Investigation Of Anti-Candida Activities And Related Mechanisms Of Natural Compounds From Traditional Chinese Medicine

It is well-known that as a major opportunistic fungal pathogen,Candida albicans can cause a series of infections in patients with compromised immune function and the morbidity of C.albicans infections is increasing.Currently,the clinically available antifungal drugs are limited to polyenes（amphotericin B）,azoles（fluconazole）and echinocandins（caspofungin）,with their targets constrained to only the biosynthesis of ergosterol in membrane andβ-1,3-glucan.Side effects and toxicity,antifungal resistance and low bioavailability,undermines the therapeutic efficacy,while the presence of C.albicans biofilms during infections makes it worse.Therefore,developing novel antifungal agents is a pressing task.Natural products from plants have always been the major sources of antifungal,antibacterial and antitumor drugs.Researches on antifungal activities of natural compounds from traditional Chinese medicine（TCM）have been increasing while their targets are diverse.So,antifungal screening with library of compounds from TCM may lead to the identification of not only new antifungal leads but also novel antifungal targets.Therefore,the purpose of this study was focused on the anti-Candida activities of compounds from TCM.Through antifungal screening against the widely-used strain,SC5314,based on clinical laboratory and standard committee recommended methods（CLSI-M27-A3）,we found eight compounds and one standard extract with good activities.They were:dracorhodin perchlorate（DP）,lycorine hydrochloride（LH）,shikonin,magnolol,honokiol,pseudolaric acid B,isobavachalcone,plumbagin and Huangshanyao saponin extract（HSE,saponin extract from Rhizomes of Dioscorea panthaica Prain et Burk）.Antifungal activities of DP and LH against C.albicans have not been reported in public databases,unlike the others,the activities of which have been reported for many times and in detail.Therefore,we choose DP,LH and HSE for further investigation.The methods included:time-killing assay,morphological transition assay,biofilm formation and development（XTT assay）,adhesion assay,phospholipase assay on the egg yolk emulsion agar,exopolysaccharide（EPS）in mature biofilms,PI staining with flow cytometry（FCM）and confocal laser scanning microscope（CLSM）,reactive oxygen species（ROS）detection,cAMP rescue assay,toxicity assay using mammalian cells and nematodes,and sorbitol rescue assay.The antifungal activity of dracorhodin perchlorate（DP）was fungistatic,with a minimal inhibitory concentration（MIC）of 64μM.DP could inhibit the biofilm formation and development of C.albicans,as well as the yeast-to-hyphal morphological transition.It could also decrease the cell surface hydrophobicity（CSH）,adhesion to polystyrene surfaces and adhesion to HUVEC cells.The production of phospholipase and EPS in mature biofilms could also be suppressed by DP.Its toxicity against mammalian cells and Caenorhabditis elegans were relatively low.The inhibition on hyphal growth may be exerted through reducing intracellular cAMP,as revealed by cAMP assay.Similar to DP,the activity of LH was also fungistatic,with a MIC of 64μM.It could inhibit the morphological transition,biofilm formation and development,and adhesion to polystyrene surfaces.The production of EPS and extracellular phospholipase can also be inhibited by LH.cAMP may play important roles in the antifungal activities of LH.The toxicity against mammalian cells was low.The activity of HSE against C.albicans was fungicidal,with both minimal fungicidal concentration（MFC）and MIC being 64μg/mL.It could inhibit the hyphal growth,biofilm formation and development,adhesion to polystyrene surfaces,extracellular phospholipase production and EPS production in mature biofilms.The toxicity against mammalian cells was low,with a half maximal inhibitory concentration(IC₅₀)higher than its MIC against C.albicans.HSE causes damages on cell membrane,as revealed by increased cells stained with PI after treatment with HSE.HSE could also increase the endogenous ROS production in C.albicans cells,while the addition of N-acetyl-cysteine（NAC）could suppress the antifungal activity of HSE against C.albicans biofilms.After the characterization of antifungal activity of HSE,we further explore the activities of dioscin,the major active compound in HSE,against virulence factors of C.albicans.As expected,dioscin could inhibit virulence factors of C.albicans,such as morphological transition,biofilm formation and development,as well as the production of extracellular phospholipase.Dioscin can increase the membrane permeability of fungal cells,consistent with previous report performed by others.However,dioscin could not induce ROS production in C.albicans,which was inconsistent with that of HSE.Hints from in vitro differences in the time-killing and ROS detection assays between HSE and dioscin have fueled the seeking for other antifungal compounds in HSE.Through antifungal screening in commercially available saponins,gracillin was found active against C.albicans,with a MIC of 16μg/mL.This fungistatic saponin could also inhibit the adhesion,filamentous growth,biofilm formation and development.This compound can increase the permeability of plasma membrane.The endogenous ROS production could also be elevated by gracillin,which was consistent with the antifungal activity of HSE.The sorbitol assay indicated gracillin caused damages to cell wall while cAMP rescue tests revealed that gracillin may inhibit biofilm formation through reducing intracellular cAMP.Moreover,gracillin could synergize with dioscin.As for marketed antifungal drugs,gracillin could produce additive effects in combination with fluconazole,as well as caspofungin,while synergism was demonstrated when gracillin and amphotericin B were used in combination.Based on C.albicans SC5314,we found several antifungal agents,among which dracorhodin perchlorate and lycorine hydrochloride were reported for the first time,to our knowledge.We explore preliminarily the inhibitory effects of those compounds on virulence factors（such as adhesion,biofilm formation,morphological transition,phospholipase production,and so on）of C.albicans.The toxicity of those compounds was relatively low.Through comparing the differences between antifungal profiles of HSE and dioscin,followed by further antifungal screening,gracillin was found to be another antifungal saponin in HSE.Gracillin exerts antifungal activity through cell wall.These natural compounds might provide important leads for development antifungal drugs and structure optimization.