PHYTOCHEMICAL CONSTITUENTS, FREE RADICAL SCAVENGING PROPERTIES AND ANTIDIABETIC POTENTIALS OF METHANOL EXTRACT OF THE STEM (BARK) OF ALSTONIA BOONEI ON ALLOXAN- INDUCED DIABETIC ALBINO RATS

ABSTRACT

This  study  was  carried  out  to  investigate  the  phytochemical  constituents  of  the methanol extract of the stem (bark) of Alstonia boonei (MEAB), in vitro antioxidant activity of the extract and its possible antidiabetic  and antioxidant  potential using alloxan- induced diabetic rats as model. The qualitative analysis of the extract showed a  wide   range   of   phytochemicals,   which   could   be   physiologically   potent   in ameliorating several diseases. Quantitative  phytochemical  analysis revealed that the extract  contains  relatively  high   concentration  on  tannin  (7.375±0.005mg/100g), flavonoid   (6.176±0.003   mg/100g)   and   phenols   (5.867±0.003   mg/100g).   The quantitative  result  of   antioxidant   vitamins  shows  that  vitamin  C  was  highest (24.91±0.005mg/100g)  compared  to  vitamin  A  (1.314±0.002µg/g)  and  vitamin  E (0.886±0.002mg/100g).  The  methanol  extract  of Alstonia  boonei  scavenged  1,  1- diphenyl-2-picrylhydrazyl  radical (DPPH.) in a concentration dependent manner with a correlation coefficient (R2) of 0.7066, indicating antioxidant activity with effective concentration  that  inhibits  50 percent  of the radicals  (EC50) of 12.33  ± 0.2µg/ml compared to ascorbic acid standard EC50   of 98 ± 0.02µg/ml. The superoxide radical scavenging  activity was concentration-dependent  with an  EC50   of 7.03±0.42µg/ml compared with ascorbic acid and rutin standards with EC50 of 812.97±0.97µg/ml and 3.47 ± 0.6µg/ml respectively. The extract also showed hydroxyl radical scavenging activity with an EC50  of 42.75±0.02µg/ml compared to α- tocophenol standard with EC50  of 232.31 ± 6.97µg/ml. The nitric oxide radical scavenging activity shows that the extract scavenged nitric oxide radical in a concentration dependent manner with 500µg/ml being more effective than 500µg/ml of ascorbic acid standard. There was a significant  increase  (P  >  0.05)  in  the  serum  glucose  level  in  group  2  (diabetic untreated) compared to group 1 (normal control). Significant decrease (P < 0.05) in glucose serum concentration was recorded in all groups treated with the extract and the standard drug compared to group 2 (diabetic untreated) . There was a significant increase (P < 0.05) in urea, creatinine, sodium ion and chloride ion concentrations of group  2  (diabetic  untreated)  compared  to  group  1  (normal  control).  Significant decrease (P < 0.05) in urea, creatinine, sodium  ion and chloride ion concentrations was  recorded  in  all  groups  treated  with  the  methanol  extract  and  the  standard drug(group 3 to 6) compared to group 2 (diabetic untreated). There was no significant increase (P > 0.05) in potassium ion concentration of group 2 when compared with group 1. There was a  significant increase (P < 0.05) in the serum concentration of Aspartate  aminotransferase,  (AST), Alanine  aminotransferase,  (ALT)  and Alkaline phosphatase, (ALP) of group 2 (diabetic untreated) when compared with group 1 and significantly reduced (P < 0.05) in all groups treated with the extract and  standard drugs when  compared  to  group 2. Serum  superoxide  and  catalase  activities  were significantly (P < 0.5) reduced in group 2 (diabetic untreated) when compared to the normal control. Serum superoxide and catalase activities increased significantly (P < 0.05) in all groups treated  with the extract and standard drugs when compared  to group 2 (diabetic untreated). There was a significant  increase (P < 0.05) in  serum malondialdehyde   (MDA)  concentration  of  group  2  (diabetic   untreated)  and  a significant reduction (P < 0.05) in all groups treated with the extract and the standard drug compare to group 2. These results suggest that  methanol extract of the stem (bark)  of  Alstonia  boonei  (MEAB),  possesses  and  antidiadetic  and  antioxidant potentials.

CHAPTER ONE

INTRODUCTION

Diabetes is one of the most challenging health problems in the twenty first century  (Rahman  et  al.,  2009).  Diabetes  currently  afflicts  171  million  people worldwide (Boden and Taggart, 2009). Normal non-diabetic patients maintain plasma glucose <100 mg/dl in the fasting and <135 mg/dl in the post prandial period (Rossetti et al., 2008).  Diabetes  mellitus  is a group of  metabolic diseases  characterized  by hyperglycemia  resulting  from  defects  in  insulin  secretion,  insulin  action  or  both (American Diabetes  Association,  2009a).  Diabetes mellitus is classified  as type 1, type 2, with other specific  examples  and gestational  diabetes  (American  Diabetes Association,  2008).  Type  1  diabetes  is  known  as  insulin  dependent  diabetes  or Juvenile-onset  diabetes  and type 2 diabetes  is known as non-insulin dependent  or adult onset  diabetes (American Diabetes Association, 2009b). A slowly progressive form of type 1 diabetes was acknowledged  as latent autoimmune diabetes in adults (LADA)   by  the  World   Health   Organization   (WHO)   and  American   Diabetes Association (ADA) (Van Deutekom et al., 2008). Classification schemes define type 1 diabetes as a state of absolute insulin deficiency and type 2 as a state of  insulin resistance combined with inadequate insulin secretion (Greenbaum et al., 2009). Type 1 diabetes is an autoimmune disease where auto reactive immune cells attack insulin producing β-cells, destroying insulin reserve leading to hyperglycemia (Eldor et al.,

2009). The rate of loss of β-cell function is affected by factors like age at diagnosis, degree  of metabolic  control,  immune  status,  genetics  and  marked  inter-individual variation (Palmer, 2009). The symptoms of type 1 diabetes are significant weight loss and ketoacidosis (Ludvigsson et al., 2008). Diabetes Type 2 diabetes is a progressive disease  characterized   by  declining  β-cell  function  that  in  concert  with  insulin resistance,  leads  to  loss  of  glycemic  control  and  eventual  diabetes  complications (Nauck et al., 2009). Type 1.5 diabetes also known as latent autoimmune diabetes in adults  (LADA)  is  an  important  form  of diabetes  although  it  is  frequently  under estimated (Mayer et al., 2007). Type 1.5 diabetes is also known as slowly progressive type I diabetes, autoimmune diabetes in adults with slowly progressive β-cell failure, autoimmune  diabetes  not  requiring  insulin  at  diagnosis,  autoimmune  diabetes  in adults and type 1.5 diabetes (Dunn et al., 2008). Type 1.5 diabetes has a later onset and slower progression towards an absolute insulin requirement (Cernea et al., 2009). Type 1.5 diabetes occurs in about 10% of patients classified as type 2 diabetes and not

initially requiring  insulin (Agardh  et al., 2009). Diagnosis  of type 1.5 diabetes  is difficult due to lack of defining features (Jasem et al., 2010).

1.1 Alstonia boonei

There  has  been  worldwide  increase  in realization  of the  use  of  medicinal plants in various traditional health systems of many countries. Recent estimates by the World  Health Organization  (WHO)  revealed  that about 80%  of  the population  in Africa  relies  on  traditional  medicine  of  which  the  botanicals  constituted  greater components (Kayode and Kayode, 2008). These  botanicals had over the years been subjected to wide and unsustainable use  (Kayode, 2006). Many of these medicinal plants  provide  relief  of  symptoms  comparable  to  that  obtained  from  allopathic medicine (Choi and Hwang, 2003). One of these plants, Alstonia boonei is a herbal medicinal plant of West Africa origin. The plant parts have been traditionally used for its  antimalarial,  aphrodisiac,  antidiabetic,  antimicrobial,  and  antipyretic  activities, which have also been proved scientifically (Kweifio-Okai, 1999). The plant parts are rich in  various bioactive compounds such as echitamidine,  Nα-formylechitamidine, boonein, loganin, lupeol, ursolic acid, and β-amyrin among which the alkaloids and triterpenoids form a major portion (Tepongning et al., 2011).

1.1.1 Morphology of Alstonia boonei

Alstonia grows into a giant tree in most of the evergreen rain forests of tropical West Africa. The plant thrives very well in damp riverbanks. It is well known to all the traditional healers practicing along the west coast of Africa.  Alstonia boonei De Wild is a deciduous  tree up to 35 meters high. It  buttresses  deep-fluted  high and narrow. Its white latexes are copious. The leaves are in whorls at nodes, oblanceolate, apex rounded to acuminate, lateral vein prominent almost at right angle to midrib. The flowers are white with lax terminal cymes. The fruits are paired with slender follicle up to 16 cm long with brown floss at each end.

1.1.2 Classification of Alstonia boonei

Alstonia comprises about 40 species and has a pantropical distribution. There are about twelve species of the genus Alstonia. Alstonia boonei De Wild belongs to the family Apocynaceae. The species are scattered all over the world of which two are indigenous to Africa. The plant is known locally as Onyame dua, Egbu e.t.c . Elsewhere,  Alstonia  is known as Australian fever bush,  Australian quinine, Devil tree,  Dita  bark,  fever  bark,  or  palimara  (Gosse   et   al.,  1999).  The  scientific classification of Alstonia boonei is presented thus;

Kingdom: Plantae Phylum: Tracheophyta Order: Gentianales Family: Apocynaceae Genus: Alstonia Species: boonei

Botanical name: Alstonia boonei

Common name: Devil tree.

Figure 2: Leaves of Alstonia boonei

1.1.3 Uses of Alstonia boonei

The bark of Alstonia tree is one of the most effective analgesic herbs available in nature. All the parts of the plant are very useful but the thick bark cut from the matured tree is the part that is most commonly used for therapeutic purposes.  The bark of the tree is highly effective when it is used in its fresh form; however, the dried one  could  equally  be  used.  Therapeutically,  the  bark  has  been  found  to  possess antirheumatic,    anti-inflammatory,    analgesic/pain-killing,    antimalaria/antipyretic, antidiabetic   (mild   hypoglycaemic),   antihelminthic,   antimicrobial   and   antibiotic properties (Abbiw, 1990). A decoction could be sweetened with pure honey and be taken  up  to  4  times  daily  as  an  effective  painkiller  for  the  relief  of  Painful menstruation (dysmenorrhoea), when associated with uterine fibroid or ovarian cysts in women;  lower abdominal  and  pelvic congestion associated  with gynaecological problems  such  as  pelvic  inflammatory  diseases;  to  relieve  the  painful  urethritis common with  gonococcus or other microbial infections in men. Alstonia decoction also  exerts  a mild  antibacterial  effect  in this  case,  relieving  the  aches  and  pains associated  with  malaria  fever.  Alstonia  is taken  in the  form  of preparations  that exhibits antipyrexia and anti-malaria effects, to combat rheumatic and arthritic pains. The decoction of Alstonia bark could be taken alone as an effective pain-killing agent. A cold infusion made from the fresh or dried bark of  Alstonia  taken orally two to three times daily exerts a mild hypoglycaemic  effect  on diabetic patients. The cold infusion  is  also  administered  orally  for  the  purpose  of  expelling  round  worms, threadworms  and other  intestinal  parasites  in children.The  fresh  bark  of  Alstonia could be used in preparing  herbal tinctures; it is particularly useful as an effective antidote against snake, rat, or scorpion poison. It is also useful in expelling retained products of conception and afterbirth when given to women. Asthma can be treated with a  drink prepared from parts of Trema orientalis and decoction of the bark of Alstonia boonei mixed with the roots and bark of cola and fruits of Xylopia parviflora with  hard  potash  .  The  bark  decoction  of  Alstonia  boonei  is  used  with  other preparations in the treatment of fractures or dislocation,  jaundice, and for inducing breast  milk.  Its latex is taken as a purgative.  The  hardened  latex is used  for the treatment of yaws. Alstonia boonei De Wild is  regarded  as one of few herbs with potential  anti-HIV  indicators  (Adotey  et  al.,  2012).  In  some  African  countries Alstonia boonei is considered  a sacred  tree and worshiped  in the forest and hence human beings in those countries do not eat its parts.

1.2 Reactive Oxygen Species (ROS)

A free radical is defined as any molecule or atom which contains one or more unpaired  electrons  in  its  outer  orbit  (Halliwell  and  Gutteridge  1989).  The  term “reactive  oxygen  species”  (ROS)  is often used  to refer  to free  radicals  and other

oxygen-related reactive compounds, such as singlet oxygen (1O2), hydrogen peroxide

(H2O2),  and  hydroperoxides  (ROOH)  (Halliwell  and  Gutteridge  1989).  All  these compounds have the potential to cause free-radical reactions (Pryor, 1986). When a free radical reacts with a non-radical molecule, the target molecule is converted to a radical,  which  may  further  react  with  another  molecule.  Oxidative  stress  is  a physiological  condition that occurs when there is a  significant  imbalance  between production of reactive oxygen species and antioxidant defenses. In humans, oxidative stress is involved in many diseases, such as atherosclerosis,  Parkinson’s disease and Alzheimer’s disease (Valko et al, 2005), but it may also be important in prevention of aging by induction of a process named mitohormesis. ROS can be beneficial, as they are used by the immune system as a way to attack and kill pathogens. ROS are also used  in cell  signaling.  This  is dubbed  redox signaling.  A particularly  destructive aspect of oxidative stress is the production of ROS, which include free radicals and peroxides.  Some of the less reactive of these species (such as superoxide)  can  be converted by oxidoreduction reactions with transition metals or other redox cycling compounds (including quinones) into more aggressive radical species that can cause extensive cellular damage. (Valko et al., 2005).

1.2.1 Superoxide Radical

Superoxide  (O2–)  is  generated  by  multiple  enzymatic  and  non-enzymatic pathways and is often at the start of the oxidative stress cascade. A major source is via the cellular electron transport chains, such as those of mitochondria, chloroplasts and the endoplasmic  reticulum  (Halliwell  and  Gutteridge,  1990) where some electrons

passing through the chain “leak” directly from the intermediate electron carriers onto O2. Since oxygen accepts one electron at a time, O2–  is formed  (Halliwell,  1992). Superoxide  anions are generated  enzymatically  by a number of  oxidases,  such as

xanthine oxidase and the oxidase that is found in the plasmalemma of phagocytic cells (Reiter  et al., 1995).  Activated  phagocytic  cells  (such as monocytes,  neutrophils, eosinophils  and macrophages  including microglia)  also produce  superoxide,  which

plays  an  important  part  in  the  mechanism  by  which  bacteria  are  engulfed  and destroyed (Colton and Gilbert, 1987). Thus excessive activation of phagocytic cells (as  in  chronic  inflammation)  can  lead  to  free  radical  damage.  The  toxicity  of superoxide is seen in its ability to inhibit certain enzymes and thereby attenuate vital metabolic  pathways,  as well as in its  effects on other  major classes  of biological molecules (McCord, 2000). E. coli deficient in superoxide dismutase (SOD) activity show increased rates of mutagenesis (Touati and Farr, 1990), illustrating the role of the radical in DNA damage.

1.2.2 Hydroxyl Radical

The OH. radical is probably the most reactive of the ROS species (Poeggler et al., 1993; Dawson and Dawson, 1996) as it will react with almost all molecules in living cells (Fridovich, 1974). Hydroxyl radicals are short-lived  and  can be formed from O2–  and H2O2  through the Haber-Weiss reaction or  through the interaction of metals such as iron or copper and H2O2, through the Fenton reaction (Halliwell and Gutteridge, 1985) as shown in the equations below.

O2– + H2O2 → H2O + OH – + OH. Haber-Weiss Reaction (1)

Fe3+ + O2- → Fe2+ + O2

Fe2+ + H2O2→ Fe3+ + OH. + OH – Fenton Reaction (2)

The hydroxyl radical has been implicated in damage to proteins, carbohydrates, DNA, and lipids(Reiter  et al., 1995; Dawson and Dawson,  1996; Volterra  et  al., 1994). Action on DNA results in strand breakage and chemical alterations of the deoxyribose and of the purine and pyrimidine bases. A main physiological target of free radicals is the polyunsaturated fatty acids of cell membranes and the resultant degradation causes alterations in membrane structure and function (Viani et al., 1991).

1.2.3 Nitric Oxide

Nitric oxide (NO) is a free radical released by several cell types, especially vascular endothelial  cells  and  phagocytes  (Moncada  et  al.,  1991).  Nitric  oxide  radical  is formed by nitric oxide synthase (NOS). The process involves the conversion of L- arginine to L-citrulline (Knowles and Moncada, 1994). NO has been suggested to be involved in both the normal functioning of excitatory amino acids such as glutamate

and in the damaging effects produced by their generation in excess (Dawson et al.,

1991; Forstermann et al., 1991).

1.3 Diabetes: Definition and Types

Diabetes   mellitus   is   a   group   of   metabolic   diseases   characterized   by hyperglycemia resulting from defects in insulin secretion, insulin action or both (American Diabetes  Association,  2009a).  Diabetes mellitus is classified  as type  1, type 2, other specific types and gestational diabetes (American Diabetes Association,

2008).  Type  1 diabetes  is known  as  insulin  dependent  diabetes  or  Juvenile-onset diabetes and type 2 diabetes is known as non-insulin dependent or adult onset diabetes (American  Diabetes  Association,  2009b).  A  slowly  progressive  form  of  type  1 diabetes was acknowledged as latent autoimmune diabetes in adults (LADA) by the World Health Organization (WHO) and American Diabetes Association (ADA) (Van Deutekom  et al., 2008).  Classification  schemes define type 1diabetes as a state of absolute insulin deficiency and type 2 as a state of insulin resistance combined with inadequate  insulin  secretion  (Greenbaum  et  al.,  2009).  Type  1  diabetes  is  an autoimmune disease where auto reactive  immune cells attack insulin producing  β- cells, destroying insulin reserve leading to hyperglycemia  (Eldor et al.,  2009).  The rate of loss of β-cell function is affected by factors like age at diagnosis, degree of metabolic  control,  immune  status,  genetics  and  marked  inter-individual  variation (Palmer,  2009). The symptoms of type 1 diabetes  are  significant  weight  loss and ketoacidosis  (Ludvigsson  et  al.,  2008).  Type  2  diabetes  is  a  progressive  disease characterized by declining β-cell function that in concert with insulin resistance, leads to loss of glycemic control and eventual diabetes complications (Nauck et al., 2009). The symptoms of type 2 diabetes are polyuria, polyphagia, blurred vision and general malaise (Goldfine, 2008).       The  criteria  for the diagnosis  of diabetes  mellitus in clinical practice is fasting plasma glucose that is equal or greater than 126 mg/dL or two-hours post  prandial  plasma glucose greater than 200 mg/dL (Nwankwo  et al., 2008). With good glycemic control, several long-term, life-threatening complications of diabetes can be prevented (Shabbidar et al., 2006).

1.3.1 Complications of Diabetes

Severe  long  term  abnormalities  can  result  from  diabetes.  Complications already identified are eye complications,  heart disease, kidney and  foot problems if blood sugar levels are poorly controlled (Brophy et al., 2007). These complications are of two types, microvascular complications that include retinopathy, nephropathy, neuropathy and peripheral vascular disorders and macrovascular  complications that include cardiovascular and cerebrovascular disorders. The complications of diabetes can  involve  multiple  systems  throughout  the  body  that  are  susceptible  to  the detrimental effects of oxidative stress and apoptotic cell injury (Maiese et al., 2010). Diabetes  also  leads  to  long-term  complications  throughout  the  body  involving cardiovascular,   renal   and   nervous   disorders   (Daneman,   2006).   The   chronic hyperglycemia  of diabetes  is  associated  with  long-term  damage,  dysfunction  and failure of various organs especially the eyes, kidneys, nerves, heart and blood vessels (Chandramohan   et  al.,  2009).   The   importance   of   protecting   the   body  from hyperglycemia  cannot  be overstated;  the direct  and  indirect  effects on the human vascular tree are the major source of morbidity and mortality in both type 1 and type 2 diabetes.  Generally,   the  injurious  effects  of   hyperglycemia   are  separated   into macrovascular complications (coronary artery disease, peripheral arterial disease and stroke)  and  microvascular  complications  (diabetic  nephropathy,  neuropathy,  and retinopathy) (Fowler, 2008).

1.3.2 Oxidative Stress and Diabetic Complications

Oxidative stress plays both microvascular and cardiovascular pivotal roles in the development of diabetes complications. The metabolic abnormalities of diabetes cause mitochondrial superoxide overproduction in endothelial cells of both large and small vessels, as well as in the myocardium (Du et al., 2000; Nishikawa et al., 2000). This  increased  superoxide  production  causes  the  activation  of  5  major  pathways involved  in  the  pathogenesis  of  complications:  polyol  pathway  flux,  increased formation of advanced  glycation end products (AGEs), increased expression of the receptor for AGEs and its activating ligands, activation of protein kinase C isoforms, and  overactivity of the hexosamine  pathway.  It also  directly inactivates  2 critical antiatherosclerotic   enzymes,   endothelial   nitric  oxide  synthase   and   prostacyclin synthase  (Nascimento  et  al.  2006  ;  Xie  et  al.,  2008).  Through  these  pathways, increased intracellular reactive oxygen species (ROS) cause defective angiogenesis in response  to ischemia,  activate  a number  of  proinflammatory  pathways,  and cause

long-lasting epigenetic changes that drive persistent expression of  proinflammatory genes after glycemia is normalized (“hyperglycemic memory”) (Yip et al., 1998; Du et al., 2006). Atherosclerosis and cardiomyopathy in type 2 diabetes are caused in part by   pathway-selective   insulin   resistance,   which   increases   mitochondrial   ROS production from free fatty acids and by inactivation of antiatherosclerosis enzymes by ROS (Brown and Goldstein, 2008; Li et al., 2010). Oxidative stress may promote the onset  of  diabetes  by  decreasing  insulin  sensitivity  and  destroying  the  insulin- producing  cells  (Wallace,  1992).  ROS can penetrate  through cell membranes  and cause damage to β-cells of pancreas (Chen et al., 2005). A high fat diet or free fatty acids also  has been shown  to release  ROS and contribute  to mitochondrial  DNA damage and impaired pancreatic β-cell function (Rachek et al., 2006). Oxidant stress and  ROS  exposure  can  result  in  the  opening  of  the   mitochondrial  membrane permeability  transition  pore,  reduce  mitochondrial   NAD+  stores  and  result  in apoptotic  cell injury (Chong et al., 2005). Free  fatty acids also  can lead  to  ROS release, mitochondrial DNA damage and impaired pancreatic β-cell function (Li et al.,

2008). The development of diabetes has been associated with a decrease in the levels of mitochondrial proteins and mitochondrial DNA (Choo et al., 2006). Long standing diabetes  mellitus is associated  with an increased  prevalence  of  microvascular  and macrovascular diseases (Mehta et al.; 2009a, Mehta et al., 2009b). Cellular pathways in  diabetes   are  closely  associated   to   cellular   energy  maintenance   and  intact mitochondrial  function  (Newsholme  et al.,  2007).  Figure 1  represents  the protein kinase  C  (PKC)  pathway  and  polyol  pathway  in  the  pathogenesis  of  diabetic complications.

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