ABSTRACT
Lead poisoning is known to cause a number of adverse effects including nephrophaty, infertility, liver, testis and heart damages in human and experimental animals. The toxic effects of lead are treated by chelation therapy whichalso depletes the body store of essential cations and as such there is need to look for alternative therapy to lead poisoning. The present study was aimed at evaluating the effects of Aqueous and Ethanol extracts of Ziziphus mauritiana leaves (AZM & EZM) on lead acetate induced liver toxicity in adult male Wistar rats. Forty (40) adult male Wistar rats were divided into 8 groups of 5 rats per group. Group 1 was administered with distilled water from 1st to 35th day, Group 2 to 7 were administered with 120mg/kg bwt of lead acetate from 1st to 21st. While the rats in Group 2 were sacrificed on the 22ndday of the administration, From the 22nd to 35th day; Group 3 was treated with 100mg/kg bwt of AZM, Group 4 was treated with 400mg/kg bwt of AZM, Group 5 was treated with 100 mg/kg bwt of EZM, Group 6 was treated with 400mg/kg bwt EZM, Group 7 was treated with 10mg/kg bwt of Succimer, Group 8 was administered with distilled water. The administrations were carried out once orally for 35 days. The rats were sacrificed and blood samples were collected via cardiac puncture for haematological and biochemical analysis. Liver tissues were removed and fixed in 10% Formalin and processed for histopathological studies using Haematoxylin and Eosin (H&E) for Histo-architecture of the Liver, Gordon and Sweets for Reticular fibre and Periodic Acid Schiff for glycogen storage. The results showed that there were increased in liver enzyme parameters AST, ALT and ALP, and the effects were reversed in rats exposed to lead acetate and treated with AZM and EZM. Oxidative stress was induced in rats exposed to lead acetate by decreasing catalase, superoxide dismutase and reduced glutathione, and the effects were ameliorated in lead exposed rats treated with AZM and EZM. There were changes in haematological indices of rats exposed to lead acetate and these changes were reversed in rats treated with AZM and
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EZM. The study showed that there were increased lead accumulations in liver of rats exposed to lead acetate and these changes were ameliorated in rats treated with AZM and EZM. The study also revealed histopathological changes in liver of rats exposed to lead acetate, changes includes Vacoulations, Necrotic Hepatocytes, Distorted Sinusoids, Degeneration of Reticular fibres and Depletion of glycogen storage and the changes were ameliorated when treated with AZM and EZM. Thus, the present study has concluded that Aqueous and Ethanol extracts of Ziziphus mauritiana leaves at doses of 100mg/kg bwt and 400mg/kg bwt was able to ameliorate the effects of lead acetate induced toxicity and may likely be beneficial to the population in endemic areas that are exposed to lead poisoning.
TABLE OF CONTENTS
Cover page ……………………………………………………………………. Error! Bookmark not defined.
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Declaration page ………………………………………………………………………………………………………… v
Certification page ………………………………………………………………………………………………………. vi
Dedication ……………………………………………………………………………………………………………….. vii
Acknowledgements ………………………………………………………………………………………………….. viii
Abstract ……………………………………………………………………………………………………………………. ix
CHAPTER ONE ………………………………………………………………………………………………………… 1
1.0 INTRODUCTION ………………………………………………………………………………………. 1
1.1 Background …………………………………………………………………………………………… 1
1.2 Statement of Research Problem …………………………………………………………………….. 3
1.3 Significance of the Study ……………………………………………………………………………… 3
1.4 Aim and Objectives of the Study …………………………………………………………………. 4
1.4.1 Aim of the study ………………………………………………………………………………………………. 4
1.4.2 Objectives of the study …………………………………………………………………………………….. 4
1.5 Scope of the Study ………………………………………………………………………………………. 5
1.6 Study Hypothesis ………………………………………………………………………………………… 5
CHAPTER TWO ……………………………………………………………………………………………………….. 6
2.0 LITERATURE REVIEW …………………………………………………………………………… 6
2.1 Lead ………………………………………………………………………………………………………….. 6
2.1.1 Sources of lead …………………………………………………………………………………………………. 7
2.1.2 Exposure routes of lead ……………………………………………………………………………………. 9
2.1.3 Toxicokinetics of lead acetate …………………………………………………………………………. 10
2.1.4 Effects of lead ……………………………………………………………………………………………….. 13
2.1.5 Molecular mechanism of lead toxicity ……………………………………………………………….. 18
2.2 Ziziphus mauritiana ………………………………………………………………………………………. 20
2.2.1 General description of Ziziphus mauritiana plant …………………………………………………. 20
2.2.2 Scientific classification ……………………………………………………………………………….. 23
2.2.3 Phytochemical constituents of Ziziphus mauritiana ……………………………………………. 23
2.2.4 Medicinal uses of Ziziphus mauritiana ……………………………………………………………… 24
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2.3 Standard Drug: Succimer (Meso-2,3-dimercaptosuccinic acid) ……………………….. 26
2.4 Liver ……………………………………………………………………………………………………….. 27
2.4.1 Morphology of the liver …………………………………………………………………………………… 27
2.4.2 Functional division of the liver …………………………………………………………………………. 28
2.4.3 Histology of liver ……………………………………………………………………………………………. 28
2.4.4 Blood Supply to liver ………………………………………………………………………………………. 31
2.4.5 Liver function test ………………………………………………………………………………………… 31
2.4.6 Antioxidant levels in liver ………………………………………………………………………………. 32
2.4.7 Liver disorders ……………………………………………………………………………………………… 34
2.5 Blood ………………………………………………………………………………………………………. 34
2.5.1 Functions of blood ………………………………………………………………………………………….. 34
2.5.2 Hematological indices ……………………………………………………………………………………… 35
2.6 Oxidative Stress ……………………………………………………………………………………….. 35
CHAPTER THREE ………………………………………………………………………………………………….. 36
3.0 MATERIALS AND METHODS ………………………………………………………………….. 36
3.1 Materials …………………………………………………………………………………………… 36
3.1.1 Experimental rats ……………………………………………………………………………………………… 36
3.1.2 Source of lead acetate ……………………………………………………………………………………… 36
3.1.3 Source and identification of Ziziphus mauritiana leaves …………………………………….. 36
3.1.4 Preparation of extract …………………………………………………………………………………….. 36
3.2 Experimental Procedure ……………………………………………………………………………… 37
3.2.1 Dosage determination ……………………………………………………………………………………. 37
3.2.2 Experimental design ………………………………………………………………………………………. 38
3.2.3 Termination of experiment …………………………………………………………………………….. 41
3.3 Tissue Processing …………………………………………………………………………………….. 41
3.3.1 Haematoxylin and Eosin (H and E) staining method ………………………………………….. 41
3.3.2 Special stain using Gordon and Sweet ……………………………………………………………… 42
3.3.3 Special stain using Periodic Acid Schiff (PAS) …………………………………………………. 42
3.4 Determination of Lead Acetate Concentration ………………………………………………. 43
3.5 Biochemical Assay …………………………………………………………………………………….. 43
3.6 Determination of Haematological Parameters …………………………………………………. 43
3.7 Determination of Oxidative Stress Parameters …………………………………………… 44
CHAPTER ONE
INTRODUCTION
1.1 Background
The diverse deleterious health effects upon exposure to heavy metals in the environment are a matter of serious concern and a global issue. Lead is the most abundant toxic metal in the environment (Patraet al., 2011). Lead is a poisonous metal, which exist in both organic (Tetraethyl lead) and inorganic (lead acetate, lead chloride) forms in the environment (Shalanetal., 2005). Both occupational and environmental exposures remain a serious problem in many developing and industrializing countries (Yücebilgicet al., 2003).The metal (lead) is primarily found in leaded gasoline, (Tong et al., 2000). Automobile emissions have been an important source of lead exposure to urban residents, particularly in areas with congested traffic. The main source of adult human exposure is through contaminated food, which is believed to account for over 60% of blood levels; while air inhalation accounts for approximately 30% and water of 10% (John et al., 1991).Lead poisoning is also known as plumbism, colicaPictonum, saturnism, Devon colic, or painter‘s colic, which is a medical condition caused by increased levels of the heavy metal lead in the body (Rossi, 2008). The manifestations of lead poisoning in humans are nonspecific. They may include weight loss, anemia (Khalil-Maneshet al., 1994), nephropathy, infertility, liver, testis and heart damages (Patocka andCerny, 2003; Gurer-Orhanet al., 2004). Symptoms of lead toxicity include abdominal pain, confusion, headache, anaemia, irritability, and in severe cases seizures, coma and death (Barbosa et al., 2005).
It has been reported that lead exposure produces free radicals, such as, Reactive oxygen species (O2, OH or lipid peroxyl radical), cause oxidative damage to lipids, proteins, and nucleic acids and may lead to oxidative stress, biological carcinogenesis, mutagenesis, aging,
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atherosclerosis, neuro-degenerative diseases and stress-induced depression (Sapolsky, 2000; Biliciet al., 2001). Lead is known to produce oxidative damage in the liver tissues by enhancing peroxidation of membrane lipids (Chaurasia and Kar, 1997), a deleterious process solely carried out by free radicals (Halliwell and Gutteridge, 1990). Lead-induced oxidative stress in blood and other soft tissues has been postulated to be one of the possible mechanisms of lead-induced toxic effects (Pandeet al., 2001). Oxidative stress leads to metabolic cellular processes in which oxidative species such as super oxide radical anions, hydrogen peroxide and lipid peroxides are generated intracellularly (Chen and Buck, 2000; Lee et al., 2012). These reactive species, if not eliminated, may damage DNA, proteins or membrane lipids and cause oxidative cell death. Endogenous antioxidative enzymes as well as antioxidants are required for cells to survive (Semenza, 2005), while exogenous antioxidants have been shown to effectively prevent oxidative cell death in cultured cells (Kurutas, 2016). The use of medicinal and aromatic plants for the treatment of diseases is as old as mankind. Medicinal plants receive attention from research centers because of their special importance in safety of communities (Najafiet al., 2010). The curative properties of medicinal plants are due to the presence of various complex chemical substances of different composition which occur as secondary metabolites (Lozoyaet al.,1989; Karthikeyanet al., 2009), secondary metabolites grouped as alkaloids, glycosides, flavonoids, saponins, tannins and essential oils. The activities of these curative plants are evaluated by their chemical components. Indian Ayurveda utilizes about 2000 plants to cure different ailments (Daniel, 2006). Some of these curative plants have natural antioxidants, which neutralize free radicals, therefore are receiving more attention from nutritionist and medical researchers for their potential effects in the prevention of chronic and degenerative changes, such as cancer, cardiovascular disease and aging (Young and Woodside, 2001).
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Ziziphusmauritiana leaf is an important part of jujube tree which is useful in treating many health problems. The leaves are applied as poultices and are helpful in liver troubles, asthma and fever (Morton, 1987; Michel, 2002).
1.2 Statement of Research Problem
Lead remains a considerable occupational and public health problem which is known to interfere with a number of body functions and it primarily affects the central nervous, heamatopoietic, hepatic and renal systems (Kaliaet al., 2005). The toxic effects of lead are treated by chelation therapy which also depletes the body store of essential cations (Ruff et al., 1996) and as such there is need to look for an alternative solution to lead poisoning.There are reports on lead induced toxicity in experimental studies but scanty works on medicinal plants as treatment options for lead induced liver damage.
1.3 Significance of the Study
The recent increase in lead exposure in our environment, especially the cases of Zamfara and Niger states (Lucia, 2015) has placed a heavy burden on health risk in both adult and children. The incessant exposure to lead and related products require investigation on a locally available mode of treatment. Ziziphusmauriitanaleaves has been reported to elicit strong antioxidative activity of which the present study intends to evaluate. The present study could be of importance in identification and evaluation of available natural remedy that may possibly be used as alternatives to currently used chelation therapy to treat lead poisoning, which is costly and not free from adverse effects. Therefore, from the results of the present study, Ziziphusmauritiana leaves could be of help in the management of lead poisoning.
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1.4 Aim and Objectives of the Study
1.4.1 Aim of the study
The aim of the study is to evaluate the effects of aqueous and ethanol extracts of Ziziphusmauritiana leaves on lead acetate induced liver toxicity in adult male Wistar Rats.
1.4.2 Objectives of the study
The objectives of the study were:
I. to study changes in the histology of the liver using routine Haemotoxylin and Eosin stain; histochemical techniques using special stains Periodic Acid Schiff (PAS) for glycogen; and Gordon and Sweet for Reticular fibres.
II. to determine the effect of the extracts on hepatotoxicity using biochemical analysis for liver enzyme markers (Alanine amino transaminase – ALT, Alkaline Phosphotase – ALP and Aspartate amino transferase – AST).
III. to examine the effect of the extracts on lead induced alterations on biomarkers of oxidative stress: Catalase (CAT), Superoxide dismutase (SOD), Malondialdehyde (MDA) and Reduced Glutathione (GSH).
IV. to study changes in heamatological parameters (WBC, RBC, HGB, PLT, LYMP) following treatment with aqueous and ethanol extracts of Ziziphus mauritiana leaves on the liver of lead acetate exposed adult male Wistar rats.
V. to examine changes in liver lead concentration following treatment with aqueous and ethanol extracts of Ziziphus mauritiana leaves on the liver of lead acetate exposed adult male Wistar rats using atomic absorption spectrometry.
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1.5 Scope of the Study
The scope of the study was limited to the study of the general histomorphological and biochemical changesfollowing treatment with aqueous and ethanol extracts of Ziziphus mauritiana leaves on the liver of lead acetate exposed adult male Wistar rats.
1.6 Study Hypothesis
Aqueous and Ethanol extracts of Ziziphusmauritiana leaves has effect on lead acetate induced changes on the liver of adult male Wistar rats.
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