Abstract: Objective To study the characteristics of inhibition on hemozoin formation by chloroquine under in vitro condition. Methods Under different concentrations（0.5-2 mol/L）of sodium acetate（NaAc）and at the pH range of 4.0-5.0, chloroquine was tested for inhibition of β-hematin（hemozion）formation by using the HPIA（heme polymerization inhibitory activity）assay. The morphology of β-hematin crystals was determined by light microscopy. Ultraviolet spectrophotometry was employed to measure β-hematin content, and the size of β-hematin crystal was analyzed by X-ray diffraction（XRD）. Results Chloroquine exhibited varied effect on β-hematin formation, depending on pH value and Na+ concentration. When the NaAc concentration increased from 0.5 mol/L （pH 4.2）to 2 mol/L（pH 4.8）, the chloroquine inhibitory effect also increased. Results suggested that there exists a threshold pH, below which the β-hematin formation escalates and chloroquine inhibition declines, and at or above which chloroquine exerts a stronger inhibitory effect on β-hematin formation. With the increase of pH from 4.4 to 4.8, the crystallinity and the size of crystal changed from 6.93% and 357 Å to 6.32% and 264Å, respectively. When pH reached to 5, no more β-hematin formed. Chloroquine could reduce the crystallinity and crystal size of β-hematin at same pH value. Morphology analysis on the samples was consistent with the above results. Conclusion Chloroquine inhibits hemozoin formation only when the pH value is at or above threshold pH.

Key words: Malaria parasite, Hemozoin, Chloroquine, Threshold pH, X-ray diffraction