Application Of Microbial Transformation By Cunninghamella Blakesleana AS3.153

Harmine has some curative effects including antitumor, anti-trypanosomes, antibacterial, and as well as cardiovascular protection. However, its toxicity constrains the clinical application, for the toxic dose is approximal to the maximum safe dose. Thus, it is necessary to study the activity and toxicity of harmine and its metabolites. Nevertheless, few reports focus on the metabolites of harmine in vivo because of its low concentraion. Microorganism transformation has the advantages of low cost, easy of handling, and scale-up capability. AS3.153 has similar drug metabolizing enzymes which can simulate both phase I and phase II metabolism reactions. In this work, filamentous fungi-cunninghamella blakeleana AS3.153 was chosen for harmine microbial transformation. Harmine's metabolites were subsequently purified and structure identified.Materials and methods:1. Mycelium spores were obtained by rinsing the culture medium with sterile water, instead of ring vaccination.2. HPLC processing was established to determine the novel harmine metabolites.3. Transformation condition was optimized for further amplification experiment.4. Transformation products were isolated through semi-preparative high performance liquid chromatography.5. The chemical structures of metabolites were identified by the mass spectrum (MS) and the nuclear magnetic resonance (NMR).Results:1. Rinsing inoculation method was easy handling, and prone to obtain pearl-shaped fungal colony.2. HPLC processing was established and used for optimizing transformation conditions.3. The total conversion rate of drugs was over 90%. The conversion rate of two transformation products was about 40%. After amplification experiment, the total yield of metabolism could reach 80%.4. Two metabolites (H1 and H2) had been isolated and collected successfully by semi-preparative HPLC.5. The transformation products of harmine were harmol and N-hydroxyl-harmine after identified by MS and NMR.Conclusions:1. Microbial transformation of harmine using cunninghamella blakeleana was established. This method could get high yield of products for further study.2. The transformation products of harmine were harmol and N-hydroxyl-harmine. In conclusion, microbial transformation of harmine using cunninghamella blakeleana AS3.153 demonstrated the high potential of Cunninghamella species to produce primary (phase I) metabolites. And the advantage of microbial models includes low cost, easy of handling, and scale-up capability. By this means, sufficient quantity of putative metabolites could be isolated and identified, then used as standards for metabolite identification and determination in further studies.