Purification Of Antifungal Substance From Saussurea Costus And Studies On Its Mechanisms

The fungal cell wall has been considered the ideal target for screening antifungal agents,based on its unique structure.In our Laboratory,three Saccharomyces cerevisiae mutation models(?fks1,CHT020,CHT003)constructed by gene disruption and wide-type WHU2a for screening,Saussurea costus was obtained from 100 kinds of 75%ethanol extract of Chinese herbal medicine using double-playered petri dishes with disc diffusion method,which has specific inhibition to?fksl strain.Its MIC on WHU2a,?fksl,CHT020 and CHT003 are 64?g/mL,16 ?g/mL,64 ?g/mL and 64 ?g/mL.It is founded that the petroleum ether extract of Saussurea costus having the strongest antifungal effect,and it is relatively stable under low(-20 ?>?4 ?)and high(60 ??80 ??100 ?)temperatures,but at 115?,the activity was partly compromised.When treated by acid and alkali solution,the substance can still keep its activity.Saussurea costus was initially analysed through the Czech eight solvents,and contains two active substances,both of which are non-water-soluble class I antibiotics.Preliminary purification of the anti-fungal active ingredients from the petroleum ether extract of Saussurea costus was carried out using TLC method,the optimal TLC diffusion solvent was determined as petroleum ether:dichloromethane:ethyl acetate:acetic acid(30:10:2:1);Futhermore,the active substance,dehydrocostus lactone was obtained by silica gel column chromatography,with the purity of 98.36%.The preliminary antifungal mechanism of dehydrocostus lactone was studied.Studies have shown that when the concentration of dehydrocostus lactone reached 4096 ?g/mL,it's inhibition on ?-glucan synthase was 94.42±6.66%which was determined by a microtiter-based aniline blue fluorescence assay;the ?-glucan content of wild-type WHU2a was reduced by 18.4%after treated with dehydrocostus lactone in 32?g/mL(IC20),the chitin content did not change.Wild-type WHU2a was protected by sorbitol when treated with dehydrocostus lactone;after treated WHU2a with dehydrocostus lactone in 36 ?g/mL(IC50),the cell wall was stained with CFW and aniline blue,the ?-glucan content was also reduced.Above all of the experimental results,dehydrocostus lactone would specifically inhibit the ?-glucan synthase of S.cerevisiae.Therefore,the dehydrocostus lactone may be a novel lead compound for the development of antifungal agent through the control of glucan synthesis.