ABSTRACT
Diabetes mellitus produces a lot of highly reactive oxygen species which have been attributed to the aetiology and pathophysiology of the disease. In view of the adverse effects associated with synthetic drugs and natural medicine being considered to be safer, cheaper and more effective, traditional antidiabetic plants can be explored. The results of the experiment showed that there were significant increases (P<0.05) in the concentrations of total cholesterol, low density lipoprotein (LDL) and triacylglycerol (TAG) in group 2 rats (diabetic untreated) compared with normal control rats (group 1). Administration of 300 mg/kg b.w. of ethanol extracts of Euphorbia hirta to rats in group 3 to 6 and 0.01mg/kg b.w of voglibose to rats in group 7 showed significant reduction (p<0.05) in total cholesterol, LDL and TAG concentrations. On the other hand, there was significant decrease (p<0.05) in high density density (HDL) concentrations in the group 2 (diabetic untreated) compared with group 1 (normal rats). However, administration of 300 mg/kg b.w of ethanol extracts of E. hirta to rats in group 3 to 6 and 0.01 mg/kg b.w to rats in group 7 showed significant increase (p<0.05) in HDL concentration. There was no significant increase (p>0.05) in sodium and bicarbonate ion concentrations but significant increase (p<0.05) in potassium and chloride ion concentrations in diabetic untreated rats (group 2) compared with rats in normal control group. There was significant increase (p<0.05) in serum urea and creatinine concentrations in diabetics untreated rats (group 2) compared with normal rats (group 1). Administration of 300 mg/kg b.w. of ethanol extract of E. hirta to groups 3 to 6 and 0.01 mg/kg b.w. of voglibose to group 7 resulted in significant decrease (p<0.05) in serum urea and creatinine concentrations. There was significant decrease (p<0.05) in serum catalase and superoxide dismutase activities and vitamin C concentration with significant increase (p<0.05) in serum malondialdehyde concentration in group 2 (diabetics untreated rats) compared with normal rats (group1). However, addition of 300 mg/kg b.w. of ethanol extract of E. hirta to Groups 3 to 6 and 0.01 mg/kg b.w. of voglibose to group 7 resulted in significant increase (p<0.05) in serum catalase and superoxide dismutase activities and vitamin C concentration, with significant decrease (p<0.05) in MDA concentration compared with the diabetic untreated rats (group 2). There was significant increase (p<0.05) in blood glucose concentration in rats of group 2 to 7 before administration of ethanol extracts of E. hirta and voglibose compared with normal rats (group 1). When 300 mg/kg b.w. of ethanol extract of E. hirta was administered to groups 3 to 6 and 0.01 mg/kg b.w. of voglibose to group 7, there was significant decrease (p<0.05) in blood glucose concentration compared with diabetic untreated (group 2). The administration of 300 mg/kg b.w. of ethanol extract of E. hirta and 0.01 mg/kg b.w. of voglibose showed significant increase (p<0.05) in the body weights of the rats in groups 4 to 7 compared with that of normal control. No significant increase (p>0.05) in the body weights of rats in group 2 and 3 compared with normal rats (group 1). When 300 mg/kg b.w. of ethanol extract of E. hirta and 0.01 mg/kg b.w. of voglibose were administered to rats in groups 3 to 7, there was significant increase (p<0.05) in the body weights of the rats compared with diabetic untreated rats (group 2).
CHAPTER ONE
INTRODUCTION
Euphorbia hirta herb is traditionally used to treat asthma, respiratory tract
infections and cough (Ogbulie et al., 2007) but has been recently reported to have antidiabetic effect which may be related to its antioxidant capacity and its alpha glucosidase inhibitory properties (Widharna et al., 2010). Some established alpha glucosidase inhibitors within the intestinal brush border, attenuates post-prandial blood glucose peaks (Balfour and Tavish, 1993). Diabetes mellitus produces a lot of highly reactive oxygen species which have been attributed to the aetiology and pathophysiology of the disease. Antioxidant enzymes such as catalase, glutathione peroxidase and superoxide dismutase help to neutralize harmful free radicals (Nelson and Cox, 2005). In view of the adverse effects associated with synthetic drugs and natural medicine being considered to be safer, cheaper and more effective, traditional antidiabetic plants can be explored (Kamboj, 2000).
1.1 HISTORY OF DIABETES MELLITUS
The term diabetes was coined by Aretaeus of Cappodocia susbruta (6th Century B.C) identified diabetes and classified it as med humelia and identified it with obesity sendentary life; hence, advising exercise to cure (Dwired et al., 2007). Medieral Persia Aricenna (980-1037) provided a detailed account on diabetes in the canon of medicine, describing the abnormal appetite and the collapse of sexual function. He also recognised a primary and secondary diabetes, and described diabetes gangrene. He treated diabetes using a mixture of lupine trigonella (Fenugreek) and zedoary seed which produced a reduction in excretion of sugar. He described diabetes insupidus very precisely for the first time but Johann Peter Frank (1745-1821) differenciated between diabetes mellitus and diabetes insupidus (Nabipour, 2003).
Diabetes was first recorded in English in the form diabete, in a medical text written around 1425. In 1675, Thomas Willis added two words mellitus from the Latin origin meaning “honey”, a reference to the sweet taste of the urine. Matthew Dabson confirmed that the sweet taste was because of an excess of a kind of sugar in the urine and of people with diabetes (Dobson, 1776). Aretaeus did attempt to treat it but could not give a good prognosis; He commented on life (with diabetes) is short and disgusting (Medvei, 1993).
The discovery of a role for the pancreas in diabetes was described by Joseph
Von Mering and Oskar Minkowski, who in 1889 found that dogs whose pancreas was
removed, developed all the signs and symptoms of diabetes and died shortly after wards (Von Mering and Minkowski, 1890). In 1910, Sir Edwin Albert sharpey- Schafer suggested that people with diabetes were deficient in a single chemical that is normally produced by the pancreas. He proposed calling this substance insulin, from the Latin insula meaning island, but the endocrine role of insulin was not clarified until 1921 when Sir Fredrick Grant Banting and Charles Herbert Best repeated the work of Von Mering and Minkowski and demonstrated they could reverse induced diabetes in dogs by giving them extracts from the pancreatic islets of Langerhans of healthy dogs (Bating et al., 1991).
In 1869, Paul Langerhans, a medical student of Berlin, was studying the structure of the pancreas under microscope when he identified some previously unnoticed tissue climps scattered throughout the bulk of the pancreas and were known as islets of Langerhans. In 1901, another major step was taken by Eugene Opie when he clearly established the link between the islet of Langerhans and diabetes. The distinction between what is now known as type 1 diabetes and type 2 diabetes was first clearly made by Sir Harold Perciral (Harry) Hims Worth and in January, 1936 (Himsworth, 1936).
Bating and Best purified the hormone insulin from Biovin pancreas at the University of Toronto (Bating et al., 1991) and in plants such as Safflower. The first synthetic insulin was produced simultaneously in the labs of Panaroitis Katsovannis at the University of Pittsburgh and Helmut, Zahn at RWTH, Aachen University in the early 1960. The first genetically engineered synthetic human insulin was produced in a Laboratory in 1977 by Herbert Boyer using E. coli, and in 1980, a U.S. biotech company Genentech, founded by Boyer and Eli Lily developed human insulin under the brand name Humulin. The insulin was isolated from genetically altered bacterial which produced large quantities of insulin.
Other land mark discoveries include:
Identification of the first of the sulfonylureas in 1942 by Marcel Janbon and co-workers (Janbon et al., 1942) and it induced hypoglycaemia in animals (Patlak, 2002).
Determination of the amino acid sequence of insulin by Sir Fredick Sanger
The radioimmuno assay for insulin as discovered by Rosaly Jallow and
Solomon Berson, gaining Jallow a Nobel Prize in Physiology or Medicine in
1977.
Dr. Gerald Reaven identified the constellation of symptoms now called metabolic syndrome in 1988.
1.2 Types of Diabetes Mellitus
There are four main types of diabetes mellitus: Type 1 diabetes, Type 2 diabetes, gestational diabetes and miscellanous types of diabetes.
1.2.1 Type 1 diabetes mellitus (insulin dependent diabetes mellitus)
Type 1 diabetes is characterised by loss of the insulin-producing beta cells of the islets of the langerhans in the pancreas leading to insulin deficiency apparently mediated by white cell production of active oxygen species (Oberley, 1988). This type of diabetes can be further classified as:
1.2.1.1 Immune-mediated type 1 diabetes mellitus
This type of diabetes mellitus accounts for majority of type 1 diabetes where the beta cell loss is a T-cell mediated autoimmune attack (Rother, 2007).
1.2.1.2 Idiopathic type 1 diabetes mellitus
In this type of diabetes mellitus, no aetiology has been clearly implicated.
1.2.2 Type 2 diabetes mellitus (Non-insulin dependent diabetes mellitus)
This type of diabetes mellitus result from insulin resistance, a condition in which cells fail to use insulin properly, sometimes combined with an absolute insulin deficiency. There is a strong inheritable genetic connection in type 2 daibetes.
1.2.3 Gestational diabetes mellitus
This type of diabetes mellitus occurs when pregnant women, who have never had diabetes before have high blood glucose level during pregnancy. Even though it may be transient, untreatable gestational diabetes can damage the health of the foetus or mother. Risks to the baby includes: Macrosomia (high birth weight), congenital
cardiac anomalies, central nervous system anomalies and skeletal muscle malformation. Infact, the rate of diabetes in expectant mothers has more than doubled in the past 6 years (Lawrence et al., 2008).
1.2.4 Miscellaneous types of diabetes mellitus
1.2.4.1 Pre-diabetes (Impaired glucose tolerance)
This is a condition that occurs when a person’s blood glucose levels are higher than normal, but not high enough for a diagnosis of type 2 diabetes. Many people destined to develop type 2 diabetes spend many years in a state of pre-diabetes and have risks of cardiovascular complications (ADA, 2002) which have been termed “America’s largest health care epidemics (Jellinger, 2009).
1.2.4.2 Genetic Type
Genetic mutations (autosomal and mitochondrial) can lead to defects in beta cell function. Abnormal insulin action may also have been genetically determined in some cases (Tominaga, 1999).
1.2.4.3 Secondary diabetes
Any disease that causes extensive damage to the pancreas may lead to diabetes. Such examples include chronic pancreatitis, cystic fibrosis, acromegaly, haemochromatosis, Cushing’s syndrome (Iwasaki et al., 2008), thyrotoxicosis, aging (Jack et al., 2004), high fat diet (Lovejoy, 2002) and less active life (Hu, 2003). Obesity has been found to contribute to approximately 55% of cases of type 2 diabetes (CDC, 2004) and decreasing consumption of saturated fats and transfatty acids, while replacing them with unsaturated fats, may decrease risks (Riserus et al.,
2009).
1.3 Clinical features of diabetes mellitus
The classical symptoms of diabetes mellitus include: Polyuria (frequency urination), polydipsia (increased thirst), polyphagia (frequent hunger), fatigue and weight loss (Cooke, 2008). Other important features include: collapse of sexual function (Nabipour, 2003), blurring of vision and skin rashes collectively known as diabetes dermadromes and develop in 30 to 70% of diabetic patients (Izaki, 2000).
1.4 Diagnosis of Diabetes Mellitus
Diabetes is characterized by recurrent or persistent hyperglycemia and is diagnosed by demonstrating any one of the following: (WHO, 1999).
Fasting plasma glucose level ≥ 7.0mmol/L (126 mg/dL)
Plasma glucose ≥ 11.1mmol/L (200 mg/dL) 2 hours after a 75g oral glucose load as in a glucose tolerance test.
Symptoms of hyperglycemia and casual plasma glucose ≥ 11.1mmol/L (200 mg/dL).
Glycerated haemoglobin (HbAIC ≥ 6.5%). Also, 2006 WHO diabetes criteria
Table 1: World Health Organisation diabetes criteria
Condition | 2 hours glucose mmol/L (mg/dL) | Fasting glucose mmol/L (mg/dL) |
Normal | < 7.8 (< 140) | < 6.1 (<110) |
Impaired Fasting glycaemia | < 7.8 (<140) | ≥ 6.1 (≥ 110) and < 7.0 (<126) |
Impaired glucose tolerance | ≥ 7.8 (≥ 140) | < 7.0 (< 126) |
Diabetes mellitus | ≥ 11.1 (≥ 200) | ≥ 7.0 (≥ 126) |
For pre-diabetes, fasting plasma glucose level from 6.1 mmol/L (110 mg/dL) to 6.9 mmol/L (125 mg/dL) (DCDM, 2005). It is preferred to measure a fasting glucose level because of the ease of measurement and the considerable time commitment of formal glucose tolerance testing which takes 2 hours to complete and offers no prognostic advantage over the fasting test (Saydah et al., 2001). Glycosylated haemoglobin (HbAIC) has emerged as an accepted marker of glycemic control and clinical efficacy in studies of diabetes (Horie, 2009).
1.5 Complications of Diabetes Mellitus
1.5.1 Acute complications diabetes mellitus
Diabetes without proper treatment can cause many complications such as hypoglycaemia, diabetes ketoacidosis or non ketotic hyperosmolar coma:
1.5.2 Chronic complications of diabetes mellitus
1.5.2.1 Diabetes Macroangiopathy
Diabetes macroangiopathy affects small muscular arteries, especially arteries of the lower leg and foot. A toe may be gangrenous in the presence of normal female and popliteal pulses due to the fact that relatively small vessels are narrowed by atheroma. This result in atherosclerotic cardiovascular disease like coronary arthery disease, cerebrovascular disease and peripheral vascular disease.
1.5.2.2 Diabetic Microangiopathy
Diabetes microangiopathy affects large arteries and affects diabetes of all types, appears to be related to the duration of the diseas and is properly aggravated by poor diabetic control. It is responsible for diabetic retinopathy, neuropathy and nephropathy (MacSween and Whaley, 1992), coronary heart disease and hypertension (Adler et al., 2000).
1.5.2.3 Infections
There is an increased susceptibility of bacterial and fungal infections. Boils, carbuncles and urinary tract infections sometimes complicated by pylonephritis and renal papillary necrosis are of frequent occurrence and may precipitate diabetic coma.
1.6 Euphorbia hirta Herb
Euphorbia hirta herb, commonly called Asthma weed is a very common annual herb. It has a hairy plane that grows up to 2 inches in height; it has numerous small flowers clustered together with opposite oblong leaves. The young yellow fruit is a small hairy capsule with 3 reddish-brown seeds. The plant flowers and fruits all year long. Fig. 1 shows the picture of the plant with its leaves, stem and flower.
1.6.1 Medicinal Application of Euphorbia hirta
Euphorbia hirta herb is traditionally used to treat asthma, bronchitis, worm infestation, conjunctivitis and dysentery (Ogbulie et al., 2007) but has been recently reported to have antidiabetic effect which may be related to its antioxidant capacity and its alpha glucosidase inhibitory properties (Widharna et al., 2010).
1.7 Alpha Glucosidase Inhibitors
Alpha glucosidase inhibitors act by delaying the absorption of complex carbohydrates and disaccharides to absorbable monosaccharide from the small intestine. They lower post prandial blood glucose and insulin levels by reversibly inhibiting glucosidases in intestinal brush (Balfour and Tavish, 1993). This process leads to a reduction of glucose absorption and subsequent reduction in postprandial hyperglycemia (Van de Larr, 2005).
1.7.1 Voglibose in the Management of Type 2 Diabetes
Voglibose is an alpha glucosidase inhibitor and is an ideal agent for the management of type 2 diabetes due to its direct hypoglycaemic effect through
decreased absorption and hypolipidemic effect via improved insulin sensitity
(Shinozaki, 1996).
Voglibose reduces cartid in time media thickness with decrease in HbAiC hence reducing the incidence of chronic vascular complications in diabetic patients (Yibchok-anun, 2009). A study conducted by Satoh and co-workers on 30 type 2 diabetic patients suggested that voglibose reduces oxidative stress generated and soluble intercellular adhesion molecule in obese type 2 diabetic patients (Satoh,
2006).
1.8 Oxidative Stress in Diabetes
Increasing evidence in both experimental and clinical studies suggest that increased oxidative stress is a widely accepted participant in the development and progression of diabetes and its complications (Baynes, 1991). Diabetes is usually accompanied by increased production of free radicals (Chang et al., 1993), or impaired antioxidant defenses (Mc Lennan et al., 1991).
Common antioxidants includes the vitamin A, C and E and the enzymes superoxide, dismutase, catalase, glutathione peroxidase and glutathione reductase (Maritin et al., 2003). Other antioxidants include α-lipoic acid, mixed carotenoids, coenzyme Q10 (Brownlee, 2001). Several bioflavonoids, antioxidants minerals (copper, zinc, manganese and selenium) and the cofactors (folic acid, vitamin B1, B2, B6, B12). They work in synergy with each other and against different types of radicals. Vitamin E suppresses the propagation of lipid peroxidation (Hensley et al., 2000). Vitamin C, with vitamin E inhibits hydroperoxide formation. Metal complexing agents, such as penicillamine bind transitional metals involve in some reactions in lipid peroxidation and inhibit Fenton and Haber-weiss-type reactions (Laight et al.,
2000); vitamins A and E scavenge free radicals (Chow, 1991).
The involvement of oxidative stress in the pathology of diabetes from its associated cardiovascular dysfunctions, nephropathy, retinopathy (leading to blindness) and embryopathy or congenital malfunctions, suggests that potential management of diabetes could benefit from use of dietary biofactors in medicinal and food plants (Okezie et al., 2007). The effects of antioxidants on oxidative stress are measured through some observable biomarkers which include:
1.8.1 Catalase
Catalase located in peroxisomes, decomposes hydrogen peroxide to water and oxygen (Winterbourn, 1993). Catalase activity is consistently found to be elevated in the heart (Sanders et al., 2001), Aorta (Kocak et al., 2000), as well as brain (Aragno et al., 1999) of diabetic rats.
1.8.2 Glutathione Peroxidase and Glutathione Reductase
Glutathione peroxidase and reductase are two enzymes that are found in the cytoplasm, mitochindria and nucleus. Glutathione peroxidase metabolizes hydrogen peroxide to water by using reduced glutathione as a hydrogen donor (Sies, 1993). Glutathione disulphide is recycled back to glutathione reductase, using the co-factor NADPH generated by glucose-6-phosphate dehydrogenase (Santini et al., 1997). Glutathione peroxidase activity is seen to be elevated in liver (Rauscher et al., 2000), kidney (Rauscher et al., 2000), aorta (Kocak et al., 2000), blood (Mohan and Das,
1998) and red blood cells (Sailaja and Suresh, 2000) whereas decreased activity was seen in heart (Kaul et al., 1996) and retina (Obrosova et al., 2000).
1.8.3 Lipid peroxidation
Hydroperoxides have toxic effects on cells both directly and through degradation to highly toxic hydroxyl radicals. They may also react with transitional metals like iron or copper to form stable aldehydes such as malondialdehydes that will damage cell membranes. Peroxyl radicals can remove hydrogen from lipids, producing hydroperoxides that further propagate the free-radical pathway (Halliwell and Guttetidge, 1990).
Induction of diabetes in rats with streptozotocin (STZ) or alloxan uniformily
result in an increase in thiobarbituric acid reactive substances (TBARS) an indirect evidence of intensified free-radical production. Increase in TBARS associated with diabetes is presented by treatment with nicotinamide (Melo et al., 2000), aspirin (Caballero et al., 2000), sodium nitroprusside (Mohan and Das, 1998), captoprin, enalapril (Kedziora-Kornatowska et al., 2000), if this treatment is given before or immediately after the diabetogen (Mekinova et al., 1995).
1.8.4 Superoxide Dismutase
Isoforms of SOD are variously located within the cells. CuZn-SOD is formed in both the cytoplasm and the nucleus. Mn-SOD is confined in the mitochodria but can be released into extracellular space (Reiter et al., 2000). SOD converts superoxide
anion radical produced in the body to hydrogen peroxide, thereby reducing the likelihood of superoxide anion interacting with nitric oxide to form reactive peroxynitrite. Alternations of SOD activity in diabetic animals are normalised by captopril (Kedziora-Kornatowska et al., 1998), lipoic acid (Obrosova et al., 2000), all of which were administered prior to or concomitant with the diabetogen. Treatment with vitamin C, vitamin E and β-carotene for eight weeks elevates hepatic SOD activity in diabetic rats, which is normal without treatment (Mekinova et al., 1995).
In the heart, which is an important target in diabetes and prone to diabetic cardiomyopathy leading to chronic heart failure, SOD and glutathione peroxidase expression as well as activities are decreased (Maritim et al., 2003), whereas catalase is increased in experimental models of diabetes (Hayden and Tyagi, 2003).
1.8.5 Vitamins
Vitamins A, C and E are diet-derived and detoxify free radicals directly. They also interact in recycling processes to generate reduced form of the vitamins. Α- tocopherol is reconstituted when ascorbic acid recycles the tocopherol radical; dihydroascorbic acid which is generated is recycled to glutathione. Vitamin E, a component of the total peroxyl radical-trapping antioxidant system, reacts directly with peroxyl and superoxide radicals and singlet oxygen and protect membranes from lipid peroxidation (Weber et al., 1997).
Treatment with vitamin C and E was shown to decrease urinary albumin excretion, glomerular basement membrane thickness and kidney weight in STZ- induced diabetic rats (Kedziora-Kornatowska et al., 2003).
1.9 Justification of the Research
Good herbal remedy for the treatment of diabetes mellitus is a welcomed development. Most of the severe complications of diabetes are due to the hyperglycaemic effects and the effects of free radicals produced as a result of the pathogenesis of diabetes mellitus. Hence, a good herbal drug with antioxidant and alpha glucosidase inhibition actions could prevent complications of diabetes.
1.10 AIM AND OBJECTIVES OF THE STUDY
1.10.1 Aim of the Study
This research is carried out to assess the effects of ethanol extracts of the leaves, flowers and stems of Euphorbia hirta on the blood glucose levels, body weight, oxidative and biochemical parameters in alloxan-induced diabetic rats.
1.10.2 Specific Objectives of the Study
This research work is therefore set out to achieve the following objectives:
1. To determine the effect of 300 mg/kg b.w. of the flower, leaf and stem ethanol extracts of E. hirta on serum electrolyte concentrations, lipid and renal profiles in comparison with 0.01 mg/kg b.w. of the standard drug, voglibose.
2. To determine the effect of 300 mg/kg of the flower, leaf and stem ethanol extracts of E. hirta on the variation of the blood glucose and body weight rats compared with 0.01 mg/kg b.w of the standard drug, voglibose.
3. To determine the effect of the 300 mg/kg b.w. of the ethanol extracts on some oxidative parameters of the rats compared with the 0.01 mg/kg b.w. of standard drug, voglibose.
This material content is developed to serve as a GUIDE for students to conduct academic research
EFFECTS OF ETHANOL EXTRACTS OF EUPHORBIA HIRTA HERB ON SOME OXIDATIVE AND BIOCHEMICAL PARAMETERS IN ALLOXAN-INDUCED DIABETIC RATS>
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