Diabetes mellitus is a metabolic disorder which affects about 346 million people globally. Synthetic antidiabetic agents are expensive and also associated with side effects. In a bid to develop cheaper and safer antidiabetic agents, this study was carried out to determine the effect of the methanol tuber extract of Chlorophytumalismifolium.It is used in folkloric medicine for the treatment of diabetes and bacterial infections. The dried powdered tubers was extracted using soxhletextraction method while acute toxicity study was carried out in rats using Lorke‟s method. Antidiabetic study was carried out using streptozotocin-induced hyperglycemia in Wistar rats and in-vitro antibacterial susceptibility testing was carried out using Agar-well diffusion method. Adult male Wistar rats weighing 150-200 g were injected with a single dose of 50 mg/kg streptozotocinintraperitoneally and the rats with fasting blood glucose between 200 and 450 mg/dL were considered hyperglycemic and selected for the study. Experimental groups were set up using control rats in group Iand hyperglycemic rats in five groups of six rats each. Group II was the diabetic control that received normal saline (1 ml/kg) while groups III, IV and V received graded doses of the extract (150, 300 and 600 mg/kg) respectively. Group VI received glimepiride (10 mg/kg). All treatments were given daily by oral route which lasted for 28 days. Blood glucose levels and weights were noted on days 0,7, 14,21 and 28 after which therats were sacrificed and blood samples collected for hematology, changes in lipid profile, liver and kidney function tests, relative organ body weights and histopathology (kidneys, liver, pancreas and heart). The LD50 in Wistar rats was determined to be >5,000 mg/kg using the oral route. The extract of C. alismifolium at all the doses tested showed blood glucose lowering effect.Statistical significant (p<0.01) blood glucose lowering effectoccurred at a dose of 150 mg/kg on day 21, at a dose of 300 mg/kg on days 21 and 28 (p<0.001 and p< 0.01 respectively) and 600
mg/kg on days 7, 14, 21 and 28 (p<0.05, p< 0.01, p<0.001 and p<0.01respectively) was produced by the extract. The extract didnot alter significantly (p > 0.05) liver enzymes, kidney and hematological parameters and relative organbody weights. The histopathological studies showed no changes in the cardiac cells of the Wistar rats.However, degenerative effects were seen in the liver and kidneys. There was regeneration of islet cells at the doses of 300 and 600 mg/kg of the extract. The antibacterial screening showed that the extract at all the concentrations tested possesses a concentration dependent inhibition ofEscherichia coli.Staphylococcusaureus, Beta-hemolytic streptococcusand Pseudomonasaeruginosa.Phytochemical screening of the methanol tuber extract of C.alismifolium revealed the presence of carbohydrates, glycosides, cardiac glycosides, saponins, flavonoids, triterpenes and alkaloids, some of which may be responsible for the observed antihyperglycemic and antibacterialproperties. In conclusion, the methanol tuber extract of C. alismifolium has antihyperglycemic activity in streptozotocin-induced hyperglycemic rats and inhibitory effect against Staphylococcusaureus, Beta-hemolytic streptococcus, Escherichia coli and Pseudomonasaeruginosa.
TABLE OF CONTENTS
Title Page—————————————————————————————— i
Dedication ———————————————————————————————- iv
Acknowledgement ———————————————————————————— v
Abstract ———————————————————————————————— vi
List of Tables —————————————————————————————– xiv
List of Figures—————————————————————————————– xv
List of Plates——————————————————————————————- xvi
List of Abbreviations——————————————————————————- xviii
CHAPTER ONE ————————————————————————————- 1
1.0 INTRODUCTION————————————————————————– 1
1.1 TraditionalMedicine———————————————————————– 1
1.2 StatementofResearchProblem——————————————————— 2
1.3 Justification———————————————————————————– 3
1.4 TheoreticalFrameWork—————————————————————— 4
1.5.1 Aim——————————————————————————————— 5
1.5.2 Specific Objectives————————————————————————— 5
1.6 Research Hypothesis————————————————————————- 6
CHAPTER TWO ———————————————————————————— 7
2.0 LITERATURE REVIEW—————————————————————– 7
2.1 DiabetesMellitus————————————————————————— 7
2.1.1 Epidemiology and etiology of type 1 diabetes mellitus—————————– 8
2.1.2 Pathophysiology of type 1 diabetes mellitus——————————————- 10
2.1.3 Epidemiology and etiology of type 2 diabetes mellitus—————————- 11
2.1.4 Pathophysiology of type 2 diabetes mellitus—————————————— 11
2.1.5 Diagnosis of diabetes mellitus———————————————————- 12
2.1.6 Glycatedhaemoglobin——————————————————————– 13
2.2 Diabetes and Hyperlipidemia————————————————————- 14
2.3 Diabetic Foot Ulcer and Urinary Tract Infections————————————– 14
2.4 Characteristics of Bacterial Species Used in the Study——————————– 16
2.4.1 Staphylococcusaureus —————————————————————– 16
2.4.2 Beta- streptococci——————————————————————— 17
2.4.4 Escherichia coli —————————————————————————— 19
2.5 Non-pharmacological Management of Diabetes Mellitus—————————– 20
2.6 Drug Management of Diabetes Mellitus————————————————- 21
2.6.1 Insulin—————————————————————————————- 21
2.6.2 Sulphonylureas—————————————————————————— 22
2.6.3 Biguanides———————————————————————————– 22
2.6.4 Thiazolidinediones————————————————————————- 23
2.6.5 Meglitinides———————————————————————————- 24
2.6.6 Alpha-glucosidase Inhibitors————————————————————– 24
2.6.7 Other Oral Antidiabetic Agents———————————————————– 25
2.6.8 Potential New Antidiabetic Agents——————————————————- 25
2.7 Medicinal Plants Used in in the Management of Diabetes Mellitus—————– 25
2.8 The Family Liliaceae———————————————————————– 26
2.8.1 The genus Chlorophytum—————————————————————– 26
2.8.2 The plant Chlorophytumalismifolium ————————————————– 27
2.9 Models for Experimental Diabetes——————————————————- 30
2.9.1 Alloxan-inducedhyperglycemicmodel———————————————— 30
2.9.2 Streptozotocin-induced hyperglycemic model—————————————- 31
2.9.3 Pancreatectomy in dogs—————————————————————— 32
2.9.4 Growth hormone induced diabetes—————————————————– 32
2.9.5 Virus induced diabetes——————————————————————- 32
2.9.6 Corticosteroids induced diabetes——————————————————- 33
2.9.7 Ferric nitrilotriaceticinduction of diabetes——————————————– 33
2.9.8 Genetic models————————————————————————— 33
CHAPTER THREE ——————————————————————————– 34
3.1 MATERIALS——————————————————————————— 34
3.1.1 Drugs and chemicals———————————————————————— 34
3.1.2 Equipment and other materials————————————————————–34
3.1.3 Experimental animals———————————————————————– 35
3.2 METHODS———————————————————————————— 35
3.2.1 Collection and Extraction of Plant———————————————————- 35
3.2.2 Acute toxicity study———————————————————————— 35
3.2.3 Pharmacological studies—————————————————————— 36
18.104.22.168 Streptozotocin-induced hyperglycaemia————————————————- 36
22.214.171.124 Experimental design————————————————————————- 37
126.96.36.199 Streptozotocin –induced hyperglycaemia(28 day studies) ————————— 37
3.2.4 Antibacterial studies————————————————————————- 38
188.8.131.52 Cultivation and standardizationof test bacteria—————————————- 38
184.108.40.206 Antibacterialassay of Chlorophytumalismifolium————————————- 39
220.127.116.11 Minimum Inhibitory ConcentrationChlorophytum alismifolium Extract———— 39
18.104.22.168 Minimum Bactericidal Concentration of Chlorophytum alismifolium Extract—— 40
3.2.5 Preliminary Phytochemical Screening of Chlorophytumalismifolium—————- 40
22.214.171.124 Test for carbohydrates———————————————————————– 40
126.96.36.199 Test for free Anthracene/Anthraquinone Derivatives———————————– 41
188.8.131.52 Test for Glycosides————————————————————————– 41
184.108.40.206 Test for Cardiac Glycosides—————————————————————- 41
220.127.116.11 Test for Saponins—————————————————————————- 42
18.104.22.168 Test for Tannins—————————————————————————– 42
22.214.171.124 Test for Flavonoid————————————————————————— 42
126.96.36.199 Test for Alkaloids————————————————————————— 43
188.8.131.52 Test for Unsaturated Steroids and Triterpenes—————————————— 43
3.2.6 Statistical Analysis————————————————————————– 43
CHAPTER FOUR———————————————————————————– 44
4.0 RESULTS————————————————————————————- 44
4.1 PercentageYield of the Extract———————————————————— 44
4.2 Result of Acute Toxicity StudyonC. alismifolium extract—————————– 45
4.3 Pharmacological Studies——————————————————————— 46
4.3.1 Effect of 28-day daily administration of methanol extract of C. alismifolium
on blood glucose level of hyperglycemic rats——————————————– 46
4.3.2 Effect of 28- day daily administration of methanol extract of C. alismifolium
on lipid profile of hypergly———————————————————- 48
4.3.3 Effect of 28-days oral daily administration of methanol extract of C.alismifolium
onHaematological parameters of hyperglycemic rats————————————49
4.3.4 Effect of 28-days oral daily administration of the methanol extract of
C.alismifolium on liver function of hyperglycemic rats——————————– 50
4.3.5 Effect of 28-day oraldaily administration of the methanol extract of
C.alismifolium on renal indices of hyperglycemic rats——————————— 51
4.3.6 Effect of 28-days oral daily administration of the methanol extract of
C.alismifoliumon body weight of hyperglycemic rats———————————- 53
4.3.7 Effect of 28-days oral dailyadministration of the methanol extract of
C.alismifoliumon relative organ weights———————————————— 54
4.3.8 Effects of 28-days oral dailyadministration of C.alismifolium on histopathology
of the kidney, liver, pancreas and heart ofSTZ-induced hyperglycemic rats——– 55
4.4 Results of Antibacterial Studies———————————————————— 60
4.4.1 Diameter of Zone of Inhibition————————————————————- 60
4.4.2 Minimum Inhibitory and Bactericidal Concentrations of C.alismifolium Extract– 62
4.5 Phytochemical Constituents—————————————————————– 64
CHAPTER FIVE————————————————————————————- 65
5.0 DISCUSSIONS——————————————————————————- 65
CHAPTER SIX————————————————————————————— 70
6.0 CONCLUSION, SUMMARYANDRECOMMENDATIONS———————– 70
APPENDIX ——————————————————————————————- 91
1.1 Traditional Medicine
Traditional medicine is defined as the knowledge, skills and practices based on the theories, beliefs and experiences indigenous to different cultures whether explicable or not, used in the maintenance of health and in prevention, diagnosis, improvement or treatment of physical and mental illness (WHO, 2000).In some Asian and African countries,80% of the population depends on traditional medicine for primary health care needs (WHO,2008). This is attributed to the fact that traditional medicine is more affordable and accessible compared to orthodox medicine (Bhushan,2005).Traditional medicine that has been adopted by other population (outside its indigenous culture) is often termed alternative or complementary medicine (WHO,2000). Traditional medicine practice utilizes plants, animals, minerals and other methods (WHO,2008).
Herbal medicine is the use of medicinal plants for the prevention and treatment of diseases and it has become a popular healthcare remedy (Fabio and Luigi, 2007). According to World Health Organization,medicinal plant is defined as „any plant in which one or more of its part contains substance that can be used for therapeutic purposes or as precursors for the synthesis of useful drugs‟ (WHO, 2003).
Traditional medicines have provided us with lead compounds some of which are very useful synthetic class of modern drugs (Gregoryetal.,2009) that were originally discovered through herbal cures and folk knowledge (Gilanietal.,2011). Many known therapeutic agents used in orthodox medicine were derived from medicinal plants, some of these include digoxin from Digitalis purpurea(cardiac activity),quinine (an
antimalarial) from cinchona bark, vincristine and vinblastine alkaloids (antileukemic agents) from Catharantusroseus (Hardman,1991).Studies have shown that increasing daily intake of green leafy vegetables (such as Moringa oleifera, Vernonia amygdalina e.t.c.)could significantly reduce the risk of type 2 diabetes (Carter etal.,2010).
1.2 Statement of Research Problem
The World Health Organization (WHO) estimated the number of Diabetic patients to be 346 million worldwide and this number is likely tobe more than double by the year 2030 (WHO,2012). Almost 80% of the Diabetic deaths occur in low and middle income countries (WHO,2012). Diabetes mellitus is ranked seventh among the leading causes of death globally and it is considered third when its complications are taken into account (Trivedietal.,2004). In 2012 diabetes was the direct cause of 1.5 million deaths and hyperglycaemia was the cause of another 2.2 million deaths(WHO,2016). Diabetes related mortality is on the increase and every six seconds a person dies from diabetes (Diabetes Atlas,2013). Nigeria has the highest number of people with diabetes in the African region with approximately 1.22 million people affected and 3.85 million people with impaired glucose tolerance (IDF,2011).
Numerous hospital-based studies in Nigeria demonstrated that the prevalence of diabetic limb ulcerations was between 11.7 % and 19.1 % among individuals with diabetes in Nigeria (Ogberaet al.,2006; Unachukwu,2007) and diabetic foot ulcer is the second leading cause of diabetes-related deaths in Nigeria, accounting for 24 % of all diabetes related mortalities (Ogberaetal.,2005). Diabetic patients are also susceptible to many other infections and of particular interest urinary tract infection such as acute
pyelonephritis which is a more common disease in patients with diabetes than in non-diabetic patients (Patterson and Andriou, 1997).
Currently available antidiabetic agents do not provide cure and their usage is accompanied by debilitating adverse reactions. Thus, there is an increasing demand by patients to use natural products which are documented to possess antidiabetic activity (Ivorraetal.,1989).Available literature indicates that there are more than 800 plant species with antidiabetic activity (Rajagopal and Sasikala,2008).
To our knowledge, no scientific data on the antidiabetic property of Chlorophytum alismifoliumis available in literature. This study is designed to explore the folkloric claim that Chlorophytum alismifolium possesses antihyperglycaemicand antibacterial activities (Personal Communication,2014).
The number of people with DM is growing worldwide and this necessitates immediate action by introducing cost-effective strategies to reverse this trend (WHO, 2013). Diabetes mellitus caused 5.1 million deaths in 2013 taking up about USD 548 billion in health spending which accounts for 11% of total money spent worldwide (Diabetes Atlas, 2013).Insulin and oral antidiabetic agents are not only expensive but are also known to produce serious side effects, therefore the search for cheaper, safer and effective antidiabetic agents has continued to be an important area of investigation (Kleinet al., 2007).In addition, traditional medicine is a more affordable and accessible healthcare system available when compared to orthodox medicine (Bhushan,2005). Although insulin is the main therapyfor type 1 diabetes mellitus there are several
drawbacks like insulin resistance (Piedrolaetal.,1999), anorexia nervosa, brain atrophy and fatty liver (Yaryuraetal., 2001) following chronic treatment. Biguanides and sulphonylureas are valuable in the management of type 2 diabetes mellitus but their use is limited by side effects such as lactic acidosis, gastrointestinal tract disturbances and hypoglycaemia (Satoskar et al., 1999). Moreover, these therapies only partially compensate for metabolic derangements seen in diabetics and do not necessarily cure diabetes (Expert Commmittee on Classification and Diagnosis of Diabetes Mellitus, 1997).
There has been a focus on the search for new chemically active compounds from medicinal plants that will be suitable for use in diabetes mellitus (Mariangelietal.,2006). The genus chlorophytum (Liliaceae), owing to the presence of pharmacologically important saponins has attracted the interest of the scientific community (Nutan,2005).This project seeks to investigate the antihyperglycaemic and antibacterial activities of the methanol tuber extract of Chlorophytum alismifolium plant to justify (or otherwise) the folkloric claim of its uses. This therefore forms the basis for evaluating its purported antihyperglycaemic and antibacterial properties.
1.4 Theoretical Framework
The focus on plant research has increased all over the world and more than 13,000 plants have been studied between 1996 and 2000 (Dahanukar et al.,2000). Over the years, several animal models have been developed for studying diabetes mellitus. These models include chemical, surgical (pancreatectomy) and genetic manipulations in several animal species. The diabetogenic agents mostly used include Alloxan monohydrate and streptozotocin (with or without nicotinamide).
The selection of these models for investigation of the antidiabetic properties of a new compound may be a difficult task (Etuk,2010). Streptozotocin which is selected for this study is a glucosamine-nitrosourea compound derived from Streptomyces achromogenes that is used clinically as a chemotherapeutic agent in the treatment of pancreatic β cell carcinoma. It damages the β cells and result in hypoinsulinemia and hyperglycemia (Lenzen, 2008). Streptozotocin is a more suitable model for non-insulin dependent diabetes mellitus or type II diabetes (Pellegrino, 1998) but there are some disadvantages to its use in chronic experiments, especially by the development of functioning insulinoma (Steiner etal., 1970) and high incidence of kidney and liver tumors which are attributed to its oncogenic action (Antiaet al., 2005).
1.5 Aim and Objectives of the study
The aim of this study isto validate the folkloric claim for the use of Chlorophytum alismifolium in the treatment of diabetes and some of its complications.
1.5.2 Specific objectives
i. To extract and conduct phytochemical analysis on the methanol extract of C. alismifoliumtuber so as to identify the various chemical constituents present.
ii. To carry out acute toxicity studies on the methanol extract of C. alismifoliumtuber.
iii. To investigate the effect of methanol extract of C. alismifoliumtuber in streptozotocin-induced hyperglycaemic rats.
iv. To investigate the effect of methanol extract of C. alismifolium on lipid profile in streptozotocin-induced hyperglycaemic rats.
v. To carry out the invitro antibacterial studies of the methanol extract of C. alismifolium
vi. To determine the effect of the methanol extract of C.alismifoliumtuber on haematological indices (RBC, WBC, PCV, LYM and PLT), renal indices (EL, U and CR) and liver enzymes (AST, ALT, ALP and TB) in hyperglycaemic rats.
vii. To carry out histopathological studies on the pancreas, liver, kidney and heart ofhyperglycaemic rats.
1.6 Research Hypothesis
The methanol crude extract of C. alismifolium does not possess antidiabetic activity.
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