Structure Identification For Phoxalone And The Optimization Of Its Fermentation Conditions

Myxobacteria have attracted much more interest as drug sources.In this work, based on the preliminary experiments of our lab, we attempted to investigate the separation, bioactivity-directed fractionation, purification and structural analysis of the anti-tumor active fermentation elution, to optimize the medium composition of significant factors to increase the yields of Phoxalone using response surface methodology, and to investigate the anti-tumor active fermentation elution Phoxalone's effect in vivo.Based on bioactivity evaluation, 7 components with higher and broad-spectrum antitumor bioactivity were directly screened via B16 and SGC7901 cell lines models in the WXNXJ-C fermentation elution. By using C-18 column chromatography, 15RPC column and C-18 preparation column, a component (named 7F) with molecular weight 424 Da was obtained. The structure of 7F component was analyzed with LC/MS, MALDI-TOF, MS/MS, IR and NMR. The molecular formula of 7F was calculated as C22H32O8. The traditional name and systematic name of compound C22H32O8 were Phoxalone and [1R,7R,8R,10S,11S,12S,13S]- 1,7,12,13-tetrahydroxy-14-methoxy-8,10-dimethyl-6-phenyl-5,15-dioxa-bicyclo[9.3.1]pentadecan-4-one, respectively. Via confirming with investigating by L08 Station of Science and Technology, Minister of Education it was confirmed as a novel 15-number ring macrolide containing an oxygen-bridge.The antitumor activity of Phoxalone were examined and evaluated with the mice transplanted B16 tumor in vivo. Phoxalone had strong antitumor activity, could inhibit tumor tumoral growth and extend survival span.The histopathology of tumors from the various groups indicated that the tumor cells of untreated mice grew vigorously, however the tumor cells from the different Phoxalone treated groups had clear nucleus pycnosis and necrosis areas in different degree.Based on Plackett-Burman design, the significant factors affecting the Phoxalone -producing fermentation were determined as follows: potato starch, CaCl₂ and skim milk. Based on Box-Behnken design, a response surface methodology (RSM) was used to optimize three critical internal factors mentioned. The optimal concentration of the variables was determined as: potato starch 8.05 g/L, CaCl2 2.72 g/L and skim milk 10.00 g/L. Under these conditions, the maximum yield of Phoxalone was 120.46mg/L theoretically. Through simulation experiment, the result of study was validated. The yield of Phoxalone was significantly increased from 67 mg/L to 119.98 mg/L.