Microbial Transformation Of Aristolochic Acid Compounds And Their Cytotoxicity Comparison On HK-2 Cell

It has been reported that Aristolochic acid can cause kidney injury and even induce renal failure. However its mechanism has not been identified, because the metabolism of Aristolochia compounds in body is very complicated and its metabolite is very difficult to be collected. So we established microbial transformation system of Aristolochia compounds by Cunninghamella blakesleana(AS3.910). Then, the metabolite was isolated and purified for its structure identification and toxicology experiment.We did the comparison of Aristolochic A,B and metabolite's cytotoxicity on HK-2 cell.Procedures and methods:1. Cultured Cunninghamella blakesleana AS3.910 and established the transformation reaction system.2. Optimized the transformation system by analyzing the multi-factors' influence to transformation reaction.3.Detected and obtained the metabolite by high performance liquid chromatography.4. Identified the chemical structure of the metabolite by mass spectra and nuclear magnetic resonance.5. Observed the morphological changes of HK-2 cell that exposed to AA Ⅰ,AAⅡ or AAⅠa.6. The inhibition rate of cell growth was determined by MTT assay after treatment with three aristolochia compounds(concentration points: 0,5,10,15, 20, 30,40, 60, 80μg.ml^-1, time points: 12, 24, 36,48h).7. Observed the expression of α-smooth muscle actin by immunohistochemistry.8. Analyzed cell cycle and apoptosis rate of HK-2 cell exposed to three Aristolochia compounds by flow cytometry.