Citrus Aurantium Effects On Uterus Biology Essay

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Background: Citrus aurantium is a small citrus tree, about five meters tall, with scented white flowers. The C. aurantium is used in Asian herbal medicine primarily to treat digestive problems.

Objective: The goal of this study is to investigate the effect of C. aurantium flower's aqueous extract on uterine contraction in presence of some known uterus stimulants.

Material and Methods: Virgin Wistar rats 200-300g were obtained. After laparatomy, a piece of uterus was dissected out and mounted in an organ bath (10ml) containing De Jalon (29°C) and contracted by KCl (60mM), oxytocin (10mU/ml) and barium chloride (4mM) then the effect of C. aurantium flower's aqueous extract on the uterine contractions was investigated. Uterus was separately incubated with propranolol (1mM), naloxone (1mM) and the role of β-adrenoceptors, opioid receptors were evaluated.

Results: Cumulative concentrations of the extract (1-8 mg/ml) decreased KCl, oxytocin and barium chloride induced uterine contractions, dose-dependently (P<0.001). Aqueous extract of C. aurantium flowers was unaffected on incubation the tissue with propranolol and naloxone.

Conclusion: It seems that the extract induced antispasmodic effect mainly via calcium influx blockade. However, neither β-adrenoceptors nor opioid receptors were involved. Since the extract has antispasmodic effect on uterus contraction therefore we can suggest that it may support the use of this extract in traditional medicine to relief dysmenorrheal.

Keywords: Citrus aurantium, propranolol, naloxone, antispasmodic, uterus.

Introduction

Citrus aurantium (also called Seville orange or sour orange) is a small citrus tree, about five meters tall, with scented white flowers. The most common use of C. aurantium is medicinal rather than culinary. The dried, entire unripe fruit is used in Asian herbal medicine primarily to treat digestive problems. There is thus a history of benign human consumption of C. aurantium fruit. This herb use as a weight-loss aid could result in very different levels of intake and perhaps a different safety profile. Several components of C. aurantium are cause for concern (1). C. aurantium is the unique species, with pharmacological applications. The Hydrolate of its flowers has been used in traditional medicine as a treatment of mild depression, sedation and as a heart tonic (2). In humans, rats, guinea pigs, cats, and/or dogs, extracts of C. aurantium have been evaluated for effects on the cardiovascular system that include changes in blood pressure, cardiovascular toxicity, contractility and excitability of the heart muscle, and/or adrenergic activity (3). Studies of the flowers of this natural medicine have reported that some constituents exhibited antioxidant activity by flavonoids containing and the aqueous extract showed inotropic effect. The C. aurantium extract had a significantly greater effect in the portal hypertention (1). But no clinical and experimental studies have been conducted on its effects on the uterus. Thus the present study was an attempt to investigate the effect of C. aurantium flower aqueous extract on uterine contraction and its possible mechanism.

Materials and Methods

Plant extraction: C. aurantium flower were purchased from local herbal shop in 2009 and were powdered by miller machine. Aqueous extract was accommodated by sokcele method using 30gr powder and 300ml distill water for 8 hours. The complex was filtered with Whatman No1 filter paper. After that solution dried at room temperature to obtain powder and C. aurantium extract powder was stored at 4°C until being used.

Animals and tissue preparation: Adult female Wistar rats (200-300g) were obtained from Ahwaz Jundishapur University of Medical Sciences animal house and kept at 12-h light/dark cycle and at 20-24°C were allowed free access to tap water and commercial chow. In this study the rats were sacrificed by a sharp below on the neck. After laparotomy uterus was removed and washed with cold and oxygenated with De Jalon solution and cut into 1.5cm long and then mounted in an organ bath containing De Jalon solution (10 ml) between two stainless steel hooks vertically. The lower hook was fixed at the bottom of the organ bath and upper one was connected to an isometric transducer (UF1 Harvard transducer, UK) connected to a recorder (Harvard Universal Oscillograph, UK). The De Jalon solution composition (pH 7.4 and 29 °C) was (in mM): NaCl (154), KCl (5.6), CaCl2 (0.3), NaHCO3 (1.7), MgCl2 (1.4) and glucose (5.55) which was continuously bubbled with air (4). Tissue was then maintained under 1g initial tension and allowed to equilibrate for 1h during which bath solution was refreshed every 15 min.

Drugs: potassium chloride and barium chloride were purchased from merck (Germany), naloxone (Tolid Daru Company, Tehran-Iran), propranolol (Tolid Daru Company, Tehran-Iran) and oxytocin (Rasht-Iran Companies). C. aurantium extract powder and all chemicals were dissolved in De Jalon and added to organ bath, degree of drugs solution was equivalent with laboratories temperature (29°C).

Experimental protocols: After equilibrium period, the uterus was contracted by KCL 60mM (5, 6) and barium chloride 4mM (7) in the plateau of contraction the concentrations of extract (1, 2, 4 and 8 mg/ml) were added cumulatively. For the sake of study uterus opioid receptors, naloxone (1 μM) and for the study of β-adrenoceptors receptors, propranolol (1 μM) (8) were added to organ bath about 30 min without any refreshing for blocking these receptors in uterus tissue and after that concentrations of extract (1, 2, 4 and 8 mg/ml) added cumulatively. Uterus tissue contracted by oxytocin (10mU/ml) (8) and since oxytocin (OT) doesn't have plateau the tissue was washed. After that extract was added to organ bath without any dose of oxytocin and then percent's differences between peak amplitude of oxytocin was calculated.

Statistical Analysis: The mean±SEM of contraction forces induced by KCl, barium chloride and OT were regarded as (0%) and calculated for each group. The results were statistically analyzed by one way ANOVA and post hoc LSD tests and P-values less than 0.05 were considered as significant. The (n) represents the number of animals used in each protocol.

Results

Effect of C. aurantium flowers aqueous extract on KCl-induced contraction

KCl (60mM) induced uterus contraction and after 2min when uterus contraction arrived to the plateau, the concentrations of C. aurantium flowers aqueous extract (1, 2, 4 and 8 mg/ml) was added cumulatively to the organ bath with 10 min intervals and without refreshing bath solution. KCl (60mM) had significantly difference with all of the concentrations of extracts (P<0.001) also the relaxation effect of 1mg/ml extract had significantly difference with 4 and 8 mg/ml (P<0.01) of extract on contraction that made by KCL (60mM) (Figure 1).

Figure1. Effect of cumulatively concentration of C. aurantium aqueous extract on KCL (60mM) induced uterus contractions (n=6, **=P<0.01, ***=P<0.001). a= difference between KCL group with another groups, b= difference between 1mg/ml group with another groups, c= difference between 2mg/ml group with another groups, d= difference between 4mg/ml group with another groups, e= difference between 8mg/ml group with another groups.

Effect of C. aurantium flowers aqueous extract on barium chloride-induced contraction

First barium chloride (4mM) induced uterus contraction and then C. aurantium flowers aqueous extract (1, 2, 4 and 8 mg/ml) was added cumulatively. Contractions barium chloride (4mM) had significantly difference with another groups (P<0.001). Relaxation effect of 1 and 2 mg/ml extract has significantly difference with 8 mg/ml of extract (P<0.001). Also 1mg/ml of C. aurantium flowers aqueous extract had significantly difference with 4 mg/ml of extract (P<0.05) (Figure 2).

Figure2. Effect of cumulatively concentration of C. aurantium aqueous extract on barium chloride (4mM) induced uterus contractions (n=6, *=P<0.05, ***=P<0.001). a= difference between barium chloride group with another groups, b= difference between 1mg/ml group with another groups, c= difference between 2mg/ml group with another groups, d= difference between 4mg/ml group with another groups, e= difference between 8mg/ml group with another groups.

Effect of C. aurantium flowers aqueous extract activity after tissue incubation with propranolol

After induced uterus contraction by KCl (60mM) and without refreshing bath propranolol (1μM) added to organ bath for 30 min without any refreshing for blocking β-adrenoceptors, then concentrations of extract (1, 2, 4 and 8 mg/ml) added cumulatively to the organ bath with 10 min intervals and without refreshing. There were significantly differences among propranolol (1μM) with 2 (P<0.01), 4 and 8 mg/ml (P<0.001) of extract. Relaxation effect of 1mg/ml extract had significantly differences with 4 and 8 mg/ml (P<0.001) also this effect of 2mg/ml extract had significantly differences with 4 (P<0.05) and 8 mg/ml (P<0.01) (Figure 3).

Figure3. Effect of cumulatively concentration of C. aurantium aqueous extract on KCL (60mM) induced uterus contractions after incubated with propranolol (1μM) (n=6, *=P<0.05, **=P<0.01 ***=P<0.001). a= difference between propranolol group with another groups, c= difference between 2mg/ml group with another groups, d= difference between 4mg/ml group with another groups, e= difference between 8mg/ml group with another groups.

Effect of C. aurantium flowers aqueous extract activity after tissue incubation with naloxone

KCl (60mM) induced uterus contraction and after 2min, naloxone (1μM) as a non-selective opioid receptor antagonist added to organ bath for 30 min without refreshing. Then 1, 2, 4 and 8 mg/ml of extract added to organ bath cumulatively with 10 min intervals. The result showed a significantly difference between naloxone (1μM) and all groups (P<0.001) and relaxation effect of 1mg/ml extract had significantly differences with 8 mg/ml (P<0.05) group (Figure 4).

Figure4. Effect of cumulatively concentration of C. aurantium aqueous extract on KCl (60mM) induced uterus contractions after incubated with naloxone (1μM) (n=6, *=P<0.05, ***=P<0.001). a= difference between naloxone group with another groups, b= difference between 1mg/ml group with another groups, c= difference between 2mg/ml group with another groups, d= difference between 4mg/ml group with another groups, e= difference between 8mg/ml group with another groups.

Effect of C. aurantium flowers aqueous extract activity on oxytocin-induced uterus contraction

Oxytocin (10mU/ml) was added to the bath for 3min and then the tissue was washed. In next stage, extract (1mg/ml) was added for 3min and then in the presence of extract, oxytocin was applied to the bath for more 3min. This protocol was repeated for 2, 4 and 8 mg/ml of extract with 15 min interval and tissue washing. All of the concentrations of extract had shown relaxation effect with Oxytocin (10mU/ml) group significantly (P<0.001). Also 1mg/ml of extract had significantly difference with 2 (P<0.05), 4 and 8 mg/ml (P<0.001) and the difference between 2 mg/ml with 8 mg/ml was significantly (P<0.05) (Figure 5).

Figure5. Effect of noncumulatively concentration of C. aurantium aqueous extract on on rat uterus contracted by oxytocin (10mU/ml) (n=6, *=P<0.05, ***=P<0.001). a= difference between oxytocin group with another groups, b= difference between 1mg/ml group with another groups, c= difference between 2mg/ml group with another groups, d= difference between 4mg/ml group with another groups, e= difference between 8mg/ml group with another groups.

Discussion

Present results of this study showed that aqueous extract of C. aurantium flowers induces dose dependently, antispasmodic effect on the uterine muscle contraction caused by KCl, barium chloride or oxytocin. Also highest dose of the aqueous extract had most anticontractile effect on the uterus contraction.

propranolol is β-adrenoceptors antagonist and in some studies it seems clear that activation of β-adrenoceptors in uterine smooth muscle results in inhibition of myometrial contraction (9). After incubation with propranolol (1μM), antispasmodic effect of C. aurantium flowers aqueous extract decreased the contractile tension of uterine smooth muscle that made by KCl 60mM and propranolol (1μM) incubated in nonpregnant virgin's rat. According to these results we can suggest that C. aurantium flowers aqueous extract doesn't have any effect from β-adrenoceptors.

Opioid system expression is collectively observed in both endometrial and myometrial regions of the uterus (10). Opioid receptors activation relaxes uterus (11). Naloxone is a nonselective opioid receptors antagonist. In these results C. aurantium flowers aqueous extract activity had relaxant effect on uterus after incubation by naloxone (1μM). Therefore we can suggest that extract activity was not mediated via these receptors.

Uterine smooth muscle contraction is mediated mainly via increased intracellular Ca2+ and is accomplished by excitation-contraction coupling mechanisms. A high K+ medium could depolarize the cellular membrane of uterine smooth muscle (12). Moreover, it is well-known that K+-induced contraction in smooth muscles is due to an increase in Ca2+ influx through voltage-operated Ca2+ channels. It has been suggested that any substance that inhibits high K+-induced contractions of smooth muscles is a blocker of Ca2+ influx (13).

KCl induced uterine smooth muscle contraction results mainly from calcium influx via voltage-sensitive calcium channels (14). The present findings suggest that aqueous extract of C. aurantium flowers inhibits KCl-induced uterine contractions acting on voltage-sensitive calcium channels, because this contraction is dependent on extracellular calcium.

Oxytocin induces its effects via OT receptors (OTRs), which are members of the heptahelical family of G protein-coupled receptors. It is now well established that OT binding to its myometrial receptors leads to an increase in intracellular free calcium via the generation of inositol trisphosphate (15). The oxytocin-stimulated increase in [Ca2+]i is rapid and declines rather quickly in comparison to that seen in some other cell types, presumably as a result of active pumping of calcium out of the cell and back into the endoplasmic reticulum by the calcium transport ATPases (16). In this study, the effect of aqueous extract of C. aurantium flowers on oxytocin-induced uterus contraction was investigated in De Jalon solution with calcium concentration which prevents the spontaneous motility in uterus and superimposing these contractions with oxytocin-induced contraction and this relaxation effect was dose-dependently that 8mg/ml of this extract had most relaxation effect in uterus muscle. OT also activates the L-type VDCCs (voltage dependent calcium channels) (15, 16). In our results, we demonstrated that aqueous extract of C. aurantium flowers reduced the OT-induced contractions in De Jalon solution with calcium. Therefore it may suggest that this extract blocks the L-type VDCCs and therefore relaxes the contracted uterus also we can say that voltage sensitive calcium channels are involved in these contractions was reduced by the extract. Therefore, it may postulate that this extract acts through blockade of these channels.

Barium chloride could induce smooth muscle contraction by blocking the potassium channels followed by depolarization and contraction (17). It has also been reported that Ba2+ promotes Ca2+ release from intracellular pools in uterus (7) and smooth muscles. Our result showed that C. aurantium flowers extract reduced the Barium chloride induced contraction dose-dependently that results is from prevention either calcium efflux or calcium release.

In Conclusion our result indicated that C. aurantium flowers aqueous extract induces antispasmodic effect on isolated rat uterus and this extract decreased the uterus contractions without involving β-adrenoceptors, and opioid receptors. These results indicate that C. aurantium flowers aqueous extract induces antispasmolytic effect on rat uterus mainly through blockage of the VDCCs. The precise mechanism of extract activity can be the study of Ca2+ alterations in the presence of the extract in animals or human myometrium. The antispasmodic effect of the plant's extract observed in the present study, support the clinical efficacy and use of C. aurantium flowers in the treatment of dysmenorrhoea and other uterine spasmodic disorders. This process appears to be the most relevant physiologically and should be concentrated on in the future research.

Conflicts of interest

Authors have no conflicts of interests regarding this paper.

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