Study On Antitumor Agents From A Fermention Broth Of Paecilomyces Militaris

In a screening of entomogenous deuteromycetes for pharmaceutical use, a fermentation broth of Paecilomyces militaris exhibited strong antitumor properties. To characterize the antitumorious compounds, the following works were done: bioactive components extraction, purification, structure identification and bioactive studies.The optimal fermentation time of the strain was determined by the yield of the bioactives which could be detected by TLC scanner in different time. The experiment showed, the yield of bioactives of the strain reached the highest at the tenth day. So 10 days was the optimal fermentation time.The best extraction and purification procedure of bioactive components were as follows:filtrate to remove condense aftermycelium condense extracting fermentation supernatant condensate crude sampleremove polysaccharid freeze-drysilica gel column sephadex LH-20 columnchromatogramphy chromatogramphy HPLC structureviuuc pure sample bioactivity testpure sample identificationIn search of an extracting technique of antitumour compounds which was lower energy consumption and pollution, extracting ratio and extracting times were optimized. The result revealed that the optimal extracting technique was the extraction of the fermentation broth with two times volume of ethylacetate for two times.The first step of purification was column chromatography. The solid phase was silica gel(200-300 mesh). The mobile phase for rough liposoluble extract (acetic ester)was trichloromethane:methanol:water=40:10:1 and the best mobile phase of rough water soluble extract was trichloromethane:methanol:water =13:7:1.Sephadex LH-20 column chromatography was used to purify the rough liposoluble extract of RCEF0718 and 4 pure components Z1-3,Z1-4,Z2-1 and Z2-2 were obtained. Thepurity of these 4 components were proved with TLC and analytical HPLC. Z1-3 and Z1-4 were subordinate components, Z2-1 and Z2-2 were the main components, which constituted more than 80% of the mass of the rough extract of the strain.The two components Z2-1 and Z2-2 were analysed by MS, NMR, ect. The result showed that they are two new unreported compounds with close structures similar to isoflavone. The name of Z2-1 is 4' -Hydroxylisoflavone-7-O- β -4" -Methoxyl glucoside (C22H22O9) and the name of Z2-2 is 5,4' -Hydroxylisoflavone-7-O- β -4" -Methoxyl glucoside (C22H22O10).The experiment of MTT showed that the inhibition of HepG2 cells by Z and the 4 pure components isolated from Z at the level of 100ug/ml and 20ug/ml were outstanding, the inhibition of common liver cells by the same components was lower than that of tumor cells. On the other hand, the inhibition of tumor cells by pure compounds was significantly lower than that of coarse extract. The detailed mechanism is to be studied further.