N-Butylphthalide Potentiates the Effect of Fluconazole Against Drug-Resistant Candida glabrata and Candida tropicalis. Evidence for Its Mechanism of Action

Yixin Liu,1 Feifei Ren,1 Shan Li,1 Xiangchen Li,1 Dongyan Shi,2 Zhiqing Zhang1 1Department of Pharmacy, the Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, People’s Republic of China; 2Department of Clinical Laboratory, the Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, People’s Republic of ChinaCorrespondence: Zhiqing Zhang, Department of pharmacy, the Second Hospital of Hebei Medical University, 215, Heping Road, Xinhua District, Shijiazhuang, Hebei, People’s Republic of China, Email 26500007@hebmu.edu.cn Dongyan Shi, Department of clinical laboratory, the Second Hospital of Hebei Medical University, 215, Heping Road, Xinhua District, Shijiazhuang, Hebei, People’s Republic of China, Email shidongyan73@126.comObjective: To define the antifungal activity of n-butylphthalide alone or in combination with fluconazole in Candida glabrata and Candida tropicalis.Methods: The antifungal activity of n-butylphthalide alone and in combination with fluconazole was investigated by the classical broth microdilution method and the time-killing curve method. The QRT-PCR method was used to determine gene expressions changes of mitochondrial respiratory chain enzymes, drug efflux pumps and drug target enzymes in Candida glabrata and Candida tropicalis after n-butylphthalide treatment.Results: The MIC values of n-butylphthalide against Candida glabrata and Candida tropicalis ranged from 16 to 64 μg·mL-1. The FICI value of the combination of n-butylphthalide and fluconazole against drug-resistant Candida glabrata and Candida tropicalis ranged from 0.5001 to 0.5315 with partial synergism. Time-killing curves showed that 256 μg·mL-1 n-butylphthalide significantly inhibited the growth of drug-resistant colonies of Candida glabrata and Candida tropicalis, and 128 μg·mL-1 n-butylphthalide combined with 1 μg·mL-1 fluconazole had an additive effect. N-butylphthalide could alter the expression of mitochondrial respiratory chain enzymes COX1, COX2, COX3, and CYTB genes in Candida glabrata and Candida tropicalis (P< 0.05) and downregulate the expression of the drug efflux pump genes CDR1 and CDR2 in drug-resistant Candida glabrata to 3.36% and 3.65%, respectively (P< 0.001), but did not affect the drug target enzyme ERG11 in drug-resistant Candida tropicalis.Conclusion: N-butylphthalide had antifungal activity against Candida glabrata and Candida tropicalis. N-butylphthalide improved the activity of fluconazole against drug-resistant Candida glabrata by affecting the expression of mitochondrial respiratory chain enzyme genes and reversing the high expression of drug efflux pump genes CDR1 and CDR2.Keywords: n-butylphthalide, fluconazole, drug resistance, Candida glabrata, Candida tropicalis, potentiation