Regulation of Murine Myometrial Contraction by Ginger Extract Via Activation of Voltage Dependent Ca2+ Channels

Ginger (Zingiber officinae Roscoe) is a flowering plant in family
Zingiberaceae whose roots is widely used as an ingredient in both
cooking and East Asian traditional medicine.


Ginger (Zingiber officinae Roscoe) is a flowering plant in family
Zingiberaceae whose roots is widely used as an ingredient in both cooking and East Asian traditional medicine. Ginger is traditionally used to treat fever, nausea, vomiting, and uterine disorders. Ginger has also been used to treat paralytic ileus in Japan [1][2][3][4]. It has been reported that ginger can regulate contractility of uterine, gastrointestinal (GI) tract, and airway smooth muscle. Rat uterine muscle tone and spontaneous contractions can also be enhanced by ginger extract [5]. Such enhancing effect on the contraction is produced by activation of voltage-dependent L-type Ca 2+ channels (VDCC L ) and release of Ca 2+ from sarcoplasmic reticulum [5].
Other reports have suggested similar enhancing effects of ginger extract on rabbit uterine smooth muscle tone and its spontaneous contractions [6]. In general, VDCC L is essential for the regulation of smooth muscle contractility in many species [7][8][9][10]. Nifedipine, a VDCC L blocker, can also block oxytocin (OXT)-induced phasic contraction in murine myometrium (unpublished data). Regulatory effects of ginger on contractility of uterine, gastrointestinal (GI) tract, and airway smooth muscle have been reported [5][6]11].
Ginger also can improve gastric emptying and motility, intestinal contractility, and irritable bowel syndrome (IBS) in the GI tract [12][13]. In addition, ginger extract and its components can also inhibit motility of GI including the lower esophageal sphincter (LES) [14][15]. Other studies have suggested that ginger extract has regulatory effects on airway and vascular smooth muscle [16][17].
However, the mechanism of action involved in the effect of ginger extract on these organs remains unclear. In this study, we found that ginger extract could inhibit uterine smooth muscle contractility by blocking VDCCs. Since ginger is safe for humans, ginger might have potential to be developed as a tocolytic agent to relieve excessive uterine contractions.

Ginger Extraction
Dried ginger powder was purchased from a local company and 300 g was extracted twice with methanol for 2 L 24 hours at room temperature. Four liters of this methanol extract was dried in a rotary evaporator to yield 21 g of precipitate. The precipitate was resuspended in 500 ml of water and mixed with 500 ml of dichloromethane. The mixture was set at room temperature until water and dichloromethane phases separated clearly. Water and dichloromethane fractions were then collected and freeze-dried.
Yields of water and dichlromethane fractions were 9.4 g and 11.4 g, respectively. We already got patent in Korea (  All drugs used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Statistics
Data are expressed as means ± standard errors of the mean (SEM). Statistical significance was measured using analysis of variance (ANOVA) and student's t-test. Any p-value less than 0.05 was regarded as statistically significant.

Smooth Muscle
Oxytocin (OXT, 10 nM) produced tri-phasic contractions, showing an initial contraction followed by a tonic contraction
However, the inhibitory effect of Gin C on spontaneous contractions was not mediated by nitric oxide (NO): Gin C at 100 mg/mL inhibited spontaneous contractions completely in the presence of NO synthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 100 µM; n = 9; Figure 3C).

C-Induced Relaxation in Mouse Uterine Smooth Muscle
We studied the effect of Gin C on uterine smooth muscle contractions in the presence of KBC and NBC to investigate whether K + channels and nerves were activated during Gin C-induced relaxation. Gin C produced relaxation (n = 2) in the presence of tetraethylammonium (TEA, 10 mM) which blocked Ca 2+ -activated K + (K ca ) channels ( Figure 3B). Gin C-induced relaxation was also observed in the presence of KBC and NBC ( Figure 3D). Gin C at 100 and 200 mg/l produced relaxation up to 14 ± 7.3 % and 0 ± 0 %, respectively, of the control (n = 3 and n = 2, respectively).

Presence of Protein Kinase Inhibitors
Inhibitory effects of Gin C on OXT-induced contractions were studied in the presence of KT 5823 and KT 5720 known to inhibit protein kinase G (PKG) and PKA, respectively. OXT-induced contraction was inhibited by Gin C at 40, 100 and 200 mg/l in the presence of KT 5823 to 50 ± 20.8%, 0 %, and 0 % of the control, respectively (n = 4, 3, and 2, respectively, p < 0.05; Figure 4C). OXT-induced contractions in the presence of KT 5720 were inhibited by Gin C at 20, 40, 100 and 200 mg/l to 78 ± 0.2 %, 82 ± 0.1 %, 31 ± 0.2 %, and 0 % of the control, respectively (p < 0.05, n = 5, 5, 5, and 0, respectively; data now shown). To evaluate the involvement of PKC in Gin C-induced inhibition of OXT-induced contractions, Gin C was applied in the presence of a PKC inhibitor. As shown in Figure 4C, in the presence of PKC inhibitor bisindolylmaleimie II (Bis II, 0.5 µM), Gin C at 40 and 100 mg/l inhibited OXT-induced contractions to 36 ± 20.9 % and 1 ± 0.8 % of the control, respectively (p < 0.05; n = 4 and n = 2, respectively).
A and C: PGF 2a produced tri-phasic contractions, such as an initial contraction, followed by a tonic contraction overlapped with a phasic contraction. PGF 2a -induced phasic contractions were inhibited by Gin C (100, 200, and 400 mg/mL) to 39 %, 20 %, and 14 % of the control. B: ACh-induced phasic contraction was completely inhibited by Gin C and data as averaged in C.

Discussion
It is well known that regulation of myometrial contractility is tightly linked to Ca 2+ influx and Ca 2+ signaling at cellular level [18].
In this study, we found that Gin C inhibited contractions of murine uterus longitudinal smooth muscle by inhibition of VDCC L . As shown in (Figures 1 & 2B), murine myometrial contraction was tightly related to the activation of VDCC. Well-known pharmacological blockers such as nifedipine inhibited myometrial contractions.
This implies that both VDCC L is important for the regulation of myometrial contraction. In this study, Gin C inhibited high K + -, OXT-, ACh-, and PGF 2α -induced phasic contractions. Its effect was independent of NO synthesis, protein kinases (PKA, PKG and PKC), K + channel, or nerve blockers (Figures 3 & 4). This finding suggests that Gin C may inhibit mouse uterine smooth muscle contractions by inhibiting VDCC L and/or VDCC T strongly. It has been reported that ginger can enhance and inhibit uterine, GI tract, and airway smooth muscle contractions by activating VDCC L [4,14,19]. In the present study, we found that murine uterine spontaneous contractions and OXT-induced phasic contractions were sensitive to BayK 8644 and nifedipine ( Figures 1B & 1C).
In smooth muscle, Ca 2+ -induced Ca 2+ release (CICR) mechanisms also known to be important to regulate smooth muscle contraction [20][21][22]. As shown in Figure 2B, effect of Gin C was studied in the presence of CPA too. Therefore, inhibition of murine myometrial by Gin C might be responsible for the inhibition of VDCC L . However, in fact, effects of constituents of ginger extract on smooth muscle contractions were reported differently depending on the diverse extract. Therefore, we will also try to do some more supplementary experiments delicately by using other extracts from ginger in the future. Primary dysmenorrhea may be caused by an increase in PGF 2α produced in the uterus that can hyper contract the uterine smooth muscle and/or locally contracts blood vessels [23][24]. However, the exact mechanism of uterine spasms is currently unclear. However, phasic contraction was also sensitive to nifedipine in murine myometrium (unpublished data), Gin C inhibited PGF 2αinduced contractions by inhibition of VDCC L . Therefore, inhibition of PGF 2α -induced contractions by Gin C might suggests it could reduce symptoms of dysmenorrhea.
It is well known that VDCC L performs a key role in the regulation of smooth muscles [7][8][9]. However, the role of VDCC T in such regulation is relatively unclear. In myometrium, VDCC T in rat and human pregnant samples has been reported. In rat pregnant myometrium, spontaneous phasic contraction has been found to be sensitive to micromolar ranges of mibefradil [11].
Meanwhile, specific subtype of VDCC T (CaV 3.1 ) and role of VDCC T in the regulation of contraction in human pregnant myometrium has been identified [25][26]. Whether the exact subtype of VDCC L and/or VDCC T might be related to various conditions of myometrial contractility is not fully understood yet. Furthermore, the role of VDCC T compared to that of VDCC L in myometrial contraction is not well studied yet. In fact, even the regulation of rat pulmonary arterial proliferation is regulated by VDCC T through activation of CaV 3.1 channel [27]. We identified functional expression of VDCC L (CaV 1.2 ) in murine myometrium by performing mechanics and immunohistochemistry (data not shown in here). However, further study is needed to identify T-type Ca 2+ channel and more functions of it in murine and human myometria.
Ginger extracts are known to have effects on the GI tract. For example, they can inhibit LES motility [14,19]. In these cases, serotonergic receptors and/or cholinergic M receptors are involved in inhibiting smooth muscle contraction [15,28]. Ginger extract can also improve gastric emptying and IBS for gastric and intestinal motility, respectively [12][13]. That implies ginger extract might produce increasing and decreasing functions of smooth muscle via affecting receptor levels too. However, we found Gin C specifically inhibited murine myometrial contraction by inhibition of VDCCL. Ginger plant has been used to treat inflammation, rheumatic disorders, and diarrhea in traditional medicine [2,4,29].
Zingerone is thought to be the active antidiarrheal component responsible for limiting endotoxin-induced diarrhea [30][31].
Therefore, some effects of ginger extracts are not direct on smooth muscle. Meanwhile, gingerol produces dual effects (enhancing and inhibitory) on ileal contractions in guinea-pig [12][13] through capsaicin-sensitive neurons [32]. Shogaol- [6] from ginger can also inhibit vascular smooth muscle proliferation by activating specific signaling pathways [16].
A cyclooxygenase-related system in vascular smooth muscle may be involved in regulating eicosanoid-induced contraction [16].
Additionally, gingerols exhibit various effects on the cardiovascular system [16] while zingerone may activate the same capsaicin receptors and/or a common pathway in trigeminal ganglion neurons [32]. Some other Gin C components such as ginerols and/or shogaol could also inhibit uterine contractility. Therefore, extracts from ginger produces diverse effects on different organs via various actions. From these results, we tried to exclude involvement of nerves and other K + channels by other ginger extracts [33]. As shown in Figure 3B & 3D, the relaxing effect of ginger extract was studied in the presence of TEA, nerve blocker cocktail. In addition, involvement of nitric oxide (NO) in the action of antinocipective activity by ginger extract was also reported [34]. Therefore, we also studied and found inhibitory effects of Gin C in the presence of and L-NAME (100 µM) on murine uterine smooth muscle ( Figure   3C). Our results suggest that Gin C may produce murine uterine relaxation by inhibiting VDCC L . This is first report showing that inhibition of uterine smooth muscle contractility by ginger extract obtained by dichloromethane fraction (Gin C). Our results revealed the possibility that Gin C inhibited High K + -, OXT-, PGF 2α -, and ACh-induced uterine contraction and spontaneous contraction of uterine longitudinal smooth muscle of mouse by inhibiting VDCC L .

Conflicts of interest
The authors declare that there is no conflict of interest.