Heliotrine extraction and detoxification by bacterial derived FAD-dependent oxidoreductase

Alkaloids are plant secondary metabolites or toxins produced by plants. Pyrrolizidine alkaloids (PA), with toxic effects on human, are widely distributed in plants. Hepatoxic pyrrolizidine alkaloids are one of the most important causes of human disease in the prevalence of liver poisoning. To remove PA from feed, enzymatic biotransformation currently is concerned. Here, heliotrine a dominant PA in Gole Gav-Zaban herbal tea, from Echium amoenum was investigated. The petals were extracted with methanol at ۶۰°C by soxhlet extractor. The impurities were removed applying diethyl-ether and ethyl-acetate, the resin debris pelleted by NaCl. The method ended by adding chloroform to collect heliotrine. The purification of heliotrine was done by silica gel column and a concentration gradient in chloroform-methanol. FAD-dependent oxidoordoxase derived from Pseudomonas aeruginosa ۲۷۸۵۳ ATCC Structurally similar to human-specific FMO۳, effective on the metabolism of toxins in the liver was used to oxidize heliotrine as a pyrrolizidine alkaloid precursor. Enzyme activity of Fmo۵p was assayed photometrically. reaction was set up in a total volume of ۲۰۰ μl, ۰.۱-۱ μm the final concentration of the recombinant enzyme (periplasmic proteins, ۵۰ mM Tris-HCl buffer pH=۸ and ۱۰۰ μM NADPH at ۲۵°C in Quartz covet. The reaction was followed by the decrease of NADPH absorption at ۳۴۰ nm with an UV/Visible Spectrophotometer in ۳۰ second steps until the change stops. Control experiments without enzymes were prepared. The difference in light absorption at ۳۴۰ nm for the test sample was ۰.۴۰۴ and for the control reaction was ۰.۰۲۷ over ۵ minutes, which was a confirmation of the oxidative activity of Fmo۵p enzyme to be used to detoxify heliotrine. This enzyme is introduced as a drug design candidate. Detoxification effects of this oxidoreductase on other types of pyrrolizidine alkaloids is suggested in the continuation of present study.

کلیدواژه ها:

Echium amoenum ، FAD-dependent oxidoreductase ، Heliotrine ، Pyrrolizidine