EFFECT OF THE ETHANOL EXTRACT OF ZAPOTECA PORTORICENSIS ROOTS ON MALARIA-INFECTED MICE

ABSTRACT

The effect of the Zapoteca portoricensis roots on malaria was determined by checking the percentage parasitaemia, some haematological parameters such as packed cell volume (PCV), haemoglobin concentration (Hb), total white blood cell count (TWBC) and red blood cell count (RBC), also some biochemical parameters such as liver enzyme markers; aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) and kidney function markers which include urea and creatinine concentrations. The analyses were carried  out  using  standard  analytical  procedures.  The  Zapoteca  portoricensis  roots  were randomly collected from Umabor Ehalumona, Nsukka, Enugu State, Nigeria. The results of this study showed that the percentage yield of the ethanol extract of Zapoteca portoricensis roots was 3.18%. The acute toxicity test of the ethanol extract showed no toxicity up to 2900mg/kg body weight. The phytochemical constituents found were alkaloids, glycosides, carbohydrate, steroid, terpenoids, saponins, flavonoids, resin, fats and oil. Treatment of infected mice with the ethanol extract of Zapoteca portoricensis roots caused mean percentage parasitaemia to reduce significantly (p < 0.05) in groups 4, 5 and 6 administered 100, 200 and 300mg/kg b.w of the extract respectively when compared to the group 2 mice (malaria untreated). Haematological parameters such as PCV, Hb concentration and RBC count significantly (p < 0.05) increased in groups 4, 5 and 6 administered 100, 200 and 300mg/kg b.w of the extract respectively when compared to group 2 (malaria untreated) but TWBC count did not change significantly (p > 0.05) in the controls, group 1 (normal control), groups 2 (malaria untreated) and 3 administered 28mg/kg b.w of artemether and lumefantrine and test groups 4 and 6 administered 100 and 300mg/kg b.w of the extract respectively. The  AST activity showed significant (p < 0.05) increase in all the groups when compared to group 1 (normal control) on day 14 but significantly (p < 0.05) reduced in all the treated groups 4,5 and 6 administered 100, 200 and 300mg/kg b.w of the extract respectively when compared to group 2 mice (malaria untreated) on day 28. The ALT and ALP activities reduced significantly (p < 0.05)  in all the groups when compared to group 2 mice (malaria untreated).   The kidney function markers urea showed significant decrease in groups  1  (normal  control),  4  and  5  administered  100  and  200mg/kg  b.w  of  the  extract respectively when  compared to  group 2  mice  (malaria untreated).  Creatinine  concentration reduced significantly (p < 0.05) in groups 1 (normal control), 3 administered 28mg/kg b.w of artemether and  lumefantrine  and  group 4  administered 100mg/kg  b.w  of the  extract  when compared to group 2 mice (malaria untreated) on day 14 but on day 28 no significant (p > 0.05) differences were observed on the creatinine concentration in all the groups when compared to group 1(normal control). These results may be useful in explaining the medicinal application of Zapoteca portoricensis roots.

CHAPTER ONE

INTRODUCTION

Malaria remains one of the most widespread infectious diseases of our time. The latest estimates reveal that ~250 million people are infected with malaria across the globe, of whom ~800,000 die  every  year  (WHO,  2010),  the  vast  majority  being  young  children.  Most  available antimalarials were designed to target the pathogenic blood stages in humans and to address the constant threat of drug resistance (Fidock, 2010).

Traditional methods of treatment and control of malaria could be a promising source of potential anti-malaria drugs (Ugwu et al., 2013; Sumalata and Sreedevi, 2012; Venkat et al., 2011; Wright and Phillipson, 1990). More than 80% of the world’s population relies on traditional medicine for their primary healthcare needs (WHO, 2008). In developing countries, low income people such as farmers, people of small isolate villages and native communities use folk medicine for the treatment of common infectious diseases. These plants are ingested as decoctions, teas or juice preparations (Gonzalez, 1980).

The aqueous and alcoholic extracts of Zapoteca portoricensis belonging to the family Fabaceae are traditionally used as anti diarrhoel, anti canvulsant, antispasmodic and in the treatment of tonsillitis. Terpenoids and steroids obtained from the column fractions of the root extracts are proved to be responsible for the production of significant anti inflammatory activity (Agbo et al.,

2010). This study was designed to investigate the antimalarial properties of the ethanol extract of

Zapoteca portoricensis root.

1.1 Malaria

Malaria is a mosquito-borne infectious disease of humans and other animals caused by protists (a type of microorganism) of the genus Plasmodium (Fairhurst and Wellems, 2010). It begins with a bite from an infected female mosquito (Anopheles mosquito), which introduces the protists via its  saliva  into  the  circulatory system,  and  ultimately  to  the  liver  where  they  mature  and reproduce. The disease causes symptoms that typically include fever and headache, which in severe cases can progress to coma or death (Nayyar et al., 2012).

Five species of Plasmodium can infect and be transmitted by humans. The vast majority of deaths are caused by P. falciparum while P. vivax, P. ovale, and P. malariae cause a generally milder form of malaria that is rarely fatal. Malaria is prevalent in tropical and subtropical regions

because of rainfall, warm temperatures, and stagnant waters provide habitats ideal for mosquito larvae (Nayyar et al., 2012).

1.1.2 Signs and Symptoms of Malaria

The signs and symptoms of malaria typically begin 8–25 days following infection (Fairhurst and Wellems, 2010). However, symptoms may occur later in those who have taken antimalarial medications as prevention. According to Nadjm and Behrens (2012), initial manifestations of the disease common to all malaria species are similar to flu-like symptoms (Bartoloni and Zammarchi, 2012) and can resemble other conditions such as septicemia, gastroenteritis, and viral diseases. The presentation may include headache, fever, shivering, arthralgia (joint pain), (Nadjm and Behrens, 2012) vomiting, haemolytic anemia, jaundice, haemoglobinuria, retinal damage and convulsions (Beare et al., 2006). Approximately 30% of people, however, will no longer have a fever upon presenting to a health care facility (Nadjm and Behrens, 2012).

The classic symptom of malaria is paroxysm which is a cyclical occurrence of sudden coldness followed by rigour and then fever and sweating, occurring every two days (tertian fever) in P. vivax  and  P. ovale  infections,  and  every  three  days  (quartan  fever)  for  P. malariae. P. falciparum infection can cause recurrent fever every 36–48 hours or a less pronounced and almost continuous fever (Ferri, 2009).

Severe malaria is usually caused by P. falciparum (often referred to as falciparum malariae). Symptoms of falciparium malaria arise 9–30 days after infection (Bartoloni and Zammarchi,

2012). Splenomegaly, severe headache, hepatomegaly (enlarged liver), hypoglycemia, and haemoglobinuria with renal failure may occur. Renal failure is a feature of blackwater fever, where haemoglobin from lysed red blood cells leaks into the urine (Bartoloni and Zammarchi,

2012). Cerebral malaria is a form of severe malaria that involves encephalopathy specifically related to P. falciparum infection. It is associated with retinal whitening, which may be a useful clinical sign in distinguishing malaria from other causes of fever (Beare et al., 2011). Individuals with cerebral malaria frequently exhibit neurological symptoms, including abnormal posturing, nystagmus, conjugate gaze palsy (failure of the eyes to turn together in the same direction), opisthotonus, seizures, or coma (Bartoloni and Zammarchi ,2012).

1.1.3 Complications

There are several serious complications of malaria. Among these is the development of respiratory distress, which occurs in up to 25% of adults and 40% of children with severe P. falciparum malaria (Taylor et al., 2012). Possible causes include respiratory compensation of metabolic acidosis,  noncardiogenic pulmonary oedema,  concomitant  pneumonia, and  severe anaemia. Acute respiratory distress syndrome (ARDS) may develop in 5–25% in adults and up to

29% of pregnant women but it is rare in young children (Taylor et al., 2012), Co-infection of HIV with malaria increases mortality (Korenromp et al., 2005). Malaria in pregnant women is an important  cause of stillbirths,  infant  mortality  and  low  birth weight  (Hartma et  al.,  2010) particularly in P. falciparum infection, but also with P. vivax (Rijken, 2010).

1.1.4 Causes of malaria

Malaria parasites belong to the genus Plasmodium (phylum Apicomplexa). In humans, malaria is caused by P. falciparum, P. malariae, P. ovale, P. vivax and P. knowlesi (Collins, 2012). Among those infected, P. falciparum is the most common species identified (~75%) followed by P. vivax (~20%) (Nadjm and Behrens ,2012). P. falciparum accounts for the majority of deaths, (Sarkar et al., 2009) non-falciparum species have been found to be the cause of about 14% of cases of severe malaria in some groups (Nadjm and Behrens, 2012). P. vivax proportionally is more common outside of Africa (Arnott et al., 2010).

1.1.5 Transmission of Malaria

The malaria parasite typically is transmitted to people by mosquitoes belonging to the genus Anopheles. In rare cases, a person may contract malaria through contaminated blood (Owusu- Ofori et al., 2010). Due to the fact that malaria parasite is found in rbcs, malaria can also be transmitted through blood transfusion, organ transplant, or the shared use of needles or syringes contaminated with blood. Malaria also may be transmitted from a mother to her fetus before or during delivery (“congenital” malaria).

1.1.6 Life cycle of malaria parasite

In the life cycle of Plasmodium, a female Anopheles mosquito (the definitive host) transmits a motile infective form (called the sporozoite) to a vertebrate host such as a human (the secondary

host), thus acting as a transmission vector. A sporozoite travels through the blood vessels to liver cells (hepatocytes), where it reproduces asexually (tissue schizogony), producing thousands of merozoites (Arrow et al., 2004). These infect new red blood cells and initiate a series of asexual multiplication cycles (blood schizogony) that produce 8 to 24 new infective merozoites, at which point the cells burst and the infective cycle begins anew(Arrow et al., 2004). In a process called gametocytogenesis, other merozoites develop into immature gametes, or gametocytes. When a fertilized mosquito bites an infected person, gametocytes are taken up with the blood and mature in the mosquito gut. The male and female gametocytes fuse and form zygotes (ookinetes), which develop into new sporozoites. The sporozoites migrate to the insect’s salivary glands, ready to infect a new vertebrate host. The sporozoites are injected into the skin, alongside saliva, when the mosquito takes a subsequent blood meal. This type of transmission is occasionally referred to as anterior station transfer. (Talman et al., 2004).

Only female mosquitoes feed on blood; male mosquitoes feed on plant nectar, and thus do not transmit the disease. The females of the Anopheles genus of mosquito prefer to feed at night. They usually start searching for a meal at dusk, and will continue throughout the night until they take a meal. (Arrow et al., 2004).

1.1.7   Malaria Diagnosis

Malaria is usually diagnosed by the microscopic examination of blood films or by antigen-based rapid diagnostic tests (RDT) (Kattenberg et al., 2011). Microscopy is the most commonly used method to detect the malaria parasite-about 165million blood films were examined for malaria in

2010. According to Wilson (2012) despite its widespread usage, diagnosis by microscopy suffers from two main drawbacks: many settings (especially rural) are not equipped to perform the test, and the accuracy of the results depends on both  the skill of the person examining the blood film and the levels of the parasite in the blood. The sensitivity of blood films ranges from 75-90% in optimum conditions, to as low as 50%. Commercially available RDTs are often more accurate than blood films at predicting the presence of malaria parasites, but they are widely variable in diagnostic sensitivity and specificity depending on manufacturer, and are unable to tell how many parasites are present (Wilson, 2012).

In regions where laboratory tests are readily available, malaria should be suspected, and tested for, in any unwell patient who has been in an area where malaria is endemic. In areas that cannot afford laboratory diagnostic tests, it has become routine to use only a history of subjective fever as the indication to treat  for malaria—a presumptive approach exemplified by the common teaching “fever equals malaria unless proven otherwise” (Perkins and Bell, 2008). The drawback of this practice, however, is overdiagnosis of malaria and mismanagement of non-malarial fever, which wastes limited resources, erodes confidence in the health care system, and contributes to drug resistance (Perkins and Bell, 2008). Although polymerase chain reaction-based tests have been developed, these are not widely implemented in malaria-endemic regions as of 2012, due to their complexity (Nadjm and Behrens, 2012).

Fig. 2: The blood film for malaria diagnosis

(Source: Abba et al., 2011)

1.1. 8 Prevention of malaria

Methods used to prevent malaria include medications, mosquito elimination and the prevention of bites. The presence of malaria in an area requires a combination of high human population density,  high  mosquito  population density  and  high  rates of transmission from humans  to mosquitoes and from mosquitoes to humans. If any of these is lowered sufficiently, the parasite will eventually disappear from that area, as happened in North America, Europe and much of the Middle East. However, unless the parasite is eliminated from the whole world, it could become re-established if conditions revert  to  a combination that  favours the parasite’s reproduction (World Health Organization, 1958).

Many researchers argue that prevention of malaria may be more cost-effective than treatment of the disease in the long run, but the capital costs required are out of reach of many of the world’s poorest people. There is  a  wide disparity in the costs of control (i.e.  maintenance of low endemicity) and elimination programs between countries. For example, in China, whose government  in  2010  announced  a  strategy  to  pursue  malaria  elimination  in  the  Chinese provinces, the required investment is a small proportion of public expenditure on health. In contrast, a similar program in Tanzania would cost an estimated one-fifth of the public health budget (Sabot et al., 2010). However malaria can be prevented by vector control, mosquito net, by the use of antimalaria medication and other methods.

1.1.8.1 Vector Control of malaria

The mosquitoes remain on the wall until they fall down dead on the floor. Vector control refers to preventative methods used to decrease malaria and morbidity and mortality by reducing the levels of transmission (Pates and Curtis, 2005).

For  individual  protection,  the  most  effective  chemical  insect  repellents  to  reduce  human- mosquito  contact  are  those  based  on  N,  N-diethyl-meta-toluamide  (DEET)  and  picaridin

(Kajfasz, 2009). Indoor Residual Spraying (IRS) is the practice of spraying insecticides on the interior walls of homes in malaria-affected areas (Lengeler, 2004). After feeding, many mosquito species rest on a nearby surface while digesting the blood meal, so if the walls of dwellings have been coated with insecticides, the resting mosquitoes can be killed before they can bite another victim and transfer the malaria parasite (Tanser et al., 2010). As of 2006, the World Health Organization advises the use of 12 insecticides in IRS operations, including dichlorodiphenyltrichloroethane (DDT) and the pyrethroids cyfluthrin and deltamethrin (World Health Organization, 2006). This public health use of small amounts of DDT is permitted under the Stockholm Convention on Persistent Organic Pollutants (POPs), which prohibits the agricultural use of DDT (Van den Berg, 2009).

1.1.8.2 Mosquito Nets for preventing malaria

Mosquito nets create a protective barrier against malaria-carrying mosquitoes that bite at night. Mosquito nets help keep mosquitoes away from people and significantly reduce infection rates and transmission of malaria. The nets are not a perfect barrier and they are often treated with an insecticide designed to kill the mosquito before it has time to search for a way past the net. Insecticide-treated nets are estimated to be twice as effective as untreated nets and offer greater than 70% protection compared with no net (Raghavendra et al., 2011). Between 2000 and 2008, the use of ITNs saved the lives of an estimated 250,000 infants in Sub-Saharan Africa (Howitt et al., 2012). Although ITNs prevent  malaria, only about  13% of households in Sub-Saharan countries own them (Miller et al., 2007). A recommended practice for usage is to hang a large “bed net” above the center of a bed to drape over it  completely with the edges tucked in. Pyrethroid-treated  nets  and   long-lasting  insecticide-treated  nets  offer  the   best   personal protection, and are most effective when used from dusk to dawn (Miller et al., 2007).

1.1.8.3 Other methods of preventing malaria

Community participation and health education strategies promoting awareness of malaria and the importance of control measures have been successfully used to reduce the incidence of malaria in some areas of the developing world (Lalloo et al., 2006). Recognizing the disease in the early stages can stop the disease from becoming fatal. Education can also inform people to cover over areas of stagnant, still water, such as water tanks that are ideal breeding grounds for the parasite

and mosquito, thus cutting down the risk of the transmission between people. This is generally used  in  urban  areas  where  there  are  large  centers of population  in  a  confined  space  and transmission would be most likely in these areas (Mehlhorn , 2008). Intermittent preventive therapy is another intervention that has been used successfully to control malaria in pregnant women and infants, (Bardají et al., 2012) and in pre-school children where transmission is seasonal (Meremikwu et al., 2012).

1.1.9 Antimalaria medications of malaria infection

Antimalarial medications, also known as antimalarials, are designed to prevent or cure malaria. Such drugs may be used for some or all of the following:

Treatment  of  malaria  in  individuals  with  suspected  or  confirmed  infection,  Prevention  of infection in  individuals visiting a  malaria-endemic region who  have  no  immunity (Malaria prophylaxis) (Jacquerioz and Croft, 2009). Routine intermittent treatment of certain groups in endemic regions (Intermittent preventive therapy). Some antimalarial agents, particularly chloroquine and hydroxychloroquine, are also used in the treatment of rheumatoid arthritis and lupus-associated arthritis.

Current practice in treating cases of malaria is based on the concept of combination therapy, since this offers several advantages, including reduced risk of treatment failure, reduced risk of developing resistance, enhanced convenience, and reduced side-effects (WHO, 2010).

Medications/Mechanism of Action

It  is  practical to  consider antimalarials  by chemical  structure since this  is  associated with important properties of each drug, such as mechanism of action.

1.1.9.1 Quinine and related agents

Quinine has a long history stretching from Peru, and the discovery of the cinchona tree, and the potential uses of its bark (Segurado et al., 1997) to the current day and a collection of derivatives that are still frequently used in the prevention and treatment of malaria (Kyle and Shampe,

1974). Quinine is an alkaloid that acts as a blood schizonticidal and weak gametocide against Plasmodium vivax and Plasmodium malariae (Sparkes and Roland, 2010). As an alkaloid, it is accumulated in the food vacuoles of Plasmodium species, especially Plasmodium falciparum. It acts by inhibiting the hemozoin biocrystallization, thus facilitating an aggregation of cytotoxic haeme (Achan et al., 2011). Quinine is less effective and more toxic as a blood schizonticidal agent than chloroquine; however, it is still very effective and widely used in the treatment of acute cases of severe P. falciparum. It is especially useful in areas where there is known to be a high level of resistance to chloroquine, mefloquine, and sulfa drug combinations with pyrimethamine. Quinine is also used in post-exposure treatment of individuals returning from an area where malaria is endemic (Sparkes and Roland, 2010).

The treatment regimen of quinine is complex and is determined largely by the parasite’s level of resistance and the reason for drug therapy (i.e. acute treatment or prophylaxis) (Fernando et al., 2011). The World Health Organization recommendation for quinine is 20 mg/kg first times and

10 mg/kg 8 hr for 5days where parasites are sensitive to quinine, combined with doxycycline, tetracycline or clindamycin. Doses can be given by oral, intravenous or intramuscular routes. The recommended method depends on the urgency of treatment and the available resources (i.e. sterilized needles for IV or IM injections) (Freedman, 2008).

Use  of quinine  is  characterized  by  a  frequently  experienced  syndrome  called  cinchonism. Tinnitus (a hearing impairment), rashes, vertigo, nausea, vomiting and abdominal pain are the most common symptoms (Freedman, 2008). Quinine can cause hypoglycaemia through its action of stimulating insulin secretion; this occurs in therapeutic doses and therefore it is advised that glucose levels are monitored in all patients every 4–6 hours. This effect can be exaggerated in pregnancy and therefore additional care in administering and monitoring the dosage is essential. Repeated or over-dosage can result in renal failure and death through depression of the respiratory system (Sparkes and Roland, 2010).

Quinimax and quinidine are the two most commonly used alkaloids related to quinine in the treatment  or  prevention of malaria.  Quinimax is  a  combination of four  alkaloids (quinine, quinidine, cinchoine and cinchonidine) (Mills and Bone, 2000). This combination has been shown in several studies to be more effective than quinine, supposedly due to a synergistic action between the four cinchona derivatives. Quinidine is a direct derivative of quinine (Sinclair et al.,

2012). It is a distereoisomer, thus having similar anti-malarial properties to the parent compound.

Quinidine is recommended only for the treatment of severe cases of malaria (Sparkes and

Roland, 2010).

1.1.9.2 Chloroquine

Chloroquine was until recently the most widely used anti-malarial. It was the original prototype from which most methods of treatment are derived (Foley and Tilley, 1997). It is also the least expensive, best tested and safest of all available drugs (Wellems and Plowe, 2001). The emergence of drug-resistant parasitic strains is rapidly decreasing its effectiveness; however, it is still the first-line drug of choice in most sub-Saharan African countries. It is now suggested that it is used in combination with other antimalarial drugs to extend its effective usage. Popular drugs based on chloroquine phosphate (also called nivaquine) are Chloroquine FNA, Resochin and Dawaquin (Jacquerioz and Croft, 2009).

Chloroquine is a 4-aminoquinolone compound with a complicated and still unclear mechanism of action. It is believed to reach high concentrations in the vacuoles of the parasite, which, due to its alkaline nature, raises the internal pH. It controls the conversion of toxic heme to hemozoin by inhibiting the biocrystallization of hemozoin, thus poisoning the parasite through excess levels of toxicity. Other potential mechanisms through which it may act include interfering with the biosynthesis of parasitic nucleic acids and the formation of a chloroquine-haem or chloroquine-DNA complex (Caudron et al., 2008). The most significant level of activity found is against  all  forms  of the  schizonts  (with  the  obvious  exception of chloroquine-resistant P. falciparum and P. vivax strains) and the gametocytes of P. vivax, P. malariae, P. ovale as well as the immature gametocytes of P. falciparum. Chloroquine also has a significant anti-pyretic and anti-inflammatory effect when used to treat P. vivax infections, and thus it may still remain useful even when resistance is more widespread (Jacquerioz and Croft, 2009).

Children  and  adults  should  receive  25 mg  of  chloroquine  per  kg  given  over  3 days.  A pharmacokinetically superior regime, recommended by the WHO, involves giving an initial dose of 10 mg/kg followed 6–8 hours later by 5 mg/kg, then 5 mg/kg on the following 2 days (WHO,

2010). For chemoprophylaxis, 5 mg/kg/week (single dose) or 10 mg/kg/week divided into 6 daily doses are advised. Chloroquine is only recommended as a prophylactic drug in regions only affected by P. vivax and sensitive P. falciparum strains. Chloroquine has been used in the

treatment  of malaria  for  many  years  and  no  abortifacient  or teratogenic effects  have  been reported during this time; therefore, it is considered very safe to use during pregnancy. However, itching can occur at intolerable level and Chloroquinine can be a provocation factor of psoriasis (Jacquerioz and Croft, 2009).

1.1.9.3 Amodiaquine

Amodiaquine is a 4-aminoquinolone anti-malarial drug similar in structure and mechanism of action to chloroquine (Winstanley et al., 1987). Amodiaquine has tended to be administered in areas of chloroquine resistance while some patients prefer its tendency to cause less itching than chloroquine. Amodiaquine is now available in a combined formulation with artesunate (ASAQ) and is among the artemisinin-combination therapies recommended by the World Health Organisation. Combination with sulfadoxine=pyrimethamine is no longer recommended (WHO,

2010). It is effective against the erythrocytic stages of all four species of Plasmodium falciparum (Olliaro and Mussano, 2003). AQ, like other 4-aminoquinolines, accumulates in the lysosomes of the  parasites  and  brings  about  loss  of  function,  making  the  parasites  unable  to  digest haemoglobin, which it depends upon for its energy. This drug binds to nucleoproteins of the parasites and inhibits its DNA and RNA polymerases (O’Neill et al., 1998).  It generates free radicals in the form of AQ quinine immine and semi quinine immine which have been implicated in lipid peroxidation (Maggs et al., 1988).

The drug should be given in doses between 25 mg/kg and 35 mg/kg over 3 days in a similar method to that used in chloroquine administration (Freedman, 2008). Adverse reactions are generally similar in severity and type to that seen in chloroquine treatment. In addition, bradycardia, itching, nausea, vomiting and some abdominal pain have been recorded. Some blood and hepatic disorders have also been seen in a small number of patients (WHO, 2010).

1.1.9.4 Pyrimethamine

Pyrimethamine is used in the treatment of uncomplicated malaria. It is particularly useful in cases of chloroquine-resistant P. falciparum strains when combined with sulfadoxine. It acts by inhibiting dihydrofolate reductase in the parasite thus preventing the biosynthesis of purines and pyrimidines, thereby halting the processes of DNA replication, cell division and reproduction. It acts primarily on the schizonts during the erythrocytic phase, and nowadays is only used in concert with a sulfonamide (Lim et al., 2009).

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