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STUDIES ON THE EFFECT OF AQUEOUS EXTRACT OF MILLETTIA ABOENSIS LEAVES ON LOMOTIL INDUCED CONSTIPATION IN WISTAR ALBINO RATS

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ABSTRACT

Millettia aboensis leaf is a rich source of reducing sugar, tannins, glycosides and flavonoids and has been reported to have medicinal property as well as some physiological potentials. The leaf also has been used by traditional herbalists for general healing of diseases  including ulcer and laxatives. This study evaluated the effect of aqueous extract of M.   aboensis leaves on lomotil- induced  constipation  in Wistar  albino rats. The qualitative  phytochemical  constituents  of the extract showed the relative presence of reducing sugars, tannins and flavonoids in relatively high concentrations;  alkaloids,  steroids  and  glycosides  in  moderately  high  concentrations;  soluble carbohydrates, saponins and hydrogen cyanides were in low concentrations while terpernoid was not detected. The median lethal dose effect  (LD50) of the extract recorded no death at dose of 5000 mg/kg b.w. Assay of Aspartate Amino transferase and Alanine Amino transferase activities in serum of treated rats (groups 2 and 3) given 100 and 1000 mg/kg b.w. of the extract showed significant increase (p<0.05) compared to the control group 1 (normal saline). The ALP activity in serum of the mice in groups 2 and 3 administered 100 and 1000 mg/kg b.w. of the extract exhibited  neither  significant  increase  nor decrease  (p>0.05)  compared  to the control  group  1 mice. Triacylglycerol  and High density lipoprotein concentrations  in serum of the mice treated with 100 and 1000 mg/kg b.w. of the extract showed non-significant increase (p>0.05) compared to the control group while the LDL and total  cholesterol  concentrations  of the groups 2 and 3 given  100  and  1000  mg/kg  b.w.  of  the  extract  showed  non-significant  decrease  (p>0.05) compared  to  the  control  group.  The  potassium  ion  concentration  showed  a  non-significant increase (p>0.05)  in the groups 2  and 3 mice administered  100 and 1000 mg/kg b.w. of the extract  compared  to the  control group  while the  serum level  of sodium  ion showed  a non- significant increase (p>0.05) in group 2 mice administered 100 mg/kg b.w. of the extract and a significant  increase  (p<0.05)  in  group  3  mice  that  received  1000  mg/kg  b.w.  of  the extract compared to the control mice. There was neither a significant decrease nor increase (p>0.05) in the serum level of glucose of the mice in groups 2 and 3 administered 100 and 1000 mg/kg b.w. of the extract compared to the control. The result of the aqueous extract of M. aboensis on the mean  value of the faecal  droppings  on lomotil-induced  constipation  in rats showed  neither a significant decrease nor increase (p>0.05) in groups 2 (standard drug of lomotil), 3 (100 mg/kg b.w. of extract), 5 (100 mg/kg of extract + 5 mg/ml of lomotil, 7 (5 mg/ml of lomotil + 200 mg/kg b.w. of extract) and 8 (5mg/ml  of lomotil + 200 mg/kg  of extract) compared to the negative control (normal saline) while group 4 mice (200 mg/kg b.w. of extract) showed a non-significant increase (p>0.05)  and   group 6 (200  mg/kg  b.w. of extract  + 5 mg/ml  of lomotil)  showed  a significant increase (p<0.05) compared to the  positive control group 2 (standard drug of 5 mg/ml of lomotil). The study of the effects of the aqueous extract of M. aboensis on transport of glucose across everted rat  intestine showed significant  increase (p<0.05) in the glucose influx into the everted intestinal sac (serosal compartment) in a dose-dependent manner in all the treated groups contained in groups 3, 4 and 5 everted in 100, 200 and 400 µg/ml of the extract compared to the control group 1 while in group 2 (standard drug metformin),  there was a significant  decrease (p<0.05) compared to the control. Sodium transport  across the everted rat  intestine  showed a significant increase (p<0.05) in the influx of sodium ions into the serosal compartment of groups 2, 3 and 4 everted in 100, 200 and 400 µg/ml of the extract compared to the control group while potassium transport across everted rat intestine showed significant increase (p<0.05) in the efflux of potassium ions into the mucosal compartment  of all the treated groups 2, 3 and 4 everted in 100, 200 and 400 µg/ml of the extract compared to the control group 1. The significant increase in the frequency  of faecal droppings  on the extract treated groups may prove that  the extract contains   some  bioactive   compounds   that   have  the  properties   of  laxative   effects   when administered  and therefore  support  the claim that  this plant is used in folk  medicine  for the treatment of constipation.

CHAPTER ONE

INTRODUCITON

Traditional medicine according to (Treben, 1998) is defined as the knowledge, skills and practices of holistic health care, recognized and accepted for its role in the maintenance of health and the treatment of disease conditions. The application of herbs to improve man’s health must have come from early man in the most non-scientific way. Since that period, the application of herbs has been known and accepted by all individuals and nations (Theiss and Peter, 2000).

Herbal medicine also called botanical medicine or phytomedicine  refers to  using  a plant’s seeds, berries, roots, leaves, bark or flowers for medicinal purposes. Herbalism has a long tradition of use outside of conventional medicine. It is becoming more mainstream as improvements  in  analysis  and  quality  control  along  with  advances  in  clinical  research showing the value of herbal medicine in the treating and preventing disease (Izzo et al., 2009) Medicinal plants are plants in which one or more of their plant contain substances that

can be used for the therapeutic purposes or which are precursors for the synthesis of useful drugs (WHO, 2005). However, some contain excipients in addition to the active ingredients. Medicinal plants as a group comprise approximately 8000 species and account for about 50% of higher flowering plant species (Treben, 1998). Some examples of medicinal plants include Napolliena imperalis, Digoxin lancta, Chenopodium ambrosoides, Morinda lucida, Zingiber officinale etc. Medicinal plants are used commonly in modern medicine and pharmacology. One of the early attempts to store information on medicinal plants have long been on going in Nigeria in an attempt to set appropriate pharmacopoeia standards and obtain the quantity of plants in our natural environment (Katzung et al., 1995).

Constipation (also known as dyschezia) refers to bowel movements that are infrequent or hard to pass. It could also be referred to as infrequent passage (less than three per week), difficulty in expulsion or unusually hard stool, feeling of incomplete evacuation and need for manual evacuation of stool (Chatoor and Emmanuel, 2009). Constipation is a common cause of painful defecation. Severe constipation includes obstipation (failure to pass stools or gas) and fecal impaction, which can progress to  bowel obstruction and become life-threatening (Walia et al., 2009).

The genus Millettia appears in the African pharmacopeia since centuries. It has a wide range of biological activities such as antitumoral,  anti-inflammatory,  antiviral,  bactericidal, insecticidal  and pest-destroying  (Burkill,  1995). Thus,  the multiplicity of  these activities, beginning to be confirmed by pharmacological studies in laboratory, confers on this genus a

great interest in traditional medicine as well as in the research of new biologically active compounds. (Okafor and Ham, 1999).

1.1  Millettia  aboensis  belongs  to  the  family  of  fabacaea.     It  is  popularly  known  as “Otoroekpo or Uturuekpa” among the indigenous people of the Nsukka senatorial district of Enugu state of Nigeria. They are perennial evergreen non-climbing trees of 30 – 40 feet high and up to 2 feet in girth but usually 12m high with reddish-brown pubescence on the petioles, branches, inflorescence and fruits (Burkill, 1995). They are found commonly in low land rain forest. The flowers are purple in erect woody racemes up to 18in long. It has conspicuously rusty-hairy leaves and handsome purple flowers in erect terminal racemes at branch (Burkill,

1995). Millettia aboensis have been used by traditional medicinal practitioners to  manage constipation,  respiratory difficulties,  colds and headaches (Neuwinger,  2000).  The ethanol extract  of  the  root  is  also  used  in  the  study  of  anti-inflammatory,   antioxidant   and antimicrobial activity and also macerated root in alcohol is used to treat hernias and jaundice (Lock, 1989).

Studies have also shown that the leaf, stem and roots mixed with other plant materials

(herbs) are used to cure veneral diseases such as gonorrhea, syphilis and so on. (Neuwinger, 2000).

Fig 1: Structural view of the plant Millettia aboensis. (Otoroekpo).

1.1.2 Scientific classification of Millettia aboensis

Millettia aboensis is a member of the family fabaceae. This dicotyledonous plant is classified scientifically as;

Domain                                                                      Eukaryota Kingdom                                                                   Plantae Subkingdom                                                              Viridaeplantae Phylum                                                                      Tracheophyta Subphylum                                                                Euphyllophytina Class                                                                          Spermatopsida Subclass                                                                     Rosidae Superorder                                                                 Rosanae

Order                                                                         Fabales Family                                                                       Leguminosae Subfamily                                                                  Giseke

Tribe                                                                         Milletieae

Genus                                                                         Millettia

Specific epithet                                                          aboensis

Botanical Name                                                         Millettia aboensis

Lock, (1989).

1.2. Phytochemistry

Phytochemicals  are  natural  bioactive  substances  that  are  found  in  plants.  These natural substances have shown to work with nutrients and dietary fibers to protect animals and man against diseases. These plant products which are derived from plant parts such as fruits, leaves, roots, stem bark, seeds and so on have been part of  phytomedicine,  thereby showing that any part of plant may contain these important  active substances (Njoku and Aku, 2007). Phytochemicals are also non-nutritive chemicals that have protective or disease preventive property. (Harborne, 1998). Phytochemicals comprise of a number of substances such  as  carotenoids  (carotene,  lutein,  and  lycopene),  alkaloids  (caffeine,  theobromine, theophylline etc), phenolics  (flavorioids, anthocyanins, catechins) and sterols (campesterol, sitosterol, and stigmasterol) (Akinmoladun et al., 2007).

1.2.1.     Phytochemical components in plants

Phytochemical compounds of plants have shown to be formed during the plant normal metabolic processes. These substances are often referred to as secondary metabolites such as Alkaloids,   flavonoid,   Saponins,   Tannins,   Glycosides,   Steroids,   Hydrogen   Cyanides, Terpenoids,  Reducing  sugars,  soluble  carbohydrates  and  so  on  (Harborne,  1998;  Okwu,

2004). The qualitative and quantitative studies of aqueous extract of Millettia aboensis leaves showed to contain the above chemical compounds (Harborne, 1998).

Phytochemicals are naturally occurring and are believed to be effective in fighting or preventing disease due to their antioxidant effect (Halliwell and Gutteridge, 1992). Thus, the medicinal values of these plants depend in their component phytochemicals that produce the definite physiological actions on the human body. The most essential of these phytochemicals are flavonoids tannins, phenolic and alkaloids compounds (Hill, 1952)

Some  of these  naturally occurring  phytochemicals  are anticarcinogenic  and  while

some have other useful properties,  some prevent oxidation  by free radicals and  therefore known  as  chemopreventers.  Among  these  chemopreventers  are  some  plant  polyphenols, vitamins and pigments such as flavorioids, carotenoids and chlorophylls (Farombi, 2000).

1.2.1.1. Flavonoids

Flavorioids   are  polyphenolic   compounds   that  are  common  in  nature  and   are categorized according to chemical structure into flavones, flavonols, flavonones,  catechins, chalcones, isoflavones and anthocyanidins. They are plant Secondary metabolites occurring at relatively  high amounts  in several kinds of fruits,  grains and  vegetables  harvested  for human  consumption  (Manach  et al., 2004). Flavonoids  are  widely distributed  throughout plants and give the flowers and fruits of many plants their  vibrant colours. They also play important roles in protecting the plants from microbes and insect attacks. At the cellular level, flavonoids have been found to exert a variety of biological effects (Middleton et al., 2000), likely mediated by specific interaction with molecular targets. Flavonoids can be nutritionally helpful by triggering enzymes that reduce the risk of certain cancers, heart diseases and age- related degenerative diseases. Some studies also shows flavonoids may help to prevent tooth decay and reduce the occurrence of common ailments such as the flu. Hence, the capacity of flavonoids to act as antioxidants  depends  upon their molecular  structure.  The position of hydroxyl groups and other features in the chemical structure of flavonoids are important for their antioxidant  and free radical activities.  For instance,  Quercetin  is the most abundant dietary flavonoid and is a potent antioxidant because of the presence of all the right structural features for free radical scavenging activity.

1.2.1.2. Tannins

Tannins are an astringent bitter plant polyphenolic compound that binds to precipitate proteins and various other organic compounds including amino acids and alkaloids, (Petridis,

2010). The word Tannin is widely applied to any large polyphenolic compound  containing sufficient hydroxyls and other suitable groups (such as carboxyl) to form strong complexes with proteins and other macromolecules.  These compounds are widely distributed in many plants where they play a role in protection from predation perhaps also as pesticides and in plant  growth  regulation  (Katie  et al., 2006).  Tannins  are also  polymerized  phenols  with defensive properties. In tanning, collagen proteins are bound together with phenolic groups to increase the hide’s resistance to water, heat  and microbes (Heldt and Heldt, 2005). Thus, property of astringency  from tannins  is  what causes the dry and unripe fruit or red wine (McGee,  2004).  Tannins  are  incompatible  with  heavy  metals,  iron,  lime  water,  alkalis, metallic salts, zinc sulfate, strong oxidizing agents and gelatin since they form complexes and precipitate in aqueous solution (Bisanda et al., 2003). Many human physiological activities,

such as stimulation of phagocytic cells, host-mediated tumour activity and a wide range of anti-infective actions, have been assigned to tannins (Haslam 1996)

1.2.1.3. Hydrogen cyanides

Hydrogen  cyanide  is an inorganic  compound  with  chemical  formal  HCN.  It  is  a colorless extremely poisonous liquid hydrogen cyanide  is a linear molecule,  with  a  triple bond between carbon and nitrogen. It has a bitter, faint and almond like color  with some people are unable to detect owing to a genetic character. The volatile  compound has been used as inhalation rodenticide and human poison. Cyanide ions interfere with iron-containing respiratory enzymes (Mathews, 2004; Morocco, 2005).

The  most  important  toxic  effect  of  hydrogen  cyanide  is  inhibition  of  the  metal containing enzymes such as cytochromoxidase.  This enzyme system is responsible  for the energy providing processes in the cell where oxygen is utilized that is respiration. Thus, when cell respiration ceases, it is no longer possible to keep normal  cell functions,  which may result to cell death (Baud et al., 2002).

1.2.1.4. Alkaloids

Alkaloids are a group of natural organic bases found in plants, characterized by their specific physiological action and toxicity used by many plants as a defense against herbivores like insects. They are one of the most diverse groups of secondary metabolites found in living organisms and have an array of structural types, biosynthetic pathways, and pharmacological activities (Robert, 1998). Alkaloids may be rated to various organic bases, the most one being quinolone,  isoquinoline  pyrrole,  pyridine  and  other  more  complicated  derivatives.  Most alkaloids  are  crystalline  solids,  others  volatile  liquids  and  some  are gums.  They contain nitrogen as part of a ring, and have general properties of amines. They also include some related  compounds with neutral and  even weakly acidic properties (Raj, 2004). Alkaloids generally exert pharmacological activity particularly in mammals. Presently, many of most commonly used drugs are alkaloids from natural sources and new alkaloid drugs are alkaloid drugs are alkaloid  drugs  are  still  being developed  for  clinical use (Robert,  1998). Most alkaloids with biological activity in humans affect the nervous system, particularly the action of neural transmitters, e.g. adrenaline, dopamine, serotonin, acetylcholine and so on (Richard, 1999)

1.2.1.5. Steroids

A steroid is a type of organic compound that contains a characteristic arrangement of four cycloalkane rings that are joined to each other. Examples of steroids are the dietary fat cholesterol,  the  sex  hormones  estradiol  and  testosterone  and  the  anti-inflammatory  drug dexamethasone.  (Zollner et al., 2006). The core of steroids is  composed of twenty carbon ions bond atoms bonded together that take the form of four fused rings, three cyclohexane rings. The steroids vary by the functional groups attached to this four-ring core and by the oxidation state of the rings. Sterols are  special forms of steroids, with a hydroxyl group at position 3 and a skeleton derived  from cholestane (Rossie, 2006). Sterols were used to be considered to be animal substances that are similar to sex hormones but in recent years, an increasing number of such compounds have detected in plant tissues. Sterols have important functions  in  all  eukaryotes.  For  instance,  free  sterols  are  integral  components  of  the membrane  lipid bilayer where they play an important role in the regulation of membrane fluidity and permeability (Corey et al., 1993). Thus, distinct of steroids are found in plants, animals and fungi. Therefore, all steroids are made in cells either from the sterols lanosterol (animals and fungi) or from cycloaterol. (Plants).

1.2.1.6. Saponins

Saponins are natural glycosides of steroid or triterpene which showed many different biological  and  pharmacological  activities.  Saponins  a  characterized  by  bitter,  foaming properties, haemolytic effect on red blood cells and cholesterol binding properties (Okwu,

2005).  Saponins  can  also  activate  the  mammalian  immune  system,  which  have  led  to significant  interest  in their potential as vaccine  adjuvants.  Thus, sapins  are  glycosides  of triterpenoid or steroidal aglycones with a varying number of sugar side chains. Saponins are especially enriched in plant epidermal cells forming a protective surfactant that forms a soapy froth when mixed with water (Wegner et al., 2002). Extracts of plants rich on saponins have been shown to have cholesterol lowering and  anticancer  properties but are also known to reduce the digestibility in ruminants and to be generally toxic to cold blooded animals and insects (Kerwin, 2004).

1.2.1.7.   Glycosides

Glycosides  are molecules  in which a sugar  is bound  to  another  functional  group through a glycosidic bond. They are also a variety of natural occurring substances in which a carbohydrate portion, consisting of one or more sugars or an ironic acid that is a sugar acid is combined with a hydroxyl compound. The hydroxyl compound, usually a non-sugar entity (aglycon), such as a derivative of phenol or an alcohol, may also be another carbohydrate as in cellulose, starch or glycogen which consist of many glucose unites thus, many glycosides occur  in  plants  often  as  fruit  pigment,  for  instance,  anthocyanins,  various  medicines, condiments and dyes from plants occurs as glycosides; of great value are the heart stimulating glycosides  of  digitalis  and  strophan,  members  of  a  group  known  as  cardiac  glycosides (Arewang et al., 2007).

Glycosides derived from glucuronic acid (the uronic acid of glucose) and steroids are constituents  of  normal  animal  urine.  Compounds  (nucleosides)  derived  from  the  partial breakdown of nucleic acids are also glycosides.  The most important synthetic  enzymes in nature are glycosyltransferases (Polakova et al., 2004).

1.2.1.8.   Reducing sugars

A reducing sugar  is any sugar that either has an aldehyde  group or is capable  of forming one in solution through isomerism. The reducing property of a reducing sugar is due to the presence of an aldehyde functional group. However, the cyclic hemiacetral forms of aldoses can open to reveal an aldehyde and certain ketoses can undergo tautomerization to become aldoses. (Campbell and Farrell, 2012).

1.3.     Constipation

1.3.1. Definition of constipation

Constipation is referred to as infrequent bowel movements (Typically three times or fewer per week). It could be referred to as the difficulty during defecation (Straining during more than 25% of bowel movement or a subjective sensation of had stools) or sensation of incomplete  bowel evacuation (Chatoor et al., 2009). Constipation  is a  common digestive complaint and often a chronic functional gastrointestinal disorder.  The estimation revealed that the occurrence is 2% to 20% of the population (Sonnenberg and Koch, 1989). It is also a common  clinical  problem  comprising  a constellation  of  symptoms  that include  excessive straining, hard stools, and feeling of incomplete evacuation or infrequent defecation (Higgins and Johanson, 2004). Constipation is not only discomforting but also a cause of abdominal distension, vomiting, restlessness, gut obstruction and perforation and may be associated with aspiration or fatal pulmonary embolism (Mostafa et al., 2003). Constipation may be caused by a number of conditions such as metabolic problems, fiber deficiency, anorectal problems, and  drugs.  But  the  three  possible  causes  of  constipation  are  congenital,  primary  and secondary while the most common cause is primary and is not life-threatening (Leung, 2007). Constipation  can  be  treated  by  water  and  fibre  intake  either  as  dietary  source  or  as supplements (Hsieh, 2005).

1.3.2. Constipation in children

Constipation in children usually occurs at three distinct points in time, after starting formula or processed foods (while an infant), during toilet training in toddlerhood, and soon after starting school (as in kinder garden) (Cohn, 2010).

Studies  have  shown  that  after  birth,  most  infants  pass  4-5  soft  liquid   bowel movements  (BM)  a  day.  Thus,  Breast  –  fed  infants  usually  tend  to  have  more  bowel movement  compare  to  formula  –  fed  infants.  Some  breast  –  fed  infants  have  a  bowel movement after each feed, whereas others have only one bowel movement every 2 -3 days. Hence, infants who are breast –fed rarely develop constipation (Cohn, 2010).

1.3.3. Types of constipation

There are two main kinds of constipation; Occational constipation and

Chronic constipation

1.3.3.1. Occational constipation

As the name of the constipation suggests, it is a type of constipation that does not happen every day. However, while uncomfortable, it is a short – term condition that may only temporarily interrupt usual – routine. Thus, this type of constipation can often be relieved through changes to diet, exercise regimen or through the use of over-the counter medications (Chang, 2006)

1.3.3.2. Chronic constipation

Chronic constipation on the other hand, almost becomes a new routine of its  own. Moreover,  most  people  who  have  chronic  constipation  still  experiences  the   “typical” symptoms – straining, hard or lumpy stool, feeling like not being empty after having a bowel movement,  but they happen on an ongoing basis (symptoms  last more  than   3 months). (Chang, 2006).

1.3.4. Causes of constipation

Primary or functional constipation is ongoing symptoms for greater than six months not due to any underlying cause such as medication side effects or and underlying medical conditions. Thus, it is not associated with abdominal pain thus distinguishing it from irritable bowel syndrome. It is the most common cause of constipation. (Longstreth et al., 2006).

1.3.4.1. Diet cause

Constipation can be caused or exacerbated by a low fibre diet, low liquid intake or dieting (Bharucha, 2007).

1.3.4.2. Medication cause

Many medications  have  constipation  as a side  effect.  Some  include  (but  are  not limited  to); opioids (e.g.  common  pain  killers),  diuretics,  antidepressants,  antihistamines, antispasmodics, anticonvulsants and aluminum antacids (Lee et al., 2010).

1.3.4.3. Metabolic and muscular cause

Constipation  has  a  number  of  structural  (mechanical,  morphological,  anatomical) causes including; spinal cord lesions, Parkinsons, colon cancer, anal fissures, proctitis and pelvic floor dysfunction.

Constipation also has functional (neurological) causes including; animas, descending, perineum syndrome, and Hirschsprung’s diseases. Infants, Hirschspruny’s disease is the most common medical disorder associated with constipation. Anismus occurs in a small minority of person with chronic constipation or obstructed defecation (Schouten et al., 1997).

1.3.4.4. Psychological cause

Voluntary withholding of the stool is a common cause of constipation. The choice to withhold can be due to factors such as fear or pain, fear of public restrooms, or  laziness. However, when a child holds in the stool a combination of encouragement, fluids, fiber and laxatives may be useful to overcome the problem (Longstreth et al., 2006).

1.3.5. Diagnosis of constipation

The diagnosis of constipation is essentially made from the patient’s description of the symptoms. Thus, bowel movements that are difficult to pass, very firm, or made up of small hard pellets (like those excreted by rabbits) qualify as constipation, even if they occur every day, thus, other symptoms related to constipation can include bloating, distension, abdominal pain,  headaches,  a  feeling  of  fatigue  and  nervous  exhaustion,  or  a  sense  of  incomplete emptying (Arce et al., 2002).

Moreover,  inquiring about dietary habits will often reveal a low intake of  dietary fibre, inadequate amounts of fluids, poor ambulation or immobility, or  medication that are associated with constipation.

Studies also showed  that during physical examination,  scybala (manually  palpable lumps of stool) may be detected on palpation of the abdomen. Rectal examination gives an impression of the anal sphincter tone and whether the lower rectum contains any faeces or not. Rectal examination also gives information on the consistency of the stool, presence of

hemorrhoids,  admixture  of  blood  and  whether  any  tumors,  polyps  or  abnormalities  are present.  Physical  examination  may  be  done  manually  be  the  physician  or  by  using  a colonoscope,  X-rays  of  the  abdomen,  generally  only  performed  if  bowel   obstruction suspected may reveal extensive impacted faecal matter in the colon and confirm or rule other causes of similar symptoms (Barish et al., 2010).

1.3.5.1. Rome II criteria for constipation

The Rome II criteria for constipation require at least two of the following symptoms for 12 weeks or more over the period of a year

Straining with more than one – fourth of defecations

Hard stool with more than one-fourth of defecations

Feelings of incomplete evacuation with more than one-fourth of defecations. Sensation of anorectal obstruction with more than one-fourth of defecations Manual maneuvers to facilitate more than one-fourth of defecations

Fewer than three bowel movements per week

Insufficient criteria for irritable bowel syndrome (Sonnenberg, 1989).

1.3.6. Prevention of constipation

Constipation  is  usually  easier  to  prevent  than  to  treat.  Following  the  relief  of constipation,  maintenance  with adequate fluid intake and high fibre diet is  recommended. Children benefit from scheduled toilet breaks, once early in the morning and 30 minutes after meals (Camilleri and Deiteren, 2010).

1.3.7. Treatment of constipation

There  are many treatments  for constipation  and the best approach  lies on a  clear understanding of the underlying cause.

1.3.7.1. Dietary fiber

The best way of adding fiber to the diet is by increasing the quantity of fruits and vegetables that are eaten. This means a minimum of five servings  of fruits or  vegetables every day

However, the amount of fruits and vegetable that are necessary may be inconveniently large or may not provide adequate relief from constipation. In this case,  fiber supplements can be useful (Bharucha, 2007).

Thus, fiber is defined as a material made by plants that is not digested by the human gastrointestinal  tract. Fiber is one of the mainstays in the treatment of  constipation.  Many types of fiber within the intestine bind to water and keep the water within the intestine. The fiber adds bulk (volume) to the stool and the water softens the stool (Bharucha, 2007). There are different sources of fiber and the type of the fiber varies from source to source. Types of fiber can be categorized  in several ways, for  instance by their source. The most common source of fiber includes: Fruits and vegetable, Wheat or oat bran.Psyllium seed (For example, Metamucil, Konsyl). Synthetic methyl cellulose (for example Citrucel) and polycarbophil (for example, Equilactin, Konsyl fiber).

However, the fiber should be started at a low dose and increased every one to  two weeks  until  either  the  desired  effect  on the  stool  is  achieved  or  troublesome  flatulence interferes. Hence when increasing amounts of fiber are used, it is recommended that greater amounts of water be consumed (for instance, a full glass with each dose). In theory, the water prevents “hardening” of the fiber and blockage (obstruction) of the intestine (Hsieh, 2005).

1.3.7.2. Lubricant laxatives

Lubricant  laxatives  contain  mineral  oil  as  either  the  plain  oil  or  an  emulsion (combination with water) of the oil. The oil stays within the intestine, coats the particles of stool and presumably prevents the removal of water from the stool. This retension of water in the stool results in softer stool (Selby, 2010).

1.3.7.3. Emollient laxatives (Stool softeners)

Emollient laxatives are generally known as stool softeners. They contain a compound called docusate (for example, Colace). Docusate is a welting agent that improves the ability of water within the colon to penetrate and mix with stool. This increased water within the stool softens the stool. Although, studies have not shown docusate to be consistently effective in relieving constipation (Emmanuel et al., 2009).

1.3.7.4. Hyperosmolar laxatives

Hyperosmolar laxatives are undigestible, unabsorbable compounds that remain within the colon and retain the water that already is in colon. The result is softening of the stool. The most commonly hyperosmolar laxatives are lactulose (for example kristalose), sorbitol and polyethylene glycol (for example Miralax) and are available by prescription only. (Emmanuel et al., 2009).

1.4. Biochemical parameters

1.4.1. Body electrolyte

Chemically, electrolytes are substances that become ions in solution and acquire the capacity to conduct electricity. Thus, they are present in the human body and the balance of electrolyte in the body is essential for normal function of the cells and  organs. Electrolyte solutions can also result from the dissolution of some biological (e.g. DNA, Polypeptides) and synthetic polymers (e.g. Polystyrene sulfonate), termed  polyelectrolytes  which contain charged functional groups.

Physiologically,  the  primary  ions  of  electrolytes  are  sodium(Na+),  potassium(K+),

calcium(Ca2+), magnesium(Mg2+),  chloride(Cl-), hydrogen phosphate(HPO42-), and hydrogen carbonate(HCO2-)  (Kamil et al., 2011).All  known  higher  life  forms  require  a  subtle  and complex   electrolyte   balance   between  the  intracellular   and   extracellular  environment. Electrolyte activity between the extracellular fluid and intracellular fluid helps in activation of muscles and neurons (Syzdek and Jaroslaw, 2000). Moreover, the maintenance of precise osmotic gradients of electrolytes is important.   Thus, such gradients affect and regulate the

hydration of the body as well as blood pH, and are critical for nerve and muscle function. In humans,  electrolyte  homeostasis  is regulated  by hormones  such as  antidiuretic  hormone, aldosterone and parathyroid hormone. (Syzdek and Jaroslaw, 2010).The common electrolytes that are measured with blood testing include; sodium, potassium, chloride and bicarbonate.

1.4.2. Depletion and absorption of sodium

Sodium  is  the  most  abundant  extracellular  cation  and  with  its  associated  anions, accounts for the most of the osmotic activity of the extra cellular fluid (ECF), it is important in  determining  water  distribution  across  cell  membranes.  Osmotic  activity  depends  on concentration, and therefore on the relative amounts of sodium and water in the extracellular fluid compartment, rather than the absolute quantity of either. An imbalance between the two causes  either  hyponatraemia  or  hypernatraemia  and  therefore  brings  about  changes  in osmolality. The daily water and sodium intakes are very variable, but in an adult amount to about 1.5 to 2 litres and 60 to 150mMol respectively (Philip, 1994). Sodium regulates the total amount of water in the body and the transmission of sodium into and out of individual cells also plays a role in critical body functions. Many processes in the body, especially in the brain  nervous  system,  and  muscles,  require  electrical  signals  for  communication.  The movement of sodium is critical in generation of these electrical signals. Too much or too little

sodium therefore can cause cells to malfunction, and extremes in the blood sodium levels (too much or too little) can be fatal.

1.4.3. Depletion and absorption of potassium

Potassium (K+) is the most abundant intracellular cation. Only about 2% of the total body K+  is extracellular. Since most intracellular K+  is contained within muscle cells, total body  K+   is  roughly  proportional  to  lean  body mass.  An  average  70kg  adult  has  about

3500mEq of K+ (Harrison et al., 2005). The normal potassium intake is about 60 to 100mMol a day (Philip, 1994).

1.5. Na+ /K+ ATPase

Na+ /K+ -ATPase (sodium-potassium adenosine triphosphatase, also known as Na+ / K+ pump, sodium-potassium pump, or sodium pump) is an antiporter enzyme (an electrogenic transmembrane  ATPase)  located  in plasma  membrane  of all animal  cells.  The Na+  /K+  – ATPase enzyme pumps sodium out of cells, while pumping potassium into cells.

1.5.1. Sodium-potassium pumps

Active transport is responsible for cells containing relatively high concentrations of potassium ions but low concentrations of sodium ions (Tian et al., 2006). The  mechanism responsible for this is the sodium-potassium pump, which moves these two ions in opposite directions across the plasma membrane. It is also showed that the concentrations of sodium and potassium ions in the two sides of the membrane are interdependent, suggesting that the same carrier transports both ions (Forrest et al., 2012). Thus, it is known that the carrier is an ATPase and that it pumps three sodium ions out of the cell for every two potassium ions pumped in (Forrest et al., 2012).

1.5.2. Functions of Na+ /K+ ATPase

The Na+ /K+ -ATPase helps to maintain resting potential, avail transport, and regulate cellular volume. It also functions as signal transducer/integrator to regulate MAPK pathway, ROS as well as intracellular  calcium. However, in most animal cells,  it is responsible  for about 1/5 of the cell’s energy expenditure (Howarth et al., 2012). For neurons, the Na+ /K+ – ATPase can be responsible for up to 2/3 of the cell’s energy expenditure (Howarth et al., 2012).

1.5.2.1. Resting potential

In order to maintain the cell membrane potential, cells keep a low concentration of sodium ions and high levels of potassium ions within the cell (intracellular).  The  sodium- potassium pump moves 3 sodium ions out and 2 potassium ions in, thus in total removing one positive  charge carrier  from the intracellular  space.  Moreover,  the  action of the sodium- potassium  pump is not the only mechanism  responsible  for the  generation  of the resting membrane potential. Also the selective permeability of the cell’s plasma membrane for the different ions plays important role (Lee et al., 2001).

1.5.2.2. Transport

Export of sodium from the cell provides the driving force for several secondary active transporters,  membrane  transport  proteins,  which  import  glucose,  amino  acids  and  other nutrients into the cell by use of the sodium gradient.  Na+  -K+  pump also  provides a Na+ gradient  that  is  used  by  certain  carrier  processes.  For  instance,  in  the  gut,  sodium  is transported out of the reabsorbing cell on the blood (interstitial fluid) side via Na+ -K+ pump,

whereas, on the reabsorbing (luminal) side, the Na+ -glucose symporter uses the created Na+

gradient as a source of energy to import both Na+ and glucose which shows to be far more efficient than simple diffusion (Lee et al., 2001).

1.5.2.3. Controlling cell volume

Failure of the Na+ -K+ pumps can result to swelling of the cell. A cell’s osmolarity is the sum of the concentrations of the various ion species and many proteins and other organic compounds inside the cell. When this is higher than the osmolarity outside of the cell, water flows into the cell through osmosis. This can result to the cell swell up and lysis. Therefore,

the Na+ -K+ pump helps to maintain the right concentrations of ions (Li et al., 2009).

1.5.3. Mechanism of Na+ -K+ ATPase pump

The pump while binding ATP, binds 3 intracellular Na+ ions. The ATP is hydrolysed leading  to  phosphorylation  of  the  pump  at  a  highly  conserved   aspartate  residue  and subsequent release of ADP. A conformational change in the pump exposes the Na+     ions to the outside. The phosphorylated form of the pump now has a low affinity for Na+ ions, and thus  they  are  released.  The  pump  binds  2  extracellular  K+  ions.  This  brings  about  the dephosphorylation   of  the  pump  and   reverting  it  to  its  previous  conformational   state transporting the K+ ions into the cell (Skou, 1957)

1.6. Blood glucose

Blood sugar concentration or blood glucose level is the amount of glucose (sugar) that is present in the blood of a human or animal (Boily et al., 2006). The body naturally tightly regulates blood glucose levels as a part of metabolic homeostasis.  Glucose is  the primary source of energy for the body’s cells, and blood lipids (in the forms of  fats and oils) are primarily a compact energy store (Eiler, 2004). Glucose is transported from the intestine or liver to body cells through the bloodstream and is made available for cell absorption through the hormone insulin, produced by the body primarily in the pancreas.

In the measurement of blood glucose, normally the values ranges may vary slightly among  different  laboratories.  Thus,  many factors  affect  a person’s  blood  sugar level.  A body’s homeostatic mechanism, when operating normally, restores the blood sugar level to a narrow array of about 4.4 to 6.1mmol/L (79.2 to 110mg/dL) as measured by a fasting blood glucose test American Diabetes Association, (2006).

1.6.1. Regulation of blood glucose

The body’s homeostatic mechanism keeps blood glucose interacting within a narrow range. It is composed of several interacting systems of which hormone regulation is the most important. Thus, there are two types of mutually antagonistic metabolic hormones affecting blood glucose levels; catabolic hormones (such as glucagon,  cortisol and catecholamines) which  increase  blood  glucose  and one  anabolic  hormone  (insulin)  which decrease  blood glucose (Comell et al., 1988).

1.6.2. Abnormality in blood glucose level

1.6.2.1. High blood sugar (Hyperglyceamia)

The high concentration of blood sugar in the blood above normal range is referred to as hyperglyceamia. If blood sugar levels remain too high, the body suppresses appetite over the short term. As a result of long term hyperglyceamia  it causes many  of the long-term health problems such as heart disease, eye, kidney, and nerve damage (Daly et al., 1998).

1.6.2.2. Low blood sugar (Hypoglyceamia)

A situation whereby the blood sugar levels drop too low, a potentially fatal condition called hypoglyceamia results. Thus, the following are the likely symptoms; lethargy, shaking, twitching,  weakness  in  arm  and  leg  muscles,  pale  complexion,  sweating,  paranoid  or aggressive mentality and loss of consciousness (Daly et al., 1998).

1.7. Liver

The liver is a vital organ present in vertebrates and some other animals. It has a wide range of functions including detoxification, protein synthesis, and production of biochemical necessary for digestion (Cotran et al., 2005). Anatomically, it is a reddish- brown organ with four lobes of unequal size and shapes. A human liver normally weighs 1.44-1.66kg (3.2-

3.7lb) and located in the right upper quadrant of the abdominal cavity, resting just below the

diaphragm (Kmiec, 2001).

1.7.1. Functions of the liver

The liver plays a major role in metabolism and has a number of functions in the body, including  glycogen  storage,  decomposition  of  red  blood  cells,  plasma  protein  synthesis, hormone production, and detoxification. It lies below the diaphragm in the abdominal-pelvic region of the abdomen.  It produces  bile,  an alkaline  compound  which  aids  in digestion through the emulsification  of lipids. The liver’s highly specialized  tissues regulate a wide variety of high-volume  biochemical  reactions,  including  the  synthesis  and  breakdown  of small  and  complex  molecules,  many of  which  are  necessary  for  normal  vital  functions (Maton et al., 1993). Due to the  unique and consideration of the reserve capacity of liver, even a moderate cell injury is not reflected by measurable change in its metabolic function. Thus, some of its functions are so sensitive that abnormalities start appearing depending on the nature and degree of initial result.

1.8. Liver markers

Liver function tests (LFTs or LFs) are groups of clinical biochemistry  laboratory blood assays designed to give information about the state of a patient’s  liver  (McClatchy,

2002).  The  liver  related  enzymes  or liver  biomarkers,  aspartate  aminotransferase  (AST), alkaline  phosphatase  (ALP),  gamma  glutamyl  peptidyl  transferase  (ALT)   are  indirect measures of liver homeostasis.Most liver diseases cause only mild symptoms initially, but it is vital that these diseases can be detected early. Hepatic (liver) involvement in some diseases can be of vital importance. Some tests are related with functionality (e.g., albumin), some with cellular integrity (e.g., transaminase) and some with conditions linked to the biliary tract (gamma-glutamyl transferase and alkaline phosphatase) (Nyblom et al., 2006).

1.8.1. Alanine transaminase

Alanine transferase is an enzyme that transfers an amino group from the amino acid alanine  to  a  ketoacid  acceptor  (oxaloacetate).  The  enzyme  was  formerly  called  serum glutamic pyruvic transaminase (SGPT) after the products formed by this reaction. Although ALT is present in other tissues besides liver, its concentration in liver is far greater than any other tissue, and blood levels in nonhepatic conditions rarely produce levels of a magnitude seen in liver disease. The enzyme is very sensitive to necrotic or inflammatory liver injury. Consequently,  if ALT or direct bilirubin is  increased,  then some form of liver disease us likely. If both are normal, then liver disease is unlikely (Henry, 2001).

1.8.2. Alkaline phosphatase

Alkaline phosphatase is increased in obstructive liver diseases, but it is not specific for the liver. Increases of a similar magnitude (three to four-fold normal) are commonly seen in  bone  diseases,  late  pregnancy,  leukemia,  and  some  other  malignancies.  The  enzyme gamma-glutamyl  transferase (GGT) is used to help  differentiate the source of an elevated ALP. GGT is greatly increased in obstructive jaundice, alcoholic liver disease, and hepatic cancer. When the increase in GGT is two or more times greater than the increase in ALP, the source of the ALP is considered to be from the liver. When the increase in GGT is five or more times the increase in ALP, these points to a diagnosis of alcoholic hepatitis. GGT, but not  AST  and  ALT,  is  elevated  in the  first  stages  of liver  inflammation  due  to  alcohol consumption, and GGT is useful   as a marker for excessive drinking. GGT has been shown to rise after acute persistent alcohol ingestion and then fall when alcohol is avoided (Wallach,

2000).

1.8.3. Aspartate transaminase

Aspartate aminotransferase, formerly called serum glutamic oxaloacetic transaminase (SGOT),  is not as specific for liver disease  as is ALT, which is  increased  in myocardial infarction  pancreatitis,  muscle  wasting  diseases,  and  many  other  conditions.  However, differentiation of acute and chronic forms of hepatocellular injury is aided by examining the ratio of ALT to AST, called DeRitis ratio. In acute hepatitis, Reye’s syndrome, and infectious mononucleosis, the ALT predominates. However, in alcoholic liver disease, chronic hepatitis and cirrhosis, AST predominates (Lee, 2009).

1.9. Lipid profile

Lipid profile or lipid panel is the collective  term given to the estimation  of  total cholesterol,  low-density  lipoprotein  cholesterol,  triglycerides  and high-density  lipoprotein. An extended lipid profile may include very low-density lipoprotein. This is used to identify hyperlipidemia (various disturbances of cholesterol and triglyceride levels), many forms of which are recognized risk factors for cardiovascular disease and sometimes pancreatitis.

1.9.1. High density lipoprotein

High-density lipoprotein (HDL) is one of the five major groups of lipoproteins, which in order of sizes, largest to smallest, are chylomicrons, LDL, HDL and VLDL, which enable lipids like cholesterol and triglycerides to be transported within the water-based bloodstream. In healthy individuals, about thirty percent of blood  cholesterol is carried by HDL (Mard- Soltani et al., 2012).

Blood tests typically report HDL-C level that is the amount of cholesterol contained in HDL particles. It is often contrasted with LDL cholesterol or LDL-C, HDL particles are able to remove cholesterol from within artery atheroma and transport it back to the liver for excretion or re-utilization, which is the main reason why the cholesterol carried within HDL particles (HDL-C) is sometimes called “good  cholesterol. Individual with higher levels of HDL-C seem to have fewer problems with cardiovascular diseases (Lewington et al., 2007), while those with low HDL-C cholesterol levels (less than 40mg/dL or about 1mmol/L) have increased rates for heart disease (Lewington et al., 2007).

1.9.2. Triacylglycerol

Triglycerides are the main constituents of vegetable oil (typically more unsaturated) and animal fats (typically more saturated).  In humans,  triglycerides  are a  mechanism  for storing unused calories, and their high concentration in blood correlates with the consumption of starchy and other high carbohydrate food. Triglycerides are a major component of human skin oils. Triglycerides are formed by combining glycerol with three molecules of fatty acid.

1.9.3. Low density lipoprotein

Low-density lipoprotein (LDL) is one of the five major groups of lipoprotein, which enable transport of multiple different fat molecules, including cholesterol, within the water around cells and within the water-based bloodstream. Studies have shown that higher levels of type-B LDL particles (as opposed to type-A LDL particles) promote health problems and cardiovascular  disease,  they are  often informally  called  the bad  cholesterol  particles,  (as opposed to HDL particles, which are frequently referred to as good cholesterol) (John et al.,

2007).Because LDL particles can also transport cholesterol into the artery wall, which are retained there by arterial proteoglycans  and thus attract macrophages  that engulf  the LDL particles   and   start   the   formation   of   plaques,   increased   levels   are   associated   with atherosclerosis.  Over time vulnerable plaques rupture, activate blood  clothing and produce arterial  stenosis,  which  can result  to  heart  attack,  stroke  and  peripheral  vascular  disease symptoms and major debilitating events, LDL particles are formed as VLDL lipoproteins lose triglyceride  through the action of lipoprotein  lipase  (LPL)  and they become  smaller  and denser  (i.e.  fewer  fat  molecules  with  same  protein  transport  shell),  containing  a  higher proportion of cholesterol esters (Segrest et al., 2001).

1.9.4. Cholesterol

Cholesterol  is important  for all animal  life,  each cell synthesizes  it from  simpler molecules,  a complex  37-step  process  which starts with the intracellular  protein  enzyme HMG-CoA  reductase.  However,  normal  and  especially  high  levels  of  fats  (including cholesterol)  within  the  blood  circulation,  depending  on  how  it   is  transported  within lipoproteins,  are  strongly  associated  with  progression  of  atherosclerosis.  However,  most ingested cholesterol is esterified, and esterified cholesterol is poorly absorbed. The body also compensates for any absorption of additional cholesterol by reducing cholesterol synthesis. For these reasons, cholesterol intake in food has little, if any effect on total body cholesterol content or concentrations of cholesterol in the blood (Lecerf and Lorgeril, 2011).

Cholesterol is recycled. Thus, the liver excretes it in a non-esterified form (via bile) into the digestive tract. Typically about 50% of the excreted cholesterol is reabsorbed by the  small bowel back into the bloodstream. Some plants make cholesterol in very small amounts. Plants manufacture  phytosterols  (substances  chemically  similar  to  cholesterol  produced  within plants),  which can compete  with cholesterol  for  reabsorption  in the intestinal  tract,  thus potentially reducing cholesterol reabsorption.  However,  phytosterols  are foreign to animal cells and, if absorbed, accelerate the  progression of atherosclerosis.  When intestinal lining cells  absorb  phytosterols,  in  place  of  cholesterol,  they  usually  excrete  the  phytosterol molecules back into the GI tract, an important protective mechanism (John et al., 2007).

1.10. Aim and objectives of the research

This study is aimed at evaluating the effect of aqueous extract of Millettia aboensis leaves on

Lomotil-induced constipation using Wistar albino rats.

The following objectives are designed to achieve the aim of this research:

  To determine both qualitatively and quantitatively the phytochemical composition of the aqueous extract of Millettia aboensis leaves.

  To determine  the effect  of the  aqueous  extract  of Millettia  aboensis  on the  liver marker enzymes.

  To determine the effect of the aqueous extract of Millettia aboensis on the serum level of some body electrolytes

  To determine  the lethal dose (acute/ sub-acute  toxicity)  of the aqueous  extract  of

Millettia aboensis.

  To determine the effect of the aqueous extract of Millettia aboensis on the  serum levels of lipid profile (HDL, LDL, Cholesterol and Triacylglyceride).

  To determine  the effect of the aqueous  extract of Millettia aboensis on the  blood glucose concentration.

  To determine  the  effect  of the  aqueous  extract  of Millettia  aboensis  on  Lomotil

induced constipation in rat

  To determine the effect of the aqueous extract of Millettia aboensis on the transport of glucose, sodium and potassium ions across everted rat intestine.



This material content is developed to serve as a GUIDE for students to conduct academic research


STUDIES ON THE EFFECT OF AQUEOUS EXTRACT OF MILLETTIA ABOENSIS LEAVES ON LOMOTIL INDUCED CONSTIPATION IN WISTAR ALBINO RATS

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