Antifungal Activity And Mechanisim Of Essential Oil From The Seeds Of Anethum Graveolens L.

The incidence of fungal infections (such as Candida spp. and Dermatophyte) has increased dramatically, causing high morbidity and mortality. The more the broad-spectrum antibiotics, corticosteroids, immunosuppressive agents are used by humans, the easlier they get fungal infections. Currently, with the large scale application of broad-spectrum antifungal agents and the introduction of protocols for antifungal prophylaxis in patients at risk, there has been a notable increase in drug resistance. The development of more effective antifungal therapies is therefore of paramount importance.Essential oil is one of secondary metabolites in plants. It is reported that essential oil has antibacterial insecticide, anti-inflammatory, antiviral, antioxidant, antitumor, antipyretic and analgesic, expectorant cough and other biological activity. A. graveolens (dill), one of species of Umbelliferae, is a traditional Chinese herb. Dill seed essential oil have antioxidant, antimicrobial and other biological activity.In this paper, the activities (in vitro and in vivo) and mechanism of the seed of Anethum graveolens L. against fungal were researched.1. The dill seed essential oil was obtained by steam distillation. The chemical composition of the DSEO was analyzed by gas chromatography-mass spectral (GC-MS) spectroscopy. Twenty-three compounds, constituting about99.4%of the total essential oil, were identified. The most abundant oil components are carvone (41.5%), limonene (32.6%), apiole (16.8%).We study the storage of dill seeds at different time on the yield and main composition of DSEO. Besides, antifungal activity of DSEO on Candida spp. and dermatophytes when dill seeds storage different time. The results showed that the yield of DSEO was reduced. The yield of DSEO was reduced39.02%after storage one year. The storage time of dill seeds had little effect on the content of carvone and limonene.2. The microbroth dilution method was used in the minimal inhibitory concentration (MIC), according to M38-A2of the guidelines of the Clinical and Laboratory Standard Institute (CLSI). The effect of DSEO on hyphal growth of dermatophyte by agar dilution method. And then, we study DSEO on spore germination and biomass of dermatophyte. The results showed that DSEO was active in vitro against all tested strains, with MICs ranging from0.156-0.312μL/mL for dermatophyte. All tested stains were completely inhibited by DSEO at1.25μL/mL. The spore germination of dermatophyte were inhibited, and the biomass of dermatophyte were reduced on dose-dependent by DSEO.The further study the effect of DSEO on ergosterol content, and the activity of the Malate Dehydrogenase (MDH), Succinate Dehydrogenase (SDH), T-ATPase of T. rubrum ATCC40051. The results showed that ergosterol biosynthesis were inhibited and the activities of MDH, SDH andT-ATPase of T. rubrum ATCC40051were reduced by DSEO. It means that DSEO antidermatophytic through mitochondrial and membrance dysfunction3. The microbroth dilution method was used in the minimal inhibitory concentration (MIC), according to M27-A3of the guidelines of the Clinical and Laboratory Standard Institute (CLSI). And then, Growth curves and germ tube formation of Candida spp. inhibited by DSEO. The results showed essential oil was active in vitro against all tested strains, with MICs ranging from0.312-0.625μL/mL for Candida spp. All tested stains were completely inhibited bydill seed essential oil at1.25-2.5μL/mL.The germ tube formation rate were3.33%,2.33%,1.67%,1.33%for C. albicans09-1522,C. krusei09-1681,C. tropicalis032,C. parpapsilosis07-305, respectively, while the dill seed essential oil concentration at0.625μL/mL, it means that the germ tube formation were inhibited by dill seed essential oil. Further,The biofilm metabolic activity of Candida spp. were measured by XTT reduction assay.The result showed that dill seed essential oil severly inhibited Candida spp.strains from forming biofilms at concentrations>0.312μL/mL. Candida spp. strains biofilms treated with dill seed essential oil showed a significant reduction in metabolic activity when viability was measured by XTT reduction assay.The susceptibility of fungal biofilms to dill seed essential oil was investigated and compared with that of planktonic yeast cells. The result showed Candida spp. biofilms were significantly more resistant to dill seed essential oil than their planktonic counterparts. 4. Efficacy evaluation of essential oil in the prophylaxis and treatment of experimental vaginal candidiasis was performed in immunosuppressed mice. The anti-Candida activity was analyzed by microbiological and histological techniques and was compared with that of fluconazole (FCZ). Essential oil (2%v/v) was highly efficacious in accelerating C. albicans09-1555clearance from experimentally infected mice vagina by prophylaxis and therapeutic treatments. In both therapeutic efficacy and prophylaxis studies, the histological findings confirmed the microbiological results. The experimental results revealed that the tested essential oil is effective against vulvovaginal candidiasis in immunosuppressed mice.5. Effect of dill seed essential oil on membrane of Candida albicans by pyridine iodine staining and UVspectrophotometry. The result showed that membrane of Candida albican were severly damaged by dill seed essential oil. We assayed the mitochondrial membrane potential (mtAy) using rhodamine123and detected the antivities of mitochondrial dehydrogenases by spectrophotometry. Besides, the mitochondrial dysfunction-induced reactive oxygen species (ROS) production was determined by a fluorometric assay, and the effects of antioxidant L-cysteine on DSEO-induced ROS production and the antifungal effect of DSEO on C.albicans were also investigated. The results showed that exposure to DSEO resulted in an elevation of (mtΔΨ) and inhibition of the mitochondrial dehydrogenases. The dysfuntions of mitochondria caused ROS accumulation in C.albicans, and the increase in the level of ROS production and DESO-induced decrease in cell viability were prevented by addition of L-cysteine, indicating that ROS was an important mediator of the antifungal action of DSEO. In summary, DSEO exert its antifungal activity through mitochondrial dysfunction induced ROS accumulation in C.albicans.6. The ROS are a key regulator to yeast apoptosis. As a consequence, the further study was designed to investigate whether DSEO induced apoptosis in C.albicans.We observed the ultrastructure by transmission electron microscopy, studied the nuclear fragmentation by DAPI staining, and investigated the expore of phosphatidylserine at the outer layer of the cytoplasmic membrane by the FITC-Annexin V staining. Besides, the activity of metacaspase was detected by FITC-V AD-FMK staining, and the release of cytochrome C from mitochondria was also determined. Furthermore, the effect of antioxidant L-cysteine on DSEO-induced apoptosis in C.albicans was also investigated. The results showed that cells treated with DSEO showed typical markers of apoptosis, such as phosphatidylserine exposure, nuclear fragmentation, chromatin condensation. Besides, DSEO promoted the cytochrome C release and activated the metacaspase, which resulted in the yeast apoptosis. The addition of L-cysteine prevented DSEO-induced nuclear fragmentation, phosphatidylserine exposure, and metacaspase activation, indicating the ROS was an important mediator of DSEO-induced apoptosis. In summary, DSEO induced apoptosis in C.albicans through a metacaspase-dependent apoptotic pathway.