Inhibitory mechanism of alkannin on Candida albicans

Abstract:

[Objective] This study aims to explore the inhibitory effect of alkannin on Candida albicans and the mechanism. [Methods] The minimal inhibitory concentration and minimal fungicidal concentration of alkannin on C. albicans were measured by microdilution method. The effect of alkannin on the membrane permeability of C. albicans was determined by ultraviolet spectrophotometry. The effect of alkannin on the morphology of C. albicans was observed by scanning electron microscope and the effect of alkannin on intracellular calcium ion concentration of C. albicans was determined by laser scanning confocal microscope. The effect of alkannin on membrane phospholipid enzyme activity of C. albicans was determined by egg-yolk agar plate medium method. The effect of alkannin on amount of gene expression of PLB1 and PLB2 was determined by RT-PCR. [Results] Alkannin had potent inhibitory effect on C. albicans, the minimal inhibitory concentration and minimal fungicidal concentration of alkannin on C. albicans were 16 mg/mL and 32 mg/mL respectively. Alkannin could damage the cytomembrane integrity of C. albicans and increased the permeability of cytomembrane, leading to the leakage of macromolecular substances such as DNA and RNA in cells and the loss of intracellular calcium ions. The concentration of macromolecular substances such as DNA and RNA in the supernatant increased by 117.32% (P<0.01) after alkannin acted on C. albicans for 16 h and the intracellular[Ca²⁺] decreased by 72.02% (P<0.01). The scanning result of electron microscopy also proved the destructive effect of alkannin on C. albicans cytomembrane. Alkannin could inhibit secretion of phospholipase by C. albicans and showed the dose-dependence. Alkannin in minimal inhibitory concentration decreased phospholipase secretion of C. albicans by 56.3% (P<0.01) compared with the control group. RT-PCR results demonstrated that alkannin could inhibit the expression of PLB1 and PLB2, which were a pair of gene encoding phospholipase B. After alkannin in 1/2 minimal inhibitory concentration acted on C. albicans for 16 h, the expression of PLB1 and PLB2 decreased by 56.4% and 61.4% respectively (P<0.01) compared with the control group. [Conclusion] Alkannin had a strong inhibitory effect on C. albicans by damaging cytomembrane integrity of C. albicans and increasing the permeability, leading to leak of intracellular macromolecule such as DNA and RNA and loss of[Ca²⁺], eventually causing cell death. The inhibition of alkannin on phospholipase secretion of C. albicans prevented cells from maintaining and repairing the damage of cytomembrane caused by alkannin, was also one of reasons of cell death.