Allicin Enhances The Effect Of Amphotericin B Against Candida Albicans

With the increasing immunocompromised patients in recent years, the incidence of systemic fungal infections has been raised dramatically with a heavy mortality. Owing to the adverce effect of antifungal drugs and the severely drug resistance of fungal pathogen, the therapy systemic fungal infections is becomes more and more difficult. Candida albicans (C.albicans) is the major opportunistic fungal pathogen of humans and investigation on the treatment of candidiasis is an important issue.Screening active component from traditional Chinese drug is an important strategy for antifungal drug investigation. Allicin (diallyl thiosulfinate) is the main biologically active component of freshly crushed garlic extract. Allicin exerts various biological activities such as antimicrobial and anticancer activities in addition to the capacity to lower serum lipid levels, particularly cholesterol levels, and ocular pressure. Previous published data indicated that allicin had certain in vitro antifungal activity, but the minimal inhibitory concentration (MIC) was relatively high, limiting its clinical utility. Researchers demonstrated that Cu2+ exerted fungicidal activity by promoting endogenous ROS production, and recent studies found that allicin could enhance the fungicidal activity of Cu2+. It is suggested that allicin may also be able to enhance the fungicidal activity of antifungal drugs.In the present study, we investgated the interaction effect beteen allicin and antifungal drugs including fluconazole, amphotericin B, terbinafine and 5-fluorouracil. The in vitro MICs of the compounds against isolates of fungal pathogen were determined by the microbroth dilution method according to the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards). The fractional inhibitory concentration index (FICI) is defined as the sum of the MIC of each drug when used in combination divided by the MIC of the drug used alone. Synergy and antagonism were defined by FICIs of≤0.5 and >4, respectively. A FICI result of >0.5 but≤4 was considered indifferent. Our results indicated that AmB+ allicin combination and FLC+allicin combination markedly reduced MIC values of either individual agent for C.albicans and synergism was observed in all isolates of C.albicans (FICI <0.5).The polyene macrolide antibiotic AmB is the gold standard of antifungal treatment for the most severe invasivemycoses. However, owing to its poor permeability across membranes, an increased amount of AmB must be administered to patients in clinical situations, often resulting in severe side effects such as renal damage. To lessen the severity of the side effects, AmB is often combined with other antifungal drugs. So in the study, we further confirmed the synergic effect beteen allicin and AmB. Our results indicated that Synergism was observed in all 40 isolates (all the FICI<0.5) .The synergic effect of allicin with AmB was also confirmed in time-kill curves. The fungistatic activity of AmB was dramatically enhanced by addition of allicin. To assess the antifungal activity of AmB combined with allicin, initial experiments were performed by challenging mice. The adddtion of allicin (1 mg/kg) to AmB (0.5 mg/kg) treatment for mice infected by C.albicans significantly improved the survival time (P<0.05) and significantly reduced the fungal burden in kidney tissues, liver tissues and spleen tissues.There is no study suggested the possible mechanism of the the synergic effect beteen allicin and AmB. So in the present study, we first investigated the change of gene expression profiling with a complementary DNA microarray and metabolite profile with GC/MS analysis in C.albicans after addtition of alllicin to AmB treatment. Our results suggested that oxidative damage, ergosterol biosynthesis inhibition and electron transport chain blocking may be involved in the mechanism of the the synergic effect beteen allicin and AmB.Our further resaeach work indicated: (1) ROS production significantly elevated in C.albicans after addtition of alllicin to AmB treatment. And MDA level, representing the phospholipid peroxidation reaction, was significantly elevated in C. albicans treated by AmB in the presence of allicin, indicating that allicin could accelerate the endogenous ROS production and thus induced oxidative damage such as phospholipid peroxidation; (2) addition of allicin to AmB treatment could inhibit ergosterol production and lanosterol, while elevated squalene level. The above results suggested that maybe target squalene epoxidase to block ergosterol biosynthesis signaling; (3) addition of allicin to AmB treatment induced ATP production inhibited, exerting synergic effectwith AmB; (4) the breakdown products of allicin, diallyl disulfide(DADS), also posees the synergic anticandidal effect with AmB.To conclusion,the present study shows that allicin has a synergistic effect with AmB against C. albicans in vitro and in vivo. Oxidative damage, ergosterol biosynthesis inhibition and inhibition of mitochondrial ATP production may be involved in the mechanism of the the synergic effect. Our study suggested that allicin was a promising and safe agent to combine with AmB for efficacy and to reduce the AmB dose to lessen side effects, although the molecular mechanisms need further study.