2.5. In Vitro Heme Polymerization Inhibitory Activity Assay

The heme polymerization inhibitory activity (HPIA) was conducted according to Basilico et al.'s modified method [6]. The 100 μL of 1 mM hematin in 0.2 M NaOH was added into a microtube. Then, 50 μL of testing compound at various concentrations (20.475; 10.238; 5.119; 2.580; 1.269 mM) was added in triplicates. Distilled water was used as a negative control. The 50 μL solution of glacial acetic acid (pH 2.6) was added into the microtube to initiate the polymerization reaction and incubated at a temperature of 37°C for 24 hours. Microtubes were centrifuged at 8000 rpm for 10 minutes and the supernatant was discarded and then washed three times using the 200 μL dimethyl sulfoxide (DMSO).Then, the deposition of the hematin crystal was dissolved with 200 μL of 0.1 M NaOH and 100 μL of solution was added into the 96-well microplate.

Absorbance was read using ELISA reader at λ 405 nm. A standard curve was made to illustrate the relationship between the concentration of hematin and its absorbance. The various concentrations of hematin (250; 125; 62.5; 31.25; 15.6; 7.8; and 3.9 mM) were used to make the standard curve. The heme polymerization inhibition was expressed as IC_50, which is the concentration of testing compounds that can inhibit 50% of the formation of β-hematin. The IC₅₀ value was calculated by probit analysis using SPSS software (IBM Corp., Chicago). A compound shows to have heme polymerization inhibition activity in if it has an IC₅₀ value lower than the IC₅₀ value of chloroquine as in reference (37.5 mM) [17].