Plant materials collection
The leaves latexes of A. elegans and A. monticola were collected from Axum and Hgumburda, Tigray regional state, Ethiopia, respectively. The root part of C. tomentosa and T. gracilipes were collected from the locality where the Kunama ethnic group has resided. The leaves of H. schulli were collected from Grakahsu, Tigray regional state, Ethiopia. All the samples were collected in November 2018. Each plant was authenticated and specimens were deposited in the National Herbarium, Addis Ababa University, Ethiopia.
Consumables and equipments
Ethanol, chloroquine (CQ), drying oven (Genlab, England), refrigerator, Roswell Park Memorial Institute (RPMI-1640) medium (EuroClone, Celbio), AlbuMax (Invitrogen, Milan, Italy), hypoxanthine, HEPES, glutamine, dimethyl sulfoxide (DMSO), 96-well flat-bottomed microplates, incubator, and UV-VIS spectrophotometer were used to conduct this study.
Preparation of the extracts
The leaf latexes of both A. elegans and A. monticola were collected and dried as per the method described and were considered as an extract [1, 13]. The leaf of H. schulli and the roots of C. tomentosa and T. gracilipes were dried under shaded open air. These plant materials were pounded separately using grinding mill. The powdered plant materials were then macerated using 70% ethanol for three days separately and filtered using What man’s filter paper. The marcs were re-macerated two times and filtered after 72 hours. The filtrates of each plant were combined separately and dried in oven at 40 ºC. The dried latexes and ethanolic extracts were stored in a refrigerator at -4 ºC until used for further investigation.
Antiplasmodial activity
P. falciparum cultures were prepared based on the method described by Trager and Jensen [14] with minor modifications. The CQ-sensitive D10 and CQ-resistant W2 strains were preserved at 5% hematocrit (human type A+ erythrocytes) in RPMI 1640 medium (EuroClone, Celbio) supplemented with 1% AlbuMax (Invitrogen, Milan, Italy), 0.01% hypoxanthine, 20 mM Hepes, and 2 mM glutamine. All the cultures were maintained at 37 °C in a standard gas mixture consisting of 1% O2, 5% CO2, and 94% N2. Test samples were dissolved in DMSO and then diluted with medium to achieve the required concentrations (final DMSO concentration <1%, non-toxic to the parasite). Drugs were placed in 96-well flat-bottomed microplates and serial dilutions made. Asynchronous cultures with parasitaemia 1-1.5% and 1% final hematocrit were aliquoted into the plates and incubated for 72 hours at 37 °C. Parasite growth was examined spectrophotometrically (OD650) by measuring the activity of the parasite lactate dehydrogenase (pLDH), according to a modified version of the method of Makler [15, 16]. The antiplasmodial activity is expressed as 50% inhibitory concentrations (IC50) as mean ± standard deviation of at least three separate experiments performed in duplicate.
Antimalarial activity against stage IV-V P. falciparum gametocytes
The 3D7elo1-pfs16-CBG99 transgenic strain expressing the CBG99 luciferase under the pfs16 gametocyte specific promoter was used and gametocytes cultures were conducted as described [17, 18]. Methylene blue was used as positive control.
To trigger gametocytogenesis, asexual parasites cultures were diluted to 0.5% parasitaemia and medium was changed daily, to obtain a parasitaemia higher than 5% when the cultures were treated for 48-72h with N-acetylglucosamine (NAG) (Sigma-Aldrich) to clear residual asexual parasites and to obtain virtually pure gametocytes cultures. Stage IV-V gametocytes were obtained and used for the experiments after 12-14 days from the addition of NAG to the culture. The luciferase activity was taken as measure of gametocytes viability [18]. Briefly, 100 μL of culture medium were removed from each well to increase haematocrit; 70μL of resuspended culture were transferred to a black 96-well plate; 70μL of D-luciferin (1mM in citrate buffer 0·1 M, pH 5·5) were added. Luminescence measurements were performed after 10 min with 500 ms integration time using a microplate reader Synergy4 (BioTek). The results are expressed as 50% inhibitory concentrations (IC50), extrapolated from the non-linear regression analysis of the concentration–response curve. Each IC50 value is the mean ± standard deviation of at least three separate experiments performed in duplicate.
Cell cytotoxicity assays
The long-term human microvascular endothelial cell line (HMEC-1) immortalized by SV 40 large T Antigen [19] was maintained in MCDB 131 medium (Invitrogen, Milan, Italy) supplemented with 10% fetal calf serum (HyClone, Celbio, Milan, Italy), 10 ng/ml of epidermal growth factor, 1 µg/mL of hydrocortisone, 2 mM glutamine and 20 mM Hepes buffer (EuroClone). For the cytotoxicity assays, cells were treated with serial dilutions of test compounds and cell proliferation evaluated using the MTT assay already described [20]. Plates were incubated for 72 h at 37°C in 5% CO2, then 20 µL of a 5 mg/mL solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (M-2128 Sigma) in PBS was added for an additional 3 h at 37°C. The supernatants were then discarded and the dark blue formazan crystals dissolved in 100 µL of lysing buffer consisting of 20% (w/v) of a solution of SDS (Sigma), 40% of N,N dimethylformamide (Merck) in H2O, at pH 4.7 adjusted with 80% acetic acid. The plates were then read on a microplate reader (Synergy 4 Bio-Tek Instruments) at a test wavelength of 550 nm and a reference wavelength of 650 nm.
Data analysis
GraphPad Prism version 8 was used to compute the data obtained in this study and results were presented as Mean ± standard deviation (M ± SD). One-way analysis of variance (ANOVA) accompanied by Tukey’s honestly significance difference (HSD) post-hoc test was also carried out to compare mean of groups to each other. P<0.05 was considered as a significant.