EFFECTS OF METHANOL EXTRACT OF CHRYSOPHYLLUM ALBIDUM SEEDS ON ALLOXAN- INDUCED-DIABETIC RATS

ABSTRACT

Methanol  seed  extract  of  Chrysophyllum  albidum  was  studied  to  ascertain  the  potential effects  of  the  extracts  on  some  biochemical   indices.  The  proximate   composition  of Chrysophyllum  albidum seed methanol extract was found to contain  crude protein (8.41±

0.02 mg/g), crude fat (19.12±0.01 mg/g), Ash content (1.42±0.01 mg/g) and moisture content (41.87± 0.03 mg/g). Results of the vitamin composition showed that it contains vitamin C (99.0± 0.03 µ/100g) vitamin A (70.0±0.01 µ/100g) and vitamin E (64.0± 0.03 µ/100g). The preliminary qualitative photochemical  screening showed that the seed  methanol extract of Chrysophyllum  albidum contains flavonoids, alkaloids, resin,  cardiac glycosides, saponins, terpenoids,   steroid  and  tannins.  The  quantitative   phytochemical   analysis  revealed  the presence  of flavonoids  (1.54± 0.11  mg/100g),  alkaloids  (1.04± 0.04  mg/100g),  glycoside (1.87± 0.02 mg/100g), saponins (3.66± 0.03  mg/100g), steroids (0.43± 0.01 mg/100g) and tannins (2.19±0.03  mg/100g).  A total of  thirty-six albino rats were assigned into nine (9) groups  of four  (4)  rats  each.  The  LD50  of  the  seed  methanol  extract  of  Chrysophyllum albidum was found to be less than 1000 mg/kg body weight. Groups 1, 2 and 3 represented normal, diabetic and standard control rats respectively. Groups 4, 5 and 6 represented diabetic rats treated with 100, 200 and 300 mg/kg b.w. of the extract respectively while Groups 7, 8 and 9 represented non-diabetic rats treated with 100, 200 and 300 mg/kg b.w. of the extract respectively. After induction of alloxan, the blood glucose levels of groups 2, 3, 4, 5 and 6 increased  significantly (p< 0.05)  compared  to group 1 (normal  control).  After  5 days of treatment with the seed methanol extract, there was a significant (p<0.05)  reduction in the glucose level of the diabetic treated except in group 3 and 5 compared to group 2 (positive control). There was no significant  change (p>0.05) in groups 7, 8  and 9 (normal treated) when  compared  to  negative  control.  Groups  3,  4,  5  and 6  (diabetic  treated)  showed  no significant change when compared with a negative control after 5 days of treatment. Result of catalase activity showed a significant  increase  (p<0.05) in group 5 when compared to the positive  control.  There was no significant  change  (p>0.05)  in catalase  activity of rats in groups 3, 4, 6, 7, 8 and 9 when compared to positive control. Result of reduced  glutathione (GSH) levels of rats in diabetic groups showed significant increase (p<0.05) compared to non-diabetic groups and the normal  groups. The histopathology studies of liver and kidney of rats treated with the seed methanol extract showed that the extract was toxic.

CHAPTER ONE

INTRODUCTION

Diabetes  mellitus  is  a  complex  disorder  characterized  by chronic  hyperglycemia  which results from malfunction in insulin secretion and / or insulin action, both causing impaired metabolism of glucose, lipid and protein (Mayfield, 1998; Kim et al., 2006). It is prevalent worldwide and is known to be one of the major causes of death. The prevalence of diabetes in urban  African  communities  is  increasing  with  ageing  of  the  population  and  changes  in lifestyle associated with urbanization and westernization (Sobngwi et al., 2001).

The chronic hyperglycemia of diabetes is associated with long term complications such as dyfunction and failure of various organs especially the eyes, kidneys, nerves, heart and blood vessels. These complications are being initiated and propagated by oxidative stress processes (Zozulinska et al., 1998). In diabetes, an altered oxidative metabolism is a consequence of either the chronic exposure to hyperglycaemia or the absolute or relative insulin deficiency. Insulin regulates several reactions involved in oxido-reductive  metabolism (Baynes, 1991). Toxic  oxygen  species  produces  serious  derange   in   cell   metabolism,   including  DNA destruction, modification of proteins and also induced damage to membrane ion transporters and / or other specific protein. Reactive oxygen species produced in the course of oxidative stress participate in the development of diabetic vascular complications (Packer, 1993). There is evidence that diabetes induces changes in the activities of antioxidant enzymes in various tissues (Ahmed, 2005).

The pathogenesis  of diabetes  mellitus  and  the possibility  of its management  by  existing therapeutic  agents without  any side effects  have stimulated  great  interest  in  recent  years (Bailey,  1999).  Management  of diabetes  without  any side effects  is still  a  challenge  for medical system. This leads to an increasing search for improved antidiabetic drugs.

.

Few plants that were used in traditional medicine for the treatment of diabetes have received little scientific scrutiny and the world health organization has recommended  that this area warrants further studies (Fattaneh, 2012). Since there is no current literature on the effect of Chrysophyllum albidum seed on diabetes, an investigation on the methanol seed extract of Chrysophyllum albidum on alloxan diabetic rats was carried out.

1.1 Chrysophyllum albidum

Chrysophyllum  albidum  is  a  tropical  edible  fruit  locally  called  udara  (Igbo),  agbalumo (Yoruba)  and commonly called African white apple or white star apple  (Kang, 1992). It belongs to the family of Sapotaceae and frequently found in many ecological zones of West Africa. It is distributed throughout the southern part of Nigeria (Idowu et al., 2006).

1.1.1    Morphological Characteristics of Chrysophyllum albidum

Chrysophyllum  albidum  is  a  flowering  plant  which  usually  flowers  from  April  to  June (Ehiagbonare et al., 2008). The flowers are sessile and occur in clusters in the leaf axil of the fruiting branch. The fruiting are normally from January to March but the fruits have been seen recently in November (Ehiagbonare et al., 2008). When ripe, the fruits are pale orange, edible, ovoid in shape and pointed at the apex as seen in Fig 1.1  below. It is a berry with cresent shaped seeds. The size of the seeds depends on the size of the fruit, with large fruits having large seeds and the small fruits with small seeds as shown in Fig 1.2 below. There are also variations among seed width, fruit length, number of seeds and mean diameter of seeds per fruits. Morphological  characteristics  indicated  likewise differences  ranging from taste, fruit colour and shape to seed colouration (Oyebade et al., 2011).

Fig. 1.1: Chrysophyllum albidum fruits

Fig. 1.2: Chrysophyllum albidum seeds

1.1.2 Taxonomical Profile of Chrysophyllum albidum

Chrysophyllum  albidum  has  up  to  800  species.  The  species  are  called  different  names depending on the locality. Nine fruit types of Chrysophyllum alibum were identified based on the ripe fruit size, colour and taste which varied from very sweet to  sour (Oyebade et al.,

2011).

Scientific classification of Chrysophyllum albidum.

Kingdom:                                          Plantae Subkingdom:                                    Tracheobionta Subdivision:                                      Spermatophyta Division:                                            Magnoliophyta Class:                                                 Magnoliopsida Subclass:                                           Dilleniidae Order:                                               Ebenales

Family:                                              Sapotuceae-Sapodilla family

Genus:                                               Chrysophyllum L-Chryysophyllum

Species:                                             Chrysophyllum Albidum

1.1.3    Habitat / Ecology of Chrysophyllum albidum

The plant is commonly found in the Central,  Eastern and Western Africa (Amusa et  al.,

2003).  They  are  distributed  in  Nigeria,  Uganda,  Niger,  Cameroon  and  Cote  d’  Ivoire Chrysophyllum albidum is a popular tropical fruit tree and widely distributed in the low land rain forest zones and frequently found in forest (Madubuike and Ogbonnaya, 2003).

1.1.4 Pharmacological / Medicinal potentials of Chrysophyllum albidum

Chrysophyllum  albidum contains some chemicals that are of pharmacological  importance. The ascorbic  acid content of C. albidum serves as antioxidant  as well as  in treatment  of scurvy (Adisa, 2000). The barks of C. albidum have been employed in folk medicine for the treatment of yellow fever and malaria, while the leaf is used as  an emollient and for the treatment of skin eruption, stomachache and diarrhoea (Adisa 2000; Idowu et al., 2006). The cotyledons from the seeds of C. albidum are used as ointments in the treatment of vaginal and dermatological infections in Western Nigeria (Abiodum, 2011). The seed cotyledon has been reported to possess anti- inflammatory and hypolipidemic effects (Olorunnisola et al., 2008).

The key constituents of the plants seed cotyledons are eleagnine (an alkaloid) tetrahydro-2- methycharman and skatole. Eleagmine was found to be the main compound responsible for its  antimicrobial  activity  (Idowu  et  al.,  2003).  Eleagnine  was  further  shown  to  exhibit, antinociecptive, anti-inflammatory and antioxidant activities (Idowu et al., 2006).

1.2 Diabetes mellitus

Diabetes  mellitus  is  a  metabolic  disorder  of  multiple  etiology  characterized  by  chronic hyperglycaemia with disturbances in carbohydrate, protein and fat metabolism resulting from defects in insulin secretion, insulin action or both (WHO, 2010).  Chronic  hyperglycaemia which is the chief symptom of diabetes mellitus has been found to enhance the generation of reactive oxygen species. Oxidative stress, through the production of reactive oxygen species (ROS) has been proposed as the root cause underlying the development of insulin resistance, β-cell  dysfunction,  and  impaired  glucose  tolerance.  It  has  also  been  implicated  in  the progression  of  long  term  diabetes  complications,  including  micro-vascular  and  macro

vascular dysfunction (Aruoma, 1999). According to Mayfield (1998), there are four  major classes of diabetes mellitus based on their aetiology and clinical manifestations.

1.2.1 Types I diabetes mellitus

Types  I  diabetes  mellitus  was  formerly  known  as  insulin-dependent  diabetes  mellitus (IDDM).  It is characterized  by loss  of  the  insulin  producing  beta  cells  of  the  islets  of langerhans in the pancreas leading to insulin deficiency.   About 5 % of total diabetic patients are of type I (Vasudevan and Sreekumari, 2007). Type I diabetes mellitus is sub classified as:

(a) Immune mediated

(b) Idiopathic

The majority of type 1 diabetes is of the immune- mediated nature, where beta cell loss is a T-cell mediated autoimmune attack (Rother, 2007). Onset of this type of diabetes is usually below 30 years of age, most commonly during adolescence (American Diabetic Association,

2003).

1.2.2 Types II diabetes mellitus

The  disease  is  due  to  decrease  biological  response  to  insulin,  otherwise  called  insulin resistance, defective insulin secretion or reduced insulin sensitivity which almost involves the insulin receptor in cell membranes. It is commonly seen in individual above 40 years. In early stage, the predominant abnormality is reduced insulin sensitivity,  characterized by elevated levels of insulin in the blood. At this stage hyperglycaemia can be reversed by a variety of measures and medication that improve insulin sensitivity or reduced glucose production by the  liver.  As  the  disease  progresses,  the  impairment  of  insulin  secretion  worsen  and therapeutic replacement of insulin often becomes necessary (WHO, 1999)

1.2.3 Gestational diabetes mellitus

Gestation  diabetes  involves  inadequate  insulin  secretion  and responsiveness.  It  resembles type 2 diabetes mellitus. Gestational diabetes starts during pregnancy. Hormones secreted by the placenta – oestrogen, progesterone, growth hormone,  corticotrophim-releasing  hormone and  prolactin-  decrease  the function  of  insulin,  resulting  in high blood  sugar  (American Diabetes Association,  2011). It affects about 1% to 3% of all pregnant women. It usually develops in the second trimester (sometimes as early as 20th weeks of pregnancy). About 20-

50%  of  women  with  gestational  diabetes  develop  type  2  diabetes  mellitus  later  in  life

(American Diabetes Association, 2011).

1.2.4    Maturity onset diabetes

This type of diabetes refers to a collection of different genetically caused forms of diabetes inherited in an autosomal dominant fashion. This type of diabetes mellitus is neither Type1 nor Type 2, though it is often misdiagnosed  by doctors not familiar  with these  kinds of genetic diabetes (American Diabetes Association, 2003).

Others in this group are:

    Diabetes induced by certain medications or chemicals.

    Diabetes caused by other diseases of the pancreas.

    Diabetes associated with abnormal conditions of hormones.

1.2.5    Risk factor for diabetes mellitus

These factors are collectively referred to as metabolic syndrome. It is characterized by

(i)        Abdominal obesity

(ii)       Hyper-triglyceridemia, low  HDL Cholesterol

(iii)      Elevated blood pressure: It has been observed that hypertension and diabetes  share predisposing  risk factors or rather  each  is a risk for the other  (Pyorala  et  al., 1987; Reaven, 1988; Laakso, 1999)

(iv) Insulin resistance  or decreased  glucose intolerance:  Insulin resistance  or  sensitivity is defined as the reduced ability of body tissues to respond to insulin.  Although, Type II diabetes mellitus results from both insulin resistance and insufficient insulin secretion (De Fronzo and Godmann, 1995), insulin resistance is probably the primary defect because those prone  to develop  Type 2 diabetes  mellitus exhibit  insulin resistance  before the development of glucose intolerance. It  is known that about 25% of non-diabetic adults manifest  this  syndrome  (Reaven,  1995).  People  with  the  metabolic  syndrome  are  at increased  risk  of  coronary  heart  disease  and  type  II  diabetes  (American  Diabetes Association, 2003).

1.2.6 Clinical presentations in diabetes mellitus

These symptoms include the following: (1) Polyuria (frequent urination).

(2) Polydipsia (increased thirst) and consequent increased fluid intake. (3) Polyphagia (increased appetite).

The symptoms may develop quite fast in type 1 particularly in children. The symptoms may be subtle or completely absent as well as developing much more slowly in type 2 diabetes mellitus (American Diabetes Association, 2003).

In type 1 diabetes, there may also be weight loss and irreducible fatigue. When the blood glucose  concentration  is  above  the  renal  threshold,  reabsorption  of  glucose  in  the proximal renal tubule is incomplete and part of the glucose remains in the urine giving rise to glycosuria (American Diabetes Association, 2003).

Prolonged high blood glucose cause glucose absorption and so changes in the shape of the lens,  leading  to  vision  changes.  Blurred  vision  is  a  common  symptom  of  diabetes mellitus. Diabetic patients may also present with diabetic ketoacidosis (DKA), which is an extreme  state  of metabolic  dysregulation  eventually  characterized  by the  smell of acetone on the patient’s breath (American Diabetes Association, 2003).

1.2.7 Complications of diabetes mellitus

The complications of diabetes mellitus are far less common and less severe in people who have well controlled  blood sugar levels (Nathan et al., 2005). Acute  complications  of diabetes mellitus include the following:

(a) Diabetic  ketoacidosis (DKA). This is an acute and dangerous complication  that  is always a medical emergency. Low insulin levels cause the liver to turn to ketone for fuel (Ketosis). Ketone bodies are intermediate substrates in the metabolic sequence.   This is normal when periodic, but can become a serious problem if sustained. Elevated levels of ketone bodies in the blood decrease the blood’s pH leading to diabetic ketoacidosis.

(b)  Nonketotic  hyperosmolar  coma.  This results  when  blood  glucose  levels  is  above

300mg/dl (16mmol/l). In this case, water is osmotically drawn out of cells into the blood and the kidneys eventually begin to dump glucose into the urine. This results in loss of water and an increase in blood osmolarity.

(c) Hypoglycemia or abnormally low blood glucose is an acute complication of several diabetes  treatments.  The  patient  may become  agitated,  sweaty,  weak and  have many symptoms  of  sympathetic  activation  of  the  autonomic  nervous  system  resulting  in feelings akin to dread and immobilized panic. Consciousness can be altered or even lost in extreme cases, leading to coma, seizures or even brain damage and death.

(d) Amputation, patients with poorly controlled diabetes often heal wound slowly, even from small cut. This can result in infection and subsequent amputation (NHS, 2010).

Chronic complications include the following:

(i)  Diabetic  neuropathy,  abnormal  and  decreased  sensation,  usually  in  a  “glove  and stocking’  distribution,  starting with the feet but potentially in other nerves,  later often fingers and hands, when combined with damaged blood vessels can lead to diabetic foot.

(ii) Diabetic retinopathy, resulting from the growth of friable and poor-quality new blood vessels in the retina as well as macular oedema which can lead to severe vision loss or blindness.  Diabetic  retinopathy  is the most frequent  cause of new  cases of blindness among adult aged 20-70 years (Fong et al., 2003).

(iii)  Diabetic  nephropathy,  damage  to  the  kidney(s)  which  can lead  to  chronic  renal failure and eventually requiring dialysis (WHO, 1999).

1.2.8 Diagnosis of diabetes mellitus

The examination begins with a medical history and a physical examination. The diagnosis of type 1 diabetes mellitus is usually prompted by onset symptom of excessive urination and excessive  thirst often accompanied  by loss of weight.  These symptoms  typically worsen over days to weeks and about 25% of people with type 1 diabetes mellitus have developed some degrees of diabetic ketoacidosis by the time the diabetes is recognized (WHO, 1999).

Diabetes  mellitus  is  characterized  by recurrent  or  persistent  hyperglycaemia,  and  is diagnosed by:

(1)  Determination   of   glucose   in  body  fluids:   The   blood   is   collected   using   an anticoagulant  (potassium  oxalate) and an inhibitor of glycolysis (sodium  fluoride). Capillary blood from finger tips may also be used for glucose  estimation by strip method (American Diabetes Association, 2003) .

(2)  Enzymatic method: This is highly specific, giving ‘true glucose” values. Present day autoanalysers can use only the enzymatic methods. The glucose oxidase method is the one most widely used. Glucose oxidase is very specific; it  converts glucose to gluconic  acid and hydrogen  peroxide.  Peroxidase  converts  the  H202  into  H20 and nascent  oxygen.  The  oxygen  oxidizes  a  colourless  chromogenic  substrate  to  a coloured one; the colour intensity is directly proportional to concentration of glucose (Weyer et al., 1999).

(3)  Oral Glucose Tolerances Test (OGTT)

Glucose tolerance test is artificial, because in day to day life, such a large quantity of glucose does not enter into blood. However, the GTT is a well standardized test and is highly useful to diagnose diabetes mellitus in doubtful cases (Weyer et al., 1999).

1.2.9   Effect of diet on glucose level

Diabetes mellitus is characterized by recurrent or persistent hyperglycaemia and is diagnosed by demonstrating any of the following (WHO, 1999):

          Blood sugar analyzed at any time of the day without any prior preparations is called

random blood sugar and at or above 200mg/dl (11.2mmol/l) indicates hyperglycaemia

           Sugar  estimated  in the early morning,  before  taking breakfast  ( 12  hr  fasting)  is fasting blood sugar and at or above 126 mg/dl (7.0mmol/l) indicates hyperglycaemia

           The test done about 2 hours after a good meal is post-prandial blood sugar and at or above 200mg/dl (11.1mmol/l) indicated hyperglycaermia.

           The ability of a person to metabolize a given load of glucose after 2 hours is referred to  as glucose  tolerance  test  (OGTT)  and  at or above  200  mg/dl  or  11.1  mmol/l indicates hyperglycaemia.

Diagnosis of diabetes mellitus should not be based on a single random test alone, it should be repeated. A blood test for hemoglobin levels can be used to follow the adequacy of blood sugar control. It provides a fairly accurate measurement of the average blood sugar during the previous two to three months. Risk of complications is lower in patients who maintain lower hemoglobin values. Type I diabetic patients can be confirmed by blood  tests that identify antibodies directed against the pancreas. However, there is no test for type 2 diabetes beyond measurement of blood sugar (American Diabetes Association, 2011)

1.2.10 Management of diabetes mellitus

Diabetes  mellitus  is  a  chronic  disease  which  cannot  be  cured  except  in  very  specific situations.  Management  concentrates on keeping blood sugar levels as close to  normal as possible without causing hypoglycemia. This can usually be accomplished with diet, exercise and use of appropriate medications (NHS, 2009).

Patient education, understanding and participation is vital since the complications of diabetes are far less common and less severe in people who have well managed blood  sugar levels (Nathan et al.,  2005). Attention is also paid to other health problems that may accelerate the deleterious effects of diabetes. These include smoking, elevated  cholesterol levels, obesity, high blood pressure and lack of regular exercise (NHS, 2009).

1.2.11 Treatment of diabetes mellitus

Diabetes mellitus is currently a chronic disease without a cure and medical emphasis must necessarily  be  on  managing/avoiding  possible  short-term  as  well  as  long-term  diabetes related  problems.  Given  the  associated  higher  risks  of  cardiovascular  disease,  lifestyle modifications should be undertaken to control blood pressure and cholesterol by exercising, consuming appropriate diet and avoiding smoking (Nathan et al., 2005).

Currently, type 1 diabetes mellitus can be treated only with insulin, with careful monitoring of blood glucose levels. Apart from the common subcutaneous injection, it is also possible to deliver insulin by a pump (Ohkubo et al., 1995) which allows continuous infusion of insulin

24 hours a day. It is also possible to deliver insulin with an inhaled powder. Type 1 diabetes mellitus treatment must be continued  indefinitely.  Type 2 diabetes mellitus is  usually first treated by attempts to increase physical activity,  decrease carbohydrate  intake and loss of body weight.  These  can restore  insulin  sensitivity  and  even  achieve  satisfactory  glucose

control; sometimes for years. However, the underlying tendency to insulin resistance is not lost and so adherence to diet and exercise must continue. Next step in type 2 diabetes mellitus treatment (if necessary)  is intake of antidiabetic drugs which can still be used  to improve insulin production (e.g. sulfonylureas), regulate inappropriate release of glucose by the liver, attenuate  insulin  resistance  to  some  extent  (metformin  biguanides),  and  attenuate  insulin resistance (e.g. thiazolidinediones) (Nathan et al., 2005).

Although there is no cure for diabetes mellitus, controlled studies have shown that intensive glycaemia  control  goes  a  long  way  to  reduce  the  associated  morbidity  and  risk  for complications (Ohkubo et al., 1995; Stratton et al., 2000).

Since the actual mechanism by which type 1 diabetes mellitus develops is not known, there are no preventive measures available for type 1 diabetes mellitus. Studies have attributed a protective effect of breast feeding on the development of type 1 diabetes mellitus (Kostraba et al., 1999). Type 2 diabetes mellitus risk can be reduced in many cases by making changes in diets and increasing physical activity (Knowler et al., 2002; Lindstrom et al., 2006).

Some of plant extract that possess hypoglycaemic activities include: Gymena sylvestre  leaf (Shanmugasundaram  et al., 1990), Trigonella foecum-graecuim seed (Sharma et al., 1990), Loranthus micranthus  (mistilloe)  (Osadabe et al., 2004). The hypoglycaemic  activities  of these plant extracts  have been demonstrated  in different  animal models.  Other studies by Ugochukwu  and  Babady  (2003),  Pushparaj  (2002),  Grover  et  al.  (2002)  described  the antidiabetic properties of Gongronema latifolium, Averrhoa bilinibi, Brassica juncea seeds and Vernonia amygdalina  leaf extracts respectively using animal models. There have also been successful clinical trials of some of these antidiabetic plants (Vuskan et al., 2000).

About 80% of the world’s populations rely on herbal medicines, and governments of Third World countries were unable to sustain a complete coverage with Western-type drugs. These have encouraged the rationale development of traditional treatments (Fattaneh et al., 2012). Presently, the World Health Organization is taking an official interest in such developments in the bid to make health care available to all.

1.3 Experimental Diabetes

1.3.1   Alloxan

Hyperglycaemia, hyperlipidaemia and depressed antioxidants are the main common clinical features of the autoimmune disease, diabetes mellitus. Diabetes mellitus could be induced in experimental animals by chemicals which selectively destroy pancreatic beta-cells. The most common diabetogenic agents are alloxan or streptozotocin (Baynes, 1991). Alloxan (2, 4, 5,

6-  tetraoxypyrimidine  5,  6-  dioxyuracil)  was  first  described  by Brugnatelli  in 1818  but

Wohler and Liebig first called it alloxan and described  its synthesis (Lengen and  Panten,

1988).

The cytotoxic action of alloxan is medicated  by reactive oxygen species (Bromme et  al.,

2006). Alloxan and the product of its reduction, dialuric acid establishes a redox cycle with the  formation  of  superoxide  radicals.  These  radicals  undergo  dismutation  to  hydrogen peroxide  with a simultaneous  massive  increase  in cytosolic  calcium  concentration  which causes  rapid  destruction  of  pancreatic  beta-cells  (Szudelski,  2001).     Alloxan  oxidizes glutathione (GSH): one molecule of alloxan oxidizes at least 50 molecules of GSH and forms about  25  molecules  of  hydrogen  peroxide  (Bromme  et al., 2001).  Induction  of diabetes mellitus by alloxan in animals was associated with a marked fall in plasma nitric oxide (NO) and insulin, a simultaneous elevation in  glucose,  lactate, ketone bodies and lipid peroxide levels  (Graier  et  al.,  1996).  Alloxan  exerts  its  diabetogenic  action  when  administered parenterally,  intravenously,  intraperitoneally  or  subcutaneously.     The  dose  required  for diabetes   induction   depends   on  the   animal   species,   nutritional   status   and   route   of administration.  According to the administered  dose of alloxan, symptoms similar to either type 1, type 2 diabetes mellitus or glucose intolerance can be induced (Lengen and Panten,

1988; Mythill et al., 2004). Human islets are considerably more resistant  to alloxan  than those of other animals. Most frequently used intravenous dose of alloxan to induce diabetes in rat is 65 mg/kg body weight (Gruppuso et al., 1990; Boycon et al., 1992). The dose should be double or tripled if it has to be given through other routes (katsumata et al., 1992). Fasted animals  are more  susceptible  to  alloxan  (kastsumata  et al., 1992)  while  increased  blood glucose provides partial protection (Szkudelski et al., 1998).

1.3.2    Free radicals and reactive oxygen species

Free  radicals  are  molecules  or  molecular  fragments  containing  one  or  more  unpaired electrons in its outermost orbital (Vasudevan and Sreekumari, 2007). Molecules and atoms

contain in their outermost orbit two electrons spinning in opposite directions. The unpaired electron in the outermost  orbit of a free radical  is unstable  and highly  reactive.  Aerobic organisms derive energy by oxidizing molecules with oxygen, finally producing water and carbon dioxide. Products of partial reduction of oxygen are highly reactive like oxygen free radicals and other oxidizing agents like hydrogen peroxide  and are called reactive oxygen species  (ROS).  They  are  formed  as  necessary  intermediates   in  a  variety  of  normal biochemical reactions, but when generated in excess or not appropriately controlled, radicals can wreak havoc on a broad range of macromolecules. Free radicals are extremely reactive which explains not only their normal biological activities, but how they infilict damages on cells.

ROS  include  free  radicals  such  as  superoxide  (.O2-),  hydrogen  (.OH),  peroxyl  (.RO2),

hygroperoxyl (.HRO2-) as well as non radical species such as hydrogen peroxide (H2O2) and hydrochlorous acid (HOCl), Ozone (O3) and singlet oxygen (O2) (Valko et al., 2007).

In the mitochondria of cells, production of superoxide and hydrogen peroxide takes place. During  energy  transduction,  a small  number  of  electrons  “leak”  to  oxygen  prematurely, forming   the   oxygen   free   radical   superoxide,   which   has   been   implicated   in   the pathophysiology of a variety of diseases (Valko et al., 2007). Cellular productions of these ROS are enhanced during stress and can pose a threat to cells but it is also thought that ROS act as signal for the activation of stress-response and defence pathways (Mittler, 2002). Thus, ROS can be viewed as cellular indicators of stress and as secondary messengers involved in the stress-response signal transduction pathway (Valko et al., 2005). Over-accumulation  of ROS  can result  in cell death  (Toykuni,  1999).  ROS-induced  cell  death  can  result  from oxidative  processes  such   as  membrane   lipid  peroxidation,   protein  oxidation,   enzyme inhibition and DNA and RNA damage (Etsuo et al., 1991).

Peroxisomes are known to produce H2O2, but not O2.-, under physiologic condition (Valko,

2004). Peroxisomes  are major sites of oxygen consumption  in the cell and participate  in several metabolic functions that use oxygen. Oxygen consumption in the peroxisome leads to H2O2 production, which is then used to oxidize a variety of molecules (Forman et al., 2010). The organelle also contains catalase, which decomposes hydrogen peroxide and presumably prevent accumulation  of this toxic compound.  Thus, the peroxisome  maintains a delicate balance with respect to the relative concentrations or activities of these enzymes to ensure no net production of ROS. When peroxisomes are damaged and their H2O2  consuming enzymes

downregulated, H2O2 releases into the cytosol which is significantly contributing to oxidative stress (Juranek and Bezek, 2005). Free radicals  are formed  disproportionately  in diabetes mellitus  by  glucose  degradation,  non  enzymatic  glycation  of  proteins,  and  subsequent oxidative degration, which may play an important role in the development of complications in diabetic patients (Soliman, 2008).

1.3.3    Oxidative stress

Oxidative  stress indicates  the balance  status of living things at the molecular  level. It  is generally  defined  as  excess  formation  and  /  or  insufficient  removal  of  highly  reactive molecules  such  as  reactive  oxygen  species  (ROS)  and  reactive  nitrogen  species  (RNS) (Johansen  et  al.,  2005).  Oxidative  stress  has  been  proven  as  the  major  factor  in  the development  of  many chronic  human  disease  (Halliwell  and  Gutteridge,  2007),  such  as cardiovascular  diseases  and  diabetes  (Finkel  and  Holbrook,  2000;  Clarke  and  Armitage,

2002). Oxidative stress leads to several events in the biological system such protein damage leading to loss of function, lipid peroxidation leading to membrane damage,  mitochondrial damage resulting in permeability transition and DNA damage leading to cell death, mutations and cancer (Vasudevan  and Sreekumari, 2007).

1.3.4    Lipid peroxidation

One of the best known toxic effects of oxygen  radical is damage to cellular  membranes (plasma, mitochondrial and endomembrane systems) which is initiated by a process known as lipid  peroxidation.  Lipid  peroxidation  is the oxidative  deterioration  of lipid  containing  a number  of carbon  bonds.  A large  number  of toxic  by–products  are formed  during  lipid peroxidation (LP). The damage caused by LP is highly detrimental to the functioning of the cell and its survival (Raha and Robinson, 2000).

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The presences of polyunsaturated fatty acid (PUFAS) in the phospholipids of the bilayer of biological membranes are the basis of their critical feature of fluidity. Since LP attacks the components  that  impart  these  properties,  it  affects  the  biophysical   properties  of  the membranes.  LP  decreases  the  membrane  fluidity,  changes  the  phase  properties  of  the membranes and decreases electrical resistance (Raha and Robinson, 2000).

Also  cross  linking  of  membrane  components  restricts  mobility  of  membrane  protein. Peroxidative attack on PUFAS of biological membrane will compromise one of its important functions, its ability to act as barrier.  LP causes lysosome latency to decrease ie they become

fragile or simply “leaky”. Peroxidative attack on the plasma membrane of hepatocytes causes extensive damage such that molecules as large as enzyme are able to leak out  (Raha and Robinson, 2000).

Lipid  peroxidation  is implicated  in the pathogenesis  of a number of disease  and  clinical conditions.  These  include  premature  birth  disorders,  diabetes,  adult  respiratory  distress syndrome,  aspects  of  shock,  Parkinson  diseases,  Alzheimer   diseases,  various  chronic inflammatory conditions, ischemia – reperfusion mediated injury to organs including heart, brain and intestine,  arthrosclerosis,  organ injury  associated  with shock and inflammation, fibrosis and cancer, preeclampsia, and eclampsia, inflammatory liver injury, type I diabetes, anthracycline   induced   cardiotoxicity,   silicosis   and   pneumoconiasis   (Halliwell      and Gutteridge, 1992).

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