Trypanosoma cruzi, a parasitic protozoan, is the causative agent of Chagas' disease in Central and South America. In our search for trypanocidal compounds in medicinal plants used in Uzbekistan, we isolated new trypanocidal diterpenes from the EtOAc extract of Dracocephalum komarovi. Since komaroviquinone, a quinone-type diterpene 1, showed potent trypanocidal activity, we tested natural quinones to find out quinone-type diterpenes 2-5 from Salvia miltiorrhiza to have potent trypanocidal activity. Several quinones have been reported to have trypanocidal activity, and these compounds are thought to exert their trypanocidal action by generation of reactive oxygen species, which is catalyzed by flavo-enzymes such as trypanothione reductase, in the parasite. Recently, an enzyme, which catalyzes the two-electron reduction of 9,11-endoperoxide PGH_2 to PGF^<2α>, was isolated from T. cruzi. This enzyme (TcOYE) was found to be a member of old yellow enzyme family, which has been isolated from yeasts, plants, and bacteria but not from animals, and to catalyze one-electron reduction of trypanocidal quinones. Thus, we investigated the role of this enzyme. Under an anaerobic condition, quinone-type diterpenes 1-5 were efficiently reduced by TcOYE in the presence of NADPH as a cofactor. NADPH was almost 2-fold more effective as the cofactor than NADH. Electron spin resonance experiments showed that, under the anaerobic condition, TcOYE catalyzed one-electron reduction of 1-5 to form semiquinone radicals, and under aerobic condition, these radicals reduced molecular oxygen leading to regeneration of the quinone and formation of superoxide anion radicals, which will cause oxidative damage to cellular components resulting in the death of the parasites. Compounds 2-5, which have larger one-electron reduction potentials, were better substrate of TcOYE than 1, which has a smaller reduction potential. However, the specific activity was not completely parallel with the reduction potential, indicating the importance of the substrate specificity of TcOYE. The specificity was not also parallel with the trypanocidal activity: komaroviquinone (1), which was not the best substrate of TcOYE, showed the strongest trypanocidal activity among the tested compounds. This suggested the involvement of other flavo-enzymes in the generation of active oxygen species. An experiment to evaluate the importance of TcOYE in this process is now in progress.
