EFFECTS OF ETHANOL EXTRACTS OF EUPHORBIA HIRTA HERB ON SOME OXIDATIVE AND BIOCHEMICAL PARAMETERS IN ALLOXAN-INDUCED DIABETIC RATS

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.

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