**1. Introduction**

Inferences into free radicals' release and their subsequent OS generation has been described as the causes and drivers of malaria [1–3]. The *Plasmodium* [4]-free radical production [5]-antioxidant defense systems [6] triumvirate axis may be elaborate in the host cell as a pathologic apparatus triggered to subside malarial infection intensity. The character of OS has scarcer shades of clarity up to date with some authors insinuating a protective facilitation against malaria disease, others claim a pathophysiologic role in the pathogenesis of the disease [6]. Studies, however, tend to associate the production of reactive oxygen (ROS) and nitrogenous species (RNS) with OS (OS) in the development of complex sequalae and systemic malarial disease and its outcomes.

Malarial parasite infection invokes hydroxyl free radical (OH˙) production by the hepatocytes which may induce OS and apoptosis of liver parenchymal cells [7]. Additionally, it has been observed that parasitized red blood cells (pRBCs) generate OH˙ radicals and H2O2 at approximately double the concentration found in nonparasitized red blood cells (npRBCs); an elicitation from the abundant intracell and endogenous redox reaction players.

Free hemoglobin (Hb), copiously available in malaria, is also a readily obtainable foundation of free radicals as the *Plasmodium* parasite uses the Fe2+-containing molecule as a fountain of amino acids crucial for its sustenance during the erythrocytic stage of disease. The main component and source of protein-bound and free Fe2+ is high levels of haeme. The haeme-Fe2+ complex induces intravascular OS with deleterious conformational changes to the red blood cells (RBC's) and the endothelial cells. Consequentially, release from pRBC's during haemolysis and subsequent internalization of malarial parasites into liver and brain tissues ensues with varied malarial syndromes presentations [8].

Generally, free haem release during cell haemolysis has a prospective capacity of increasing OS through the Fenton-Fritz Haber-Joseph Joshua Weiss reaction [9, 10] which iron catalyzes, mainly: Fe3+ + ˙O2 <sup>−</sup> → Fe2+ + O2.

The second step is the Fenton reaction: Fe2+ + H2O2 → Fe3+ + OH<sup>−</sup> + ˙OH.

Net reaction: ˙O2 <sup>−</sup> + H2O2 → ˙OH + OH<sup>−</sup> + O2.

And OS created by excess hydroxyl radicals (˙OH), superoxide (O2˙ <sup>−</sup>), reactive non-radical compounds including singlet oxygen (1 O2), hydrogen peroxide (H2O2), lipid hydroperoxides, hypochlorous acid (HOCl), chloramines (RNHCl), and ozone (O3) [11, 12] potentiates malarial infection patency and snowballing risk for fulminant disease. In the non-immune individuals, cell-mediated immune response is the initial defense mechanism which generates OS as well and subsequently, aberrant immune system response with disease traction increases.

Reactive nitrogen radical compounds such as nitric oxide (˙NO), nitrogen dioxide (˙NO2), and non-radical nitrogen-based compounds which include peroxynitrite (ONOO<sup>−</sup>) and dinitrogen trioxide (N2O3), make up the collective group of reactive nitrogen species (RNS). The unpaired electrons in their outer electron obit make these species very unstable and highly reactive. Reactive nitrogen species have direct linkages to ROS, especially in the formation of ONOO<sup>−</sup> which gives rise to nitrosative stress (NS).

The combination of OS and NS have been associated with the etiology of an extensive variety of disease processes and states to include aging, infections, ischemia-reperfusion (I/R) injury [13], acute kidney injury (AKI) and chronic kidney diseases [14], diabetic neuropathies [15], inflammatory disease [16], vascular dysfunction and hypertension [17], atherosclerosis, neurological diseases [18] including Alzheimer's disease [19, 20]. Most of these conditions and diseases are displayed as syndromes and facets of malaria disease.

Management of malaria, will of necessity therefore, require the inclusion of anti-disease remedies that will concurrently suppress or eradicate the pathophysiology associated with malarial. There have been strides to invent remedial treatment for malaria by improving the potency of current antimalarials. However, this avenue does not correspond to requirement of ameliorating the disease aspect of malaria. Phytochemicals, some in basic research stage of investigation or clinical stages, show promising outcomes that are worth promulgating, formulating and pre-empting strides towards attempts to eradicate both the parasite and the sequalae of malaria.

**19**

this chapter.

anti-disease action in malaria.

*Malarial Pathophysiology and Phytochemical Interventions: A Current Discourse on Oxidative…*

There are five main Phylum Apicomplexa (Sporozoa) *Plasmodium* strains inflicting human malarial infection with changing disease outcomes, mainly *P. falciparum*, *P. vivax*, *P. malariae*, *P. ovale* and a zoonotic parasite *P. knowlesi*. Accordingly, parasite species, sub-species, host genetics and host demographics at point of infection, determine malaria disease presentations of varying intensities [21]. A disease process and time-lines with differing syndromic mediators is generated [22, 23]. The *P. falciparum* infection has the highest fatalities with strains having developed multidrug resistance. Artemisinin derivatives are the latest additions to the rug-casualties list. *P. vivax* and *P. ovale* present chronic disease with quiescent liver stage parasite hypnozoites driving disease relapses onwards of 7 years duration from initial infection reported [24]. However, there is divergent

Malaria disease displays manifestations in adults from non-endemic areas as a different disease phenotype when compared to pregnant women and to children under the age of 5-years. Accordingly, the pathophysiology of malaria, or malaria disease [26] displays immunological idiosyncrasies, inflammatory aberrations, haemolysis which may lead to severe malaria anemia (SMA) [27], acute kidney injury (AKI) [28], and malaria cachexia leading to cardiac failure [29], hypoglycaemia [30, 31], acute respiratory distress syndrome (ARDS) [32], acute lung injury (ALI) [33], cerebral malaria [34], hyperlactaemia with non-respiratory acidosis. Of these pathophysiology, children invariably develop SMA [35], hypoglycaemia [36], hyperlactaemia with non-respiratory acidosis and cerebral malaria while adults presents with severe malaria, AKI [37] and non-respiratory acidosis [38]. Pregnant women present with placental malaria with SMA also a common feature [39]. The bottom line to all these manifestations is the OS mediation to the disease process driven by various species emanating from the parasite-human host interactions. The perceived relationship between the antioxidant capacities displayed by phytochemicals and phytotherapeutics and the oxidant-driven malaria disease motivates

**2. Malarial systemic disease and phytochemicals administration**

**3. Oxidative stress and artemisinin malarial treatment**

The terms phytotherapeutics, phytotherapeutics are commonly used in the branch of science involved in the use of plant natural products and their derivatives and their use as disease management alternatives and ameliorates. The discovery that phytochemicals like the artemisinin, asiatic acid (AA), oleanolic acid and masilinic acid (MA) have both anti-inflammatory (and other physiological influences) activity and antimalarial activities have led to the exploration of their

Artemisinin is a tetracyclic 1,2,4-trioxane containing an endoperoxide bridge

(C─O─O─C), the key pharmacophore of the antimalarial [40] (**Figure 1**). Increasing solubility and pharmacological of the drug has been achieved when semi-synthetic compounds were synthesized through modification of C10 in the

*DOI: http://dx.doi.org/10.5772/intechopen.83529*

of opinion straying from this recrudescence dogma [25].

**1.2 Phenotypic presentations of malaria**

**1.1 Malaria causes**

*Malarial Pathophysiology and Phytochemical Interventions: A Current Discourse on Oxidative… DOI: http://dx.doi.org/10.5772/intechopen.83529*

#### **1.1 Malaria causes**

*Malaria*

OH˙

however, tend to associate the production of reactive oxygen (ROS) and nitrogenous species (RNS) with OS (OS) in the development of complex sequalae and

Malarial parasite infection invokes hydroxyl free radical (OH˙) production by the hepatocytes which may induce OS and apoptosis of liver parenchymal cells [7]. Additionally, it has been observed that parasitized red blood cells (pRBCs) generate

 radicals and H2O2 at approximately double the concentration found in nonparasitized red blood cells (npRBCs); an elicitation from the abundant intracell and

Free hemoglobin (Hb), copiously available in malaria, is also a readily obtainable foundation of free radicals as the *Plasmodium* parasite uses the Fe2+-containing molecule as a fountain of amino acids crucial for its sustenance during the erythrocytic stage of disease. The main component and source of protein-bound and free Fe2+ is high levels of haeme. The haeme-Fe2+ complex induces intravascular OS with deleterious conformational changes to the red blood cells (RBC's) and the endothelial cells. Consequentially, release from pRBC's during haemolysis and subsequent internalization of malarial parasites into liver and brain tissues ensues with varied

Generally, free haem release during cell haemolysis has a prospective capacity of increasing OS through the Fenton-Fritz Haber-Joseph Joshua Weiss reaction [9, 10]

The second step is the Fenton reaction: Fe2+ + H2O2 → Fe3+ + OH<sup>−</sup> + ˙OH.

lipid hydroperoxides, hypochlorous acid (HOCl), chloramines (RNHCl), and ozone (O3) [11, 12] potentiates malarial infection patency and snowballing risk for fulminant disease. In the non-immune individuals, cell-mediated immune response is the initial defense mechanism which generates OS as well and subsequently, aberrant

Reactive nitrogen radical compounds such as nitric oxide (˙NO), nitrogen dioxide (˙NO2), and non-radical nitrogen-based compounds which include peroxynitrite (ONOO<sup>−</sup>) and dinitrogen trioxide (N2O3), make up the collective group of reactive nitrogen species (RNS). The unpaired electrons in their outer electron obit make these species very unstable and highly reactive. Reactive nitrogen species have direct linkages to ROS, especially in the formation of ONOO<sup>−</sup> which gives rise

The combination of OS and NS have been associated with the etiology of an extensive variety of disease processes and states to include aging, infections, ischemia-reperfusion (I/R) injury [13], acute kidney injury (AKI) and chronic kidney diseases [14], diabetic neuropathies [15], inflammatory disease [16], vascular dysfunction and hypertension [17], atherosclerosis, neurological diseases [18] including Alzheimer's disease [19, 20]. Most of these conditions and diseases are

Management of malaria, will of necessity therefore, require the inclusion of anti-disease remedies that will concurrently suppress or eradicate the pathophysiology associated with malarial. There have been strides to invent remedial treatment for malaria by improving the potency of current antimalarials. However, this avenue does not correspond to requirement of ameliorating the disease aspect of malaria. Phytochemicals, some in basic research stage of investigation or clinical stages, show promising outcomes that are worth promulgating, formulating and pre-empting strides towards attempts to eradicate both the parasite and the sequa-

<sup>−</sup> + H2O2 → ˙OH + OH<sup>−</sup> + O2. And OS created by excess hydroxyl radicals (˙OH), superoxide (O2˙

<sup>−</sup> → Fe2+ + O2.

<sup>−</sup>), reactive

O2), hydrogen peroxide (H2O2),

systemic malarial disease and its outcomes.

endogenous redox reaction players.

malarial syndromes presentations [8].

which iron catalyzes, mainly: Fe3+ + ˙O2

non-radical compounds including singlet oxygen (1

immune system response with disease traction increases.

displayed as syndromes and facets of malaria disease.

Net reaction: ˙O2

to nitrosative stress (NS).

**18**

lae of malaria.

There are five main Phylum Apicomplexa (Sporozoa) *Plasmodium* strains inflicting human malarial infection with changing disease outcomes, mainly *P. falciparum*, *P. vivax*, *P. malariae*, *P. ovale* and a zoonotic parasite *P. knowlesi*. Accordingly, parasite species, sub-species, host genetics and host demographics at point of infection, determine malaria disease presentations of varying intensities [21]. A disease process and time-lines with differing syndromic mediators is generated [22, 23]. The *P. falciparum* infection has the highest fatalities with strains having developed multidrug resistance. Artemisinin derivatives are the latest additions to the rug-casualties list. *P. vivax* and *P. ovale* present chronic disease with quiescent liver stage parasite hypnozoites driving disease relapses onwards of 7 years duration from initial infection reported [24]. However, there is divergent of opinion straying from this recrudescence dogma [25].

#### **1.2 Phenotypic presentations of malaria**

Malaria disease displays manifestations in adults from non-endemic areas as a different disease phenotype when compared to pregnant women and to children under the age of 5-years. Accordingly, the pathophysiology of malaria, or malaria disease [26] displays immunological idiosyncrasies, inflammatory aberrations, haemolysis which may lead to severe malaria anemia (SMA) [27], acute kidney injury (AKI) [28], and malaria cachexia leading to cardiac failure [29], hypoglycaemia [30, 31], acute respiratory distress syndrome (ARDS) [32], acute lung injury (ALI) [33], cerebral malaria [34], hyperlactaemia with non-respiratory acidosis. Of these pathophysiology, children invariably develop SMA [35], hypoglycaemia [36], hyperlactaemia with non-respiratory acidosis and cerebral malaria while adults presents with severe malaria, AKI [37] and non-respiratory acidosis [38]. Pregnant women present with placental malaria with SMA also a common feature [39]. The bottom line to all these manifestations is the OS mediation to the disease process driven by various species emanating from the parasite-human host interactions. The perceived relationship between the antioxidant capacities displayed by phytochemicals and phytotherapeutics and the oxidant-driven malaria disease motivates this chapter.

### **2. Malarial systemic disease and phytochemicals administration**

The terms phytotherapeutics, phytotherapeutics are commonly used in the branch of science involved in the use of plant natural products and their derivatives and their use as disease management alternatives and ameliorates. The discovery that phytochemicals like the artemisinin, asiatic acid (AA), oleanolic acid and masilinic acid (MA) have both anti-inflammatory (and other physiological influences) activity and antimalarial activities have led to the exploration of their anti-disease action in malaria.

#### **3. Oxidative stress and artemisinin malarial treatment**

Artemisinin is a tetracyclic 1,2,4-trioxane containing an endoperoxide bridge (C─O─O─C), the key pharmacophore of the antimalarial [40] (**Figure 1**). Increasing solubility and pharmacological of the drug has been achieved when semi-synthetic compounds were synthesized through modification of C10 in the

#### **Figure 1.** *Artemisinin, structure of the endoperoxides [42].*

**Figure 2.** *Dihydroartemisinin, structure of the endoperoxides [42].*

original backbone to generate hemi-acetal or ester derivative such as dihydroartemisinin (**Figure 2**), artemether (**Figure 3**) and artesunate (**Figure 4**) [41].

The debate remains unresolved on the mode(s) of activation and consequent biological target(s) of endoperoxides [43]. Activation of the endoperoxide bridge is believed to be the source of artemisinin antimalarial activity. Cleavage of the bridge, which is located at the core of the structure, generates short-lived cytotoxic oxyradicals in the presence of haem iron or free iron Fe2+ [44, 45]. However, two different mechanisms of action premised on the endoperoxide bioactivation, have been proposed.

Rearrangement of the oxygen-centred radicals, to produce more stable carboncentred radicals, have been hypothesized by the Poster Laboratory using 18O-labeled trioxane analogues [46, 47]. The 'reductive scission' model, has ferrous iron binding to either O1 or O2 cleaving the endoperoxide bond and generating oxyradical intermediates which subsequently rearrange to primary or secondary carbon-centred radicals via either b-scission or a [1,5]-H shift. This hypothesis has been supported through evidence of the formation of these carbon-centred radical intermediates using electron paramagnetic resonance spin-trapping techniques [48, 49].

**21**

**Figure 3.**

**Figure 4.**

*Malarial Pathophysiology and Phytochemical Interventions: A Current Discourse on Oxidative…*

Capabilities of the C-centred radicals to drive haem and or proteins alkylation have been proposed. However, the only evidence, so far provided, has been for haem alkylation [50] and a few reported model studies on protein alkylation with ferrous

The concept of free radical generation and protein alkylation points towards creation of OS as the parasite killing apparatus of the artemisinin derivatives but also indicates the deleterious effect of the drugs on the human host and possibility of initiating or exacerbating malarial disease in the course antiparasitic activity. However, paradoxically artemisinin has been reported to be pluripotent with anti-inflammatory activity [52] although post treatment artemisinin haemolysis has also been observed in people that would have been cleared of malaria. The latter adverse reaction has been seen several weeks after successful treatment with artemisinin drugs.

Artemisinin and its derivatives require conversion to the biologically active dihydroartemisinin (DHA **Figure 2**) to exert their activity. Besides the excellent antimalarial effects, there is clinical and experimental evidence that suggests potent

salts reactions in the presence of cysteine (iron-sulfur chelates) [51].

**3.1 Artemisinin and anti-oxidative stress disease inductions**

*DOI: http://dx.doi.org/10.5772/intechopen.83529*

*Artemether, structure of the endoperoxides [42].*

*Artesunate, structure of the endoperoxides [42].*

*Malarial Pathophysiology and Phytochemical Interventions: A Current Discourse on Oxidative… DOI: http://dx.doi.org/10.5772/intechopen.83529*

**Figure 3.** *Artemether, structure of the endoperoxides [42].*

#### **Figure 4.**

*Malaria*

**Figure 1.**

*Artemisinin, structure of the endoperoxides [42].*

*Dihydroartemisinin, structure of the endoperoxides [42].*

**20**

been proposed.

**Figure 2.**

original backbone to generate hemi-acetal or ester derivative such as dihydroartemisinin (**Figure 2**), artemether (**Figure 3**) and artesunate (**Figure 4**) [41].

The debate remains unresolved on the mode(s) of activation and consequent biological target(s) of endoperoxides [43]. Activation of the endoperoxide bridge is believed to be the source of artemisinin antimalarial activity. Cleavage of the bridge, which is located at the core of the structure, generates short-lived cytotoxic oxyradicals in the presence of haem iron or free iron Fe2+ [44, 45]. However, two different mechanisms of action premised on the endoperoxide bioactivation, have

Rearrangement of the oxygen-centred radicals, to produce more stable carboncentred radicals, have been hypothesized by the Poster Laboratory using 18O-labeled trioxane analogues [46, 47]. The 'reductive scission' model, has ferrous iron binding to either O1 or O2 cleaving the endoperoxide bond and generating oxyradical intermediates which subsequently rearrange to primary or secondary carbon-centred radicals via either b-scission or a [1,5]-H shift. This hypothesis has been supported through evidence of the formation of these carbon-centred radical intermediates using electron paramagnetic resonance spin-trapping techniques [48, 49].

*Artesunate, structure of the endoperoxides [42].*

Capabilities of the C-centred radicals to drive haem and or proteins alkylation have been proposed. However, the only evidence, so far provided, has been for haem alkylation [50] and a few reported model studies on protein alkylation with ferrous salts reactions in the presence of cysteine (iron-sulfur chelates) [51].

The concept of free radical generation and protein alkylation points towards creation of OS as the parasite killing apparatus of the artemisinin derivatives but also indicates the deleterious effect of the drugs on the human host and possibility of initiating or exacerbating malarial disease in the course antiparasitic activity. However, paradoxically artemisinin has been reported to be pluripotent with anti-inflammatory activity [52] although post treatment artemisinin haemolysis has also been observed in people that would have been cleared of malaria. The latter adverse reaction has been seen several weeks after successful treatment with artemisinin drugs.

#### **3.1 Artemisinin and anti-oxidative stress disease inductions**

Artemisinin and its derivatives require conversion to the biologically active dihydroartemisinin (DHA **Figure 2**) to exert their activity. Besides the excellent antimalarial effects, there is clinical and experimental evidence that suggests potent immune-suppressive against autoimmune and allergic disease of artemisinin and its derivatives [52]. Derivatives of artemisinin possessing lower range toxicity, higher bioavailability, and compelling immunosuppressive activity have been studied and some commercialized. These are 3-(12--artemisininoxy) phenoxyl succinic acid (SM735) [53], 1-(12--dihydroartemisinoxy)-2-hydroxy-3-tert-butylaminopropane maleate (SM905) [54, 55], ethyl 2-[4-(12--artemisininoxy)] phenoxylpropionate (SM933) [56], and 2′-aminoarteether ()maleate (SM934) [57, 58].

To date, it is generally accepted that artemisinin wields antimalarial properties through (i) haeme or free iron breaking the peroxide bridge resulting in the degradation of artemisinin molecular structure to form the nucleophilic radical metabolite with the centre at C4. (ii) subsequently, the free radical, acting as an alkylating agent, will attack macromolecular bearing electrophilic groups or centres, which ultimately leads to parasitic demise [50, 59]. In fact, the pRBC's have increased concentrations of OS due to the parasitic infection by *Plasmodium*. In the intervening time, OS driven free radicals and lipid peroxidation concentrations dramatically increase intracellularly. Apparently, pRBC's are rendered more susceptible to artemisinin than npRBC's. In vivo artemisinin is effective in killing pRBC's at nM concentrations which differs sharply when contrasted to marginal effects of artemisinin on resting RBC's even at high mM concentrations [60, 61].

#### **3.2 Anti-inflammation and immunoregulatory effect of artemisinin**

There are three fundamental steps by which T cells perform pivotal role in acquired immune reaction [62, 63]: (i) G0 to G1 transition of T cells is driven by TCR cross-linking which leads to the secretion of T cell growth factor IL-2 and expression of high-affinity receptor IL-2R chain (CD25); (ii) through autocrine and or paracrine proliferative loop, IL-2 influences clone expansion and maintains activated T cells survival; (iii) after efficacious clearance of the pathogen, the inducement for cytokines production is lost and activated T cells will undergo apoptosis.

Nevertheless, in autoimmune diseases, due to the tenacity of autoantigen, autoreactive T cells will be activated and with better survival [52]. Autoreactive T cell proliferation is involved in the pathogenesis of various immune-related diseases, such as rheumatoid arthritis (RA) and multiple sclerosis (MS) [20, 21] as well as malaria.

Artemether is a powerful antimalarial drug [64] found to significantly suppress the proliferation and synthesis of IL-2 and interferon- (IFN-) by T cells through the TCR engagement influence [65]. The TCR engagement-triggered MAPKs signaling pathway as well as phosphorylation of ERK1/2, Jnk, and P38 were significantly inhibited by artemether. Discovery was made that artemether greatly affects T cells function as compared to that of the antigen presenting cells (APCs) to exert the immunosuppressive effects [65].

A series of artemisinin derivatives, with higher water solubility and lower toxicity, have been created by inserting, to the parent artemisinin structure, new functional groups like ethylene glycol [66, 67]. These have immunosuppressive targeted at T cell activation suppression, combat inflammation through substantial inhibition of the proliferation and production of IFN-, IL-12 and IL-6. While there is no direct influence of artemether and its derivatives on IL-2 and CD25 upregulation of T cells, there is remarkable suppression of IL-2-mediated proliferation and survival of activated T cells alluding to blocking of IL-2 induced phosphorylation of Akt [68]. Additionally, artemether derivative SM934-driven preferential early apoptosis activated of T cells, with no effect on resting T cells, has been observed through staining of CD69 and annexin V.

**23**

*Malarial Pathophysiology and Phytochemical Interventions: A Current Discourse on Oxidative…*

to inhibit phosphodiesterase activity, which causes the increase of intracellular cAMP concentration, and therefore to exert the immunosuppressive activity have

Furthermore, studies suggesting that artemisinin derivatives bind to calmodulin

Additionally, oral treatment of SM905 has been shown to skew the T cell subset from pathogenic Th17 to protective Th2 subset in an arthritic model with increased IL-4 production and suppression of the RORt mRNA expression together with IL-17 production [71]. The artemisinin derivatives' effects hinge on the antiinflammatory properties which play an antimalarial role. To back this assertion up, artesunate has minor effects on inflammatory responses downstream of antibody production demonstrating that highly proliferative germinal centre B cells are the most sensitive cellular targets to the treatment. Significantly, *in vitro* artesunate inhibits IL-1, IL-6, and IL-8 production by way of stimulation by TNF-α as well as the expression of vascular endothelial growth factor and hypoxia-inducible factor-1 [72]. Moreover, artesunate inhibits Akt phosphorylation and IB degradation by blocking PI3K/Akt signaling pathway downstream of TNF- [73] making it an efficient adjunct to malarial inflammatory response characterized by increased

The intriguing anti-inflammatory properties of the artemisinin (SM933) are observed through the regulatory mechanism involving the NFKB and Rig-G/JAB1 pathways which regulation alters cell cycle activity of activated T cells selectively. In contrast to SM933, SM934 and DHA treatment is majorly through the regulation of the balance between effector T cells and regulatory T cells. Administering of DHA significantly decreases effectors CD4 T cells and increases in Treg cells in a reciprocal regulatory process through modulation of the mTOR pathway which character-

Macromolecules bearing electrophilic groups or centres are prone to alkylating nucleophilic radicals, metabolites of artemisinin, which eventually leads to cell damages [61]. Furthermore, artemisinin inhibits endoplasmic reticulum Ca2+ ATPase (SERCA) with consequent cytoplasmic calcium accumulation and second-

*P. falciparum* cellular apoptosis [75]. The same effect has been shown by thapsigargin (TG), a specific SERCA inhibitor with structural similarity to artemisinin, which induces cellular calcium accumulation leading to apoptosis. While artemisinin and TG have the same binding sites on SERCA, there are structural biology differences in the binding pocket for different mammalian and *Plasmodium* species resultantly conferring differential susceptibility to the drug. A single amino acid (Leu263) in the transmembrane segment 3 of SERCA in *P. vivax* SERCA (PvCERCA) has a 3-fold sensitivity to artemisinin while introduction of the same residue in *P. berghei*

Interestingly, while the peroxide bridge plays a necessary role in the artemisinin biological activity, artemisinin-SERCA binding does not involve this moiety [77] but play a catalytic role in the inhibition [52]. Naturally, stereochemistry and transitional state theory have it that the intact peroxide bridge makes the spatial configuration of artemisinin to be relatively rigid and making the sesquiterpene lactone unable to flexibly rotate and fold. This results in lower affinity for SERCA. Reduction and breaking of the peroxide bridge by divalent iron ion, however, releases the sesquiterpene lactone increasing its flexibility and binding affinity to SERCA enhancing inhibitory effect of artemisinin on the enzyme. Moreover, the concentration of Fe2+ in pRBC's and activated lymphocytes is significantly increased

*DOI: http://dx.doi.org/10.5772/intechopen.83529*

and decreased Th1 and Th2 type cytokines, respectively.

**3.3 Artemisinin structure: activity relationship in oxidative stress**

ary activation of cellular calcium influx. In this way artemisinin induces

SERCA (PbSERCA) decreases sensitivity 3-fold [75, 76].

izes its regulatory mechanism [56, 74].

been reported [69, 70].

*Malarial Pathophysiology and Phytochemical Interventions: A Current Discourse on Oxidative… DOI: http://dx.doi.org/10.5772/intechopen.83529*

Furthermore, studies suggesting that artemisinin derivatives bind to calmodulin to inhibit phosphodiesterase activity, which causes the increase of intracellular cAMP concentration, and therefore to exert the immunosuppressive activity have been reported [69, 70].

Additionally, oral treatment of SM905 has been shown to skew the T cell subset from pathogenic Th17 to protective Th2 subset in an arthritic model with increased IL-4 production and suppression of the RORt mRNA expression together with IL-17 production [71]. The artemisinin derivatives' effects hinge on the antiinflammatory properties which play an antimalarial role. To back this assertion up, artesunate has minor effects on inflammatory responses downstream of antibody production demonstrating that highly proliferative germinal centre B cells are the most sensitive cellular targets to the treatment. Significantly, *in vitro* artesunate inhibits IL-1, IL-6, and IL-8 production by way of stimulation by TNF-α as well as the expression of vascular endothelial growth factor and hypoxia-inducible factor-1 [72]. Moreover, artesunate inhibits Akt phosphorylation and IB degradation by blocking PI3K/Akt signaling pathway downstream of TNF- [73] making it an efficient adjunct to malarial inflammatory response characterized by increased and decreased Th1 and Th2 type cytokines, respectively.

The intriguing anti-inflammatory properties of the artemisinin (SM933) are observed through the regulatory mechanism involving the NFKB and Rig-G/JAB1 pathways which regulation alters cell cycle activity of activated T cells selectively. In contrast to SM933, SM934 and DHA treatment is majorly through the regulation of the balance between effector T cells and regulatory T cells. Administering of DHA significantly decreases effectors CD4 T cells and increases in Treg cells in a reciprocal regulatory process through modulation of the mTOR pathway which characterizes its regulatory mechanism [56, 74].

#### **3.3 Artemisinin structure: activity relationship in oxidative stress**

Macromolecules bearing electrophilic groups or centres are prone to alkylating nucleophilic radicals, metabolites of artemisinin, which eventually leads to cell damages [61]. Furthermore, artemisinin inhibits endoplasmic reticulum Ca2+ ATPase (SERCA) with consequent cytoplasmic calcium accumulation and secondary activation of cellular calcium influx. In this way artemisinin induces *P. falciparum* cellular apoptosis [75]. The same effect has been shown by thapsigargin (TG), a specific SERCA inhibitor with structural similarity to artemisinin, which induces cellular calcium accumulation leading to apoptosis. While artemisinin and TG have the same binding sites on SERCA, there are structural biology differences in the binding pocket for different mammalian and *Plasmodium* species resultantly conferring differential susceptibility to the drug. A single amino acid (Leu263) in the transmembrane segment 3 of SERCA in *P. vivax* SERCA (PvCERCA) has a 3-fold sensitivity to artemisinin while introduction of the same residue in *P. berghei* SERCA (PbSERCA) decreases sensitivity 3-fold [75, 76].

Interestingly, while the peroxide bridge plays a necessary role in the artemisinin biological activity, artemisinin-SERCA binding does not involve this moiety [77] but play a catalytic role in the inhibition [52]. Naturally, stereochemistry and transitional state theory have it that the intact peroxide bridge makes the spatial configuration of artemisinin to be relatively rigid and making the sesquiterpene lactone unable to flexibly rotate and fold. This results in lower affinity for SERCA. Reduction and breaking of the peroxide bridge by divalent iron ion, however, releases the sesquiterpene lactone increasing its flexibility and binding affinity to SERCA enhancing inhibitory effect of artemisinin on the enzyme. Moreover, the concentration of Fe2+ in pRBC's and activated lymphocytes is significantly increased

*Malaria*

immune-suppressive against autoimmune and allergic disease of artemisinin and its derivatives [52]. Derivatives of artemisinin possessing lower range toxicity, higher bioavailability, and compelling immunosuppressive activity have been studied and some commercialized. These are 3-(12--artemisininoxy) phenoxyl succinic acid (SM735) [53], 1-(12--dihydroartemisinoxy)-2-hydroxy-3-tert-butylaminopropane maleate (SM905) [54, 55], ethyl 2-[4-(12--artemisininoxy)] phenoxylpropionate

To date, it is generally accepted that artemisinin wields antimalarial properties through (i) haeme or free iron breaking the peroxide bridge resulting in the degradation of artemisinin molecular structure to form the nucleophilic radical metabolite with the centre at C4. (ii) subsequently, the free radical, acting as an alkylating agent, will attack macromolecular bearing electrophilic groups or centres, which ultimately leads to parasitic demise [50, 59]. In fact, the pRBC's have increased concentrations of OS due to the parasitic infection by *Plasmodium*. In the intervening time, OS driven free radicals and lipid peroxidation concentrations dramatically increase intracellularly. Apparently, pRBC's are rendered more susceptible to artemisinin than npRBC's. In vivo artemisinin is effective in killing pRBC's at nM concentrations which differs sharply when contrasted to marginal effects of

(SM933) [56], and 2′-aminoarteether ()maleate (SM934) [57, 58].

artemisinin on resting RBC's even at high mM concentrations [60, 61].

**3.2 Anti-inflammation and immunoregulatory effect of artemisinin**

There are three fundamental steps by which T cells perform pivotal role in acquired immune reaction [62, 63]: (i) G0 to G1 transition of T cells is driven by TCR cross-linking which leads to the secretion of T cell growth factor IL-2 and expression of high-affinity receptor IL-2R chain (CD25); (ii) through autocrine and or paracrine proliferative loop, IL-2 influences clone expansion and maintains activated T cells survival; (iii) after efficacious clearance of the pathogen, the inducement for cytokines production is lost and activated T cells will undergo

Nevertheless, in autoimmune diseases, due to the tenacity of autoantigen, autoreactive T cells will be activated and with better survival [52]. Autoreactive T cell proliferation is involved in the pathogenesis of various immune-related diseases, such as rheumatoid arthritis (RA) and multiple sclerosis (MS) [20, 21] as well as

Artemether is a powerful antimalarial drug [64] found to significantly suppress the proliferation and synthesis of IL-2 and interferon- (IFN-) by T cells through the TCR engagement influence [65]. The TCR engagement-triggered MAPKs signaling pathway as well as phosphorylation of ERK1/2, Jnk, and P38 were significantly inhibited by artemether. Discovery was made that artemether greatly affects T cells function as compared to that of the antigen presenting cells (APCs) to exert the

A series of artemisinin derivatives, with higher water solubility and lower toxicity, have been created by inserting, to the parent artemisinin structure, new functional groups like ethylene glycol [66, 67]. These have immunosuppressive targeted at T cell activation suppression, combat inflammation through substantial inhibition of the proliferation and production of IFN-, IL-12 and IL-6. While there is no direct influence of artemether and its derivatives on IL-2 and CD25 upregulation of T cells, there is remarkable suppression of IL-2-mediated proliferation and survival of activated T cells alluding to blocking of IL-2 induced phosphorylation of Akt [68]. Additionally, artemether derivative SM934-driven preferential early apoptosis activated of T cells, with no effect on resting T cells, has been observed

**22**

apoptosis.

malaria.

immunosuppressive effects [65].

through staining of CD69 and annexin V.

compared to resting state cells increasing the opportunity for peroxide bridge to be broken. In this manner, the pRBC's and activated leukocytes, rather than npRBC's and resting cells, become more vulnerable to artemisinin activity. Nevertheless, the mammalian SERCA is not susceptible to artemisinin inhibition [75]. Therefore, the biochemical mechanism and artemisinin molecular target to exert immunosuppression in malaria and other disease driven by OS requires further perusal and investigations.
