EFFECTS OF AQUEOUS LEAF EXTRACTS OF LORANTHUS MICRANTHUS LINN. (LORANTHACEAE) ON BIOCHEMICAL AND HAEMATOLOGICAL PROFILES OF ALBINO RATS INFECTED WITHTRYPANOSOMA BRUCEI BRUCEI

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ABSTRACT


The effects of oral administration of aqueous crude seed extracts of Loranthus micranthus on the biochemical and haematological parameters of albino rats infected with Trypanosoma brucei brucei were investigated for 28days. A total of seventy-two adult-male albino rats were divided into six groups: 3 treatment groups (400 mg/kg and 800 mg/kg and 1200mg/kg), infected and untreated control, one standard drug group and one normal control groups of twelve rats each. Each group was further replicated three times with four rats per replicate. The treatment groups were administered 400 mg/kg and 800 mg/kg and 1200mg/kg of aqueous leaf extracts of L. micranthus according to their body weights. Level of parasitaemia was ascertained in rats that were inoculated with T. brucei brucei at Day zero and subsequently every two days until the end of treatment. Blood samples were collected on weekly basis for various biochemical and haematological parameters using standard methods and assay kits. Two-way ANOVA was used to determine interactive effects of treatment and time while One-way ANOVA was used to test the effect of treatment. The phytochemical screening revealed high concentrations of tannins and flavonoids; moderate concentrations of alkaloids, saponins and steroids; and trace concentrations of glycosides and terpenes to be present in the aqueous crude extracts of L. micranthus leaves. The LD50 of the crude leaf extracts of L. micranthus showed no mortality at dose levels of 5000 mg/kg after twenty four hours. A significant decrease (P < 0.05) was observed in the mean values of body weights of the infected and treated animals throughout the duration of the experiment. Data from the study revealed that the level of parasitaemia in all the infected animals together with the infected and untreated control group was significantly high (P < 0.05) in comparison with those of the standard drug and normal control groups throughout the duration of the experiment. Liver marker enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) of the infected and treated rats as well as those of the infected and untreated control group showed marked significant increases (P < 0.05) in comparison with the standard drug and normal control groups. Similarly, data obtained show that serum levels of bilirubin, urea, creatinine and cholesterol were significantly high (P < 0.05) in the infected animals and in the infected and untreated control group. Serum albumin was significantly lower (P > 0.05) in the treatment groups with minimal increases in the group administered 800mg/kg of the aqueous leaf extract. The result obtained from the haematological analysis revealed that the haemoglobin (HB), packed cell volume (PCV), red blood cell (RBC) and its indices (mean cell haemoglobin, MCH, mean cell volume, MCV and mean cell haemoglobin, MCHC) of the infected rats significantly decreased (P < 0.05) at various dose levels of the aqueous leaf extract of L. micranthus when compared with the standard drug and normal control groups. The white blood cells (WBC) of the infected and treated rats and those of the infected and untreated control group showed significant increases (P < 0.05) in WBC counts when compared with the normal control group in all the weeks. WBC differentials revealed that neutrophils were significantly higher (P < 0.05) in the infected and treated animals in comparison to the standard drug control group in week 3, however lymphocytes, eosinophils, basophils were not significantly different (P > 0.05) from the standard drug control group in week 3 of extract administration. Furthermore, minimal increases in the WBC differentials were observed in the group administered 800mg/kg of the aqueous leaf extract of L. micranthus. Data generated in the present study showed that all infected and treated rats as well as the infected and untreated control group died from the resultant overwhelming parasitaemia unlike the case of those administered the standard drug. This is an indication that the extract lacks any anti-tryopanocidal activity. Thus, the aqueous leaf extract of Loranthus micranthus inhabiting the host plant Kola acuminata is an inadequate anti-trypanosomal agent.


TABLE OF CONTENTS

Title Page
Table of Content
List of tables
List of figure
Abstract

CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW
1.1       Introduction
1.2       Aims and objectives of the study
1.3       Literature Review
1.3.1    Epidemiology
1.3.2    Disease Burden of Trypanosomiasis
1.3.3    Life-cycle of Trypanosoma
1.3.4    Pathology/Pathogenesis of the disease
1.3.5    Immunology
1.3.6    Immunopathology
1.3.7    Signs and symptoms
1.3.8    Diagnosis
1.3.9    Treatment of trypanosomiasis
1.3.9.1 First stage/phase treatment
1.3.9.2 Second stage/phase treatment
1.3.10  Resistance to drugs
1.3.11  Herbal medicine

CHAPTER TWO: MATERIALS AND METHODS
2.1       Experimental Plant
2.2       Preparation of Aqueous Extract
2.3       Phytochemical Screening of the Plant Material
2.3.1    Determination of Alkaloids (General Tests)
2.3.2 Determination of Saponins (Fehling’s Method)
2.3.3    Determination of Tannins (Ferric Chloride Method)
2.3.4    Determination of Flavonoids (Ammonium Test Method)
2.3.5    Determination of Resins (Precipitation Test)
2.3.6    Determination of Steroids and Terpenoids
2.4   Acute Toxicity Studies of the Plant Extract
2.5       Procurement and Management of Experimental Animals
2.6       Experimental Design
2.7   Determination of Parasitaemia in Rats Infected with Trypanosoma
            brucei brucei
2.8       Collection of Blood Samples
2.9       Biochemical Analysis
2.9.1    Alanine transaminase (ALT)
2.9.2    Aspartate transaminase (AST)
2.9.3    Alkaline phosphatase (ALP)
2.9.4    Serum albumin
2.9.5    Total bilirubin
2.9.6    Total serum cholesterol
2.9.7    Blood urea
2.9.8    Serum creatinine
2.10     Haematological Analysis
2.10.1 Haemoglobin estimation
2.10.2 Haematocrit (Packed Cell Volume) estimation
2.10.3 Red blood cell count (RBC)
2.10.4 Red blood cell indices
2.10.4.1 Mean cell volume (MCV)
2.10.4.2 Mean cell haemoglobin (MCH)
2.10.4.3 Mean cell haemoglobin concentration (MCHC)
2.10.5 White blood cell count (WBC)
2.10.5.1. Differential white cell count
2.11 Statistical analysis

CHAPTER THREE: RESULTS
3.1       Quantity of Phytochemicals Present in the Crude Extract of L. micranthus leaves
3.2       Acute Toxicity Studies of Extracts of L. micranthus
3.3       Weekly Effects of Aqueous Extracts of L. micranthus on the Body Weights of Albino Rat
3.4       Effects of Aqueous Extracts of L. micranthus on the Level of Parasitaemia
3.5       Effects of Different Treatments of the Aqueous Extracts of L. micranthus
            leaves on the Biochemical Parameters of Albino Rats
3.5.1    Weekly effects of the extracts on the aspartate transaminase (AST) level
3.5.2    Weekly effects of the extracts on the alanine transaminase (ALT) level
3.5.3    Weekly effects of the extracts on the alkaline phosphatase level
3.5.4    Weekly effects of the extracts on the albumin level
3.5.5    Weekly effects of the extracts on bilirubin level
3.5.6    Weekly effects of the extracts on urea
3.5.7    Weekly effects of the extracts on creatinine level
3.5.8    Weekly effects of the extracts on cholesterol level
3.6       Effect of the Aqueous Extract of L. micranthus on the Haematological Indices of Albino Rats
3.6.1    Weekly effects of aqueous leaf extracts of L. micranthus on the haemogloblin levels of albino rats
3.6.2    Weekly effects of aqueous leaf extracts of L. micranthus on the packed cell volume (PCV) of albino rats
3.6.3    Weekly effects of aqueous leaf extract of L. micranthus on the red blood cell, (RBC) of albino rats
3.6.4    Weekly effects of aqueous leaf extracts of L. micranthus on the mean cell haemoglobin (MCH) of albino rats
3.6.5    Weekly effects of aqueous leaf extracts of L. micranthus on the mean cell volume (MCV) of albino rats
3.6.6    Weekly effects of aqueous leaf extracts of L. micranthus on the mean cell haemoglobin concentration (MCHC) of albino rats
3.6.7    Weekly effects of aqueous leaf extracts of L. micranthus on the white blood cell (W.B.C) of albino rats
3.6.8    Weekly effects of aqueous leaf extracts of L. micranthus on the      neutrophil levels of albino rats
3.6.9    Weekly effects of aqueous leaf extracts of L. micranthus on the lymphocyte levels of albino rats
3.6.10 Weekly effects of aqueous leaf extracts of L. micranthus on the eosinophil levels of albino rats
3.6.11  Weekly effects of aqueous leaf extracts of L. micranthus on the basophil levels of albino rats
3.6.12 Weekly effects of aqueous leaf extracts of L. micranthus on the monocyte levels of albino rats

CHAPTER FOUR: DISCUSSION AND CONCLUSION
4.1       Discussion
4.2       Conclusion
References
Appendices


CHAPTER ONE

1.1        Introduction
The incidence of trypanosomiasis remains a source of concern in the tropics and other parts of the world. Trypanosomiasis is a lethal disease which affects both man and animals; and caused by a parasitic protozoa of the Genus Trypanosoma (Adeiza et al., 2010). This parasite is transmitted by a vector, tsetse fly, most of which belong to the species Glossina palpalis. The distribution of trypanosomiasis corresponds roughly with that of tse-tse flies. Trypanosomiasis, which is also known as sleeping sickness is caused by Trypanosoma brucei gambiense and or Trypanosoma brucei rhodesiense following an infective bite from tse-tse fly (Welburn et al.,2001; Haydon et al., 2002; WHO, 2006;).

Transmission of this parasite is mostly through the bite of an infected tse-tse fly; however there are other ways in which people can be infected. These include – mother to child transmission through the placenta, mechanical transmission through other blood sucking insects (though it is difficult to assess the epidemiological impact of this mode of transmission), accidental transmissions/infection due to pricks from contaminated needles in the laboratory (Seed, 1998; WHO, 2006; Kennedy, 2006).


Trypanosomiasis and its vectors occur in vast areas of the sub-Saharan Africa with devastating impact on livestock productivity as well as posing a serious threat to the lives and livelihood of entire communities (Doua and Yapo, 1993; Rates, 2001; Engels and Savioli, 2006). Trypanosomiasis infestation constitutes the greatest single constraint to livestock and crop production thereby directly contributing to hunger, poverty, protein malnutrition and suffering of entire communities in Africa. This is as its name suggest, infected people tend to sleep a lot, leading to loss of man hours that could have been applied to productive farm work (Murray, 1994; Aroke et al.,1998 Jodi et al., 2011).

Treatment of trypanosomiasis infection is dependent on the stage of infection (that is whether it is chronic and or acute infection). Normally the drugs used in the initial stage of the disease infection are of lower toxicity and easier to administer (Burri et al., 2001; Chappius et al., 2005). The earlier the disease is identified the better the prospect of a cure. Treatment success in the second stage of infection (that is the advanced stage) depends on a drug that can cross the bloodbrain barrier to reach the parasite. Some of the drugs used include; Pentamidine, Suramin, and Eflornithine (Burri et al., 2001; Chappius et al., 2005; WHO, 2008).

Despite the efficacious nature of these drugs, researchers have directed their energies into research projects designed to screen local medicinal plants as potential trypanocides. Although some herbal formula are already in circulation (such as Jubi herbal formula, African herbal formula among others) efforts are made to develop effective drugs from medicinal plants for both the management and treatment of trypanosomiasis (Okochi, et al., 2003; Erah, et al., 2003).


The African mistletoe, Loranthus micranthus Linn is an ubiquitous hemiparasitic plant that thrives well in tropical climates (Obatomi et al., 1996). It depends on its host for mineral salts and water but can photosynthesize its own carbohydrates. Mistletoe grows on a wide range of evergreen and deciduous trees. Host plants of mistletoe include Persea americana, Kola accuminata, Baphia nitida, Treculia africana etc (Nzekwe et al., 2009). Mistletoe has a long history of traditional use for a wide range of diseases such as diabetes, diarrhea, epilepsy etc. Due to its medicinal value/uses and pharmacological activities, mistletoe have been revealed to have a great potential for its use in various systemic and non-systemic infections due to bacteria and fungi (Osadebe and Ukwueze, 2004).....

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