ABSTRACT
Mercury is a highly toxic metal that exerts its adverse effects on the health of humans and animals worldwide. The present study was aimed at evaluating the protective effects of aqueous and ethanol extracts of Phoenix dactylifera L. on mercury induced liver and kidney damage in Wistar rats. A total of forty five (45) male Wistar rats of (80 – 125g) were randomly divided into nine groups (I-IX) of five (5) rats each. Group 1 served as the Control and was administered distilled water (0.5 ml), Group II was administered mercury chloride (HgCl2) only at 5 mg/kg body weight (bwt); Group III was pretreated with silymarin at 100 mg/kgbwt then followed by HgCl2 at5 mg/kgbwt; Groups IV and V were pretreated with aqueous fruit extract of Phoenix dactylifera (AFPD) at 500 mg/kgbwt and 1000 mg/kgbwt, respectively, followed by HgCl2 at 5 mg/kgbwt; Groups VI and VII were pretreated withethanol fruit extract of Phoenix dactylifera(EFPD) at 500mg/kgbwt and 1000 mg/kgbwt, respectively, followed by HgCl2 at 5 mg/kgbwt, while Groups VIII and IX were pretreated with AFPD and EFPD only at 1000 mg/kgbwt, respectively. After two weeks of oral administration, the animals were sacrificed using cervical dislocation method and blood samples were collected through the jugular vein for biochemical studies (liver serum enzymes: Aspartate transaminase (AST), Alaninetransaminase (ALT) and Alkaline phosphatase (ALP); and oxidative stress markers, Malondialdehyde (MDA), Superoxide dismutase (SOD), Catalase (CAT) and Glutathione peroxide (GPx). Tissue samples of the liver and kidney were collected and processed for light microscopy using routineHaematoxylin and Eosin (H&E), Periodic Acid Schiff (PAS) and Gordon and Sweet (GS) stains. The results of the present study showed that toxicity and oxidative stress were induced by the significant increased levels of AST, ALP and MDA (p<0.05)
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and decreased levels of SOD, CAT and GPx when compared to the Control. Histological and histochemical changes in the liver and kidney revealed severe degenerative changes, such as hepatocellular vacuolation, sinusoidal dilatation, pyknotic nuclei and cytoplasmic vacuolation in rats exposed to HgCl2 when compared to the Control. The administration of extracts of AFPD and EFPDpreserved liver serum enzymes, kidney electrolytes and antioxidant enzyme activities to levels similar to that of the Control and provided protection against mercury-induced changes in the liver and kidney of Wistar rats. The protective activities of the extracts of Phoenix dactylifera could be attributed to the antioxidant properties such as flavonoids. Thus, these extracts could be potential candidates for use in the management and treatment of mercury-related liver and kidney diseases in our localities.
TABLE OF CONTENTS
Title Page….………………………………………………………..……………….i Declaration….………………………………..……………………………………ii Certification………………………………………………………….………..……iii Dedication………..………………………………………………………….………iv Acknowledgements…………..…………………………….……………………….v Abstract……………………………………………………..…………….…….….vii Table of contents………………………………………………….…………………..ix List of Figures…………………..……………….…………………………………..xv List of Tables………………………………………………………………………xv List of Plates …………………………………….………………………………….xvi CHAPTER ONE
1.0 Introduction………………………………….……………………….………..………….1
1.1 Background Information……………………………………………………………………….…..………..1 1.2 Statement of the research problem..…………………….………………………………….4 1.3 Significance of the study…………………………………..……..…………………………5 1.4 Hypothesis………………………………………..………………………………………..….5 1.5 Aim and Objectives of the Study ………………………………….…………………………5 1.5.1 Aim……………………………………….……………………………..…………5 1.5.2 Objectives…………….………………………….………………………..……….5
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CHAPTER TWO
2.0 Literature Review……………………………………………..………….…….…7
2.1 Mercury………………………………………………………………………..…..7 2.1.1 Chemistry of mercury.…………………………………………..………………11 2.1.2 History of mercury…….……………………………………………..…………..13 2.1.3 Occurrence…………..………………………………….………………………..15 2.1.4 Uses of mercury……..………………………………………….………………..15 2.1.5 Mercury poisoning………….……………….……….………………………….16 2.1.6 Sources of mercury exposure………………………………………..……………18 2.1.7 Diagnosis of mercury poisoning………………..………………………………..20 2.1.8 Prevention…………………………………………………………..……………20 2.1.9 Treatment…….…………………………………………………………..………21 2.1.10 Prognosis…….…………………………………………………………………22 2.2 Plants………….……………………………………………………………………22 2.2.1. Medicinal Plants………………………………………….………………………22 2.2.2 Phoenix dactylifera (Date Palm)………………………………….……………….24
2.2.3 Taxonomy.……………………………………………………………………….26
2.2.4 Botanical Description…………………………..……………………….……………27 2.2.5 Varieties of Date Palm…………………………………………..…………………29 2.2.6 Nutritional Composition…………………………………………………….……31 2.2.7 Phytochemistry………………………………….…………………………………32
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2.2.8 Medicinal Applications of Phoenix dactylifera………..………..……….……….33 2.3 The Liver………………………..……………………………………….…………39 2.3.1 Embryology…………………………………………………….…………………40 2.3.2 Fetal Blood Circulation……….…………………………………………..………41 2.3.3Gross Anatomy………………………………………..………..…………………42 2.3.4 Anatomical Lobes of the Liver…………………………………..………………..45 2.3.5 Functional Subdivision of the Liver………..……………………………….…….45 2.3.6 Blood Vessels of the Liver………………………………………………………..48 2.3.7 Lymphatic Drainage of the Liver……………………………..………….……….49 2.3.8 Innervation of the Liver………………………………………….……………….50 2.3.9 Histology of the Liver………………………..…………………………………..50 2.3.10 Functions of the Liver……………………………..…………………..……….51 2.3.11 Liver Enzymes and Liver Function Test..……………….……………………..55 2.4 The Kidney………….…………………………………………….……….……..54 2.4.1 Embryology………………………………………………………………………55 2.4.2 Gross Anatomy…………………………………………………………………..57
2.4.3 Blood Supply, Venous Drainage and Lymphatics of the Kidney………….…..60
2.4.4 Histology of the Kidney…..……………………………………………………61
2.4.5 Functions of the Kidney……………….…….…………..….………….………..65 2.4.6 Kidney Function Test…………………….….……………..………….…………65 2.5 Silymarin……………………………….…..………………………..…….……….66 2.5.1 Pharmacodynamics………………..……………..………………..…….………67 2.5.2 Stimulation of Liver Regeneration………………………………..……………..67
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2.5.3 Antioxidant Property………………………………………..…….……………..68 2.5.4 Hepatoprotective Activity………………………………………………………..68 2.5.5 Pharmacokinetics……………………………………………..…………………69 CHAPTER THREE 3.0 Materials and Methods………………………………………………..……………….….71 3.1 Plant Material……………………………………….………………………….……….…..71 3.2 Experimental Animals…………………………………….…………………..…….……..71 3.3 Drugs……………………………………………………………….……………….………..72 3.4 Animal Feed…………………………………………………..….….…….……….………..72 3.5 Other Materials………..………………………………………..………………….……….72 3.6 Plant Extract Preparation……….…………………………………………………………..73 3.6.1 Phoenix dactyliferaPhytochmical Screening……………..…………………..…..74 3.6.2 Dose Preparation of P.dactylifera……………………………..……..……………..74 3.6.3 Dose Preparation of Mercury……………..………………………..…….………..75 3.6.4 Dose Preparation of Silymarin………………….….………………..……………76
3.7 Experimental Design…..…………………………………….……….…………………78
3.8 Experimental Procedure………………………….………………..………………….80
3.8.1 Animal Sacrifice ……………….………………………….…………….……….80
3.8.2 Weight Change…………………………………………….…….……….…….80
3.8.3 Biochemical Studies……………………………….…………………………..….80 3.8.4 Histological and Histochemical Studies………..………………………….…….92
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3.8.5 Tissue Processing……..…………………………………………………….……92 3.8.6 Preparation of Stains and Staining………………………………………….……95 3.8.7 Data Analysis…………………………………………………..………….……..97 CHAPTER FOUR 4.0 Results……………………………………………………………..…………………….…..98 4.1Phytochemical Analysis…………………………………………………………..…………98 4.2 Physical Observation…………………………………………………………..…………..98 4.2.1 Weight Change…………………………………………………………….………98 4.2.2 Organ Weight/Body Weight Ratio ……………………………………………..101 4.4 Biochemical Studies…..………………………………………………..………..………104
4.4.1 Serum Liver Enzymes…………….………………………………………..…104
4.4.2 Kidney Electrolytes…………………….……..……………………….….………108 4.4.3 Lipid Peroxide Level and Antioxidant Enzyme Activity…….…………..……..111 4.5 Histopathological and Histochemical Studies of the Liver……………………………117 4.6 Histological and Histochemical studies of the Kidney…………………..….…….…..146 CHAPTER FIVE 5.0 Discussion…………………………………………………………………………..……..166 CHAPTER SIX 6.0 Conclusion, Recommendation and contribution to knowledge…………………..….173
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6.1 Conclusion…….………………………..………………………………………………..173 6.2 Recommendations…………………………………………………………..…………..174 6.3 Contribution to knowledge….……………………………………………….………….174 REFERENCES…………………………………………………………….………..175
CHAPTER ONE
1.0 Introduction 1.1 Background of the Study Mercury is a widespread environmental and industrial pollutant that exerts toxic effect on a variety of vital organs; it induces severe alterations in the tissues (Lund et al., 1993; Mahboob et al., 2001; Sener et al., 2007; Wadaan, 2009; Xu et al., 2012). Mercury will cause severe disruption of any tissue with which it comes into contact in sufficient concentration, but the two main effects of mercury poisoning are neurological and renal disturbances. The former is characteristic of poisoning by methyl- and ethyl mercury (II) salts while the latter is characteristic of poisoning by inorganic mercury such as mercuric chloride in which liver and renal damage is of relative significance.
Inorganic mercury compounds are rapidly accumulated in the kidney, the main target organ for these compounds. The biological half-time is very long, probably years, in both animals and humans. Mercury salts are excreted via the kidney, liver, intestinal mucosa, sweat glands, salivary glands and milk; the most important routes are via the urine and faeces (IPCS, 2003). Mercury is absorbed through inhalation, ingestion and dermal contact, its absorption is related to the type of mercury compound and the duration of exposure (Collasiol et al., 2004). In general, mercurial are attracted to sulfhydryl radicals in the body and are bound to proteins, membranes and enzymes, altering their normal functioning (Yee et al., 1996; WHO, 2003). Mercury poisoning has been reported in humans following exposure to metallic mercury and its organic and inorganic derivatives (Kelly et al., 2006). Inorganic mercury accumulates primarily in the kidneys, liver, spleen,
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bone marrow, intestine, skin and respiratory mucosa (EUESA, 2007). Mercury poisoning can result from inhalation, ingestion, or absorption through the skin and may be highly toxic and corrosive once absorbed into blood stream (Wadaan, 2009). High exposures to inorganic mercury may result in damage to the gastrointestinal tract, the nervous system, and the kidneys (US EPA, 2010). Both inorganic and organic mercury compounds are absorbed through the gastrointestinal tract and affect other systems via this route. However, organic mercury compounds are more readily absorbed via ingestion than inorganic mercury compounds. Symptoms of high exposures to inorganic mercury include: skin rashes and dermatitis; mood swings; memory loss; mental disturbances; and muscle weakness (US EPA, 2010). Despite massive efforts in search of new drugs that could counteract mercurial toxicity, there is no effective treatment available that can completely abolish its toxic effects. In mercurial poisoning, supportive care is given when necessary to maintain vital functions. In addition, the use of chelating agents assists the body‘s ability to eliminate mercury from the tissues (Carvalho et al., 2007). However, these drugs appear to be of limited use, because of their adverse side effects (Tchounwou et al., 2003). Since these medicines have certain serious side effects, there is an urgent need to systematically evaluate plants for their activities. Such research could also lead to new discovery or advance the use of indigenous herbal medicines for orthodox treatment (Uma et al., 2012).
Date palm (Phoenix dactylifera) fruits, an important component of diet in the arid and semiarid regions of the world (Biglari et al., 2008) is a good source of energy, vitamins
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such as vitamin A, vitamin B6 (pyridoxine), vitamin K; and a group of elements like phosphorus, iron, potassium, and a significant amount of calcium (Abdel-Hafez et al., 1980; Usama et al., 2009). Dates palm friuts contain vitamins and are widely used in traditional medicine for the treatment of various disorders, e.g., memory disturbances, fever, inflammation, paralysis, loss of consciousness, nervous disorders (Nadkarni, 1976). It is also used in the treatment of sore throat, to relieve fever, cystitis, gonorrhea, edema, liver and abdominal troubles and to counteract alcohol intoxication (Barh and Mazumdar, 2008; Al-daihan and Bhat, 2012). The cultural use of medicinal plants is wide spread in Africa (Ashafa and Olunu, 2011). The World Health Organization (WHO) estimates that up to 80% of the world‘s population relies on traditional medicinal system for some aspect of primary health care (Farnsworth et al., 1985). Phoenix dactylifera L. (date palm) is known to be one of the oldest cultivated trees in the world (Dowson, 1982; Abdullah, 2008). It has been an important crop in arid and semiarid regions of the world. It has always played an important part in the economic and social lives of the people of these regions. The fruit of the date palm is well known as a staple food. It is composed of a fleshy pericarp and mesocarp which encloses a single pit or stone (seed), therefore, it is a drupe. Date palms have been cultivated in the Middle East over at least 6000 years ago (Copley et al., 2001). Phoenix dactylifera Linn is well known in northern Nigeria (with almost desert-like climate); the states in this axis are Jigawa, Borno, Kebbi, Yobe, Sokoto, Katsina and Zamfara) (Okere et al.,, 2010).
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Many pharmacological studies have been conducted on Phoenix dactylifera and it has been demonstrated to have anti-ulcer activity; anti-cancer activity; anti-diarrhoeal activity; hepatoprotective activity; anti-mutagenic activity; anti-inflammatory activity; in vitro antiviral activity; effect on reproductive system; anti-hyperlipidemic activity; nephroprotective activity and antioxidant activity (Vyawahare et al., 2009). Several researchers have also documented the antioxidant property of Phoenix dactylifera (Mohamed and Al-Okbi, 2004; Allaith and Abdul, 2005; Al-Qarawi et al., 2008). Researchers pay more attention to the protective or therepeutic role of antioxidants, mainly phenolic compounds, found in natural plants as they may exhibit better antioxidant activity than already established antioxidant drugs such as vitamins C and E (Vinson et al.,, 1998; Haslam, 2006).
1.2 Statement of the Research Problem
Mercury is present in various environmental media and food, and has caused a variety of documented, significant adverse impacts on human health and wildlife throughout the world. Some studies have focused their efforts on the protective effects of natural compounds in various nephropathological and hepatopathological conditions (Esmaeili et al., 2012; Uma et al., 2012). However, the nephroprotective and hepatoprotective potential of higher plants as sources for new drugs is still largely unexplored. There is paucity of literature on the potential beneficial effects of aqueous and ethanolic fruit extracts of P. dactylifera against the nephrotoxicity and hepatotoxicity induced by mercuric chloride in Wistar rats.
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1.3 Significance of the Study The results of the present study could lead to the discovery of P. dactylifera as a more effective, affordable and safe agent in combating the problems of liver and kidney related disorders caused by mercury poisoning in developing countries which may be used as an alternative to currently used synthetic drugs, often accompanied by side effects. 1.4 Hypothesis Aqueous and ethanol fruit extracts of Phoenix dactylifera has protective effect on mercury-induced alterations in the liver and kidney of Wistar rats. 1.5 Aim and Objectives of the Study 1.5.1 Aim The aim of the study was to evaluate the protective effects of aqueous and ethanolic fruit extracts of Phoenix dactylifera L. on mercury-induced nephrotoxicity and hepatotoxicity in Wistar rats. 1.5.2 Objectives The objectives of the present study were:
1. To investigate the protective effects of aqueous and ethanolic extracts of P. dactylifera on mercury-induced hepatotoxicity and nephrotoxicity in Wistar rats using routine histological and histochemical techniques.
2. To investigate the protective effect of aqueous and ethanolic extracts of P. dactylifera on mercury-induced hepatotoxicity and nephrotoxicity in Wistar rats using biochemical analyses for liver serum markers, kidney electrolytes, lipid
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peroxide and antioxidant enzyme activity namely Malondialdehyde (MDA), Superoxide dismutase (SOD), Catalase (CAT) and Glutathione peroxidase (GSHPx).
3. To investigate the protective effects of aqueous and ethanolic extracts of P. dactylifera on mercury-induced hepatotoxicity and nephrotoxicity in Wistar rats using an established antioxidant (Silymarin) as a standard.
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