N 2 hydroxyethylpiperazine n 2 ethanesulfonic acid hepes buffer

Plant materials were dried under the shade and grinded using an electric grinder. One hundred gram of individual material was macerated into 1 L of each solvent and thoroughly mixed using a sterilized glass rod at room temperature. The solution was kept for 24 h and mixed after every 2 h. Extracts were filtered through Whatman No. 1 filter paper, freeze dried and kept at 4 °C in well-closed containers for use in the anti-plasmodial assay. The extracts were dissolved in dimethylsulfoxide (DMSO) at concentration of 1 mg/ml and then diluted with incomplete medium (without serum) to achieve required concentrations of 0.1, 0.5, 1, 5, 10, 20 and 30 μg/ml for evaluation against known P. falciparum chloroquine sensitive strain, 3D7 (SS) and chloroquine resistance strains, LS1 (RS). P. falciparum culture was maintained in vitro on human erythrocytes (blood group O⁺) in RPMI-1640 medium (Sigma) supplemented with 10 % AB⁺ human serum, 25 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES buffer; Sigma Aldrich), 25 mM NaHCO₃ and 60 µg/ml gentamicin sulfate (7.2 pH) at 37 °C and 5 % CO₂ in a CO₂ incubator (HERA cell 240i, Thermo Scientific).

McCoy’s modified medium, RPMI-1640, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) buffer, sodium pyruvate, d-glutamine, resazurin, rhodamine 123 (RHO 123) and neutral red were supplied by Sigma-Aldrich (Prague, Czech Republic). Fetal bovine serum and gentamicin sulfate were purchased from Invitrogen (Carlsbad, CA, USA) and bovine serum albumin (BSA) from Fluka (Prague, Czech Republic). Doxorubicin and pure sesquiterpenes (α-humulene, β-caryophyllene oxide, trans-nerolidol, valencene) were supplied by Sigma-Aldrich. Stock solutions of sesquiterpenes were prepared in dimethyl sulfoxide (DMSO) and stored in the dark at 4 °C. All other chemicals used were of HPLC or analytical grade and provided by Sigma-Aldrich.