020 Elsevier B.V. All rights reserved.
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Calcium-free solution was prepared exactly as the Krebs solution but without CaCl2 and adding 1 mM calcium-chealator ethyl glycol-bis (βaminoethyl ether)-N,N,N′,N′-tetra acetic acid (EGTA) (Zafrah and Alotaibi, 2017). The desired concentrations of PAE were dissolved directly in the Krebs solution and maintaining the pH at 7.4.
other studies (Özcan and Chalchat, 2006; Shojaii and Abdollahi Fard, 2012). Anethole, estragole, and eugenol have been shown to cause relaxation of different types of smooth muscle (Lima et al., 2011; Cabral et al., 2014; Soares et al., 2007). Despite the reported significant effects of Pimpinella anisum in reducing postpartum pain associated with uterine cramping and involution in vivo (Simbar et al., 2015), its direct effects on uterine contractions have not been tested in vitro to support its therapeutic use in reproductive medicine. Therefore, this study was designed to explore the potential effects of Pimpinella anisum extract (PAE) on term-pregnant uterine contractility and to investigate its possible underlying mechanisms in vitro.
2.4. Experimental protocols 2.4.1. Effects of PAE on spontaneous uterine contractions Uterine strips were left contracting in oxygenated buffered Krebs solution until regular spontaneous activity was established and this was used as a control period (100%). Cumulative concentrations of PAE (1, 3, 5, and 7 mg/mL) were determined while uterine strips were contracting spontaneously and a concentration-response curve was obtained.
2. Materials and methods 2.1. Plant materials and extraction
2.4.2. Effects of PAE on uterine contractions induced by oxytocin After obtaining steady spontaneous contractions in Krebs solution, uterine strips were stimulated with oxytocin until regular phasic contractions were established. After equilibrium period, cumulative concentrations of PAE (1, 3, 5, and 7 mg/mL) were determined in the continued presence of oxytocin and a concentration-response curve was obtained. To investigate the effect of PAE on calcium release from the intracellular calcium stores, calcium-free medium was used. After equilibrium of spontaneous contractions, Krebs solution was replaced with the calcium-free medium and was left until complete abolition of spontaneous activity and oxytocin was applied in the absence (control) and presence of 5 mg/mL PAE.
Fresh Pimpinella anisum seeds were purchased from a medical herb store in August 2017 and authenticated by Dr. Basodaan, Department of Pharmacognosy, King Saud University (Riyadh, Saudi Arabia) and a voucher specimen (0912) was deposited. Seeds were extracted as previously described (Alotaibi, 2016). Briefly, seeds were washed with distilled water, air-dried at room temperature, and ground, 400 g of the yield powder was extracted with a 50:50 water-ethanol solution for 36 h with occasional shaking. After filtration, the extract was desiccated by a rotary evaporator to eliminate the solvents which finally yielded a viscous gummy dark extract (50 g). The extract was kept in a sterile bottle and kept at 4 °C until used.
2.4.3. Effects of PAE on uterine depolarization induced by high-KCl solution High-KCl solution depolarizes the cell membrane leading to calcium influx via voltage gated calcium channels (VGCCs) and sustained tonic contraction (Amédée et al., 1986). To investigate the effect of PAE on uterine depolarization, uterine strips were pre-contracted for 15 min with high-KCl solution until tonic contraction was established. Uterine strips were then washed out with Krebs solution until complete recovery and then pre-contracted again with high-KCl solution containing 5 mg/mL PAE for 15 min.
2.2. Animals and tissue preparations Term-pregnant Wistar rats (~22 days of gestation) were used in this study. Animals were killed by carbon dioxide (CO2) asphyxia followed by decapitation in accordance with the guidelines of the Institutional Animal Care Committee (IACC), College of Medicine, King Saud University and in accordance with the principles and procedures of laboratory animal use and care published by the European Communities Council Directives (2010/63/EU). The abdomen was opened and the uterus was removed and immediately immersed in Krebs solution. Longitudinal uterine strips were dissected and the endometrium was carefully removed by gentle scrape using a blade. Uterine strips were tied from both ends by surgical threads, and placed in a 5-mL tissue bath chamber (Radnoti, Monrovia, CA, USA) containing a pre-warmed Krebs solution and aerated with 95% O2 and 5% CO2 at 37 °C. Strips were attached to a sensitive isometric force transducer (ADInstruments Ltd, Sydney, Australia) which measures any changes in tension and displays it on a monitor using LabChart software (AD Instruments Ltd, Sydney, Australia). The strips were allowed to equilibrate in the Krebs solution for at least 30 min before performing any experiment to establish regular and steady contractile activity. The tissue bath is continually superfused with warmed Krebs solution at a rate of 4 mL/min using Dynamax RP-1 peristaltic pump (Rainin Instrument Co., USA).
2.4.4. Effects of PAE on uterine contractions induced by carbachol To investigate if the inhibitory effect of the PAE involves muscarinic receptors, carbachol (a non-selective and cholinesterase-resistant muscarinic receptor agonist; CCh) was used. After obtaining steady spontaneous contractions, uterine strips were stimulated with CCh until phasic contractions were established. After equilibrium period, cumulative concentrations of PAE (1, 3, 5, and 7 mg/mL) were determined in the continued presence of CCh and a concentration-response curve was obtained. 2.4.5. Effects of PAE on uterine contractions induced by Bay K8644 To investigate if the inhibitory effect of the PAE was due to blockade of calcium influx via L-type VGCCs, Bay K8644 (a specific L-type calcium channel agonist) was used. After obtaining steady spontaneous contractions, uterine strips were stimulated with Bay K8644 until phasic contractions were established. After equilibrium period, cumulative concentrations of PAE (1, 3, 5, and 7 mg/mL) were determined in the continued presence of Bay K8644 and a concentration-response curve was obtained.
2.3. Chemicals and solutions All drugs and reagents used in this study were supplied by SigmaAldrich (St. Louis, MO, USA) and were of analytic grade. Krebs solution was made with the following compositions [mmol/L]: 115 NaCl, 4.7 KCl, 2 CaCl2, 1.16 MgSO4, 1.18 KH2PO4, 22 NaHCO3, and 7.88 dextrose, pH was adjusted to 7.4 (Alotaibi, 2016). Oxytocin and Carbachol (CCh) were prepared in distilled H2O and dissolved directly in Krebs solution at a final concentration of 5 nmol/L and 10 μmol/L, respectively. Bay K8644 was dissolved in absolute ethanol and used at a final concentration of 1 μmol/L. High-potassium chloride (KCl) solution (60 mmol/L) was prepared by equimolar substitution of NaCl for KCl.
2.5. Analysis of PAE by mass spectrometry Fresh PAE was characterized by a high resolution Direct Analysis in Real Time – Time of Flight – Mass Spectrometry (DART-ToF-MS) and the ESI-MS spectra were recorded on the MS system (AccuTOF LC-Plus; JEOL Co., Tokyo, Japan). The volatile components of the PAE were evaporated in a stream of helium heated at 300 °C, then ionized by the 2
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excited metastable helium atoms, before entering the ion source of the time of flight mass spectrometer. In the positive ionization mode, each molecule is transformed into either a protonated ion [M+H]+ or a nonprotonated radical molecular ion M+.. Since the ionization process is considered as “soft”, no or little fragmentation occurs, such that each peak in the spectrum corresponds to a given compound. The experimental conditions used for analysis of PAE by DART-ToF-MS are as follow: Vacuum (Pirani gauge): 1.8x10+2 Pa, Vacuum (Analyzer): 1.3x10−5 Pa, Heater temperature: 350 °C, Ionization mode: positive, Heating and ionization gas: helium, Ring lens voltage: 4 V, Orifice 1 voltage: 5 V, Orifice 2 voltage: 5 V, Peaks voltage: 500 V, Mass range: 50–1000 Da, Mass resolution range: 3600–4900.
Table 1 Effects of different concentrations of Pimpinella anisum extract (PAE) on contractile parameters during spontaneous contraction in term-pregnant rats (n = 7). Contraction parameters
0 mg/mL (control)
1 mg/mL
3 mg/mL
5 mg/mL
7 mg/mL
AUC Frequency
100% 100%
99 ± 2% 99 ± 1%
67 ± 3%* 112 ± 8%
49 ± 2%* 68 ± 6%*
2 ± 2%* 0%*
*P < 0.01 compared with control or with preceding concentration (ANOVA/ Bonferroni).
concentrations. Mean data for the effects of different concentrations of PAE on contractile parameters during spontaneous contractions are shown in Table .1.
2.6. Data and statistical analyses Data were imported and analysed using Microcal Origin Software 9.0 (Northampton, MA, USA). The main parameters measured were force integral (area under the curve; AUC) and frequency of contractions. AUC (in arbitrary units) was calculated by measuring the entire uterine activity for 10 min before and after application of the extract, whereas, the frequency was calculated by counting the number of contractions occurring in 10 min. The effect of a known agent or drug can be determined by measuring the changes in these parameters, as they considered an index for the entire contractile activity by the uterine strip at a given time (Arrowsmith et al., 2018). The half maximal inhibitory concentration (IC50) which produced 50% of maximum reduction of AUC was calculated. The contractile activity in the last 10 min before adding PAE was precisely measured and taken as 100% control and the results were expressed as a percentage of this control. Data are presented as means ± SEM and “n” represents the number of uterine strips, one from different rat. Differences between control and treatment groups were evaluated using one-way analysis of variance (ANOVA)/Bonferroni tests. P values < 0.05 were considered significant.
3.2. Dose-response effects of PAE on uterine contractions induced by oxytocin
3. Results
Stimulation of uterine strips with oxytocin significantly increased the contractile parameters including AUC and frequency of contractions compared with the preceding spontaneous activity (Alotaibi et al., 2015). When PAE was applied in the continued presence of oxytocin, the force significantly and progressively decreased in a concentrationdependent manner (Fig. 2A and B; n = 7). The frequency of contractions transiently but insignificantly increased upon application of 3 mg/ mL PAE and progressively decreased and abolished with higher concentrations of PAE. Mean data for the effects of different concentrations of PAE on contractile parameters in the presence of oxytocin are shown in Table .2. In calcium-free medium, spontaneous uterine activity ceases and oxytocin can produce a transient small contraction in the continued absence of external calcium indicating calcium release from the sarcoplasmic reticulum (Munglue et al., 2013; Sukwan et al., 2014). Application of 5 mg/mL PAE in the continued absence of external calcium prevented oxytocin-induced transient contraction (Fig. 3A; n = 7).
3.1. Dose-response effects of PAE on spontaneous uterine activity
3.3. Effects of PAE on uterine depolarization induced by high-KCl solution
Application of PAE inhibited spontaneous uterine activity in a concentration-dependent manner in all samples tested (Fig. 1A&B; n = 7). The AUC progressively decreased after addition of rising concentrations of PAE and the force completely abolished with the highest dose (7 mg/mL). The IC50 for the AUC was 5 mg/mL which decreased the force to 49 ± 2% compared with 100% control (0 mg/mL). The frequency of contraction transiently but insignificantly increased and then progressively decreased until abolished after increasing PAE
Application of high-KCl solution caused depolarization and sustained tonic contraction which was maintained as long as it was applied. We have shown previously that repeated applications of high-KCl solution on the same uterine strip produced no significant difference in tonic force in term-pregnant rats (Zafrah and Alotaibi, 2017). Interestingly, application of 5 mg/mL PAE significantly reduced the force integral from 100% (before adding the extract) to 44 ± 3% (Fig 3B; n = 7).
Fig. 1. (A) Original recording for the effect of cumulative concentrations of PAE on spontaneous uterine contractions in term-pregnant rats. (B) Concentrationresponse curve for the effect of PAE on spontaneous uterine contractions. Vertical bars represent SEM (n = 7). *P < 0.01 compared with the preceding dose. 3
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Fig. 2. (A) Original recording for the effect of cumulative concentrations of PAE on uterine contractions induced by 5 nmol/L oxytocin in term-pregnant rats. (B) Concentration-response curve for the effect of PAE on oxytocin-induced uterine contractions. Vertical bars represent SEM (n = 7). *P < 0.01 compared with the preceding dose.
3.4. Effects of PAE on uterine contraction induced by carbachol and Bay K8644
Table 2 Effects of different concentrations of Pimpinella anisum extract (PAE) on contractile parameters in the presence of 5 nmol/L oxytocin in term-pregnant rats (n = 7). Contraction parameters
0 mg/mL (control)
1 mg/mL
3 mg/mL
5 mg/mL
7 mg/mL
AUC Frequency
100% 100%
99 ± 2% 100%
67 ± 2%* 106 ± 5%
46 ± 3%* 71 ± 3%*
2 ± 1%* 0%*
Stimulation of uterine strips with agonists (CCh or Bay K8644) resulted in a marked increase in force integral and contraction frequency compared with the preceding spontaneous activity (Wu et al., 2015). Application of PAE significantly and progressively decreased uterine contractile activity in a concentration-dependent manner in the continued presence of CCh and Bay K8644 (Fig 3C; n = 7). Mean data for the effects of different concentrations of PAE on contractile parameters in the presence of CCh and Bay K8644 are shown in Table s 3 and 4, respectively.
*P < 0.01 compared with control or with preceding concentration (ANOVA/ Bonferroni).
Fig. 3. (A) A typical trace for the effect of 5 mg/mL PAE on uterine contraction induced by 5 nmol/L oxytocin in calcium-free medium in term-pregnant rats. (B) A typical trace for the response of uterine strip to high-KCl solution (60 mmol/L) before and after addition of 5 mg/mL PAE in term-pregnant rats. (C) Concentrationresponse curve for the effects of PAE on uterine contractions pre-contracted with 10 μmol/LCarbachol (CCh) and 1 μmol/L Bay K8644 in term-pregnant rats. Control did not receive any PAE dose. Vertical bars represent SEM (n = 7). *P < 0.01 compared with the preceding dose. 4
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be increased following stimulation by oxytocin, Bay K8644, or CCh (Wu et al., 2015). Extracellular Ca2+ is vital for uterine spontaneous contractions and blocking calcium entry via VGCCs can significantly abolish spontaneous activity (Promprom et al., 2010). In this study, PAE is able to inhibit spontaneous uterine contractions in a concentration-dependent manner suggesting inhibition of Ca2+ entry via Ltype VGCCs. To further investigate the effect of PAE on calcium influx pathway, we stimulated the uterine strips with high-KCl solution and Bay K8644 to activate calcium entry via L-type VGCCs. High-KCl solution causes tonic and maximum uterine contraction by changing the reversal potassium potential resulting in membrane depolarization and opening of L-type VGCCs, thus further elevating [Ca2+]i (Wray, 2007). Pharmacological blockade of VGCCs or absence of external Ca2+ can markedly abolish KCl-induced tonic contraction (Perez and Sanderson, 2005; Maggi and Meli, 1984), suggesting that Ca2+ entry via VGCCs is a major pathway for KCl-induced smooth muscle contraction. Bay K8644 is a stimulator of L-type VGCCs being able to elevate [Ca2+]i by Ca2+ influx via L-type VGCCs leading to increased amplitude and frequency of contraction (Alotaibi et al., 2017). In the presence of PAE, KCl-induced tonic contraction was significantly attenuated which is consistent with the reported relaxant effects of PAE on contractions induced by high-KCl in tracheal smooth muscle (Boskabady and Ramazani-Assari, 2005), and in depolarized portal vein (Pontes et al., 2019). Furthermore, PAE progressively and significantly decreased the force induced by Bay K8644 in a concentration dependent manner, indicating blockade of Ca2+ entry mechanism. It has been shown recently that PAE exerted a relaxation effect on cardiac and vascular smooth muscles by blocking calcium influx pathway (Pontes et al., 2019). Therefore, our findings suggest the ability of PAE to inhibit Ca2+ influx via VGCCS in pregnant uterine smooth muscle. Oxytocin augments uterine activity and increases the force amplitude, duration, and frequency compared to spontaneous activity (Alotaibi, 2017). The principal mechanism of oxytocin-increased force is due to the activation of L-type VGCCs and OTRs stimulation. Activation of OTRs (which belong to G proteincoupled receptor superfamily) causes phospholipase C activation and increased production of inositol 1,4,5-triphosphate (IP3) resulting in Ca2+ release from the SR and augmentation of contractions (Vrachnis et al., 2011). Our findings show that PAE can significantly decrease and abolish oxytocin-induced uterine contraction in the presence of external calcium in a concentration-dependant manner. Oxytocin can cause transient contraction in entire absence of external Ca2+ suggesting IP3induced Ca2+ release as uterine smooth muscle lacks functional ryanodine receptors responsible for Ca2+-induced Ca2+release (Matsuki et al., 2017). Our findings show that PAE can significantly prevent oxytocin-induced transient release of Ca2+from the SR, suggesting inhibition of IP3 messenger or its receptors on the SR. The myometrium expresses muscarinic receptors which mediate CCh-induced uterine contraction (Kitazawa et al., 2008). The action of CCh on rat uterine muscarinic receptors is mediated mainly via M3 receptor subtype (Abdalla et al., 2000, 2004; Kitazawa et al., 2008) which is coupled primarily to PLC-mediated IP3 production and Ca2+ release from the SR (Caulfield and Birdsall, 1998). We observed that CCh can markedly increase the force amplitude and frequency of contraction compared to spontaneous uterine activity. PAE can significantly reduce the CCh-induced uterine contraction in a concentration-dependent manner which further characterizes the relaxant effects of PAE on uterine activity. This finding is consistent with previous studies where PAE attenuated acetylcholine-induced contraction in rat anococcygeus (Tirapelli et al., 2007) and guinea pigs tracheal (Boskabady and Ramazani-Assari, 2001) smooth muscles. Our MS analysis revealed that the active constituents of PAE are anethole and eugenol which are consistent with the previous phytochemical studies (Orav et al., 2008; Özcan and Chalchat, 2006). Previous works have confirmed the relaxant effects of anethole and eugenol on smooth muscle contraction (Lima et al., 2011; Cabral et al., 2014; Damiani et al., 2003; Soares et al., 2007), and we suggest that the relaxant effects of PAE on uterine contractions could be due to the
Table 3 Effects of different concentrations of Pimpinella anisum extract (PAE) on contractile parameters in the presence of 10 μmol/L CCh in term-pregnant rats (n = 7). Contraction parameters
0 mg/mL (control)
1 mg/mL
3 mg/mL
5 mg/mL
7 mg/mL
AUC Frequency
100% 100%
100% 100%
70 ± 4%* 108 ± 5%
52 ± 3%* 75 ± 3%*
3 ± 2%* 0%*
*P < 0.01 compared with control or with preceding concentration (ANOVA/ Bonferroni). Table 4 Effects of different concentrations of Pimpinella anisum extract (PAE) on contractile parameters in the presence of 1 μmol/L Bay K8644 in term-pregnant rats (n = 7). Contraction parameters
0 mg/mL (control)
1 mg/mL
3 mg/mL
5 mg/mL
7 mg/mL
AUC Frequency
100% 100%
98 ± 2% 100%
64 ± 5%* 109 ± 3%
41 ± 4%* 67 ± 2%*
2 ± 2%* 0%*
*P < 0.01 compared with control or with preceding concentration (ANOVA/ Bonferroni).
3.5. Main constituents of PAE MS analysis revealed that PAE contains anethole (principal component) and other compounds as shown in Table 5. 4. Discussion Although previous clinical trials have confirmed the efficacy of Pimpinella anisum in reducing migraine attacks (Mosavat et al., 2019) and relieving postpartum depression (Ghoshegir et al., 2015), there are no previous studies showing the direct effect of PAE on uterine activity. Our current study shows for the first time that Pimpinella anisum possesses a clear tocolytic effects on uterine strips isolated from pregnant rats which can cause relaxation in a concentration-dependent manner. PAE can significantly reduce the uterine contractions irrespective how the force was produced which may support the reported in vivo effects of Pimpinella anisum in alleviating the postpartum pain associated with uterine cramping and involution (Simbar et al., 2015). Our in vitro findings are in agreement with previous studies showing the relaxant effects of PAE on tracheal (Boskabady and Ramazani-Assari, 2001) and anococcygeus (Tirapelli et al., 2007) smooth muscles contractility. The uterus is a spontaneously active tissue and contraction of its smooth muscle is controlled primarily by the intracellular calcium concentration [Ca2+]i. In smooth muscle, Ca2+ can enter the cell via L-type VGCCs or it can be released from its intracellular stores (sarcoplasmic reticulum; SR) (Thorneloe and Nelson, 2005). In addition, [Ca2+]i can Table 5 Composition of Pimpinella anisum extract. No
Percentage
Compound
1 2 3 4 5 6 7 8 9 10 11 12
0.08 90.5 1.4 1.12 0.19 0.22 2.9 0.03 0.02 0.04 0.01 3.49
p-cymene anethole γ-himachalene carvone dihydrocarvone methylcinnamic acid eugenol 2,7-octadienyl acetate cuparene caryophyllene 1,2-diphenyl-1-butanol Not identified
5
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Fig. 4. Scheme showing the possible underlying mechanisms of decreased uterine force by Pimpinella anisum extract (PAE) in term-pregnant rats. (1) calcium entry via L-type voltage gated calcium channels (VGCCs) is blocked by PAE. (2) PAE blocks uterine activity stimulated by L-type VGCCs activator and depolarization by high-KCl. (3) PAE blocks oxytocin/ muscarinic receptors which reduces membrane depolarization/calcium influx and inhibits PLC-mediated IP3 production and calcium release from the SR. SM; smooth muscle. SR; sarcoplasmic reticulum. MLCK; myosin light-chain kinase. MLC20; myosin light chain.
the plant extraction and identification of the active ingredients of PAE.
presence of these compounds. Therefore, our data clearly demonstrate that the tocolytic effects of Pimpinella anisum on uterine activity are partially attributed to the disruption of calcium entry and/or release mechanism. In addition, we suggest that PAE could act as a competitive antagonism on oxytocin or muscarinic receptors to reduce uterine contractions. As this is the first study showing the dose-dependent relaxation effect of PAE on pregnant rat uterine activity in vitro, the same dosage we used cannot be necessarily extrapolated to pregnant human uterus. Therefore, clinical trials and in vivo experiments are required to find the optimal and safe dose for the use in the clinical practice in order to control the abnormal uterine hyper-activity that occurs during dysmenorrhea or before term.
References Abdalla, F.M., Abreu, L.C., Porto, C.S., 2000. Effect of estrogen on intracellular signaling pathways linked to activation of M2-and M3-muscarinic acetylcholine receptors in the rat myometrium. Mol. Cell. Endocrinol. 160, 17–24. Abdalla, F.M., Maróstica, E., Picarelli, Z.P., Abreu, L.C., Avellar, M.C.W., Porto, C.S., 2004. Effect of estrogen on muscarinic acetylcholine receptor expression in rat myometrium. Mol. Cell. Endocrinol. 213, 139–148. Al Mofleh, I.A., Alhaider, A.A., Mossa, J.S., Al-Soohaibani, M.O., Rafatullah, S., 2007. Aqueous suspension of anise “Pimpinella anisum” protects rats against chemically induced gastric ulcers. World J. Gastroenterol.: WJG 13, 1112. Alotaibi, M., 2016. The effect of cinnamon extract on isolated rat uterine strips. Reprod. Biol. 16, 27–33. Alotaibi, M., Arrowsmith, S., Wray, S., 2015. Hypoxia-induced force increase (HIFI) is a novel mechanism underlying the strengthening of labor contractions, produced by hypoxic stresses. Proc. Natl. Acad. Sci. Unit. States Am. 112, 9763–9768. Alotaibi, M., Kahlat, K., Nedjadi, T., Djouhri, L., 2017. Effects of ZD7288, a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker, on term-pregnant rat uterine contractility in vitro. Theriogenology 90, 141–146. Alotaibi, M.F., 2017. The response of rat and human uterus to oxytocin from different gestational stages in vitro. Gen. Physiol. Biophys. 36, 75–82. Amédée, T., Mironneau, C., Mironneau, J., 1986. Isolation and contractile responses of single pregnant rat myometrial cells in short‐term primary culture and the effects of pharmacological and electrical stimuli. Br. J. Pharmacol. 88, 873–880. Arrowsmith, S., Keov, P., Muttenthaler, M., Gruber, C.W., 2018. Contractility measurements of human uterine smooth muscle to aid drug development. JoVE 26, 56639. Boskabady, M., Ramazani-Assari, M., 2001. Relaxant effect of Pimpinella anisum on isolated Guinea pig tracheal chains and its possible mechanism (s). J. Ethnopharmacol. 74, 83–88. Boskabady, M., Ramazani-Assari, M., 2005. Possible mechanism for the relaxant effect of Pimpinella anisum. on Guinea pig tracheal chains. Pharmaceut. Biol. 42, 621–625. Cabral, P.H.B., De Morais Campos, R., Fonteles, M.C., Santos, C.F., Cardoso, J.H.L., Do Nascimento, N.R.F., 2014. Effects of the essential oil of Croton zehntneri and its major components, anethole and estragole, on the rat corpora cavernosa. Life Sci. 112, 74–81. Caulfield, M.P., Birdsall, N.J., 1998. International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol. Rev. 50, 279–290. Cook, J.L., Zaragoza, D.B., Sung, D.H., Olson, D.M., 2000. Expression of myometrial activation and stimulation genes in a mouse model of preterm labor: myometrial activation, stimulation, and preterm labor. Endocrinology 141, 1718–1728. Damiani, C.E.N., Rossoni, L.V., Vassallo, D.V., 2003. Vasorelaxant effects of eugenol on rat thoracic aorta. Vasc. Pharmacol. 40, 59–66.
5. Conclusion The current study provides clear evidence for the relaxant effects of PAE on pregnant rat uterus which may potentially be used as a tocolytic herbal medicine to control unwanted uterine contractions occurring in preterm labours or to reduce strong uterine contractions during menstruation (dysmenorrhea). The possible underlying mechanisms of PAEinduced relaxation could be due to the blockage of calcium entry via Ltype VGCCs and/or inhibition of calcium release from the SR in termpregnant rat uterus. Proposed mechanisms of PAE-induced uterine relaxation are depicted in Fig. 4. Declaration of competing interest None declared. Acknowledgments I would like to thank the Deanship of Scientific Research at King Saud University for supporting this project. Special thanks to Dr. Basodaan and Professor A. Yacine Badjah-Hadj-Ahmed for their help in 6
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(Pimpinella anisum L.) fruit oil at ripening stage. Ann. Microbiol. 56, 353–358. Pereira-De-Morais, L., De Alencar Silva, A., Da Silva, R.E.R., Da Costa, R.H.S., Monteiro, Á.B., Dos Santos Barbosa, C.R., De Souza Amorim, T., De Menezes, I.R.A., Kerntopf, M.R., Barbosa, R., 2019. Tocolytic activity of the Lippia alba essential oil and its major constituents, citral and limonene, on the isolated uterus of rats. Chem. Biol. Interact. 297, 155–159. Perez, J.F., Sanderson, M.J., 2005. The contraction of smooth muscle cells of intrapulmonary arterioles is determined by the frequency of Ca2+ oscillations induced by 5-HT and KCl. J. Gen. Physiol. 125, 555–567. Pontes, V.C.B., Rodrigues, D.P., Caetano, A., Gamberini, M.T., 2019. Preclinical investigation of the cardiovascular actions induced by aqueous extract of Pimpinella anisum L. seeds in rats. J. Ethnopharmacol. 237, 74–80. Promprom, W., Kupittayanant, P., Indrapichate, K., Wray, S., Kupittayanant, S., 2010. The effects of pomegranate seed extract and β-sitosterol on rat uterine contractions. Reprod. Sci. 17, 288–296. Shojaii, A., Abdollahi Fard, M., 2012. Review of pharmacological properties and chemical constituents of Pimpinella anisum. ISRN pharmaceutics 2012. Simbar, M., Shadipour, M., Salamzadeh, J., Ramezani-Tehrani, F., Nasiri, N., 2015. The combination of “Pimpinella anisum, Apium graveolens and Crocus sativus (PAC)” is more effective than “mefenamic acid” on postpartum after-pain. J. Herb. Med. 5, 20–25. Soares, P.M.G., Lima, R.F., De Freitas Pires, A., Souza, E.P., Assreuy, A.M.S., Criddle, D.N., 2007. Effects of anethole and structural analogues on the contractility of rat isolated aorta: involvement of voltage-dependent Ca2+-channels. Life Sci. 81, 1085–1093. Stanton, C., Lawn, J.E., Rahman, H., Wilczynska-Ketende, K., Hill, K., 2006. Stillbirth rates: delivering estimates in 190 countries. Lancet 367, 1487–1494. Sukwan, C., Wray, S., Kupittayanant, S., 2014. The effects of Ginseng Java root extract on uterine contractility in nonpregnant rats. Phys. Rep. 2. Thorneloe, K.S., Nelson, M.T., 2005. Ion channels in smooth muscle: regulators of intracellular calcium and contractility. Can. J. Physiol. Pharmacol. 83, 215–242. Tirapelli, C.R., De Andrade, C.R., Cassano, A.O., De Souza, F.A., Ambrosio, S.R., Da Costa, F.B., De Oliveira, A.M., 2007. Antispasmodic and relaxant effects of the hidroalcoholic extract of Pimpinella anisum (Apiaceae) on rat anococcygeus smooth muscle. J. Ethnopharmacol. 110, 23–29. Vrachnis, N., Malamas, F.M., Sifakis, S., Deligeoroglou, E., Iliodromiti, Z., 2011. The oxytocin-oxytocin receptor system and its antagonists as tocolytic agents. International journal of endocrinology 2011. Wray, S., 2007. Insights into the uterus. Exp. Physiol. 92, 621–631. Wu, C.-H., Shieh, T.-M., Wang, K.-L., Huang, T.-C., Hsia, S.-M., 2015. Quercetin, a main flavonoid in onion, inhibits the PGF2α-induced uterine contraction in vitro and in vivo. Journal of Functional Foods 19, 495–504. Zafrah, H.A., Alotaibi, M.F., 2017. The effect of extracellular ATP on rat uterine contraction from different gestational stages and its possible mechanisms of action. J. Basic Clin. Physiol. Pharmacol. 28, 209–217.
Ghoshegir, S.A., Mazaheri, M., Ghannadi, A., Feizi, A., Babaeian, M., Tanhaee, M., Karimi, M., Adibi, P., 2015. Pimpinella anisum in the treatment of functional dyspepsia: a double-blind, randomized clinical trial. J. Res. Med. Sci. 20, 13–21. Gülçın, İ., Oktay, M., Kıreçcı, E., Küfrevıoǧlu, Ö.İ., 2003. Screening of antioxidant and antimicrobial activities of anise (Pimpinella anisum L.) seed extracts. Food Chem. 83, 371–382. Jamshidzadeh, A., Heidari, R., Razmjou, M., Karimi, F., Moein, M.R., Farshad, O., Akbarizadeh, A.R., Shayesteh, M.R.H., 2015. An in vivo and in vitro investigation on hepatoprotective effects of Pimpinella anisum seed essential oil and extracts against carbon tetrachloride-induced toxicity. Iranian journal of basic medical sciences 18, 205. Karimzadeh, F., Hosseini, M., Mangeng, D., Alavi, H., Hassanzadeh, G.R., Bayat, M., Jafarian, M., Kazemi, H., Gorji, A., 2012. Anticonvulsant and neuroprotective effects of Pimpinella anisum in rat brain. BMC Compl. Alternative Med. 12, 76. Kim, A., Shim, J.Y., 2006. Emerging tocolytics for maintenance therapy of preterm labour: oxytocin antagonists and calcium channel blockers. BJOG An Int. J. Obstet. Gynaecol. 113, 113–115. Kitazawa, T., Hirama, R., Masunaga, K., Nakamura, T., Asakawa, K., Cao, J., Teraoka, H., Unno, T., Komori, S.-I., Yamada, M., 2008. Muscarinic receptor subtypes involved in carbachol-induced contraction of mouse uterine smooth muscle. N. Schmied. Arch. Pharmacol. 377, 503–513. Lee, J.-B., Yamagishi, C., Hayashi, K., Hayashi, T., 2011. Antiviral and immunostimulating effects of lignin-carbohydrate-protein complexes from Pimpinella anisum. Biosci. Biotechnol. Biochem. 75, 459–465. Lima, F.C., Peixoto‐Neves, D., Gomes, M.D.M., Coelho‐De‐Souza, A.N., Lima, C.C., Araújo Zin, W., Magalhães, P.J.C., Saad, L., Leal‐Cardoso, J.H., 2011. Antispasmodic effects of eugenol on rat airway smooth muscle. Fundam. Clin. Pharmacol. 25, 690–699. Maggi, C., Meli, A., 1984. The effect of nifedipine and verapamil on KCl-induced rhythmic contractions of Guinea pig ureter in vitro. Experientia 40, 681–686. Matsuki, K., Takemoto, M., Suzuki, Y., Yamamura, H., Ohya, S., Takeshima, H., Imaizumi, Y., 2017. Ryanodine receptor type 3 does not contribute to contractions in the mouse myometrium regardless of pregnancy. Pflueg. Arch. Eur. J. Physiol. 469, 313–326. Monji, F., Lau, L.C., Siddiquee, A.a.-M., Said, B.B., Yang, L.-K., Yoganathan, K., Choolani, M.A., Adaikan, P.G., 2018. Potent tocolytic activity of ethyl acetate fraction of Ananas comosus on rat and human uteri. Biomed. Pharmacother. 105, 824–834. Mosavat, S.H., Jaberi, A.R., Sobhani, Z., Mosaffa-Jahromi, M., Iraji, A., Moayedfard, A., 2019. Efficacy of Anise (Pimpinella anisum L.) oil for migraine headache: a pilot randomized placebo-controlled clinical trial. J. Ethnopharmacol. 236, 155–160. Munglue, P., Eumkep, G., Wray, S., Kupittayanant, S., 2013. The effects of watermelon (Citrullus lanatus) extracts and L-citrulline on rat uterine contractility. Reprod. Sci. 20, 437–448. Orav, A., Raal, A., Arak, E., 2008. Essential oil composition of Pimpinella anisum L. fruits from various European countries. Nat. Prod. Res. 22, 227–232. Özcan, M.M., Chalchat, J.C., 2006. Chemical composition and antifungal effect of anise
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Pimpinella anisum extract attenuates spontaneous and agonist-induced uterine contraction in term-pregnant rats
T
Mohammed F. Alotaibi Department of Physiology, College of Medicine, King Saud University, P.O Box 2925, Riyadh, 11461, Saudi Arabia
A R T I C LE I N FO
A B S T R A C T
Keywords: Anise Uterus Contraction Term-pregnant Rats Oxytocin
Ethnopharmacology relevance: Pimpinella anisum is a well-known traditional medicinal herb which has been used in folk medicine as an antiulcer, anticancer, antibacterial and as a muscle relaxant. Aim of the study: This study was performed to explore the modulatory effects of Pimpinella anisum on termpregnant rat uterine contractility and to investigate its possible underlying mechanisms. Material and methods: Intact uterine strips without endometrial layer were isolated from female term-pregnant Wistar rats (22 days of gestation) and mounted in a tissue bath apparatus for in vitro isometric force recording. The effects of different concentrations of Pimpinella anisum extract (PAE) (1, 3, 5, and 7 mg/mL) were examined on uterine contractions generated spontaneously or induced with oxytocin (5 nmol/L), Bay K8644 (1 μmol/L), and carbachol (10 μmol/L). In some experiments, PAE was applied on depolarized myometrium in the presence of high-KCl solution (60 mmol/L). The effect on Ca2+ release was also examined. Results: Application of PAE significantly reduced uterine contractions generated spontaneously or induced with oxytocin, Bay K8644, and carbachol in a concentration-dependent manner (n = 7; P < 0.01). In depolarized myometrium, PAE significantly reduced the tonic force induced by high-KCl solution (n = 7; P < 0.01). PAE prevented oxytocin-induced transient contraction in the entire absence of external calcium (n = 7; P < 0.01). Conclusion: The present findings demonstrate the potentials of PAE to relax pregnant uterine contractions possibly by blocking Ca2+ entry via L-type calcium channels and inhibiting Ca2+ release from the internal store. The tocolytic effects of PAE may be a potential adjuvant against strong premature uterine contractions which threaten early pregnancy although clinical studies are required.
1. Introduction The uterus is a relatively quiescent organ throughout pregnancy which transforms to a very excitable tissue at the onset of term or preterm labours. Preterm birth (delivery of a baby before 37 gestational weeks) is the most likely contributing factor to neonatal morbidity and mortality (Stanton et al., 2006). The underlying mechanisms of premature uterine stimulation during preterm labour are not fully understood but it involves a premature activation of contractile associated proteins including connexin-43, oxytocin and prostaglandin F2α receptors (Cook et al., 2000). Available medications to restrain forceful uterine activity during preterm labor are still questioned regarding their effectiveness, maternal/neonatal side effects, and long-term safety (Kim and Shim, 2006). Due to these considerations and due to socio-economic problems in different populations, several studies have been
performed in reproductive medicine to better find safe and cost-effective tocolytic medicinal plants to control uterine activity (Pereira-DeMorais et al., 2019; Monji et al., 2018). One of the oldest traditional plant that has been used worldwide in folk medicine is Pimpinella anisum L. (anise) that belongs to the Apiaceae family. Pimpinella anisum possesses antibacterial (Gülçın et al., 2003), antiviral (Lee et al., 2011), anticonvulsant (Karimzadeh et al., 2012), antitoxic (Jamshidzadeh et al., 2015), antioxidant (Gülçın et al., 2003), antiulcer (Al Mofleh et al., 2007), and muscle relaxant (Tirapelli et al., 2007) effects. Pimpinella anisum is grown for its seeds (aniseeds) and their beneficial properties are derived mainly from its essential oil which contains transanethole, cis-anethole, estragole, γ-himachalene, eugenol, α-cuparene, β-bisabolene, p-anisaldehyde, and terpenic compounds (Özcan and Chalchat, 2006; Orav et al., 2008; Gülçın et al., 2003). A full description of chemical compositions of Pimpinella anisum has been reported in
Abbreviations: PAE, Pimpinella anisum extract; IACC, Institutional Animal Care Committee; CCh, Carbachol; KCl, Potassium chloride; VGCCs, voltage gated calcium channels; OTRs, oxytocin receptors; DART-ToF-MS, Direct Analysis in Real Time – Time of Flight – Mass Spectrometry; AUC, area under the curve; ANOVA, analysis of variance E-mail address: [email protected]. https://doi.org/10.1016/j.jep.2020.112730 Received 13 September 2019; Received in revised form 13 February 2020; Accepted 29 February 2020 Available online 04 March 2020 0378-8741/ © 2020 Elsevier B.V. All rights reserved.
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Calcium-free solution was prepared exactly as the Krebs solution but without CaCl2 and adding 1 mM calcium-chealator ethyl glycol-bis (βaminoethyl ether)-N,N,N′,N′-tetra acetic acid (EGTA) (Zafrah and Alotaibi, 2017). The desired concentrations of PAE were dissolved directly in the Krebs solution and maintaining the pH at 7.4.
other studies (Özcan and Chalchat, 2006; Shojaii and Abdollahi Fard, 2012). Anethole, estragole, and eugenol have been shown to cause relaxation of different types of smooth muscle (Lima et al., 2011; Cabral et al., 2014; Soares et al., 2007). Despite the reported significant effects of Pimpinella anisum in reducing postpartum pain associated with uterine cramping and involution in vivo (Simbar et al., 2015), its direct effects on uterine contractions have not been tested in vitro to support its therapeutic use in reproductive medicine. Therefore, this study was designed to explore the potential effects of Pimpinella anisum extract (PAE) on term-pregnant uterine contractility and to investigate its possible underlying mechanisms in vitro.
2.4. Experimental protocols 2.4.1. Effects of PAE on spontaneous uterine contractions Uterine strips were left contracting in oxygenated buffered Krebs solution until regular spontaneous activity was established and this was used as a control period (100%). Cumulative concentrations of PAE (1, 3, 5, and 7 mg/mL) were determined while uterine strips were contracting spontaneously and a concentration-response curve was obtained.
2. Materials and methods 2.1. Plant materials and extraction
2.4.2. Effects of PAE on uterine contractions induced by oxytocin After obtaining steady spontaneous contractions in Krebs solution, uterine strips were stimulated with oxytocin until regular phasic contractions were established. After equilibrium period, cumulative concentrations of PAE (1, 3, 5, and 7 mg/mL) were determined in the continued presence of oxytocin and a concentration-response curve was obtained. To investigate the effect of PAE on calcium release from the intracellular calcium stores, calcium-free medium was used. After equilibrium of spontaneous contractions, Krebs solution was replaced with the calcium-free medium and was left until complete abolition of spontaneous activity and oxytocin was applied in the absence (control) and presence of 5 mg/mL PAE.
Fresh Pimpinella anisum seeds were purchased from a medical herb store in August 2017 and authenticated by Dr. Basodaan, Department of Pharmacognosy, King Saud University (Riyadh, Saudi Arabia) and a voucher specimen (0912) was deposited. Seeds were extracted as previously described (Alotaibi, 2016). Briefly, seeds were washed with distilled water, air-dried at room temperature, and ground, 400 g of the yield powder was extracted with a 50:50 water-ethanol solution for 36 h with occasional shaking. After filtration, the extract was desiccated by a rotary evaporator to eliminate the solvents which finally yielded a viscous gummy dark extract (50 g). The extract was kept in a sterile bottle and kept at 4 °C until used.
2.4.3. Effects of PAE on uterine depolarization induced by high-KCl solution High-KCl solution depolarizes the cell membrane leading to calcium influx via voltage gated calcium channels (VGCCs) and sustained tonic contraction (Amédée et al., 1986). To investigate the effect of PAE on uterine depolarization, uterine strips were pre-contracted for 15 min with high-KCl solution until tonic contraction was established. Uterine strips were then washed out with Krebs solution until complete recovery and then pre-contracted again with high-KCl solution containing 5 mg/mL PAE for 15 min.
2.2. Animals and tissue preparations Term-pregnant Wistar rats (~22 days of gestation) were used in this study. Animals were killed by carbon dioxide (CO2) asphyxia followed by decapitation in accordance with the guidelines of the Institutional Animal Care Committee (IACC), College of Medicine, King Saud University and in accordance with the principles and procedures of laboratory animal use and care published by the European Communities Council Directives (2010/63/EU). The abdomen was opened and the uterus was removed and immediately immersed in Krebs solution. Longitudinal uterine strips were dissected and the endometrium was carefully removed by gentle scrape using a blade. Uterine strips were tied from both ends by surgical threads, and placed in a 5-mL tissue bath chamber (Radnoti, Monrovia, CA, USA) containing a pre-warmed Krebs solution and aerated with 95% O2 and 5% CO2 at 37 °C. Strips were attached to a sensitive isometric force transducer (ADInstruments Ltd, Sydney, Australia) which measures any changes in tension and displays it on a monitor using LabChart software (AD Instruments Ltd, Sydney, Australia). The strips were allowed to equilibrate in the Krebs solution for at least 30 min before performing any experiment to establish regular and steady contractile activity. The tissue bath is continually superfused with warmed Krebs solution at a rate of 4 mL/min using Dynamax RP-1 peristaltic pump (Rainin Instrument Co., USA).
2.4.4. Effects of PAE on uterine contractions induced by carbachol To investigate if the inhibitory effect of the PAE involves muscarinic receptors, carbachol (a non-selective and cholinesterase-resistant muscarinic receptor agonist; CCh) was used. After obtaining steady spontaneous contractions, uterine strips were stimulated with CCh until phasic contractions were established. After equilibrium period, cumulative concentrations of PAE (1, 3, 5, and 7 mg/mL) were determined in the continued presence of CCh and a concentration-response curve was obtained. 2.4.5. Effects of PAE on uterine contractions induced by Bay K8644 To investigate if the inhibitory effect of the PAE was due to blockade of calcium influx via L-type VGCCs, Bay K8644 (a specific L-type calcium channel agonist) was used. After obtaining steady spontaneous contractions, uterine strips were stimulated with Bay K8644 until phasic contractions were established. After equilibrium period, cumulative concentrations of PAE (1, 3, 5, and 7 mg/mL) were determined in the continued presence of Bay K8644 and a concentration-response curve was obtained.
2.3. Chemicals and solutions All drugs and reagents used in this study were supplied by SigmaAldrich (St. Louis, MO, USA) and were of analytic grade. Krebs solution was made with the following compositions [mmol/L]: 115 NaCl, 4.7 KCl, 2 CaCl2, 1.16 MgSO4, 1.18 KH2PO4, 22 NaHCO3, and 7.88 dextrose, pH was adjusted to 7.4 (Alotaibi, 2016). Oxytocin and Carbachol (CCh) were prepared in distilled H2O and dissolved directly in Krebs solution at a final concentration of 5 nmol/L and 10 μmol/L, respectively. Bay K8644 was dissolved in absolute ethanol and used at a final concentration of 1 μmol/L. High-potassium chloride (KCl) solution (60 mmol/L) was prepared by equimolar substitution of NaCl for KCl.
2.5. Analysis of PAE by mass spectrometry Fresh PAE was characterized by a high resolution Direct Analysis in Real Time – Time of Flight – Mass Spectrometry (DART-ToF-MS) and the ESI-MS spectra were recorded on the MS system (AccuTOF LC-Plus; JEOL Co., Tokyo, Japan). The volatile components of the PAE were evaporated in a stream of helium heated at 300 °C, then ionized by the 2
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excited metastable helium atoms, before entering the ion source of the time of flight mass spectrometer. In the positive ionization mode, each molecule is transformed into either a protonated ion [M+H]+ or a nonprotonated radical molecular ion M+.. Since the ionization process is considered as “soft”, no or little fragmentation occurs, such that each peak in the spectrum corresponds to a given compound. The experimental conditions used for analysis of PAE by DART-ToF-MS are as follow: Vacuum (Pirani gauge): 1.8x10+2 Pa, Vacuum (Analyzer): 1.3x10−5 Pa, Heater temperature: 350 °C, Ionization mode: positive, Heating and ionization gas: helium, Ring lens voltage: 4 V, Orifice 1 voltage: 5 V, Orifice 2 voltage: 5 V, Peaks voltage: 500 V, Mass range: 50–1000 Da, Mass resolution range: 3600–4900.
Table 1 Effects of different concentrations of Pimpinella anisum extract (PAE) on contractile parameters during spontaneous contraction in term-pregnant rats (n = 7). Contraction parameters
0 mg/mL (control)
1 mg/mL
3 mg/mL
5 mg/mL
7 mg/mL
AUC Frequency
100% 100%
99 ± 2% 99 ± 1%
67 ± 3%* 112 ± 8%
49 ± 2%* 68 ± 6%*
2 ± 2%* 0%*
*P < 0.01 compared with control or with preceding concentration (ANOVA/ Bonferroni).
concentrations. Mean data for the effects of different concentrations of PAE on contractile parameters during spontaneous contractions are shown in Table .1.
2.6. Data and statistical analyses Data were imported and analysed using Microcal Origin Software 9.0 (Northampton, MA, USA). The main parameters measured were force integral (area under the curve; AUC) and frequency of contractions. AUC (in arbitrary units) was calculated by measuring the entire uterine activity for 10 min before and after application of the extract, whereas, the frequency was calculated by counting the number of contractions occurring in 10 min. The effect of a known agent or drug can be determined by measuring the changes in these parameters, as they considered an index for the entire contractile activity by the uterine strip at a given time (Arrowsmith et al., 2018). The half maximal inhibitory concentration (IC50) which produced 50% of maximum reduction of AUC was calculated. The contractile activity in the last 10 min before adding PAE was precisely measured and taken as 100% control and the results were expressed as a percentage of this control. Data are presented as means ± SEM and “n” represents the number of uterine strips, one from different rat. Differences between control and treatment groups were evaluated using one-way analysis of variance (ANOVA)/Bonferroni tests. P values < 0.05 were considered significant.
3.2. Dose-response effects of PAE on uterine contractions induced by oxytocin
3. Results
Stimulation of uterine strips with oxytocin significantly increased the contractile parameters including AUC and frequency of contractions compared with the preceding spontaneous activity (Alotaibi et al., 2015). When PAE was applied in the continued presence of oxytocin, the force significantly and progressively decreased in a concentrationdependent manner (Fig. 2A and B; n = 7). The frequency of contractions transiently but insignificantly increased upon application of 3 mg/ mL PAE and progressively decreased and abolished with higher concentrations of PAE. Mean data for the effects of different concentrations of PAE on contractile parameters in the presence of oxytocin are shown in Table .2. In calcium-free medium, spontaneous uterine activity ceases and oxytocin can produce a transient small contraction in the continued absence of external calcium indicating calcium release from the sarcoplasmic reticulum (Munglue et al., 2013; Sukwan et al., 2014). Application of 5 mg/mL PAE in the continued absence of external calcium prevented oxytocin-induced transient contraction (Fig. 3A; n = 7).
3.1. Dose-response effects of PAE on spontaneous uterine activity
3.3. Effects of PAE on uterine depolarization induced by high-KCl solution
Application of PAE inhibited spontaneous uterine activity in a concentration-dependent manner in all samples tested (Fig. 1A&B; n = 7). The AUC progressively decreased after addition of rising concentrations of PAE and the force completely abolished with the highest dose (7 mg/mL). The IC50 for the AUC was 5 mg/mL which decreased the force to 49 ± 2% compared with 100% control (0 mg/mL). The frequency of contraction transiently but insignificantly increased and then progressively decreased until abolished after increasing PAE
Application of high-KCl solution caused depolarization and sustained tonic contraction which was maintained as long as it was applied. We have shown previously that repeated applications of high-KCl solution on the same uterine strip produced no significant difference in tonic force in term-pregnant rats (Zafrah and Alotaibi, 2017). Interestingly, application of 5 mg/mL PAE significantly reduced the force integral from 100% (before adding the extract) to 44 ± 3% (Fig 3B; n = 7).
Fig. 1. (A) Original recording for the effect of cumulative concentrations of PAE on spontaneous uterine contractions in term-pregnant rats. (B) Concentrationresponse curve for the effect of PAE on spontaneous uterine contractions. Vertical bars represent SEM (n = 7). *P < 0.01 compared with the preceding dose. 3
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Fig. 2. (A) Original recording for the effect of cumulative concentrations of PAE on uterine contractions induced by 5 nmol/L oxytocin in term-pregnant rats. (B) Concentration-response curve for the effect of PAE on oxytocin-induced uterine contractions. Vertical bars represent SEM (n = 7). *P < 0.01 compared with the preceding dose.
3.4. Effects of PAE on uterine contraction induced by carbachol and Bay K8644
Table 2 Effects of different concentrations of Pimpinella anisum extract (PAE) on contractile parameters in the presence of 5 nmol/L oxytocin in term-pregnant rats (n = 7). Contraction parameters
0 mg/mL (control)
1 mg/mL
3 mg/mL
5 mg/mL
7 mg/mL
AUC Frequency
100% 100%
99 ± 2% 100%
67 ± 2%* 106 ± 5%
46 ± 3%* 71 ± 3%*
2 ± 1%* 0%*
Stimulation of uterine strips with agonists (CCh or Bay K8644) resulted in a marked increase in force integral and contraction frequency compared with the preceding spontaneous activity (Wu et al., 2015). Application of PAE significantly and progressively decreased uterine contractile activity in a concentration-dependent manner in the continued presence of CCh and Bay K8644 (Fig 3C; n = 7). Mean data for the effects of different concentrations of PAE on contractile parameters in the presence of CCh and Bay K8644 are shown in Table s 3 and 4, respectively.
*P < 0.01 compared with control or with preceding concentration (ANOVA/ Bonferroni).
Fig. 3. (A) A typical trace for the effect of 5 mg/mL PAE on uterine contraction induced by 5 nmol/L oxytocin in calcium-free medium in term-pregnant rats. (B) A typical trace for the response of uterine strip to high-KCl solution (60 mmol/L) before and after addition of 5 mg/mL PAE in term-pregnant rats. (C) Concentrationresponse curve for the effects of PAE on uterine contractions pre-contracted with 10 μmol/LCarbachol (CCh) and 1 μmol/L Bay K8644 in term-pregnant rats. Control did not receive any PAE dose. Vertical bars represent SEM (n = 7). *P < 0.01 compared with the preceding dose. 4
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be increased following stimulation by oxytocin, Bay K8644, or CCh (Wu et al., 2015). Extracellular Ca2+ is vital for uterine spontaneous contractions and blocking calcium entry via VGCCs can significantly abolish spontaneous activity (Promprom et al., 2010). In this study, PAE is able to inhibit spontaneous uterine contractions in a concentration-dependent manner suggesting inhibition of Ca2+ entry via Ltype VGCCs. To further investigate the effect of PAE on calcium influx pathway, we stimulated the uterine strips with high-KCl solution and Bay K8644 to activate calcium entry via L-type VGCCs. High-KCl solution causes tonic and maximum uterine contraction by changing the reversal potassium potential resulting in membrane depolarization and opening of L-type VGCCs, thus further elevating [Ca2+]i (Wray, 2007). Pharmacological blockade of VGCCs or absence of external Ca2+ can markedly abolish KCl-induced tonic contraction (Perez and Sanderson, 2005; Maggi and Meli, 1984), suggesting that Ca2+ entry via VGCCs is a major pathway for KCl-induced smooth muscle contraction. Bay K8644 is a stimulator of L-type VGCCs being able to elevate [Ca2+]i by Ca2+ influx via L-type VGCCs leading to increased amplitude and frequency of contraction (Alotaibi et al., 2017). In the presence of PAE, KCl-induced tonic contraction was significantly attenuated which is consistent with the reported relaxant effects of PAE on contractions induced by high-KCl in tracheal smooth muscle (Boskabady and Ramazani-Assari, 2005), and in depolarized portal vein (Pontes et al., 2019). Furthermore, PAE progressively and significantly decreased the force induced by Bay K8644 in a concentration dependent manner, indicating blockade of Ca2+ entry mechanism. It has been shown recently that PAE exerted a relaxation effect on cardiac and vascular smooth muscles by blocking calcium influx pathway (Pontes et al., 2019). Therefore, our findings suggest the ability of PAE to inhibit Ca2+ influx via VGCCS in pregnant uterine smooth muscle. Oxytocin augments uterine activity and increases the force amplitude, duration, and frequency compared to spontaneous activity (Alotaibi, 2017). The principal mechanism of oxytocin-increased force is due to the activation of L-type VGCCs and OTRs stimulation. Activation of OTRs (which belong to G proteincoupled receptor superfamily) causes phospholipase C activation and increased production of inositol 1,4,5-triphosphate (IP3) resulting in Ca2+ release from the SR and augmentation of contractions (Vrachnis et al., 2011). Our findings show that PAE can significantly decrease and abolish oxytocin-induced uterine contraction in the presence of external calcium in a concentration-dependant manner. Oxytocin can cause transient contraction in entire absence of external Ca2+ suggesting IP3induced Ca2+ release as uterine smooth muscle lacks functional ryanodine receptors responsible for Ca2+-induced Ca2+release (Matsuki et al., 2017). Our findings show that PAE can significantly prevent oxytocin-induced transient release of Ca2+from the SR, suggesting inhibition of IP3 messenger or its receptors on the SR. The myometrium expresses muscarinic receptors which mediate CCh-induced uterine contraction (Kitazawa et al., 2008). The action of CCh on rat uterine muscarinic receptors is mediated mainly via M3 receptor subtype (Abdalla et al., 2000, 2004; Kitazawa et al., 2008) which is coupled primarily to PLC-mediated IP3 production and Ca2+ release from the SR (Caulfield and Birdsall, 1998). We observed that CCh can markedly increase the force amplitude and frequency of contraction compared to spontaneous uterine activity. PAE can significantly reduce the CCh-induced uterine contraction in a concentration-dependent manner which further characterizes the relaxant effects of PAE on uterine activity. This finding is consistent with previous studies where PAE attenuated acetylcholine-induced contraction in rat anococcygeus (Tirapelli et al., 2007) and guinea pigs tracheal (Boskabady and Ramazani-Assari, 2001) smooth muscles. Our MS analysis revealed that the active constituents of PAE are anethole and eugenol which are consistent with the previous phytochemical studies (Orav et al., 2008; Özcan and Chalchat, 2006). Previous works have confirmed the relaxant effects of anethole and eugenol on smooth muscle contraction (Lima et al., 2011; Cabral et al., 2014; Damiani et al., 2003; Soares et al., 2007), and we suggest that the relaxant effects of PAE on uterine contractions could be due to the
Table 3 Effects of different concentrations of Pimpinella anisum extract (PAE) on contractile parameters in the presence of 10 μmol/L CCh in term-pregnant rats (n = 7). Contraction parameters
0 mg/mL (control)
1 mg/mL
3 mg/mL
5 mg/mL
7 mg/mL
AUC Frequency
100% 100%
100% 100%
70 ± 4%* 108 ± 5%
52 ± 3%* 75 ± 3%*
3 ± 2%* 0%*
*P < 0.01 compared with control or with preceding concentration (ANOVA/ Bonferroni). Table 4 Effects of different concentrations of Pimpinella anisum extract (PAE) on contractile parameters in the presence of 1 μmol/L Bay K8644 in term-pregnant rats (n = 7). Contraction parameters
0 mg/mL (control)
1 mg/mL
3 mg/mL
5 mg/mL
7 mg/mL
AUC Frequency
100% 100%
98 ± 2% 100%
64 ± 5%* 109 ± 3%
41 ± 4%* 67 ± 2%*
2 ± 2%* 0%*
*P < 0.01 compared with control or with preceding concentration (ANOVA/ Bonferroni).
3.5. Main constituents of PAE MS analysis revealed that PAE contains anethole (principal component) and other compounds as shown in Table 5. 4. Discussion Although previous clinical trials have confirmed the efficacy of Pimpinella anisum in reducing migraine attacks (Mosavat et al., 2019) and relieving postpartum depression (Ghoshegir et al., 2015), there are no previous studies showing the direct effect of PAE on uterine activity. Our current study shows for the first time that Pimpinella anisum possesses a clear tocolytic effects on uterine strips isolated from pregnant rats which can cause relaxation in a concentration-dependent manner. PAE can significantly reduce the uterine contractions irrespective how the force was produced which may support the reported in vivo effects of Pimpinella anisum in alleviating the postpartum pain associated with uterine cramping and involution (Simbar et al., 2015). Our in vitro findings are in agreement with previous studies showing the relaxant effects of PAE on tracheal (Boskabady and Ramazani-Assari, 2001) and anococcygeus (Tirapelli et al., 2007) smooth muscles contractility. The uterus is a spontaneously active tissue and contraction of its smooth muscle is controlled primarily by the intracellular calcium concentration [Ca2+]i. In smooth muscle, Ca2+ can enter the cell via L-type VGCCs or it can be released from its intracellular stores (sarcoplasmic reticulum; SR) (Thorneloe and Nelson, 2005). In addition, [Ca2+]i can Table 5 Composition of Pimpinella anisum extract. No
Percentage
Compound
1 2 3 4 5 6 7 8 9 10 11 12
0.08 90.5 1.4 1.12 0.19 0.22 2.9 0.03 0.02 0.04 0.01 3.49
p-cymene anethole γ-himachalene carvone dihydrocarvone methylcinnamic acid eugenol 2,7-octadienyl acetate cuparene caryophyllene 1,2-diphenyl-1-butanol Not identified
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M.F. Alotaibi
Fig. 4. Scheme showing the possible underlying mechanisms of decreased uterine force by Pimpinella anisum extract (PAE) in term-pregnant rats. (1) calcium entry via L-type voltage gated calcium channels (VGCCs) is blocked by PAE. (2) PAE blocks uterine activity stimulated by L-type VGCCs activator and depolarization by high-KCl. (3) PAE blocks oxytocin/ muscarinic receptors which reduces membrane depolarization/calcium influx and inhibits PLC-mediated IP3 production and calcium release from the SR. SM; smooth muscle. SR; sarcoplasmic reticulum. MLCK; myosin light-chain kinase. MLC20; myosin light chain.
the plant extraction and identification of the active ingredients of PAE.
presence of these compounds. Therefore, our data clearly demonstrate that the tocolytic effects of Pimpinella anisum on uterine activity are partially attributed to the disruption of calcium entry and/or release mechanism. In addition, we suggest that PAE could act as a competitive antagonism on oxytocin or muscarinic receptors to reduce uterine contractions. As this is the first study showing the dose-dependent relaxation effect of PAE on pregnant rat uterine activity in vitro, the same dosage we used cannot be necessarily extrapolated to pregnant human uterus. Therefore, clinical trials and in vivo experiments are required to find the optimal and safe dose for the use in the clinical practice in order to control the abnormal uterine hyper-activity that occurs during dysmenorrhea or before term.
References Abdalla, F.M., Abreu, L.C., Porto, C.S., 2000. Effect of estrogen on intracellular signaling pathways linked to activation of M2-and M3-muscarinic acetylcholine receptors in the rat myometrium. Mol. Cell. Endocrinol. 160, 17–24. Abdalla, F.M., Maróstica, E., Picarelli, Z.P., Abreu, L.C., Avellar, M.C.W., Porto, C.S., 2004. Effect of estrogen on muscarinic acetylcholine receptor expression in rat myometrium. Mol. Cell. Endocrinol. 213, 139–148. Al Mofleh, I.A., Alhaider, A.A., Mossa, J.S., Al-Soohaibani, M.O., Rafatullah, S., 2007. Aqueous suspension of anise “Pimpinella anisum” protects rats against chemically induced gastric ulcers. World J. Gastroenterol.: WJG 13, 1112. Alotaibi, M., 2016. The effect of cinnamon extract on isolated rat uterine strips. Reprod. Biol. 16, 27–33. Alotaibi, M., Arrowsmith, S., Wray, S., 2015. Hypoxia-induced force increase (HIFI) is a novel mechanism underlying the strengthening of labor contractions, produced by hypoxic stresses. Proc. Natl. Acad. Sci. Unit. States Am. 112, 9763–9768. Alotaibi, M., Kahlat, K., Nedjadi, T., Djouhri, L., 2017. Effects of ZD7288, a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker, on term-pregnant rat uterine contractility in vitro. Theriogenology 90, 141–146. Alotaibi, M.F., 2017. The response of rat and human uterus to oxytocin from different gestational stages in vitro. Gen. Physiol. Biophys. 36, 75–82. Amédée, T., Mironneau, C., Mironneau, J., 1986. Isolation and contractile responses of single pregnant rat myometrial cells in short‐term primary culture and the effects of pharmacological and electrical stimuli. Br. J. Pharmacol. 88, 873–880. Arrowsmith, S., Keov, P., Muttenthaler, M., Gruber, C.W., 2018. Contractility measurements of human uterine smooth muscle to aid drug development. JoVE 26, 56639. Boskabady, M., Ramazani-Assari, M., 2001. Relaxant effect of Pimpinella anisum on isolated Guinea pig tracheal chains and its possible mechanism (s). J. Ethnopharmacol. 74, 83–88. Boskabady, M., Ramazani-Assari, M., 2005. Possible mechanism for the relaxant effect of Pimpinella anisum. on Guinea pig tracheal chains. Pharmaceut. Biol. 42, 621–625. Cabral, P.H.B., De Morais Campos, R., Fonteles, M.C., Santos, C.F., Cardoso, J.H.L., Do Nascimento, N.R.F., 2014. Effects of the essential oil of Croton zehntneri and its major components, anethole and estragole, on the rat corpora cavernosa. Life Sci. 112, 74–81. Caulfield, M.P., Birdsall, N.J., 1998. International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol. Rev. 50, 279–290. Cook, J.L., Zaragoza, D.B., Sung, D.H., Olson, D.M., 2000. Expression of myometrial activation and stimulation genes in a mouse model of preterm labor: myometrial activation, stimulation, and preterm labor. Endocrinology 141, 1718–1728. Damiani, C.E.N., Rossoni, L.V., Vassallo, D.V., 2003. Vasorelaxant effects of eugenol on rat thoracic aorta. Vasc. Pharmacol. 40, 59–66.
5. Conclusion The current study provides clear evidence for the relaxant effects of PAE on pregnant rat uterus which may potentially be used as a tocolytic herbal medicine to control unwanted uterine contractions occurring in preterm labours or to reduce strong uterine contractions during menstruation (dysmenorrhea). The possible underlying mechanisms of PAEinduced relaxation could be due to the blockage of calcium entry via Ltype VGCCs and/or inhibition of calcium release from the SR in termpregnant rat uterus. Proposed mechanisms of PAE-induced uterine relaxation are depicted in Fig. 4. Declaration of competing interest None declared. Acknowledgments I would like to thank the Deanship of Scientific Research at King Saud University for supporting this project. Special thanks to Dr. Basodaan and Professor A. Yacine Badjah-Hadj-Ahmed for their help in 6
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(Pimpinella anisum L.) fruit oil at ripening stage. Ann. Microbiol. 56, 353–358. Pereira-De-Morais, L., De Alencar Silva, A., Da Silva, R.E.R., Da Costa, R.H.S., Monteiro, Á.B., Dos Santos Barbosa, C.R., De Souza Amorim, T., De Menezes, I.R.A., Kerntopf, M.R., Barbosa, R., 2019. Tocolytic activity of the Lippia alba essential oil and its major constituents, citral and limonene, on the isolated uterus of rats. Chem. Biol. Interact. 297, 155–159. Perez, J.F., Sanderson, M.J., 2005. The contraction of smooth muscle cells of intrapulmonary arterioles is determined by the frequency of Ca2+ oscillations induced by 5-HT and KCl. J. Gen. Physiol. 125, 555–567. Pontes, V.C.B., Rodrigues, D.P., Caetano, A., Gamberini, M.T., 2019. Preclinical investigation of the cardiovascular actions induced by aqueous extract of Pimpinella anisum L. seeds in rats. J. Ethnopharmacol. 237, 74–80. Promprom, W., Kupittayanant, P., Indrapichate, K., Wray, S., Kupittayanant, S., 2010. The effects of pomegranate seed extract and β-sitosterol on rat uterine contractions. Reprod. Sci. 17, 288–296. Shojaii, A., Abdollahi Fard, M., 2012. Review of pharmacological properties and chemical constituents of Pimpinella anisum. ISRN pharmaceutics 2012. Simbar, M., Shadipour, M., Salamzadeh, J., Ramezani-Tehrani, F., Nasiri, N., 2015. The combination of “Pimpinella anisum, Apium graveolens and Crocus sativus (PAC)” is more effective than “mefenamic acid” on postpartum after-pain. J. Herb. Med. 5, 20–25. Soares, P.M.G., Lima, R.F., De Freitas Pires, A., Souza, E.P., Assreuy, A.M.S., Criddle, D.N., 2007. Effects of anethole and structural analogues on the contractility of rat isolated aorta: involvement of voltage-dependent Ca2+-channels. Life Sci. 81, 1085–1093. Stanton, C., Lawn, J.E., Rahman, H., Wilczynska-Ketende, K., Hill, K., 2006. Stillbirth rates: delivering estimates in 190 countries. Lancet 367, 1487–1494. Sukwan, C., Wray, S., Kupittayanant, S., 2014. The effects of Ginseng Java root extract on uterine contractility in nonpregnant rats. Phys. Rep. 2. Thorneloe, K.S., Nelson, M.T., 2005. Ion channels in smooth muscle: regulators of intracellular calcium and contractility. Can. J. Physiol. Pharmacol. 83, 215–242. Tirapelli, C.R., De Andrade, C.R., Cassano, A.O., De Souza, F.A., Ambrosio, S.R., Da Costa, F.B., De Oliveira, A.M., 2007. Antispasmodic and relaxant effects of the hidroalcoholic extract of Pimpinella anisum (Apiaceae) on rat anococcygeus smooth muscle. J. Ethnopharmacol. 110, 23–29. Vrachnis, N., Malamas, F.M., Sifakis, S., Deligeoroglou, E., Iliodromiti, Z., 2011. The oxytocin-oxytocin receptor system and its antagonists as tocolytic agents. International journal of endocrinology 2011. Wray, S., 2007. Insights into the uterus. Exp. Physiol. 92, 621–631. Wu, C.-H., Shieh, T.-M., Wang, K.-L., Huang, T.-C., Hsia, S.-M., 2015. Quercetin, a main flavonoid in onion, inhibits the PGF2α-induced uterine contraction in vitro and in vivo. Journal of Functional Foods 19, 495–504. Zafrah, H.A., Alotaibi, M.F., 2017. The effect of extracellular ATP on rat uterine contraction from different gestational stages and its possible mechanisms of action. J. Basic Clin. Physiol. Pharmacol. 28, 209–217.
Ghoshegir, S.A., Mazaheri, M., Ghannadi, A., Feizi, A., Babaeian, M., Tanhaee, M., Karimi, M., Adibi, P., 2015. Pimpinella anisum in the treatment of functional dyspepsia: a double-blind, randomized clinical trial. J. Res. Med. Sci. 20, 13–21. Gülçın, İ., Oktay, M., Kıreçcı, E., Küfrevıoǧlu, Ö.İ., 2003. Screening of antioxidant and antimicrobial activities of anise (Pimpinella anisum L.) seed extracts. Food Chem. 83, 371–382. Jamshidzadeh, A., Heidari, R., Razmjou, M., Karimi, F., Moein, M.R., Farshad, O., Akbarizadeh, A.R., Shayesteh, M.R.H., 2015. An in vivo and in vitro investigation on hepatoprotective effects of Pimpinella anisum seed essential oil and extracts against carbon tetrachloride-induced toxicity. Iranian journal of basic medical sciences 18, 205. Karimzadeh, F., Hosseini, M., Mangeng, D., Alavi, H., Hassanzadeh, G.R., Bayat, M., Jafarian, M., Kazemi, H., Gorji, A., 2012. Anticonvulsant and neuroprotective effects of Pimpinella anisum in rat brain. BMC Compl. Alternative Med. 12, 76. Kim, A., Shim, J.Y., 2006. Emerging tocolytics for maintenance therapy of preterm labour: oxytocin antagonists and calcium channel blockers. BJOG An Int. J. Obstet. Gynaecol. 113, 113–115. Kitazawa, T., Hirama, R., Masunaga, K., Nakamura, T., Asakawa, K., Cao, J., Teraoka, H., Unno, T., Komori, S.-I., Yamada, M., 2008. Muscarinic receptor subtypes involved in carbachol-induced contraction of mouse uterine smooth muscle. N. Schmied. Arch. Pharmacol. 377, 503–513. Lee, J.-B., Yamagishi, C., Hayashi, K., Hayashi, T., 2011. Antiviral and immunostimulating effects of lignin-carbohydrate-protein complexes from Pimpinella anisum. Biosci. Biotechnol. Biochem. 75, 459–465. Lima, F.C., Peixoto‐Neves, D., Gomes, M.D.M., Coelho‐De‐Souza, A.N., Lima, C.C., Araújo Zin, W., Magalhães, P.J.C., Saad, L., Leal‐Cardoso, J.H., 2011. Antispasmodic effects of eugenol on rat airway smooth muscle. Fundam. Clin. Pharmacol. 25, 690–699. Maggi, C., Meli, A., 1984. The effect of nifedipine and verapamil on KCl-induced rhythmic contractions of Guinea pig ureter in vitro. Experientia 40, 681–686. Matsuki, K., Takemoto, M., Suzuki, Y., Yamamura, H., Ohya, S., Takeshima, H., Imaizumi, Y., 2017. Ryanodine receptor type 3 does not contribute to contractions in the mouse myometrium regardless of pregnancy. Pflueg. Arch. Eur. J. Physiol. 469, 313–326. Monji, F., Lau, L.C., Siddiquee, A.a.-M., Said, B.B., Yang, L.-K., Yoganathan, K., Choolani, M.A., Adaikan, P.G., 2018. Potent tocolytic activity of ethyl acetate fraction of Ananas comosus on rat and human uteri. Biomed. Pharmacother. 105, 824–834. Mosavat, S.H., Jaberi, A.R., Sobhani, Z., Mosaffa-Jahromi, M., Iraji, A., Moayedfard, A., 2019. Efficacy of Anise (Pimpinella anisum L.) oil for migraine headache: a pilot randomized placebo-controlled clinical trial. J. Ethnopharmacol. 236, 155–160. Munglue, P., Eumkep, G., Wray, S., Kupittayanant, S., 2013. The effects of watermelon (Citrullus lanatus) extracts and L-citrulline on rat uterine contractility. Reprod. Sci. 20, 437–448. Orav, A., Raal, A., Arak, E., 2008. Essential oil composition of Pimpinella anisum L. fruits from various European countries. Nat. Prod. Res. 22, 227–232. Özcan, M.M., Chalchat, J.C., 2006. Chemical composition and antifungal effect of anise
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