Redox Reaction of Artemisinin with Ferrous and Ferric Ions in Aqueous Buffer

Nathawut SIBMOOH, Rachanee UDOMSANGPETCH, Anake KIJJOA, Udom CHANTHARAKSRI, Samlee MANKHETKORN
2001 Chemical and pharmaceutical bulletin  
Artemisinin (qinghaosu), a sesquiterpene lactone with endoperoxide bond, possesses potent antimalarial activity against chloroqine resistant Plasmodium falciparum, widely used for therapy of malaria in China and Southeast Asia. It was demonstrated that artemisinin can kill parasites in the ring and late stage, both in vitro and in vivo. 1) It is believed that the mode of action of artemisinin is its iron-dependent mechanism, involving cleavage of the peroxide bridge by heme iron, yielding
more » ... ron, yielding carbon centred free radicals 2-4) which in turn alkylates some parasite-specific proteins. 5) Although the chemical reaction of artemisinin with free iron or heme has been extensively studied, and the end-products have been characterized in many laboratories, 6-13) the nature of the biological active intermediates is still unclear. Many biomolecules are well suited for metal chelation and are considered to be prooxidant or antioxidant or both (e.g. ascorbate). 14) The prooxidant activity of these compounds is related to the production of reactive oxygen species such as O 2 ·Ϫ or H 2 O 2 . This event occurs in the presence of transition metals. 14,15) Coordination of transition metals to biomolecules almost always involve d orbitals of the metal. In addition, dioxygen can also ligate to transition metals primarily through the d orbitals of the metals. Therefore, transition metals may simultaneously bind to biomolecules and dioxygen and may often act as a bridge between the molecule and dioxygen, for example, the formation of active O 2 -Fe 2ϩbleomycin as an intermediate in bleomycin-induced DNA cleavage. 16) In this study, we have shown that in an aqueous buffer artemisinin generates a cycle of iron oxidation-reduction reaction and consumption of dioxygen, yielding an active product, dihydroartemisinin. Dihydroartemisinin binds favorably to ferric ion, yielding an intermediate, ferric-dihydroartemisinin complex. The complex is oxidized in the presence of oxygen, yielding artemisinin. Decontamination of Adventitious Metals and Buffer Used The presence of contaminating metals in reagents can lead to unpredictable change in the redox chemistry of iron under study due to exchange of contaminating metals with iron, which may affect the rates of oxidation or reduction of iron. Therefore, in order to remove trace amounts of adventitious catalytic metals in water, chelating resin (Chelex 100 resin) was used as described previously. 14,15) Chelex 100 resin was added into double-distillated water (5 mg/100 ml) and stirred gently for 1 h. Then, the water was filtered from the resin using filter paper for quantitative analyses (MN 615, Macherey-Nagel). The buffer solutions were prepared in glassware. The glassware was washed with 10% nitric acid and rinsed three times with metal-free water. The buffer solution contained 20 mM Hepes buffer plus 132 mM NaCl, 3.5 mM KCl, 0.5 mM MgCl 2 , and 5 mM glucose, pH 7.25. All other reagents were prepared in disposable plastic wares. The stock solution of 0.01 M ferrous or ferric ions (in 0.4 M H 2 SO 4 ) was freshly prepared before being used. The stock solutions of 0.01 M artemisinin were prepared in dimethyl sulfoxide (DMSO) and stored at Ϫ20°C. To obtain the desired concentration of artemisinin, the stock solution was diluted with a buffer. The absorption spectra were recorded on a Hewlett Packard HP 8435 and a Shimadzu, UV 2501 PC spectrophotometer. Experiments were conducted in a 1-cm quartz cuvette containing 2 ml of solution under continuous stirring. The temperature was controlled at 25°C using a peltier temperature control, cell holder model 89090A. The 1 H-NMR spectra were recorded on a Brucker model WM 250 spectrometer. The mass spectra were recorded on an API 100 spectrometer. Determination of Ferrous and Ferric Concentration Ferrous concentration was determined by colorimetric method, using 1,10-phenanthroline as chelator. The reaction was performed in 50 ml air-saturated or N 2 -saturated buffer solution. At desired time intervals, 2 ml of the reaction mixture were removed and added to 0.1 ml of 25 mM 1,10-phenanthroline in 0.25 M sodium acetate buffer pH 5.3 containing 50 mM sodium arsenite. The ferrous-phenanthroline complex was monitored by the absorbance at 515 nm with the molar extinction coefficient (e) equal to 11000 M Ϫ1 · cm Ϫ1 . 17) Artemisinin, a sesquiterpene with endoperoxide bond, possesses potent antimalarial activity against the ring and late stage of chloroqine-resistant Plasmodium falciparum malaria both in vitro and in vivo. The mode of antimalarial activity of artemisinin is iron-dependent. The aim of this study was to investigate the reactions of artemisinin with ferrous and ferric ions in aqueous buffer. Artemisinin generated a cycle of iron oxidation and reduction. It oxidized ferrous and reduced ferric ions with similar rate of reaction (k‫5.0؎01؍‬ M ؊1 · s ؊1 for ferrous and k‫0.2؎5.8؍‬ M ؊1 · s ؊1 for ferric ion). The major active product was dihydroartemisinin which exhibited antimalarial activity at least 3 times more potent than artemisinin. Dihydroartemisinin preferably binds to ferric ion, forming ferric-dihydroartemisinin complex. The re-oxidation of the complex gives artemisinin and ferric ion. This suggests that in aqueous buffer, the reaction of artemisinin with iron may give rise to the active reaction products, one of them being dihydroartemisinin, which is responsible for antimalarial activity.
doi:10.1248/cpb.49.1541 pmid:11767072 fatcat:ngli3rjekzhhvmtaof2psj5ag4