Inhibitory Effect Of Osmanthus Fragrans Flavonoids On Candida Albicans And Its Application In Antibacterial Dental Paste

Dental caries has become a global problem threatening human oral health,and the research on the control of Candida albicans（C.albicans）,one of its main causative organisms,is very hot.So far,the resistance of C.albicans to various antibiotics has gradually increased,and the natural antibacterial agent has become a new development direction for the clinical control of caries,which is of great significance.Osmanthus fragrans Lour.（O.fragrans）,a plant with high ornamental and medicinal value,is rich in flavonoids,phenols,terpenoids,polysaccharides and other effective components,which have inhibitory effects on a variety of bacteria and fungi,some of which may have anti-caries effects.At present,the types of anti-caries oral products on the market are relatively single and the effective ingredients used have some limitations.Therefore,in this study,the natural ingredients with antifungi activity from O.fragrans were selected to evaluate the antifungi effect of C.albicans and a new type of antifungi dental paste product was designed.The main research contents and results are as follows:1.To investigate the antifungi effect of active ingredients in O.fragrans on C.albicans,optimize the extraction process of antifungi ingredients,and analyze the components of antifungi ingredients the following experiments were conducted.To determine the antifungi effect of O.fragrans flavonoids,triterpenes and polysaccharides on C.albicans,the minimum inhibitory concentration(MIC₅₀)was used as the judgment standard.The components with the lowest MIC₅₀ values among them were selected for the optimization of the extraction process,and single-factor and response surface process optimization experiments were conducted for ultrasound-assisted extraction with the extraction rate as the response value.The components of four varieties of O.fragrans flavonoids were identified by high performance liquid chromatography-mass spectrometry（UPLC-MS/MS）.The inhibition effects of four varieties of O.fragrans flavonoids on C.albicans in terms of MIC₅₀,cell membrane integrity and biofilm formation were determined.The results showed that the MIC₅₀ values of O.fragrans flavonoids,triterpenes and polysaccharides were 1.5 mg/m L,3.0 mg/m L and>3.0 mg/m L,respectively.After the single-factor and response surface optimization experiments,the best extraction process of cinnamon flavonoids was 50℃,the solid-liquid ratio was 17 m L/g,and the ethanol concentration was 52%.Under these conditions,the extraction rates of thunbergii group,latifolius group,aurantiacus group and semperflorens group were 13.88%,8.79%,13.16%and10.28%,respectively.The antifungal effect of different varieties of O.fragrans flavonoids on C.albicans was different,among which,Osmanthus fragrans var.thunbergii had the best antifungal effect with MIC₅₀ value of 1.5 mg/m L,which could significantly improve the permeability of the cell membrane of C.albicans within 24 h,leading to the efflux of cell contents,and the inhibition rate of biofilm formation could reach up to 46.59%.Therefore,O.fragrans flavonoids can be studied and developed as natural antibacterial agents in depth.2.Several flavonoid monomers from O.fragrans including rutin,baimaside,naringenin and rhoifolin were selected to investigate the antifungi effect and mechanism of inhibition of C.albicans by O.fragrans flavonoid monomers.The results showed that the MIC₅₀ values of the four components were 125μg/m L,250μg/m L,250μg/m L and 250μg/m L,respectively.The four O.fragrans flavonoid monomer components at 2MIC₅₀,MIC₅₀ and 1/2MIC₅₀ concentrations could disrupt the cell membrane integrity of C.albicans within 24 h,causing different leakage of intracellular substances such as nucleic acid and protein,and increasing the uptake of propidium iodide of C.albicans.High concentrations of O.fragrans flavonoid monomer were able to inhibit the formation of initial biofilm by more than 40%,in contrast to the weaker inhibition of mature biofilm.The treatment of O.fragrans flavonoid monomers could induce the production of reactive oxygen species in C.albicans,and the levels of reactive oxygen species produced by C.albicans were 5.08,5.06,4.88 and 4.83 times higher than the control after treatment with high level of rutin,baimaside,naringenin and rhoifolin,respectively.O.fragrans flavonoid monomer treatment could induce changes in mitochondrial membrane potential of C.albicans,and the rate of cells with altered mitochondrial membrane potential was 2.44,2.27,1.75 and 1.75 times more than the control group.In a comprehensive comparison,the inhibitory effects of rutin and baimaside on C.albicans were better than that of naringin and rhoifolin among the four O.fragrans flavonoid monomer components.3.Two natural macromolecular compounds,chitosan and pectin,were selected to combine with O.fragrans flavonoid to develop a kind of antifungi dental paste with anti-caries effect.The results showed that the gel reached a stable state after the mixing of chitosan and pectin at a proportion of 1:5.And the zeta potential,infrared spectra and X-ray diffraction patterns demonstrated at the structural level that the gels were successfully formed by covalent bonding between chitosan and pectin.The gel dental paste after the addition of O.fragrans flavonoids has good microscopic morphology,mechanical properties and thermal stability.The antibacterial experiment showed that the dental paste containing 3 mg/m L of O.fragrans flavonoids had 77.11%antibacterial effect on C.albicans.In conclusion,flavonoid is the best active ingredient in O.fragrans for C.albicans inhibition,which can achieve antifungi effect by damaging the cell membrane,increasing the production of reactive oxygen species and changing the mitochondrial membrane potential.Based on this,O.fragrans flavonoid antifungi dental paste was developed,with a view to laying the foundation for the prevention and treatment of dental caries.