Flucrypyrim, a novel uterine relaxant, has antinociceptive and anti-inflammatory effects in vivo

Consequences of primary dsysmenorrhea (PD) can be severe. Increased prostaglandin production leads to uterine contraction and insufficient blood flow to the endometrium causing ischemia and pain symptoms. Protein tyrosine kinase/phosphatase activities contribute to the modulation of uterine contraction. In our previous study, we found the synthetic β-methoxyacrylates compound Fluacrypyrim (FAPM), significantly increased protein tyrosine phosphatases (PTPs) activity, resulting in dephosphorylation of tyrosine kinases. In the present study, we found that FAPM near completely inhibited prostaglandin F2α (PGF2α)-, oxytocin-, acetylcholine-, and high K+-induced uterine contractions in rats in vitro, and decreased rat myometrial myosin light chain (MLC20) phosphorylation induced by PGF2α. A structure–activity relationship assay indicated that the β-methoxyacrylates structure of FAPM is crucial for the inhibition of PGF2α-induced uterine contractions. FAPM caused a concentration-dependent parallel rightward shift of the concentration–response curve induced by oxytocin, dose-dependently reduced the number of abdominal constrictions and increased the latency time in PGF2α- and acetic acid-induced writhing test in mice in vivo. Furthermore, FAPM considerably inhibited the development of Carr-induced rat paw edemas and thexylene-induced mouse ear edemas. Taken together, our results indicate that FAPM exerts antinociceptive and anti-inflammatory effects in vivo with considerable potential as a novel uterine relaxant.

contractions elicited by pervanadate, a protein tyrosine phosphatase inhibitor, which can potentiate contractile receptor-mediated contraction 15,16 . Similarly, Imatinib mesylate suppresses the contractile activity of human uterus by inhibiting the tyrosine kinase activity of c-kit, PDGFR, and abl 17,18 . Tyrosine phosphorylation results from the dynamic equilibrium between phosphorylation and dephosphorylation reactions, the latter of which is regulated through the activity of protein tyrosine phosphatases (PTPs) 19 . In our previous work, we found fluacrypyrim (FAPM), identified as a potent STAT3 activation inhibitor, significantly increases PTPs activity in a dose-dependent manner. Furthermore, FAPM-induced suppression of STAT3 tyrosine phosphorylation can be reversed via sodium pervanadate, which highlights the importance of FAPM in regulating PTPs 20 . In the study described herein, we examined the direct effects of FAPM on uterine contraction in a rat model and evaluated the analgesic and anti-inflammatory effects in vivo. Moreover, we report a novel, inhibitory role of FAPM on rat uterine contractions induced by stimulators and evaluate the analgesic and anti-inflammatory activities of this agent in vivo.

Effects of FAPM and its analogs on PGF 2α -induced uterine contractions in the rats. To investi-
gate the in vivo inhibition of PGF 2α -induced uterine contraction by FAPM, we first examined the effect of FAPM on PGF 2α -induced uterine contraction in vitro. As shown in Fig. 1b, application of nanomolar concentration of PGF2α (450 nM final concentration in the bath solution) produced phasic contractions of constant amplitude and frequency. FAPM exerted a relaxant effect on PGF 2α -induced uterine contraction in a concentration-dependent manner (0.625~10 μ mol/L). A representative recording of the inhibition of both frequency and amplitude of contractions induced by FAPM is shown in Fig. 1c. The contractions expressed as percentage of the response to 450 nM PGF 2α were measured as the AUC at 10-min intervals to characterize the inhibitory activity of FAPM. On the basis of the dose-response curve (Fig. 1b), FAPM induced a complete (100%) reduction in contractility vs. 6.41 ± 2.68% for Vehicle (P < 0.001), with the average pD 2 value of (− log10 of the concentration of FAPM achieving half maximal inhibition) 5.72.
To characterize the structure of FAPM, a structure-activity relationship assay was performed using FAPM analogs against PGF 2α -induced uterine contraction (Fig. 1a). As showed in Fig. 1b, removing the isopropyl group at O14-site in FAPM (HTFAPM) or hydrolyzing the methyl acrylate of FAPM into acrylic acid (FAPMA) both leads to non-significant inhibition against PGF 2α -induced uterine contraction. When we changed the connection mode of benzene and pyrimidine in FAPM, where the benzyl was moved to N9-position from O7-position (IFAPM), the inhibitory activity of IFAPM was significant (P < 0.001), but lower than that of FAPM. On the basis of the dose-response curve (Fig. 1c), the net maximum reduction in contractility for HTFAPM, FAPMA and IFAPM was 7.72 ± 3.52%, 6.84 ± 3.11% and 42.15 ± 4.49% respectively. The structure-activity relationship assay indicated that both methyl group (C19) at O18-position and isopropyl group at O14-site in FAPM are critical for the effect, while the bonding site of arylmethyl group to the pyrimidine is also critical.

Effects of FAPM on oxytocin-induced uterine contractions in rats.
Oxytocin is a nonapeptide hormone that stimulates uterine and mammary gland contractions via oxytocin receptor 21 . To determine whether FAPM exerted an inhibitory effect on oxytocin-induced contractions, different doses of FAPM were administered along with oxytocin (1 mU/ml). As showed in Fig. 2a, FAPM exerted an inhibitory effect on uterine contractile force (amplitude) and frequency. On the basis of the dose-response curve (Fig. 2b), the percentage of net maximum reduction in contractility for FAPM was 100% vs. 13.81 ± 2.68% for Vehicle (P < 0.001), with the average pD 2 value of 5.79.
To characterize the antagonism of oxytocin-induced contraction by FAPM, increasing doses of oxytocin were administered along with FAPM. As showed in Fig. 3a, the presence of FAPM caused a concentration-dependent parallel rightward shift without any reduction in maximal contractile response. The slope and pA 2 values obtained from the Schild plot analysis were 1.78 ± 0.34 and 6.72 ± 0.03, respectively, indicating a competitive antagonism by FAPM against the oxytocin-induced contraction (Fig. 3b).

In vitro analysis of the effects of FAPM on acetylcholine and KCl -induced uterine contractions.
Acetylcholine (Ach) behaves as an excitatory neurotransmitter at neuromuscular junctions in uterine smooth muscle. As shown in Fig. 4a, application of 0.25 μ M Ach produced phasic contractions of constant amplitude and frequency, and administration of FAPM along with Ach exerted an inhibitory effect on uterine contractile force and frequency. Based on the dose-response curve (Fig. 4b), the percentage of net maximum reduction in contractility for FAPM was 100% vs. 25.21 ± 3.15% for Vehicle (P < 0.001), with the average pD 2 value of 5.86.
Previous reports have suggested that high-K + depolarizing solution can induce uterine contractions. Therefore, we investigated the effects of FAPM on K + depolarization-induced uterine contractions. The administration of different doses of FAPM along with KCl (16 mM) resulted in a dose-dependent inhibition of uterine contraction. On the basis of the dose-response curve (Fig. 4c), the percentage of net maximum reduction in contractility for FAPM was 100% vs. 10.58 ± 4.28% for Vehicle ( P < 0.001), with the average pD 2 value of 5.92.  Analgesic activity of FAPM on the acetic acid-induced writhing test. The writhing test was used to evaluate the analgesic activity of FAPM. Administration of FAPM (50, 100 and 200 mg/kg) 1 h before acid injection produced a significant and dose-dependent inhibition of acetic acid-induced abdominal constrictions in mice (Fig. 6a). The latency time was also found to be increased significantly in a dose dependent manner in the FAPM group (Fig. 6b). FAPM (100 mg/kg) demonstrated a significant inhibition of acetic acid-induced writhing response, similar to that of indomethacin (50 mg/kg), a standard NSAID used as positive control. The FAPM group (200 mg/kg) exhibited the maximum inhibiting effect of acetic acid-induced writhing and the longest latency time (12.2 min) -almost six fold long as that in the vehicle control group (2.2 min).  Anti-inflammatory activities of FAPM in mouse and rat swelling models. Prostaglandin release is thought to be a pathogenic factor associated with PD. Therefore, the anti-inflammatory activities of FAPM were evaluated using the mouse ear swelling model. As shown in Fig. 7a, treatment of mice with FAPM (100 mg/ kg) significantly inhibited the xylene-induced ear edema by 43.76% (p < 0.001). Treatment with indomethacin (10 mg/kg) also reduced the ear edema for 17.95% (p > 0.05).

Effects of FAPM on PGF
As expected, the volume of the injected hind paw of rats was increased by subplantar injection of carrageenan (edema), which peaked 3 h post-injection. The time course for the development of paw-edema following administration of FAPM (100 mg/kg, i.p.) is shown in Fig. 7b. Dexamethasonea (4 mg/kg, i.g.), a steroid used to treat many inflammatory and autoimmune conditions, was used as a positive control. Two agents were able to significantly reduce the paw edema at 2 and 3 h post-injection. The inhibition of paw swelling by FAPM and dexamethasone was 42.12% (p < 0.01) and 64.05% (p < 0.001) respectively (Fig. 7c).
Effect of FAPM on a mouse model of PD. PGF 2α plays an important role in the pathogenesis of PD. PGF 2α -induced writhing response occurred within 30 min after injection of PGF 2α in the females pretreated with estrogen while male mice did not experience any reactions. Administration of FAPM (100 and 200 mg/ kg), 1 hour prior to PGF 2α injection, highlighted a significant and dose-dependent inhibition of PGF 2α -induced abdominal constrictions in mice (Fig. 8a). Indomethacin, the positive control, showed a significant inhibition of

Discussion
Protein tyrosine kinase/phosphatase activities contribute to the modulation of uterine contractions induced by contractile agonists or stretch via tyrosine phosphorylation and dephosphorylation reactions [12][13][14][15] . We previously reported that fluacrypyrim (FAPM) significantly increases the protein tyrosine phosphatases (PTPs) activity in a dose-dependent manner, inhibiting protein tyrosine phosphorylation which can be reversed by PTP inhibitor sodium pervanadate 20 . In the present study, we firstly demonstrated that FAPM treatment near completely inhibited PGF 2α -, oxytocin-, Ach-, and high K + -induced uterine contractions in rats in vitro, with similar pD2 value from 5.72 to 5.92.
Uterine contraction is realized by the phosphorylation of 20 kDa myosin light chain (MLC 20 ) at Ser 19 , stimulating the ATPase activity of the smooth muscle myosin 22 . The levels of MLC 20 are regulated by opposing activities of MLC kinase (MLCK) and MLC phosphatase (MLCP) 23 . To stimulate MLCK by contractile agonists, such as PGF 2α , oxytocin, et al., are mainly dependent upon increasing cytosolic Ca 2+ and inhibiting MLCP 24,25 . As expected, we found FAPM, a novel inducer of PTPs, significantly reduced the PGF 2α -induced MLC 20 phosphorylation in dose-dependent manner, which indicated that FAPM inhibited smooth muscle contraction by affecting MLCK activity.
It has been reported that activated phospholipase C γ 1 (PLC-γ 1), produced in response to tyrosine phosphorylation, appears to be an important factor in uterine contractions, and Lck and c-Src kinases act an important role in regulating tyrosine phosphorylation of PLC-γ 1 and uterine contractions in rats 14,15 . The sustained smooth muscle contraction induced by vascular smooth muscle cell membrane depolarization involves genistein-sensitive tyrosine phosphorylation, leading to phosphorylation of MYPT1 (the myosin-targeting subunit of MLCP) and MLC 20 26 . The Ca 2+ -dependent tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2) was then identified as the major tyrosine-phosphorylated protein in response to membrane depolarization 13 . Tyrosine kinase inhibitors, such as genistein, PP1, PP2, among others, attenuated uterine contractions elicited by the stimulation of contractile receptors, whereas the protein tyrosine phosphatase inhibitors, such as pervanadate, bpV(phen), et al., potentiated receptor-mediated contraction 15,16 . Hear we found FAPM, a novel inducer of PTPs, caused a concentration-dependent parallel rightward shift in the oxytocin-induced concentration-response curve without any reduction in maximal contractile response, demonstrating a competitive antagonism by FAPM against the oxytocin-induced contraction. These data indicated that inhibition of FAPM on myometrial MLC 20 phosphorylation and contraction induced by contractile agents may be mediated through the increase of the PTPs activity.
Reversible oxidation of PTPs has emerged as an important regulatory mechanism whereby reactive oxygen species (ROS) inactivates PTPs, promotes phosphorylation and induction of the signaling cascade 27,28 . Mitochondrial reactive oxygen species (ROS), a major source of ROS in eukaryotic cells, are generally involved in oxidative stress, whereby complex III derived ROS especially has been involved in cellular redox signaling pathways 29 . However, a detailed understanding of the molecular mechanisms that trigger the generation of 'signaling ROS' from mitochondrial cytochrome bc1 complex is missing 30 . FAPM, with a β -methoxyacrylates structure, is proposed to be an inhibitor of mitochondrial cytochrome bc1 complex 31 . A structure-activity relationship assay indicated that the β -methoxyacrylates structure, a pharmacophore of class Ia inhibitors of mitochondrial cytochrome bc1 complex, is crucial for the inhibition of PGF 2α -induced uterine contraction. At present, we do not know whether the effects of FAPM on uterine contraction are directly related to the inhibition of mitochondrial cytochrome bc1 complex. The biochemical mechanism by which FAPM operates is still needed and should be evaluated in future studies.
Clinical studies have demonstrated high PGF2a levels are associated with dysmenorrhea. Moreover, prostaglandin's signaling has been shown to directly affect uterine contraction, and therefore may represent the most significant contributor to PD 6,32 . In China, the PGF 2α or oxytocin-induced writhing mouse models, represent frequently-used pain models of PD. We established this model and evaluated the anti-dysmenorrhea effects of FAPM. We noted that intraperitoneal administration of FAPM reduced the number of abdominal constrictions in a dose-dependent manner and increased the latency time when assessed in PGF 2α -induced writhing in mice in vivo. These results indicate that FAPM may be potentially useful in the release of PD.
Inflammation is also believed to be involved in the pathogenesis of PD. A previous study reported that non-steroidal anti-inflammatory drugs could be utilized in the treatment of PD 8 . Several studies have evaluated the effect of a cyclooxygenase-2 (COX-2) inhibitor as a treatment for PD 33,34 . Our study evaluated the anti-inflammatory effects of FAPM, using classical xylene-induced ear-edema model 35 . As a chemical agent, xylene can cause severe vasodilation and edematous changes of skin partially associated with phospholipase A 2 (PLA 2 ) 35,36 . Our results indicate that FAPM significantly inhibited xylene-induced ear edema. This effect may be produced by inhibiting the release of PLA 2 thereby reducing the concentrations of prostanoids and leukotrienes. For the further evaluation of the anti-inflammatory effects of FAPM, we performed a carrageenan-induced paw-edema assay in rats, which induces the development of edema in three phases: the early phase (the first 90 min) involving the release of histamine and serotonin; the second phase (90-150 min) mediated by kinin and the third phase (after 180 min) mediated by prostaglandin 35,37 . As shown in our results, FAPM significantly inhibited the development of the edemas induced by Carr after 2 h of treatment, while dexamethasonea, a steroidal anti-inflammatory agent as a positive control, inhibited the development of edema 1-3 h after treatment. Together these results further suggest that the anti-inflammatory mechanism of FAPM may be related to the inhibition of prostanoids and leukotrienes production.
The acetic acid-induced writhing test, an assay widely employed to assess anti-inflammatory and analgesic activities, is considered to be a model for visceral inflammatory pain 35,38 . It is generally thought that acetic acid lead to stimulation of nociceptive neurons by increasing endogenous mediators, such as prostaglandin and histamine, in peritoneal fluids. We found FAPM reduced the abdominal constrictions in a dose-dependent manner and increased the latency time when assessed in acetic acid-induced writhing in mice. These results suggest the effect of FAPM could be attributed, at least in part, to a reduction of inflammatory mediators.
Above all, we found FAPM, a novel inducer of PTPs, significantly reduced the PGF 2α -induced MLC 20 phosphorylation, near completely inhibited PGF 2α -, oxytocin-, Ach-, and high K + -induced uterine contractions in rats in vitro, and caused a concentration-dependent parallel rightward shift in the oxytocin-induced concentrationresponse curve. In consistent with the in vitro activity, we demonstrated FAPM dose-dependently reduced the number of abdominal constrictions and increased the latency time in PGF 2α -and acetic acid-induced writhing test in mice in vivo. It was then found that FAPM inhibited the development of Carr-induced rat paw edemas and thexylene-induced mouse ear edemas. A structure-activity relationship assay based on the inhibition of PGF 2α -induced uterine contraction indicated that the mitochondrial cytochrome bc1 complex may be the target of FAPM for its uterine relaxant effect as well as its antinociceptive and anti-inflammatory activity, through the inhibition of ROS production from mitochondria. Therefore, rational modifications of FAPM by medicinal chemistry techniques are anticipated to obtain derivative uterine relaxants with better specificity and therapeutic potential for patients suffering from PD.

Ethics StatementN. This study was approved by Beijing Experimental Animal Ethics Committee (2006)
No. 5118 set by the Beijing People's Government.
Animals. SPF KM male and female mice, weighting from 22 to 28 g, and female Sprague Dawley (SD) rats, weighing 120-140 g were purchased from the Laboratory Animal Center, Chinese Academy of Medical Sciences. The animals were kept in an environmentally controlled room at 22 ± 2 °C with free access to pellet food and water on a 12-h light/dark cycle. All animal procedures were performed in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals. The method of euthanasia was cervical dislocation and the method of anesthesia which we used in some tests was intraperitoneal administration of sodium pentobarbital.

Assessment of Uterine Contractility in vitro.
Female SD rats were administered intraperitoneally with estradiol benzoate (0.1 mg/kg) for 2 days before the experiments. Rats were sacrificed by cervical dislocation. Uteri were removed and then cut into 10 × 2 × 2 mm 3 strips along the longitudinal axis. The uterine strips mounted vertically in an organ bath containing 5 ml organ baths of Krebs' solution (136 mM NaCl, 2.68 mM KCl, 1.8 mM CaCl 2 , 0.5 mM MgCl 2 , 11.9 mM NaHCO 3 , 0.32 mM NaH 2 PO 4 and 5.04 mM glucose, pH 7.2), aerated continuously with 95% O 2 /5% CO 2 and maintained at 37 ± 0.2 °C. The tension of the myometrial rings was measured isometrically with a tension transducer connected to a polygraph system (Beijing Microsignalstar Techology Development Co., Ltd, China). To ensure contractile viability and to determine maximum contraction, the solution for each strip was first changed to 40 mM K + for 10 min. The strips which did not respond to KC1 were discarded. The recorded value to 40 mM K + for 10 min was taken as the control 39 . Each uterine strip was allowed to equilibrate at 1 g tension for 20 min until a steady tension was achieved and then treated with different agents (PGF 2α , oxytocin, Ach, or KCl) to stimulate uterine contraction. Various concentrations of FAPM were then added to the bath solution in a cumulative manner at 10-min intervals. Mechanical responses of uterine strips were analyzed by the area under the curve (AUC) according to the response curve of each uterine strip tested and expressed as a percentage of the control.
Antagonism assasyment of oxytocin-induced contraction. As described above, uterine strips were prepared and disposed before the experiment. Each uterine strip was allowed to equilibrate at 1 g tension for And pA 2 = lgK M when lg(dr − 1) = 0 in the linear plot.
Culture of Dispersed Myometrial Cells. Primary cultures of uterine smooth muscle cells were prepared as previously described by us 25 . Briefly, uteri was removed from estrostilben primed rats and dissected free of fat and endometrium. The tissues were cut into 1 × 1 × 1 mm 3 pieces and placed in culture flasks containing DMEM medium supplemented with 15% fetal bovine serum (FBS) and maintained at 37 °C in 5% CO 2 atmosphere. Cells were subcultured every 3-4 days prior to reaching confluence. When most of the cells had contracted and become rounded, the semi-dispersed cells were washed with PBS twice, and trypsin (0.25% w/v) was added. FBS was added to neutralize the effect. The dissociated myometrial cells were collected by centrifugation (200 g for 15  and resuspended in DMEM medium containing 10% FBS, then plated on glass coverslips for 24 h at 37 °C in 5% CO 2 atmosphere before the experiments. Total Protein Extraction. Protein extraction was performed according to the method as previously described by us 25 . Briefly, myometrial cells were plated at a cell density of 5 × 10 4 cells/well in 60-mm culture plates and cultured in serum-free DMEM for 24 h. After treatment with varying concentrations of FAPM and PGF 2α for 5 min, myometrial cells were washed with cold PBS three times, and then 2 × SDS loading buffer was added. The cell lysates were then boiled for 10 min and pelleted by centrifugation (10,000 g, 5 min). The samples were aliquoted and stored at − 20 °C for further use.
MLC 20 Phosphorylation Analysis. MLC 20 phosphorylation was analyzed by Western blot as previously described 25 . In brief, the cell lysates were centrifugated at 12,000 × g for 10 min at 4 °C. Equal amounts of protein (20 μ g) were separated on 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes. The membranes were probed with the phospho-Ser-19-specific antibody, followed by an HRP-conjugated secondary antibody. The enhanced chemiluminescence system detection kit (Cell Signaling) was used to visualize the immunoreactive proteins on PVDF membranes.
Acetic acid-induced abdominal writhing in mice. Acetic acid-induced writhing test was performed according to the previously described methods with minor modification 35 . Fifty mice were divided randomly into five groups. One hour before the test, a single dose of FAPM (50, 100 and 200 mg/kg) and indomethacin (50 mg/kg) were administered intraperitoneally. The mice were then injected intraperitoneally with acetic acid (0.6%, v/v in saline, 10 ml/kg b w., i.p.) and placed in separated boxes. The number of abdominal writhing was recorded for 30 min and the time that passed until the first appearance of writhing was recorded as the latency of pain response. For the precision and accuracy of the results, two well-trained lab technicians performed the test in a double-blind manner.
Xylene-induced ear edema in mice. The xylene-induced ear edema test was performed according to the previously described methods 38 . In brief, fifty mice were divided randomly into five groups. 1 h before the test, a single dose of FAPM (50, 100 and 200 mg/kg) and indomethacin (50 mg/kg) were administered intraperitoneally. The test animal was received 20 μ L xylene to the inner and outer surface of the right ear, while the left ear kept untreated. One hour later, mice were sacrificed and the central sections of the right and left ears were obtained using a stainless steel 6 mm punch. The weight difference between the sections of the right and left ears was used to evaluate ear edema. The ear edema percent was calculated using the following formula: Carrageenan induced paw edema in rats. Carrageenan induced paw edema in rats was conducted according to the method described previously 35 , with minor modification. Eighty rats were divided randomly into three groups. Before edema induction, the test groups were orally administered with dexamethasone (4 mg/kg), intraperitoneally injected with FAPM (100 mg/kg) and vehicle (DMSO, 20%) for 3 consecutive days. 30 min after the last administration, the rat left hind paw was injected sub-plantar with 0.1 ml of 1% carrageenan to induce edema, while the right hind paw kept untreated. Paw thickness was measured with a digital micrometer at 0, 1, 2 and 3 hours following injection. The rats were sacrificed at 3 h after carrageenan injection. Both hind paws were removed from the knee joint and weighed with an analytical balance. An increase in paw weight (in g) between the treated and the untreated paw was used to evaluate the degree of the hind paw swelling and the inhibition percent of paw swelling was calculated by using the following equation: where C and T indicate the non-treated and drug treated edema, respectively. In vivo mouse model of PD. In brief, fifty female mice were randomly separated into five groups. Before the test, each mouse was intragastrically treated with 0.2 mg diethylstilbestrol daily for 12 days. On the twelfth day, 30 min after the last administration, the test groups were intraperitoneally injected with FAPM (50, 100 and 200 mg/kg), indomethacin (50 mg/kg) and vehicle (DMSO, 20%). 1 hour after drug administration, the mice were treated with PGF2α (i.p. 1.3 mg/kg) and placed in separated boxes. The writhing responses were observed for 30 min and the time that passed until the first appearance of writhing was recorded as the latency of pain response. For the precision and accuracy of the results, two well-trained lab technicians performed the test in a double-blind manner.
Statistical Analysis. Results are expressed as means + /− SEM for n samples, and statistical analyses were performed using the GraphPad Prism 5 software (GraphPad Software, San Diego, CA). Student's t-test was used for comparison of the means of two groups. One-factor ANOVA followed by Dunnett-t test was applied for MLC 20 phosphorylation analysis (Fig. 5b), acetic acid-induced abdominal writhing assay (Fig. 6) and PGF 2α -induced pain response assay (Fig. 8). Two-factor ANOVA followed by Student-New-Keuls (SNK-q) test was applied for the inhibition experiment of uterine contraction (Figs 1c, 2b and 4b,c) and paw edema assay (Fig. 7b). Values of P < 0.05 were considered significant.