Effect Of Four Antifungal Drugs On Two Denture Base Materials

Objective:The diseases of denture stomatitis often caused by infection of candida albicans. It is a common problem of the patients of wearing denture. In our research, by using method of micro calorimetry, we systematically studied the pharmacology and pharmacodynamics of four antifungal drugs curcumin, chlorhexidine, econazole and miconazole, observed the growth of albicans that adhesion on the surface of the denture metal components and evaluated the influence of four antifungal drugs on adhering albicans. The research is divided into three parts. First, we monitored the metabolic heat of candida albicans and evaluated the influence of four antifungal drugs on albicans. From the metabolic heat spectrogram, a great variety of candida albicans growth dynamics parameters and thermodynamic parameters can be calculated. Secondly, we studied the candida albicans adhesion on the denture and the inhibiting effect on candida albicans that adhered on the surface of the denture metal components. Finally, by using fluorescence spectroscopy and circular dichroism, we have discussed the interaction between antifungal drugs curcumin, chlorhexidine and of serum albumin.Part One:the research of the efficacy of four kinds of antifungal drugs.Exp.l Detection of inhibiting effects on candida albicans of four drugs by micro calorimetryMaterials and methods:In Salouraud Liquid Medium, the candida albicans were cultivated at 37?. Then bacteria solution was collected from the medium and diluted to 108 CFU/mL(CFU, Colony-Forming Units) as stoke solutions. Stoke solutions, Salouraud Liquid Medium and test drugs with different concentrations were mixed in the ampoule. Put the ampoule in TAM air, and obtained the thermal variation curves of reaction process.Results:According to the metabolic heat spectrogram, we found that all four of curcumin, chlorhexidine, econazole and miconazole were potential good antifungal drugs. The inhibition effectiveness order is chlorhexidine> miconazole> econazole> curcumin.Exp.2 Observation the absorbance of candida albicans suspension after adding four drugs by visible spectrophotometer.Materials and methods:Diluted the candida albicans suspension and added different concentration drugs in it. After 48 hours, the absorbance of bacteria suspension were observed.Results:After adding antifungal drugs, changes of absorbance indicated that the number of candida albicans was decreased. The inhibition of trend is consistent with micro calorimetry.Part Two:Compare the inhibiting effect of three antifungal drugs on candida albicans which adhesion on the surface of Ni-Cr alloy and Co-Cr alloy.Exp.3 Detection of candida albicans adhesion on Ni-Cr alloy and Co-Cr alloy. Materials and methods:Bacteria cultured with test pieces (10mm×10mm×1mm) for 24 hours at 37?, and then washed surface of it for counting the fungus number that adhesion on surface.Results:The number of candida albicans on Co-Cr alloy is less than Ni-Cr alloy.Exp.4 Detect three antifungal drugs inhibiting effects on candida albicans that adhesion on surface of metal pieces by micro calorimetryMaterials and methods:Mixed metal pieces that cultured with candida albicans, Salouraud Liquid Medium and different concentration of antifungal drugs in the ampoule. Put the ampoules in the TAM air, and record thermal variation curves of reaction process.Results:Curcumin, chlorhexidine and miconazole are potential antifungal drugs for adsorbent candida albicans. In terms of the inhibition effectiveness, chlorhexidine is the best and the curcumin is the least. Miconazole and curcumin have a better inhibition effect on Ni-Cr alloy than Co-Cr alloy.Part Three:Investigating the interaction between two kinds of drugs and BSAExp.5 Fluorescence spectra for investigating the interaction between two kinds of drugs and BSAMaterials and Methods:Before the titration, BSA was dissolved in PBS solution (pH=7.4) and the concentration of BSA was set to 10 ?M. During the titration, different amount of drugs was added into the BSA solution at 25,31 and 37?, respectively. The result was analyzed by the fluorescence intensity around 350 nm.Results:At three different temperatures, two kinds of drugs can effectively quench the fluorescence of BSA. According to the quenching constant, it is obvious that the addition of curcumin forms the curcumin-BSA complex, while the fluorescence quenching caused by chlorhexidine is dynamic quenching.Exp.6 Site competition method for investigating the interaction siteMaterials and Methods:BSA was dissolved in PBS solution (pH=7.4) and the concentration of BSA was set to 10 ?M. Warfarin and ibuprofen were added into the BSA solution respectively. Then, curcumin was added. Through the changes of fluorescence spectra of BSA before and after the addition of competition reagent, it is possible to obtain the interaction site.Results:Association constant when warfarin exist is much bigger than that of BSA. When adopting ibuprofen, association constant is close to the blank, which indicates that the binding site is site I.Exp.7 CD spectra for investigating the change of structure of BSA caused by drugsMaterials and Methods:During the experiment, the concentration of BSA was maintained and different amount of drugs was added. After being incubated for several minutes, the solution was sustained to CD measurement.Results:For curcumin, when increasing its concentration, ?-helix firstly increases and then decreases. While the addition of chlorhexidine leads to the decrease of a-helix.Exp.8 Molecular docking for investigating the binding mode of two antifungal drugs on BSAMaterials and Methods:Molecular docking for binding modes of curcumin and chlorhexidine on BSA was studied by Surflex DOCK program in Sybyl 8.1 software. The binding modes were analyzed and visualized in VMD software.Results:Curcumin can effectively cause the change of microenvironment near TRP213 residues, which might be the mechanism of fluorescence quenching. Chlorhexidine is close to luminescent residues TRP213, TYR156 and TYR451, fluorescence resonance energy transfer may occur.Conclusions:1. Curcumin, chlorhexidine, miconazole and econazole, all of them are potential antifungal drugs. The order of inhibition effectiveness:chlorhexidine> miconazole> econazole> curcumin.2. Under the same condition, the number of candida albicans on Co-Cr alloy is less than that on Ni-Cr alloy. As denture matrials, Co-Cr alloy is more likely be able to reduce the incidence of denture stomatitis than Ni-Cr alloy.3. On Ni-Cr alloy, Miconazole and curcumin have better inhibition effect than that on Co-Cr alloy. On Co-Cr alloy, chlorhexidine has better inhibition effect than that on Ni-Cr alloy. Therefore, target drugs can be supplied to cure denture stomatitis given different denture matrials.4. The interaction mode between curcumin and chlorhexidine is different. Curcumin and BSA tend to form curcumin-BSA complex, while fluorescence quenching of BSA caused by chlorhexidine is the result of molecule collision.5. The association constant between curcumin and BSA can is stable. Albumin plays the role of storage and transportation in vivo, helping the drug achieve the acting site through blood circulation and take effect.6. Curcumin has positive effect on secondary structure of BSA, while chlorhexidine may suppress this secondary structure. This may influence the dosage in clinic as science evidences.