Recent progress in antimalarial drug research has prompted the search for potent chemotherapeutic agents within the phytochemical sanctuary, particularly following the discovery of artemisinins; a potent antimalarial phytochemical discovered in Artemisia annua . Artemisinin is now recognized as one of the conventional antimalarials, moreover Artemisinin-based combination therapy (ACTs) is now the front-line treatment for P. falciparum instead of mono-therapies, especially in the regions wherein multidrug resistant strains of P. falciparum persist .
It is worthy of mention that several phytochemicals have been investigated for their anti-plasmodial activity in the past, however, the majority of these phytochemicals failed to produce considerable antimalarial effects comparable to that of existing antimalarial drugs. Nevertheless, the possibility exists of developing therapeutically active pharmacophores from phytochemical precursors. In addition to a compound’s antimalarial activity its selectivity toward the parasite and safety to individuals infected with malaria are also of major importance . Several studies fail to assess the time-dependent effects of pharmacologically active antimalarial principles derived from phytochemicals on the progression of various stages of the plasmodium parasite’s reproductive cycle within erythrocytes or incidence of morphologic and dimensional changes on different stages of the parasite .
This study screened the anti-plasmodial activity of AG at different time points during the intraerythrocytic cycle of P. falciparum 3D7 while simultaneously determining its impact on the development of different stages of the parasite, its morphology, and related dimensional characteristics. AG has previously been reported to exert anti-plasmodial activity against P. falciparum 3D7 [20, 21]. Furthermore, Megantara et al., (2015) reported the promising potential of AG as an effective pharmacophore for developing new antimalarial drugs  .
In this experiment, the pure compound of AG showed a good anti-plasmodial activity (IC50 = 4148.61 nM). However, the anti-plasmodial activity of AG was about 1000-fold less than that of chloroquine. This suggests that further structural modifications could possibly be beneficial towards improving its anti-plasmodial activity. Furthermore, the results obtained corroborate previous observations reported by Zaid et al., . The anti-plasmodial activity of AG was attributed to its inhibitory effect against plasmepsins I, II, and IV by Megantara et al., (2015) . Plasmepsin is an aspartate protease enzyme present abundantly in the acidic digestive vacuoles of Plasmodium . It takes part in the degradative pathway that breaks down globin into essential amino acids. It is well known that Plasmodium relies on haemoglobin as its sole source of protein . Haemoglobin is broken down by hemoglobinase enzymes into globin and heme, where the former is subjected to a series of degradative aspartate proteases to be broken down into its essential amino acids . This pathway was also suggested as a candidate target for drugs used in malaria chemotherapy .
It is well known that CQ produces its effect by targeting the haemoglobin degradation pathway. Its mechanism of action is slightly different from that proposed for AG. It interferes with pathways that detoxify heme, an obnoxious waste product that is released after the breakdown of haemoglobin. It is detoxified inside the digestive vacuoles of plasmodia into hemozoin; an innocuous inert waste product of heme. Consequently, inhibition of heme detoxification results in its accumulation and induction of a cascade of heme-induced oxidative pathways .
In Plasmodium, haemoglobin degradation pathway starts during the ring stage and continues throughout early and mature trophozoite development. Its activity reaches its utmost level during the early trophozoite stage within the period of 6 to 12 h after cultivation of the synchronized ring stage .
The results showed that most of the parasiticidal effect due to chloroquine was observed within the first 12 h of the cycle wherein the ring stage predominated (Figs. 1 and 2). This finding is in good agreement with a report by Zhang (1986)  describing the time-dependent effect of CQ during the course of the intraerythrocytic cycle. Furthermore, CQ produced noticeable morphological changes during this period on both rings and trophozoites. These changes were characterized by cellular shrinkage, cellular disfiguration, and loss of chromatin materials (Fig. 3). In addition, morphological changes were absent at the schizont stage in agreement with reports by Zhang et al., (1986). Overall, morphological changes were observable and persisted up to the end of the cycle indicating that CQ compromised the cellular activity of the mature trophozoites without inducing prominent lethality.
The stage wherein breakdown of haemoglobin and release of heme commences is still being debated. Some reports have pointed out to the possible incidence of this pathway during the ring stage , while its maximum incidence occurs during early trophozoite development . This suggests that the maximum effect of CQ should occur during the trophozoite, not the ring stage. The study contradicts this notion as it emphasizes that a prominent anti-plasmodium effect for CQ can be observed before the trophozoite stage and during the ring stage. Nevertheless, there are studies reporting that inhibition of heme detoxification and release of heme is not the only mechanism of anti-plasmodial activity of CQ. Some studies declared that CQ might interfere with the integrity of lysosomal membranes resulting in the increased release of degrading enzymes like caspase . This, in turn, may affect the mitochondrial membrane and increase the release of cyt C, which in turns triggers cellular apoptosis .
Andrographolide failed to produce similar changes as compared to CQ during the first 12 h. Its impact was however notable after 12 h of the cycle, wherein dead parasites and morphologic changes in intact cells were observed (Figs. 3 and 4). However, these changes were less prominent as compared to those induced by CQ. This is because AG failed to produce noticeable morphological changes during the early stages of the parasite inoculation and did not exhibit any effect on cellular viability during the late stages of inoculation. Also, the delayed lethality can be attributed to its impact on targets that showed their utmost expression during this stage. For instance, AG was suggested as a potent inhibitor of the plasmepsin enzyme , which is involved in haemoglobin breakdown .
Andrographolide is derived from a labdane diterpenoid (a terpene derivative), Goulart et al., (2004) elucidated that some terpene compounds (nerolidol, farnesol, and linalool) show inhibitory effect(s) on the isoprenoid biosynthesis of trophozoite and schizont of P. falciparum . Furthermore, the mono-terpenes have been reported to inhibit the growth of P. falciparum by inhibition of protein prenylation . From these, it was surmised that AG might have exerted a negative impact on the functional characters of isoprenoid synthesis pathway, which begins in apicoplasts. Apicoplasts are relict organelles that occur as parts of the cellular structure of apicomplexan protozoa. They (apicoplasts) possess several functions ranging from pathways inavolved in fatty acid, isoprenoid, iron-sulphur, and heam biosynthesis . The isoprenoid synthesis pathway is considered the most important pathway within mature trophozoites and can be set as an essential drug target for malaria chemotherapy . This pathway is essential, as it fuels the cell with the required energy .
After commencement of the synchronized cycle, the results showed that the number of viable parasites subsequent to CQ exposure was decreased compare to the control, moreover, this number remained the same after 24 h as compared to that after 12 h. In the drug sensitivity assay, only a small decline in the inhibitory growth parameters was observed. This was attributed to the pLDH assay being performed at the same time as the drug sensitivity assay, which overestimates the parasitemia when measuring the enzyme activity of non-viable parasites. The number of viable cells decreased prominently as per the results of the Giemsa-stained thin blood film, but the changes in the growth inhibitory parameters as per the results of the sensitivity assay were less. The pLDH enzyme is involved in the conversion of lactate to pyruvate, and helps in the release of energy in the form of Adenosine Triphosphate ATP. Its activity may persist even after parasite death, and is responsible for the overestimation of parasite growth while using the well-known pLDH technique [34, 35], unlike SYBR Green I Malaria Drug Sensitivity Assay: which detect the existence of malaria DNA of the parasite inside infected erythrocytes .
A similar situation was observed subsequent to AG exposure. However, in this case, dead parasites were absent during the first 12 h. whereas, the dead parasites started to appear after 24 h of drug exposure. This implies that the anti-plasmodial activity of AG began 12 h after commencement of the cycle. Similarly, inhibitory growth parameters due to AG were prominently lesser after 48 h as compared to that at 24 h. However, results of Giemsa-stained thin blood smears for AG treated flasks showed a comparable number of the parasites after 24 and 48 h. The morphological features of the cells exposed to CQ suggested the incidence of necrosis and apoptosis within the parasites. Meanwhile, the death crisis features of the AG-treated flasks were suggestive of a mild form of apoptosis. The incidence of apoptosis is expected when the mitochondrial membrane starts losing its integrity, resulting in the release of cytochrome C and other apoptosis initiators into the cytosol. This, in turn, induces a cascade of the apoptotic pathway that is characterized by the activation of caspase enzymes and loss of cellular content . Induction of the apoptosis pathway was detected for AG in most cancer mammalian cells models . In addition to that, Luo et al., (2013) mentioned that during treatment of lung cancer cells, the nuclear transcription factor-kappa B (NF-κB) targets inhibition using AG . However, further studies are warranted to elucidate the role of AG in apoptotic pathways in the context of malaria.