EFFECT OF MORINDA LUCIDA ON THE LIVER STATUS OF LACTOSE INDUCED OSMOTIC DIARRHEA IN WISTAR RATS

ABSTRACT

Diarrhea is the condition of having at least three loose or liquid bowel movements each day. It can result in dehydration due to fluid loss. Morinda lucida  belongs to the family Rubiaceae. M. lucida extracts have been reported to have antioxidant and reducing activities and anti-microbial activity. This medicinal plant has also been reported to be effective in treatment or management of diarrhea. This study shows the effect of aqueous leaf extract of morinda lucida (dry and fresh extracts) on the liver status of lactose induced osmotic diarrhea of albino wistar rats using the liver function test such as; serum albumin analysis, serum total protein and serum bilirubin. In this study the effectiveness of morinda lucida is tested for in albino rats induced with osmotic diarrhea using 25% lactose enriched diet. The animals were treated thereafter with both dry and fresh extracts of Morinda lucida and also a standard drug, loperamide. Findings reveal that there was a significant change in albumin level, the total protein and bilirubin comparing the control (normal) with the lactose induced rats, level. Although on administration of the extracts, there level of albumin, total protein and bilirubin were not restored to the valves in the control. The study shows that the aqueous extract of the leaf of morinda lucida is not as effective as claimed in the management of the liver status of lactose induced osmotic diarrhea in albino wistar rats, although the dry extract showed some degree of effectiveness than the fresh extract. 

TABLE OF CONTENTS

CHAPTER ONE

1.0     INTRODUCTION AND LITERATURE REVIEW

1.1     INTRODUCTION

1.1.1  SECRETORY DIARRHEA

1.1.2  OSMOTIC DIARRHEA

1.1.3  EXUDATIVE DIARRHEA

1.1.4  INFLAMMATORY DIARRHEA

1.1.5  DYSENTERY

1.1.6  INFECTIOUS DIARRHEA

1.1.7  MALABSORPTION

1.1.8  OTHER CAUSES,

1.2     PREVENTION

1.3     MANAGEMENT

1.3     LITERATURE REVIEW

1.3.1  DESCRIPTION OF MORINDA LUCIDA

1.3.2  TAXONOMY OF MORINDA LUCIDA

1.3.3  OTHER NAMES FOR MORINDA LUCIDA

1.3.4  HABITAT OF MORINDA LUCIDA

1.3.5  TOXICITY OF M.LUCIDA

1.4     LACTOSE INTOLERANCE

1.4.1 SYMPTOMS

1.4.2  DIAGNOSIS

1.4.3  HYDROGEN BREATH TEST

1.4.4  BLOOD TEST

1.4.5  INTESTINAL BIOPSY

1.4.6  MANAGEMENT

1.5     THE LIVER

1.5.1  ANATOMY

1.5.2  CELL TYPES

1.5.3  BLOOD FLOW

1.5.4  PHYSIOLOGY

1.5.5  SYNTHESIS:

1.5.6  BREAKDOWN

1.5.6  OTHER FUNCTIONS

1.5.2  DISEASES OF THE LIVER

1.5.3  LIVER FUNCTION TESTS

1.5.3.1        ALBUMIN

1.5.3.2        TOTAL BILIRUBIN

1.5.3.3        SERUM TOTAL PROTEIN

1.6     AIM AND OBJECTIVE OF RESEARCH

1.7     AIM

1.8     OBJECTIVE

CHAPTER TWO

2.0     MATERIALS AND METHODS

2.1     APPARATUS

2.2     REAGENT

2.3     EXPERIMENTS

2.4     PREPARATION OF PLANT EXTRACTS USING STANDARD METHOD (WILLIAMS ET AL., 1996)

2.5     COLLECTION OF ANIMALS

2.6     EXPERIMENTAL DESIGN

2.7     MODE OF SACRIFICE

2.8     TREATMENT OF LACTOSE INDUCED RATS

2.9     ASSAY METHODS

2.9.1  Determination of liver bilirubin by Evelyn Malloy method(Magos,1960).

Principle:

2.9.2  Estimation of liver Albumin by Bromocresol Purple Method (Lasky et al., 1985)

2.9.3  ESTIMATION OF LIVER TOTAL PROTEIN BY BIURET METHOD (OKUTUCU ET AL., 2007).

CHAPTER THREE

3.0     RESULTS

3.1     OBSERVATIONS

3.2     FINAL RESULTS OF ALBUMIN, BILIRUBIN AND TOTAL PROTEIN AS MEAN + S.E.M

CHAPTER FOUR

4.0     DISCUSSION AND CONCLUSION

REFERENCES

APPENDIX I:      ABSORBANCE FOR ALBUMIN FOR VARIOUS GROPUS

APPENDIX II:     CONCENTRATION OF ALBUMIN FOR EACH RAT (g/l)

APPENDIX III:    CONCENTRATION OF ALBUMIN FOR VARIOUS GROUPS AS IN MEAN AND S.E.M (g/l)

APPENDIX IV:    ABSORBANCE FOR BILIRUBIN OF VARIOUS GROUPS (g/l)

APPENDIX V:     CONCENTRATION OF BILIRUBIN FOR EACH RAT (mg/dl)

APPENDIX VI:    CONCENTRATION OF BILIRUBIN FOR VARIOUS AS IN MEAN AND S.E.M (IN mg/dl)

APPENDIX VII: STANDARD CURVE FOR TOTAL PROTEIN

APPENDIX VIII: ABSORBANCE FOR TOTAL PROTEIN OF VARIOUS GROUPS

APPENDIX IX:    CONCENTRATION OF TOTAL PROTEIN FOR EACH RAT (mg/ml)

APPENDIX X:     CONCENTRATION OF TOTAL PROTEIN OF VARIOUS GROUP AS IN MEAN AND S.E.M (mg/ml)

APPENDIX XI:    WEIGHT OF RATS BEFORE SACRIFICE (IN GRAM)

APPENDIX XII:  ANOVA FOR ALBUMIN

APPENDIX XIII: ANOVA FOR BILIRUBIN

APPENDIX XIV:          ANOVA FOR TOTAL PROTEIN

CHAPTER ONE

1.0     INTRODUCTION AND LITERATURE REVIEW

1.1     INTRODUCTION

Diarrhea or diarrhoea (from the Ancient Greek from dia "through" and rheo "flow") is the condition of having at least three loose or liquid bowel movements each day. It often lasts for a few days and can result in dehydration due to fluid loss. Signs of dehydration often begin with loss of the normal stretchiness of the skin and changes in personality. This can progress to decreased urination, loss of skin color, a fast heart rate, and a decrease in responsiveness as it becomes more severe. Loose but non watery stools in babies who are breast fed, however, may be normal (WHO, 2014).The most common cause is an infection of the intestines due to a virus, bacteria, parasite, or a condition known as gastroenteritis. These infections are often acquired from food or water that has been contaminated by stool, or directly from another person who is infected. It may be divided into three types: short duration watery diarrhea, short duration bloody diarrhea, and if it lasts for more than two weeks persistent diarrhea. The short duration watery diarrhea may be due to an infection by cholera. If blood is present it is also known as dysentery (WHO, 2014). A number of non-infectious causes may also result in diarrhea including: hyperthyroidism, lactose intolerance, inflammatory bowel disease, a number of medications, and irritable bowel syndrome among other (Doyle et al., 2013). In most cases stool cultures are not required to confirm the exact cause (Dupont, 2014).Prevention of infectious diarrhea is by improved sanitation, clean drinking water, and hand washing. Breastfeeding for at least six months is also recommended as is vaccination against rotavirus. Oral rehydration solution (ORS), which is clean water with modest amounts of salts and sugar, along with zinc tablets are the treatments of choice (WHO, 2014). This treatment has been estimated to have saved 50 million children in the past 25 years (WHO, 2014). When people have diarrhea it is recommended that they continue to eat healthy food and babies continue to be breastfeed. If commercial ORS are not available, homemade solutions may be used (Sarah et al., 2012). In those with severe dehydration, intravenous fluids maybe required. Most cases; however, can be managed well with fluids by mouth.  Antibiotics, while rarely used, may be recommended in a few cases such as those who have bloody diarrhea and a high fever, those with severe diarrhea following travelling, and those who grow specific bacteria or parasites in their stool (Dupont, 2014). Loperamide may help decrease the number of bowel movement but is not recommended in those with severe disease. About 1.7 to 5 billion cases of diarrhea occur per year (Doyle, 2013). It is most common in developing countries, where young children get diarrhea on average three times a year (WHO, 2014). Worldwide, as of 2012, it is the second most common cause of deaths in children younger than five (0.76 million or 11%) (CDC, 2014). Frequent episodes of diarrhea are also a common cause of malnutrition and the most common cause in those younger than five years of age (WHO, 2014). Other long term problems that can result include poor physical and intellectual development (CDC, 2014).Diarrhea is defined by the World Health Organization as having three or more loose or liquid stools per day, or as having more stools than is normal for that person. (WHO)Acute diarrhea is defined as an abnormally frequent discharge of semi solid or fluid fecal matter from the bowel, lasting less than 14days, by World Gastroenterology Organization (WGO, 2011).

1.1.1  SECRETORY DIARRHEA

Secretory diarrhea means that there is an increase in the active secretion, or there is an inhibition of absorption. There is little to no structural damage. The most common cause of this type of diarrhea is a cholera toxin that stimulates the secretion of anions, especially chloride ions. Therefore, to maintain a charge balance in the lumen, sodium is carried with it, along with water. In this type of diarrhea intestinal fluid secretion is isotonic with plasma even during fasting. It continues even when there is no oral food intake.

1.1.2  OSMOTIC DIARRHEA

Osmotic diarrhea occurs when too much water is drawn into the bowels. If a person drinks solutions with excessive sugar or excessive salt, these can draw water from the body into the bowel and cause osmotic diarrhea (WHO, 2005). Osmotic diarrhea can also be the result of mal digestion (e.g., pancreatic disease or Coeliac disease), in which the nutrients are left in the lumen to pull in water. Orit can be caused by osmotic laxatives (which work to alleviate constipation by drawing water into the bowels). In healthy individuals, too much magnesium or vitamin C or undigested lactose can produce osmotic diarrhea and distention of the bowel. A person who has lactose intolerance can have difficulty absorbing lactose after an extraordinarily high intake of dairy products. In persons who have fructose mal absorption, excess fructose intake can also cause diarrhea. High-fructose foods that also have high glucose content are more absorbable and less likely to cause diarrhea. Sugar alcohols such as sorbitol (often found in sugar-free foods) are difficult for the body to absorb and, in large amounts, may lead to osmotic diarrhea. In most of these cases, osmotic diarrhea stops when offending agent (e.g. milk, sorbitol) is stopped.

1.1.3  EXUDATIVE DIARRHEA

Exudative diarrhea occurs with the presence of blood and pus in the stool. This occurs with inflammatory bowel diseases, such as Crohn's disease or ulcerative colitis, and other severe infections such as E. coli or other forms of food poisoning.

1.1.4  INFLAMMATORY DIARRHEA

Inflammatory diarrhea occurs when there is damage to the mucosal lining or brush border, which leads to a passive loss of protein-rich fluids and a decreased ability to absorb these lost fluids. Features of all three of the other types of diarrhea can be found in this type of diarrhea. It can be caused by bacterial infections, viral infections, parasitic infections, or autoimmune problems such as inflammatory bowel diseases. It can also be caused by tuberculosis, colon cancer, and enteritis.

1.1.5  DYSENTERY

If there is blood visible in the stools, it is also known as dysentery. The blood is trace of an invasion of bowel tissue. Dysentery is a symptom of, among others, Shigella, Entamoebahistolytica, and Salmonella.

1.1.6  INFECTIOUS DIARRHEA

There are many causes of infectious diarrhea, which include viruses, bacteria and parasites (Navaneethan et al., 2008). Norovirus is the most common cause of viral diarrhea in adults, (Patel et al., 2009). But rotavirus is the most common cause in children under five years old (Greenberg et al., 2009). Adenovirus types 40 and 41, (Uhoo et al., 1990) and astroviruses cause a significant number of infections.

1.1.7  MALABSORPTION

Mal-absorption is the inability to absorb food fully, mostly from disorders in the small bowel, but also due to mal-digestion from diseases of the pancreas.

1.1.8  OTHER CAUSES,

Diarrhea can be caused by chronic ethanol ingestion (Kasper et al., 2005). Ischemic bowel disease usually affects older people and can be due to blocked arteries. Microscopic colitis is a type of inflammatory bowel disease where changes are only seen on histological examination of colonic biopsies. Bile salt mal-absorption (primary bile acid diarrhea) is where excessive bile acids in the colon produce a secretory diarrhea. Hormone-secreting tumors: some hormones (e.g. serotonin) can cause diarrhea if excreted in excess (usually from a tumor). Chronic mild diarrhea in infants and toddlers may occur with no obvious cause and with no other ill effects; this condition is called toddler's diarrhea.

1.2     PREVENTION

A rotavirus vaccine decreases the rates of diarrhea in a population (WHO). New vaccines against rotavirus, Shigella, ETEC, and cholera are under development, as well as other causes of infectious diarrhea. Probiotics decrease the risk of diarrhea in those taking antibiotics (Hempel et al., 2012). In institutions and in communities, interventions that promote hand washing lead to significant reductions in the incidence of diarrhea (Ejemot et al., 2008).

1.3     MANAGEMENT

In many cases of diarrhea, replacing lost fluid and salts is the only treatment needed. This is usually by mouth – oral rehydration therapy – or, in severe cases, intravenously (WHO). Diet restrictions such as the BRAT diet are no longer recommended (King et al., 2003). Research does not support the limiting of milk to children as doing so has no effect on duration of diarrhea. To the contrary, WHO recommends that children with diarrhea continue to eat as sufficient nutrients are usually still absorbed to support continued growth and weight gain and that continuing to eat speeds also recovery of normal intestinal functioning. CDC recommends that children and adults with cholera also continue to eat (CDC, 2010).Medications such as loperamide (Imodium) and bismuth subsalicylate may be beneficial; however they may be contraindicated in certain situations (Schiller, 2007).

1.3     LITERATURE REVIEW

 In the world, mostly in the rural areas of the developing countries, people depend on local medicinal plant as remedy for their diseases and illness probably either because of the absence of modernized functional health facilities or because of ancestral and traditional beliefs. Plants produce a diverse range of bioactive molecules, making them rich sources of different types of medicines (Nair et al., 2005).

In different parts of Nigeria, different varieties of plants are used in the treatment of different types of diseases. Roots, barks or leaves of Newbolbea leavis are used in the treatment of dysentery, syphilis, ear ache, ringworm and scrotal elephantiasis (Azoro, 2002.) Morinda lucida known as Oruwo in the South-Western part of Nigeria is a medium sized tree with a crooked hole and rather short twisted branches. It belongs to the family Rubiaceae. It has a rough bark, grey in colour, flaking off in irregular patches. Its leaves are about 7 to 15 cm long by 3.5 to 7.5 cm broad, and flowers are white with a narrow glabrous corolla-tube about 2.5 cm. Stem bark, roots and leaves infusion is used as an anti-malarial, anti-diabetic, jaundice and dysentery treatment (Burkill, 1997), and it is used in anti-malarial activity (Tona et al., 1999; Agomo et al., 1992; Asuzu and Chineme, 1990; Koumaglo et al., 1992), anti-Salmonella typhi activity (Akinyemi et al., 2005), effect on contractility of isolated uterine smooth muscle of pregnant and non-pregnant mice (Elias et al., 2007), toxicity and mutagenic studies (Sowemimo et al., 2007; Akinboro and Bakare, 2007; Raji et al., 2005), and it has anti-diabetic property (Olajide et al., 1999). M. lucida extracts have been reported to have antioxidant and reducing activities (Ogunlana et al., 2008), and anti-microbial activity (Ogundare and Onifade, 2009; Adomi, 2006, 2008).

Several drugs including some antibiotics are no longer active against targeted organisms. It has been reported that the effective life span of these therapeutics agents are limited (Cowan, 1999). Therefore, we experience antibiotic resistant organisms, and ineffective malarial drugs. More so, majority of the orthodox drugs are both expensive and display dangerous side effects in the users. Hence, discovering and identifying new safe drugs without severe side effects has become an important goal of research in biomedical science. It is in this context that the aqueous and ethanolic extracts of stem bark, leaves and roots of M. lucida were screened for possible anti-typhoid effect in vitro in comparison with known standard antimicrobial or anti-typhoid agents.

1.3.1  DESCRIPTION OF MORINDA LUCIDA

Morinda lucida occurs from Senegal to Sudan and Southwards to Angola and Zambia, sometimes planted around villages in Nigeria. It grows in grassland, exposed hillsides, thickets, forest, often on termite mounds, sometimes in areas, which are regularly flooded, from sea level up to 1300m altitude. Evergreen small to medium-sized tree, up to 9-18m or 25m tall in coastal areas of Ivory Coast, with bole and branches often crooked or gnarled or straight, sometime short, 20-30cm in diameter. Bark is smooth to roughly scaly, grey to brown colour often with distinct purple layers. Leaves are opposite, simple and entire. Inflorescence is a staked cup-shaped gland. Flowers are bisexual and the fruits are drupe several arranged together into an almost globuse succulent syncarp. Morinda lucida comprises about 80 species and occurs throughout the tropics. In Africa, 5 species are found. The comparatively small flowering and fruiting heads on long slender peduncles are distinct characteristics of Morinda lucida. Many species including those from Africa are important medicinal plants, widely applied against various kinds of fevers and infections. 

1.3.2  TAXONOMY OF MORINDA LUCIDA

(sambamurty, 2005).

1.3.3  OTHER NAMES FOR MORINDA LUCIDA

Sangogo(cote d’ ivoire)

Kno kroma(Ghana)

Atak ake(togo)

Egbogo(esan, Nigeria)

Oruwo (Yoruba, Nigeria)

Eze ogu or njisi (Igbo, Nigeria) (adeneye and agbaje, 2008).

1.3.4  HABITAT OF MORINDA LUCIDA

Morinda lucida is a mesophytic plant that can be found in tropical regions, coastal zone or coastal savannah and also in northern savannah.

1.3.5  TOXICITY OF M.LUCIDA

Toxicity test is done on animals to assess the potential adverse effect of chemical compounds or plant extracts on administration to humans. In research done by ashafa and olumu, 2001, it was shown that the ethanolic extract of morinda lucida was not toxic to the liver, kidney or heart and the hematological parameters were not affected by administration to study animal.

1.4     LACTOSE INTOLERANCE

Lactose intolerance is the inability of adults individual to digest lactose, a sugar found in milk and dairy product. Lactose intolerance individual has a low level of lactase, an enzyme that break down lactose into glucose and galactose in their digestive system.

Lactase deficiency has a number of causes and is classified as one of three types:Primary lactase deficiency is genetic, only affects adults, and is caused by the absence of a lactase persistence allele (Heyman, 2006 and Enattah et al., 2002). It is the most common cause of lactose intolerance, as a majority of the world's population lacks these alleles (Swallow, 2003).Secondary, acquired, or transient lactase deficiency is caused by an injury to the small intestine, usually during infancy, from acute gastroenteritis, diarrhea, chemotherapy, intestinal parasites, or other environmental causes (Lawson et al., 2002 and Swagerty et al., 2002).Congenital lactase deficiency is a very rare, autosomal recessive genetic disorder that prevents lactase expression from birth (Heyman, 2006). It is particularly common in Finland (Behrendt et al., 2009). People with congenital lactase deficiency cannot digest lactose from birth, so cannot digest breast milk. Lactose intolerance is not an allergy, because it is not an immune response, but rather a problem with digestion caused by lactase deficiency. Milk allergy is a separate condition, with distinct symptoms that occur when the presence of milk proteins trigger an immune reaction.

1.4.1 SYMPTOMS

The principal symptom of lactose intolerance is an adverse reaction to products containing lactose (primarily milk), including abdominal bloating and cramps, flatulence, diarrhea, nausea, borborygmi and vomiting (particularly in adolescents). These appear one-half to two hours after consumption (NDDIC, 2011). The severity of symptoms typically increases with the amount of lactose consumed; most lactose-intolerant people can tolerate a certain level of lactose in their diets without ill effects (Savaiano et al., 1987 and Madry et al., 2011).

1.4.2  DIAGNOSIS

To assess lactose intolerance, intestinal function is challenged by ingesting more dairy products than can be readily digested. Clinical symptoms typically appear within30 minutes, but may take up to two hours, depending on other foods and activities (Bowen, 2006). Substantial variability in response (symptoms of nausea, cramping, bloating, diarrhea, and flatulence) is to be expected, as the extent and severity of lactose intolerance varies among individuals. Lactose intolerance is distinct from milk allergy, an immune response to cow's milk proteins. They may be distinguished in diagnosis by giving lactose-free milk, producing no symptoms in the case of lactose intolerance, but the same reaction as to normal milk in the presence of a milk allergy. A person can have both conditions. If positive confirmation is necessary, four tests are available (Olivier et al., 2012).

1.4.3  HYDROGEN BREATH TEST

In a hydrogen breath test, the most accurate lactose intolerance test, after an overnight fast, 25 g of lactose (in a solution with water) are swallowed. If the lactose cannot be digested, enteric bacteria metabolize it and produce hydrogen, which, along with methane, if produced, can be detected on the patient's breath by a clinical gas chromatograph or compact solid-state detector. The test takes about 2.5 hours to complete. If the hydrogen levels in the patient's breath are high, they may have lactose intolerance. This test is not usually done on babies and very young children, because it can cause severe diarrhea.

1.4.4  BLOOD TEST

In conjunction, measuring blood glucose level every 10 to 15minutes after ingestion will show a "flat curve" in individuals with lactose mal absorption, while the lactase persistent will have a significant "top", with a typical elevation of 50% to100%, within one to two hours. However, due to the need for frequent blood sampling, this approach has been largely replaced by breath testing. After an overnight fast, blood is drawn and then 50 g of lactose (in aqueous solution) are swallowed. Blood is then drawn again at the 30-minute, 1-hour, 2-hour, and 3-hourmarks. If the lactose cannot be digested, blood glucose levels will rise by less than 20 mg/dl. Stool acidity test can be used to diagnose lactose intolerance in infants, for whom other forms of testing are risky or impractical (NDDIC, 2006). The infant is given lactose to drink. If the individual is tolerant, the lactose is digested and absorbed in the small intestine; otherwise, it is not digested and absorbed, and it reaches the colon. The bacteria in the colon, mixed with the lactose, cause acidity in stools. Stools passed after the ingestion of the lactose are tested for level of acidity. If the stools are acidic, the infant is intolerant to lactose (Jay et al., 2011). Stool pH in lactose intolerance is less than 5.5.

1.4.5  INTESTINAL BIOPSY

An intestinal biopsy can confirm lactase deficiency following discovery of elevated hydrogen in the hydrogen breath test (Hargrove et al., 1993). Modern techniques have enabled a bedside test, identifying presence of lactase enzyme on upper gastrointestinal endoscopy instruments (Kuokkanen et al., 2006). However, for research applications such as mRNA measurements, a specialist laboratory is required.

1.4.6  MANAGEMENT

Lactose intolerance is not considered a condition that requires treatment in societies where the diet contains relatively little dairy produce. However, those living among societies that are largely lactose-tolerant may find lactose intolerance troublesome. Although no way to reinstate lactase production had been found as of 2013, some individuals have reported their intolerance varies over time, depending on health status and pregnancy (Roy et al., 2006). About 44% of lactose-intolerant women regain the ability to digest lactose during pregnancy. This might be caused by slow intestinal transit and intestinal flora changes during pregnancy. Lactose intolerance can also be managed by ingesting live yogurt cultures containing lactobacilli that are able to digest the lactose in other dairy products. This may explain why many South Asians, though genetically lactose intolerant, are able to consume large quantities of milk without many symptoms of lactose intolerance. Consuming live yogurt cultures is very common in the South Asian population. Lactose intolerance is not usually an absolute condition: The reduction in lactase production, and the amount of lactose that can therefore be tolerated, varies from person to person. Since lactose intolerance poses no further threat to a person's health, the condition is managed by minimizing the occurrence and severity of symptoms. Berdanier and Hargrove recognized four general principles in dealing with lactose intolerance—avoidance of dietary lactose, substitution to maintain nutrient intake, regulation of calcium intake and use of enzyme substitute (Hargrove et al., 1993).

Avoiding lactose-containing products

Since each individual's tolerance to lactose varies, according to the U.S. National Institutes of Health (NIH),"Dietary control of lactose intolerance depends on people learning through trial and error how much lactose they can handle." (NDDIC) Label reading is essential, as commercial terminology varies according to language and region (Hargrove et al., 1993).Lactose is present in two large food categories—conventional dairy products, and as a food additive (casein, caseinate, whey), which may contain traces of lactose.

1.5     THE LIVER

The liver is a vital organ of the digestive system present invertebrates and some other animals. It has a wide range of functions, including detoxification, protein synthesis, and production of biochemical substances necessary for digestion. The liver is necessary for survival; there is currently no way to compensate for the absence of liver function in the long term, although new liver dialysis techniques can be used in the short term. This gland 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 via 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). Estimates regarding the organ's total number of functions vary, but textbooks generally cite it at around 500 or so (Zakim et al., 2002).Terminology related to the liver often starts in hepar- or hepat-from the Greek word for liver, hēpar ( root hepat).

1.5.1  ANATOMY

The liver is a reddish brown organ with four lobes of unequal size and shape. A human liver normally weighs 1.44–1.66 kg(3.2–3.7 lb), (Cotran et al., 2001) and is a soft, pinkish-brown, triangular organ. It is both the largest internal organ (the skin being the largest organ overall) and the largest gland in the human body. It is located in the right upper quadrant of the abdominal cavity, resting just below the diaphragm. The liver lies to the right of the stomach and overlies the gall bladder. It is connected to two large blood vessels, one called the hepatic artery and one called the portal vein. The hepatic artery carries blood from the aorta, whereas the portal vein carries blood containing digested nutrients from the entire gastrointestinal tract and also from the spleen and pancreas. These blood vessels subdivide into capillaries, which then lead to a lobule. Each lobule is made up of millions of hepatic cells which are the basic metabolic cells. Lobules are the functional units of the liver.

1.5.2  CELL TYPES

Two major types of cells populate the liver lobes; parenchymaland non-parenchymal cells. 80% of the liver volume is occupied by parenchymal cells commonly referred to as hepatocytes. Non-parenchymal cells constitute 40% of the total number of liver cells but only 6.5% of its volume. Sinusoidal hepatic endothelial cells, Kupffer cells and hepatic stellate cells are some of the non-parenchymal cells that line the liver sinusoid (Kmie, 2001).

1.5.3  BLOOD FLOW

The liver gets a dual blood supply from the hepatic portal vein and hepatic arteries. Supplying approximately 75% of the liver's blood supply, the hepatic portal vein carries venous blood drained from the spleen, gastrointestinal tract, and its associated organs. The hepatic arteries supply arterial blood to the liver, accounting for the remainder of its blood flow. Oxygen is provided from both sources; approximately half of the liver's oxygen demand is met by the hepatic portal vein, and half is met by the hepatic arteries (Shneider et al., 2008).Blood flows through the liver sinusoids and empties into the central vein of each lobule. The central veins coalesce into hepatic veins, which leave the liver.

1.5.4  PHYSIOLOGY

The various functions of the liver are carried out by the liver cells or hepatocytes. Currently, there is no artificial organ or device capable of emulating all the functions of the liver. Some functions can be emulated by liver dialysis, an experimental treatment for liver failure. The liver is thought to be responsible for up to 500 separate functions, usually in combination with other systems and organs.

1.5.5  SYNTHESIS:

Proteins produced and secreted by the livera large part of amino acid synthesis. The liver performs several roles in carbohydrate metabolism:

Gluconeogenesis (the synthesis of glucose from certain amino acids, lactate or glycerol)

Glycogenolysis (the breakdown of glycogen into glucose)

Glycogenesis (the formation of glycogen from glucose) (muscle tissues can also do this)

The liver is responsible for the mainstay of protein metabolism, synthesis as well as degradation. The liver also performs several roles in lipid metabolism: Cholesterol synthesis Lipogenesis, the production of triglycerides (fats).A bulk of the lipoprotein is synthesized in the liver. The liver produces coagulation factors I (fibrinogen), II(prothrombin), V, VII, IX, X and XI, as well as protein C, protein S and antithrombin. In the first trimester fetus, the liver is the main site of red blood cell production. By the 32nd week of gestation, the bone marrow has almost completely taken over that task. The liver produces and excretes bile (a yellowish liquid) required for emulsifying fats and help the absorption of vitamin K from the diet. Some of the bile drains directly into the duodenum, and some is stored in the gallbladder. The liver also produces insulin-like growth factor 1 (IGF-1),a polypeptide protein hormone that plays an important role in childhood growth and continues to have anabolic effects in adults. The liver is a major site of thrombopoietin production. Thrombopoietin is a glycoprotein hormone that regulates the production of platelets by the bone marrow.

1.5.6  BREAKDOWN

The liver breaks down insulin and other hormones. The liver breaks down or modifies toxic substances (e.g. methylation) and most medicinal products in a process called drug metabolism. This sometimes results in toxication, when the metabolite is more toxic than its precursor. Preferably, the toxins are conjugated to avail excretion in bile or urine. The liver converts ammonia to urea (urea cycle).

1.5.6  OTHER FUNCTIONS

The liver stores a multitude of substances, including glucose (in the form of glycogen), vitamin A (1–2 years' supply), vitamin D(1–4 months' supply), vitamin B12 (1–3 years' supply), vitamin K, iron, and copper. The liver is responsible for immunological effects—the mononuclear phagocyte system (MPS) of the liver contains many immunologically active cells, acting as a 'sieve' for antigens carried to it via the portal system. The liver produces albumin, the major osmolar component of blood serum. The liver synthesizes angiotensinogen, a hormone that is responsible for raising the blood pressure when activated by renin, an enzyme that is released when the kidney senses low blood pressure. The liver also functions as a blood reservoir, being an expandable organ. Large quantities of blood can be stored in its blood vessels, its normal blood volume in the hepatic veins and that in the hepatic sinuses is about 450ml. During cardiac failure with peripheral congestion, the liver expands, and 0.5 to 1 liter of extra blood is occasionally stored in the hepatic veins and sinuses, due to high pressure in right atrium which causes back pressure in the liver.

1.5.2  DISEASES OF THE LIVER

The liver supports almost every organ in the body and is vital for survival. Because of its strategic location and multi dimensional functions, the liver is also prone to many diseases (NDDIC, 2010).The most common include: Infections such as hepatitis A, B,C, D, E, alcohol damage, fatty liver, cirrhosis, cancer , drug damage (particularly by acetaminophen (paracetamol) and cancer drugs).Many diseases of the liver are accompanied by jaundice caused by increased levels of  bilirubin in the system. The bilirubin results from the breakup of the hemoglobin of dead red blood cells; normally, the liver removes bilirubin from the blood and excretes it through bile. There are also many pediatric liver diseases including biliary atresia, alpha-1 antitrypsin deficiency, alagille syndrome, progressive familial intra hepatic cholestasis, and Langerhans cell histiocytosis, to name but a few. Diseases that interfere with liver function will lead to derangement of these processes. However, the liver has a great capacity to regenerate and has a large reserve capacity. In most cases, the liver only produces symptoms after extensive damage. Liver diseases may be diagnosed by liver function tests, for example, by production of acute phase proteins.

1.5.3  LIVER FUNCTION TESTS

Liver function tests (LFTs or LFs) are groups of blood tests that give information about the state of a patient's liver (Lee, 2009). These tests include prothrombin time (PT/INR), aPTT, albumin, bilirubin (direct and indirect), and others. Liver transaminases (AST or SGOT and ALT or SGPT) are useful biomarkers of liver injury in a patient with some degree of intact liver function (Johnston, 1999). Most liver diseases cause only mild symptoms initially, but these diseases must be detected early. Hepatic (liver) involvement in some diseases can be of crucial importance. This testing is performed on a patient's blood sample. Some tests are associated 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). Several biochemical tests are useful in the evaluation and management of patients with hepatic dysfunction. These tests can be used to detect the presence of liver disease, distinguish among different types of liver disorders, gauge the extent of known liver damage, and follow the response to treatment. Some or all of these measurements are also carried out (usually about twice a year for routine cases) on those individuals taking certain medications, such as anticonvulsants, to ensure the medications are not damaging the person's liver.

1.5.3.1        ALBUMIN

Albumin is a protein made specifically by the liver, and can be measured cheaply and easily. It is the main constituent of total protein (the remaining from globulins). Albumin levels are decreased in chronic liver disease, such as cirrhosis. It is also decreased in nephrotic syndrome, where it is lost through theurine. The consequence of low albumin can be edema since the  intravascular oncotic pressure becomes lower than the extravascular space. An alternative to albumin measurement is prealbumin, which is better at detecting acute changes (half-lifeof albumin and prealbumin is about 2 weeks and about 2 days, respectively).

1.5.3.2        TOTAL BILIRUBIN

Measurement of total bilirubin includes both unconjugated and conjugated bilirubin. Unconjugated bilirubin is a breakdown product of heme (a part of hemoglobin in red blood cells). It is very hydrophobic and is mainly transported bound to albumin circulating in the blood. Addition of high-concentration hydrophobic drugs (certain antibiotics, diuretics) and high free fatty acids can cause elevated unconjugated bilirubin. Heme can also come from myoglobin, found mostly in muscle, cytochromes, found mostly in mitochondria, catalase, peroxidase, and nitric oxide synthase. The liver is responsible for clearing the blood of unconjugated bilirubin, and about 30% of it is taken up by a normal liver on each pass of the blood through the liver by the following mechanism: bilirubin is taken up into hepatocytes, 'conjugated' (modified to make it water-soluble) by UDP-glucuronyl-transferase, and secreted into the bile by CMOAT (MRP2), which is excreted into the intestine. In the intestine, conjugated bilirubin may be metabolized by colonic bacteria, eliminated, or reabsorbed. Metabolism of bilirubin into urobilinogen followed by reabsorption of urobilinogen accounts for the yellow color of urine, as urine contains a downstream product of urobilinogen. Further metabolism of urobilinogen into stercobilin while in the bowels accounts for the brown color of stool. Thus, having white or clay-colored stool is an indicator for a blockage in bilirubin processing and thus potential liver dysfunction or cholestasis .Increased total bilirubin (TBIL) causes jaundice, and can indicate a number of problems:

1. Prehepatic: Increased bilirubin production can be due to a number of causes, including hemolytic anemias and internal hemorrhage.

2. Hepatic: Problems with the liver are reflected as deficiencies in bilirubin metabolism (e.g., reduced hepatocyte uptake, impaired conjugation of bilirubin, and reduced hepatocyte secretion of bilirubin). Some examples would be cirrhosis and viral hepatitis.

3. Posthepatic: Obstruction of the bile ducts is reflected as deficiencies in bilirubin excretion. (Obstruction can be located either within the liver or in the bile duct).

DIRECT BILIRUBIN

The diagnosis is narrowed down further by evaluating the levels of direct bilirubin. If direct (conjugated) bilirubin is normal, then the problem is an excess of unconjugated bilirubin (indirect bilirubin), and the location of the problem is upstream of bilirubin conjugation in the liver. Hemolysis, or internal hemorrhage can be suspected. If direct bilirubin is elevated, then the liver is conjugating bilirubin normally, but is not able to excrete it. Bile duct obstruction by gallstones, hepatitis, cirrhosis or cancer should be suspected.

1.5.3.3        SERUM TOTAL PROTEIN

Serum total protein, also known as total protein, is a biochemical test for measuring the total amount of protein in serum. Protein in the plasma is made up of albumin and globulin. The globulin in turn is made up of α1, α2, β, and γ globulins. These fractions can be quantitated using protein electrophoresis, but the total protein test is a faster and cheaper test that estimates the total of all fractions together. The traditional method for measuring total protein uses the biuret reagent, but other chemical methods such as Kjeldahl method, dye-binding and refractometry are now available. The measurement is usually performed on automated analysers along with other laboratory tests.

1.6     AIM AND OBJECTIVE OF RESEARCH

Extracts of morinda lucida have been used over the years for the treatment of several diseases and ailment including malaria, diabetes and typhoid fever.

1.7     AIM

This study is aimed at investigating biochemically the effect of the aqueous leaves extracts of morinda lucida on the liver of lactose induced wister albino female rat with a view to justifying the use of the plant in forklore medicine.

1.8     OBJECTIVE

1.     To assess the effect of aqueous extracts of morinda lucida on the total protein, bilirubin and albumin

2.     To assess the capability of the liver in the presence of the aqueous extracts of morinda lucida

3.     To assess the detoxifying function of liver on treatment with the aqueous extract of morinda lucida.

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