Approval to conduct this study was granted by the Institute of Primate Research (IPR) Institutional Scientific and Ethics Review Committee (ISERC/02/18). The care and handling of experimental mice was done according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. The Institute of Primate Research facility is accredited by the National Commission for Science, Technology and Innovation (NACOSTI) and Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International.
Plant Collection, Extraction And Processing
Senna occidentalis roots were collected from Migori county (0.9366o S, 34.4198o E), Kenya in the months of August and September. This plant was identified by a taxonomist and a voucher specimen (38/81) deposited at the National Museums of Kenya’s East African Herbarium for future reference. Further, the plant name was verified with http://www.theplantlist.orgon10/05/2022.
The collected plant roots were air dried in the shade, ground into powder and stored in airtight plastic containers at 4oC until extraction.
Hexane, chloroform, ethyl acetate, methanol and distilled water were used for extraction by maceration. The aqueous decoction was included to mimic the traditional method of preparing the antimalarial therapy as used by the locals. The filtrates were concentrated by rotary vaporization (BÜCHI R-200 rotary evaporator) for organic solvents and lyophilization (NANBEI freeze dryer: NBJ-10-1, Zhengzhou, China) for aqueous solutions. Upon drying, the extracts were stored in sealed sample bottles at 4oC until required for use.
Plant secondary metabolites are excellent indicators of their bioactivity potential (24). As such, standard procedures were used to screen the extracts for the presence of saponins, tannins, alkaloids, flavonoids and sterols (25) in order to predict the bioactivity of S. occidentalis root.
Plasmodium falciparum propagation and extracts preparation
Senna occidentalis root extracts were tested for antiplasmodial efficacy, in vitro, using P. falciparum, strain 3D7 obtained from Kenya Medical Research Institute (KEMRI) repository. A modified version of a previously used method (26) was utilized to establish a continuous culture of these malaria parasites. In summary, the parasites were propagated at 37oC in blood group O + red blood cells (RBCs) maintained in RPMI 1640 growth medium (Life technologies Ltd., Paisley, UK) supplemented with 1 molar 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), (Gibco, Life technologies Ltd.,Paisley, UK), 1 molar Sodium hydroxide (EMD Millipore corporation, Darmstadt, Germany), 20% D-glucose (PANREAC QUIMICA SA Barcelona, Spain), 200mM L-glutamine (Gibco, Life technologies Ltd.,Paisley, UK), 10% human serum (group O+) and a gas mixture of 90% N2, 5% O2 and 5% CO2. The culture was refreshed every 48 hours at 3% haematocrit.
Dilutions of the extract and standard drug were performed as described (27), with minor modifications. To make a stock solution (5mg/ml), each extract was first dissolved in dimethyl sulfoxide (DMSO), vortexed and RPMI 1640 (incomplete medium) added to the required volume. For extract combinations, each respective extract was weighed and blended in a 1:1 ratio. A 0.5mg/ml stock solution of pyrimethamine was also prepared.
In vitro growth inhibition assay
Growth inhibition of P. falciparum malaria parasites by S. occidentalis root extract was assessed as described (28), with minor modification. A ten-fold dilution was prepared from the stock solutions using complete RPMI 1640 and serially diluted 7-fold across a 96-well cell culture plate. This provided a dose-titration range of 250µg/ml to 1.95µg/ml for the extracts and 25µg/ml to 0.20µg/ml for pyrimethamine. Sorbitol synchronized ring stage parasitized RBCs (1% parasitemia) suspended in complete RPMI at 3% hematocrit was then added to respective wells. Non-infected RBCs as well as DMSO were also included in the assay as controls. The plates were incubated at 37°C. After 72 hours, thin smears were prepared and parasites quantified by light microscopy. Percentage parasitemia suppression was computed and 50% inhibitory concentration (IC50) values determined for each extract (29, 30).
Evaluation Of The Extract For Cytotoxicity
To assess the in vitro cytotoxicity of S. occidentalis root extract, Vero cell line (sourced from KEMRI) was grown to confluent monolayer in Minimum Essential Medium Eagle (MEM) containing sodium bicarbonate and l-glutamine. The growth medium was supplemented with 1% pen-strep (Sigma), 1% HEPES (Gibco) and 10% Fetal bovine serum (FBS). The assay was performed as previously described (31, 32).
Experimental Animals And Rodent Malaria Parasites
In this study, male and female inbred BALB/c mice aged 8–9 weeks were used The rodent facility at IPR provided the animals. They were housed in standard Macron type II cages within 12 hours’ dark/light cycle at 23oC. Food and water was provided ad libitum according the IPR’s Animal Science Department standard operating procedures.
Plasmodium berghei, strain ANKA (sourced from KEMRI) was retrieved from the IPR repository and maintained in mice (33) for use in this study. This parasite strain was utilized because it causes severe disease in BALB/c mice with clinical characteristics similar to P. falciparum infection (34). Despite the phylogenetic distance between rodent and human malaria parasites, P. berghei and the human malaria parasites possess conserved genes that have over time allowed their use in Peters’ model of antimalarial drug efficacy testing (35).
Mice Infection, Treatment And Monitoring Of Parasitemia And Survivorship
A modification of the method by Ryley and Peters (1970) was used to evaluate the curative potency of the extract against P. berghei in mice (36, 37). Mice were inoculated intraperitoneally with 1×106 P. berghei infected erythrocytes and assigned randomly to 5 experimental groups (5 mice per group). At approximately 5% parasitemia, treatment was started. Group 1 was treated with 1mg/kg pyrimethamine (Sigma – Aldrich Chemie, Steinheim, Germany). Group 2 (placebo group) was administered with 1% DMSO in phosphate buffered saline (vehicle). Groups 3 and 4 were treated with 200mg/kg and 100mg/kg of the methanolic extract, respectively. Group 5 was treated with 200mg/kg of the aqueous extract. The treatment was done orally for 4 consecutive days. The oral route was chosen based on documented ethnomedical usage of the plant (22). The mice were monitored for survival till day 30 post infection. Meanwhile, parasitemia suppression was determined. The administered extract quantity was based on dose recommendations for in vivo administration of crude extracts (38) and, previous antimalarial studies regarding Senna family (5, 39).
Determination Of Parasite Growth Suppression
The effect of the extract on P. berghei growth inhibition in mice was determined as described previously (36). Tail blood was used to prepare thin smears that were fixed and observed under ×100 lens of a light microscope. The number of parasitized red blood cells (pRBCs) was examined per at least 1000 RBCs. Percentage parasitemia and parasite suppression was then computed using the following formulas (40);
Determination Of Mean Survival Time
To assess the effect of the extract on survival time of the infected mice, the number of days that each mouse lived from the day of parasite inoculation to death was recorded in a 30 days’ period. The following formula was used to determine the mean survival time of each group (41);
The collected data was recorded as means ± standard error of the means (M ± SEM). In vivo parasite suppression was analyzed through Ordinary One-Way ANOVA followed by Tukey’s multiple comparisons test at 95% confidence level (alpha = 0.05). Log rank analysis was used to compare survival time of mice in the different treatment groups. Graph Pad prism (Version 7.00, California, USA) was used for the analyses.