Study On The Antifungal Effect Of Macleaya Cordata Alkaloids On Cryptococcus Neoformans And The Preparation Process Of Liposomes

Cryptococcus neoformans is a conditional pathogenic fungus containing capsule,which can cause fatal cryptococcal meningitis.The problem of fungal resistance has become further serious due to the excessive use of broad-spectrum antibiotics and antifungal drugs.Thus,it is urgent to search for novel antifungal drugs.In recent years,traditional Chinese herbals has become the focus of all over the world owing to its wide-range of sources and rich ingredients.In this study,the anti-fungal and anti-biofilm activities of sanguinarine and chelerythrine against C.neoformans were explored against C.neoformans H99.Additionally,the potential molecular mechanism and the dosage forms of sanguinarine were examined.Our results will lay a theoretical foundation for people to develop antifungal drugs based on sanguinarine and chelerythrine,as well as provide the candidate lead compounds for the treatment of C.neoformans-asscociated infections.The main contents are divided as follows:The effective ingredients of traditional Chinese herbals posessing better anti-fungal effect were screened.The active components of selected three traditional Chinese herbels including Macleaya cordata,Fructus Mume and Hawthorn Fruit were extracted.The antifungal activity of crude extracts was further tested using the paper diffusion method,indicating that the antifungal activity of Macleaya cordata extracts was better than that of Fructus Mume and Hawthorn Fruit.Then,column chromatography,thin-layer chromatography and high-performance liquid chromatography were carried out to separate and identify target active ingredients,suggesting that sanguinarine and chelerythrine were the candidate antifungal ingredients.The antifungal and antibiofilm mechanism of the combination of sanguinarine and chelerythrine against C.neoformans was evaluated in this study.The minimum inhibitory concentration(MIC)of sanguinarine-chelerythrine were measured using the microdilution method;the effects of sanguinarine-chelerythrine on the metabolic activity and antibiotic uptake ability of C.neoformans were analyzed via the confocal laser confocal microscope(CLSM);the antibiofilm effect of sanguinarine-chelerythrine on C.neoformans was investigated by CLSM and field emission scanning electron microscopy(FESEM),including biofilm inhibition and dispersal,the effect on the content of biofilm matrix and the activity of cells within biofilm.These results showed that the MIC of sanguinarine-chelerythrine against C.neoformans was 16 μg/mL.The decrease of cell membrane permeability and cell metabolic activity were observed by CLSM,whereas the antibiotic uptake ability was enhanced.Moreover,FESEM observation showed that sanguinarine-chelerythrine at 1/2 MIC could sharply inhibit the biofilm formation.And sanguinarine-chelerythrine at 16 MIC could eradicate the mature biofilm.The results indicate that sanguinarine-chelerythrine exhibited robust antifungal and antibiofilm activities against C.neoformans.Transcriptome sequencing technology was utilized to examine the influence of sanguinarine on the expression of C.neoformans genome.Between the treated group and the control group,a total of 2044 genes were differentially expressed,of which 1057 were up-regulated genes and 987 were down-regulated genes.The results of GO function enrichment and KEGG pathway enrichment showed that the antifungal mechanism of sanguinarine was mainly involved in ATP synthesis ability,melanin synthesis,pathogenicity,drug resistance and proteins function.Preparation of RDP-FITC modified sanguinarine liposomes.The RDP-FITC sanguinarine liposomes were prepared by the thin film dispersion method combined with ultrasonic technology,where the encapsulation efficiency was used as the evaluation index.The preparation formula was optimized using single factor and response surface method.The results showed that the optimum condition was defined as follows:the film-to-material ratio was 10.87:1,the drug-to-lipid ratio was 12.57:1,and the pH value of the hydration medium was 7.07.The prepared RDP-FITC sanguinarine liposome has an encapsulation efficiency of 84.92%,a particle size of 140.5 nm±1.3 nm,PDI value of 15.69%±0.93%,and an average potential size of-5.97 mv±0.13 mv.Futhermore,it has Tyndall effect and a regular circular structure,and good stability of various physical and chemical properties.Finally,the antifungal activity was further evaluated,indicating that RDP-FITC sanguinarine liposomes displayed better antifungal activity than sanguinarine.