Chloroquine biphosphate

This bioassay was performed to evaluate the inhibition of FP biocrystallization in presence of the essential oils and the pure compounds [22 (link)]. The bioassay was carried out in a non-sterile 96-well plate flat-bottom at 37 °C for 18–24 h. The EOs and pure compounds were tested at several concentrations (EOs at 10, 5, and 2.5 mg/mL; and compounds at 100, 50, and 25 µg/mL). Chloroquine biphosphate (Sigma-Aldrich) was used as the reference drug. A series of solutions were added to the plate, namely: 0.5 mg/mL of hemin chloride (Sigma-Aldrich) freshly dissolved in dimethylsulphoxide (DMSO) (50 µL), 100 µL of 0.5 M sodium acetate buffer (pH 4.4), and 50 µL of EOs or compounds dissolved in DMSO. After 18–24 h, the plates were centrifuged at 3000 rpm for 5 min, and the supernatant was discarded. The remaining pellet was resuspended in 200 µL of DMSO so as to remove the unreacted FP. The plate was centrifuged again and the supernatant was discarded. The pellet was dissolved in 150 µL of 0.1 M NaOH, and the absorbance measured at 405 nm. The percentage of inhibition of the FP biocrystallization was calculated as follows:

This bioassay was performed to evaluate the inhibition of FP biocrystallization in presence of the essential oils as described [52 (link)]. The bioassay was carried out in a non-sterile 96-well plate flat-bottom at 37 °C for 24 h. The EOs were tested at 10, 5, 2.5, 1.25, 0.63 0.31 and 0.16 mg/mL; Chloroquine biphosphate (Sigma-Aldrich) was used as the reference drug at 0.1 mg/mL. The following solutions were added to the plate: 0.5 mg/mL of hemin chloride (Sigma-Aldrich) in dimethylsulphoxide (DMSO) (50 µL), 100 µL of 0.5 M sodium acetate buffer (pH 4.4), and 50 µL of EOs in DMSO. After 24 h, the plates were centrifuged at 3000 rpm for 5 min, and the supernatant was discarded. The remaining pellet was resuspended in 200 µL of DMSO so as to remove the unreacted FP. The plate was centrifuged again, and the supernatant was discarded. The pellet was dissolved in 150 µL of 0.1 M NaOH, and the absorbance measured at 405 nm. The percentage of inhibition of the FP biocrystallization was calculated as follows: Inhibition (%) = 100 × [(OD control − OD treatment)/OD control].