Evaluating the Time Dependent Anti-plasmodium Activity of Andrographolide and Chloroquine on Different Stages of the Intraerythrocytic Cycle of Plasmodium Falciparum 3D7 in Vitro (original) (raw)

Background: The increasing incidence of drug resistance among various strains of Plasmodium falciparum has compelled researchers to search for new improved therapeutic alternatives to current antimalarials. Consequently, the study aimed to investigate the effect of varying the duration of andrographolide exposure on its antiplasmodial effect against intra erythrocytic stages of the P. falciparum 3D7 parasite. Although andrographolide has demonstrated prior anti-plasmodial effect against P. falciparum 3D7, its time-dependent effect subsequent to different durations of drug exposure in addition to the impact of relevant pharmacologically active concentrations on the cellular morphology of various intraerythrocytic stages of the P. falciparum 3D7 parasite cycle are limited. Methods: P. falciparum 3D7 parasites cultivated in vitro in blood cultures were individually incubated with different concentrations of andrographolide, chloroquine and drug-free parasite culture which served as the representative control. Suppression of parasite growth was determined by parasite lactate dehydrogenase (pLDH) based drug sensitivity assay. The inhibition of parasite growth and changes in morphology of intraerythrocytic parasites subsequent to treatment initiation with andrographolide or chloroquine were assessed upon commencement of a synchronized cycle at 12, 24 and 48 h respectively. Results: Andrographolide showed satisfactory growth inhibitory effect however its inhibitory activity was substantially lower when compared to that of chloroquine. Unlike chloroquine which showed maximal inhibitory activity within the rst 12 h of the cycle, suppression of parasite growth by andrographolide was most prominent during the development of early trophozoites (viz the second 12 hours). Andrographolide failed to produce any effect on the morphology of ring stage parasites, it however produced a noticeable change in the morphological appearance and sizes of mature trophozoites. Whereas, with chloroquine notable changes to ring and trophozoite stages of the parasites were evident. Conclusion: The data obtained indicates the potential role of andrographolide as an adjunctive treatment in malaria subject to further clinical evaluations. malaria caused by P. vivax or P. ovale [5]. Both of the afcorementioned species have hepatic schizonts that may persist for long intervals of time intra-hepatically [6]. The discovery of novel antimalarial principles from plant sources in the past coupled with the emergence of drugresistance among species of P. falciparum to current antimalarials has prompted researchers to extensively explore phytochemicals as potential sources of anti-malarial pharmacophore candidates against antigenically variant malaria parasites as potential substitutes for the conventional anti-malarial drugs. Ethnomedicinal plants offer potential as a source of viable cost-effective anti-malarial principles in the realm of malaria chemotherapy particularly in underprivileged populations where access to healthcare facilities is limited. Andrographolide (AG) is a labdane diterpenoid derivative present abundantly in the herbaceous plant Andrograpcchis paniculata which is extensively cultivated in Southern Asia, China and some parts of Europe [7]. AG is an active principle of A. paniculate, and several publications have appeared in recent years highlighting the biological characteristics of AG including its antimicrobial, anti-in ammatory and antioxidant properties [8, 9]. The in vitro and in vivo anti-plasmodial activity of andrographolide was screened previously by Mishra et al., [10]. Additionally, Zaid et al., (2015) [11] reported the probable mechanism of action of AG as via permeation pathways channels visible on the membrane of infected RBCs as well as its impact on merozoite invasion. However, the impact of AG against different stages of the plasmodium parasite has not been su ciently explored. This study aimed to determine the time-dependent effect(s) of AG and CQ on the various stages of the P. falciparum 3D7 parasite's intraerythrocytic cycle (as a means to assess the stage of the parasite's reproductive cycle wherein maximum antiparasitic effect can be achieved) in addition to its impact on size and morphology of parasite forms (variants) constituting the intraerythrocytic cycle. Methods Red blood cells Uninfected RBCs (type O-negative) was donated by the rst author under the supervision of a haematologist. The blood was mixed with citrate phosphate buffer as anticoagulant (in the ratio of 1:9 anticoagulant/ blood); the blood was subsequently washed thrice using washing medium to remove plasma and white blood cells and resuspended to obtain a suspension of RBCs. The washing medium contained RPMI-1640, 25 mM HEPES (4-(2hydroxyethyl)-1-piperazine-ethan-sulphonic acid) buffer (pH 7.4), 24 mM sodium bicarbonate, 11 mM glucose and 50 µg/L gentamicin. The standard protocol for blood washing was adopted as previously described [12]. Chemicals and consumables Human O-erythrocytes were pelleted from blood suspended in RPMI-1640 medium.