In vitro anti-malarial activity

Two P. falciparum strains, MRC-2 (sensitive to chloroquine) and RKL-9 (resistant to chloroquine), obtained from National Institute of Malaria Research (NIMR), New Delhi, India, were used in this study. These strains were perpetuated in vitro in continuous culture according to the method of Trager and Jensen [26] with slight modifications. Briefly, both sensitive and resistant strains of P. falciparum were maintained in A+ erythrocytes in RPMI-1640 medium (having glutamine, but without any sodium bicarbonate) comprising 1.00 g of dextrose, 5.94 g of HEPES buffer, 40.00 mg of gentamycin. Additionally supplemented with 5% sodium bicarbonate and 10% (v/v) inactivated human AB+ serum then incubated in gas mixture of 5% CO2, 5% O2 and 90% N₂ at 37 °C. Parasitized erythrocytes at initial 5% haematocrit were suspended in above mentioned culture medium and parasitaemia was regularly checked to maintain level between 2 and 4% with further sub-culturing for parasitaemia beyond 5%. Growth and multiplication of parasite was monitored by microscopy using Giemsa-stained slides.

To obtain ring stages of the parasite, the cultures were synchronized using d-sorbitol [27]. The cultures, with majority of ring stages, were treated with equal volume of aqueous 5% d-sorbitol for 5 min and then after centrifugation pellet were suspended in complete medium and fresh erythrocytes synchronized culture with 1% parasitaemia and 5% haematocrit were used for compound concentration response assay.

The concentration of each test compound needed to hinder multiplication of parasites by 50% (IC₅₀) against P. falciparum strains were obtained through concentration response assay performed in 96-well sterile tissue culture plates. Synchronized parasite cultures were applied to different doses of each compound. Dilutions were performed in gentamycin-free culture medium, and incubated at 37 °C having gaseous mixture (5% CO₂, 5% O₂ and 90% N₂) supply for 24 h. The results were expressed as IC₅₀ values computed from HN-NonLin Regression analysis [28], as well as mean percentage inhibition ± standard error examined by thick smear Giemsa stained slides [29, 30].

The degree of resistance was determined by comparing the activity of chalcones on the chloroquine sensitive and chloroquine resistant strains of P. falciparum using the following formula [31]: