Anti-trichomonas Vaginalis Activity In Vitro And Mechanism Of Essential Oil From The Seeds Of Anethum Graveolens L. And Its Main Components

Trichomoniasis is one of the most non-viral sexually transmitted diseases worldwide,and it is caused by Trichomonas vaginalis parasitic on human’s urogenital canal.Trichomoniasis may lead to some inflammatory reaction and problems such as painful urination,premature rupture of membranes,preterm labor,abortion and increased risk of human immunodeficiency virus(HIV)infection.Metronidazole is the commonly used clinical drug with a lot of side effects to human body.What’s worse is that the cure rate has deepened down gradually these years due to drug resistance.As a consequence,it is a very important way to find out new and highly effective drugs among traditional medical plant resources against Trichomonas vaginalis without evident side effects.Dill seeds,are the dry and ripe fruits from Anethum graveolens L.(Dill)which is a traditional Chinese herb.Dill seed essential oil(DSEO)have antioxidant,antimicrobial and other biological activity.In this study,we first proved that the DSEO and its main components carvone and limonene were active against Trichomonas vaginalis by in vitro tests.Then based on the in vitro results on the activity against Trichomonas vaginalis of the this three essential oils,the mechanism of anti-trichomonas effects were studied,which provides experimental bases for the therapies of clinical infectinon with Trichomonas vaginalis and the co-infection with Trichomonas vaginalis and other pathogenic bacteria.In this article,our main research contents and results are demonstrated from the perspectives of three parts following:1.The activities of the DSEO,carvone and limonene as well as the combination of two components against Trichomonas vaginalis in vitro were tested through three different methods including trypan blue,MTT and bromocresol purple assays.All the results showed the good activity in vitro against ATCC Trichomonas vaginalis PRA-98 of the three essential oils(EOs).In the qualitative test by Trypan blue staining,the MICs of DSEO,carvone and limonene were 0.5 μl/ml,1μl/ml and 0.25μl/ml.The IC50 of these three EOs is 0.19 μl/ml,0.367 μl/ml and 0.101 μl/ml by MTT,and 0.229 μl/ml,1.382 μl/ml,0.12 μl/ml by Bromocresol purple method.The combination of carvone and limonene against T.vaginalis shows no synergistic effect.2.Mechanism of three EOs on Trichomonas vaginalis was focused on the researchs of membrane,morphology and cell cycle.Effect of the DSEO,carvone and limonene on membrane of Trichomonas vaginalis was tested by pyridine iodine staining and LDH assays.The result showed that membrane of Trichomonas vaginalis were severly damaged by dill seed essential oil,carvone and limonene.The observation of the morphology of treatment groups under inverted microscope illustrated that the shape of Trichomonas vaginalis turned round,the movements slowed down or even stopped,the contents of cells flew out,and finally the treatment lead to the decomposition of the parasites.The G2/M phase of the cell cycle was arrested by the EOs.3.The further study was aimed to investigate whether dill seed essential oil,carvone as well as limonene induced apoptosis in Trichomonas vaginalis.The JC-1 was applied to estimate the hydrogenosome membrane potential after giving three EOs.We observed that all DSEO,carvone and limonene could interfere the energy metabolism of Trichomonas vaginalis through reduce the hydrogenosome membrane potential.In addition,we tested the expore of phosphatidylserine at the outer layer of Trichomonas vaginalis cytoplasmic membrane by the Annexin V-FITC staining and flow cytometry.Then,we studied the nuclear fragmentation by DAPI staining and found nuclear fragmentation,chromatin condensation after giving the three EOs.The results above showed that cells treated with DSEO,carvone and limonene showed typical markers of apoptosis,such as phosphatidylserine exposure,nuclear fragmentation,chromatin condensation.In summary,T.vaginalis treated with DSEO and its main components carvone and limonene showed typical signs of apoptosis.To sum up,dill seed essential oil and its main components all have good effects against Trichomonas vaginalis,and they inhibited the growth of T.vaginalis by damaging the integrity of the cell membrane and entering the cells,leading to the abnormal morphology of the cells as well as arresting the G2/M phase of the cell cycle.Meanwhile,the three EOs induced apoptosis in Trichomonas vaginalis.All the studies above provide a significant foundation and basis for dill seed essential oil,carvone and limonene to the clinical therapy for Trichomonas vaginitis.