| Objective:In vitro observation of the dynamic characteristics of Candida albicans andCandida tropicalis biomembrane growth. We measured respectively the Bupleurumsaponin D MIC80of two Candida and the formation of two Candida’s biomembrane atdifferent stages of the MIC50.Method:Part One: Standard strains of Candida albicans ATCC32354and Candidatropicalis CBS94were activated with bacterial suspension of1×106/ml and100μl/holeinoculated into96-well microtiter plates. MTT assay analysis determination ofinoculation,3h,6h,12h,24h,48h,72h,96h Candida biological activity and weredrawn Candida albicans and Candida tropicalis growth curve.Part Two: The activation of Candida albicans and Candida tropicalis dilutionoscillator oscillation15s,blood cell count board to adjust its concentration to1×10~5～5×10~5/ml, fungi RPMI1640medium1:100dilution of a bacteria concentration of1×10~3～5×10~3/ml, by microdilution method for the determination of saikosaponin Dminimum inhibitory concentration (MIC80) of Candida albicans and Candida tropicalis.Using96-well microtiter plates to set the concentration of8to an increase of only thecorresponding bacterial suspension and fungal RPMI1640medium each100μl as apositive control, an increase of only fungal RPMI1640culture solution200μl with ahole as a blank hole zero.Part Three: The activation of the standard strains of Candida albicans’ andCandida tropicalis’ concentration were1×106/ml. With100μl in a hole inoculated into96-well microtiter plates, the plates at37℃incubator discard the original culturemedium after2h incubation and gentle washing away free bacteria cells with PBS. Adding100μl of fresh fungi RPMI1640culture medium, and continue to be placed at37℃incubator culture, every24h to replace a fresh fungi RPMI1640medium.Vaccination remove corresponding plates discard the last to medium concentration ofthe1280μg/ml~10μg/ml of saikosaponin D after3h,6h,12h,24h,72h,and at37℃incubator for24h. MTT assay was measured by two kinds of Candida activity in theabove point in time and draw the curve, select saikosaponin D against Candidaalbicans and Candida tropicalis,50%inhibitory concentration (MIC50) of wereanalyzed and compared. Setting the concentration of8with96-well microtiter plates,added to the corresponding bacterial suspension and100μl with a well as a positivecontrol, and the fungal RPMI1640culture solution each hole with100μl as a blankhole zero.Results:Part One: Albicans group and tropical groups with significant difference, P <0.05statistically significant difference in the ability to3h,6h,12h,24h,48h,72h,96h eachtime point on both biomembrane formation. Candida albicans and Candida tropicalisbiomembrane activity increased with the incubation time was dependent. Candidaalbicans24h in culture biomembrane activity increased most significantly for48h afterthe biomembrane activity is maintained at a relatively high level platform of noobvious growth; Candida tropicalis in the6hours prior to culture biomembraneactivity increased significantly, after6h incubation, the biomembrane activity ismaintained at a relatively low level platform of no obvious growth. In vitro the candidaalbicans biomembrane formation capacity was stronger with the candida tropicalisbiomembrane formation capacity.Part Two: The microplate reader saikosaponin D measured the candida albicansand candida tropicalis MIC80were80μg/ml and320μg/ml. The naked eye observationwas the same with the result of MTT assay.Part Three: Candida albicans and candida tropicalis biomembrane formationinhibition with the saikosaponin D extension of the formation time. Compared the twocandidas50%inhibitory concentration found by saikosaponin d,after3h,6h,12h,24h,72h each time point was significantly stronger inhibition of Candida albicansbiomembrane to its inhibition of Candida tropicalis biomembrane, P <0.05differencewas statistically significant. Microgram levels have exceeded the50%inhibitoryconcentration of saikosaponin D on Candida tropicalis biomembrane at72h. Conclusion:1.Candida albicans and candida tropicalis were able to form biomembrane in96-well plates, biomembrane formation can be maintained at a relatively high level.The ability of biomembrane formation candida albicans’ were stronger than candidatropicalis’.2.Saikosaponin D significantly inhibited the growth of candida albicans andcandida tropicalis. And the inhibition of saikosaponin D with candida tropicalis wasbigger than candida albicans. The microplate reader saikosaponin D measured thecandida albicans and candida tropicalis MIC80were80μg/ml and320μg/ml. It is rarehave the effective within reach microgram levels in Chinese herbal medicine. It has thehigh value of further research and development.3.Saikosaponin D has the inhibition of biomembrane formation to candidaalbicans and candida tropicalis with different levels. Saikosaponin D the ability ofinhibition biomembrane formation is differ. With the candida albicans was strongerthan the candida tropicalis biomembrane forming ability. |
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