I SAFETY ASSESSMENT of the ETHANOLIC LEAF EXTRACT OF

SAFETY ASSESSMENT OF THE ETHANOLIC LEAF EXTRACT OF LAUNAEA

TARAXACIFOLIA (WILLD) OF THE FAMILY ASTERACEAE IN RODENTS

A THESIS SUBMITTED IN FULFILLMENT OF THE

REQUIREMENTS FOR THE DEGREE OF

MASTER OF PHILOSOPHY

In the

Department of Pharmacology

Faculty of Pharmacy and Pharmaceutical Sciences

LYDIA ENYONAM KUATSIENU

KWAME NKRUMAH UNIVERSITY OF SCIENCE & TECHNOLOGY

KUMASI

AUGUST, 2012

DECLARATION

The experimental work described in this thesis was carried out at the Department of

Pharmacology, KNUST. This work has not been submitted for any other degree.

…………………………..

Lydia Enyonam Kuatsienu

……………………………

Rev. Prof. Charles Ansah

......

Prof. Eric Woode

(Head Of Department)

DEDICATION

TO GOD ALMIGHTY, MY FAMILY AND MY MOTHER FOR THEIR ABSOLUTE

SUPPORT.

ABSTRACT

Launaea taraxacifolia is a very important herb used as medicine and / or food in the West

African Sub-region. This study therefore investigated its safety in rodents. Administration of the extract (10-5000 mg/kg) to mice and rats in acute and sub-acute toxicity studies did not have any negative effect on the physical, haematological and serum biochemical parameters assessed. However, serum creatinine and urea were significantly decreased in extract-treated animals compared to the vehicle treated control group indicating possible reno-protective ability. The liver was not affected by treatment with the extract. The reno-protective effect of the extract was investigated against gentamicin sulphate (GS)-induced nephrotoxicity (160 mg/kg, i.p). Plasma creatinine, urea, total protein, electrolyte and malondialdehyde (MDA) levels increased significantly as a result of gentamicin treatment but these increases were reversed by treatment with the extract. Photomicrographs of the kidney showed less damage in the extract-treated groups especially at higher doses indicating a possible protective effect of the extract on the kidney. In a pentobarbitone-induced sleeping time experiment the extract

(1000 and 2000 mg/kg) shortened sleep latency and increased the duration of sleeping time significantly in mice but not in rats. Similarly, treatment with the extract for 14 days shortened sleep latency and increased the duration of sleeping time at 2000 mg/kg significantly. The extract at 300 and 1000 mg/kg, p.o. given for 7 days did not cause any significant difference in the levels of hepatic cytochrome P450 enzymes but caused significant decrease in spontaneous locomotor activity in mice at higher doses. Treatment with the extract at (1, 2, 5 and 10 mg/kg,

iii

p.o.), showed a possible antidiarrhoeal property. In castor oil-induced defecation, enteropooling and intestinal transit, there were statistically significant decrease shown in defecation and small intestinal transit but not in enteropooling compared to the vehicle-treated control groups. The remarkable antidiarrhoeal effect showed in small intestinal transit and castor oil-induced defecation shows that it could be useful in the management of diarrhoea.

ACKNOWLEDGEMENT

My first and foremost gratitude goes to the Almighty God for sustaining me throughout these years. I exalt his holy name, praise him and glorify his existence forever and ever.

My gratitude goes to my supervisor Rev. Prof. Charles Ansah for his guidance and supervision in this work. My heartfelt gratitude also goes to Prof. Eric Woode for his helps, suggestions and inputs.

For the great success of this project I am highly indebted to many people. In this regard, I must acknowledge a number of them without whose support and encouragement this work would have been much more difficult than it really was.

My greatest appreciation goes to my family especially my husband Abraham Adri, my wonderful children Senyo Bulelani and Seli Maliaka Chris-Adri my parent especially my mother Stella Klutse, Rev. Fr. David Ahiahornu, my siblings and everybody for their unflinching support, love, prayers and advices.

I am very grateful to the laboratory technicians of the Department of Pharmacology especially

Mr. Thomas Ansah (aka uncle T). It is only with exceptional luck that one gets the opportunity to work with such rare individuals who are so dedicated to their work.

Many thanks to all my wonderful colleague, especially Donatus, Jemima, Valens, Mbobila and Kyei for all the laughs we shared in the course of our studies which in itself was enough motivation to carry on as a family.

TABLE OF CONTENT

DECLARATION...... I

DEDICATION...... II

ABSTRACT...... III

ACKNOWLEDGEMENT...... V

TABLE OF CONTENTS...... VI

LIST OF TABLES...... XI

LIST OF FIGURES...... XIII

ABBREVIATIONS...... XV

CHAPTER 1...... 1 1.0 GENERAL INTRODUCTION...... 1

1.1 OVERVIEW...... 1

1.2 THE PLANT Launaea taraxacifolia...... 4

1.2.1 Taxonomy...... 5

1.2.2 Geographical distribution...... 6

1.2.3 Traditional uses...... 6

1.3 IMPORTANCE OF TOXICITY STUDIES...... 7

1.3.1 Blood and target organ toxicity...... 8

1.3.2 Enzyme inhibition and induction in the liver...... 10

1.4 NEPHROTOXICITY...... 11

1.5 INVESTIGATION OF THE POTENTIAL EFFECT OF LAUNAEA ON THE GASTROINTESTINAL TRACT...... 14

1.6 JUSTIFICATION OF PROJECT...... 16

1.7 GENERAL AND SPECIFIC OBJECTIVES...... 19

1.7.1 General objective...... 19

1.7.2 Specific objectives of the study...... 19

CHAPTER 2...... 20

2.0 MATERIALS AND METHODS...... 20

2.1PLANT MATERIALS...... 20

2.2 ANIMALS USED FOR THE STUDY...... 21

2.3 CHEMICALS AND DRUGS USED...... 21

2.4 QUALITATIVE ANALYSIS OF PHYTOCHEMICALS...... 22

2.5 LD 50 AND ACUTE TOXICITY STUDIES IN RODENTS...... 24

2.6 SUB-ACUTE TOXICITY STUDIES IN MALE RATS...... 25

2.7 EFFECT OF EXTRACT ON PENTOBARBITONE–INDUCED SLEEPING TIME IN RODENTS...... 27

1.7.1 Pentobarbitone - induced sleeping time in male ICR mice...... 27

1.7.2 Pentobarbitone - induced sleeping time in male SD rats...... 28

2.8 EFFECT OF EXTRACT ON SPONTANEOUS LOCOMOTOR ACTIVITY...... 29

2.9 EFFECT OF EXTRACT ON TOTAL CYTOCHROME P450 ENZYME LEVELS....29

2.10 NEPHROTOXICITY STUDIES...... 33

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2.10.1 Effect of extract on gentamicin – induced nephrotoxicity...... 33

2.10.2 Malondialdehyde (MDA) measurement in the right kidney...... 34

2.10.3 Kidney photomicrograph...... 36

2.11 DIARRHOEA STUDIES...... 36

2.11.1 Effect of extract on gastrointestinal system using the charcoal meal...... 36

2.11.2 Effect of extract on castor oil – induced defecation in rats...... 38

2.11.3 Effect of extract on castor oil – induced enteropooling...... 39

2.11.4 Effect of extract on castor oil – induced small intestinal transit time in rats...... 39

2.12 STATISTICAL ANALYSIS...... 41

CHAPTER 3...... 42 3.0 RESULTS...... 42 3.1 ASSESSMENT ON PHYTOCHEMICAL CONSTITUENTS OF THE EXTRACT...42

3.2 LD 50 AND TOXICITY STUDIES IN RODENTS...... 43

3.3 SUB – ACUTE TOXICITY STUDIES IN MALE RATS...... 43

3.3.1 Weight of male SD rats before and after treatment...... 44

3.3.2 Effect of extract on organ to body ratio...... 45

3.3.3 Effect of extract on haematological parameters...... 46

3.3.4 Effect of extract on serum biochemical parameters...... 47

3.3.5 Photomicrograph of the liver cells...... 48

3.4 EFFECT OF EXTRACT ON PENTOBARBITONE–INDUCED SLEEPING TIME IN RODENTS...... 50

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3.4.1 Pentobarbitone – induced sleeping time in ICR mice...... 50

3.4.2 Pentobarbitone – induced sleeping time in Sprague–Dawley rats...... 51

3.5 EFFECT OF EXTRACT ON SPONTANEOUS LOCOMOTOR ACTIVITY IN ICR MICE...... 52

3.6 EFFECT OF EXTRACT ON TOTAL CYTOCHROME P450 ENZYME LEVELS IN RATS...... 53

3.7 EFFECT OF EXTRACT ON GENTAMICIN – INDUCED NEPHROTOXICITY IN SD RATS...... 54

3.7.1 Serum creatinine levels...... 54

3.7.2 Serum urea levels...... 55

3.7.3 Serum total protein levels...... 56

3.7.4 Serum electrolyte levels...... 57

3.7.5 Malondialdehyde (MDA) levels...... 58

3.7.6 Photomicrograph showing the effect of the extract on the left kidney of SD rats treated with gentamicin...... 59

3.8 EFFECT OF EXTRACT ON THE GASTROINTESTINAL TRACT MOTILITY IN RATS...... 61

3.8.1 Small intestinal transit time...... 61

3.8.2 Castor oil – induced defecation...... 62

3.8.3 Castor oil – induced enteropooling...... 63

3.8.4 Castor oil – induced small intestinal transit time...... 64

CHAPTER 4...... 65 4.0 DISCUSSION AND CONCLUSIONS...... 65

4.1 DISCUSSION...... 65

4.2 CONCLUSIONS...... 74

4.3 FUTURE WORK...... 75

REFERENCES...... 76

LIST OF TABLES

Table 1: Phytochemical constituents found in the extract...... …...... ……...... …42

Table 2: Effect of the extract on 24-hour treated mice and rats in acute toxicity studies...... 43

Table 3: Effect of the extract on organ to body ratio in rats treated for 14 days in sub-acute toxicity studies...... ………...... 45

Table 4: Effect of extract on haematological parameters in male rats during sub-acute toxicity studies...... ……46

Table 5: Effect of extract on serum biochemical parameters in male rats during sub-acute toxicity studies...... ……...... …...... ………...... …….…..47

Table 6: Photomicrograph report on the liver cells during the sub-acute toxicity studies for 14 days...... …………...... ……49

Table 7: Effect of extract on serum electrolyte levels in rats after 10 days treatment period in nephrotoxicity studies...... ………………...... ………...... ……………...... ….57

Table 8: Effect of extract on small intestinal transit time in SD rats in gastrointestinal tract motility experiment...... ………………………….....………………...... ……..61

Table 9: Effect of extract on castor oil - induced defecation in rats in gastrointestinal tract motility experiment...... …...... …...... …..62

Table 10: Effect of extract on Castor oil - induced enteropooling in rats in gastrointestinal tract motility test...... ……….....……...... …63

Table 11: Effect of extract on Castor oil - induced transit time in rats in gastrointestinal tract motility experiment...... ………….…...... 64

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LIST OF FIGURES

Figure 1: Launaea taraxacifolia plant……………...... ………………………...... 4

Figure 2: Weight of male SD rats before and after sub-acute toxicity studies…...... 44

Figure 3: Photomicrograph of SD rats liver cells during sub -acute toxicity studies...... 48

Figure 4A: Effect of extract on the onset of sleep in ICR mice in pentobarbitone - induced sleeping time...... ……...... 50

Figure 4B: Effect of extract on the duration of sleep in ICR mice in pentobarbitone-induced sleeping time...... 50

Figure 5A: Effect of extract on the onset of sleep in SD rats in pentobarbitone-induced sleeping time...... ….51

Figure 5B: Effect of extract on the duration of sleep in SD rats in pentobarbitone-induced sleeping time …...... ….....51

Figure 6: Effect of extract on spontaneous locomotor activity in ICR mice...... …....52

Figure 7: Effect of extract on total CYP450 enzyme levels in SD rats after 7 days treatment period...... …..53

Figure 8: Effect of extract on serum creatinine level after 10 days treatment period in SD rats in nephrotoxicity studies...... 54

xiii

Figure 9: Effect of extract on serum urea level after 10 days treatment period in SD rats in nephrotoxicity studies...... ………….....………...... 55

Figure 10: Effect of extract on serum total protein level after 10 days treatment period in SD rats in nephrotoxicity studies...... …………………………………...... …...... 56

Figure 11: Effect of extract on Malondialdehyde (MDA) level on the right kidney after 10 days treatment period in SD rats in nephrotoxicity studies...... ………...... 58

Figure 12 A & B: Photomicrograph showing the effect of the extract on the left kidney of SD rats treated with gentamicin for10 days in nephrotoxicity studies...…...... …...... ….59

xiv

ABBREVIATIONS

WHO World Health Organization

SD Sprague–Dawley

MDA malondialdehyde

ICR Inprint Control Region mice

CNS central nervous systerm

AKI acute kidney injury

GGT gamma glutamic transpeptidase

ALT alanine aminotransferase

ALP alkaline phosphatase

AST aspartate aminotransferase

T b total bilirubin

Db direct bilirubin

INb indirect bilirubin

RBC red blood cell

TWBC total white blood cells

HB haemoglobin

PCV packed cell volume

MCH mean cell haemoglobin

LYM lymphocytes

NEUT neutrophils

CO castor oil

DTT dithiothreitol

SEM standard error of mean

RBF renal blood flow

GS gentamicin sulphate

CYP cytochrome P450

ROS reactive oxygen species

GFR glomerular filtration rate

RDA recommended dietary allowance

EDTA ethylenediaminetetraacetic acid

EXTRACT ethanolic leaf extract of Launaea taraxacifolia

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CHAPTER 1

1.0 GENERAL INTRODUCTION

1.1 OVERVIEW

Recent studies have shown that between 70 % and 95 % of individuals in most developing countries use traditional medicine, including herbal medicines, for the management of diseases and to address their primary health-care needs (WHO, 2002-2005). In some developed nations, the use of traditional medication is equally significant. In Canada,

France, Germany and Italy for instance, it was reported that between 70 % and 90 % of the people use traditional medicines under the titles “complementary”, “alternative”, or

“nonconventional” medications (WHO, 2005; Traditional Medicine, 2008 ; Barnes et al.,

2007).

In America the use of complementary and integrative medicine approaches in health care delivery is on the increase. Forty-two percent of Americans were reported using alternative therapies in 1997; 40 % for treatment of chronic illness and 60 % for disease prevention. In

2007, the most commonly used complementary and alternative medicines therapy was non vitamins, non minerals and natural products making 17.7 % (Barnes et al., 2007). Despite this rapid growth in the use of herbal medicines, limited evidence exists for their effectiveness and toxicity. Much more needs to be done to develop the evidence base for herbals, botanicals and dietary supplements (Turner, 1965).

On the other hand natural products either as pure compounds, fractions or whole plant extracts have remained useful and unmatchable sources of molecules for effective treatment and mitigation of disease burdens of men and animals (Perry, 1980). It is very essential to assess natural products for their efficacy in the treatments of diseases. Many plants synthesize substances that are useful to the maintenance of health in humans and other animals. These include aromatic substances most of which are phenols, tannins and secondary metabolites of which at least 10 % have been isolated. In many cases, substances such as alkaloids serve as plant defence mechanisms against predators by microorganisms, insects and herbivores. Many of the herbs and spices used by human to season food yield useful medicinal compounds (Lai and Roy, 2004; Tapsell et al., 2006). Similar to prescribed drugs, a number of herbs are thought to cause adverse effects (Talalay and Talalay, 2001). Furthermore, adulteration, inappropriate formulation or lack of understanding of plants and mechanisms of drugs or interactions has led to adverse reactions that are sometimes life threatening (Elvin-Lewis, 2001).

The use of herbs to treat disease is almost universal among non-industrialized societies (Edgar et al., 2002). Many of the pharmaceuticals currently available to physicians have a long history of herbs as a dominant component over other natural resources because of the notion that they have no side effects (Ram et al., 1997; Veale et al., 1992). The use of such drugs and dietary supplement derived from plants has accelerated in recent years. Pharmacologists,

Microbiologists, Botanists and Natural-product chemists are researching into photochemical for leads that could be developed for treatment of various diseases. According to WHO, approximately 25 % of modern drugs used in the US have been derived from plant products. 2

Over the past century, approximately 121 pharmaceutical products have been discovered based on information derived from traditional healers (Anesini and Perez, 1993). In some

Asian and African countries, 80 % of the prescriptions are based on traditional medicine

(Traditional medicine, 2008). Research needs to be done on these herbals to determine the safety and efficacy of each plant before they are recommended for medical use (Vickers, 2007).

Although many consumers believe that herbal medicines are safe because they are „natural‟, herbal medicine and synthetic drugs may act together causing toxicity to the patient. It can also be dangerously contaminated and without established efficacy may unknowingly be used to replace medications that do have corroborated efficacy (Ernst, 2007).

The use of Launaea taraxacifolia as a remedy is scarcely documented. Though not much work has been done scientifically on the plant, claims from the public and herbalists suggested that the plant is very useful in the management of diabetes, as a mild laxative and for other health problems. To date there is limited data on the efficacy and safety of Launaea. This study seeks to investigate the possible safety of the leaves of the plant using rodent models.

1.2 THE PLANT (HERB) Launaea taraxacifolia (Willd)

Figure 1: Picture of Launaea taraxacifolia plant

Launaea taraxacifolia (Willd) (Figure 1) is an annual West Tropical Africa herb commonly known as wild lettuce. The plant is known in French as langue de vache which implies tongue of the cow. There are some common names of the plant; Ga; agblòke, Ewe (anlo); aŋòto, Twi; dadedru, Akan Akuapem; nne noa (boil today) Hausa; namijin dayii (applied loosely) Yoruba; efo yanrin, Sierra Leone-Kissi; bekuhoa-pomboe. The plant has leaves which are in basal rosette form of 3–5, pinnately lobed with ultimate margins dentate. The

stem is erect up to about 1–3 meters high from a woody rhizome which is solitary branched and borne with 25–30 floret flowers with yellow corollas in a convex receptacle at the apex slightly narrowed, which produces a white 7–8 mm long pappus air-borne seeds

(Burkill,1985).

1.2.1 Taxonomy

Domain: Eukaryota – Whittaker and Margulis, 1978 Eukaryote

Kingdom: Plantae – Haeckel, 1866 plants

Subkingdom: Viridaeplantae – Cavalier-Smith, 1981

Phylum: Tracheophytae – Sinnott, 1935 Ex Cavalier-Smith, 1998 Vascular plant

Subphylum: Euphyllophytina

Infraphylum: Radiatopses – Kenrick and Crane, 1997

Class: Magnoliiopsida – Brogniart, 1843 Dicotyledons

Subclass: Asteridae – Takhtajan, 1967

Superorder: Asteranae – Takhtajan, 1967

Order: Asterales – Lindley, 1833

Family; Asteraceae – Dumortier, 1822 Sunflower family

Genus: Launaea – Cassini in F. Cuvier,Dict. Sci. Nat,ed 2.25:60,321.1822

Specific epithet: taraxacifolia (Willd) Amin ex C. Jeffrey

Botanical name: Launaea taraxacifolia (Willd) Amin ex C. Jeffrey (GBIF data portal, 2007)

1.2.2 Geographical distribution

The plant is found in the Tropical West Africa, Mexico, West Indies, Central and South

America, Europe, North Africa, Atlantic Islands, South, West and Central Asia (Burkill,

1985). It grows in an open habitat and is considered as weed because it invades fields and farmlands.

1.2.3 Traditional uses

There are several traditional uses of the plant. The plant is used for food, medicine and social purposes. The leaves are eaten fresh as a salad or used in soups and sauces preparation. The plant is often grown for the purpose of harvesting the leaves which are sold in the markets as 6

uncooked or cooked because several studies have revealed that wild or semi-wild plants are nutritionally important due to high levels of vitamins, minerals, proteins, essential fatty acids and fibre contents (Adinortey et al., 2012). The leaves are given to cows-in-milk in Northern

Nigeria to increase their yield, and to rabbits, sheep and goats to induce multiple births. The leaves of L. taraxacifolia have been reported to have hypolipidaemic effect and the ability to treat water retention disorders (Wichtl, 1994; Adebisi, 2004). L. taraxacifolia leaf was estimated to have a total energy value per 100 g of 287.47 kcal/100 g (1202.78 kJ/100 g) of the dry sample. This low calorific value and high protein content (26.67 %) may be recommended to individuals suffering from overweight and obesity. The leaves mixed with fine ash are rubbed onto the sores of yaws by Ghanaians and the boiled leaves are applied to the head of a newly-born baby to promote proper fixing of the bones. It also features in a

Yoruba‟s invocation for someone to become well-known in the community (Burkill, 1985).

1.3 IMPORTANCE OF TOXICITY STUDIES

Worldwide, people are exposed to various forms of drugs to enhance their quality of life including natural products. An adverse effect on the individual as a result of an introduction of new product is very possible and significant. Institutional bodies have been established such as

Food and Drugs Board (FDB), Ghana Standard Board (GSB) and various research centres to regulate and establish the safety and efficacy of products for safe consumption. These

institutions normally use animal models to provide the prospective information on the safety and efficacy of those products.

Toxicity studies may be acute, sub-acute or chronic depending on the period of studies. Acute toxicity studies are for short periods of investigations on the effect of the drug or product to establish the safety and efficacy profile of the drug. It is studied by administering a single large dose as possible. The results are not only relevant in the determination of toxicity but used for planning sub-acute and chronic toxicity studies. Usually multiple dose studies are used in predicting the maximum tolerant levels for the species involved during potential lifetime exposure. The results of chronic toxicity studies with acute and sub-acute toxicity studies help in the evaluation and the establishment of any possible toxic effect of the product.

1.3.1 Blood and Target Organ Toxicity

 Toxicity to Blood

During sub acute toxicity studies, the blood as the transport system of the body is assessed. The blood components such as white blood cells, red blood cells, platelets are the first to be exposed to toxins. Blood assessment is very important (Olson et al., 1984) since its parameters carry higher prognostic values for toxicity in humans. Contemporary drugs have been implicated in various forms of blood toxicity (Synder et al., 1977; Yunis et al., 1980). From simple to sophisticated analyzers are used to detect and analyze blood components for any possible destruction of its structure and function. Some components detected and analyzed 8

include total white blood cells (TWBC), red blood cells (RBC), haemoglobin (Hb), platelets and lymphocytes.

 Role of CYP450 in liver toxicity

Cytochrome P450 (CYP) isoenzymes are a group of heme-containing enzymes located in the lipid bilayer of the endoplasmic reticulum of hepatocytes, (Guengerich, 1992). They mediate a wide range of oxidative reactions, including xenobiotic biotransformation and endogenous substrate synthesis. Their expression and action are influenced by numerous agents such as genetic, endogenous, environmental, or pathophysiological factors (Denis et al., 2005). The effect of drugs on CYP activity is important to avoid drug–drug interactions (Park et al.,

1995). One of the most distinguished features of this enzyme system is its selective induction

(i.e., induced de novo biosynthesis) of definite CYP isoforms in animal or human cells following exposure to certain chemicals such as phenobarbital, tetrachlorodibenzo-p-dioxin

(TCDD), or polycyclic aromatic hydrocarbons (Waxman and Azaroff, 1992; Conney, 1967)

An area of concern in clinical pharmacology is possible drug-drug interactions, resulting from the co-administration of more than two drugs leading to therapeutic inappropriateness as well as toxic effects. Inhibition of CYP enzymes by drugs and exogenous compounds and chemicals can lead to the inhibition of drug metabolism through many mechanisms such as the destruction of previously existing enzymes. Cimetidine, for example inhibits CYP

enzymes and can lead to increase in the plasma levels of drugs metabolized exclusively by the

CYP enzymes.

1.3.2 Enzyme inhibition and induction in the liver

Inhibition is the reduction of enzyme activities as a result of direct interaction with a drug.

This development usually starts on the first administration of the inhibitors, the period of inhibition depending on the half-lives of the drugs implicated (Markowitz et al., 1995). There are three basic types of enzyme inhibition (competitive, non-competitive and uncompetitive), and their clinical effects are predisposed by these basic mechanisms (Bossche et al., 1995;

Murray, 1997). The most typical example of the competitive inhibition occurs with terfenadine and erythromycin. This interaction is known to involve a nitro compound, namely a metabolite demethylated by P450 which forms a complex with P450. Since the metabolism of macrolides example erythromycin are catalysed by CYP3A, which selectively formed stable enzyme-substrate complex with CYP3A (Nahata, 1996).

In induction there is an increase in the synthesis of CYP450 enzyme activities which occurs as a result of exposure to drugs. It can occur when a drug stimulates the biotransformation of co- administered drugs either through the same enzyme pathway or through an alternative pathway (Meyer and Rodvold, 1996). The effects of induction can be seen within the first two

days of therapy, but it usually takes more than a week for new enzymes to be synthesized and the maximal effect to occur.

Phenobarbitone is one of the cytochrome P450 enzyme inducers which are well documented and its drug-drug interaction has been assessed in patients treated with phenobarbitone and warfarin. The time course of enzyme production and degradation of induction processes is marked by the beginning and end of the plasma concentration of the inducer, as well as their half-life (Dossing et al., 1983).

1.4 NEPHROTOXICITY

Nephrotoxicity can be defined as renal dysfunction that arises as a direct result of contact to external agents such as drugs and environmental chemicals. Many therapeutic agents have been shown to induce clinically significant nephrotoxicity. Gentamicin sulphate is one of the most effective aminoglycosides which causes nephrotoxicity (Ajami et al., 2010). The goal of reducing or preventing the development of gentamicin sulphate - induced nephrotoxicity has attracted considerable efforts (Sayed-Ahmed, 2007).

Acute kidney injury (AKI) is a very difficult problem in Africa because of the burden of diseases, human immunodeficiency virus [HIV]-related AKI in sub-Saharan Africa, diarrhoea, malaria and nephrotoxicity. The late management of patients at health care facilities and the lack of or inadequate resources to sustain patients with known AKI in many countries (El Matri et al., 2008). The pattern of AKI in the sub-Sahara region is very much different from that in 11

more developed countries. There are no reliable statistical data about the prevalence of AKI in

Africa (Naicker et al., 2008). Acute kidney injury (AKI), as it is now referred to in the literature, is defined as a sudden or rapid turn down in renal filtration function. This situation is usually noticeable by a significant rise in serum creatinine concentration or by a rise in blood urea nitrogen concentration (Schrier et al., 2004). The driving force for glomerular filtration is the pressure gradient generated from the glomerulus to the Bowman space. Glomerular pressure is primarily reliant on renal blood flow (RBF) and is controlled by combined resistances of renal afferent and efferent arterioles. In spite of the cause of acute kidney injury

(AKI), reductions in RBF signify a common pathologic pathway for decreasing GFR. The aetiology of AKI consists of three main mechanisms.

 Pre-renal failure - Defined by conditions with normal tubular and glomerular function;

GFR is depressed by compromised renal perfusion

 Intrinsic renal failure - Includes diseases of the kidney itself, predominantly affecting

the glomerulus or tubule, which are associated with release of renal afferent

vasoconstrictors; ischemic renal injury is the most common cause of intrinsic renal

failure.

 Post-obstructive renal failure - Initially causes an increase in tubular pressure,

decreasing the filtration driving force; this pressure gradient equalizes and maintenance

of the depressed GFR.

Some studies have confirmed that various agents, including lycopene (Karahan et al., 2005),

Spirulina platensis (Karadeniz et al., 2008) and fish oil (Priyamvada et al., 2008) can prevent gentamicin sulphate - induced renal damage. However, some plant products with high therapeutic effect may be harmful to the kidney. These include hydroalcoholic extract of the stem bark of Alstonia boonei, which could be potentially nephrotoxic, especially when the dose is high and the duration of use is extended (Oze et al., 2007), Aristolochia species containing aritolochic acid constituents have been implicated in nephrotoxic issues (EA, 2000), fruits of

Pithecellobium labatum, roots of Callilepis laureola, fruit of Morinda citrifolia and (Averrhoa carambola) star fruit (Combest et al., 2005). In view of this Launaea taraxacifolia leaf is being investigated on the effect of gentamicin - induced renal dysfunction. Numerous individual studies and meta-analyses have shown that the prevalence of aminoglycoside nephrotoxicity is quite inconsistent with a reported range from 0 to 50% (Galloe et al., 1995; Rybak et al., 1999;

Baciewicz et al., 2003). There are quite a number of explanations for this marked inconsistency in occurrence. In the first place, the various studies differed in the parameters used to characterize nephrotoxicity. Some studies used increases and percentage increases of serum creatinine as the threshold for nephrotoxicity. Further, not all of the studies used the same aminoglycosides or treated similar client populations. This is significant since aminoglycosides differ in their nephrotoxic activity. Smith et al., (1980) noted renal impairment in 26 % of patients who received gentamicin, but only 12 % of patients who received tobramycin. Several lines of evidence have suggested that reactive oxygen species are implicated in the pathogenesis of agent-induced renal tubular injury, both in vivo and in vitro (Hye et al., 2007). 13

ROS are formed during a variety of biochemical reactions and cellular functions. Most ROS come from the endogenous sources as by-products of normal and essential metabolic reactions, such as energy generation from mitochondria or the detoxification reactions involving the cytochrome P-450 enzyme system (Halliwell and Gutteridge, 1989). The effects of aminoglycosides on mitochondrial (Weinberg et al., 1980) and lysosomal function (Cojocel et al., 1983) appear to play the main role in aminoglycoside – induced nephrotoxicity.

1.5 INVESTIGATION OF THE POTENTIAL EFFECT OF LAUNAEA ON THE

GASTROINTESTINAL TRACT

Antidiarrhoea1 drugs decrease the symptoms of diarrhoea (loose stool consistency, frequency of defecation and excessive stool weight) by effects on intestinal transit, mucosal transport or luminal contents. Opiates have major effects on intestinal transit, pro-absorptive and antisecretory effects are barely documented (Schiller, 2007). Also there are drugs or dietary fibre-forming agents that relieve the symptoms of diarrhoea. The most effective antidiarrhoeal drugs are opioid derivatives, which slow intestinal motility to permit greater time for the absorption of water and electrolytes (Li et al., 2008). Dietary fibre-forming agents improve stool consistency but may not decrease fluid and electrolyte loss. There are two forms of antidiarrhoeal medicines. One works by slowing down the gut movement, which reduces the speed at which the contents pass through the gut. Bulk-forming agents result in firmer stools

that are passed less often and are used by people with irritable bowel syndrome (Ford et al.,

2008).

Adverse effects of antidiarrhoeal medicines are constipation and dizziness. Occasionally they cause abdominal cramps, bloating, skin rashes and itching. Some children have very severe side-effects after they have taken these medicines. Example, necrotising enterocolitis, delirium, depressed breathing, coma and even death which is very rare (Koo et al., 2009). It may be used in patients with mild to moderate acute diarrhoea. However, these agents should not be used in patients with bloody diarrhoea, high fever or systemic toxicity because of the high risk of deteriorating the underlying conditions. Patients whose diarrhoea is worsening in spite of treatment should not use them (Camilleri, 2004; Baker, 2007; Bertram et al., 2009).

Diarrhoea disease is the second principal cause of death in children under five years old and can last a number of days, leaving the body devoid of water and salts that are very essential for survival (WHO, 2009). There are three clinical types of diarrhoea;

 Acute watery diarrhoea – lasts several hours or days, and includes cholera;

 Acute bloody diarrhoea – also called dysentery; and

 Persistent diarrhoea – lasts 14 days or longer.

In developing countries, almost all the population depends on the use of herbs for their health care needs. Diarrhoea a death threatening disease has long been known as one of the most important gastrointestinal health problems in developing countries. It is a major cause of childhood morbidity and mortality in socio-economically developing countries. In Ghana, the 15

diarrhoea incidence obtained from hospitals represent only a small proportion of all illnesses because most of the cases do not seek medical attention or utilize community pharmacy service but may be drawn to the use of common herbs and other belief practices (Osam-Tewiah, 2010).

Diarrhoea is a marker of infection by water or food – borne parasites, resulting in dehydration and electrolyte imbalances. More than one billion episodes of diarrhoea occur every year among children under five years of age causing approximately 2.5 million deaths, however, the

WHO Child Health Epidemiology (Kosek et al., 2003; Inácio et al., 2007) group estimates that

16 % of deaths in African children younger than five years are directly attributable to diarrhoea diseases (Reither et al., 2007). Diarrhoea killed about 1.3 million children aged 1–59 months with 1 % happening in newborns in 2008 (Black et al., 2010). Contaminated water causing 90

% of diarrhoea cases revealed that children are more susceptible than adults to the effects of diarrhoea because their immune systems are less able to respond to these infections (UN,

2010). Secretory diarrhoea which is the most dangerous gastrointestinal problem which is associated with too much stool outputs with unusually loose uniformity. Each year there are alarming reports of about 2 billion cases of acute diarrhoea disease worldwide (WHO, 2009). It could be managed by giving the affected individual exclusive breastfeeding, complementary feeding, safe water, good sanitation and hygiene, zinc tablets and vitamin A supplementation, oral rehydrated salt (ORS) which is a solution of clean water, sugar and salt, and antibiotics for dysentery (Jones et al., 2003). In different parts of the world, several studies have been done to evaluate effectiveness of some traditional medicines in treating diarrhoea which is the number one cause of mortality especially in children (Havagiray et al., 2004). 16

Dokosi (1998) states Launaea taraxacifolia plant possessed a mild laxative property which could pose a threat or make it unsafe for consumption. As part of the safety assessment therefore, this study aimed at investigating the diarrhoea activity of the ethanol extract from the leaves of Launaea taraxacifolia in rat models of diarrhoea.

1.6 JUSTIFICATION OF PROJECT

The global demand for medicinal plants is increasing (Yadav et al., 2010). In developing countries, adaptation of traditional medicine as a complementary to orthodox medicine is fast advancing (Salahdeen and Yemitan, 2006) however, these traditional medicines usually lack insufficient proof for their efficacy or safety (WHO, 2000; Patwardhan, 2005; Mosihuzzaman and Choudhary, 2008).

Plant extracts and phytochemicals are of great importance in therapeutic treatments

(Varalakshmi et al., 2011). Safety assessments on these plants for medicines are undisputable since plants have several phytochemicals which may cause toxicity on their own or by interacting with other chemicals (Teixera et al., 2003).

Some examples of toxic phytochemicals or plants include chaya leaves (Cnidoscolous acontifolius) in which when uncooked are toxic as they contain cyanogenic glycosides that can release toxic cyanide (HCN) upon tissue damage (Adebiyi et al., 2012). Anthraquinones can lead to electrolyte imbalance especially hypokalemia (Westendorf, 1993). It was found out that

an increased risk of lung cancer occurred in those taking combined beta-carotene and vitamin E supplements (Goodman et al., 2004). Plants of the genus Euphorbia produces caustic lattices, which constitute a health problem to humans and livestocks (Adedapo et al., 2004).

Launaea taraxacifolia is a plant employed as a herbal medicine for the treatment of skin and eye diseases–conjunctivitis, measles, diabetes mellitus and also rubbed on the limbs of toddlers to facilitate walking (Ayensu, 1978; Adebisi, 2004; Obi et al., 2006), abdominal disorders, heartburn, dyslipidaemia, liver diseases and also as food (Adinortey, 2012). However, there is little evidence to support these uses and to ascertain its safety. The traditional use of the plant as a mild laxative by (Dokosi, 1998) needs to be investigated, since it could present as a side effect in the use of the plant. Also as a traditional claim for treatment of diabetes, the plant could be exerting its effect on some target organs like the pancreas or the kidney. While there is no record of adverse effect with the use of the plant, the possibility of toxicity cannot be ruled out. The World Health Organisation encourages studies into agents for the treatment, management and prevention of diseases especially diarrhoea depending on traditional medical practices (Atta and Mouneir, 2004). Here, the plant Launaea taraxacifolia is evaluated for safety or toxicity. It is expected that the findings from this work may add to the overall value of the medicinal potential of the plants especially Launaea taraxacifolia.

1.7 GENERAL AND SPECIFIC OBJECTIVES

1.7.1. General Objective

The objective of the present study is to assess the effect of Launaea in rodent models for safety

1.7.2 Specific objectives of the study in vivo include;

 Determination of the acute effects of the extract in rodents.

 Determination of the safety of the extract by:

 Determining the organ–body ratio of liver, kidney, heart, spleen, testes and

gastrointestinal tract.

 Assessing changes in haematological parameters following the administration of the

extract.

 Studying changes in serum biochemical markers

 Examining the histoarchitecture of the liver in male Sprague-Dawley rats after

administration of the extract.

 Determination of the effect of the extract on pentobarbitone - induced sleeping time in

ICR mice and Sprague–Dawley rats.

 Determination of the effect of the extract on spontaneous motor activity in ICR mice.

 Determination of the effect of the extract on Cytochrome P450 enzymes in rats.

 Investigating the effect of extract on gentamicin sulphate-induced nephrotoxicity in

Sprague-Dawley rats.

CHAPTER 2

2.0 MATERIALS AND METHODS

2.1 PLANT MATERIAL

Fresh tender leaves of Launaea taraxacifolia were collected from the KNUST

Pharmacognosy garden in the month of November 2010 at 2:30 GMT. The samples were authenticated at the Department of Pharmacognosy, Faculty of Pharmacy and

Pharmaceutical Sciences KNUST Kumasi Ghana, with a voucher herbarium specimen number KNUST/HM1/2012/L060 which was also preserved in the same department.

The harvested leaves were washed, air dried, ground using laboratory blender and weighed.

Ethanol was used to extract the sample by adding 5 litres to 475 g of the powdered leaves for 72 hours. The supernatant was filtered and evaporated using the rota evaporator. The sample was then oven dried at 50 ºC and the yield obtained was 34.5 % w/w. It was then kept in a refrigerator.

The extract was prepared for dosing by dissolving it in freshly prepared 2 % w/v tragacanth which was used as vehicle for the control groups throughout the acute and sub-acute toxicity studies. The doses of the extract were given to the animals orally by gavage. The concentrations were prepared such that no animal received more than 1.0 ml/kg body weight.

2.2 ANIMALS USED FOR THE STUDIES

Sprague-Dawley rats (100 - 300 g) and ICR mice (15 - 30 g) of either sex were used for the experiments. The animals were either obtained from the Animal house of the Department of Pharmacology, KNUST or purchased from the Human Biology Department, University of Cape Coast. Animals were quarantined and observed for 14 days before an experiment.

All animals were kept in aluminium laboratory cages of dimension (34 x 47 x 18) cm with fine wood shavings as bedding. The animals were given the standard diet which is the normal commercial pellet feed from Tema GHAFCO and fresh water ad libitum.

2.3 CHEMICALS AND DRUGS USED

Tragacanth, normal saline, ethanol, EDTA tubes, serum biochemical tubes, activated charcoal and pentobarbitone sodium were purchased from BDH chemicals Ltd Poole

England. Castor oil from Bell sons and Co Ltd, South Port, England. Atropine sulphate purchased from Sigma chemical Co, St Louis MO, USA. Diazepam, ketoconazole (Ernest chemist Ltd Ghana), gentamicin sulphate (Roche Pharmaceutical Ltd, China) and gum acacia.

2.4 QUALITATIVE ANALYSIS OF PHYTOCHEMICALS

Phytochemicals are bioactive constituents of medicinal plants which are not nutrients but very useful to the plants. Some bioactive constituents analysed from the crude extract qualitatively included alkaloids, glycosides, saponins, tannins, flavonoids, steroids and tritepenoids. The methods used were as described by Sofowora, (1982).

Test for alkaloids

The plant extract (0.5 g) was added to 5 ml of 1 % w/v dilute sulphuric acid on a steam bath. The solution was filtered, and the filtrate was treated with a few drops of

Dragendorff‟s reagent.

Test for saponins

Distilled water 5 ml was added to 0.5 g of the extract and shaken vigorously and the content warmed over water bath.

Test for tannins

To 0.5 g of extract was dissolved in 10 ml of distilled water. The mixture was filtered, and the filtrate was treated with 1 % ferric chloride.

Test for steroids

The plant extract of 0.5 g was dissolved in 2 ml chloroform and filtered. To about 2 ml of the filtrate, acetic acid anhydride (2 ml) was added. Two millilitres of concentrated H2SO4 was also added.

Test for terpenoids

The extract 0.5 g was mixed with 2 ml of chloroform and filtered. To the filtrate three drops of concentrated H2SO4 was then carefully added to form a thin layer.

Test for flavonoids

About 2 ml dilute ammonia solution was added to 0.5 g of the extract and then concentrated H2SO4.

Test for glycosides

About 2 ml dilute H2SO4 solution was added to 0.5 g of the extract and filtered. To the filtrate 20 % of potassium hydroxide was added to de-acidify the filtrate which was confirmed by turning red litmus paper blue. Fehling‟s solution A and B was added and heated over a warmed water bath.

2.5 LD 50 AND ACUTE TOXICITY STUDIES IN RODENTS

The LD 50 is the dose of a toxic agent that is sufficient to kill 50 percent of a population of animals usually within 24 hour period. Acute toxicity studies usually show the first line of defence against potentially harmful substances such as drugs and chemicals. Acute toxicity bioassay was conducted according to the World Health Organization guidelines for the evaluation of safety and efficacy of herbal medicines (WHO, 1993).

LD50 and acute toxicity in male and female mice

Thirty ICR male mice (25 – 30 g) were grouped into six (n =5). The animals were allowed to acclimatise to their new environment for one week with adequate fresh water and feed.

The animals were then grouped as follows;

 Group 1 served as the control, received 2 % w/v tragacanth, the vehicle.

 Group 2 received 10 mg/kg extract

 Group 3 received 100 mg/kg extract

 Group 4 received 300 mg/kg extract

 Group 5 received 1000 mg/kg extract

 Group 6 received 5000 mg/kg were given extract orally by gavage.

After treatment, the animals were observed every 30 minutes over a period 24 hours for general behavioural, physiological, pharmacological changes and lethality. The changes

monitored included vomiting, excitement, salivating, sedation, diarrhoea, eating and drinking and death. The animals were observed for 14 days for possible latent toxicity of the extract.

LD 50 and acute toxicity in male rats

Thirty male rats (250 – 300 g) were grouped into six (n =5). The animals were left to acclimatise to their new environment for one week with adequate fresh water and feed.

Group 1 served as the control and received 2 % w/v tragacanth as vehicle. Groups 2 - 6 received (10, 100, 300, 1000 and 5000 mg/kg) of extract orally by gavage. After treatment, the animals were observed every 30 minutes for 24 hours for general behavioural, physiological, pharmacological changes and lethality. The changes monitored included aggressiveness, respiratory movements, vomiting, excitement, salivating, sedating, diarrhoea, eating, drinking and death. The animals were observed for 14 days for possible latent toxicity of the extract.

2.6 SUB-ACUTE TOXICITY STUDIES IN MALE RATS

Twenty-five male rats (250-300 g) were grouped into five (n =5). The animals were allowed to acclimatise to their new environment for one week with adequate fresh water and feed. Group 1 served as the control and received 2 % w/v tragacanth as vehicle. Groups

2 – 5 received (10, 100, 300, 1000 mg/kg) of extract orally by gavage. The treatments were repeated to the animals daily at 10.00 am and the animals were observed for 14 days for any general behavioural, physiological, pharmacological changes and lethality. The changes monitored included mobility, aggressiveness, sensitivity to sound and pain, as well as respiratory movements, vomiting, excitement, salivating, sedation, diarrhoea, eating, drinking and death. The animals were starved on day 15 but were given access to water.

Their weights were determined and recorded. Blood samples were then taken for haematological and biochemical analysis. Selected target organs, liver, kidney, heart, testes and spleen were excised, weighed and preserved for histopathology analysis.

Haematological analysis of SD rats during the sub-acute toxicity studies

Blood samples were collected by cutting the throat with surgical knife and scissors into sterile haematological tubes containing EDTA and sample tubes containing anticoagulant and analysed using Flexor junior auto analyser.

Organ Ratios assessment in rats during the sub-acute toxicity studies

These organs were excised weighed, the liver, kidney, heart, testes and the spleen by cutting the abdominal cavity of the rats. Relative organ/body weight ratios were calculated.

Histopathological Analysis of SD rats during the sub-acute studies

Livers of control and treated animals were collected for histopathological examination. The tissue samples were fixed in 10 % neutral buffered formalin, embedded in paraffin wax, cut into 3 µm sections and stained with haematoxylin and eosin. The tissues were examined under Nikon Eclipse E 200 light microscope of X 10 magnification and images were graded on a scale of normal limit, moderate limit and severe limit of injury.

2.7 EFFECT OF EXTRACT ON PENTOBARBITONE-INDUCED SLEEPING TIME IN

RODENTS

2.7.1 Pentobarbitone – induced sleeping time in male mice

Thirty-six ICR mice (15 – 20 g) were divided into six groups (n =6) and treated with single dose of the extract as follows;

 Group 1 – normal saline (0.5 ml, p.o.),

 Group 2 – (0.5 mg/kg, i.p.) diazepam, and

 Group 3 – 10 mg/kg extract (p.o.)

 Group 4 – 100 mg/kg extract (p.o.)

 Group 5 – 300 mg/kg extract (p.o.)

 Group 6 – 1000 mg/kg extract (p.o.).

After 60 minutes, the groups were given pentobarbitone sodium (40 mg/kg, i.p.). The onset of sleep was the time of loss of righting reflex and duration of sleep was the time interval between loss and regain of righting reflex.

2.7.2 Pentobarbitone – Induced Sleeping Time in male SD Rats

Male Sprague-Dawley (SD) rats (100–125 g) were grouped into six (n =5). The groups received a single dose of treatment as follows;

 Group I – (1 ml/100 g, p.o.) normal saline.

 Group II – 100 mg/kg extract (p.o.)

 Group III – 300 mg/kg extract (p.o.)

 Group IV – 1000 mg/kg extract (p.o.)

 Group V – 2000 mg/kg extract (p.o.).

After 60 minutes, the groups were given pentobarbitone (50 mg/kg, i.p.) the onset and duration of sleep were noted and recorded in seconds.

In another experiment, SD rats (125 – 130 g) were grouped into six (n = 5) and treated as above except that the animals were treated daily for 14 days before the pentobarbitone challenge.

2.8 EFFECT OF EXTRACT ON SPONTANEOUS LOCOMOTOR ACTIVITY

Spontaneous locomotor activity was performed using Activity Cage 7401, Ugo Basile

(Milan–Italy). Male mice of (25–30 g) were grouped into six (n =5). Group one received

0.5 ml normal saline, group 2 received diazepam (0.5 mg/kg, i.p.), groups 3, 4, 5, 6 received extract (p.o.) at 10, 100, 300 and 1000 mg/kg respectively. Activity was automatically recorded 30 minutes after treatment. The activity was recorded at an interval of 5 minutes, for a total of 50 minutes. Results of the treated groups were compared with those of control group at each time interval (Amos et al., 2001).

2.9 EFFECT OF EXTRACT ON TOTAL CYTOCHROME P450 ENZYME LEVELS

Sprague–Dawley rats (95–115 g) were grouped into five (n =5). The animals were treated as follows;

 Group I – distilled water (2 ml / 100) g body weight.

 Group II – 100 mg/kg body weight of ketoconazole (p.o.).

 Group III – 80 mg/kg pentobarbitone (p.o.).

 Group IV – 300 mg/kg extract (p.o.).

 Group V – 1000 mg/kg extract (p.o.).

Treatments were repeated at the same concentrations daily for 7 days. 29

Preparation of Liver Homogenates

After the seven days treatment period, the rats were sacrificed with a sharp surgical knife and scissors. The excised livers were quickly placed in an ice- cold 0.25 M sucrose solution in order to rinse excess blood and cool the tissue. The liver was then blotted dry using filter papers. It was weighed and placed in 0.25 M sucrose solution, containing 60 ml/L of 0.2 M dithiothreitol (DTT), which was four times the weight of the liver, so that a concentrated homogenate can be obtained. The liver was chopped with scissors and homogenized with

Ultra Trarax E20 homogenizer. The homogenate was centrifuged at 4000 g for 30 minutes in a refrigerated centrifuge at 4 ºC for the preparation of liver homogenate

Preparation of post mitochondrial supernatant

The post mitochondrial supernatant was prepared by centrifuging the homogenate in a refrigerated centrifuge at 4000 g for 30 minutes using Mikro 220R Hettich Zentrifugen to pellet intact cells. The resultant supernatant (the post-mitochondrial supernatant) was carefully decanted. This contains mitochondrial supernatant.

Preparation of subcellular tissue fractions

Aliquots of post-mitochondrial supernatant were mixed were added 1.0 ml of 88 mM CaCl2 per ml of the supernatant and was placed on ice for 5 minutes, with occasional gentle swirling. The mixture was centrifuged at 6000 g for 15 minutes, the supernatant was

discarded and the microsomal pellet was resuspended in 5 ml of 0.1M phosphate buffer at pH 7.4 and homogenized to yield the microsomal suspension.

Protein determination by Biuret method

The protein concentration was estimated by using the biuret method. The biuret method

(Gornall et al., 1949) relies on the principles of complexation of Cu2+ by the functional groups involved in the peptide bond. A minimum of two peptide bonds is needed for the complexation to occur. Upon complexation, a violet colour is observed. Bovine Serum

Albumin (BSA) was used as the standard protein to obtain a calibration plot.

Biuret reagent was prepared to a final volume of 1000 ml in 9.0 gm sodium potassium tartrate, 3.0 gm copper sulphate x 5 H2O, 5.0 gm potassium iodide, all dissolved in order in

400 ml 0.2 M NaOH before bringing to final volume.

The standard protein was prepared from 100 µg/ml bovine serum albumin stock. Then 0.0–

1.0 ml of the stock solution were pipetted into different test tubes and made up to a final volume of 1 ml with the normal saline. Biuret reagent (4.0 ml) was added to each sample including the blank (1.0 ml normal saline). The solutions were mixed vigorously by vortexing for 5 minutes and incubated at room temperature for 30 minutes. The spectrophotometer Celcil CE 2041was switched on for 15 min prior to its reading. The absorbance of each of the solutions was read at 540 nm after zeroing it with the blank. The

values obtained were used to plot a standard plot of absorbance against concentration of bovine serum albumin per assay.

The tissue sample (0 - 0.9 ml) was diluted with the normal saline in a test tube to a final volume of 1.0 ml. Normal saline (1.0 ml) was used as the blank. Biuret reagent (4.0 ml) was added to each sample and mixed thoroughly by vortexing for 5 minutes and incubated at room temperature for 30 minutes. The absorbance was read at 540 nm with the same spectrophotometer, after zeroing the instrument with the blank. The tissue protein content was determinated by direct interpolation from the standard plot of the bovine serum albumin.

Cytochrome P450 determination

Microsomes of 2.0 mg/ml were diluted in 0.05 M Tris HCl buffer, pH7.4.0 to 5.0 ml.

About 4.0 ml of the Tris HCl buffer without the samples was used to set the baseline recorded between 400 and 500 nm. A few grains of sodium dithionite were then added to both sample and reference cuvettes with gentle stirring with plastic stirrer for 1 minute and carbon monoxide (produced by mixing 20 ml concentrated sulphuric acid and 40 ml formic acid in a conical flask plugged with rubber with bent glass tube) was then bubbled through the sample cuvettes for 30 seconds. The samples were measured at 450 nm and 500 nm and cytochrome P450 content was calculated using the Beer‟s Law with cuvette path length of

1.0 cm.

Cytochrome P450 (nmol/ml diluted sample) = Absorbance difference (nm) X 1000 Extinction coefficient Mm-1 cm-1

The specific content of cytochrome P450 in the original tissue sample was then calculated knowing the dilution factor used and the percentage protein content of the original sample as follows:

Cytochrome P450 (nmol/mg protein)

= Cytochrome P450 (nmol/ml diluted sample) X dilution factor X 25 % microsome

2.10 NEPHROTOXICITY STUDIES

2.10.1 Effect of extract on Gentamicin-induced nephrotoxicity

Adult male Sprague–Dawley rats weighing (150–200 g) were used. Animals were divided randomly into eight groups, (n =5). The animals were treated as follows;

 Group I was given normal saline (0.5 ml/100 g) for 10 days.

 Group II was given saline from day1 to day10 and gentamicin sulphate 160 mg/kg/d

(i.p.) from days 6 to 10.

 Group III was given extract of 10 mg/kg/d (p.o.) for 10 days

 Group IV was given extract of 100 mg/kg/d (p.o.) for 10 days

 Group V was given extract of 300 mg/kg/d (p.o.) for 10 days.

 Group VI was given extract 10 mg/kg/d orally for 10 days and 160 mg/kg/d

gentamicin sulphate (GS) (i.p.) from day the 6 to 10.

 Group VII was given extract 100 mg/kg/d orally for 10 days and 160 mg/kg/d

gentamicin sulphate (GS) (i.p.) from day 6 to 10.

 Group VIII was given extract 300 mg/kg/d orally for 10 days and 160 mg/kg/d

gentamicin sulphate (GS) (i.p.) from day 6 to 10.

After the 10 days of treatment, experimental animals were anaesthetized and euthanized.

Blood samples were collected into sample tubes and samples were separated by centrifugation and used for measurement of serum creatinine (SCr), urea, total protein and electrolytes levels.

2.10.2 Malondialdehyde (MDA) measurement in the right kidney

Malondialdehyde (MDA) levels, as an index of lipid peroxidation, were measured in renal tissues. MDA, as a thiobarbituric acid reactive substance (TBARS), reacts with thiobarbituric acid (TBA) to produce a red colour complex with peak absorbance at 532 nm

(Fernandez et al., 1997).

Preparation of the kidney for malondialdehyde (MDA) determination

The right kidneys were removed and maintained at -80 °C (Fernandez et al., 1997). On the day of analysis, the right kidney was homogenized in cold potassium Chloride solution (1.5

%) to give a well concentrated homogenate suspension, at 200 – 300 mg tissue per 1.0 ml of buffer, and used for the malondialdehyde determination.

Phosphoric acid (3.0 ml; 1%) and TBA (1.0 ml; 0.6%) were added to 0.5 ml of homogenate in a centrifuge tube and the mixture was heated for 45 minutes in a boiling water bath.

After cooling, 4.0 ml of n–butanol was added to the mixture and vortex-mixed for 1 minute followed by centrifugation at 4000 for 25 minutes. The organic layer was transferred to a fresh tube and its absorbance was measured at 532 nm. Absorbance was measured in the visible range using a UV Win spectrophotometer model 5.

The standard plot of MDA was constructed over the concentration range of 0–40 µM

(Uchiama and Miahara, 1978) using tetramethoxypropane of concentration 20 µM. The free MDA concentration in the sample was calculated from the relation below;

[MDA] = A532 – b/a * df

Where MDA = [MDA] of the sample

A532 = Absorbance at 532 nm of the tissue samples; df = Sample dilution factor; a = Regression coefficient (slope)

* = Multiplication sign b = Intercept

2.10.3 Kidney photomicrograph

The abdomens of rats were opened and kidneys were dissected out. The left kidneys were processed and viewed under light microscopic (Nikon Eclipse E200, Japan). The photomicrographs were captured by Infinity 1 camera microscope according to standard procedures. The left kidneys were fixed in 10 % formalin and then embedded in paraffin wax, sectioned (3.0 µm) and stained with haematoxylin and eosin.

2.11 DIARRHOEAL STUDIES

2.11.1 Effect of extract on gastrointestinal system using the charcoal meal

This method determines the effects of drugs on the extent of intestinal transit during a fixed time period following a single oral administration of the extract. The measured endpoint is the extent of passage of a charcoal suspension through the small intestine. Atropine sulphate is the reference compound and was used as a positive control.

Carboxymethylcellulose served as the vehicle and was administered to one of the groups as negative control. Six groups of rats (195 – 205 g) each consist of 5 rats (n =5) were used.

Animals were fasted for 18 hours prior to the administration of the compounds. The animals were treated as follows;

 Group 1 – control carboxymethylcellulose 0.5 % w/v.

 Group 2 – extract (1 mg/kg, p.o.).

 Group 3 – extract (2 mg/kg, p.o.).

 Group 4 –extract (5 mg/kg, p.o.).

 Group 5 – extract (10 mg/kg, p.o.)

 Group 6 – atropine sulphate. (0.8 mg/ml, i.p).

One hour after drug administration, charcoal suspension, 10 % activated charcoal and 2.5

% gum Arabic, prepared overnight was administered orally at a dose of 1.0 ml/100 g body weight to each animal in all the groups.

The animals were euthanized by cervical dislocation 20 min after administration of the charcoal suspension. Immediately, the small intestine was removed from the duodenum to the ceacum and was carefully uncoiled without stretching. The distance covered by charcoal and the total length of the small intestine were measured with a rule in centimetres. The percentage transit was calculated from the formula: % transit = C/SI * 100 where C is the distance (cm) covered by the charcoal, SI the total length of the small intestine (cm) and * the multiplication sign. The result of each group of animals was

expressed as mean distance covered by charcoal meal and the mean percentage transit time together with the standard errors of the means (SEM) were determined.

2.11.2 Effect of extract on castor oil - induced defecation in rats

The method described by Awouters et al., (1978) was used to induce defecation. Six groups of rats (100 – 180 g) each consist of 5 rats per group (n =5). Diarrhoea was induced by administering 1.0 ml of castor oil orally to the rats for an hour. The animals were pre- treated with the extract before they were induced for defecation as follows;

 Group 1 served as the negative control and received (2 ml/kg) normal saline

(p.o.).

 Group 2 received loperamide (5 mg/kg, p.o.) which served as a standard

antidiarrhoeal drug.

 Group 3 received extract (1 mg/kg, p.o.).

 Group 4 received extract (2 mg/kg, p.o.).

 Group 5 received extract (5 mg/kg, p.o.).

 Group 6 received extract (10 mg/kg, p.o.).

The frequency of both dry and wet diarrhoea droppings were counted every hour for a period of 4 hours, mean number of the stools passed out by the treated groups were compared to the negative control group and the percentage inhibition was calculated as;

100 x Mean value of control – mean value of test agents

Mean value of control

2.11.3 Effect of extract on castor oil - induced enteropooling

Intestinal fluid accumulation was determined by the method of (Robert et al., 1976).

Overnight fasted rats were grouped into six (105 – 120 g) each consist of 5 rats per group

(n =5).

 Group 1 – normal saline (2 ml/kg, i.p.) which served as a control.

 Group 2 – atropine sulphate (3 mg/kg, i.p.).

 Group 3 – extract (1 mg/kg, p.o.).

 Group 4 – extract (2 mg/kg, p.o.).

 Group 5 – extract (5 mg/kg, p.o.).

 Group 6 – extract (10 mg/kg, p.o.).

After an hour, 1.0 ml castor oil was administered orally to all the groups. Two hours later the rats were sacrificed, their small intestines were ligated at both the pyloric sphincter and the ileocaecal junction and they were dissected and weighed. The intestinal contents were collected by milking into a graduated tube and their volumes measured. The intestine was reweighed and the differences between full and empty intestines were calculated.

2.11.4 Effect of extract on castor oil - induced intestinal transit time in rats

Overnight fasted male rats were grouped into seven (170 – 200 g) each consist of 5 rats per group

(n =5). The animals were treated as follows;

 Group 1 received 2 ml/kg of normal saline (i.p.)

 Group 2 received 2 ml of castor oil orally.

 Group 3 received atropine (3 mg/kg, i.p.).

 Group 4 received plant extract (1 mg/kg, p.o.).

 Group 5 received plant extract (2 mg/kg, p.o.).

 Group 6 received plant extract (5 mg/kg, p.o.).

 Group 7 received plant extract (10 mg/kg, p.o.).

After an hour, 1.0 ml castor oil was administered orally to the treated groups that were groups (3 – 7). The marker (1.0 ml) of (10 % charcoal suspension in 2.5 % gum acacia) was

administered orally, 1 hour after the charcoal meal treatment. The rats were sacrificed after

1 hour and the distance travelled by the charcoal meal from the pylorus to the caecum was measured and was expressed as the percentage of the whole length of the intestine.

2.12 STATISTICAL ANALYSIS

GraphPad Software was used and One-Way and Two-Way Analysis of Variance (ANOVA) for intergroup comparison (group effect).Then the data was analysed using Newman–Keuls post hoc test and Bonferroni post test for the group effects were used for the evaluation of the data and (P < 0.05) considered as significant. The experimental results were represented as mean ± SEM (standard error of mean)

CHAPTER 3

3.0 RESULTS

3.1 ASSESSMENT ON PHYTOCHEMICAL CONSTITUENTS OF THE EXTRACT

The extract showed the presence of the following bioactive substances, saponins, flavonoids, alkaloids, glycosides, condensed tannins and steroids but not terpenoid (Table 1).

Table 1: Phytochemical constituents found in the extract

PHYTOCHEMICALS OBSERVATIONS RESULTS

ALKALOIDS Reddish brown ppt +

CONDENSED TANNIS Black – green ppt +

FLAVONOIDS Yellow solution +

GLYCOSIDES Reddish brown ppt +

SAPONINS Frothing persisted over 5 min +

STERIODS Blue green solution +

TERPENOIDS No reddish brown formed at the -

interface ppt implies precipitate, + means present, - means not present.

3.2 LD 50 AND ACUTE TOXICITY STUDIES IN RODENTS

During the acute toxicity studies, the LD50 was above 5000 mg/kg in the treated rodents. Assessments made included aggressiveness, vomiting, excitement, salivation, sedation, diarrhoea, eating, drinking and death. These were observed in the treated groups and compared to the vehicle-treated control group. The treated groups showed signs of sedation compared to the control. Doses 10 - 5000 mg/kg body weight of the extract administered did not result in lethality over the 24 hour period. No latent toxicity was observed in the animals after keeping them for extra 14 days (Table 2).

Table 2: Effect of the extract on 24 - hour treated mice and rats

TREATMENT OBSERVATIONS MADE IN RATS AND MICE IN ACUTE TOXICITY

aggressive eating excitement death diarrhoea drinking salivation sedation vomiting

Control - + - - - + - - -

10 mg/kg - + - - - + - + -

100 mg/kg - + - - - + - + -

300 mg/kg - + - - - + - + -

1000 mg/kg - + - - - + - + -

5000 mg/kg - + - - - + - + -

(-) Implies not observed (+) implies observed

3.3 SUB-ACUTE TOXICITY STUDIES IN MALE RATS

3.3.1Weight of male SD rats before and after treatment

There were no significant differences in the weight of the animals treated with the extract at doses of (10

– 1000 mg/kg, p.o.) compared to the vehicle treated control group (Fig: 2).

Before experiment After experiment

350

300

250

200

150

Weight (g)

100

0 control 10 100 300 1000

Treatment groups

Figure 2: Body weight of rats before and after treatment with Launaea taraxacifolia extract for 14 days. The values are presented as mean ± SEM, (n =5).

3.2.2 Effect of the extract on organ to body ratio

In assessing the effect of extract on organ to body weight ratio, the target organs including the liver, heart, kidney, testes and spleen did not show significant differences compared to the vehicle treated control group (Table 3).

Table 3: Effect of extract on Relative Organ – Body Ratio in rats treated for 14 days

TREATMENT ORGAN – BODY RATIO IN PERCENTAGES

LIVER HEART KIDNEY TESTES SPLEEN

CONTROL 2.89 ± 0.11 0.56 ± 0.02 0.33 ± 0.02 0.99 ± 0.05 0.21 ± 0.01

10 mg/kg 2.14 ± 0.41 0.51 ± 0.02 0.35 ± 0.06 0.91 ± 0.07 0.22 ± 0.02

100 mg/kg 3.11 ± 0.20 0.53 ± 0.33 0.36 ± 0.04 0.84 ± 0.08 0.22 ± 0.01

300 mg/kg 2.98 ± 0.11 0.51 ± 0.02 0.34 ± 0.01 0.99 ± 0.13 0.22 ± 0.00

1000 mg/kg 2.78 ± 0.51 0.48 ± 0.03 0.32 ± 0.02 0.83 ± 0.07 0.22 ± 0.05

The values are mean ± SEM, (n =5). Statistical analysis was done by Newman- Kuels‟ test in One Way Anova compared to vehicle – treated group.

3.3.3 Effect of extract on haematological parameters

There were no significant differences in the treatment groups in the total white blood cells (TWBC), red blood cell (RBC) count, blood haemoglobin concentration (HB), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet (PLT), lymphocytes (LYM) and neutrophils (NEUT) compared to the vehicle treated control group (Table 4).

Table 4: Haematological parameters of rats treated with the extract during sub acute toxicity studies

PARAMETERS CONTROL 10 mg/kg 100 mg/kg 300 mg/kg 1000 mg/kg

TWBC(×103/µl) 6.867±0.584 9.300 ± 0.873 9.567 ± 0.887 8.733 ± 2.267 7.267 ± 0.176

RBC (×106/µl) 7.927±0.145 7.693 ± 0.154 7.590 ± 0.066 7.493 ± 0.159 7.647 ± 0.149

HB (g/dl) 14.870±0.240 13.670± 0.318 13.600± 0.153 14.100±0.416 13.770±0.318

PCV (%) 48.470±1.214 44.500± 1.514 43.77 ± 0.991 45.470±1.934 46.170±0.754

MCV (fl) 61.130±0.406 57.800± 1.168 58.000± 1.401 60.630±1.272 59.330±0.731

MCH (pg) 18.770±0.088 17.770±0.203 17.930±0.318 18.830±0.145 18.270±0.176

MCHC (g/dl) 30.700±0.306 30.730 ± 0.338 30.930 ± 0.233 31.030±0.433 30.800±0.666

PLT (×103/µl) 787.70±57.18 731.30 ± 21.67 831.30 ± 33.38 789.00±87.00 691.70±21.98

LYM (%) 70.800±4.681 75.200 ± 5.237 72.870 ± 0.837 72.870±0.837 69.730±9.427

NEUT (%) 29.200±4.681 29.800 ± 2.700 27.850 ± 0.750 38.450±0.750 39.600±2.300

The values are mean ± SEM, (n =5). Animals received doses orally for 14 days. Statistical analysis was done by Newman - Kuels‟ test in One Way Anova when compared to vehicle treated control group.

3.3.4 Effect of extract on serum biochemical parameters

The effect of extract on serum biochemical parameters after a period of 14 days treatment revealed no significant differences in albumin, globulin, total protein, bilirubin, alanine aminotransferase (ALT), alkaline phosphatase (ALP) and gamma glutamic transpeptidase (GGT), compared to the vehicle treated control group. Creatinine was significantly decreased (P < 0.05) at doses 10 and 300 mg/kg and also at doses 100 and 1000 mg/kg (P < 0.01). Urea showed a general decrease at all doses but with no statistically significant difference compared to the vehicle treated control (Table 5).

Table 5: Serum biochemical parameters of rats treated with the extract during sub acute toxicity studies

PARAMETERS CONTROL 10 mg/kg 100 mg/kg 300 mg/kg 1000 mg/kg Albumin (g/dl) 38.87±0.713 34.33 ± 2.173 35.07 ± 1.260 38.80 ± 2.193 35.00 ± 2.401 Globulin (mg/dl) 36.81±2.437 32.93 ± 4.707 34.44 ± 3.226 31.93 ± 0.476 29.40 ± 1.372 Totalprotein(g/dL) 75.70±2.937 67.27 ± 2.976 69.53 ± 1.994 70.77 ± 1.876 70.10 ± 4.272 ALP (U/L) 689.0±43.21 643.0 ± 57.83 558.3 ± 58.87 504.3 ± 60.51 491.0 ± 54.50 ALT (U/L) 108.3±4.604 73.00 ± 23.76 120.2 ± 4.550 132.1 ± 12.99 155.8 ± 21.02 AST (U/L) 206.6 ±16.86 146.0± 8.807 172.6 ± 9.968 202.0 ± 7.663 184.7 ± 5.686 GGT (U/L) 0.167± 0.033 0.133 ± 0.088 0.100 ± 0.100 0.133 ± 0.033 0.100 ± 0.057 Dbilirubin(mmol) 1.100±0.058 1.000 ± 0.058 0.967 ± 0.067 0.700 ± 0.116 0.800 ± 0.173 IDbilirubin(mmol) 0.407± 0.134 0.483 ± 0.136 0.683 ± 0.185 0.670 ± 0.085 0.927 ± 0.495 Tbilirubin(mmol) 1.500± 0.153 1.467 ± 0.120 1.433 ± 0.088 1.333 ± 0.088 1.367 ± 0.033 Creatinine (mg/dl) 90.10±1.069 70.80±0.924* 66.20±0.945** 76.10±2.050* 71.00±7.578** Urea (mg/dl) 9.237±0.491 6.393 ± 1.083 6.377 ± 0.157 7.733 ± 0.519 7.130 ± 0.220 Values presented are the mean ± SEM, (n = 5). Statistical analysis was done by Newman- Kuels‟ test in One Way Anova compared to vehicle treated control group. * means P < 0.05, ** means P < 0.01.

3.3.5 Photomicrograph of the liver cells Histology of the liver confirmed no cellular damage in all the treated groups, compared to the vehicle treated control group. Indices assessed included reactive changes, apoptosis, necrosis, inflammation and steatosis (Figure 3).

A B

C D

Figure 3: Photomicrographs of rat liver X 10 (A) vehicle–treated control group, (B, C, D, E) rats received

10, 100, 300, 1000 mg/kg Launaea taraxacifolia extract respectively for 14 days.

Table 6: Photomicrograph report on rats liver cells

TREATMENT LIVER HISTOLOGY DESCRIPTION Apoptosis Necrosis Inflammation Steatosis Control group - - - -

10 mg/kg Launaea extract - - - -

100 mg/kg Launaea extract - - - -

300 mg/kg Launaea extract - - - -

1000 mg/kg Launaea extract - - - -

– implies within normal limit

3.4: EFFECT OF EXTRACT ON PENTOBARBITONE–INDUCED SLEEPING TIME IN RODENTS

3.4.1: Pentobarbitone - induced sleeping time in ICR mice

The effect of extract on the onset and duration of sleep was assessed in pentobarbitone - induced sleeping time model in ICR male mice over 24 hour period. In Figure (4 A), the extract showed a general decrease in the onset of sleeping time (P < 0.05) but this was only significant at 1000 mg/kg compared to the vehicle treated control group. There was a dose dependent increase in the duration of sleep (B), with significant differences of (P < 0.001) at doses 300 and 1000 mg/kg, (P < 0.01) at 100 mg/kg and (P <

0.05) at 10 mg/kg compared to the vehicle treated control group (Figure 4 B).

A B

600

20000 ### *** 400 15000 *** ** * 200 * 10000 *

5000

0 0 10 Onset of sleeping time (seconds) time sleeping Onset of 100 300 10 1000 100 300 control (seconds) time sleeping of Duration 1000 control diazepam diazepam Doses (mg/kg) Doses (mg/kg)

Figure 4: Effect of extract (p.o.), on the onset (A) and duration (B) of sleep in mice in pentobarbitone – induced sleeping time for 24 hour period. An hour prior to the administration of (40 mg/kg, i.p.) pentobarbitone, the extract at doses (10 -1000 mg/kg, p.o.) and reference compound (0.5 mg/kg, i.p.) diazepam were given. The onset of sleep was recorded as the time they loss their righting reflex and the duration as the time between the loss and regain of righting reflex. Values are presented as the mean ± SEM (n = 6) compared to the vehicle treated control group by Newman- Kuels‟ post hoc test using One- Way Anova statistical tool. * indicates (P < 0.05), ** indicates (P < 0.01) and *** indicates (P < 0.001) significant differences compared to control treated group. # indicates (P < 0.05) and ### indicates (P < 0.001) significant differences compared to diazepam treated group.

3.4.2: Pentobarbitone - induced sleeping time in Sprague - Dawley rats

As observed in (Fig: 5) there appeared to be no effect in the onset of sleep (A) in extract treated male rats groups compared to the vehicle treated control group. Similarly the extract did not have significant effect on the duration of sleep (B). Treatment of the male rats for 2 weeks did not affect the onset and duration of sleep (data not shown).

A B

300 15000

200 10000

100 5000

0 0

Onset of sleeping time (seconds) time sleeping Onset of con 100 300 con 100 300

1000 2000 (seconds) time sleeping Duration of 1000 2000 Doses (mg/kg) Doses (mg/kg)

Figure 5: Effect of extract (p.o.) on the onset (A) and duration (B) of sleep in SD rats in pentobarbitone – induced sleeping time for 24 hour period. Pentobarbitone (50 mg/kg, i.p.) was challenged after an hour. The onset was recorded as the time they loss their righting reflex and the duration as the time between the loss and regain of righting reflex. Values are presented as the mean ± SEM (n = 5). * indicates significant (P < 0.05) compared to vehicle-treated control group by Newman –Kuels‟ post hoc test using One -Way Anova statistical tool.

3.5: EFFECT OF EXTRACT ON SPONTANEOUS LOCOMOTOR ACTIVITY IN ICR MICE

The extract showed significant decrease of locomotor time at dose 1000 mg/kg (P < 0.001) compared to the vehicle–treated control group (Figure: 6).

200

150

*** *** 100

locomotor time (minutes) time locomotor 0 10 100 300 1000 control diazepam Doses (mg/kg)

Figure 6: Effect of extract (p.o.) on spontaneous locomotor activity in male mice. Thirty minutes after treatment with extract and reference drug diazepam, (0.5 mg/kg, i.p.). The activities of the mice were determined using the activity cage at 5 minute intervals over a period of 50 minutes. Values are presented as the mean ± SEM (n = 5). *** indicates (P < 0.001) significance compared to vehicle treated control group by Newman-Kuels‟ post hoc test by one-way ANOVA.

3.6 EFFECT OF EXTRACT ON TOTAL CYTOCHROME P450 ENZYME LEVELS IN RATS

The extract did not show any significant difference on total Cytochrome P450 enzyme levels in male rats compared to the vehicle treated control group (Figure 7).

2.5

2.0 ***

1.5

1.0

0.5

CYT P450 (nmol/mgprotein) P450 CYT 0.0

control 300 mg/kg 1000 mg/kg pento 80 mg/kgketo 100 mg/kg doses

Figure 7: Effect of extract, pentobarbitone (pento) and ketoconazole (keto) on the total cytochrome P450 enzyme level, after a week treatment. The rats received extract (300 and 1000 mg/kg p.o.), pentobarbitone (80 mg/kg p.o.) or ketoconazole (100 mg/kg p.o). The protein content of the livers were determined using Biuret method and cecil CE 2041 spectrophotometer for the absorbance at 540 and that of the cytochrome P450 enzymes at 450. The values were mean ± SEM (n= 5). *** indicates (P < 0.001) compared to the control treated group by Newman–Kuels‟ post hoc test by one-way ANOVA.

3.7 EFFECT OF EXTRACT ON GENTAMICIN – INDUCED NEPHROTOXICITY IN SD RATS

3.7.1 Serum creatinine Levels

Treatment with the extract significantly (P < 0.001) inhibited the gentamicin-induced increase in creatinine levels in a dose dependent manner. Two-way analysis showed that treatment with gentamicin significantly (P < 0.001) increased creatinine levels (figure 8).

800 ### untreated control # gentamicin treated control 600 extract + gentamicin treated extract treated

400 *

***

creatinine (umol/l) creatinine 200 ***

10 100 300 control extract (mg/kg)

Figure 8: Effect of extract on serum creatinine levels of male rats after 10 days treatment period by gavage. Normal saline (0.5 ml/100 g, p.o) was given to the control group. Gentamicin sulphate (GS), (160 mg/kg, i.p) was treated 1 hour after extract treatment and the gentamicin group for 5 days that is, from day 6 to day 10 using Flexor junior auto analyzer. The values were mean ± SEM (n= 5). * indicates (P < 0.05) *** indicates (P < 0.001) significant difference compared to the vehicle-treated group by Newman–Kuels‟ post hoc test in one-way ANOVA and # indicates (P<0.05), ### indicates (P < 0.001) significant difference compared to the vehicle-treated group by Bonferroni post test in two-way ANOVA.

3.7.2 Serum Urea levels Treatment with the extract significantly (P < 0.001) inhibited the gentamicin-induced increase in urea levels in a dose dependently. Two-way analysis showed that treatment with gentamicin significantly (P <

0.001) increased urea levels (figure: 9).

### untreated control 40 gentamicin treated control # extract +gentamicin treated 30 extract treated 20

** urea urea (mmol/L) 10 **

10 100 300 control extract (mg/kg)

Figure 9: Effect of extract on serum urea levels of male rats after 10 days oral treatment period. Normal saline (0.5 ml/100 g, p.o.) was given to the control group. Gentamicin sulphate (GS), (160 mg/kg, i.p.) was treated 1 hour after extract treatment and the gentamicin group for 5 days that is, from day 6 to day 10 using Flexor junior auto analyzer. The values were mean ± SEM (n= 5). ** indicates (P < 0.01) significances compared to the vehicle-treated group by Newman–Kuels‟ post hoc test in one-way ANOVA. # indicates (P < 0.05) and ### indicates (P < 0.001) significant difference compared to the vehicle-treated group by Bonferroni post test in two-way ANOVA.

3.7.3 Serum total protein levels

Treatment with the extract significantly (P < 0.001) inhibited the gentamicin-induced increase in total protein levels in a dose dependent manner. Two-way analysis showed that the treatment with gentamicin significantly (P < 0.001) increased total protein levels. However (figure: 10).

### untreated control 1000 ### gentamicin treated control 800 extract + gentamicin treated extract treated 600

400 **

total protein (g/l) total protein 200 *** ***

0 10 100 300 control Doses (mg/kg)

Figure 10: Effect of extract on serum total protein levels of male rats after 10 days oral treatment period. Normal saline (0.5 ml/100 g, p.o.) was given to the control group. Gentamicin sulphate (GS), (160 mg/kg, i.p) was treated 1 hour after extract treatment and the gentamicin group for 5 days that is, from day 6 to day 10 using Flexor junior auto analyzer. The values were mean ± SEM (n= 5). ** indicates (P < 0.01) and *** indicates (P < 0.001) significances compared to the vehicle- treated control group by Newman–Kuels‟ post hoc test by two-way ANOVA and ### indicates (P < 0.001) significant difference compared to the vehicle-treated group by Bonferroni post test by two-way ANOVA.

3.7.4 Serum electrolyte levels

Treatment with the extract significantly (P < 0.001) inhibited the gentamicin-induced increase in sodium and chloride levels and showed an increase in potassium levels dose dependently. Two-way analysis showed that the treatment with gentamicin significantly (P < 0.001) increased sodium and chloride levels but decreased potassium level (Table: 7).

Table 7: Serum Electrolyte Levels TREATMENT ELECTROLYTES CHLORIDE POTASSIUM SODIUM CONTROL 124.9 ± 9.818 1.054 ± 0.021 139.7 ± 15.62 10 mg/kg 120.8 ± 13.25 1.256 ± 1.202 123.8 ± 6.391 100 mg/kg 104.4 ± 3.251 1.276 ± 0.092 122.0 ± 5.827 300 mg/kg 102.0 ± 1.286 1.202 ± 0.041 112.2 ± 5.986 GS 160 mg/kg 315.3 ± 35.70### 0.600 ± 0.510### 404.4 ± 46.20### 10 mg/kg & GS 235.1 ± 35.94* 1.153 ± 0.098* 288.9 ± 27.34** 100 mg/kg & GS 122.7 ± 13.40*** 1.448 ± 0.034** 142.9 ± 6.882*** 300 mg/kg & GS 121.7 ± 18.98** 1.346 ± 0.061** 145.6 ± 9.989***

Effect of extract at 10, 100, 300 mg/kg, on serum electrolyte levels of male rats treated orally for 10 days. Normal saline (0.5 ml/100 g, p.o) was given to the control group. Extract was given was given 1 hour after the Gentamicin sulphate (GS), (160 mg/kg, i.p.) for 5 days that is, from day 6 to day 10 using 9180 electrolyte auto analyzer. The values were mean ± SEM (n= 5). * indicates (P < 0.05), ** indicates (P < 0.01) and *** indicates (P < 0.001) significances compared to the control treated group by Newman–Kuels‟ post hoc test by one-way ANOVA and ### indicates (P < 0.001) significant difference by Bonferroni post test by two-way ANOVA.

3.7.5 Malondialdehyde levels

Treatment with the extract significantly (P < 0.01) inhibited the gentamicin-induced increase in MDA levels in a dose dependent manner. In two-way analysis it showed that gentamicin significantly (P <

0.001) increased MDA levels. However, (figure: 11).

untreated control ### 2.5 gentamicin treated control

2.0 extract + gentamicin treated extract treated 1.5 *

1.0 ** ** MDA (umol/g) MDA 0.5

0.0 10 100 300 control extract (mg/kg)

Figure 11: Effect of extract after 10 days treatment period. These animals received extract (10,100, 300 mg/kg, p.o.) and the gentamicin (160 mg/kg, i.p.) after one hour, MDA content was determined in the right kidney by using UV Win spectrophotometer model 5 and the absorbance was read at 565 and 532 for the standard and the samples respectively. The values were mean ± SEM (n = 5). * indicates (P < 0.05), ** (P < 0.01) significance compared to the vehicle–treated control group by Newman–Kuels‟ post hoc tests by one -way ANOVA and ### indicates (P < 0.001) significant difference by Bonferroni post test by two-way ANOVA.

3.7.6 Photomicrograph showing the effect of the extract on the left kidney of SD rats treated with gentamicin

The left kidney of male rats treated with the extract alone for 10 days did not differ from the control as shown in the photomicrograph (Figure 12 A). Treatment with gentamicin alone for 5 days showed severe damage to the kidney (Figure 12 B, panel A). The extract reversed the renal damage caused by gentamicin (Figure 12 B, panel B, C and D).

control 10 mg/kg

100 mg/kg 300 mg/kg

Figure: 12 A Photomicrograph showing the effect of extract alone on the left kidney compared to vehicle treated control after 10 days treatment.

(A) Gentamicin 160 mg/kg (B) 10 mg/kg & gentamicin

Figure 12 B: Photomicrographs of gentamicin sulphate treated group exhibited complete tubular degeneration and desquamation, tubular necrosis, mononuclear cells infiltration, intertubular haemorrhage and tubular brush boarder loss compared to the vehicle treated control group and the extract treated groups (A). However, animals treated with gentamicin and extract showed a protective effect especially at 300 & gentamicin group.

3.8 EFFECT OF EXTRACT ON GASTROINTESTINAL TRACT MOTILITY IN RATS

3.8.1 Small intestinal transit time

The extract did not show any significant difference in the mean distance moved by charcoal and the mean % transit time compared to the vehicle treated control group on the small intestinal transit time in SD rats.

Table 8: Small intestinal transit time in rats

Treatment Doses Distance moved by % transit time mean ± charcoal mean ± SEM SEM (cm) Control 0.5 % CMC 71.75 ± 5.74 82.11 ± 7.24

Atropine 2 mg/kg (i.p.) 49.25 ± 4.70* 67.44 ± 9.72

Extract 1 mg/kg (p.o.) 57.50 ± 7.53 62.71 ± 1.57

Extract 2 mg/kg (p.o.) 67.63 ± 3.61 73.25 ± 2.01

Extract 5 mg/kg (p.o.) 58.63 ± 1.59 65.52 ± 2.94

Extract 10 mg/kg (p.o.) 59.38 ± 7.59 63.46 ± 5.36

A single oral dose (p.o) of vehicle and extract, atropine sulphate (i.p.) were administered 1 hour prior to the administration of charcoal suspension (p.o.). Values presented are mean ± SEM (n =5). Control received 0.5 % w/v carboxymethylcellulose CMC. Statistical analysis by Newman–Kuels‟ tests and One Way Anova statistical tool. * indicates (P < 0.05) significant to the control.

3.8.2 Castor oil- induced defecation

The extract–treated groups showed significant decrease (P < 0.05) at a dose of 5 mg/kg and (P < 0.01) 10 mg/kg in the mean frequency of defecation hourly for 4 hours. The extract–treated groups showed a dose dependent increase in percentage inhibition with significant differences at 5 mg/kg (P < 0.05) and 10 mg/kg (P< 0.01).

Table 9: Castor oil induced defecation

Treatment Mean frequency of defecation in Percentage inhibition

4 hours

CO + Control(-ve) 4.350 ± 0.516 ------

CO +Loperamide 2.550 ± 0.539* 41.38*

CO + 1 mg/kg 3.250 ± 0.474 25.29

CO + 2 mg/kg 3.200 ± 0.429 26.44

CO + 5 mg/kg 2.400 ± 0.245* 44.83*

CO + 10 mg/kg 1.950 ± 0.348** 55.17**

The extract and the reference drug were administered orally to overnight fasted male rats. After 1 hour 1.0 ml castor oil was administered orally by gavage. Values were expressed as mean ± SEM (n =5). * indicates (P < 0.05) and ** indicates (P < 0.01) considered significant compared to the castor oil (CO) and vehicle treated control group. Analysis by Newman–Kuels‟post hoc tests by One - Way ANOVA.

3.8.3 Castor oil-induced enteropooling

Effects of the extract on castor oil induced enteropooling in male rats did not exhibit significant difference compared to the vehicle treated control group in the mean intestinal content and the mean intestinal weight.

Table 10: Castor oil-induced enteropooling in SD rats

Treatment Doses Mean of intestinal Mean of difference in

content (ml) intestinal weight (g)

Control 2 ml/100g ns + CO 2.06 ± 0.051 2.11 ± 0.157

Atropine 3 mg/kg (i.p) + CO 1.96 ± 0.144 2.06 ± 0.241

Extract 1 mg/kg (p.o) + CO 2.12 ± 0.318 1.79 ± 0.145

Extract 2 mg/kg.(p.o) + CO 1.92 ± 0.248 2.04 ± 0.324

Extract 5 mg/kg (p.o )+ CO 1.70 ± 0.203 1.77 ± 0.314

Extract 10 mg/kg (p.o )+ CO 1.60 ± 0.428 1.86 ± 0.425

Extract was administered (p.o.) and atropine sulphate was given (i.p.) to overnight fasted rats. After an hour, 1.0 ml castor oil was administered orally by gavage. After another two hours rats were sacrificed and their small intestinal content milked and measured in (ml), the weight in (g). Values were expressed as mean ± SEM (n =5), were compared with castor oil (CO) and vehicle treated control. Analysis by Newman–Kuels‟ post hoc tests by One - Way ANOVA.

3.8.4 Castor oil-induced small intestinal transit time

The extract–treated groups showed significant decrease at a dose of 2 mg/kg (P < 0.05), 5 and 10 mg/kg

(P < 0.01) in the mean distance moved by charcoal compared to control treated group. In the mean % transit time, the extract at 5 mg/kg and 10 mg/kg showed significant decrease (P < 0.05) compared to vehicle treated control group.

Table 11: Castor oil-induced small intestinal transit time in rats

Treatment Doses Distance moved by % transit time mean ±

charcoal mean ± SEM SEM

Control 2 ml normal saline 81.30 ± 3.470 77.91 ± 2.004

Castor oil 2 ml CO + 2 ml ns(i.p) 66.10 ± 5.102* 66.03 ± 4.840

Atropine CO + 3 mg/kg (i.p) 53.30 ± 7.937** 55.46± 8.690*

Extract CO + 1 mg/kg (p.o) 61.80 ± 5.578 70.72 ± 5.651

Extract CO + 2 mg/kg (p.o) 58.80 ± 2.301* 63.16 ± 1.816

Extract CO + 5 mg/kg (p.o) 55.10 ± 3.100** 57.65 ± 4.240*

Extract CO + 10 mg/kg (p.o) 56.38 ± 3.384** 56.76 ± 2.452*

A single dose (p.o.) of vehicle, normal saline (ns), castor oil (CO) 1.0 ml and extract were administered by gavage and atropine sulfate (i.p.). An hour prior to the administration of castor oil .After two hours 1.0 ml charcoal suspension (p.o.) given by gavage. Values are mean ± SEM (n = 5). * indicates (P < 0.05) and ** indicates (P < 0.01) compared to vehicle treated control group. Analysis by Newman– Kuels‟ post hoc tests by One - Way ANOVA.

CHAPTER 4

4.0 DISCUSSION AND CONCLUSIONS

4.1DISCUSSION

Acute toxicity testing is the first step in the toxicological assessment of an unknown substance. Establishment of safety is very essential for the assessment of herbal remedies which usually do not undergo any experimental safety before use with the notion that anything natural is devoid of toxicity. Launaea taraxacifolia is a very important herbal remedy and food for both humans and animals but has not been scientifically validated.

Launaea taraxacifolia was therefore investigated for any potential toxicity in vivo on haematological and serum biochemical parameters, target organs, renal system, central nervous system and gastrointestinal tract in laboratory rodents. The results in this study showed that the acute administration (p.o.) of the ethanolic extract of Launaea taraxacifolia did not cause death of animals at all tested doses, which probably suggests safety in the acute toxicity stage.

The index for the acute toxicity is the LD50 (Abdullah, 2011). There were no deaths recorded in rodents tested during the period, placing the lethal dose above 5000 mg kg-1.

-1 According to Clarke et al. (1975) substances that show LD 50 at 1000 mg kg body weight should be considered safe or of low toxicity when administered orally.

Haematological parameters are usually associated with health status and are of diagnostic importance in clinical assessment of the state of health of a patient. Blood parameters are good indicators of physiological, pathological and nutritional status of an animal and changes in haematological parameters have the potential to explicate the impact of

therapeutic drug testing and toxicological factors. The results of the haematological studies show that Launaea taraxacifolia has little or no adverse effect on RBC, Hb, platelet number, haematocrit, and lymphocyte. However, the TWBC and neutrophils showed an increase compared to the control group suggesting an improvement in the immune system of the extract-treated animals. This property of the extract may contribute to cellular inflammatory processes, which might not be compromised as a result of increase in neutrophils and TWBC levels (Adedapo et al., 2004). Serum biochemical parameters are diagnostic markers of liver and kidney function. The globulin, albumin and total protein levels were not affected by the extract. According to Adeoye and

Oyedapo, (2004), an increase in plasma bilirubin is suggestive of a possible injury to the liver. The excess production of bilirubin may be due to failure of the liver to effectively conjugate it for excretion. The extract, caused a decrease in total bilirubin levels of treated animals at all the doses used. This decrease however, did not show statistical significance.

Adverse interaction of the plant extract with the major organs would cause cellular constriction and inflammation, which usually reflects in the organ to body ratio by the significant increase or decrease in weight (Devaki et al., 2012). In this study, no significant differences were found in the organ to body ratio and this supports the non- toxic nature of the extract. Creatinine and urea are the markers for renal function. High levels of creatinine are found in renal dysfunction or muscle injury and urea is a waste product of protein breakdown (Sodipo et al., 2012). From this study the extract showed a

remarkable decrease in creatinine and urea levels suggesting that the extract could be a possible reno-protective agent.

This property of the extract was further investigated using chemical (gentamicin) induced kidney injury. In spite of their beneficial effects, aminoglycosides induce nephrotoxicity.

Gentamicin sulphate – induced nephrotoxicity occurred in 10 - 20 % of its therapeutic cases (De Souza, et al., 2009). Launaea taraxacifolia extract-treated animals showed a significant improvement in serum Cr, urea, total protein and electrolyte levels compared to the vehicle-treated group and the toxin control groups on the 11th day. In agreement with previous results, Safa et al., (2010), serum creatinine and urea levels were high in the rats treated with gentamicin. Also, Ajami et al., (2010) showed that treatment with gentamicin for five days induced renal functional deficiency as demonstrated by significant increase in serum Cr and urea levels. An increase in serum protein level may be due to cell damage as a result of drug intake or cell proliferation (Ashang and Utu-

Baku, 2009). The decrease in the markers shows that the extract is possibly reversing cell damage caused by the gentamicin.

The balance of electrolytes is important for normal cellular function. It promotes fluid balance, maintains blood volume, facilitates fluid absorption and generates impulses.

Electrolyte reduction may lead to destruction of nerve conduction and reduced performance (Feroz et al., 2009). Electrolyte imbalance occurs with increased sodium and chloride and decreased potassium levels as in the case of gentamicin treated rats compared to the extract-treated group. The extract caused significant electrolyte balance

thereby having the potential to improve nerve conduction (Baniata et al., 2009; Feroz et al., 2009).

A relationship between oxidative stress and nephrotoxicity has been well confirmed in many experimental animal models. In gentamicin-treated rats, there was an increase in lipid peroxidation products (MDA) suggesting the involvement of oxidative stress

- - (Kumar et al., 2000). Reactive oxygen species (ROS) like O2 or OH · can assault and oxidize membrane lipids, resulting in loss of membrane integrity and function. Pre- treatment of rats with the extract reduced the levels of MDAs. The rise in MDA could be due to increase generation of ROS or to excessive oxidative damage leading to toxicity

(Ajami et al., 2010; Randjelovic et al., 2012). These results clearly indicate that gentamicin exerts its main effect on proximal tubular cells which cause electrolyte imbalance (Osamu et al., 1988). Measurement of specific ion concentrations (Na+, K+ and Cl-) may be used as potential biomarkers of chemical exposure and effects (Bheeman et al., 2012) such as gentamicin. Pretreatment with extract at doses 10, 100 and 300 mg/kg/d for five days reduced gentamicin-induced high tissue MDA levels. This decrease could possibly be due to less free-radical in the system. At these doses the extract exerted possible protection against gentamicin-induced nephrotoxicity especially at 100 and 300 mg/kg dose levels. Launaea might have antagonized the biochemical activity of gentamicin on phospholipids in the proximal tubules via possible antioxidant and free- radical scavenging properties (Nasim et al., 2006; Eslami et al., 2011). This could probably also be due to a reduction in injury caused by oxygen-free radicals in the presence of the extract (Dur-Zong et al., 2011). Histopathological analysis of gentamicin- treated animals showed that there is significant renal damage in the kidneys. This result

confirmed that the kidney is very sensitive to gentamicin toxicity as observed by others

(Nakakuki et al., 1996; Kumar et al., 2000). Shi et al., (2004) showed that a high saponin diet has the ability to manage renal problems. Since phytochemical analysis of the extract revealed the presence of saponins, it helps to suggest its possible role in renal protection.

The liver is the primary target organ of toxicity which is expressed as an increase in activity of liver associated enzymes, fatty degeneration, and necrosis (Barnes et al., 1985, &

Freundt et al., 1977). In the sub-acute toxicity study, there was no significant change in the relative liver weight of the animals, AST and ALT levels indicating that the extract is probably safe. The photomicrograph of the liver confirmed the extract is safe because there was no damage caused to the livers architecture. The sedative effect observed, however, called for further investigation into the effect of extract on the pentobarbitone - induced sleeping time in rodents. This was done to determine whether the metabolic ability of the liver was affected. Substances that have CNS depressant activity either decrease the time for onset of sleep or prolong the duration of sleep or both (Sama et al., 2011). Treatment of rodents with extract 60 minutes before pentobarbitone challenge caused significant increase in the sleeping time dose dependently. This could be due to a possible enzyme inhibition or

CNS depressant activity by the plant extract. It is likely that there exists a pharmacokinetic interaction between pentobarbitone and the extract which might have resulted in ineffective metabolism of pentobarbitone (Gilman and Goodman, 1995) hence the prolongation of the sleeping time. Barbiturates, example pentobarbitone generate many of their actions by interacting with gamma aminobutyric acid (GABA) receptors through apparent interaction with discrete allosteric sites (MacDonald, et al., 1989). It could also suggest that the extract of launaea might interact with barbiturate allosteric site on GABA receptors, potentiating

the action of pentobarbitone. This effect might cause the significant prolonged sleeping time observed in mice. Alkaloids, flavonoids and saponins for instance were shown to produce sedation and prolong sleeping time in mice (Jiang et al., 2007). It is possible that the extract exert its sedative effect via GABA receptor owing to the presence of flavonoids

(Wasowski and Marder, 2012).

However, during the cytochrome P450 content determination as described by (Omura and

Sato, 1964), the extract did not show any significant difference when compared to the vehicle-treated group. CYP3A2 which is one of the most profusely expressed CYPs in the rat liver. It metabolizes several drugs including barbiturates (Nelson et al., 1993). With this finding it suggests that, enzyme inhibition which occurred by the administration of the extract did not stimulate the synthesis of more metabolising enzymes (Dossing et al.,

1983).

Spontaneous locomotor activity was assessed. The extract showed significant decrease in activity compared to the vehicle–treated group at higher dose. The decreased spontaneous motor activity and potentiating of pentobarbitone induced sleep could be attributed to the

CNS depressant activity (Mishra et al., 2011).

Although antidiarrhoeal agents are effective in the management of diarrhoea, they come with some forms of challenges. These adverse effects include constipation and dizziness

(BNF, 2011). Antidiarrhoeal drugs such as loperamide should not be used in patients with severe ulcerative colitis, since toxic megacolon may be precipitated (Baker, 2007; Bertram et al., 2009). It may also prolong the duration of diarrhoea in patients with shigella or salmonella infection (Bertram, 1994). Loperamide is metabolised in the liver and is

virtually non-toxic to the central nervous system. However, it could be toxic to individuals with liver dysfunction (Weaver et al., 1983). Paralysis of the gut with pooling of fluid in the intestinal lumen are regarded as potentially harmful especially in infectious secretory diarrhoea (von Muhlendahl et al., 1980).

Diarrhoea occurs as a consequence of unevenness between the absorptive and secretory mechanisms in the intestinal tract accompanied with excess loss of fluid in the faeces

(Havagiray et al., 2004). The use of castor oil – induced diarrhoea model in this study was logical because the autocoids and prostaglandins are involved which have been the cause of diarrhoea in man to some extent (Greenbargarna et al., 1978). Castor oil causes decreased fluid absorption, increased secretion in the small intestine and colon, and increased smooth muscle contractility in the intestine (Senthil, 2011). Castor oil produces diarrhoea effect due to its active constituent, recinoleic acid (DiCarlo et al., 1994), inhibition of intestinal

Na+, K+-ATPase activity to decrease normal fluid absorption (Capasso et al., 1994), activation of adenylyl cyclase (DiCarlo et al., 1994), stimulation of prostaglandin formation

(Phillips et al., 1965), platelet activating factor and recently, nitric oxide release (Nell and

Rummel,1984, Galvez et al., 1993, Mascolo et al., 1996). In spite of the fact that these mechanisms have been involved in the diarrhoea effect of castor oil, it has been difficult to define its correct mechanism of action (Nwafor, 2005). The effect of the extract was similar to loperamide, which is at present one of the most efficacious and widely employed antidiarrhoeal drugs. Loperamide effectively antagonizes diarrhoea induced by castor oil significantly (Karim and Adaikan, 1997). The therapeutic effect of loperamide is due to antimotility and antisecretory properties (Couper, 1987). The extract might be acting via any one of the above mechanisms. It was also noted that the extract significantly decreased

castor oil–induced enteropooling, intestinal fluid accumulation and the volume of intestinal content. Secretory diarrhoea is associated with an activation of Cl- channels, causing Cl- efflux from the cell. Massive secretion of water into the intestinal lumen cause profuse watery diarrhoea (Ammon and Soergel, 1985). The involvement of the muscarinic receptor effect was confirmed by increased production of both gastric secretion and intraluminal fluid accumulation induced by castor oil. The extract showed decrease in the secretion of water into the intestinal lumen and this result could partially be mediated by both α2- adrenoceptor and muscarinic receptor systems (Karimulla and Kumar, 2011). The significant inhibition of the castor oil - induced enteropooling in rats suggested that the extract of Launaea causes constipation not spasmolytic activity in vivo and antienteropooling effects. (Nwodo and Alumanah, 1991). The extract of Launaea taraxacifolia significantly reduced the castor oil – induced intestinal transit as compared to control group as a confirmation. This effect was also seen in the intestinal transit without castor oil induction. In this study, atropine increased intestinal transit time possibly due to its anti-cholinergic effect (Ammon & Soergel, 1985). In castor oil – induced diarrhoea, the liberation of ricinoleic acid result in irritation and inflammation of the intestinal mucosa, leading to the release of prostaglandins, which stimulate secretion (Greenbargena et al.,

1978) by preventing the reabsorption of NaCl and water (Phillips et al., 1965). Probably, the extract increases the reabsorption of NaCl and water by decreasing intestinal motility as observed by the decrease in intestinal transit in the charcoal meal test. Anti-dysentric and antidiarrhoeal properties of medicinal plants were found to be due to the presence of tannins, alkaloids, saponins, flavonoids, sterols and reducing sugars (Brown and Taylor,

2000). Flavonoids and alkaloids are also known for inhibiting the release of autocoids and

prostaglandins, thereby inhibiting secretion induced by castor oil (Vimala et al., 1997;

Veiga et al., 2001). The phytochemical analysis of the extract revealed the presence of alkaloids, flavonoids, tannins and saponins. These constituents might have therefore mediated the antidiarrhoeal property of the Launaea taraxacifolia extract.

The present study has shown that Launaea taraxacifolia has a possible antidiarrhoea effect.

This may be undesirable especially if patients are taking drugs that are likely to cause constipation. This does not justify its widespread use by the local population for its mild laxative purpose.

4.2 CONCLUSIONS

Launaea taraxacifolia extract is safe on the liver as the liver weight, AST, GGT, ALT, lymphocytes and the liver histology did not show any differences between control and treated animals.

Launaea taraxacifolia extract has no adverse effect on blood and its cellular constituents.

Launaea taraxacifolia extract has a possible reno-protective action since it reversed the increase in creatinine, urea, total protein, electrolytes and malondialdehyde levels caused by gentamicin sulphate.

However;

The extract caused a sedative effect as observed in the reduced CNS activity and increased sleeping time.

The extract appears to induce constipation.

The extract inhibits the CYP450 level which is likely to affect the metabolism of other drugs.

4.3 FUTURE WORK

Efforts should be made to fully investigate the mechanisms involved in the pharmacological activities of Launaea taraxacifolia. Isolation and characterization of the active constituents of Launaea taraxacifolia should be done. The isolated compound may serve as useful prototype of antidiarrhoeal and reno-protective drugs of natural origin.

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