Study On Separation And Purification Of Echinococcus B And Its Nuclei

With the situation that morbidity and mortality rate of diseases which are infected byFungi, especially by invasive fungal has increased in recent years, the R&D ofanti-fungal drugs now has become a hot area of research. As anti-fungal drugs,Echinocandins are a class of cyclic hexapeptides having some different lipid side chainsmodiifcations, which was found in the1970s. They inhibit the synthesisof (3-(1,3)-D-glucan in the cell wall noncompetitively. They have a novel mechanism,alow adverse reaction rate and a strong antibacterial activity for some azoles andamphotericin B resistant ifingi. At present，there are three such drugs includingcaspofungin, micafungin and anidulafungin. Anidulaflingin is the third generation ofechinocandin semi-synthetic antifungal drugs, which was approved for use in the UnitedStates in2006. Compared with other antifungal agents,anidulafungin has a greatervolume of distribution and a more broad-spectrum antimicrobial activity..The lipid side chain of ECB (Echinocandin B) was removed by microbialconversion, and produce the product-ECBN (Echinocandin B Nucleus). Then usechemical synthesis method add a new side chain， produce the ifnalproduct-Anidulafungin. Both ECB and ECBN are important intermediates, so it’simportant to improve its method of separation and purification.We did some research about the ECB’s crude extract, pigment removal, purificationand reifning, finanlly established a complete and feasible purification process for ECB.First, Added60%ethanol solution (twice the volume of mycelium) to the culture，thenadjust the pH was4.0. Then sonicate extracted three hours before the extraction wereifltered,and then repeated it once. Removed pigments of ECB’s extract by cationexchange resin FPC1INa (pH of5.0，5mL/min); and the SP825was selected as theeffective macroporous adsorption resin ifller for purification(pH5.0, lfow rate of4mL/min). then use20%ethanol to remove strong polar impurity and pigments, thendesorbed with70%ethanol; finally NM100resin was used to reifne (pH5.0, flow rate of3mL/min), added30%ethanol to remove strong polar impurity and pigments then desorbed with80%ethanol. HPLC dectation for purity is93%, and total yield of theentire process of separation and purification is about50%.Meanwhile the separation and puriifcation process of ECBN were studied. We madesure the optimize PH of ECBN (PH4.5), chose macroporous adsorption resin XAD-18topurify (PH4.5, flow rate of3ml/min) for the ifrst column separation，added water(twicethe volume of column) to remove highly polar impurities and salt, and then desorbed with17%methanol(pH4.5); ifnally, the hydrophilic CI8packing was selected to refine ECBNwith the the natural flow rate, after that，water(pH4.5) was used to remove highly polarimpurities and salt. After ECBN was leaked, used3%methanol(pH4.5) to desorb.Eventually, HPLC dectation for purity is97%, and total yield of the entire process ofseparation and puriifcation is about65%. Finally, we analyzed the quality of theseparated ECBN.