Abstract
In this study, the comparative efficacy study of kaempferol, diminazene aceturate and their combination against experimental Trypanosoma brucei brucei infection in mice was determined. Thirty six adult swiss albino mice both sexes weighing between 18 and 22 grams were randomly divided into six groups (I, II, III, IV, V and VI) of six mice each. Mice in group I were neither treated nor infected (un-infected, normal control). Mice in group II were pre-treated prophylactically with kaempferol (1 mg/kg per os) for 14 consecutive days prior to infection. Mice in groups II to VI each were inoculated with blood containing Trypanosoma brucei brucei (106 trypanosomes/ml of blood/animal) intraperitoneally (I.P). Following detection of parasitaemia, mice in group III were treated once with diminazene aceturate (3.5 mg/kg) I.P only. Mice in group IV were treated with diminazene aceturate (3.5 mg/kg) once I.P, and then continued with kaempferol (1 mg/kg per os) for nine consecutive days. Mice in group V were treated with kaempferol (1 mg/kg per os) only for nine consecutive days. Mice in group VI were given normal saline (5 ml/kg per os) only for nine consecutive days. Nine days post-infection, all mice were sacrificed by severing their jugular veins, blood samples were collected for haematological and biochemical analyses, while tissue samples were collected for histopathological examination. The results obtained showed significant differences between the levels of parasitaemia and survival rate of mice in groups II and VI. Following infection with Trypanosoma brucei brucei mean packed cell volume, haemoglobin concentration and red blood cell count significantly (P < 0.05) decreased in groups II and VI when compared to groups III, IV and V. Similarly, Mean corpuscular volume and mean corpuscular haemoglobin concentration significantly (P < 0.05) decreased in groups II and VI when compared to groups III, IV and V. The percentage of red blood cell haemolysis at 0.5%, 0.7% and 0.9% NaCl concentrations was significantly (P < 0.05) higher in groups II and VI when compared to groups III, IV and V. Total leucocyte count significantly (P < 0.05) decreased in groups II and VI when compared to groups III, IV and V. The mean lymphocytes and neutrophil count reduced significantly (P < 0.05) in groups II and VI when compared to groups III, IV and V. There were significant (P <0.05) increases in the mean alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase in groups II and VI when compared to groups III, IV and V. Antioxidant enzymes; superoxide dismutase, catalase and glutathione peroxidase increased significantly (P < 0.05) in groups III, IV and V when compared to groups II and VI. Serum level of malondialdehyde significantly (P < 0.05) increased in groups II and VI when compared to groups III, IV and V. In conclusion, kaempferol possesses antitrypanosomal activity and probably stimulate host immunity to control the proliferation of the parasite in the blood.
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background of the Study
Trypanosomosis is a disease caused by the protozoan parasite from genus Trypanosoma and transmitted by the tse- tse fly (Glossina species) and other biting flies (Masocha et al., 2012), making the incidence of the disease to be of great concern in the tropics (Anosa et al., 1983). The trypanosomes which affect both man and animals have been subdivided into two, namely the haematic groups (Trypanosoma congolense and T. vivax) which always remain in the plasma of the host‘s blood and the tissue invading group (T.brucei, T.evansi, T.rhodesiense, T.gambiense and T. equiperdum) which are found extravascularly and intravascularly (Anosa et al.,1977). African animal trypanosomosis or nagana is a disease caused by T. congolense, T. vivax and T. brucei spp. In wild animals, these parasites cause relatively mild infections while in domestic animals they cause a severe, often fatal disease (Molyneux et al., 1996). All domestic animals can be affected by nagana and the symptoms are fever, listlessness, emaciation, hair loss, discharge from the eyes, oedema, anaemia, and paralysis (Masocha et al., 2012). As the illness progresses the animals become progressively weak and eventually become unfit for work, hence the name of the disease “N'gana” which is a Zulu word that means “powerless/useless” (Molyneux et al., 1996). Because of nagana, stock farming is very difficult within the tsetse belt. The available means of control involves tsetse control, chemoprophylaxis, chemotherapy and use of trypanotolerant livestock. At present, control of trypanosomosis is chiefly done by chemotherapy and chemoprophylaxis using the following drugs: Diminazine, Homidium, and Isometamidium (Leach and Roberts, 1981; ILRAD Reports, 1990). Diminazene aceturate is probably the most commonly used therapeutic agent for trypanosomosis in livestock in Sub- Saharan Africa (Geerts and Holmes, 1998), even in Nigeria. However, complete dependence on drugs in many situations of trypanosomosis has been hampered in many areas by their toxic effects, high cost and frequent development of resistance to these drugs by the parasites. This is considered a very serious problem in trypanosomosis control in Africa (Geerts and Holmes, 1998). This results from the fact that the drugs effectively eliminate the parasites from the blood stream and the animal appears recovered, and then undergoes relapse infection which may be characterized by severe neurological infection leading to the death of the affected animal. In Nigeria, the occurrence of drug resistance to available trypanocides has been attributed to the presence of fake drugs, abuse of the existing drugs and inadequate dosing of the drugs in trypanosomosis therapy (Ezeokonkwo et al., 2010). Therefore, the current challenge to the majority of African pastoralists is to optimize the use of the relatively old existing drugs (Ezeokonkwo et al., 2010). In view of this, the use of drug combinations, new therapeutic regimens and the use of Slow Release Devices (SRD) of existing trypanocides have been suggested (Geerts and Holmes, 1998). Secondary metabolites in plants including flavonoids are responsible for a variety of pharmacological activities (Mahomoodally et al., 2005; Pandey, 2007), recent interest in these substances has been stimulated by the potential health benefits arising from the antioxidant activities of these polyphenolic compounds (Pandey, 2007). The beneficial health effects of flavonoids have also been related to their antioxidant, anti-inflammatory, antiestrogenic, cardioprotective, cancer chemopreventive, neuroprotective, antidepressant and anxiolytic effects (Butterweck et al., 2000). Kaempferol is a natural flavonol, a type of flavonoid, found in a variety of plants and plant-derived foods. Kaempferol acts as an antioxidant by reducing oxidative stress. Many studies suggest that kaempferol may reduce the risk of various cancers, and it is currently been studied for possible treatment of cancer (Leopoldini et al., 2006).
Figure 1.1: Chemical Structure of kaempferol, adopted from (Calderon-montano et al., 2011) Diminazene aceturate is an odourless complex compound (yellow powder) which is relatively soluble in water but slightly soluble in organic solvent. Diminazene aceturate is effective against infection with T. vivax and T. congolense but less effective against T. brucei brucei, necessitating an increased dose against this species. Diminazene aceturate is used at dose of 7 mg/kg against T. brucei brucei instead of 3.5 mg/kg against other species (Brander et al., 1991; Kuriakose et al., 2012). Treatment with diminazene aceturate in dogs infected with trypanosomes is often caused acute clinical signs including vomiting and diarrhoea (Mamman et al., 1993). Diminazene aceturate overdose causes fatal nervous complications after 24-48 hours of administration in dogs. Clinical signs in dogs with diminazene aceturate toxicity that is due to overdose are depression or stupor, continuous vocalization, ataxia, opisthotonos, extensor rigidity, nystagmus and seizures (Boozer and Macintire, 2003).
1.2 Statement of Research Problems
Trypanosomosis is one of the neglected parasitic diseases that affects human health and also largely accounts for the low livestock productivity of the African continent (Welburn et al., 2006). It is estimated that about 70 million people distributed over 1.55 million km2 in Africa are at risk of the disease (Simarro et al., 2012). On the other hand, animal trypanosomosis is distributed over approximately 25 million km2 in Africa, where it reduces livestock productivity by up to 50 % (Umar et al., 2007). Pathogenic trypanosome infections of domestic animals are still a major scourge in sub-Saharan Africa and they largely account for the low livestock productivity of the continent (Umar et al., 2001), thus making it an important priority for biomedical and public agencies, agricultural sector and the scientific community (Kuriakose et al., 2012). The disease caused by the Trypanosoma brucei subgroup is associated with anaemia, hepatocellular degeneration and glomerulonephritis (Umar et al., 2008) which is largely attributed to the large amount of free radicals and superoxides generated by the trypanosomes that attack membrane polyunsaturated fatty acids and proteins, resulting in cellular injuries and consequently affecting vital tissues and organs of the infected animals (Umar et al., 2001; Igbokwe, 1994; Umar et al., 2008) There is little or no chance for production of antitrypanosomal vaccine in the near future because of antigenic variation exhibited by the parasites. The search for vaccines against African trypanosomosis remains elusive due to antigenic variation in trypanosomes. Hence, prevention of the disease is by vector control which includes the use of targets and traps, bush clearing, spraying of insecticides and sterile insect technique. However, these control measures though effective, are difficult to sustain (Rogers and Randolph, 1985).
Chemotherapy and chemoprophylaxis by trypanocides using diminazene aceturate and the salts of isometamidium and homidium formed the most important aspect of control and eradication of trypanosomoses (Leach and Roberts,1981; Kinabo, 1993; Anene, et. al. 2001). Reports of surveys in Eastern and Southern Africa (Ndung‘u et al., 1999) and in West Africa (McDermott et al., 2000; Maikai et al., 2007) have shown that the rate of trypanocidal drug resistance to be very high due to the parasites and exhibition of antigenic variation which hampers vaccine production (Kuzoe, 1993). In addition, the use of these trypanocides is beset by numerous limitations, including toxicity and unaffordability of the drugs to the resource poor rural farmers (Kuzoe, 1993; Donald, 1994; Anene, et. al., 2001).
1.3 Justification of the Study
The negative impact of trypanosomosis on animals, human health, nutrition and economy is enormous, thereby necessitating continuous research for better ways of controlling the disease (Atawodi, 2005). Also in the absence of effective vaccination, control of trypanosomosis could principally be achieved by means of either chemoprophylactic or chemotherapeutic agents (Maser et al., 2003; Antia et al., 2009). Most of these drugs for the control of both animal and human trypanosomoses are chemically related (Bizimana et al., 2006) and have been in use for decades (Maser et al., 2003; Antia et al., 2009). The repeated use of these trypanocides has led to the development of drug-resistant trypanosome populations. Also, most of these available trypanocides are bedevilled by many undesirable toxic side effects (Deterding et al., 2005) that are serious and even life-threatening and therefore the need for new, effective, safe, easy-to-administer and inexpensive trypanocides (Hoet et al., 2007). Unfortunately, the high cost of drugs with increasing adverse effects and the emergence of resistance strains have rendered existing chemotherapy inadequate (Atouguia and Costa, 1999). Therefore, there is need to explore other agents, especially of plant origin for use as new generations of anti-trypanocidal agents that could be more effective, less toxic, and readily available. Antioxidants are important inhibitors of oxidative species generation and scavengers of free radicals. Antioxidants provide protection to humans and animals against infections and degenerative diseases with oxidative stress aetiology. Kaempferol is a flavonoid and antioxidant, has good peroxynitrite scavenging activity when compared with other phenols and flavonoids (Heijnen et al., 2001; Santos and Mira, 2004). It has been shown to to have neuroprotective effect against L-glutamate toxicity to mouse hippocampal neurons in culture (Li and Pu, 2011), and it also blocked the apoptosis induced by K+ deprivation of rat cerebellar granule neurons in culture, in parallel to the blockade of the cellular oxidative stress caused by the reactive oxygen species burst observed at the very early stages of this apoptosis just before it becomes an irreversible process (Samhan-Arias et al., 2004). Melisa et al (2014) reported that kaempferol isolated from the leaves of Schima wallichii at a concentration of 250 μM inhibited the growth of Plasmodium falciparum in a time-dependent manner both in vivo and in vitro. Kaemperol is also reported to be effective against hepatic fibrosis induced by schistosoma egg (Zhou et al., 2013)
1.4 Aim of the Study
The general aim of the study was to determine the comparative efficacy of kaempferol, diminazene aceturate and their combination against experimental Trypanosoma brucei brucei infection in mice‖
1.5 Specific Objectives of the Study
The objectives of the study were to determine the:
- Prophylactic effect of kaempferol against experimental Trypanosoma brucei brucei infection in mice.
- Therapeutic effect of kaempferol in mice experimentally infected with Trypanosoma brucei brucei.
- Effect of kaempferol in combination with diminazene aceturate in mice infected with Trypanosoma brucei brucei.
1.6 Research Questions
- Does kaempferol does possess anti-trypanosomal and antioxidant activity against brucei brucei infection in mice?
- Does combination of kaempferol and diminazene aceturate have superior anti-trypanosomal activity than diminazene aceturate alone in mice?
