Over 500 million cases of malaria are reported annually among the world’s poorest populations. Millions of children die each year in Africa alone. The parasite that causes the most deadly form of malaria, plasmodium falciparum, is spread by the highly prevalent mosquitos’ anopheles gambiae and anopheles. funestus. After decades of neglect, funding from the international community to fight malaria has increased substantially in recent years (Johansson E.W et al., 2010). Increased funding has supported the scale-up of malaria control interventions such as the procurement and distribution of artemisinin-based combination therapy (ACT), the anti-malarial drug class of choice, and insecticide-treated bed nets (ITNS), as well as other mosquito vector control strategies. In certain areas of Africa, these interventions have been linked temporally to recent declines in the incidence of malaria of more than 50% (O’meara W.P, 2009). However, the incidence of malaria in other areas of Africa and other regions of the world, such as Amazonia, is static or increasing. Unfortunately, the widespread implementation of ACTS and ITNS is hampered by the poor health care infrastructure of many malaria-endemic countries. Moreover, plasmodium falciparum has proven adept at acquiring and rapidly spreading resistance to anti-malarial drugs, and even now resistance may have been acquired in Asia to the artemisinin derivatives (Dondrop A.M et al., 2010). Vector control is also threatened by the inevitability of the emergence of insecticide-resistant mosquitoes (N’guessan R et al., 2007). There is no question that a key tool for the control, elimination, or even possible eradication of malaria, in addition to anti-malarial drugs and vector control, is an effective vaccine, but is still not available. Research on malaria vaccines is complicated by the complexity of the parasite life cycle. To complicate matters, reported mechanisms of protection against the liver stage of the parasite differ not only between human and rodent malaria species, but also between different rodent malaria species. Therefore, infection of vaccinated animals with live parasites, as has previously been used to evaluate anti-malarial drugs, remains the most, if not only, meaningful readout of vaccine efficacy (Egan, 1987, Cox, 1988).
Traditional herbal medicines have been used to treat malaria for thousands of years in various parts of the world. The first antimalarial drug used in the Occident was extracted from the bark of the Cinchona (Rubiaceae) species, the alkaloid quinine, still largely used. Infusions of the plant bark were used to treat human malaria as early as 1632 (Baird et al., 1996). Years later quinine was isolated and characterized (Saxena et al., 2003), thus becoming the oldest and most important antimalarial drug. Another ancient medicinal plant of millenium use in the West is Artemisia annua, rediscovered in China in the seventies as an important source of the antimalarial artemisinin (Bruce-Chwatt, 1982; Klayman, 1985). Artemisinin-combined therapies (ACT) were formally adopted as first-line treatment of uncomplicated malaria in Nigeria from 2005 onwards (Mokuolu et al., 2007). However, ACT use is limited due to its high costs, limited production of artemisinin derivatives to Good Manufacturing Practices (GMP) standards and toxicity (Haynes, 2001; Malomo et al., 2001; Borstnik et al., 2002; Adebayo and Malomo, 2002; Afonso et al., 2006; Boareto et al., 2008).
Phyllanthus niruri, is one of the medicinal plants traditionally used to treat malaria in Nigeria. Phyllanthus niruri has been used traditionally to treat various illnesses, including renal stones, gastrointestinal disturbances, cough, hepatitis, gonorrhea, fever and malaria. This plant was reported to possess hypoglycemic activity (Hukuri et al, 1988), angiotensin-converting enzyme inhibition (Ueno et al, 1988), lipid lowering activity (Khanna et al, 2002), anticancer activity (Giridharan et al, 2002) and anti-HIV activity (Qian-Cutrone et al, 1996). However, very little scientific information is available about its activity against P. falciparum although this plant is extensively used to treat malaria.
In our attempt to find new natural compounds with antimalarial activity that may provide an alternative to chloroquine, the report here on the in vivo antiplasmodial activity of extracts of P. niruri herb. In this preliminary study, ethyl acetate fraction of methanolic extracts of P. niruri were evaluated for its antiplasmodial activity against P. burghei in infected albino mice.
1.1 Aims and Objective
To determine the effect of ethyl acetate partition fraction of methanolic extract of phyllanthus niruri on plasmodium bergei infected mice on plasmodium parasite count, packed cell volume and body weight.
In view of the Fact that malaria caused by plasmodium falciparum is becoming resistant to most anti malaria drugs, there is a need for more research into discovering active compounds from plant sources that would be effective against the parasite. Hence the need for this project work.
2.0 Literature review
2.1 Malaria an Overview
Malaria is caused by species of parasites of the genus Plasmodium that affect humans (P. falciparum, P. vivax, P. ovale, and P. malariae). Malaria caused by P. falciparum is the most deadly form. It predominates in Africa; P. vivax is less dangerous but more widespread, and the other three species are found much less frequently. Malaria parasites are transmitted to humans by the bite of infected female mosquitoes of more than 30 anopheline species. Globally, an estimated 3.3 billion people were at risk of malaria in 2011, with populations living in sub-Saharan Africa having the highest risk of acquiring malaria. Approximately 80% of cases and 90% of deaths are estimated occur on the African continent, with children less than five years of age and pregnant women most severely affected.
Malaria is an entirely preventable and treatable disease, provided the currently recommended interventions are properly implemented. These interventions include; vector control through the use of insecticide-treated nets (ITNs), indoor residual spraying (IRS) and, in some specific settings, larval control, chemoprevention for the most vulnerable populations, particularly pregnant women and infants, confirmation of malaria diagnosis through microscopy or rapid diagnostic tests (RDTs) for every suspected case, and timely treatment with appropriate antimalarial medicines (according to the parasite species and any documented drug resistance).
Malaria is caused by species of parasites of the genus Plasmodium that affect humans (P. falciparum, P. vivax, P. ovale, and P. malariae). The Anopheles vector is the link between man and the malaria parasite.
2.1.2 The parasites life cycle
Malaria is caused by an infection from the intracellular Apicomplexan parasites of the Plasmodium genus. The genus consists of unicellular, eukaryotic protozoan parasites with a number of different species affecting humans including P. falciparum (the most severe form), P. malariae, P. vivax and P. ovale (Hoffman et al., 2002). The parasites of the Apicomplexan phylum have complex life cycles and are all characterised by the presence of a special apical complex that is involved in host-cell invasion and which includes the microneme, dense granules and rhoptries (Figure 1.2) (Cowman and Crabb, 2006).
Figure 2.1: The P. falciparum merozoite showing the apical complex and other major cellular organelles and structures.
Taken from (Cowman and Crabb, 2006).
P. falciparum parasites invade host cells in order to acquire a rich source of nutrients. At the same time, these cells protect the parasites from host immune responses. The parasites are transmitted by the female Anopheles gambiae and A. funestus (Southern Africa) mosquitoes, which serve as vectors for the sexual reproduction of the parasites while the mammalian host provides the parasites with a niche for asexual development. The mosquitoes inject a sporozoite form of the parasites into the subcutaneous layer of the host skin during a blood meal. The sporozoites rapidly move to the liver where they infect the hepatocytes and differentiate into thousands of merozoites. These are subsequently released into the bloodstream where they invade erythrocytes. This invasion characterises the onset of the intra-erythrocytic asexual blood stage of the parasitic life cycle. The parasite cycles through ring, trophozoite and schizont stages and in so doing produce between 16 and 32 daughter merozoites per erythrocyte egression. This is accompanied by the characteristic bursts of fever and anaemia associated with the disease. The daughter merozoites repeat the asexual cycle by invading free erythrocytes. Some intra-erythrocytic stages, however, develop into male or female gametocytes that are ingested by the mosquito during its next blood meal. These develop into male and female gametes inside the mosquito’s gut where they fuse to form diploid zygotes. The zygotes differentiate into ookinetes that subsequently cross the midgut and develop into oocysts from which sporozoites are released. These sporozoites are stored in the salivary glands and are once again injected into the human host by the mosquito to repeat the parasitic life cycle, and thus increasing the number of malaria infectious cases (Wirth, 2002).
Figure 2.2: The life cycle of the P. falciparum parasite.
(a) The asexual stage of the P. falciparum life cycle within the human host, (b) the sexual stage within the mosquito host (Wirth, 2002).
2.1.3 Signs and Symptoms
The signs and symptoms of malaria typically begin 8’25 days following infection (Mandell et al., 2010). However, symptoms may occur later in those who have taken antimalarial medications as prevention (Nadjm et al., 2012). The presentation may include fever, shivering, arthralgia (joint pain), vomiting, hemolytic anemia, jaundice, hemoglobinuria, retinal damage, (Beare et al., 2006) and convulsions. Approximately 30% of people however will no longer have a fever upon presenting to a health care facility (Nadjm et al., 2012).
The classic symptom of malaria is cyclical occurrence of sudden coldness followed by rigor and then fever and sweating lasting about two hours or more, occurring every two days in P. vivax and P. ovale infections, and every three days for P. malariae. P. falciparum infection can cause recurrent fever every 36’48 hours or a less pronounced and almost continuous fever. For reasons that are poorly understood, but that may be related to high intracranial pressure, children with malaria frequently exhibit abnormal posturing, a sign indicating severe brain damage (Idro et al., 2010). Cerebral malaria (encephalopathy specifically related to P. falciparum infection) is associated with retinal whitening, which may be a useful clinical sign in distinguishing malaria from other causes of fever (Beare et al 2011).
Severe malaria is usually caused by P. falciparum, and typically arises 6’14 days after infection (Trampuz et al., 2003). Non-falciparum species have however been found to be the cause of approximately 14% of cases of severe malaria in some groups (Nadjm et al., 2012). Consequences of severe malaria include coma and death if untreated’young children and pregnant women are especially vulnerable. Splenomegaly (enlarged spleen), severe headache, cerebral ischemia, hepatomegaly (enlarged liver), hypoglycemia, and hemoglobinuria with renal failure may occur. Renal failure is a feature of blackwater fever, where hemoglobin from lysed red blood cells leaks into the urine (Trampuz et al., 2003).
Malaria is typically diagnosed by the microscopic examination of blood using blood films or using antigen-based rapid diagnostic tests (Abba et al., 2011). Rapid diagnostic tests that detect P. vivax are not as effective as those targeting P. falciparum. They also are unable to tell how many parasites are present (Nadjm et al., 2012).
Areas that cannot afford laboratory diagnostic tests often use only a history of subjective fever as the indication to treat for malaria (Perkin et al., 2008). Polymerase chain reaction based tests have been developed, though these are not widely implemented in malaria-endemic regions as of 2012, due to their complexity (Nadjm et al., 2012).
2.2 Global burden of malaria
Malaria is the most important parasitic disease in the world and remains of highest public health importance. In 1994, the global incidence of malaria has been estimated at 300-500 million clinical cases annually, causing 1.5 to 2.7 million deaths each year (WHO, 1997). More than 90 % of this malaria burden occurs in sub-Saharan Africa (SSA), where severe malaria disease and death mainly occur among young children of rural areas with little access to health services (Greenwood et al. 1987a; Snow et al.1999). In SSA malaria accounts for an estimated 25% of all childhood mortality below age of five excluding neonatal mortality (WHO 1997). Recent studies suggest that this percentage might even be higher because of the contribution of malaria as indirect cause of death (Alonso et al. 1991, Molineaux 1997).
According to WHO and the World Bank, malaria is responsible for an annual loss of 35 million disability adjusted life years (DALYs) worldwide (World Bank, 1993). It has furthermore been estimated that about 40% of all fever episodes in SSA are caused by malaria (Brinkmann & Brinkmann 1991).
The epidemiological situation of malaria is worsening with the spread of drug resistance in the parasite and insecticide resistance in the vector. More evidence points to significantly increasing malaria morbidity and mortality is SSA due to the development by Plasmodium falciparum of resistance to existing first-line drugs such as chloroquine and sulphadoxine/pyrimethamine (Trap?? 2001).
2.3 The vector
The Anopheles vector is the link between man and the malaria parasite. Because the sexual cycle takes place in the mosquito, it is sometimes called the definitive host. There are about 400 different species of anopheles, but there are only about 60 that are vectors of malaria and of these, about 40 are important. The most important vectors in the afrotropical region (Africa south of the Sahara, Madagascar, Seychelles and Mauritius) are the A gambiae complex (which includes A gambiae, A. arabiensis, A. melas, A. merus, A. bwambae, and A. quadriannulatus) and A. funestus (Service, 1996). Among the A. gambiae complex, A gambiae sensu stricto is the most important malaria vector and it is probably the world most efficient vector (Service, 1996). It breeds in sunlit pools, puddles, borrow pits and rice fields. It bites humans both indoors (endophagic) and outdoors (exophagic), and rests mainly indoors (endophilic) but may also rest outdoors. The other important species of the A gambiae complex, A arabiensis has similar breeding and biting habits to A gambiae s.s. except that it tends to occur in drier areas and it is more likely to bite cattle and rest outdoors (exophilic).
A. funestus, the other major vector in the afrotropical zone, prefers shaded habitats and breeds in permanent waters, especially with vegetation, such as marshes, edges of streams, rivers and ditches, and rice fields with mature plants providing shade. It bites humans predominantly but also domestic animals, and is exophagic and endophagic. Because of seasonality in climate, especially rainfall, mosquito abundance and malaria transmission tends to be seasonal. During the wet season, breeding sites are created in stagnant water leading to high mosquito populations and hence increased malaria transmission.
2.3.1 Vector control
Alternative strategies to reduce the prevalence of malaria include the use of insecticide treated bed nets and reduction of the vector population with insecticide spraying. As far as indoor insecticide spraying is concerned, bis(4-chlorophenyl)-1,1,1-trichloroethane (better known as DDT) remains the most powerful and successful pesticide to date and is responsible for the eradication of malaria from the United States and Europe. In South Africa, the discontinued use of DDT in the 1990s resulted in the worst malaria epidemic the country has experienced since the introduction of indoor spraying in the 1950s. The reintroduction of DDT in 2000 resulted in an overall decrease in the number of malaria cases of approximately 50% in 2002 (Maharaj et al., 2005). DDT is not only effective against malaria vectors but is equally potent at alleviating various other arthropod-borne diseases such as yellow fever, African sleeping sickness, dengue fever and typhus. However, DDT was also extensively used in agriculture where enormous quantities were aerially sprayed onto crops to curb pests. This widespread and uncontrolled use of DDT raised concerns in the 1960s amongst environmentalists who described possible catastrophic consequences for both the environment and humans leading to the ban of DDT use in the 1980s (Weissmann, 2006). Little scientific evidence, however, exists to support these concerns and no toxic effects caused by DDT in humans have been noted when used in low concentrations as required for the control of malaria vectors inside houses (Rogan and Chen, 2005). Since then, the efforts of several public health officials and malaria experts have resulted in the restricted use of DDT in Africa for malaria vector control only and not in agriculture (Dugger, 2006).
Malaria parasite transmission can additionally be prevented by the treatment of parasite vectors with a drug that blocks the sexual development of the parasites within the mosquito. Coleman tested the effect of 8-aminoquinolines on the sexual development of P. berghei and P. falciparum parasites in A. stephensi mosquitoes. The drug-fed mosquitoes produced fewer oocysts than the control-fed group, and in addition, the sporozoites that did manage to develop from the oocysts could not enter the salivary glands of the mosquito, and therefore prevented the parasites from being transmitted back to the mice (Coleman et al., 1994). The antifolate drugs proguanil and pyrimethamine have also been shown to be sporontocidal.
These drugs caused a reduction in oocysts in sensitive strains and pyrimethamine was also reported to directly damage ookinetes (Bray et al., 1959). Gillet et al. showed that ??- difluoromethylornithine (DFMO), a polyamine pathway inhibitor interferes with P. berghei sporozoite development in A. stephensi mosquitoes. Only one mouse out of 16 exposed to DFMO-treated mosquitoes contracted malaria. DFMO thus exerts its most deleterious effects on the stages where active cell division is taking place, namely the exo-erythrocytic schizogonous and sporogonous stages (Gillet et al., 1983).
2.4 Malaria vaccine development.
The development of a safe and effective vaccine against infection represents an alternative method for treating parasitic diseases. Despite extensive efforts, not a single vaccine against any of the human parasitic diseases is currently available. Some malaria experts, however, remain adamant that vaccination may be the most valuable strategy for reducing mortality associated with malaria (Miller and Hoffman, 1998). People living in malaria endemic areas develop low levels of protective immunity against P. falciparum infection after five years of age but this immunity is never complete and seems to be specific for the parasite strain residing in a specific area. Protective immunity is therefore lost once the host moves into an area where a different strain resides and also once the host is no longer chronically infected (Bull and Marsh, 2002).
The complex life cycle of the malaria parasite, which allows it to co-exist with the host’s immune response, is largely responsible for the absence of a successful vaccine (Todryk and Hill, 2007). Current vaccine development strategies focus on different protein antigens that are expressed during the different stages of the life cycle, namely the pre-erythrocytic (sporozoite and schizont-infected hepatic cells), the asexual intra-erythrocytic (merozoiteinfected erythrocytes) and sexual exo-erythrocytic (gametocyte) stages (Figure 1.6) (Todryk and Hill, 2007). An ideal vaccine against malarial infection should therefore induce immune responses against every stage of the life cycle. Such a multistage, multivalent and multi immune response vaccine presents the best strategy for a successful vaccine in the treatment of malaria (Doolan and Hoffman, 1997).
Antibodies directed against antigens on the surface of extracellular sporozoites (e.g. circumsporozoite protein, CSP) would result in the neutralisation of sporozoite infectivity in the bloodstream. Preliminary studies of the RTS,S/AS02 malaria vaccine (GlaxoSmithKline Biologicals) in African infants showed that the vaccine is safe, well-tolerated and reduces parasite infection and clinical illness due to malaria. The vaccine consists of two polypeptides; RTS corresponds to CSP amino acids 207-395 of P. falciparum 3D7 fused to the N-terminus of the hepatitis B surface antigen (HBsAg) and S consists of 226 amino acids of HBsAg (Stoute, 2007). A Phase II trail conducted in Mozambique reported that the vaccine is 65% effective against new infections over a three-month follow-up period after infants received three doses of the vaccine and reduced clinical malaria episodes by 35% over a sixmonth follow-up period starting after the first dose (Aponte et al., 2007).
2. 5 Treating malaria
The development of a highly efficient, novel drug against any disease is in itself a challenging process but the development of an anti-malarial is an exceptionally daunting task. The areas where malaria is prevalent at epidemic proportions are mostly devoid of trained physicians and health workers who possess the skills necessary for the early diagnosis of the disease as well as its efficient treatment. Novel antimalarials must be orally bioavailable, as the diseased individuals will most probably not have access to facilities such as hospitals and clinics. The treatment time period must also be less than a week to reduce the risks associated with the development of parasite resistance to the drugs, which may also be reduced with the use of combination-based therapy. Finally, the drugs must be cheap and have a relatively extended shelf life (Nwaka and Hudson, 2006).
2.5.1 Current anti-malarials
Various drugs have been developed and used in the fight against malaria. As with the vaccines, anti-malarials target different stages of the parasite life cycle within the human host and specifically interfere with processes that are essential to parasite survival. Eradication of malaria with the use of anti-malarials is, however, continuously compromised by the increased prevalence of parasite resistance to the small amount of available commercial drugs. Figure 1.7 shows the different stages of the parasite life cycle and drugs that specifically target these stages of parasite development.
Figure 2.3: A schematic diagram of the parasite life cycle within the human host showing the targets of different anti-malarials during the developmental stages.
The pre-erythrocytic, asexual intra-erythrocytic and sexual exo-erythrocytic stages are shown. The different intra-erythrocytic phases of malaria parasite development are also given. Finally, drugs that have been used at each stage are shown in the dashed boxes (Chauhan and Srivastava, 2001; Olliaro, 2001; Korenromp et al., 2005).
Table 2.1: Different antimalarial drug classes together with their mechanisms of action
Compiled from (Chauhan and Srivastava, 2001; Olliaro, 2001; Korenromp et al., 2005).
Stage Drug class Drug compounds Mechanism of action
Primaquine (also gametocytocidal)
Atovaquone (also sporontocidal) Interferes with cytochrome electron transport
Aminoquinolines Chloroquine (also gametocytocidal) Inhibits haem detoxification
Quinine (also gametocytocidal) Inhibits haem detoxification
Sulfonamides Sulfadoxine Inhibits DHPS
Sulfones Dapsone Inhibits DHPS
Amidines Proguanil (active as cycloguanil, also active against pre-erythrocytic forms
Pyrimethamine (also sporontocidal and interferes with sexual reproduction) Inhibits DHFR (used in combination with
sulfadoxine or dapsone)
4-Methanolquinoline Mefloquine Inhibits haem detoxification
Sesquiterpene lactone Artemisinin and derivatives (also gametocytocidal) Inhibits calcium adenosine triphosphatase
Tetracycline (also active against intra- erythrocytic forms) Inhibitors of aminoacyl-tRNA binding during protein synthesis
Doxycycline (also active against intra- erythrocytic forms) Inhibitors of aminoacyl-tRNA binding during protein synthesis
The bark of the Cinchona tree has been used for centuries to treat fever associated with malarial infection from which the active ingredient is quinine. It remained the anti-malarial of choice until the 1940s, when other anti-malarials such as its chloroquine derivative replaced quinine. Quinine, however, is still used today to treat clinical malaria and not as a prophylaxis due to its side effects and poor tolerability. Chloroquine is a 4-aminoquinoline derivative of quinine and for many years it was the main anti-malarial drug used in the treatment of malaria until parasite resistance developed in the 1950s. It remains the most popular anti-malarial developed to date due to its safety, low cost and efficacy. (Bjorkman and Phillips-Howard,
1990; Yeh and Altman, 2006). Today, the widespread resistance to the drug has rendered its use as a therapeutic agent useless, even though it still shows some efficacy in the treatment of the other human malaria parasites (Korenromp et al., 2005).
Despite more than three decades of research, the exact molecular mechanism of chloroquine action remains a controversial topic. It is believed that the weak-base drug accumulates in the acidic food vacuole of the parasite where it prevents haem detoxification (Bray et al., 2005). Chloroquine resistance in malaria parasites has been attributed to reduced concentrations of the drug in the food vacuole possibly due to drug efflux, pH modification in the vacuole, the role of a Na+/H+ exchanger and transporters (Foley and Tilley, 1998; Bray et al., 2005). In P. falciparum, two genes have been implicated in resistance, namely Pfmdr1 and Pfcrt, which encodes Pgh1 and PfCRT, respectively. Both these proteins are localised to the food vacuole membrane. Mutations in these genes could lead to small increases in the food vacuole pH thus reducing the amount of chloroquine that can accumulate, rendering the drug ineffective (Spiller et al., 2002). Alternatively, PfCRT may increase the efflux of chloroquine by directly interacting with the drug (Cooper et al., 2007). Resistance is associated with several mutations in the PfCRT protein, while the loss of a Lys residue at position 76 has been shown as the critical mutation rendering the P. falciparum parasites resistant to the drug. This residue is located within the first transmembrane segment of PfCRT and may therefore play an important role in the properties of the channel or transporter (Cooper et al., 2005). Mutations in the Pfmdr1 gene is associated with resistance to mefloquine, quinine and halofantrine (Reed et al., 2000).
A number of related aminoquinolines have since been developed and applied, including:
‘ Amodiaquine: effective against chloroquine-resistant strains, possible cross- resistance with chloroquine, safety limitation (Korenromp et al., 2005; Bathurst and Hentschel, 2006)
‘ Atovaquone: usually used in combination with proguanil (Malarone??), resistance reported in 1996, cost limitation (Korenromp et al., 2005; Bathurst and Hentschel,
‘ Lumefantrine: usually coformulated with artemether (Co-Artem’) and is highly effective against multi-drug resistant P. falciparum (Korenromp et al., 2005)
‘ Halofantrine (Halfan): resistance reported in 1992, cost and safety limitations
(Bathurst and Hentschel, 2006)
‘ Mefloquine (Lariam??): resistance reported in 1982, cost and safety limitations
(Bathurst and Hentschel, 2006)
‘ Primaquine: used for its gametocytocidal effect (P. falciparum) and its efficacy against intra-hepatic forms of all types of malaria, no resistance, safety limitations (Chauhan and Srivastava, 2001; Bathurst and Hentschel, 2006)
The antifolates are some of the most widely used anti malarials. However, their role in malaria prevention is hampered by the rapid emergence of resistance once the parasites are placed under drug pressure. The direct effect of folate biosynthesis inhibition is a reduction in the synthesis of the amino acids serine and methionine as well as in pyrimidines, which leads to decreased synthesis of DNA. The antifolate drugs target the intra-erythrocytic stages as well as the gametocytes of P. falciparum (Olliaro, 2001).
The antifolates can generally be divided into two classes; the type-1 antifolates mimic the p- aminobenzoic acid (pABA) substrate of dihydropteroate synthase (DHPS) and include the sulfonamides (sulfadoxine) and sulfones (dapsone), while the type-2 antifolates (pyrimethamine and proguanil) inhibit dihydrofolate reductase (DHFR) (Olliaro, 2001). Interestingly both of these classes of target proteins are arranged on separate bifunctional enzymes (hydroxymethyldihydropterin pyrophosphokinase/dihydropteroate synthase or PPPK/DHPS and dihydrofolate reductase/thymidylate synthase or DHFR/TS) (Ivanetich and Santi, 1990). In addition, malaria parasites are capable of in vivo folate salvage from the extracellular environment as well as synthesising folate derivatives from simple precursors. The mechanism of exogenous folate uptake by a carrier-mediated process has important implications in increasing the sensitivity of the antifolate inhibitors and is being investigated as a novel drug target (Wang et al., 2007).
Pyrimethamine is a diaminopyrimidine and is mostly used in combination with sulfadoxine (Fansidar’) or dapsone leading to the simultaneous inhibition of DHFR and DHPS. Pyrimethamine crosses the blood-brain barrier as well as the placenta. Resistance to sulfadoxine-pyrimethamine combination therapy, however, emerged rapidly due to the appearance of point mutations in the active sites of the target enzymes resulting in reduced drug binding capacity (Cowman and Lew, 1990; Plowe et al., 1997).
Artemisinin is a sesquiterpene lactone extracted from the leaves of Artemisia annua. It is a potent, fast acting blood schizontocide that shows efficacy against all Plasmodium species. Its efficacy is especially broad and shows activity against all the asexual stages of the parasites including the gametocytes (Figure 1.7) (Korenromp et al., 2005). The latter makes this class of antimalarials especially important as they reduce the transmission potential through its gametocytocidal activity.
Originally the mechanism of action of artemisinin was thought to be mediated by the peroxide ring of the drug, which is cleaved and activated by ferrous iron in the heme stores into toxic free radicals that can subsequently damage intracellular targets via alkylation (Meshnick et al., 1991). Recently, however, this theory was challenged by evidence that artemisinin exerts its inhibitory effects on the malarial sarcoplasmic-endoplasmic reticulum calcium ATPase
(SERCA) resulting in an alteration of intracellular calcium levels (Eckstein-Ludwig et al., 2003). The exact mechanism of action, however, remains elusive and different studies have produced contradicting results [reviewed in (Krishna et al., 2004)].
Several derivatives of artemisinin have been developed since artemisinin itself is poorly absorbed and include dihydroartemisinin, artesunate (sodium salt of the hemisuccinate ester of artemisinin), artemether (methyl ether of dihydroartemisinin) and artemether (ethyl ether of artemisinin) (Korenromp et al., 2005). Currently, the WHO recommends artemisinin-based combination therapy (ACT) as the first-line treatment against malaria infections where resistance to other antimalarials is prevalent. One of the obvious disadvantages of using ACT for malaria case-management in Africa is the increased cost involved in combining therapies. Even so, several reasons exist for combining antimalarials with an artemisinin derivative, namely:
1) An increase in the efficacy of the antimalarials;
2) A decrease in the treatment time period; and
3) A reduced risk of resistant parasites arising through mutation (Kremsner and Krishna, 2004).
Several ACTs that have been developed are listed below
‘ Pyramax: artesunate and the 4-aminoquinoline pyronaridine
‘ Co-Artem’: artemether and lumefantrine
‘ Artekin’: dihydroartemisinin and the quinoline-based drug piperaquine
‘ Lapdap’: artesunate, chlorproguanil and dapsone
‘ ASAQ: artesunate and amodiaquine
There are several reasons why the appearance of parasite resistance to artemisinin was originally thought to be unlikely or at least delayed:
1) Parasites are not exposed to the drug for prolonged periods due to the short half-life of the drug;
2) Artemisinin is gametocytocidal, which reduces the transmission potential and spread of the parasite; and
3) The frequent use of artemisinin combined with other anti-malarials (ACT) was specifically introduced to delay the onset of resistance (Meshnick, 2002).
Evidence for in vitro resistance to an artemisinin derivative, however, appeared in field isolates from French Guiana in 2005. The increased artemether IC50s were ascribed to the presence of a mutation in the SERCA PfATPase6 gene and was attributed to inappropriate drug use that exerted selection pressures, favouring the emergence of parasites with an artemether-resistant in vitro profile. Even though reduced in vitro drug susceptibility is not tantamount to diminished therapeutic effectiveness, it could lead to complete resistance and thus called for the rapid deployment of drug combinations (Jambou et al., 2005). Lapdap’, a combination of chlorproguanil (targets DHFR), dapsone (targets DHPS) and the artemisinin derivative artesunate, was introduced in 2003 as malaria therapeutic to replace sulfadoxine-pyrimethamine treatment in Africa (Edwards and Biagini, 2006). No resistance has been detected to date (Bathurst and Hentschel, 2006).
Several antibiotics such as tetracycline, doxycycline and minocycline are active against the exo-erythrocytic as well as the asexual blood stages of the P. falciparum parasite. The tetracyclins are antibiotics that were originally derived from Streptomyces species, but are usually synthetically prepared. They interfere with aminoacyl-tRNA binding and therefore inhibit protein synthesis in the parasite’s apicoplast or mitochondrion (Korenromp et al., 2005). This is due to the presence of genomes in the mitochondrion and apicoplast that encode prokaryote-like ribosomal RNAs, tRNAs and various proteins (Wilson et al., 1996). Doxycycline is a synthetic tetracycline derivative with a longer half-life than tetracycline. A disadvantage of these type of antibiotics as antimalarials is the development of photosensitivity during treatment, which is an obvious drawback for tourists entering malaria areas (Korenromp et al., 2005).
2.6 Phyllanthus niruri
Phyllanthus niruri, popularly known as ‘stone-breaker’ (‘quebra-pedras’) found in most tropical and subtropical regions, commonly in fields, grasslands and forests. It is a small herb that grows up to 60 cm in height. The plant is quite herbaceous unlike P. urinaria, P. simplex or P. maderaspantesis which are woody at base (Unader et al., 1995; Calixto et al., 1998; Ridley, 1967). Its leaves are small and appear oblong with very short or absent petiole. The flowers are numerous, white to greenish in colour and minute, grouping at the axillary with a pedicel. The fruit is a smooth surface and glabose capsule, (Wiart, 2002). It belongs to the Euphorbiaceae family with a worldwide distribution. More than 50 compounds were identified in the Phyllanthus niruri, including alkaloids, flavanoids, lignans and triterpenes (14)
Kingdom : Plantae
Division : Magnoliophyta
Class : Magnoliopsida
Order : Euphorbiales
Family : Euphorbiaceae
Genus : Phyllanthus
Species : niruri
Figure 1.4: showing fresh leaves of phyllanthus niruri
It grows 50 to 70 centimeters tall and bears ascending herbaceous branches. The bark is smooth and light green. It bears numerous pale green flowers which are often flushed with red. The fruits are tiny, smooth capsules containing seeds.
2.6.3 Traditional Use
P. niruri is an important plant of Indian Ayurvedic system of medicine which is used for problems of the stomach, genitourinary system, liver, kidney and spleen (Patel et al., 2011).The plant has also been used in Brazil and Peru as a supposed herbal remedy for kidney stones. A clincial study with Phyllanthus niruri, indicated that it has no significant effect on either stone voiding or pain levels, but it may reduce the levels of urinary calcium.
2.6.4 Phytochemicals of p.niruri
P. niruri has been the subject of much phytochemical studies since the mid 1960s.
Different classes of organic compounds with various medical interests have been
reported, the major being the lignans, tannins, polyphenols, alkaloids, flavonoids,
terpenoids and steroids (Calixto et al., 1998). The following chemical constituents have been isolated from P. niruri.
Lignans isolated from P. niruri mostly belongs to two groups, the 1,4-diarylbutane and 1-aryltetralin though neolignans and lignans with other skeleton were also reported from this plant. The following lignans have been isolated from P. niruri: 1,4-diarylbutane skeleton 1-aryltetralin skeleton
Coumarins, tannins and related polyphenols
The following coumarins, tannins and polyphenols have been isolated from P. niruri: gallic acid, ellagic acid, brevifolin carboxylic acid, ethyl brevifolin carboxylate (Shmizu et al., 1989), methyl brevifolin carboxylate (Iizuka et al., 2006), geraniin (Ueno et al., 1988), corilagin (Shmizu et al., 1989), phyllanthusiin D (Foo and Wong, 1992), amariin, amariinic acid, elaeocarpusin, geraniinic acid B, repandusinic acid, amarulone, furosin (Foo, 1995), 1,6-digalloyl glucopyranoside (Foo, 1993), catechin, epicatechin, gallocatechin, epigallocatechin, epicatechin 3-O-gallate, epigallocatechin 3-O-gallate (Ishimaru et al., 1992).
Flavonoids reported from P. niruri plant belongs to the flavonols and flavanone
Subclasses and their respective glycosides. The following flavonoids have been
isolated from P. niruri: quercetin, rutin, astragalin, quercitrin, isoquercitrin (Nara et al., 1977), kaempferol-4′-rhamnopyranoside, eridictyol- 7-rhamno pyranoside (Chauhan et al., 1977), fisetin-4′-O-glucoside (Gupta and Ahmed, 1984), quercetin-3-O-glucopyranoside (Foo, 1993), kaempferol-3-Orutinoside (Qian-Cutrone et al., 1996).
2.7 Plasmodium berghei
Plasmodium berghei is a unicellular parasite (protozoan) that infects mammals other than humans. P. berghei is one of the four Plasmodium species that have been described in African murine rodents.Others includes; Plasmodium chabaudi, Plasmodium vinckei, Plasmodium yoelii.
This species was first described by Vincke and Lips in 1948 in the Belgian Congo (Vincke et al., 1948).
P. berghei is found in the forests of Central Africa, where its natural cyclic hosts are the thicket rat (Grammomys surdaster) and the mosquito (Anopheles dureni).
Rodent malaria parasites are used in many research institutes for studies aiming at the development of new drugs or a vaccine against malaria.
In the laboratory the natural hosts have been replaced by a number of commercially available laboratory mouse strains, and the mosquito Anopheles stephensi, which is comparatively easily reared and maintained under defined laboratory conditions.
Rodent parasites are recognised as valuable model organisms for the investigation of human malaria because they are similar in most essential aspects of morphology, physiology and life cycle and the manipulation of the complete lifecycle of these parasites, including mosquito infections, is simple and safe.
Like all malaria parasites of mammals, including the four human malaria parasites, P. berghei is transmitted by Anopheles mosquitoes and it infects the liver after being injected into the bloodstream by a bite of an infected female mosquito. After a short period (a few days) of development and multiplication, these parasites leave the liver and invade erythrocytes (red blood cells). The multiplication of the parasite in the blood causes the pathology such as anaemia and damage of essential organs of the host such as lungs, liver, spleen. P. berghei infections may also affect the brain and can be the cause of cerebral complications in laboratory mice. These symptoms are to a certain degree comparable to symptoms of cerebral malaria in patients infected with the human malaria parasite Plasmodium falciparum.
The complete genome of P. berghei has been sequenced and it shows a high similarity, both in structure and gene content, with the genome of the human malaria parasite Plasmodium falciparum.
P. berghei can be genetically manipulated in the laboratory using standard genetic engineering technologies. Consequently this parasite is often used for the analysis of the function of malaria genes using the technology of genetic modification (Janse et al., 2006).
A number of genetically modified P. berghei lines have been generated which express fluorescent reporter proteins such as Green Fluorescent Protein (GFP) or bioluminescent reporters such as Luciferase. These transgenic parasites are important tools to study and visualize the parasites in the living host (Amino et al., 2005).
The use of this model malaria parasite has provided biologists and medical researchers with more insight into the interactions of malaria parasites with the immune system, the process of infection of the liver by malaria parasites, the cause of severe pathology, such as cerebral complications in malaria patients, the infection of the mosquito and transmission of the parasite by the mosquito.
Moreover, P. berghei is used in research programs for development and screening of anti-malarial drugs and for the development of an effective vaccine against malaria.
3.0 MATERIALS AND METHODS
3.1.1 Apparatus and Equipment
Apparatus include; Beakers, conical flasks, sterile bottles, retord stand, syringe, basket (cage), microscopic slides, cotton wool, razor blades, measuring cylinder, Dropper.
Equipment include; Rotatory Evaporator, water bath, Refrigerator, Blender, Microscopes, Haematocrite Machine, weighing balance.
Chemicals include; Distilled water, Normal Saline, Gemsa Stain, Methanol, ethyl acetate.
3.1.2 Plant Collection
Fresh leaves of Phyllanthus niruri were collected from Bosso, Niger State, Nigeria in the month of June, 2012.
3.1.3 Experimental Animals
Swiss albino mice of either sex weighing between 24-30g obtained from National Institute for Pharmaceutical Research and Development, Idu, Abuja- Nigeria were used for this study. They were fed with standard diet and water and maintained under standard conditions.
Plasmodium berghei was obtained from National Institute for Pharmaceutical Research and Development, Idu, Abuja- Nigeria, where the parasite was maintained through weekly passage in mice.
3.2.1 Preparation of Crude Extract
The whole plant were cleaned, dried and blended into coarse powder. This was filtered using a cheese cloth and the filtrate evaporated using rotary evaporator and concentrated using water bath.
3.2.2 Preparation of Partition fraction
The crude extract was poured into a separating funnel it was then dissolved with 50ml of distilled water. Partition was done with n- hexane, chloroform and Ethyl Acetate in order of increasing polarity.
3.2.3 Weight Determination
All the Swiss albino mice were weighed with a weighing balance two weeks after acclimatization.
The Swiss albino mice were grouped and labeled for proper identification of each mouse.
3.2.5 Estimation of Percentage Packed cell volume (%PCV)
Blood sample was collected from the tail of each mouse with a capillary tube by capillary action. The tube was sealed with plastestrine at one end. The sealed capillary tubes were then arranged on the haematocrit centrifuge and set to spin at 1200revolution per minute for five minutes. The PCV was read with haematocrit reader and recorded.
3.2.6 Parasite Inoculation
The inoculums consist of Plasmodium berghei parasitized erythrocytes. This was prepared by determining both the percentage parasitemia and the erythrocyte count of the donor mouse and diluting the blood in normal saline in proportions indicated by both determinations. Each mouse was inoculated on day 0, intraperitoneally with 0.2ml of infected blood containing approximately 1×107 p. berghei parasitized red blood cells. In addition, the newly inoculated animals were monitored daily to determine the level of parasitemia.
3.2.7 Curative Test
It involved treatment 72 hours with the extract after mice have been inoculated. Eighteen mice were divided into six group of three each. A mouse infected with P. berghei (parasitemia of about 20-30%) was anaesthetized with chloroform and sacrificed to collect the blood. The blood was diluted with normal saline such that 0.2ml containing about 1×107 infected cells. Each of the eighteen mice was inoculated intraperitoneally with 0.2ml diluted blood. The extract at dose levels of 50, 100, 200, and 400mg/kgbw respectively were administered orally once daily for six days (D0, D1, D2, D3, D4 and D5). A parallel test with Chloroquine (5mg/kgbw) in the fifth group serves as positive control. The sixth group was left untreated and served as negative control. Thin films were made from tail blood from D0- D5, fixed with methanol and stained with 4% Giemsa (PH 7.2) for 20 minutes before been examined on the microscope. Five fields were examined on each slide and number of infected red blood cells (RBC) counted and means taken (Peter, 1970).
3.2.8 Data Analysis
The data obtained from this experiment was subjected to statistical analysis using SPSS 16.0, 2006 version. One way Anova and Independent t-test were carried out to determine significant difference in parasitemia level of mice treated with different doses of crude extract of phyllanthus niruri and standard drug (chloroquine).
4.1 Extract yield
Total weight of milled plant sample = 1,348g
Total weight of crude extract = 106.28g
Total weight ethyl acetate fraction = 26.71g
4.2 Curative antimalarial Activiy
Figure 1 shows the curative potential of P. niruri at all concentrations administered to the mice with the standard drug having the highest percentage degree of parasitaemia.
4.3 Effect on body weight
Figure 2 shows significant difference in the weight before and after treatment as a result of the extract administered to the mice.
4.4 Packet Cell Volume (PCV) Values
Figure 3 shows significant difference in the PCV before and after treatment. The PCV value reduced after treatment.
Figure 4.1: Curative antiplasmodial activity of phyllanthus niruri after 72 hours as compared to the standard drug (chloroquine) and negative control in plasmodium berghei infected mice. Significant difference at p
INT= Infected not treated (negative control), Chq= Chloroquine (positive control)
Figure 4.2: Effect of Ethyl Acetate fraction of Phyllanthus niruri on body weight before and after treatment for curative group in plasmodium berghei infected mice. Significant difference at P<0.05
INT= Infected not treated (negative control), Chq= Chloroquine (positive control)
Figure4.3. Effect of Ethyl Acetate fraction of Phyllanthus niruri on Percentage Packed Cell Volume (%PCV) before and after treatment for curative group in plasmodium berghei Infected mice Significant difference at P<0.05
5.1 Discussion of Result.
From the phytochemicals shown to be present in the crude extract of phyllanthus niruri in the review of literature is indicative of its pharmacological properties (Haidet, 2003).
There was significant curative effect on the mice infected with plasmodium burgei and dosed with the ethyl acetate fraction from the chat in Fig 4.1. But more effective at 50mg/kgbw.
The effects of the crude extracts on Percentage Packed cell Volume (%PCV) is note worthy. The %PCV was seen to reduce after treatment. These phenomenon is normal due to the effect of the extract on the cells in the blood especially the red blood cells (RBCs). This condition is usually temporary as the cells begin to gradually divide, days after treatment.
The effect of the crude extract on the weight was also taken into consideration and it showed that the weight significantly reduced before and after treatment as shown in figure 4.2 and gradually increased days after treatment. This may be attributed to the signs and symptoms attributed to the disease (malaria) which includes loss of weight.
5.2 Conclusion and Recommendation.
Due to the curative effect of the ethyl acetate fraction of phyllanthus niruri on plasmodium burgei it can be used for the treatment of malaria upon further purification to isolate the compound responsible for the activity.
It is suggested also that the ethyl acetate fraction be further subjected to fractionation i.e. vacuum liquid chromatography and column chromatography to further exploit the active components in the crude extract, increase its yields and can be used as templates in the synthesis of therapeutically more effective antimalarial
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