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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