Acute Toxicity of Ethanolic Leaf Extract of Myrianthus Arboreus on the Liver Enzymes of Wistar Rats

Inhalt

LIST OF TABLES

LIST OF FIGURES

LIST OF PLATES

APPENDIX

CHAPTER ONE
1.0 INTRODUCTION AND LITERATURE REVIEW
1.1 PLANT PRODUCTS AS DRUGS
1.2 TOXICITY TESTS
1.3 THE USE OF EXPERIMENTAL ANIMAL
1.4 THE LIVER
1.4.1 STRUCTURE AND FUNCTIONS
1.4.2 HEPATOTOXICITY
1.4.3 DETOXIFICATION FUNCTION OF THE LIVER
1.4.4 MARKER MOLECULES AND LIVER TOXICITY
1.5 MYRIANTHUS ARBOREUS, ORIGIN, CLASSIFICATION AND MEDICAL USES
1.6 TAXONOMICAL CLASSIFICATION OF MYRIANTHUS ARBOREUS
1.7 COMPOSITIONS AND PHYTOCHEMISTRY OF MYRIANTHUS ARBOREUS
1.8 AIM AND OBJECTIVES OF THE STUDY

CHAPTER TWO
2.0 MATERIALS AND METHODS
2.1 MATERIALS
2.1.1 LABORATORY APPARATUS
2.1.2 LABORATORY EQUIPMENT
2.1.3 CHEMICAL AND REAGENTS
2.1.4 SOURCE OF ANIMAL
2.2 METHODS
2.2.1 COLLECTION AND IDENTIFICATION OF PLANT MATERIALS
2.2.2 PREPARATION OF EXTRACT
2.2.3 PHYTOCHEMICAL SCREENING
2.2.4 SELECTION AND SORTING (INTO SIZE AND SEX) OF ANIMALS
2.2.5 RECONSTITUTION OF EXTRACT
2.2.6 LETHAL DOSE (LD50) STUDY OF THE ETHANOLIC LEAF EXTRACTS OF MYRIANTHUS ARBOREUS
2.2.7 EXPERIMENTAL DESIGN
2.2.8. SACRIFICE OF THE ANIMALS
2.2.9 BIOCHEMICAL PARAMETERS STUDIES
2.2.10 HISTOPATHOLOGICAL STUDIES
2.2.11 STATISTICAL ANALYSIS

CHAPTER THREE
3.0 RESULTS
3.2 RESULTS OF HISTOLOGICAL ANALYSIS
3.3 RESULTS OF PHYTOCHEMICAL SCREENING OF THE CRUDE LEAF POWDER OF MYRIANTHUS ARBOREUS (P.BEAUV)

CHAPTER FOUR
4.0 DISCUSSIONS AND CONCLUSIONS
4.1 DISCUSSIONS
4.2 CONCLUSIONS
4.3 LIMITATIONS TO THE STUDY
4.4 SUGGESTIONS FOR FURTHER RESEARCH

REFERENCE

APPENDIX 1

APPENDIX 2

LIST OF TABLES

Table 2.1. Experimental Design

Table 2.2 Administration of Myrianthus arboreus (MA) Extracts

Table 3.1 Effects of ethanolic leaf extract of Myrianthus arboreus (MA) on liver enzymes of wistar rats for 7days

Table 3.2 Effects of ethanolic leaf extract of Myrianthus arboreus (MA) on liver enzymes of wistar rats for 14days

Table 3.3 Results of Phytochemical Screening

LIST OF FIGURES

Figure 1.1 Diagram of a Wistar Rat

Figure 1.2 Diagram of the liver

Figure 1.3a Diagram of the Tree of Myrianthus arboreus

Figure 1.3b Diagram of the leaves of Myrianthus arboreus

LIST OF PLATES

PLATE 3.1. Histology of the Liver Tissue of Wistar Rat Administered With DMSO only for 7days (H&E Staining) X400

PLATE 3.2. Histology of the Liver Tissue of Wistar Rat Administered With DMSO + 1500mg/Kg B.W of Extract of Myrianthus arboreus for 7days (H&E Staining) X400

PLATE 3.3. Histology of the Liver Tissue of Wistar Rat Administered With DMSO + 1000mg/Kg B.W of Extract of Myrianthus arboreus for 7days (H&E Staining) X400

PLATE 3.4. Histology of the Liver Tissue of Wistar Rat Administered With DMSO + 500mg/Kg B.W of Extract of Myrianthus arboreus for 7days (H&E Staining) X400

PLATE 3.5. Histology of the Liver Tissue of Wistar Rat Administered With DMSO + 1000mg/Kg B.W of Extract of Myrianthus arboreus for 14days (H&E Staining) X400

PLATE 3.6. Histology of the Liver Tissue of Wistar Rat Administered With DMSO + 500mg/Kg B.W of Extract of Myrianthus arboreus for 14days (H&E Staining) X400

APPENDIX

Appendix 1. Preparation of the Ethanolic Extract Stock

Appendix 2. Experimental Design

Appendix 3. Herbarium reference number document

Appendix 4. Raw Data of Biochemical analysis from Lively stone Diagnostic Medical Laboratory

Appendix 5. SPSS ANOVA Descriptive Result

Appendix 6. SPSS ANOVA multiple Comparisms

LIST OF ABBREVIATIONS

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ABSTRACT

The present study was done to evaluate the acute (14 days) toxicity of the ethanolic leaf extract of Myrianthus arboreus on the liver enzymes of wistar rats. In the acute (14 days) toxicity studies, 24 rats were grouped into 1- 8groups (n=3rats/cage) and administered with 1500, 1000 and 500mg/kg body weight for 7days and 14days. The rats were sacrificed after 7days and 14day of administration and blood samples and liver organ were collected for investigations. The biochemical parameters such as the Alkaline phosphatase (ALP), Alanine transaminase (ALT) and Aspartate aminotransferase (AST) were determined and the liver histology analysed. The mean values of ALP showed significant increase (P≤0.05), the ALT showed a non-significant increase (P≥0.05) at groups 2, 3 and 4 and a significant increase (p≤0.05) at groups 6, 7 and 8. The AST showed a non-significant increase (P≥0.05) at all dosages and times except for group 2. The histological analysis showed microvesicular steatosis at groups 2 and 3 and a ballooning hepatic necrosis at group7. The phytochemical analysis of Myrianthus arboreus shows the presence of alkaloids, flavonoids, tannins, anthraquinones, triterpenoids, carbonhydrate, cardenolide and saponins in detectable limits but fixed oils and cyanogenic glycosides were not determined. In this investigation, we can conclude that the ethanolic leaf extract of Myrianthus arboreus was unsafe at all doses considered for a period of 14days. However, at a dose below 500mg for 7days could be considered safe.

CHAPTER ONE

1.0 INTRODUCTION AND LITERATURE REVIEW

1.1 PLANT PRODUCTS AS DRUGS

Historically, natural products have remained the major and potent source of new drugs. The use of natural products with therapeutic properties is as ancient as human civilisation and, for a long time, mineral, plant and animal products were the main sources of drugs (De Pasquale, 1984 ). Natural products which included herbs, animals and minerals serve as the lead compounds for the development of new medicines and also for the treatment and prevention of various human ailments .( Prasanth et al., 2014).

Plant products are classified into; Primary metabolites and secondary metabolite.

i. Primary metabolites are chemical compounds (metabolites) that are essential for the basic metabolism (anabolism and catabolism) of the plants, which results to assimilation, respiration, transport, and differentiation. They incudes starch, amino acids, lipids, minerals and vitamins.

ii. Secondary metabolites are chemical compounds (metabolites) that are non-essential for the basic metabolism of the plants. These products accounts for the colour, flavours and smells and are source of fine chemicals such as drugs, insecticides, dyes, fragrances and the phyto-medicines found in medicinal plants. They include alkaloids, glycosides, terpenes/isoprenoids, amines and phenolics.

Secondary metabolites (which are the potent forms of the natural products) and their derivatives, serve as the structural template for discovery and development of new drugs. The discovery and development of drug remains a challenging scientific task, which is the transition from a screening hit to a drug candidate, requires expertise and experience (Mouhssen, 2013). Although products derived from natural sources may not necessarily represent active ingredients in their final form, the majority of all drugs in the market have their origin in nature. (Chin et al., 2006 , Newman and Cragg 2012).

The growing number of herbal drug users around the globe and lack of scientific data on the safety profile of herbal products make it necessary to conduct toxicity study of herbal products. (Saad, 2006).

1.2 TOXICITY TESTS

Toxicity tests are most widely used to examine specific adverse events or specific endpoints such as cancer, cardio-toxicity and skin/eye irritation.(Prasanth et al., 2014) .

Acute, sub-acute and chronic toxicity tests are routine toxicity tests carried out by the pharmaceutical companies in the development of new medicines. Acute, Sub-acute and chronic toxicity tests are carried out to evaluate the toxic nature of a compound.

Acute toxicity testing involves the determination of lethal dose, the dose that kills 50% of the tested group of animals, whereas sub-acute and chronic toxicity testing involves the determination of long term effects of the test compound upon repeated administration ( Prasanth et al., 2014).

Acute toxicity describes the adverse effects of a substance that result either from a single exposure (The MSDS Hyper-Glossary, 2006) or from multiple exposures in a short space of time (usually less than 24 hours).

To be described as acute toxicity, the adverse effects should occur within 14 days of the administration of the substance. (IUPAC, 1997).

The acute toxicity test in which a single dose is used in each animal on one occasion only for the determination of gross behaviour and LD50 or median lethal dose. (Bhardwaj et al., 2012).

Sub-acute toxicity tests are employed to determine toxicity likely to arise from repeated exposures or daily dose of several weeks to several months (usually 4 weeks to 6 months).(Bhardwaj et al., 2012).

Chronic toxicity tests determine toxicity from exposure for a substantial portion of a subject's life, (usually 12 to 24 months). (Bhardwaj et al., 2012).

Acute toxicity testing involves the determination of lethal dose (LD­50), the dose that kills 50% of the tested group of animals. ( Prasanth et al., 2014).

Standardized tests or routes of administration of the test substance are available for oral, intravenous, intra-peritoneal, subcutaneous and inhalation exposures.

1.3 THE USE OF EXPERIMENTAL ANIMAL

It is widely considered unethical to use humans as test subjects for acute (or chronic) toxicity research. Otherwise, most acute toxicity data comes from animal testing or, more recently, in vitro testing methods and inference from data on similar substances. (Walum, 1998).

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Fig.1.1 A Wistar Rat

The Wistar rats are currently one of the most popular rat strains used for laboratory research. It is characterized by its wide head, long ears, and having a tail length that is always less than its body length. Wistar rats are an outbred strain of albino rats belonging to the species Rattus norvegicus. This strain was developed at the Wistar Institute in 1906 for use in biological and medical research, and is notably the first rat strain developed to serve as model organism at a time when laboratories primarily used Mus musculus. (Iliuţã, 2011).

Again, owing to the fact that the albino rats and other experimental animals have great similarities with human, they are used for experimental purposes. Thus, experimental results generated from them can be extrapolative (applicable) to humans while considering the differences in their organismal structure.

1.4 THE LIVER

1.4.1 STRUCTURE AND FUNCTIONS

The human liver is a dark red-brown organ with a soft, spongy texture. (Encarta, 2009). The liver has four lobes of unequal size and shape, weighing between 1.44- 1.6kg (Crook, 2009). It is the largest internal organ in the human body, and is located in the right upper quadrant of the abdominal cavity, resting just below the diaphragm.

The liver is connected to two large blood vessels which accounts for the blood flow in the liver;

i. The Hepatic artery, which deliver oxygen-rich blood from the heart supplying about 25 per cent of the liver's blood.
ii. The Hepatic Portal vein, which carries blood to the liver that has travelled from the digestive tract, where it collects nutrients as food is digested. These nutrients are delivered to the liver for further processing or storage. This vein is the source of 75 per cent of the liver's blood supply. (Encarta, 2009).

The liver plays an astonishing array of vital functions in the maintenance, performance and regulating homeostasis of the body. It is involved with almost all the biochemical pathways to growth, fight against disease, nutrient supply, energy provision and reproduction (Sharma et al., 1991). The liver, which is part of the digestive system, performs more than 500 different functions, all of which are essential to life. (Encarta,2009).

The major functions of the liver are carbohydrate, protein and fat metabolism, detoxification, secretion of bile, decomposition of red blood cells, plasma protein synthesis, hormone production, cholesterol synthesis and storage of vitamins. Thus, it is now clear to say that maintaining a healthy life, the liver is a crucial factor for the overall health and wellbeing of an individual.

The liver is unique among the body’s vital organs in that it can regenerate, or grow back, cells that have been destroyed by some short-term injury or disease. However, if the liver is damaged repeatedly over a long period, it may undergo irreversible changes that permanently interfere with function. (Encarta, 2009)

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Fig. 1.2 Diagram of the liver (Encyclopaedia Britannica, 2003)

The liver is composed of cell called hepatocytes. There are two major types of cells that populates the liver globes;

i. The karat parenchymal cell, which covers 80% of the liver.
ii. The non-parenchymal cell which includes, sinusoidal hepatic endothelial cells, kupffer cells and hepatic stellate cells, covers 40% of the liver (Kmie,2001).

1.4.2 HEPATOTOXICITY

The liver is the most common site of damage in laboratory animals administered drugs and other chemicals. There are many reasons including the fact that the liver is the first major organ to be exposed to ingested chemicals due to its portal blood supply. Although chemicals are delivered to the liver to be metabolized and excreted, this can frequently lead to activation and liver injury. (Andy, 2001).

Hepatotoxicity implies chemical-driven liver damage. Certain medicinal agents, when taken in overdoses and sometimes even when introduced within therapeutic ranges, may injure the organ. Chemicals that cause liver injury are called hepatotoxins. Other chemical agents, such as those used in laboratories and industries, natural chemicals (e.g. microcystins) and herbal remedies can also induce hepatotoxicity. (Aashish et al., 2011).

Several mechanism are responsible for either inducing hepatic injury or worsening the damage process. Herbal remedies, synthetic drugs and some chemicals act by damaging the mitochondria of the hepatocytes (Pak et al., 2004). Its dysfunction releases excessive amount of oxidants, which in turn injure the hepatic cells. Activation of some enzyme in the cytochrome P450 systems also leads to oxidative (Lynch et al., 2007). Injury to the hepatocyte and bile duct cells leads to accumulation of bile acid inside the liver this promotes further liver damages.

1.4.3 DETOXIFICATION FUNCTION OF THE LIVER

The various functions of the liver are carried out by the liver cells or hepatocytes (Benjamin and Sherman, 2008). Amongst the many metabolic functions of the liver, the most important and probably essential function is in the detoxification of xenobiotic or foreign chemicals. Today, most of xenobiotic to which humans are exposed to come from sources that includes; environmental pollution, food additives, cosmetic products, agrochemicals, processed foods and drugs. The detoxification system of liver involves two phases viz; Phase I and Phase II.

The Phase I Process involves exposure of the xenobiotic to functional groups (amino, carboxylic, hydroxyl, thiol, etc.) which brings about reduction and oxidation of the xenobiotic. The process is catalysed by Cytochrome P450 isoenzymes (cyps), flavin monoxygenases (FMO) and Epoxide hydrolases (ER).

The Phase II process involve conjugation of the phase one products and consequently making them hydrophilic (water soluble). The conjugation enzyme includes; gluthation-s-transferase (GST), UDP-glucuronosyl transferase (UGT), Sulphotranferases (SULT), n-acetyl transferases (NAT) and methyl transferases (MT).

1.4.4 MARKER MOLECULES AND LIVER TOXICITY

Every cellular organisational level (organelle, cell, tissue, organ and system) have molecules that are specific to it. These molecules such as enzymes, hormones, proteins are called ‘Marker Molecules’. The distribution of marker enzymes in the cell reflects the compartmentation of the processes they catalyze. (koolman et al., 2005).

The liver has marker molecules such as enzymes, hormones and proteins. Marker enzymes of the liver are determinant factor in assessing the levels of liver injury (liver injury test).

Liver toxicity can be classified into Cholestasic Injury, Cytotoxic Injury and Disturbances of hepatic function/clearance.

Similarly, Evaluation of liver toxicity in vivo is carried out by; Serum enzyme tests, Hepatic excretory tests, Alterations in chemical constituents of the liver and Histological analysis of liver injury.

The marker enzymes involved in the liver injury test include; Alkaline Phosphatase [AP, ALP], γ-Glutamyl Transpeptidase [GGT], Alanine aminotransferase [ALT], Aspartate aminotransferase [AST], Lactate Dehydrogenase [LDH], Ornithine carbamyl transferase [OCT], Sorbitol dehydrogenase [SDH], and Choline Esterase [ChE]. (Enzyme markers of Enzyme markers of Toxicity, 2015).

i. Alkaline phosphatase (ALP) (EC 3.1.3.1) is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation. As the name suggests, alkaline phosphatases are most effective in an alkaline environment. In humans, alkaline phosphatase is present in all tissues throughout the entire body, but is particularly concentrated in liver , bile duct, kidney, bone, intestinal mucosa and the placenta.

ii. Gamma-glutamyl transpeptidase (GGT) is an enzyme that catalyses the reaction between a peptide and an amino acid. High concentrations are found in the liver and kidney. GGT is measured in combination with other tests. ALP is increased in hepatobiliary disease and bone disease; GGT is elevated in hepatobiliary disease, but not in bone disease.

iii. Alanine transaminase (ALT) is a transaminase enzyme (EC 2.6.1.2). It is also called Alanine aminotransferase (ALAT) and was formerly called serum glutamate-pyruvate transaminase (SGPT) or serum glutamic-pyruvic transaminase (SGPT). ALT is found in plasma and in various body tissues, but is most common in the liver. It catalyses the two parts of the Alanine Cycle. ALT is commonly measured clinically as a part of a diagnostic evaluation of hepatocellular injury, to determine liver health. When used in diagnostics, it is usually measured in international units/litre (IU/L). Significantly elevated levels of ALT often suggest the existence of other medical problems such as viral hepatitis, diabetes, congestive heart failure, liver damage, bile duct problems, infectious mononucleosis, or myopathy, so ALT is commonly used as a way of screening for liver problems. Haemolysis has a negligible effect on ALT activity

iv. Aspartate transaminase (AST) or Aspartate aminotransferase (EC 2.6.1.1). AST catalyses the reversible transfer of an α-amino group between aspartate and glutamate and, as such, is an important enzyme in amino acid metabolism. AST is found in the liver, heart, skeletal muscle, kidneys, brain, and red blood cells. Serum AST level, serum ALT (alanine transaminase) level, and their ratio (AST/ALT ratio) are commonly measured clinically as biomarkers for liver health. The tests are part of blood panels. AST is commonly measured clinically as a part of diagnostic liver function tests, to determine liver health. However, it is important to keep in mind that the source of AST (and, to a lesser extent, ALT) in blood tests may reflect pathology in organs other than the liver. In fact, when the AST is higher than ALT, a muscle sourcing of these enzymes should be considered. For example, muscle inflammation due to dermatomyositis may cause AST>ALT. This is a good reminder that AST and ALT are not good measures of liver function because they do not reliably reflect the synthetic ability of the liver and they may come from tissues other than liver (such as muscle).

v. Lactate dehydrogenase (LDH or LD) is an enzyme found in nearly all living cells (animals, plants, and prokaryotes). LDH catalyses the conversion of pyruvate to lactate and back, as it converts NADH to NAD+ and back. A dehydrogenase is an enzyme that transfers a hydride from one molecule to another. LDH has been of medical significance because it is found extensively in body tissues, such as blood cells , liver and heart muscle. Because it is released during tissue damage, it is a marker of common injuries and disease such as heart and liver failure.

vi. Sorbitol dehydrogenase (SDH) is a cytosolic enzyme. Sorbitol dehydrogenase is an enzyme in carbohydrate metabolism converting sorbitol, the sugar alcohol form of glucose, into fructose. Organs that use it most frequently include the liver and seminal vesicle. It is a Sensitive enzyme marker for liver necrosis but shall be combined with measurements of ALT or other enzymes. It is elevated after acute obstruction of bile flow.

vii. Cholinesterase is a family of enzymes that catalyse the hydrolysis of the neurotransmitter acetylcholine into choline and acetic acid, a reaction necessary to allow a cholinergic neuron to return to its resting state after activation. There are two forms of cholinesterase;

- Acetyl cholinesterase (EC 3.1.1.7) (AChE) is found primarily in the blood on red blood cell membranes, in neuromuscular junctions, and in neural synapses. Acetyl cholinesterase exists in multiple molecular forms.

- Pseudocholinesterase (EC 3.1.1.8) (BChE or BuChE), also known as plasma cholinesterase, butyrylcholinesterase, or (most formally) acylcholine acylhydrolase, is produced in the liver and found primarily in plasma. Pseudocholinesterase levels may be reduced in patients with advanced liver disease

viii. Ornithine transcarbamylase (OTC) (also called Ornithine carbamoyl transferase) is an enzyme that catalyzes the reaction between carbamoyl phosphate (CP) and Ornithine to form Citrulline and Phosphate. In plants and microbes, OTC is involved in Arginine (Arg) biosynthesis, whereas in mammals it is located in the mitochondria and is part of the urea cycle. It is found in liver (>97%) and small intestine (<2%) and its activity increases in both acute and chronic liver disease.

Notably, when elevated ALT levels are found in the blood, the possible underlying causes can be further narrowed down by measuring other enzymes. For example, elevated ALT levels due to hepatocyte damage can be distinguished from bile duct problems by measuring alkaline phosphatase. In addition, myopathy-related elevations in ALT should be suspected when the aspartate transaminase (AST) is greater than ALT; the possibility of muscle disease causing elevations in liver tests can be further explored by measuring muscle enzymes, including Creatine Kinase. Many drugs may elevate ALT levels, including Zileuton, omega-3-acid ethyl esters (Lova za), anti-inflammatory drugs, antibiotics, cholesterol medications, some antipsychotics such as risperidone, and anticonvulsants. Paracetamol may also elevate ALT levels. (Wikipedia, 2015).

In fact, abnormal level (especially elevation) of these liver marker enzymes in the serum interprets liver injury.

1.5 MYRIANTHUS ARBOREUS, ORIGIN, CLASSIFICATION AND MEDICAL USES

Myrianthus arboreus is a common tree in the forest area of West and Central Africa, occurring in rain forest, semi-deciduous forest and swamp forest. (Okafor, 2004). It belongs to the family Cecropiaceae and it is a Dioecious shrub or tree up to (14–20) m tall. (P.beauv.).The indigenous wild plant is used for food and medicine.

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Fig.1.3a.The Tree of Myrianthus arboreus Fig.1.3b. The leaves of Myrianthus arboreus

Source: Camera Photograph. Source: Camera Photograph.

The sweet or acidulous pulp around the seed is edible and the young leaves are eaten as vegetable. The leaves, bark, leafy shoots and roots are used for medicine. In Rumuji, Emohua local Government area of Rivers state of Nigeria, the leaves are cooked, the steam from the pot is inhaled, and afterwards the hot aqueous extract is used for bathing and this offers a wide range pain (analgesic) relief. Ethno-botanically, it is called ‘Uzere’ in Ikwerre- Rumuji language in Rivers state of Nigeria.

A bark decoction is drunk to treat malaria, fever and cough. Extracts of the leaves or leafy shoots of Myrianthus arboreus are used in Sierra Leone, Nigeria and the Mount Cameroon area in preparations to treat dysentery, diarrhoea and vomiting.

In the Igala area of Nigeria, the leaves are an ingredient of a febrifuge given to young children. (Okafor, 2004).

Within the continents of African such as Nigeria and Congo, chopped leaves are eaten raw with salt for heart troubles, pregnancy complications, dysmenorrhea, incipient hernia and a plaster made of beaten leaf applied to boils. Sap from the leaves is applied topically for toothache, to the chest for bronchitis or as throat pain for sore throat.

In Tanzania, an infusion of the leaves is taken to improve lactation in women. (Okafor, 2004).

1.6 TAXONOMICAL CLASSIFICATION OF MYRIANTHUS ARBOREUS

Abbildung in dieser Leseprobe nicht enthalten

Data from source Arctos Plants"http://arctos.database.museum/includes/style.min.css"

1.7 COMPOSITIONS AND PHYTOCHEMISTRY OF MYRIANTHUS ARBOREUS

The composition of fresh fruit pulp of Myrianthus sp. per 100 g edible portion is: water 85.5 g, energy 205 kJ (49 kcal), protein 1.9 g, carbohydrate 11.8 g, Ca 44 mg, P 70 mg, Fe 1.1 mg.

The composition of dried seeds per 100 g is: water 13.5 g, energy 1972 kJ (471 kcal), protein 23.6 g, fat 33.4 g, carbohydrate 27.0 g, fibre 3.5 g, Ca 132 mg, P 371 mg, Fe 6.6 mg (Leung et al., 1986).

The oil consists almost exclusively of linoleic acid (93%). The protein is rich in the amino acid cysteine, which is important in a region where chronic deficiency of sulphur-bearing amino acids occurs. Several pentacyclic triterpenoids have been isolated from the wood and the roots. Euscaphic acid, myrianthic acid, tormentic acid, ursolic acid and a derivative of ursenoic acid have been isolated from stems. Myrianthinic acid was isolated from the bark. The wood also contains myrianthiphyllin, a lignan cinnamate. Bark extracts of Myrianthus arboreus showed antiplasmodial, antimycobacterial and antitrypanosomal. (Okafor, 2004). The methanol leaf extract of Myrianthus arboreus as well as the aqueous and ethanol exhibited antimicrobial and antioxidant activities.(Agyare et al.,2014).

The phytochemical screening done by (Oyeyemi et al., 2014) revealed that the leaves of Myrianthus arboreus has;

i. Flavonoids(45.62±0.07)
ii. Alkaloids(40.56±0.05)
iii. Saponins(0.00±0.00)
iv. Tannins(6.88±0.02)
v. Phenols(7.02±0.02)
vi. Glycosides(4.60±0.01)

Similarly, the proximate analysis done by (Oyeyemi et al., 2014) showed that the leaves of Myrianthus arboreus has;

i. Protein (4.20 ± 0.00%)
ii. Crude fat/Lipid (6.01 ± 0.10%)
iii. Crude fibre (4.71 ± 0.00%)
iv. Moisture content (35.20 ± 0.84%)
v. Ash content (3.52 ± 0.18%)
vi. Carbohydrate (7.20 ± 0.14%)
vii. Dry matter (8.10 ± 0.14%)
viii. Calcium (90.08 ± 0.60 mg/100g)
ix. Magnesium(2.68 ± 0.17 mg/100g)
x. Sodium (17.95 ± 0.07 mg/100g)
xi. Potassium (70.62±0.39 mg/100g)
xii. Iron (7.44±0. 15 mg/100g).

Despite the various uses over long time, no toxicological data is available regarding the safety of repeated exposure to Myrianthus arboreus. Considering the numerous nutritional and health benefits, it is therefore necessary to conduct a toxicological study on Myrianthus arboreus and ascertain data regarding its safe use.

1.8 AIM AND OBJECTIVES OF THE STUDY

AIM:

Aim is centred on the acute toxicity of ethanolic leaf extracts of Myrianthus arboreus (P.Beauv) on the liver enzymes of wistar rats

OBJECTIVES:

The objectives of this study are to;

i. Evaluate the LD50 of Myrianthus arboreus on wistar rats.
ii. Qualitatively determine the phytochemicals of the Myrianthus arboreus.
iii. Determine the activities of Alkaline Phosphatase (ALP), Alanine Transaminase (ALT) and Aspartate Aminotransaminase (AST) in the liver of wistar rats.

CHAPTER TWO

2.0 MATERIALS AND METHODS

2.1 MATERIALS

2.1.1 LABORATORY APPARATUS

Aluminium foil, Beakers, Cotton wool, Crucibles, Desiccator, Dissecting kits, Face mask, Filter paper(Whatman), Funnel, Gavage syringe, Glass rod, Hand gloves(plastic and elastic), Laboratory coat, Marceration Jar (5000ml), Microtome, Reagent bottles, Spatula, Sterile specimen tubes and Syringes.

2.1.2 LABORATORY EQUIPMENT

Analytic weighing Balance (HCK LN 0708)

Centrifuge (Universal 320 laboratory century Hettich Zentrifugen)

Digital water bath (TT-6 Techmel & Techmel USA)

Digital weighing balance (Satorius TE1535, CANADA)

Electronic Blender

Heating incubator (DHP-9053A)

Microscope (Photomax (LB) Premier universal microscope 1966 OLYMPUS TOKYO)

Microtome (American Optical Microtome 820)

Rotary evaporator (RE-52A: ENGLAND LAB SERVICES)

Spectrophotometer (Surgispec 5M-23D: SURGIFEILD MEDICAL, ENGLAND).

2.1.3 CHEMICAL AND REAGENTS

Absolute Ethanol (99.7%) (JHD), Chloroform, Concentrated Dimethyl Sulfoxide (DMSO), Einosine, Formalin (10%), Heparin blood tube, Paraffin, Tap water, Xylene.

2.1.4 SOURCE OF ANIMAL

Thirty five (35) Albino Rats animals were used for the study. The rats were purchased from the Animal house of Pharmacology Department of the Institution where the experiment was also carried out.

Animal feed and tap water within the animal house were used to feed the animals.

The animals were restrained in a Plastic cage matted with sawdust.

2.2 METHODS

2.2.1 COLLECTION AND IDENTIFICATION OF PLANT MATERIALS

The fresh leaves of Myrianthus arboreus were collected from Rumuji-odegu in Emohua Local Government Area, Rivers State, Nigeria in August 2015. Dr. Chimezie Ekeke, a botanist at the Department of Plant Science and Biotechnology, University of Port Harcourt, Rivers State, Nigeria identified and taxonomically authenticated the plant sample. A voucher-specimen of the collected sample with voucher-specimen number UPH/V/1,224 has been deposited in the herbarium of the institution for subsequent references. See Appendix 3 for herbarium number.

2.2.2 PREPARATION OF EXTRACT

The leaves of Myrianthus arboreus were rinsed in clean water, shade-dried for two (2) weeks, milled using electronic blender into coarse powder, weighed and extracted with 99.7% ethanol (JHD) by cold maceration method for 72hours with intermittent shaking.

The extract was prepared in a ratio of 1: 4 i.e. 1gram of the powder to 4ml of ethanol. 200g of the fine powder was soaked in 800ml of the solvent (absolute Ethanol) inside a 5000ml maceration jar at room temperature (28±20C), shook every 30mins for 6hours and allowed to stand for about 72hours. Using a sterile muslin cloth followed by a funnel-filter paper filtration, the solution was filtered into a sterile container. The marc was re-soaked twice in ethanol (with ethanol covering the marc) for 24hours in each case. The entire solution was concentrated and left to evaporate to dryness using a Rotary evaporator (45oC) at the Pharmacognosy and Phytotherapy laboratory, Faculty of Pharmaceutical Sciences of the institution. The extract was purified in a crucible on a water bath to dry (45oC) for 24hours. The dark green to black gel-like crude extract was preserved in an airtight sterile container and refrigerated until required. (A modified method described by Agwa, O.K. et al).

2.2.3 PHYTOCHEMICAL SCREENING

Phytochemical screening of Myrianthus arboreus leaf extract was done in the Pharmacognosy and Phytotherapy Department, Faculty of Pharmaceutical Sciences of the institution. This was done to identify the secondary metabolite using standard procedures.

The following parameters were determined in the crude extract of Myrianthus arboreus;

i. Alkaloids: Drangedorff’s test, Mayer’s test, Hager’s test.
ii. Flavonoids: Shinodu’s test, Leadacetaete test, AICB test, NaOH.
iii. Tannins: FeCB test, Phlobatannins, Gelatin test, Albumin test.
iv. Anthraquinone (BontragersTest): Free Anthraquinone, Combined Anthraquinone.
v. Triterpenoids/Steroids: Liebermann-Buchard test, Salwoski test.
vi. Fixed oils
vii. Carbonhydrates: Molisch test. Fehlings test.
viii. Cardenolide: Keller Killani test Kedde test
ix. Cyanogenic glycosides
x. Saponins: Frothing test, Haemolysis test, Emulsion test.

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