{"paper_id":"c35e3217-0ef2-451c-8ea7-dd7766bfbe30","body_text":"1Scientific  RepoRtS  |         (2020) 10:7063  | https://doi.org/10.1038/s41598-020-63477-8\nwww.nature.com/scientificreports\nEffect of Tahiti lime (Citrus \nlatifolia) juice on the Production \nof the PGF2α/PGE2 and Pro-\nInflammatory Cytokines involved in \nMenstruation\nThaiane Robeldo1,5, Edione Fatima Canzi2,3, Priscila Maria de Andrade4,5, \nJhonne Pedro Pedotte Santana1, Felipe Roberti Teixeira1, Valentine Spagnol1, \nBeatriz Helena Lameiro Noronha Sales Maia2, Maristela Carbol4, Erika Gonçalves Caneira4, \nMaria Fátima das Graças Fernandes Da Silva  3 & Ricardo Carneiro Borra  1 ✉\nTahiti lemon juice (Citrus latifolia) (TLJ), as a natural source of flavonoids, has been used as an \nalternative to anti-inflammatory drugs for the treatment of dysmenorrhea and menstrual excessive \nbleeding, often associated with an imbalance of the prostaglandins (PG) levels. However, despite \nthe positive effects, the mechanisms that rule menstruation control are still unknown. Therefore, the \nobjectives were to characterize the TLJ and analyze its effect on the production of PGF2α, PGE2 and \npro-inflammatory cytokines involved inmenstruation. Flavonoids from TLJ were discriminated by UPLC-\nDAD-MS/MS (Qq-TOF) and the effects of TLJ were studied in vitro by quantification of the contraction \nof myoblasts in culture and PGF2α and PGE2 productions. Further, the systemic and menstrual fluid \nlevels of PGF2α, PGE2, IL-1β, TNF- α, IL-6, AK1B1 and AK1C3 enzymes produced by women during the \nmenstrual period were compared after exposition or not to TLJ or meloxicam. The results showed that \nTLJ induces an increase in the contraction of myoblasts and the PGF2α supernatant level. Regarding \nin vivo analysis, a higher concentration of PGF2α and an unaltered PGE2 level was also found in the \nmenstrual blood of women treated with TLJ, in contrast with a lower level of PGE2 and PGF2α observed \nin the meloxicam group. Concerning cytokines, only menstrual TNF-α levels decrease after treatment \nwith TLJ or meloxicam. In conclusion, TLJ may favor the control of menstruation events via a PGF2α \nmediated muscle contractile response.\nToday’s women experience more menstrual cycles than their predecessors. In the past, late menarche, multiple \npregnancies, longer periods of breastfeeding and the occurrence of menopause at 40 years of age acted as inhib-\niting factors of the menstruation. However, due to lifestyle changes combined with longer life expectancies and \nmenostasis at 45 years of age, modern women are more predisposed to developing menstrual disorders charac-\nterized by prolonged and excessive bleeding along with dysmenorrhea, which compromise health and the quality \nof life\n1,2.\nStudies have shown that both excessive menstrual bleeding and primary dysmenorrhea are related to an \nimbalance in the uterine concentration of the F2α  (PGF2α ) and E2 (PGE2) prostaglandins 3–6. In women with \ndysmenorrhea, the increased PGF2α  production has been associated with the sensitivity of the muscle fibers in \nthe uterus due to impaired blood flow caused by strong contractions of the myometrium 7,8. PGF2α  is a potent \nvasoconstrictor that acts directly on smooth muscle fibers, reducing blood vessel caliber9. In the uterus, PGF2α, \n1Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, SP , Brazil. 2Department of \nChemistry, Federal University of Paraná, Polytechnic Center, Curitiba, PR, Brazil. 3Department of Chemistry, Federal \nUniversity of São Carlos, São Carlos, SP , Brazil. 4Department of Medicine, Federal University of São Carlos, São \nCarlos, SP , Brazil. 5These authors contributed equally: Thaiane Robeldo and Priscila Maria de Andrade. ✉e-mail: \nrcborra@gmail.com\nopen\n\n2Scientific  RepoRtS  |         (2020) 10:7063  | https://doi.org/10.1038/s41598-020-63477-8\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nproduced by the endometrium, acts as an agonist in the myometrium, inducing contractions by activating FPs \nreceptors and mobilizing intracellular Ca2+. Meanwhile, the PGE2 activity in the myometrium depends on the \ntype of receptor-activated. The PGE2 binding to EP1 and mobilizing the intracellular Ca2+, or to EP3 and mod-\nulating cAMP and IP3, produces contractions. On the other hand, the binding of PGE2 to EP2 or EP4 induces \nrelaxation via elevating intracellular cAMP accumulation\n10–12. The direct involvement of prostaglandins in the \npain pathways associated with menstrual disorders, mainly the PGE2, should notbe discarded8.\nDepending on the magnitude of the menstrual disorder, non-steroidal anti-inflammatory drugs are used to \nblock the synthesis of PGs and control the clinical conditions. However, this therapeutic choice produces many \nside effects, restricting its use for long periods. Considering this, ethnomedicine has been proposed in the present \nstudy as an alternative treatment for menstrual problems.\nSeveral authors have reported the existence of the modulator effects of flavonoids on the regulation of \ncytokines, prostaglandins and other mediators that drive immune and inflammatory reactions\n13–18. Citrus fruits \nand juices (oranges, mandarins, grapefruits, lemons, bergamots, and limes) are an important source of flavo -\nnoids, mainly glycosylflavonoids19,20. Among a variety of compounds present in citrus, luteolin, for example, can \nmodulate the expression of COX-2, nitric oxide synthase, and inflammatory cytokines21. Hesperitin, naringenin \nand, rutin inhibit the COX activity as well as PGE2 production 22–24. Naringin acts on the immune system to \nprevent tissue damage, while naringenin can inhibit key enzymes in the oxidation of fatty acids, as well as the \nNF-κβ transcription factor, reducing the production of pro-inflammatory cytokines\n25,26. Diosmin and hesperidin \npossess inhibitory activity over E2 and F2α prostaglandins27. The combination of hesperidin, nobiletin and tan-\ngeretin show a potent suppression over iNO2, TNF-α , IL-1β, and IL-6 cytokines28. Furthermore, in the human \nbody, the glycosylflavonoids can be converted into their aglycone form, which has shown strong antioxidant and \nanti-inflammatory activities when compared to the glycosyl form\n29.\nBased on this knowledge, our research group conducted a pilot study in 2014 to analyze the citrus-therapy  \neffects of Tahiti lime (Citrus latifolia) juice (TLJ) in patients with menstrual disorders. The results showed that \nthis Citrus reduced the duration and intensity of excessive bleeding, the occurrence of dysmenorrhea and the \npresence of clots30. The choice of Tahiti lime was based on preliminary empirical tests that showed the best results \nfor Citrus latifolia over other limes species. In this pilot, a gynecologist used different types of lime juice during \ndiverse menstrual cycles and identified an advantage in the activity of C. latifolia. This pilot also showed that the \njuice from one lemon fruit was capable to reduce the menstrual bleeding soon as 30 min after its consumption.\nDespite the positive effects, the mechanisms that rule menstruation control are still unknown. The present \nresearch aimed to characterize by ultra-high-performance liquid chromatography,the main compounds present \nin the TLJ and to analyze in vitro and in vivo,the effectof TLJ in the cellular contraction and on level of prosta-\nglandins (E2 and F2α ), enzymes involved in the arachidonic acid pathway and pro-inflammatory cytokines on \nmenstrual fluid and peripheric blood samples from women during the menstrual period.\nResults\nCharacterization of Tahiti lime juice. The TLJ metabolites were identified based on fragmentation stand-\nards and the UV spectra obtained. Experiments were carried out in both negative (ESI − ) and positive (ESI +) \nmodes, varying the collision energy. Only glycosylated flavonoids were found in the TLJ. For these, the main \nfragment ions observed in ESI\n- were related to sugar moiety elimination, showing the aglycone ion as the most \nintense. Besides, fragments from the Retro-Diels-Alder reaction and CO and CO 2 loss were also observed 31–33. \nBased on this, hesperidin, eriocitrin, rutin, and naringenin were identified in TLJ, as shown in Table 1.\nEffect of TLJ on apoptosis rate of C2C12 cell line.  In the experiment for apoptosis detection (FACS \nanalysis: Annexin V - PI), there was not a significant difference in the percentage of viable cells submitted to 5 h \nof citrus treatment in comparison with the Control group (Fig. 1C). However, in 24 h culture, there was a small \ndifference (<3%), statistically significant between the Control and TLJ groups that could be considered with-\nout importance in context (Fig. 1C). The citrus treatment did not induce any apoptotic or necrotic effects over \nmyoblasts in a significant way in comparison with the Control without treatment. When analyzing the micro-\nphotographs, it was observed that the groups exposed to 1 or 2% of TLJ for 5 or 24 h showed cells with features of \nnormality such as spreading and multiplication (Fig. 2C–F ,G,H). Concerning the H\n2O2 treated group (Control+), \nthe cells exposed for 5 h start to show signals of loss of viability such as detaching and rounding (Fig. 2B).\nIn vitro Effect of TLJ on the production of PGF2α  induced or not with LPS or AA. Comparing the \nresults of the production of PGF2α from C2C12 cells treated with different concentrations of buffered TLJ (0, 1 \nand 2%) at different times (2, 5, 24 h), it was possible to see that there are positive correlations between TLJ con-\ncentration (p < 0.05), the time of exposition (p < 0.01) and the PGF2α level present in the supernatant (Fig. 3A). \nThe mean level of PGF2α from the cell line exposed to 2% TLJ was significantly higher (p < 0.05) than the Control \nand 1% TLJ, mainly after 5 h of exposition (Fig. 3A).\nAbout the effect produced by treatment with TLJ and stimulation with LPS, the concentration of PGF2α \nfrom C2C12 exposed to citrus and LPS (Fig. 3B) were comparable in level from cells treated exclusively with TLJ \n(Fig. 3A). The mean level of PGF2α from the cells exposed to 2% TLJ and LPS (p < 0.01) was also higher than the \nControl and 1% TLJ after 5 h or 24 h of stimulation. On the other hand, the TLJ treatment augmented the produc-\ntion of PGF2α induced by AA in an addictive way (Fig. 3C).\nAnalyzing the Resazurin assay results, it was possible to see that there was not a significant difference between \ncellular viability of the Control group to those treated with 1% or 2% of buffered TLJ for 5 h or 24 h. The viability \nof the myoblasts submitted to AA or LPS in combination or not with lime juice was also not different from the \nControl (Fig. 3D).\n\n3Scientific  RepoRtS  |         (2020) 10:7063  | https://doi.org/10.1038/s41598-020-63477-8\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nConcerning the NF−κβ  reporter activity, HEK293 cells expressing pBIIx-luc under the control of NF−\nκβ increased the luciferase production in response to TNF-α after 12 h of stimulation, but when treated with TLJ \n(1 or 2%) did not suffer any modulation of the activity. Besides, the cells treated only with TLJ (1 or 2%) did not \nalso stimulate significantly the NF−κβ gene reporter.\nEffect of TLJ on collagen gel contraction mediated by L929 and C2C12 cell lines. The contraction \nassays were performed within a 24-well plate containing 1 × 105/mL of L929 and C2C12 cells trapped inside of \ncollagen gel. As shown in Fig. 4, the TJL significantly stimulated (p < 0.001) the contraction of gels with C2C12 \nand L929 at about 2- and 5-days post addition of treatments in comparison with the Control (Fig. 4).\nPGF2α  and PGE2 levels in menstrual fluid and peripheral serum. Figure 5 and Table 2 display the \ndata relating to the concentrations of PGF2α and PGE2 in the menstrual fluid and in the peripheral serum from \nvolunteers from the Control, Meloxicam, and TLJ groups. The results showed that the median concentrations \nof PGE2 from the Control (310.2 ng.mL\n− 1) and TLJ (316.3 ng.mL− 1) groups were very similar and significantly \nhigher in comparison with the Meloxicam group (71.8 ng.mL− 1). PGF2α  values were higher in the TLJ Group \n(2953.1 ng.mL− 1) than the Control (1806.3 ng.mL− 1) and Meloxicam (1131.2 ng.mL− 1) groups, between which \nthe values also differed. The PGE2 concentration values from the peripheral serum samples from all groups were \nbelow the sensitivity of laboratory testing. However, in terms of PGF2α measurements, the Meloxicam group had \na significantly lower value (1.32 ng.mL\n−1 ) than the TLJ (2.44 ng.mL−1 ) and Control (3.16 ng.mL−1 ) groups. There \nwas not any participant that had harm or unintended effects in the groups.\nFigure 6 shows the dispersion curves formed by combining the concentration values of the PGE2 and PGF2α \nof the menstrual fluid of each volunteer from the three groups. The PGF2α  values in the TLJ group increased \nmore intensely than the PGE2 values, causing an increase in the slope of the curve of the group in comparison \nwith the Control and Meloxicam groups. In the Meloxicam group, there were reductions of PGE2 and PGF2α , \nwhich maintained the slope of the Meloxicam curve equivalent to that of the Control group. This behavior can be \nobserved in the scatter plot (6B) which shows only a difference in the median of the ratio (PGF2α/PGE2) between \nthe Control and Meloxicam groups (p < 0.01) independent of the level of PGF2α.\nTNF-α , IL-1β and IL-6 cytokines in menstrual fluid and peripheral serum. The Fig. 7 shows the \nstatistical analysis of the TNF-α, IL-1β and IL-6 cytokines present in the menstrual fluid and peripheral serum of \nthe volunteers of the Control, Meloxicam, and TLJ groups. In terms of the concentrations of TNF-α in the men-\nstrual fluid, the group that used the TLJ had a significantly lower median value (2717 pg.mL\n−1 ) than the Control \ngroup (10950 pg.mL− 1). However, in comparison with the Meloxicam group (4335 pg.mL− 1), there was no sta-\ntistical difference. In relation to the IL-1β  and IL-6 cytokines, no difference in the median concentrations was \nobserved between the groups analyzed. Concerning the TNF-α cytokine concentration present in the peripheral \nMF CM tR\nExact \nmass [Mn-H]- [M + H]+ MS/MS-\nMass Error \n(ppm)\nλmax \n(nm)\nEriocitrin C\n27H32O15\n 13.7 595.1670 609.1461 611.1648 459; 287; 151 −0,2 255; 353\nRutin C27H30O16\n 14.3 609.1459 609.1825 611.1647 300/301; 271; 255; 179; 151 0.4 284; 220\nNaringin C27H32O14\n 16.0 579.1719 595.1668 597.1829 313; 271; 151 1.8 284; 225\nHesperidin C28H34O15\n 17.8 609.1819 579.1709 581.1890 301; 286; 257; 242; 151; 134 1.0 282; 213\nTable 1. Glycosylated flavonoids present in Tahiti lime juice by UPLC-DAD-MS/MS (Qq-TOF). MF: molecular \nformula. CM: chemical structure. tR: retention time.\n\n4Scientific  RepoRtS  |         (2020) 10:7063  | https://doi.org/10.1038/s41598-020-63477-8\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nserum, the results showed that there was no difference in median cytokine levels between the TLJ (258.4 pg.\nmL− 1), Meloxicam (210.3 pg.mL− 1), and Control (229.6 pg.mL− 1) groups. The IL-6 and IL-β  values measured \nwere below the quantification sensitivity of the ELISA kits and were therefore not computed in the analysis.\nAKR1B1 and AKR1C3 in menstrual fluid and peripheral serum. Figure 7 shows the statistical analysis \nof the concentration of the AKR1B1 and AKR1C3 present in the menstrual fluid from the Control, Meloxicam \nand TLJ group volunteers. It was observed that the median concentration of AKR1B1 in the menstrual fluid did \nnot differ in any of the three groups (Control: 0.5 pg.mL\n− 1; Meloxicam: 0.7 pg.mL− 1; TLJ: 0.6 pg.mL− 1) . On the \nother hand, the treatment with the Meloxicam induced a significant reduction in the concentration of AKR1C3 \nin comparison with the Control group  (Control: 9.6 pg.mL\n− 1, Meloxicam: 3.9 pg.mL − 1; TLJ: 9.1 pg.mL − 1). \nConcerning the serological level of AKR1C3, there were no differences between the three groups (Control: 3.4 \npg.mL\n−1 ; Meloxicam: 2.5 pg.mL−1 ; TLJ: 3.9 pg.mL−1 ). The peripheric blood AKR1B1 levels from all groups were \nbelow the sensitivity of ELISA kit and therefore it was not computed in the analysis.\nDiscussion\nThe human endometrium is a dynamic tissue, whose function is mainly regulated by the activities of the estrogen \nand progesterone hormones. During menstruation, many of the events that affect the endometrium tissue are \ninflammatory in nature. Among several mediators, prostaglandins stand out as important inflammation agents \ndue to their activity in the control of the uterine musculature contraction, caliber, and vascular permeability. \nSeveral studies have associated disorders of the menstrual cycle, such as excessive menstrual flow and dysmenor-\nrhea with abnormalities in the production of prostaglandins, especially E2 and F2α\n3,4,7,34 (Fig. 8).\nThe discovery that certain in vitro flavonoids are selective modulators of prostaglandin has led to speculation \nthat these compounds, which are present in citrus fruits, could be primarily responsible for an anti-inflammatory \nmechanism. In our analysis, Citrus latifolia exhibited some flavonoids that are typically found in other limes, such \nas hesperidin, eriocitrin, rutin and naringin (Table 1). Some authors have found the hesperidin, diosmin, and eri-\nocitrin flavonoids in different citrus juices\n39,40. Another study found hesperidin to be the main component of the \nTahiti lime, followed by eriocitrin, rutin, naringenin, narirutin and diosmin41. In relation to its biological features, \nhesperidin and eriocitrin metabolites have antioxidant and anti-inflammatory activity capable of eliminating free \nradicals and inhibiting inflammation in vitro. Hesperidin significantly decreased the production of PGE2 without \nFigure 1. Absence of apoptotic and necrotic effects on C2C12 exposed to 1% or 2% of buffered TLJ for 5 or \n24 h, analyzed by flow cytometry using AnnexinV and propidium iodide (PI) staining. (A) Filters used for \nclassification of the cells; (B) Contour plot showing the distribution of cells classify as viable (I: PI−/Annexin \nV−), early apoptosis (II: PI−/Annexin V+), late apoptosis (III: PI+/Annexin V+) and necrosis (IV: PI+/\nAnnexin V−) of the groups treated with 11 µM of H\n2O2 (control+), 1% or 2% of TLJ buffered for 5 or 24 h. \n(C) Bar charts showing the numeric results of the classified groups (mean ± SEM, n = 3). Statistical Analysis: \nANOV A accompanied by the Dunnett’s Multiple Comparison test using the control (−) group as reference; \n*p < 0.05.\n\n5Scientific  RepoRtS  |         (2020) 10:7063  | https://doi.org/10.1038/s41598-020-63477-8\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\naltering the COX-2 level. Rutin is capable of inhibiting IL-1β cytokine production, apoptosis and edema, reducing \nthe inflammatory response42–45.\nIn the present study, the in vitro level of PGF2α and PGE2 produced by murine myoblast cells (C2C12) when \nexposed at different concentrations of TLJ (1% and 2%) and times (2, 5 , and 24 h) were measured (Fig. 3). The \nPGE2 concentration was not enough to be detected in our analyzes. The PGF2α level at both 1 and 2% TLJ con-\ncentrations, mainly at 5 h exposure time, was significantly higher compared to the Control (0% TLJ) (Fig. 3A). In \nrelation to experiments of co-stimulation, the results showed that there was no interaction between TLJ and LPS \nstimulation on the production of PGF2α  indicating that the action mechanism of TLJ may be independent of the \nFigure 2. Absence of degenerative signalsof the C2C12 cells exposed to TLJ (1% or 2%) until 24 h, analyzed \nby phase contrast (A–F) or panoptic stain by bright field microscopy (A–G). (A) Control cells (5 h) without \ntreatment showing normal morphology with some cells with signals of proliferation (small round bright cells). \n(B) Positive control cells treated with H\n2O2 (11 µM) at 5 h showing features of loss of viability (detachment). \n(C,E) cells treated at 5 h with 1% or 2% TLJ showing normal morphology (spreading) with some cells in \nproliferation. (D,F) cells treated for 24 h with 1% or 2% TLJ also showing normal morphology with some cells \nin proliferation. (G,H) micrographs of C2C12 cells stained by panoptic showing the normal characteristics in 5 \nand 24 h after TLJ exposition. (*) C2C12 in proliferation showing chromosome condensation.\n\n6Scientific  RepoRtS  |         (2020) 10:7063  | https://doi.org/10.1038/s41598-020-63477-8\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nproduction of pro-inflammatory mediators (Fig. 3B). However, the co-stimulation TLJ/AA showed an additive \ninteraction that was responsible for the augmentation of the level of PGF2α , indicating that the TLJ could act \ndirectly over the main branch of the AA pathway (Fig. 3C). In endometrial tissue, prostaglandins are synthesized \nfrom the cell membrane phospholipid precursor, which is undergoing the death process (Fig. 8). In our case, the \nFigure 3. Demonstration of the augmentation of the production of PGF2α by C2C12 cells treated with \nTLJ without association with lost of cell viability or NF-kβ activity induction. The productions of PGF2α by the \nC2C12 cell line treated with different concentration of buffered TLJ (1% or 2%; pH.7.0) and exposed or not (A) \nto 10 ng of LPS (B) or 10 µM of Arachidonic Acid (C) at different time intervals (2, 5 or 24 h),were quantified \nby ELISA. Line graph (A) shows the variation of the production of PGF2α (fold change) induced by different \nconcentrations of TLJ (n = 9; *p < 0.05 in relation to the 0% Control) and bar graph shows the dynamic of the \nproduction of PGF2α (ng.mL\n−1 ) induced by TLJ (n = 3) at different times (*#p < 0.05 in relation to the 1% \nand 0% Controls). Line graph (B) shows the parity in the production of PGF2α (fold change)by cells treated \nby TLJ (n = 9) and stimulated or not with LPS. Bar graph shows the dynamic of the production of PGF2α (ng.\nmL\n−1 ) induced by LPS and TLJ (n = 3) treatments at different times (*#p < 0.01 in relation to the 1% and 0% \nControls). Line graph (C) shows the difference in the production of PGF2α (fold change) after stimulation with \narachidonic acid (AA) and treatment with TLJ (n = 9; p < 0.05) in relation to the AA(−). The bar graph (C) \nshows the change of the production of PGF2α induced by TLJ (n = 3) at different times after AA stimulation. \nBar graph (D) shows the results of the viability of the C2C12 cell line (n = 4) after 5 h or 24 h of exposition \nto diverse concentrations (0, 1 and 2%) of buffered TLJ quantified by resazurin assay. Bar graph (E) showing \nthe activity of the NF−κβ reporter from HEK293 cells (n = 5) treated only with buffered TLJ (1 or 2%) or \nstimulated with TNF-α and TLJ for 12 h, in comparison with Controls group (CT-; TNF-α: 10 ng/mL and \nLPS: 10 ug/mL). NS: non-significant. In all the analyses, the results were represented by mean ± SEM and the \nANOV A and Newman-Keuls Multiple Comparison statistic tests were used.\n\n7Scientific  RepoRtS  |         (2020) 10:7063  | https://doi.org/10.1038/s41598-020-63477-8\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nresults of Figs. 1 and 2 showed that the viability of C2C12 when exposed up to 2% of TLJ at 24 h was not affected \n(Figs.1, 2 and 3D), guaranteeing the cellular integrity and discarding the possibility that PGF2α production was \ndue to any cell destruction caused by TLJ exposure.\nOne of the proposed mechanisms that would explain the control of menstruation using citrus would be the \npossibility of stimulating smooth muscle contraction. Up to this point, we determined that PGF2 α  production \ncould be equally stimulated by TLJ in vitro as in vivo. But we do not know whether such stimulation could induce \neffective contraction. To respond to this challenge, we performed a collagen contraction assay using two cell \nlines (C2C12 and L929) comparing various concentrations of TLJ with the synthetic PGF2α  and SFB (positive \nControls), known to stimulate contraction46. The results showed that the TLJ induced a contraction of the colla-\ngen disc, mainly in a concentration above 1%. In relation to cell lines, the contraction of myoblast (C2C12) was \nmore intense and faster (5 days vs2 days) than fibroblast lineage (L929). These data may explain the relationship \nbetween lime juice consumption and decreased menstrual flow since PGF2α could age in the contraction of both \nthe blood vessel and myometrium.\nThe stimulatory assay using the myoblast cell showed the ability of TLJ to induce PGF2α  production in \nvitro, but the question over its activity in the uterus remained open. To address this issue, during the menstrual \nperiod, we compared the production of PGs, enzymes and inflammatory mediators associated with the AA \npathway, using menstrual fluid and peripheric blood samples from women submitted to TLJ or non-steroidal \nanti-inflammatory drugs (NSAID); or without any intervention. The results showed that the concentration of \nPGE2 in the menstrual fluid of the volunteers from the Control and of the TLJ treated groups remained prac-\ntically equivalent (Table 2). However, in the group treated with Meloxicam, the PGE2 concentration was lower \nthan in the other groups, although the volunteers did not present any type of menstrual disorder (Table  2). In \ncontrast, the expressive increase in PGF2α levels was found in the menstrual serum of the women from the TLJ in \ncomparison with the participants from the Control or Meloxicam groups (Fig. 5). These results were corroborated \nby another work of our group, whose objective was to evaluate a methodology based on SPE-LC-MS/MS for the \nsimultaneous quantification of prostaglandins (PGE2 and PGF2α ) from the menstrual fluid. The experiments \nwere carried out using independent samples in relation to present study: group Control (n = 15) and a group of \nwomen exposed to TLJ (n = 7). The PGF2α levels were also found to be higher in the treated group, reinforcing \nan effect found previously of the intake of TLJ\n47.\nFigure 4. Demonstration of the contraction of collagen gel discs populated by L929 or C2C12 induced by \ndifferent concentrations of TLJ. (A) Bar graphic comparing the mean area from collagen discs populated by the \nL929 cell line treated with different concentrations of buffered TLJ (0, 0.5, 1 or 2%) at 5 days in relation to the \npositive Controls (10 µM of cloprostenol or 10% of FBS) and negative Control (media without FBS and TLJ: \n0%). (B) Image of the discs showing the intensity of contraction of the diverse groups after 5 days of treatment. \n(C) Bar graphic comparing the mean area from collagen discs populated by the C2C12 cell line treated with \ndifferent concentration of buffered TLJ (0, 0.5, 1 or 2%) at 2 days in relation to the positive Controls (10 µM of \ncloprostenol or 10% of FBS) and negative Control (media without FBS and TLJ: 0%). (B) Image of the discs \nshowing the intensity of contraction of the diverse groups after 2 days of treatment. Quantitative data were \nexpressed as the mean area (mm\n2) ± SEM (n = 3). ***p < 0.001 vs. the negative Control group; Statistical \nanalysis was performed using one-way ANOV A and the Dunnett’s post-hoc test.\n\n8Scientific  RepoRtS  |         (2020) 10:7063  | https://doi.org/10.1038/s41598-020-63477-8\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nRegarding the regression analysis, the results of the relationship between the PGF2α  and PGE2 (Fig. 6A,B) \nrevealed that the slope of the best adjust curve was more accentuated in the TLJ than the other two groups. This \nresult is mainly related to the increased production of PGF2α in comparison with the PGE2 in the TLJ group. On \nthe other hand, the individual concentrations of PGF2α and PGE2 in the Meloxicam group were generally lower \nthan in the other two groups which are in accordance with the literature\n48. The slope of the PGF2α/PGE2 curve \nof the Meloxicam group followed the control pattern.\nThe in vitro and in vivo data indicated that TLJ may exert a regulation of menstrual flow, inducing an increase \nof uterine PGF2α and maintaining the PGE2 level unchanged. PGF2α produced by citrus therapy may increase \nthe capillary resistance acting on prostaglandins receptors 49, reducing the menstrual flow. Studies showed \nthat naringin, rutin, eriocitrin and hesperidin, four of the found compounds of TLJ were unable to inhibit the \nFigure 5. Demonstration of the augmentation of concentration of PGF2α in the menstrual fluid of women \nthat consumed TLJ at the beginning of the menstrual phase. Scatter Plot showing the individual values and \nthe median of the F2α and E2 Prostaglandins concentrations (ng.mL\n−1 ) present in the menstrual serum from \nthe Control (n = 13), Meloxicam (n = 15) and TLJ volunteers (n = 16), collected on the second day after the \nbeginning of the menstruation cycle. The PGE2 was analyzed by the Kruskal-Wallis and Dunn’s statistical tests \nfor multiple comparisons and the data from PGF2α was log transformed and analyzed by ANOV A and the \nNewman-Keuls statistic test for multiple comparisons.\nPG GROUP N MEAN SEM MEDIAN P25 P75\nMenstrual Fluid\nPGE2 (ng.mL−1 )\nTLJ (a) 16 328.3 43.6 316.3 199.1 438.3\nMeloxicam (b) 15 146.3 48.9 71.8 44.9 109.5\nControl (a) 13 369.4 80.6 310.2 133.3 561.7\nPGF2α (ng.mL −1 )\nTLJ (a) 16 3165.1 426.9 2953.1 2057.0 3789.3\nMeloxicam(b) 15 1402.3 213.3 1131.2 801.8 1827.2\nControl (c) 13 1955.1 194.8 1806.3 1474.5 2315.0\nPeripheral Blood PGF2α (ng.mL −1 )\nTLJ (a) 16 3.37 1.05 2.44 1.34 3.32\nMeloxicam (b) 15 1.86 0.37 1.32 0.87 2.59\nControl (a) 13 5.25 1.21 3.16 1.42 10.65\nTable 2. Comparison of the concentrations of the prostaglandins E2 and F2α present in the menstrual fluid and \nthe peripheral serum from volunteers from the TLJ, Meloxicam and Control groups. Legend: SEM - Standard \nError Mean, P25 - 25% percentil, P75 - 75% percentile, The letters (a), (b) and (c) represent groups considered \nstatistically different. Statistical tests: Kruskal-Wallis and Dunn for multiple comparisons. PGE2 values from all \nperipheral blood samples were below the sensibility of the ELISA kit.\n\n9Scientific  RepoRtS  |         (2020) 10:7063  | https://doi.org/10.1038/s41598-020-63477-8\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nPGE2 production induced by the LPS in vitro50. This effect could benefit women suffering from menstrual disor-\nders associated with lower production of PGF2α  in a superior manner in comparison with the treatment based on \nthe use of NSAIDs. Anti-inflammatory drugs concomitantly inhibited PGE2 (menstrual) and PGF2α (peripheral \nand menstrual) production, which could lead to an increase in the incidence of side effects, since PGE2 has a \nsignificant physiological role in the uterus and other organs51.\nAnalyzing the endometrial concentration of PGs from normal women or those with menorrhagia, Smith et \nal. (1981) proposed that excessive blood loss may be related to the possible conversion of PGF2α  to PGE26,52. \nThe exact mechanism leading to increased production of PGF2α by TLJ remains unknown. In the present study, \ntreatment with TLJ did not significantly affect the concentration of AKR1B1 and AKR1C3 (serological or men-\nstrual) in comparison with the Control group. The Meloxicam-treated group experienced a significant decrease \nin menstrual AK1C3. As a result, the increase in the slope of the PGF2α /PGE2 curve in the menstrual blood of \nthe TLJ-treated volunteers is likely to be unrelated to a change in the concentration of the AKR1B1 and AKR1C3 \nenzymes.\nDuring inflammatory events, there is an increase in the production of cytokines, mainly TNF- α  and IL-1β, \nwhich have important functions in maintaining the pro-inflammatory uterine profile. The IL-1β  cytokine can \nstimulate the  NF−κβ transcription factor, increasing the expression of genes related to COX-1 and COX-2. In a \npro-inflammatory situation, as occurs in the menstrual process, we could expect greater production of COX-2, \nwhich would potentiate the production of PGs. Several studies have shown that flavonoids modulate AA metab-\nolism through the inhibition of COX-2, PLA2 and nitric oxide, producing enzymes (iNOS), which consequently \ninhibit the synthesis of eicosanoids and prostaglandins\n53.\nMonitoring of NF−κβ activation after TLJ treatment could help to a better understanding of the mechanism \nof action of the citrus. In our case, the data showed that the exposition to TLJ did not contribute to inhibit or \nstimulate the NF−κβ  signaling (Fig.  3E). We speculated that the direct modulation of NF−κβ /COX2 activity \nmay not be the main mechanism responsible for the control of menstrual dysfunction mediated by TLJ. The \nresults of the present study showed that the concentration of PGE2 in the menstrual blood did not change to the \nControl group. The concentrations of IL-1β and IL-6 did not differ significantly among the three groups. Only \nthe menstrual TNF-α concentration showed a decrease in the TLJ and Meloxicam groups in comparison with the \nControl group (Fig. 7). The in vitro experiments with NF−κβ reporter showed the TLJ treatment was not capable \nto inhibit or stimulated the transcription of the reporter gene. Besides, if inhibition of NF-kβ/COX-2 activity had \noccurred, as reported in the in vitro studies, likely lower values of the in vitro (PGF2α) and in vivo (PGF2α, IL-1β, \nand IL-6) pro-inflammatory mediators analyzed would have been observed in the TLJ group. However, specific \nbiochemical studies are needed to assess the effects of the treatment with TLJ in the arachidonic acid pathway.\nUnlike most studies that use the epicarp to obtain the essential oils or pure flavonoids in high concentra-\ntions, we used juice extracted from the endocarp as a therapeutic component. This specificity may explain the \ndifferences in the results related to the inhibition of COX and pro-inflammatory cytokines found in vitro studies. \nThe composition of edible and inedible parts of citrus are different and the flavonoids present in the edible part \nare more beneficial than those in the peel\n39. The literature shows that the chemical structure of the flavonoidsis \nFigure 6. Demonstration of the augmentation of concentration of PGF2α without alteration of PGE2 in \nthe menstrual fluid of women that consumed TLJ at the beginning of the menstrual phase in contrast with \nthose were treated with Meloxicam. (A) Best fitting curve (PGF2α vs PGE2) drawn from PGs concentrations \nquantified from menstrual fluid collected on the second day after the beginning of the menstrual cycle, from \nthe Control (n = 13), Meloxicam (n = 15) and TLJ (n = 16) volunteer groups. (B) Scatter graphic showing the \nindividual values and medians of the ratio (PGF2α/PGE2) in relation to change fold of PGF2α of the Control, \nMeloxicam and TLJ groups. The medians were compared using the Kruskal-Wallis and Dunn´s statistical tests \nfor multiple comparisons.\n\n10Scientific  RepoRtS  |         (2020) 10:7063  | https://doi.org/10.1038/s41598-020-63477-8\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nassociated with the capacity of COX2 activity inhibition. A study showed that the 50 µM of hesperidin (a major \ncompound of TLJ), in contrast with other types of flavonoids absent in the TLJ (quercetin, luteolin, disometin, \ngenistein..), does not change the expression of COX2 and inhibits the Iκ B-α  phosphorylation and NF-κβ p50 \nand p65 nuclear translocation stimulated by LPS54. Eriocitrin, another abundant flavonoid of TLJ, in concentra-\ntion of 100 µM also showed a lower capacity of COX2 inhibition in contrast with the other group of flavonoids \noften absent in TLJ55.\nBesides, the inhibitory activity of flavonoids is dose-dependent. LPS-induced gene expression of COX-2 was \ninhibited by hesperidin at a concentration superior to 250 µM56. Studies in animals often use a concentration of \nhesperidin around 100 mg/kg/day57. In a human clinical trial, volunteers consumed two capsules of 146 mg of \nhesperidin daily (~50 µM of serum level, considering the blood volume of 5 L) for 4 weeks and the leukocyte \ncytokine production after PHA stimulation was analyzed. Results showed that hesperidin consumptions do not \ninduce immunomodulation of basal immune cell functions and their activation capacities\n58. In our case, the vol-\nunteers consumed 1 lime (40 ml of TLJ)/ day, which contains ~20 mg of hesperidin (http://phenol-explorer.eu/\ncontents/polyphenol/207).\nAt this point, we can show the intake of the lime juice induces the production of the PGF2α uterine, which is \nprobably responsible for decreasing of the menstrual bleeding. In the TLJ exist compounds of unknown classes, \nFigure 7. Reduction of the menstrual level of TNF-α from women that consumed TLJ at beginning of the \nmenstruation. Box-plot showing the comparing of the levels of TNF-α, IL-6, IL-1β and of the enzymes AKR1B1 \nand AKR1C3 from menstrual fluid or peripheral serum of women submitted or not (n = 14) to TLJ (n = 16) \nor meloxicam (n = 16). The medians were compared using the Kruskal-Wallis and Dunn´s statistical tests for \nmultiple comparisons.\n\n11Scientific  RepoRtS  |         (2020) 10:7063  | https://doi.org/10.1038/s41598-020-63477-8\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nthat individually or in combination can stimulate in vitro and in vivo the cascade of the arachidonic acid and \nother pro-inflammatory pathways. In the pilot study, the results showed that in general, volunteers with increased \nmenstrual flow who underwent citrus therapy benefited from the treatment. Some of the volunteers exhibited \na reduction in just days (23%), bleeding (72%) and clot reduction (43%) while some had complete remission of \ndysmenorrhea (21%)\n30. The modulation of the prostaglandin production provided by citrus-therapy, probably \ninduced a reduction of the vessel caliber and menstrual flow, without potentiating the uterine contractions related \nto painful symptomatology. It may be that the increase in PGF2α was not enough to increase contractions of the \nmyometrium and intensify cramps during menstruation. Moreover, it is likely the decrease in cytokines produc-\ntion, such as TNF-α, has contributed with the inhibition of the inflammatory process and nociceptive activities. \nCorroborating with the findings of the present study, other authors showed that the treatment with Rosemary \n(Rosmarinus officials L.), a medicinal plant involved in modulation of prostaglandins from the Lamiaceae family, \nalso reduces the amount of menstrual bleeding and dysmenorrhea similar to mefenamic acid\n59. Hesperidin and \neriocitrin (eriodictyol), present in the TLJ, were found between the 57 (poly) phenolic profiles identified in the \nRosemary\n60.\nBased on the results found in vitro  and in vivo, we propose that the components present in the Tahiti lime \nendocarp facilitated action in the first route of PG synthesis, since the increase of PGF2α  in the menstrual flow \nFigure 8. Pathways responsible for producing E2 and F2α prostaglandins. PGF2α and PGE2 are produced \nfrom arachidonic acid (AA) by cyclooxygenase enzymes (COX-1 and COX-2). In the first step, AA is \nmetabolized in PGG2 intermediate by cyclooxygenase and, sequentially, in PGH2 by peroxidase. The PGH2 \ncan be synthesized directly into PGF2α by the AKR1B1 or AKR1C3 enzymes. Both AKRs are present in the \nendometrium during the menstrual cycle: AKR1B1 is in the glandular epithelium and stromal cells, and \nAKR1C3 is situated in the epithelial cells. PGH2 can also be converted into 9α,11β-PGF2α by the AKR1C3 and \nto a lesser extent by the AKR1C1 and AKR1C2 enzymes. The 9α,11β-PGF2α is a stereoisomer of PGF2α and \nhas the same potency in the contraction of smooth muscle fibers. Both the AKRs and F2α prostaglandins \n(PGF2α and 9β, 11β-PGF2α) act on the transcription of the NF−κβ factor, inducing the production of COX-2. \nFurthermore, the AKR1B1 and AKR1C3 activities in the endometrium can be stimulated by pro-inflammatory \ncytokines, which can lead to a vicious inflammatory cycle. Finally, PGH2 can be metabolized into PGE2 by \nthe PGE synthases (PGES, PGES2 or PGES3) followed by conversion into PGF2α by PGE2,9-ketoreductase \n(AKR1C1 and AKR1C2)\n35–38.\n\n12Scientific  RepoRtS  |         (2020) 10:7063  | https://doi.org/10.1038/s41598-020-63477-8\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\ndoes not seem to be accompanied by alterations in the PGE2 concentration. The effects of citrus therapy shown \nby our results were restricted to the menstrual fluid and did not have any systemic repercussion. During men-\nstruation, the desquamation process produces a high level of inflammatory mediators, and It is unlikely that the \nTLJ could  potentialize a rise. We can speculate that the TLJ action is more related to modulation of the PGF2α \nmetabolization than its own production.\nConclusion\nGlycosylated flavonoids present in the Tahiti lime juice appear to act in the PGF2α uterine pathway, favoring the \ncontrol of menstruation mediate by a muscular contraction.\nMethods\nCharacterization molecular of Tahiti lime juice.  Chemicals and reagents. All organic solvents were \nHPLC grade and purchased from Mallinckrodt Baker (St. Louis, MO, USA). The water used for the mobile phase \npreparation was purified by a Milli-Q purification system (Millipore, São Paulo, Brazil). The formic acid (≥95%) \nwas purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade. Flavonoid \nand organic acids standards, hesperidin (hesperetin 7-O-rutinoside), eriocitrin (eriodictyol 7-O-rutinoside), \nnarirutin (naringenin 7-O-rutinoside), naringin (naringenin 7-O-neohesperidoside), rutin (quercetin \n3-O-rutinoside), naringenin (4′,5,7-trihydroxyflavanone), quercetin (3,5,7,3′, 4′-pentahydroxyflavone), caffeic \nacid (3,4-dihydroxycinnamic acid) and gallic acid (3,4,5-trihydroxybenzoic acid) (≥97.0%) were all HPLC grade \nand purchased from Sigma-Aldrich. The standard and stock solutions were stored at −20 °C during the analyses. \nThe mobile phases were prepared in a volume/volume ratio.\nLC–MS system. The ultra-high performance liquid chromatographic (UHPLC) system (Shimadzu Kyoto, \nJapan) consists of two LC-30AD quaternary pumps, both equipped with a low-pressure gradient unit, a SIL-30AC \nself-injector with a 500 μL sampling loop, two DGU-20ASR degassers, a CTO-20AC oven, and a CBM 20A con-\ntroller; coupled to a DAD detector (SPD-M30A) and a QqToF Impact HD mass spectrometer (BrukerDaltonics \nGmbH, Germany) equipped with an electrospray ionization (ESI) source. Total and production (auto-MS/MS) \nexperiments were carried out in the two ionization modes: positive and negative, within a mass range of m/z 80 \nto 1300. The ionization condition sources were nebulizer 1.7 bar, drying gas flow 9.3 L.min\n− 1, drying temperature \n200 °C, end plate and capillary voltage of 500 V and 4500 V , respectively. The analyses were performed in reverse \nelution mode using the Waters XSelect HSST3 column (3.5 μm, 2.1 × 100 mm).\nSample preparation: Tahiti lime juice and standards solutions. The fruits were purchased from \nlocal retail outlets and washed. The juice of five limes was hand-squeezed and mixed. It was then centrifuged \nfor 10 min at 8000 g and 20 °C. The supernatant was submitted to off-line Solid Phase Extraction (SPE) using \nOASIS-HLB cartridges. Cleanup was performed with H\n2O (0.1% formic acid) and the analytes were eluted with \nACN:MeOH (50:50 v/v) (organic fraction). The organic fraction was evaporated in a Speedvac, 40 °C, for 12 hours. \nSubsequently, 15 mg of the organic fraction was re-suspended in 1 mL of ACN:H2O (5:95 v/v, 0.1% formic acid) \nand injected into the chromatographic system (15 mg.mL−1 ). The standard solutions were prepared in ACN:H2O \n(5:95 v/v, 0.1% formic acid) (1 μg.mL−1 ).\nIn vitro effect of tahiti lime juice on the production of prostaglandins and in the cellular con-\ntraction. Cells culture. Immortalized mouse fibroblast (L929) and myoblast (C2C12) cells were cultured \nat 37 °C and 5% CO2 in DMEM-high glicose medium supplemented with L-glutamin 2 mM, 10% FBS (Cultilab, \nSão Paulo, Brazil) and 1% antibiotics Pen/Strep 10,000 U (Vitrocell-Embriolife, Campinas, SP , Brazil). Adherent \ncells were harvested with 0.25% trypsin (Thermo Scientific) and viable cells were determined by the trypan blue \nexclusion method. The experiments were only carried out with 90% of cell viability.\nPreparation of TLJ for cell culture treatment. Tahiti lime fruit was washed, cut, squeezed, centrifuged \nat 1000 G and filtered using a sterile syringe filter with a 0.22  µm pore size (MillexSyringer-driven Filter PVDF \n– Millipore, Cork, Ireland). Subsequently, the filtered juice was diluted with sterile deionized H 2O at 10% con-\ncentration and the pH of the solution was adjusted using NaOH (1 M). The buffered citrus solution was used to \nproduce completed DMEM media with the lime juice at final work concentrations of 1.0 and 2%.\nEffect of Tahiti lime juice on viability of C2C12 analyzed by resazurin. The viability of the C2C12 \ncells treated for 5 and 24 h with 1 or 2% of TLJ were determined by Resazurin assay as described previously 61. \nC2C12 at concentrations of 1 ×  105 cells.ml − 1 was seeded in a 96-well (Corning Incorporated, NY , USA) and \nincubated in quadruplicate. At the end of the exposures periods and after washing of wells with PBS, 200 ul of \nDMEM culture media containing Resazurin (70 µM final concentration – Sigma-Aldrich, cat. R7017) was added \nto each well. The plates were gently shaken and incubated for 4  h at 37 °C in 5% CO\n2 atmosphere. By moni-\ntoring the absorbance at 570 nm and 600 nm using the UVM340 monochromator, the relative viability of the \ngroups was determined using as reference, the cells incubated only with media or DMEM with resazurin without \nmetabolization.\nApoptotic Effects of TLJ on C2C12 myoblast cell line.  For analysis of apoptosis, C2C12 cells (1 × \n106 cells.mL− 1) were cultivated in triplicate in 6 wells plate (Corning Incorporated, NY , USA). After 5 h and 24 h \nof treatment with 1 or 2% of TLJ, detached and adherent cells, harvested with ethylenediaminetetra-acetic acid \n(EDTA, Sigma-Aldrich), were collected in the same tube, washed with PBS and centrifuged at 900 G for 5 min. \nAfter discarding the supernatant, the pelleted cells were re-suspended in 400 µL binding buffer. An aliquot of sus-\npension (200 ul) was transferred to tubes and stained with 2.5 µL of Annexin V and 5ul of propidium iodide (PI) \n\n13Scientific  RepoRtS  |         (2020) 10:7063  | https://doi.org/10.1038/s41598-020-63477-8\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nsolutions (APOAF , Sigma-Aldrich, São Paulo, Brazil). The tubes were incubated for 10 min at room temperature \nin the dark and the cell suspensions were featured by flow cytometry (BD Accuri™ C6 Plus Flow, San Jose, CA, \nUSA)62. The distribution of cells classified as viable (PI−/Annexin V−), early apoptosis (PI−/Annexin V+), late \napoptosis (PI+/Annexin V+) and necrosis (PI+/Annexin V− ) was performed by the Express 6 Plus software \n(De Novo, Glendale, CA, USA) using as controls, cells grown only in completed DMEM (control− ) or treated \nwith 11 µM of H2O2 (control+)62.\nEffect of TLJ on the production of PGF2α  by myoblasts co-stimulated  or not with LPS or ara-\nchidonic acid by myoblast.  The modulatory effect of TLJ on the production of PGF2α  was analyzed, \nstimulating C2C12 with LPS (Sigma: Escherichia coli O111:B4) or arachidonic acid (AA) (Sigma: A3611), using \nthe non-exposed cell as a control. Next, C2C12 cells, at a concentration of 10 5 cells.well− 1 (96 well-microplate \n- Corning Incorporated, NY , USA), were cultivated for 24 h in DMEM media supplemented with 10% of SFB \n(serum fetal bovine) and antibiotics (penicillin/streptomycin), using an incubator at 5% of CO2 at 37 °C. After \nthis period, the C2C12 were previously (30 min) treated with different concentrations of 1 or 2% of TLJ pre -\npared just before use and co-stimulated or not with LPS (10 ng.mL− 1) or AA (10 µM) at different times (2, 5 and \n24 h). PGF2α values from the supernatant of the C2C12 cultures were quantified using ADI-901-069 ELISA kit \n(EnzoLife Sciences, Farmingdale, NY , USA).\nEffect of TLJ on In vitro monitoring of NF-kB activation and inhibition. HEK293 cells expressing \npBIIx-luc63, were cultivated in 96-well Corning Costar ® plates at 5 × 105 cells.well− 1. After 24 h, the cells were \ntreated with TNF-α 10 ng.mL− 1 (Sigma), TJL (1% and 2%), TJL (1% and 2%) + TNF-α 10 ng.mL− 1, LPS 10 µg.mL− 1  \n(Sigma), in serum deprived DMEM without phenol red (Gibco, Carlsbad, CA, USA) by 12 h. Cells were lysed by \nDual-Glo Luciferase assay kit (Promega, Madison, WI, USA), transferred to white plate 96-well Corning Costar® \nplate and Firefly and Renilla luciferase activity measurement were obtained in a SpectraMax i3 luminometer \n(Molecular Devices, Sunnyvale, CA, USA).\nEffect of TLJ on collagen gel contraction mediated by L929 and C2C12 cell lines.  Tubes with \n400 μL of DMEM medium, 1 × 105 cells.mL− 1 (L929 or C2C12) and 200 μL of collagen solution (3 mg.mL− 1 in \n0.1% acetic acid) (ChemCruz, Dallas, Texas, USA: SC136157) were prepared. After the addition of collagen, 4 μL \nof 1 M NaOH was immediately used for activation of the polymerization. The volume of 500 μL of the mixture was \ntransferred to each well of a 24-well plate and the gel was left for 20 minutes at room temperature until its solidifi-\ncation. Next, 600 μL of DMEM medium without FBS was added to each well and the gel was dissociated from the \nwell by gently running the tip of a 200-μL pipet tip along the gel edges without shearing or tearing the gels. The \nplate was placed into an incubator at 37 °C and 5% CO2 overnight. After this time, the supernatant was removed \nand fresh DMEM medium with Citrus latifolia  juice was added to the culture at three different concentrations \n(0.5%, 1%, and 2%), in triplicate. Cells cultivated in medium with FBS were considered as a positive control, as \nwell as cells cultivated in medium with 10 μM of a synthetic PGF2α, Cloprostenol Sodium (Cioprostinn, Vetecia \nLab, Jacareí, SP , Brazil). Cells cultivated in the absence of FBS were considered as a negative control. The contrac-\ntion of gels with L929 and C2C12 cells were observed in several time-points (0 h, 12 h, 24 h, 48 h, 96 h, and 120 h) \nand the diameter changes of collagen disks were recorded using a digital camera at a fixed distance in order to \nobtain images at each time-point.\nIn Vivo: - effect of Tahiti lime juice on level of prostaglandins (E2 and F2α ), enzymes involved \nin the arachidonic acid pathway,and pro-inflammatory cytokines on menstrual fluid and \nperipheric blood samples during menstruation period. Selection of volunteer participants. Forty-five \nvolunteers aged 18 to 40 years from the city of São Carlos (São Paulo State, Brazil) were enrolled in the present \nstudy after agreeing and signing the informed consent form. The Research Ethics Committee (CEP) of the Federal \nUniversity of São Carlos (n° 48233715.8.0000.5504) specifically approved the present study. The inclusion and \nexclusion participation criteria were women that did not use any type of hormonal contraceptive, with no history \nof gynecological diseases, immunodeficiency, autoimmune diseases, malignant neoplasms or history of chemo-\ntherapy or radiotherapy.\nThe G1 group (TLJ group; n = 15) was instructed to ingest one dose of Tahiti Lime juice (~20–30 mL) diluted \nin water: at the beginning of bleeding (30 min before meals) and on the second day of menstruation in the morn-\ning. The G2 group (Meloxicam group; n = 15) received two tablets of 15 mg of meloxicam drug (Meloxicam\n®, \nNovamed - ProdutosFarmacêuticosLtda, Manaus, Amazonas, Brazil) and took the medication in the same man-\nner as the TLJ: at the beginning of bleeding and another on the second day of menstruation. The G3 group \n(Control group; n = 15) was instructed not to use any type of medication and not to consume any type of citrus \nfruits in the first two days of menstruation. Most of the prostaglandin release occurs during the first 48 h of the \nmenstruation, which corresponds to the period of greatest exacerbation of the symptoms\n64.\nRegistration number and name of the clinical trial. The clinical data from the present work was \ndeposed in a Clinical Trial Database and referred toInfluence of lemon on the inflammatory activity of healthy \nwomen during menstruation (number: ReBeC - RBR-3tknxy), registered in REBEC (http://www.ensaiosclinicos.\ngov.br/).\nPeripheral blood and menstrual fluid. Peripheral blood samples were collected using non-anticoagulant \nvacuum tubes (Vacutainer Biosciences, Franklin Lakes, NJ, USA). After being left to stand for 20 minutes, samples \nwere centrifuged at 1000 G for 10 minutes at 25 °C, and the supernatant was kept at − 80 °C until analysis. The \nmenstrual fluid was collected using a menstrual collector (Menstrual Lunette Collector, Kevosai, Curitiba, Brazil), \nwith each volunteer receiving a menstrual collector for individual use. Sampling was performed on the morning \n\n14Scientific  RepoRtS  |         (2020) 10:7063  | https://doi.org/10.1038/s41598-020-63477-8\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nof the second day of menstruation, and the samples were collected at the analysis lab. The samples were immedi-\nately centrifuged for 10 minutes (1000 G), at 25 °C, and the supernatant was stored at −80 °C until analysis.\nProstaglandins, enzymes involved inthe arachidonic acid pathway and pro-inflammatory \ncytokines in peripheral blood and menstrual fluid.  PGF2α  and PGE2 quantification in menstrual \nfluid, peripheral serum and supernatant were performed using KHL1731 and KHL1701 ELISA kits (Biosource, \nCaramillo, CA, USA) from samples diluted at 1:100 and 1:10, respectively. The pro-inflammatory cytokines: \nIL-1β, IL-6, TNF-α ; and the enzymes: AKR1B1 and AKR1C3 were measured using CHC1213, CHC1263 and \nCHC1753 kits (Invitrogen-Caramillo, CA, USA) and LS-F10794 and LS-F14889 kits (LifeSpanBioSciences, Inc. \nSeattle, W A, USA) from samples diluted at a proportion of 1:10, always following the manufacturer’s instructions. \nThe absorbance of the samples was detected using a monochromator (Asys UVM 340, Holliston, MA, USA) at a \nwavelength of 450 nm.\nStatistical analysis. Data were represented by the mean and standard error of the mean (SEM). In cases \nwhere the data followed a normal distribution, according to the Shapiro-Wilk’s test, differences between groups \nwere determined using the Anova and Newman-Keuls statistic tests for multiple comparisons. When the nor -\nmality could not be guaranteed, the data were analyzed by the Kruskal-Wallis non-parametric test accompanied \nby the Dunn´s test for multiple comparisons. For cases with two factors, Anova Two Way or Mann-Whitney U \ncorrected by the Bonferroni’s method were employed. The software used for analysis was the GraphPad Prism \nversion 5.00 for Windows (GraphPad Software, San Diego California USA). The tests were considered statistically \nsignificant when the p-value was less than 0.05.\nEthical approval. All procedures performed in this study involving human participants were in accordance \nwith the ethical standards of the institutional and/or national research committee (Comitê de Ética em Pesquis\na em Seres Humanos da Universidade Federal de São Carlos http://www.propq.ufscar.br/etica/descricao-cep-n ° \n48233715.8.0000.5504) and with the 1964 Helsinki declaration and its later amendments or comparable ethical \nstandards.\nReceived: 30 October 2019; Accepted: 31 March 2020;\nPublished: xx xx xxxx\nReferences\n 1. Schoep, M. E., Nieboer, T. E., van der Zanden, M., Braat, D. D. M. & Nap, A. W . The impact of menstrual symptoms on everyday life: \na survey among 42,879 women. Am. J. Obstet. Gynecol. 220, 569 e561–569e567, https://doi.org/10.1016/j.ajog.2019.02.048 (2019).\n 2. Estanislau do Amaral, M. C., Hardy, E., Hebling, E. M. & Faundes, A. Menstruation and amenorrhea: opinion of Brazilian women. \nContraception 72, 157–161, https://doi.org/10.1016/j.contraception.2005.02.013 (2005).\n 3. Lundstrom, V . & Green, K. Endogenous levels of prostaglandin F2alpha and its main metabolites in plasma and endometrium of \nnormal and dysmenorrheic women. Am. J. Obstet. Gynecol. 130, 640–646, https://doi.org/10.1016/0002-9378(78)90320-4 (1978).\n 4. Lumsden, M. A., Kelly, R. W . & Baird, D. T. Primary dysmenorrhoea: the importance of both prostaglandins E2 and F2 alpha. Br. J. \nObstet. Gynaecol. 90, 1135–1140, https://doi.org/10.1111/j.1471-0528.1983.tb06460.x (1983).\n 5. Smith, S. K., Abel, M. H., Kelly, R. W . & Baird, D. T. Prostaglandin synthesis in the endometrium of women with ovular dysfunctional \nuterine bleeding. Br. J. Obstet. Gynaecol. 88, 434–442, https://doi.org/10.1111/j.1471-0528.1981.tb01009.x (1981).\n 6. Kelly, R. W ., Lumsden, M. A., Abel, M. H. & Baird, D. T. The relationship between menstrual blood loss and prostaglandin \nproduction in the human: evidence for increased availability of arachidonic acid in women suffering from menorrhagia. \nProstaglandins Leukot. Med. 16, 69–78, https://doi.org/10.1016/0262-1746(84)90087-8 (1984).\n 7. Jabbour, H. N., Sales, K. J., Smith, O. P ., Battersby, S. & Boddy, S. C. Prostaglandin receptors are mediators of vascular function in \nendometrial pathologies. Mol. Cell Endocrinol. 252, 191–200, https://doi.org/10.1016/j.mce.2006.03.025 (2006).\n 8. Ito, S., Okuda-Ashitaka, E. & Minami, T. Central and peripheral roles of prostaglandins in pain and their interactions with novel \nneuropeptides nociceptin and nocistatin. Neurosci. Res. 41, 299–332, https://doi.org/10.1016/s0168-0102(01)00289-9 (2001).\n 9. Maigaard, S., Forman, A. & Andersson, K. E. Different responses to prostaglandin F2 alpha and E2 in human extra- and \nintramyometrial arteries. Prostaglandins 30, 599–607, https://doi.org/10.1016/0090-6980(85)90024-3 (1985).\n 10. Sugino, N., Karube-Harada, A., Taketani, T., Sakata, A. & Nakamura, Y . Withdrawal of ovarian steroids stimulates prostaglandin \nF2alpha production through nuclear factor-kappaB activation via oxygen radicals in human endometrial stromal cells: potential \nrelevance to menstruation. J. Reprod. Dev. 50, 215–225 (2004).\n 11. Sales, K. J. & Jabbour, H. N. Cyclooxygenase enzymes and prostaglandins in reproductive tract physiology and pathology. \nProstaglandins other lipid mediators 71, 97–117 (2003).\n 12. Hata, A. N. & Breyer, R. M. Pharmacology and signaling of prostaglandin receptors: multiple roles in inflammation and immune \nmodulation. Pharmacol. Ther. 103, 147–166, https://doi.org/10.1016/j.pharmthera.2004.06.003 (2004).\n 13. Benavente-García, O. & Castillo, J. Update on uses and properties of citrus flavonoids: new findings in anticancer, cardiovascular, \nand anti-inflammatory activity. J. Agric. food Chem. 56, 6185–6205, https://doi.org/10.1021/jf8006568 (2008).\n 14. Pérez-Cano, F . J. & Castell, M. Flavonoids, Inflammation and Immune System. Nutrients 8, 659, https://doi.org/10.3390/nu8100659 \n(2016).\n 15. Chen, X.-M., Tait, A. R. & Kitts, D. D. Flavonoid composition of orange peel and its association with antioxidant and anti-\ninflammatory activities. Food Chem. 218, 15–21, https://doi.org/10.1016/j.foodchem.2016.09.016 (2017).\n 16. Impellizzeri, D. et al. Anti-inflammatory and Antioxidant Effects of Flavonoid-Rich Fraction of Bergamot Juice (BJe) in a Mouse \nModel of Intestinal Ischemia/Reperfusion Injury. Front. pharmacology 7, 203, https://doi.org/10.3389/fphar.2016.00203 (2016).\n 17. Morwood, C. J. & Lappas, M. The citrus flavone nobiletin reduces pro-inflammatory and pro-labour mediators in fetal membranes \nand myometrium: implications for preterm birth. PLoS One 9, e108390 (2014).\n 18. Manthey, J. A., Grohmann, K. & Guthrie, N. Biological properties of citrus flavonoids pertaining to cancer and inflammation. Curr. \nMed. Chem. 8, 135–153, https://doi.org/10.2174/0929867013373723 (2001).\n 19. Gattuso, G., Barreca, D., Gargiulli, C., Leuzzi, U. & Caristi, C. Flavonoid composition of Citrus juices. Molecules 12, 1641–1673 \n(2007).\n 20. Lin, Y . et al. Molecular structures of citrus flavonoids determine their effects on lipid metabolism in HepG2 cells by primarily \nsuppressing apoB secretion. J. Agric. food Chem. 59, 4496–4503, https://doi.org/10.1021/jf1044475 (2011).\n 21. Ziyan, L., Y ongmei, Z., Nan, Z., Ning, T. & Baolin, L. Evaluation of the Anti-inflammatory Activity of Luteolin in Experimental \nAnimal Models. Planta Medica 73, 221–226, https://doi.org/10.1055/s-2007-967122 (2007).\n\n\n15Scientific  RepoRtS  |         (2020) 10:7063  | https://doi.org/10.1038/s41598-020-63477-8\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\n 22. Ren, H. et al. Hesperetin Suppresses Inflammatory Responses in Lipopolysaccharide-Induced RAW 264.7 Cells via the Inhibition of \nNF-kappaB and Activation of Nrf2/HO-1 Pathways. Inflammation 39, 964–973, https://doi.org/10.1007/s10753-016-0311-9 (2016).\n 23. Ali, R. et al. Amelioration of Benzo[a]pyrene-induced oxidative stress and pulmonary toxicity by Naringenin in Wistar rats: A \nplausible role of COX-2 and NF-kappaB. Hum. Exp. Toxicol. 36, 349–364, https://doi.org/10.1177/0960327116650009 (2017).\n 24. Choi, S. et al. Rutin inhibits B[a]PDE-induced cyclooxygenase-2 expression by targeting EGFR kinase activity. Biochem. Pharmacol. \n86, 1468–1475, https://doi.org/10.1016/j.bcp.2013.08.066 (2013).\n 25. Liu, Y . et al. Naringin attenuates acute lung injury in LPS-treated mice by inhibiting NF-kappaB pathway. Int. Immunopharmacol. \n11, 1606–1612, https://doi.org/10.1016/j.intimp.2011.05.022 (2011).\n 26. Zeng, W ., Jin, L., Zhang, F ., Zhang, C. & Liang, W . Naringenin as a potential immunomodulator in therapeutics. Pharmacol. Res. 135, \n122–126, https://doi.org/10.1016/j.phrs.2018.08.002 (2018).\n 27. Damon, M. et al. Effect of chronic treatment with a purified flavonoid fraction on inflammatory granuloma in the rat. Study of \nprostaglandin E2 and F2 alpha and thromboxane B2 release and histological changes. Arzneimittel-Forschung 37, 1149–1153 (1987).\n 28. Ho, S.-C. & Kuo, C.-T. Hesperidin, nobiletin, and tangeretin are collectively responsible for the anti-neuroinflammatory capacity of \ntangerine peel (Citri reticulatae pericarpium). Food Chem. toxicology: an. Int. J. published Br. Ind. Biol. Res. Assoc. 71, 176–182, \nhttps://doi.org/10.1016/j.fct.2014.06.014 (2014).\n 29. Braune, A. & Blaut, M. Bacterial species involved in the conversion of dietary flavonoids in the human gut. Gut Microbes 7, 216–234, \nhttps://doi.org/10.1080/19490976.2016.1158395 (2016).\n 30. Andrade PriscilaM, P . T. Y ., Belsito IzabellaPB, Silva CarolinaC, Caneira ÉrikaG, Santos MaynaraZ, Garcia GabrielaS, Oliveira \nLetíciaC, Farias Elias, Borra, Ricardo C. Citrus therapy controlling the menstrual flow. International Journal of Phytotherapy, 98-101 \n(2015).\n 31. Ferreres, F ., Llorach, R. & Gil-Izquierdo, A. Characterization of the interglycosidic linkage in di-, tri-, tetra- and pentaglycosylated \nflavonoids and differentiation of positional isomers by liquid chromatography/electrospray ionization tandem mass spectrometry. \nJ. Mass. Spectrom. 39, 312–321 (2004).\n 32. Cuyckens, F . & Claeys, M. Mass spectrometry in the structural analysis of flavonoids. J. Mass. Spectrom. 39, 1–15 (2004).\n 33. Demarque, D. P ., Crotti, A. E., Vessecchi, R., Lopes, J. L. & Lopes, N. P . Fragmentation reactions using electrospray ionization mass \nspectrometry: an important tool for the structural elucidation and characterization of synthetic and natural products. Nat. Prod. \nRep. 33, 432–455 (2016).\n 34. Hapangama, D. K. & Bulmer, J. N. Pathophysiology of heavy menstrual bleeding. Womens Health 12, 3–13, https://doi.org/10.2217/\nwhe.15.81 (2016).\n 35. Sinreih, M., Anko, M., Kene, N. H., Kocbek, V . & Rižner, T. L. Expression of AKR1B1, AKR1C3 and other genes of prostaglandin \nF2α  biosynthesis and action in ovarian endometriosis tissue and in model cell lines. Chemico-biological Interact. 234, 320–331, \nhttps://doi.org/10.1016/j.cbi.2014.11.009 (2015).\n 36. Hevir, N., Šinkovec, J. & Lanišnik Rižner, T. Decreased levels of AKR1B1 and AKR1B10 in cancerous endometrium compared to \nadjacent non-cancerous tissue. Chemico-Biological Interact. 202, 226–233, https://doi.org/10.1016/j.cbi.2012.11.001 (2013).\n 37. Rižner, T. L. Enzymes of the AKR1B and AKR1C Subfamilies and Uterine Diseases. Front. Pharmacology  3, 34, https://doi.\norg/10.3389/fphar.2012.00034 (2012).\n 38. Bresson, E. et al. The human aldose reductase AKR1B1 qualifies as the primary prostaglandin F synthase in the endometrium. J. \nClin. Endocrinol. Metab. 96, 210–219, https://doi.org/10.1210/jc.2010-1589 (2011).\n 39. Kawaii, S., Tomono, Y ., Katase, E., Ogawa, K. & Y ano, M. Quantitation of flavonoid constituents in citrus fruits. J. Agric. Food Chem. \n47, 3565–3571 (1999).\n 40. Saeidi, I. et al. HPLC determination of hesperidin, diosmin and eriocitrin in Iranian lime juice using polyamide as an adsorbent for \nsolid phase extraction. J. Pharm. Biomed. Anal. 56, 419–422 (2011).\n 41. Nogata, Y . et al. Flavonoid composition of fruit tissues of citrus species. Biosci. Biotechnol. Biochem. 70, 178–192 (2006).\n 42. Sakata, K., Hirose, Y ., Qiao, Z., Tanaka, T. & Mori, H. Inhibition of inducible isoforms of cyclooxygenase and nitric oxide synthase \nby flavonoid hesperidin in mouse macrophage cell line. Cancer Lett. 199, 139–145 (2003).\n 43. Parhiz, H., Roohbakhsh, A., Soltani, F ., Rezaee, R. & Iranshahi, M. Antioxidant and anti-inflammatory properties of the citrus \nflavonoids hesperidin and hesperetin: an updated review of their molecular mechanisms and experimental models. Phytother. Res. \n29, 323–331 (2015).\n 44. Lee, J. K. Anti-inflammatory effects of eriodictyol in lipopolysaccharide-stimulated raw 264.7 murine macrophages. Arch. Pharm. \nRes. 34, 671–679, https://doi.org/10.1007/s12272-011-0418-3 (2011).\n 45. Fikry, E. M., Hasan, W . A. & Mohamed, E. G. Rutin and meloxicam attenuate paw inflammation in mice: Affecting sorbitol \ndehydrogenase activity. J Biochem Mol Toxicol 32 (2018).\n 46. Lijnen, P ., Petrov, V . & Fagard, R. In vitro assay of collagen gel contraction by cardiac fibroblasts in serum-free conditions. Methods \nFind. Exp. Clin. pharmacology 23, 377–382 (2001).\n 47. Canzi, E. F . et al. Prostaglandins E2 and F2alpha levels in human menstrual fluid by online Solid Phase Extraction coupled to Liquid \nChromatography tandem Mass Spectrometry (SPE-LC-MS/MS). J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 1109, 60–66, \nhttps://doi.org/10.1016/j.jchromb.2019.01.025 (2019).\n 48. Ribeiro, M., Cella, M., Farina, M. & Franchi, A. Effects of Aminoguanidine and Cyclooxygenase Inhibitors on Nitric Oxide and \nProstaglandin Production, and Nitric Oxide Synthase and Cyclooxygenase Expression Induced by Lipopolysaccharide in the \nEstrogenized Rat Uterus. Neuroimmunomodulation 11, 191–198, https://doi.org/10.1159/000076768 (2004).\n 49. Ekström, P ., Alm, P . & Akerlund, M. Differences in vasomotor responses between main stem and smaller branches of the human \nuterine artery. Acta obstetricia et. gynecologica Scandinavica 70, 429–433 (1991).\n 50. Takano-Ishikawa, Y ., Goto, M. & Y amaki, K. Structure-activity relations of inhibitory effects of various flavonoids on \nlipopolysaccharide-induced prostaglandin E2 production in rat peritoneal macrophages: comparison between subclasses of \nflavonoids. Phytomedicine 13, 310–317, https://doi.org/10.1016/j.phymed.2005.01.016 (2006).\n 51. Patel, F . A. & Challis, J. R. Prostaglandins and uterine activity. Front. hormone Res. 27, 31–56 (2001).\n 52. Smith, S. K., Abel, M. H., Kelly, R. W . & Baird, D. T. Prostaglandin synthesis in the endometrium of women with ovular dysfunctional \nuterine bleeding. Br. J. Obstet. Gynaecol. 88, 434–442 (1981).\n 53. Nijveldt, R. J. et al. Flavonoids: a review of probable mechanisms of action and potential applications. Am. J. Clin. Nutr. 74, 418–425, \nhttps://doi.org/10.1093/ajcn/74.4.418 (2001).\n 54. Lopez-Posadas, R. et al. Flavonoids exert distinct modulatory actions on cyclooxygenase 2 and NF-kappaB in an intestinal epithelial \ncell line (IEC18). Br. J. Pharmacol. 160, 1714–1726, https://doi.org/10.1111/j.1476-5381.2010.00827.x (2010).\n 55. Ribeiro, D. et al. Flavonoids inhibit COX-1 and COX-2 enzymes and cytokine/chemokine production in human whole blood. \nInflammation 38, 858–870, https://doi.org/10.1007/s10753-014-9995-x (2015).\n 56. Hirata, A., Murakami, Y ., Shoji, M., Kadoma, Y . & Fujisawa, S. Kinetics of radical-scavenging activity of hesperetin and hesperidin \nand their inhibitory activity on COX-2 expression. Anticancer. Res. 25, 3367–3374 (2005).\n 57. Tejada, S. et al. Potential Anti-inflammatory Effects of Hesperidin from the Genus Citrus. Curr. Med. Chem. 25, 4929–4945, https://\ndoi.org/10.2174/0929867324666170718104412 (2018).\n 58. Perche, O. et al. Orange juice and its major polyphenol hesperidin consumption do not induce immunomodulation in healthy well-\nnourished humans. Clin. Nutr. 33, 130–135, https://doi.org/10.1016/j.clnu.2013.03.012 (2014).\n\n16Scientific  RepoRtS  |         (2020) 10:7063  | https://doi.org/10.1038/s41598-020-63477-8\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\n 59. Tahoonian-Golkhatmy, F ., Abedian, Z., Emami, S. A. & Esmaily, H. Comparison of Rosemary and Mefenamic Acid Capsules on \nMenstrual Bleeding and Primary Dysmenorrhea: A Clinical Trial. Iran. J. Nurs. Midwifery Res. 24, 301–305, https://doi.org/10.4103/\nijnmr.IJNMR_99_18 (2019).\n 60. Mena, P . et al. Phytochemical Profiling of Flavonoids, Phenolic Acids, Terpenoids, and Volatile Fraction of a Rosemary (Rosmarinus \nofficinalis L.) Extract. Molecules 21, https://doi.org/10.3390/molecules21111576 (2016).\n 61. Borra, R. C., Lotufo, M. A., Gagioti, S. M., Barros, Fd. M. & Andrade, P . M. A simple method to measure cell viability in proliferation \nand cytotoxicity assays. Braz. oral. Res. 23, 255–262 (2009).\n 62. Xu, H. et al. Low frequency pulsed electromagnetic field promotes C2C12 myoblasts proliferation via activation of MAPK/ERK \npathway. Biochemical Biophysical Res. Commun. 479, 97–102, https://doi.org/10.1016/j.bbrc.2016.09.044 (2016).\n 63. Kopp, E. et al. ECSIT is an evolutionarily conserved intermediate in the Toll/IL-1 signal transduction pathway. Genes. Dev. 13, \n2059–2071, https://doi.org/10.1101/gad.13.16.2059 (1999).\n 64. Kim, H. G., Jung, G. Y ., Park, S. B., Cho, Y . J. & Han, M. Assessment of the effects of prostaglandins on myometrial and leiomyoma \ncells in vitro through microRNA profiling. Mol. Med. Rep. 18, 2499–2505, https://doi.org/10.3892/mmr.2018.9160 (2018).\nAcknowledgements\nThis work was supported by the Fundação e Amparo à Pesquisa do Estado de São Paulo (FAPESP - www.\nfapesp.br/) [grant numbers: 2015/23679-4; 2015/23509-1; PROEM 2014/50244-6 and CEPID 2013/07296-\n2] and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES - www.capes.gov.br/) \n[Finance Code 001]. The funders had no role in study design, data collection and analysis, decision to publish, or \npreparation of the manuscript.\nAuthor contributions\nT.R., E.F .C., P .M.A.B., J.P .P .S., F .R.T., V .S., F .R.T., V .S., B.H.L.N.S.M., M.C., E.G.C., M.F .G.F .S., R.C.B. contributed \nequally to perform the required experiments, analyze the data and write the paper.\nCompeting interests\nThe authors declare no competing interests.\nAdditional information\nCorrespondence and requests for materials should be addressed to R.C.B.\nReprints and permissions information is available at www.nature.com/reprints.\nPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and \ninstitutional affiliations.\nOpen Access This article is licensed under a Creative Commons Attribution 4.0 International \nLicense, which permits use, sharing, adaptation, distribution and reproduction in any medium or \nformat, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-\native Commons license, and indicate if changes were made. The images or other third party material in this \narticle are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the \nmaterial. If material is not included in the article’s Creative Commons license and your intended use is not per-\nmitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the \ncopyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.\n \n© The Author(s) 2020","source_license":"CC0","license_restricted":false}