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SANGUINARINE IS REDUCED BY NADH THROUGH A COVALENT ADDUCT

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SÁNDOR Roman SLANINA Jiří MIDLIK Adam ŠEBRLOVÁ Kristýna NOVOTNÁ Lucie TÁBORSKÝ Petr TÁBORSKÁ Eva PEŠ Ondřej

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Fakulta / Pracoviště MU

Lékařská fakulta

Citace
Popis Benzo[c]phenanthridine alkaloids are a family of compounds with cytotoxic, anti-fungal and antimicrobial properties, which are heavily metabolized by the CYP 450 system. Most of the research of these compounds has been focused rather on their biological effects than their metabolism; however, it has already been found that the first step of their biotransformation is a reduction to their respective dihydro forms. Interestingly, this reaction step may proceed with NAD(P)H even when no additional enzymatic apparatus is present. We decided to study this interaction with sanguinarine (SA), as this effect has been noted but never examined any further. From the results of Matkar et al.[1], who found an unexpected compound on RP-TLC and 1D electrophoresis of NADH and SA, we hypothesized that SA forms a negatively charged, covalent adduct with NADH. We used TLC and LC-MS to identify the compound, observed at 997 Da (singly charged) and 499 Da (doubly charged). The kinetics of the equimolar mixture and the kinetics of the adduct isolated by semi-preparative chromatography was studied by the UV-Vis and MS/MS-MS spectrometry to infer the state of all reaction compounds at different time points. The kinetics of the equimolar mixture showed a decrease in concentration of SA while the concentration of the reduced form, dihydrosanguinarine, and the adduct were increasing. The kinetics of the isolated adduct was more complicated, as the adduct partially decomposed to the reactants. The results strongly supported the hypothesis that during the non-enzymatic reduction of SA by NADH, a covalent semi-stable adduct was present, and its decomposition to products was the rate-limiting step.
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