Study On The Activity And Mechanism Of Allicin Against Aspergillus

[Background]Aspergillus fumigatus is an opportunistic pathogenic fungus that is widely present in nature and causes pulmonary infections mainly by inhaled conidia.With the increase in immunocompromised patients in recent years,the incidence of pulmonary aspergillosis has risen to the first of all pulmonary fungal infections,with approximately 3.25 million cases of pulmonary aspergillosis annually worldwide,of which Aspergillus fumigatus infections account for approximately 90%of invasive pulmonary aspergillosis(IPA).As the current library of commonly used clinical anti-Aspergillus drugs is very limited,including only polyenes,azoles and echinocandins,and drug side effects and resistance problems have not been effectively solved,and in recent years,the emergence of new resistance mechanisms and multidrug-resistant Aspergillus spp.have greatly limited the clinical application of anti-Aspergillus drugs.Therefore,it is urgent to study new anti-Aspergillus drugs,reduce the morbidity and mortality rate of IPA patients and improve the quality of life of IPA patients.Natural products with biological activity are less susceptible to drug resistance and can be widely used.Studies have shown that common active ingredients of Chinese herbal medicines such as sodium ichthyate(SH),berberine(BER),jasmine(JAT),and cinnamaldehyde(CIN)have antifungal activity against Candida albicans and Cryptococcus.Garlic has been regarded as a natural antibiotic since ancient times,with abundant sources and low production costs.When garlic is crushed,the resulting alliin and alliase catalyze the reaction to produce allicin,which is one of the most important functional components of garlic.Studies have shown that allicin is effective in inhibiting the growth of bacteria and viruses,and its related components also have antifungal properties.However,there is a lack of studies on the activity of allicin against Aspergillus.Therefore,studying the activity and mechanism of action of the natural product allicin against Aspergillus fumigatus will help to reveal and supplement the material basis of garlic as a "natural antibiotic",improve the new understanding of Chinese herbal medicine as antifungal drugs,and also provide new key basis and reference for the secondary development and in-depth utilization of other herbal anti-fungal drugs and the development of anti-Aspergillus drugs.It also provides a new key basis and reference for the secondary development and in-depth utilization of other herbal anti-fungal drugs,as well as the research and development of anti-trichoderma drugs.[Object]1.To investigate the activity characteristics of allicin against Aspergillus fumigatus in vitro and in vivo;2.To explore the action mechanism of allicin against Aspergillus fumigatus.[Methods]1.In vitro study of allicin against Aspergillus fumigatus:Firstly,the purity of allicin was determined by liquid chromatography.Secondly,to determine the minimum inhibitory concentration(MIC)of allicin against Aspergillus fumigatus clinical isolates and standard strains to clarify its in vitro drug sensitivity,and to observe its antifungal effect dynamically by time-killing curve.2.In vivo study of allicin against Aspergillus fumigatus:6-8 week old Balb/c male mice were taken,with a weight control of about 20g,were given 150mg/kg/day cyclophosphamide intraperitoneally 4 days before,1 day before and 1 day after infection.After 5 days incubation of Aspergillus fumigatus,conidia of 1 × 107 cfu/ml were harvested,and 50 μl was taken for intranasal infection,and drug intervention was performed 2 hours after infection.Voriconazole(30 mg/kg/day)and allicin(1.5 mg/kg/day,3 mg/kg/day,6 mg/kg/day)were administered orally for 5 consecutive days as the positive control group and the experimental group,respectively.The protective effect of allicin on IPA mice in vivo was clarified by measuring mouse survival rate,lung index,pulmonary fungal load and lung pathology.3.Preliminary exploration of the mechanism of action of allicin against Aspergillus fumigatus:First,the XTT method was used to confirm that the antifungal activity of allicin against Aspergillus fumigatus spores was stronger than that of mycelium.And the effect of allicin on spore germination state at different time points of spore development was detected by fluorescence microscopy and scanning electron microscopy and transmission electron microscopy.It is clear that allicin exerted its antifungal activity in vitro and in vivo by inhibiting the germination of Aspergillus fumigatus spores into mycelium.[Results]1.In vitro activity of allcin against Aspergillus fumigatus:① The purity of allicin produced by reaction of allicin and allicin was 97.5%.②The MIC of allicin against the standard strain of Aspergillus fumigatus was 2μg/mL,while the MIC of allicin and allicin were both greater than 32μg/mL.Allicin showed effective activity against 35 clinical isolates of Aspergillus fumigatus with MICs of 2-16μg/mL.The MICs activity of amphotericin B and VRC insensitive aspergillus fumigatus was 2-16μg/mL and 4-16μg/mL,respectively.③The time sterilization curve showed that allicin had a dose-dependent effect on Aspergillus fumigatus,and 0.5MIC-4MIC of allicin had an inhibition of Aspergillus fumigatus,while 8MIC of allicin had a fungicidal effect.2.In vivo activity of alliin against Aspergillus fumigatus:①Construction of immunosuppression model:after immunosuppression with cyclophosphamide given at-4d,-1d and+3d,mice showed persistent decrease in peripheral blood leukocyte,neutrophil and lymphocyte counts,which were significantly lower than those in the immune normal group.②The mortality rate of the model group was 83.3%in 14 days,and the MST was 4 days.VRC treatment group can significantly reduce mortality,which was 16.7%,and the MST was greater than 14 days.The mortality rate in the 6 mg/kg allicin group was 33.3%,and the MST was greater than 14 days either.However,the mortality rates of the 3 mg/kg and 1.5 mg/kg allicin groups were both 50%,and the MST was 9.5 days and 11 days,respectively.③The lung index of the mice in the model group increased significantly,while the VRC and 6 mg/kg and the 1.5mg/kg allicin treatment group could significantly reduce the lung index of mice on day 2.On the 5th day of infection,the lung index of mice in the VRC and the 6mg/kg and 3mg/kg allicin treatment groups were significantly decreased.④ The fungal load of the lung tissue on the second day of infection was counted on the SDA plate,except for the 1.5mg/kg of allicin treatment,the pulmonary fungal load in the other drug treatment groups was significantly lower than that in the model group.On the 5th day of infection,only the VRC group and 6 mg/kg allicin could significantly reduce the pulmonary fungal load.⑤Pulmonary fungal load was counted by q-PCR,and was significantly reduced only in the VRC treatment group on day 2 of infection.On the 5th day,the VRC and 6 mg/kg of allicin treatment group could significantly reduce the pulmonary fungal load.⑥ Lung pathological of HE,PAS and GMS staining showed that the VRC group and 6 mg/kg allicin could significantly improve the lung Aspergillus fumigatus infiltration and lung pathological damage.The results suggest that allicin has a effective protection on IPA mice,and its anti-Aspergillus fumigatus activity in vivo is dose-dependent.⑦Lung pathological injury score showed only mild lung injury in each group on day 2 of infection,and extensive bleeding and necrosis,edema,and multifocal invasive hyphae on day 5 of infection,in which the VRC and 6mg/kg allicin group significantly reduced lung injury.3.Allicin inhibited spore germination:① The XTT assay showed that allicin had stronger anti-Aspergillus fumigatus spore activities than anti-mycelium.②After 6h by fluorescence microscope,a large number of spores adhered and aggregated in normal control group,and a few spores germinated,and spore adhesion and aggregation decreased significantly after 6h of 1MIC of allicin.While the 2 MIC group significantly inhibited spore adhesion and aggregation,most of which showed single spore state.After 14h,a large number of hyphae had been formed in the normal control group.Some spores of 1MIC allicin group had begun to develop into hyphae,while in the 2MIC,the spores were still adhered and clustered,and not yet hyphae had been formed.③It was found by scanning electron microscope that allicin could reduce the diameter of spores and inhibit the development of resting conidia into expansive conidia.After incubation for 14 hours,a large number of hyphae and a little biofilm were formed in the normal group,and only a few spores developed into hyphae in the 1MIC of allicin group,while most of the conidia in the 2MIC were adhered and aggregated spores.④As shown from transmission electron microscope,after 8h of A.fumigatus spores,the normal group spores had developed into expansion stage conidia,larger in diameter than spores developing 6h,and a few germinated,and nuclei,mitochondria,and vacuoles had matured.However,the 1MIC and 2MIC groups were still quiescent conidia after 8h,with greatly reduced diameter,and the organelles were not fully developed.[Conclusions]1.Allicin showed effective anti-aspergillus fumigatus activity in vitro and in mice in dose-dependent manner.2.Allicin exerts its anti-Aspergillus fumigatus activity in vitro and in vivo mainly by inhibiting spore germination of Aspergillus fumigatus.