they are present in the normal flora ( in moist places like the intestinal system , mouth etc . ) in the human body . the dimorphic yeast candida albicans ( c. albicans ) is the most prevalent opportunistic pathogen in humans . candidiasis ranges from superficial and mucosal infections to life - threatening septicaemic mycosis in debilitated patients [ 1 - 3 ] , and this fungus is known to be the fourth leading cause of nosocomial infections [ 4 , 5 ] . over the last few decades , the incidence of candidal infections due to c. albicans has been increasing , paralleling the growing numbers of immune - compromised patients [ 4 - 7 ] . to the patients , for medical treatment of fungal infections , amphotericin b has been considered as the drug of choice [ 9 , 10 ] , and the azoles are mainly used in common clinical situations . however , the toxicity of and the resistance to these antifungal drugs are major problems [ 4 , 5 ] . in the case of amphotericin b , an increased amount of amphotericin b must be administered to patients due to its poor permeability across the membrane , which can result in severe side effects , for example , renal damage [ 12 , 13 ] . to overcome these problems of side - effects , natural products have been considered to be promising antifungal agents with less profound adverse effects . the antifungal activities of several phytochemicals including polyphenols , phenolics , terpenoids , and alkaloids have been reported . bee venom ( bv ) from honey bees ( apis mellifera l. ) has been utilized for centuries as a pain reliever , anti - coagulant and anti - inflammatory agent for the treatment of chronic diseases , such as arthritis , rheumatism , tendonitis , bursitis , fibrosis and multiple sclerosis [ 15 - 18 ] . in korea , apitherapy , which uses the stings of live honey bees , has shown therapeutic value for treating piglets , calves and dairy cows with several respiratory diseases . bv , called apitoxin , is a mixture of proteins : melittin ( main component 52% ) , apamin , adolapin , phospholipase a2 , hyaluronidase , histamine , dopamine and protease inhibitor . two major components of bv , melittin and phospholipase a2 , are generally thought to play an important role in the induction of the irritation and the allergic reactions associated with bee stings . also , sweet bee venom ( sbv ) , in which hyper - molecular materials , such as enzymes and histamines that act as allergens in bv pharmacopuncture , have been removed , has been developed . melittin , a 26 amino acid polypeptide , is known to have anti - bacterial effects [ 22 - 24 ] . however , information on the antifungal efficacies of bv and sbv is not available . this study was performed to investigate the antifungal activities of bv and sbv against c. albicans clinical isolates cultured from blood and the vagina . the bv and the sbv used in this study were produced at the korean pharmacopuncture institute ( seoul , korea ) . lyophilized whole bv and sbv were dissolved in distilled water at different concentrations , and were then used in this experiment . to examine the antifungal activities of bv and sbv , c. albicans ( kctc 7965 ) as a standard strain from the korean collection for type cultures ( kctc ) at the korea research institute of bioscience and biotechnology ( kribb ) , daejeon , korea ; the 10 c. albicans clinical isolates used as test organisms were furnished from the korean collection of medical fungi ( kcmf ) at the konyang institute of medical mycology ( kimm ) , daejeon , korea . fungal cells were cultured on sabouraud dextrose agar ( sda ; bd , difco , sparks , md , usa ) plates , and then incubated for 48 hours at 35c . louis , mo , usa ) were dissolved in 2% dimethyl sulfoxide ( dmso ) as a stock solution , and then subsequently diluted . disk diffusion was carried out with mueller - hinton agar glucose methylene blue ( mh - gmb ; bd , difco , detroit , mi , usa ) . the 0.5 mcfarland inoculum was swabbed in three directions on the mh - gmb agar plate . a disk ( advantec filter paper no . 26 , size 6 mm ) was placed on the surface agar , and a 20 l sample ( 10 mg / ml each of bv and sbv ) was loaded . amphotericin b ( 100 g ) and fluconazole ( 100 g ) were used as a positive control . the diameters ( cm ) of the zone of inhibition were measured by using a metric ruler . a minimun inhibitory concentration ( mic ) assay using a broth microdilution method was performed as described in m27-a2 ( clsi ) with modifications . the medium used was roswell park memorial institute ( rpmi ) 1640 medium ( sigma chemical co. , missouri , usa ) with l - glutamine buffered to ph 7.0 by using 0.165 mol / l of morpholino- propanesulfonic acid ( mops ) , supplemented with 2% glucose . the strain suspension was prepared in 0.85% saline , with an optical density equivalent to a 0.5 mcfarland standard , and was diluted to 1 : 100 in rpmi 1640 medium to obtain a final concentration of 1  5  10 colony - forming units per milliliter ( cfu / ml ) . this suspension was inoculated into each well of a microtiter plate previously prepared with bv and sbv at concentrations from 500 g / ml down to 0.9 g / ml by using rpmi medium . the control drug for each strain was amphotericin b and fluconazole diluted in dmso ; the concentrations tested ranged from 50 g / ml down to 0.09 g / ml in rpmi 1640 medium . the mic was defined as the lowest concentration at which the optical density ( od ) was reduced to 50% of the od in the growth control well as measured by using an enzyme - linked immunosorbent assay ( elisa ) microplate reader ( biotek ) . the kinetics of the antifungal actions of bv and sbv against c. albicans cells were evaluated by using killing curve assays . log - phased fungal cells ( 2  10 cfu / ml ) in rpmi 1640 medium were incubated with 125 g / ml of bv and 62.5 g / ml of sbv or with 0.1 g / ml of amphotericin b ( at the mic ) , which was used as a positive control . the culture was obtained and spread on a sda agar plate , then , the cfus were counted after incubation for 24 hours at 35c . all data were taken in triplicate , and statistical analyses were performed by using the student s t - test , with ( p < 0.05 ) being considered significant . the antifungal efficacies of bv and sbv against c. albicans ( kctc 7965 ) as a standard strain and 10 c. albicans clinical isolates were evaluated by the disk diffusion method with mh - gmb medium , and the results are shown in table 1 and fig . the results showed that the diameters of the inhibition zones for c. albicans blood isolates and c. albicans vaginal isolates were 9  12 mm and 10  12 mm , respectively , for bv and 12  18 mm and 12  19 mm , respectively , for sbv . the diameters of the inhibition zones for bv and sbv for the c. albicans standard strain were measured as 11 mm and 15 mm , respectively . in the blood isolates and the vaginal isolates , the diameters of the inhibition zones for bv and sbv were not significantly different ( p > 0.05 ) . however , the inhibition zone diameters of sbv compared with that of bv in clinical isolates were significantly different ( p < 0.05 ) . for the clinical isolates , the mic values , which were obtained by using the broth microdilution method , varied from 62.5 g / ml to 125 g / ml for bv and from 15.63 g/ ml to 62.5 g / ml for sbv . the mic values for bv and sbv for the c. albicans standard strain were 125 g / ml and 62.5 g / ml , respectively . the mic values for bv and sbv for all clinical isolates were similar to those for the standard strain ( table 2 ) . a killing - curve assay against c. albicans kctc 7965 cells was conducted to assess the killing potencies of bv and sbv . the results showed that bv and sbv exhibited antifungal activities through fungicidal effects . c. albicans cells treated with 50 g / ml of sbv rapidly decreased in number in a manner similar to the decrease observed with 1 g / ml of amphotericin b after incubation for 4 hours , however , for 100 g / ml of bv , the decrease in the number of c. albicans was slow ( fig . bv , called apitoxin , is a mixture of proteins : melittin ( main component 52% ) , apamin , adolapin , phospholipase a2 , hyaluronidase , histamine , dopamine and protease inhibitor . two major components of bv , melittin and phospholipase a2 , are generally thought to play important roles in the induction of the irritation and the allergic reactions associated with bee stings . also , sbv which has hyper - molecular materials , such the enzymes and the histamines that act as allergens in bv pharmacopuncture , removed , has been developed . the frequent uses of anti - microbial and antifungal agents such as anti - biotics enable many pathogens to acquire resistances to multiple drugs . the emergence of anti - bacterial - resistant strains of animal pathogens and their potential health risks to humans have captured great attention [ 28 , 29 ] . therefore , new anti - bacterial or antifungal agents with fewer adverse effects must be developed for the successful treatment of dermatophytes infections . the anti - bacterial properties of bv as a natural antibacterial agent have been extensively studied , and bv therapy has been suggested for use as an alternative to anti - biotic therapy [ 31 - 33 ] . strong antibacterial activities of bv against both gram negative and gram positive bacteria have been reported [ 34 , 35 ] . nakatuji et al   reported that bv could control the growth of staphylococcus aureus , which plays an important role in the pathogenesis of inflamed lesions in the case of acne vulgaris . moreover , bv exhibited antibacterial activities against skin bacteria such as propionibacterium acnes , staphylococcus epidermidis , and streptococcus pyogenes . bv and sbv anti - candidal activities were observed by using the disk diffusion method and the broth microdilution method , confirming that the compounds have a potential for use as anti - candidal agents , although their activities are less efficient as compared with amphotericin b and fluconazole , also , in the disk diffusion assay of c. albicans isolates from blood and the vagina , the antifungal activites of bv and sbv were similar ( tables 1,2 , fig . 1 ) . however , yu et al   reported that the antifungal activities of bv against trichophyton mentagrophytes and trichophyton rubrum were much stronger than those of fluconazole , one of the commercial antifungal drugs that is currently being used . in the current study , amphotericin b was used as a positive control : amphotericin b is a fungicidal agent that is widely used to treat serious systemic infections . according to the mic values , the anti - candidal activities of bv and sbv were less potent than those of amphotericin b and fluconazole as a positive control ( table 2 ) . most anti- biotics show an anti - microbial effect caused by a cidal or a static action . sbv exhibited an effect similar to that of amphotericin b , which is known a cidal agent for fungi ; the anti - candidal activities of sbv are due to its killing action , as described by the time killing curve assay with c. albicans ( fig . the mic values for c. albicans blood and viginal isolates , as obtained by using the broth microdilution method , varied from 62.5 g / ml to 125 g / ml for bv and from 15.63 g / ml to 62.5 g / ml for sbv ( table 2 ) . seoung   reported that methanol extracts from galla rhois showed antifungal activity against c. albicans isolated from patients with recurrent vaginal candidiasis and the mic value was 50 mg / ml for c. albicans . also , dichloromethane extract from paeonia japonica showed antifungal activity against c. albicans , and the mic value was 50 mg / ml for c. albicans . thus , the anti - candidal activities of bv and sbv are thought to be much stronger than those of extracts from galla rhois and paeonia japonica . we demonstrated that sbv had very strong antifungal activity , the antifungal activity of sbv being much stronger than that of bv . moreover , the anti - candidal activities of bv and sbv were much higher than those of phytochemicals , judging by their effective anti - candidal concentration ranges . this study raises the possibility that bv and sbv can be used as an alternative strategy for treating fungal pathogens , which would reduce anti - biotic use . bv and sbv may be candidates for new antifungal agents against c. albicans clinical isolates . although this study , demonstrated that bv and sbv had considerable antifungal activites , further experiments for evaluating the in vivo efficacies of bv and sbv for clinical applications are warranted . the antifungal activities of bv and sbv against 10 clinical isolates of c. albicans that were cultured from blood and the vagina were determined by using the disk diffusion assay , the broth microdilution assay and the killing - curve assay . bv and sbv showed antifungal activities against those clinical isolates of c. albicans , and the antifungal activites of bv and sbv showed similar results on the disk diffusion assay . the mic values obtained for clinical isolates by using the broth microdilution method varied from 62.5 g/ ml to 125 g / ml for bv and from 15.63 g / ml to 62.5 g/ ml for sbv . in the killing - curve assay , sbv behaved as amphotericin b , which was used as positive control , did . bv , bee venom ; sbv , sweet bee venom ; kctc , korean collection for type cultures ; cfus , colony forming units ; sd , standard deviation . bv , bee venom ; sbv , sweet bee venom ; kcmf , korean collection of medical fungi ; kctc , korean collection for type cultures . mic , minimal inhibitory concentrations ; bv , bee venom ; sbv , sweet bee venom ; kcmf , korean collection of medical fungi ; kctc , korean collection for type cultures .
objectives : the purpose of this study was to investigate the antifungal effect of bee venom ( bv ) and sweet bee venom ( sbv ) against candida albicans ( c. albicans ) clinical isolates.methods:in this study , bv and sbv were examined for antifungal activities against the korean collection for type cultures ( kctc ) strain and 10 clinical isolates of c. albicans . 
 the disk diffusion method was used to measure the antifungal activity and minimum inhibitory concentration ( mic ) assays were performed by using a broth microdilution method . 
 also , a killing curve assay was conducted to investigate the kinetics of the anti- fungal action.results:bv and sbv showed antifungal activity against 10 clinical isolates of c. albicans that were cultured from blood and the vagina by using disk diffusion method . 
 the mic values obtained for clinical isolates by using the broth microdilution method varied from 62.5 g/ ml to 125 g / ml for bv and from 15.63 g / ml to 62.5 g / ml for sbv . in the killing - curve assay , sbv behaved as amphotericin b , which was used as positive control , did . 
 the antifungal efficacy of sbv was much higher than that of bv.conclusion:bv and sbv showed antifungal activity against c. albicans clinical strains that were isolated from blood and the vagina . 
 especially , sbv might be a candidate for a new antifungal agent against c. albicans clinical isolates .