ANTHELMINTIC EFFICACY OF CRUDE METHANOL EXTRACT OF DONNETIA TRIPETALA G. BAKER FRUITS WITH ITS VARIOUS CHEMICAL FRACTIONS IN MICE EXPERIMENTALLY INFECTED WITH HELIGMOSOMOIDES BAKERI


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TABLE OF CONTENT

Title page
Abstract
Table of content
List of Abbreviations

CHAPTER ONE
INTRODUCTION
1.1 Statement of Research Problem
1.2 Justification
1.3 Aim and Objectives
1.3.1    Aim
1.3.2    Specific objectives
1.4       Null Hypothesis

CHAPTER TWO
LITRATURE REVIEW
2.1       Donnetia tripetala G. Baker (Annonaceae) (Pepper Fruit)
2.1.1    Plant description
2.1.2    Domestic uses of D. tripetala
2.1.3    Traditional uses of D. tripetala
2.2       Pharmacological Effects of D. tripetala
2.2.1    Treatment of glaucoma
2.2.2    Effect on bile secretion and biliary sodium and potassium ions
2.2.3    Antinociceptive and anti-inflammatory effect
2.2.4    Effect on oxidative stress and antioxidant activities
2.2.5    Antimicrobial and antihistaminic effect
2.2.6    Insecticidal effect
2.2.7    Hypnotic, anti-convulsant and anxiolytic effects
2.2.8    Antihyperglycaemic effect of ethyl acetate extract of D. tripetala
2.2.9    Other pharmacologic effects2.3          Phytochemical Constituents
2.3.1    Health benefits of dietry nutrients and elements obtained from D. tripetala
2.4       Toxicity Studies of D. tripetala
2.5       Heligmosomoides bakeri
2.5.1  Life cycle of H. bakeri
2.6       Parasitic Biomarkers of Gastrointestinal Nematodes Infection
2.6.1    Faecal Egg Count (FEC)
2.6.2    Pepsinogen
2.6.3    Antibodies
2.6.4    Pasture Larval Count
2.6.5    Gastrin
2.7.1   In vitro anthelmintic assay
2.7.2   In vivo anthelmintic activity
2.8       Control of Helminth Infection
2.8.1   The use of anthelmintics
2.8.2    Non-Chemical Control methods

CHAPTER THREE
MATERIALS AND METHODS
3.1       Location of the Experiment
3.2       List of Materials Used
3.2.1    Equipment and apparatus
3.2.2   Reagents, drugs and chemicals
3.3       Plants Collection, Identification and Preparation
3.3.1    Plants extraction
3.3.2   Solvent partitioning of extracts
3.4       Phytochemical Screening of the Extracts
3.4.1    Test for carbohydrates
3.4.2   Test for free anthraquinones (Modified Borntrager‘s test)
3.4.3   Test for cardiac glycosides
3.4.4    Test for saponins (frothing test)
3.4.5    Test for steroids triterpenes (Lieberman-Burchards test)
3.4.6    Test for flavonoids
3.4.7    Test for tannins (ferric chloride test)
3.4.8    Test for alkaloids
3.5       Experimental Animals
3.6       Acute Toxicity Studies
3.7       Post-Mortem and Histopathological Studies
3.8       Egg Culture and Larval Recovery
3.8.1    Infection of worm free mice
3.8.2    Recovery of H. bakeri eggs
3.8.3    Culture of eggs
3.8.4    Harvesting and preservation of infective H. bakeri larvae
3.8.5    Determination of larval count for artificial infection and larvicidal test
3.9       In vitro Anthelmintic Screening of D. tripetala Fruit Extract and Fractions
3.9.1    Evaluation of ovicidal activity of the extracts (Egg hatch inhibition test)
3.9.2    Evaluation of larvicidal activity of the extracts
3.10     In vivo Anthelmintic Screening of the Extracts
3.10.1  Faecal egg count reduction test (FECRT)
3.10.2 Post mortem worm counts
3.11     Data Analysis

CHAPTER FOUR
RESULTS
4.1       Extraction Yield of D. tripetala fruits
4.2       Phytochemical Screening of D. tripetala Extracts
4.3       Acute Toxicity Study of Crude Methanol Extract of D. Tripetala Fruits
4.4       In vitro Anthelmintic Activities of D. tripetala Fruits
4.4.1    Egg hatch inhibition
4.4.3    Efficacy of the extracts on egg hatch inhibition
4.4.4    Efficacy of the extracts on larvicidal activity
4.5       In vivo Anthelmintic Activity of D. tripetala Fruit extracts in Mice
4.5.1    Fecal egg count (FEC) reduction
4.5.2    Post-mortem worm counts

CHAPTER FIVE
DISCUSSION

CHAPTER SIX
CONCLUSION AND RECOMMENDATIONS
6.1       Conclusion
6.2       Recommendations
REFERENCES


Abstract

The effects of in vitro and in vivo anthelmintic activity of crude methanol extract (CME), hexane extract (HE), ethylacetate extract (EAE), butanol extract (BT) and aqueous methanol extract (AME) of Donnettia tripetala fruits on mice experimentally infected with Heligmosomoides bakeri were evaluated. The fruits were washed dried, pounded and the powder was extracted with methanol in a soxhlet apparatus. Fifty grams of the crude methanol extract (CME) was partitioned with hexane (HE), ethylacetate (EAE) and butanol (BT). The extracts were subjected to preliminary phytochemical screening and acute toxicity test (LD50). The in vitro anthelmintic studies involved the evaluation of egg hatch inhibition and larvicidal activities of the extracts. Different concentrations (0.07, 0.15, 0.3, 0.6, 1.25 and 2.5 mg/ml) of the CME, HE, EAE, BT and AME were prepared in distilled water. Two hundred microlitres of each concentration were incubated with the eggs and larvae of H. bakeri contained in 0.2 ml solution in 96 well microtiter plates and incubated at room temperature for 48 and 24 hours, respectively. Distilled water (0.2ml) and albendazole (0.07, 0.15, 0.3, 0.6, 1.25 and 2.5 mg/ml) were used as untreated and treated controls, respectively. In the in vivo trial, 85 mice infected with H. bakeri were randomly allocated into 17 groups of 5 mice each. Groups 1-3, 4-6, 7-9, 10-12, 13-15 were treated with CME, HE, EAE, BT and AME, respectively at doses of 200, 400 and 800 mg/kg. Groups 16 and 17 were treated with albendazole (10 mg/kg) once and distilled water (5 ml/kg) respectively, and served as treated and untreated controls. All treatments were administered orally on the 16th, 17th and 18th day post infection. Anthelmintic activity was assessed by comparing the faecal egg count reduction rates and the worms recovered from the treated groups to the non- treated group. The extraction with methanol yielded 0.07 % of the powder. Furthermore, fifty grams of the crude methanol extract yielded 24.87 g (49.7%) of HE, 2.11 g (4.2%) of EAE, 3.69 g (7.4%) of BT and 16.80 g (3.4%) of AME on partitioning. Phytochemical screening revealed the presence of carbohydrate, cardiac glycosides, saponins, steroids, triterpenes, flavonoids, tannins and alkaloids in the CME, EAE, BT and AME. The HE however did not contain saponins. Acute toxicity study of the CME in mice showed that the extract was relatively non-toxic and safe at a dose of ≤ 5000 mg/kg. At 2.5 mg/ml the CME, EAE, AME and BT inhibited the hatching of H. bakeri eggs by 93.5, 92.4, 87.9 and 52.4%, respectively. Similarly, CME, EAE, AME and BT caused death of the larve of H. bakeri by 100, 92.6, 85.1 and 77.8%, respectively. However, HE did not produce significant in-vitro anthelmintic activity when compared with distilled water (untreated control). In the in vivo anthelmintic trial, the CME, HE, AME, BT and EAE at a dose of 800 mg/kg reduce fecal egg count by 100, 99, 98, 80 and 65%. Similarly, CME, EAE, AME, BT and HE caused deparasitization of 93, 93, 88, 88 and 82%, respectively, in mice infected with H. bakeri which is significant (p<0 .05="" concluded="" i="" study="" that="" the="">D. tripetala fruit is safe and non toxic when administered acutely and posses anthelmintic activity.





CHAPTER ONE

INTRODUCTION




Parasitic diseases have been noted as one of the major constraints to livestock productivity (Biu et al., 2006) causing enormous economic losses through morbidity and mortality (Waruiru et al., 2001). The direct losses caused by these parasites are attributed to acute illness and death, premature slaughter and condemnation of some animal parts at meat inspection. Indirect losses include the diminution of productive potential such as decreased growth rate, weight loss in young growing calves and late maturity of slaughter stock (Hansen and Perry, 1994).

Infections with parasitic helminths represent a significant economic and welfare burden to global livestock industry. The increasing prevalence of anthelmintic drug resistance means that current control programmes are costly and unsustainable in the long term (Van Dijk et al., 2010). Another factor of increased disease and production loss due to helminths is treatment failure, which is being reported even more frequently. Sustainable control of helminth infections requires detailed knowledge of these factors. There is a need to devise new, sustainable strategies for the effective control of helminthoses (Jackson and Miller, 2006) as frequent and widespread use and misuse of current control methods has resulted in the emergence of resistant parasite populations, such that anthelmintic resistance is now a major global problem, (Kaplan, 2004), and is the greatest threat to the sustainable control of helminthoses (Familton et al., 2001; Sangster and Dobson, 2002; Sutherland and Leathwick, 2010).


Traditional medicinal practices are known to be an important component of everyday life (Bussmann and Sharon, 2006). It has been a major aspect of the sociocultural........

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