A study on the antioxidant and hepatoprotective activities of Cleome Viscosa Seed Extract

TABLE OF CONTENT

DECLARATION

CERTIFICATION

DEDICATION

ACKNOWLEDGEMENTS

ABSTRACT

TABLE OF CONTENT

LIST OF TABLES

LIST OF FIGURES

ABBREVIATIONS

CHAPTER ONE
INTRODUCTION
1.1 BACKGROUND
1.2 PROBLEMS STATEMENT/JUSTIFICATION
1.3 AIM OF PROJECT WORK
1.4 OBJECTIVES OF PROJECT WORK
1.5 SIGNIFICANCE OF PROJECT WORK

CHAPTER TWO
LITERATURE REVIEW
2.1 ANTIOXIDANT STUDY
2.1.1 Mechanism of Antioxidants
2.1.2 Classification of Antioxidants
2.1.3 Concept of Oxidative Stress
2.1.5 Generation of Free Radicals
2.1.6 Uses of Antioxidants in Technology
2.1.7 Methods of Antioxidant Capacity Determination
2.2.1 Mechanism of Hepatotoxicity
2.2.3 Drug metabolism in the liver
2.2.5 Diagnosis of Liver Damage
2.2.9 Over View of Carbon Tetrachloride Induced Hepatoprotective Model
2.3.1 Alkaloids
2.3.2 Glycosides
2.3.6 Terpenes
2.3.7 Steroids

CHAPTER THREE
MATERIALS AND METHODS
3.1 MATERIALS
3.1.1 Plant samples, experimental animals and DNA sample
3.2.1 Collection and Processing Of Sample
3.2.2 Identification and authentication
3.2.3 Preparation of ethanolic seed extract of cleome viscosa plant
3.2.4 Qualitative phytochemical screening
3.2.5 Hepatoprotective Test

CHAPTER FOUR
RESULTS AND DISCUSSION
4.1 QUALITATIVE PHYTOCHEMICAL SCREENING
4.2.1 DPPH Radical Scavenging assay
4.2.3 DNA Protection Assay
4.3 HEPATOPROTECIVE ACTIVITY
4.3.2 Weight of Wistar Albino Rats before the Experiment
4.3.3 Weight of Wistar Albino Rats after the Experiment Period
4.3.4 Effect of Cleome viscosa extract on serum level (AST) against carbon tetrachloride induced liver toxicity in normal albino rats
4.5 CONCLUSION

REFERENCES

APPENDICES

DEDICATION

This thesis is dedicated to our family and friends for their prayers and support throughout these years.

ACKNOWLEDGEMENTS

We are most appreciative to the Lord Almighty for guidance and protection throughout the period of this project. Our heartfelt thanks go to our supervisor, Dr. Addai-Mensah Donkor for his suggestions and corrections which made this work a success. We would like to give special thanks to the Department of Applied chemistry and Biochemistry for encouragement and assistance. We also want to express our gratitude to all the staffs of pharmacology department of Kwame Nkrumah University Science and Technology, Kumasi for their guidance and encouragement throughout the project. Last but not the least; we would like to thank our families for their help and unconditional support.

ABSTRACT

Cleome viscosa belongs to the family “Capariceae”, which is also known as wild mustard, dog mustard. It is an annual plant which is found all over the world and used as a medicinal plant due to its many biological properties. Phytochemical, In vitro antioxidant and in vivo hepatoprotective studies were carried out on the ethanolic extract of Cleome viscosa seeds. Preliminary phytochemical screening of extract revealed the presence of various secondary metabolites, namely: flavonoids, saponins, glycosides, tannins, steroids and terpenoids. The antioxidant activity of the extract was carried out using the DPPH radical scavenging and DNA protection assay and the results suggest that, Cleome viscosa seeds could have a great importance as therapeutic agent in preventing the progress of oxidative stress associated with degenerative diseases. The results of carbon tetrachloride induced liver toxicity experiment showed that rats treated with plant extract (200 mg/kg and 400 mg/kg), showed a significant decrease in ALT, AST, and ALP which were all elevated in carbon tetrachloride treated group (p < 0.05). Histopathological studies were observed in the photomicrographs of liver for treated and control groups to confirm the hepatoprotective activity of the extract. This project work on Cleome viscosa seeds concluded that the ethanolic extracts possess both antioxidant and hepatoprotective properties which could be attributed to the biologically relevant phytoconstituents found in the seeds

LIST OF TABLES

Table 2.1: The classification of Cleome viscosa has been reported

Table 3.1: List of animal groups in cch induced hepatotoxicity

Table 4.1: Results for qualitative phytochemical screening

Table 4.2: The DPPH inhibition potential of the various concentration of the plant extract

Table 4.3: IC50 values of standard and test extract of DPPH radical assay

Table 4.4: Weight of animals before experimental period

Table 4.4: Weight of animals before experimental period

Table 4.5: Weights of animals after experimental period

Table 4.6: Clinical biochemistry reference values

Table 4.7: Effect of Cleome viscosa seed extract on serum level (T. PRO, ALP, AST, ALP) against carbon tetrachloride induced liver toxicity in normal albino rats

LIST OF FIGURES

Figure 2.1: Classification of antioxidants

Figure 2.2: Implication of oxidative stress in multiple pathologies

Figure 2.4 : Drug metabolism in the liver

Figure 2.5: Structure of acetaminophen

Figure 2.6: Structure of silymarin

Figure 2.3 Healthy and damaged liver

Figure 2.7: Cleome viscosa Linn Plant

Figure 4.1: Scavenging effects of ethanolic extract of C. viscosa seeds and standard ascorbic acid on 2, 2-diphenyl-1-picrylhydrazyl radicals

Figure 4.2: Effect of ethanolic extract C. viscosa seed fractions on the protection of supercoiled pUC18 DNA against hydroxyl radical generated by the H2O2

Figure 4.3: Effect of Cleome viscosa seed extract on AST in carbon tetrachloride induced hepatotoxicity

Figure 4.4: Effect of Cleome viscosa seed extract on ALP in carbon tetrachloride induced hepatotoxicity

Figure 4.5: Effect of Cleome viscosa seed extract on total protien in carbon tetrachloride induced hepatotoxicity

Figure 4.6: Effect of Cleome viscosa seed extract on ALT in carbon tetrachloride induced hepatotoxicity

Figure 4.7: Photomicrograph (x100) showing histopathological profile of the livers of rats treated with CCL Cleome viscosa seed extract treatment

ABBREVIATIONS

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CHAPTER ONE

INTRODUCTION

1.1 BACKGROUND

Cleome viscosa which is also known as wild mustard, dog mustard, is an annual plant which is found in all over the world and used as a medicinal plant due to its many biological activities (Chandra, 2012). The herb has yellow flowers and the pods are long and cylindrical and contains seeds. The color of seeds of the plant Cleome viscosa is brown to slightly black (Anburaj, 2011). The whole plant of Cleome viscosa consists of a wide variety of clinical constituents which have different pharmacological actions. Its pantropical weed occurs in woodland, grassland, and wasteland and often occurs on sandy soil. This plant is found in both seasonal and humid conditions, sometimes on rocky and calcareous soil. This herb grows up to 1m high. The young shoots and leaves of Cleome viscosa are eaten as a cooked vegetable (Lakshmi, 2012). Antioxidants found in plants play an important role in the treatment and the prevention of the oxidative stress diseases (Bangou, 2012).

Plants have been an important source of molecule for thousands of years. Even today the World Health Organization (WHO) estimate that up 80 % of people still rely on traditional remedies such as herbs for their medicine (World Health Organization, 1997). The World Health Organization (2001) defines medicinal plants as herbal products produced by subjecting plant materials to extraction, fractionating, purification, concentration or other physical or biological processes 'which may be produced for immediate consumption or as basis for herbal products.

The increasing acceptability of the use of herbal remedies in the treatment of various condition of oxidative stress, including diabetes, obesity, cancer, atherosclerosis and ageing must be closely accompanied by the assessment of the safety of these remedies. Despite the availability of known antioxidant medicine in the pharmaceutical market, oxidation and the related complications continue to be a major medical problem. This therefore has call for research into natural sources for antioxidants. The medicinal values of Cleome viscosa are due to the presence of flavonoids and other polyphenols compounds which also gives it an antioxidant property. Antioxidant is an inhibitor of the process of oxidant, even at relatively small concentration and thus have diverse physiological role in the body (Sharma, 2014). Antioxidant constituent of plant materials acts as radicals’ scavengers and converts the radicals to less reactive species. Spices and herbs in food as medicine is a current hot trend that is capturing everyone’s imagination with images of a new magic bullet or fountain of youth. The intake of antioxidant compounds present in food is an important health-protecting factor. The increasing interesting in the search for natural replacements for synthetic antioxidant has led to the antioxidant evaluation of a number of plants foods. Plants and other organism have in built range of mechanism to combat these free radicals’ problem. In plant and animals these free radicals are deactivated by antioxidants. These antioxidants act as an inhibitor of the process of oxidation, even at relatively small concentration and thus have diverse physiological role in the body (Molyneux, 2004).

Liver is an important component of the mammalian system. It is involved in many functions related to digestion, metabolism, immunity, and the storage of nutrient within the body. Despite these physiological functions, the liver is also involved in the metabolism and excretion of xenobiotic substances such as drug. It is also plays an effective role in the protection of the human system against the hazard of harmful drugs and chemicals (xenobiotics and toxins). Excessive free radicals are generated from these harmful drugs and chemicals during their metabolism (Ward and Delay, 1999). Metabolism of these foreign molecules generates free radicals which if the biological system is not able to scavenge, attack the liver and cause injury to it.

Many different mechanisms lead to hepatoxicity which include disruption of the cell membrane and cell death resulting from covalent binding of the drugs to cell protein that creates new adduct that serve as immune targets, thus inciting immunologic reactions: inhibition of cellular pathways of drug metabolism ; abnormal bile flow resulting from disrupting of subcellular actin filament or interruption of transport pumps, leading to cholestasis’ and jaundice, sometimes with minimal cell injury; programed cell death, occurring through tumor necrosis-factor and Fas signal pathways, and inhibition of mitochondria function, with accumulation of reactive oxygen species and lipid peroxidation, fat accumulation and cell death. Plants and other derivatives play an important role in world health and have long being identified to possess biological activity. 30 % of all modern drugs are derived from plants (Victor and john, 2006).

Liver diseases are mainly caused by toxic chemicals, overdose of drugs (paracetamol, carbon tetrachloride, anti-cancer drugs, antibiotic and oral contraceptives) excessive consumption of alcohol, infections and auto immune disorders. Most of the hepatotoxic chemicals damage liver cells mainly by inducing lipid peroxidation and other oxidative damages (Rosario, 2009).

1.2 PROBLEMS STATEMENT/JUSTIFICATION

Liver diseases are mainly caused by toxic chemicals, over dose of drugs such as carbon tetrachloride and excessive consumption of alcohol. Previous studies have shown that liver damages have been induced by hepatotoxic chemicals such as carbon tetrachloride. This give rise to serum level AST and ALT and cholesterol which give rise to damage of structural integrity of the liver cells. (Rajaraman, 2016).

Cellular ageing and inflammatory diseases have been caused by free radicals (Saravanan, 2016). Free radicals are reactive species of oxygen. Free radicals also lead to oxidative stress which is defined as loss of balance between oxidizing and antioxidant agents within the cell.

The plant has traditionally been used for hepatoprotective activities, hence this research intends to validate the plant hepatoprotective activity. The abundance and the numerous medicinal properties of the plant has also urge us to undertake this study.

1.3 AIM OF PROJECT WORK

To study the antioxidant and hepatoprotective activity of Cleome viscosa seed extract.

1.4 OBJECTIVES OF PROJECT WORK

- To determine the Phytochemical constituents of the seed extract
- To evaluate the hepatoprotective activity of Cleome viscosa seed extract against carbon tetrachloride induced hepatotoxicity in experimental animal models.
- To determine the antioxidant activity of the seed extract of Cleome viscosa.
- To compare the antioxidant potential of the extract with the standard.

1.5 SIGNIFICANCE OF PROJECT WORK

According to WHO, about 46 % of the global disease and 59 % of the mortality is because of liver disease and almost 350 million people die in the world of liver diseases (Murray and Lopez, 1996). Liver diseases are increasing over year; recognized as the second leading cause of mortality among all digestive diseases in the United States (Murray and Lopez, 1996). The number of people that depend on drugs such as acetaminophen for the relief of pain and other ailments are great. The success of this research will provide an alternative means to curb and treat this liver diseases caused during the metabolism of these drugs.

Antioxidants significantly delay or prevent oxidation of oxidizable substrates when present at lower concentrations than the substrate. Oxidative stress has been identified as the root cause of the development and progression of several diseases. Supplementation of exogenous antioxidants or boosting endogenous antioxidant defenses of the body is a promising way of combating the undesirable effects of reactive oxygen species (ROS) induced oxidative damage. Cleome viscosa plants have an innate ability to biosynthesize a wide range of non-enzymatic antioxidants capable of attenuating ROS- induced oxidative damage (Halliwell, 2007).

CHAPTER TWO

LITERATURE REVIEW

2.1 ANTIOXIDANT STUDY

An antioxidant is a substance that slows down or inhibits oxidation reaction, especially in biological materials or within cells thereby reducing spoilage or preventing cell damage. The main function of antioxidant is trapping the free radical particularly reactive oxygen species and reactive nitrogen species which are involved in the pathogenesis of several chronic and degenerative diseases such as cardiovascular diseases, inflammation, neurodegenerative diseases, aging- related disorders and cancer. Antioxidant works by slowing or preventing the oxidation of other molecules by free radicals. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reaction produces free radicals, which starts chain reaction that damage cells. Antioxidant terminates these chain reactions by removing free radicals intermediate and inhibits other oxidation reaction by being oxidized themselves (Droge, 2002).

In healthy individuals, free radicals and other reactive oxygen species are neutralized by antioxidant defense mechanism in the body, including superoxide dismutase and glutathione. However, endogenous systems may not provide sufficient protection in individuals suffering from certain disease; in such cases help from exogenous sources is important (Lee et al., 2004). To overcome this problem, antioxidant compounds in plants have been promoted as having an important role as a health-protecting factor. Hence in recent years, lots of researches have been conducted in order to investigate the natural antioxidant activity of plant extracts.

Phenolic compounds are among phytochemicals in plant extracts that may render their effects via inhibiting the oxidation reaction caused by oxidative stress and relief its consequences. The antioxidant effect of phenolic compound in plant is due to a direct free radical scavenging activity, reducing activity and an indirect effect arising from chelating of pro-oxidant metal ions. The chelating of metal ions generally requires ortho­dihydroxylation on the phenyl ring in phenolic acids and flavonoids or the presence of 3- or 5-hydroxyl group in flavonoids (Halliwell, 1996). Among plant materials, fruits and vegetables contain phenolic, mainly belonging to the flavonoids family.

2.1.1 Mechanism of Antioxidants

Traditionally, antioxidants have been grouped into two categories, primary (chain­breaking antioxidants) and secondary (preventive antioxidants). Secondary antioxidants are chemical compounds that hinder the oxidation rate. This can be achieved in several ways which include singlet oxygen quenching or removal of substrate (Antolovich et al., 2002). Antioxidants exert their activity by scavenging the free-oxygen radical thereby giving rise to a fairly stable radical. These radicals if not scavenged in time may damage crucial biomolecules such as lipids, proteins including those present in all membranes, mitochondria and the DNA resulting in abnormalities leading to disease conditions (Uddin et al., 2008).

2.1.2 Classification of Antioxidants

Antioxidants are molecules that inhibit cellular damage. They exist in both enzymatic and non-enzymatic forms in the intracellular and extracellular environment. Intake of xenobiotics increase the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Nimse and Pal, 2015). They create oxidative stress and this oxidative stress can be neutralized by enhancing cellular defenses in the form of antioxidants. Antioxidants can be grouped based on their activity as being enzymatic and non-enzymatic antioxidants (Nimse and Pal, 2015).

- Enzymatic antioxidant

Enzymatic antioxidants work by breaking down and removing free radicals and enzymes such as glutathione peroxidase (GSHPx), Catalase (CAT), Superoxide dismutase (SOD) and peroxiredoxin (I-IV) (Nimse and Pal, 2015). These enzymes function as antioxidant by converting dangerous oxidative product to hydrogen peroxide (H2O2) in a multistep process in the presence of cofactors.

- Non-enzymatic antioxidant

Non- enzymatic antioxidants work by interrupting free radical chain reaction. Examples are carotenoids, vitamin C, plant polyphenol, and bioflavonoid.

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Figure 2.1: Classification of antioxidants (Carocho and Ferreira, 2013)

2.1.3 Concept of Oxidative Stress

Oxygen is essential for organisms. It is also harmful because of its ability to form reactive oxygen species (ROS) which lead to oxidative stress. Oxidative stress is defined as a misbalance in cell redox reactions (Sies, 1985). This is as a result of either overproduction of reactive oxygen or when there is decrease in antioxidant defense. Reactive oxygen species include all radical species such as superoxide anion ('O2-), hydroxyl radical (" OH) and also include non-radical species, such as hydrogen peroxide (H2O2), singlet oxygen (1O2) hipochloric acid (HOCL) and ozone (O3). Radicals are produced in controlled or uncontrolled manner. Oxidative stress affects all cell macromolecules, including DNA, causing mutations, proteins causing inactivation, and lipids causing lipid peroxidation (Wiseman, 1996). Oxidative stress leads to diseases such as ageing, cancer, atherosclerosis, and Diabetes mellitus.

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Figure 2.2: Implication of oxidative stress in multiple pathologies (Pham-Huy et al. 2008).

2.1.4 Concept of Nitrosative Stress

Nitric oxide is involved in many physiological conditions of the body. Under stress conditions, a family of NO-derived molecules, called reactive nitrogen species (RNS), can cause nitrosative stress. It is a state resulting from exposure to excessive levels of NO or the highly redox active proxy nitrite produced following interaction of NO with superoxide anions. The NO plays a pivotal role in plants and mammals, including the human organism, as a negative or positive regulator of cell apoptosis. The cytotoxicity of NO has been studied in various tumor models, both in vitro and in vivo (Corpas et al., 2014). Thus, the RNSs are fundamental regulators of oxidative metabolism in the cell. NO reacts rapidly with O2 and with superoxide radical (O2-) to generate a wide spectrum of RNSs that are highly damaging to cells (Errol, 2015). Studies indicate that mitochondrial permeability transition and NS represent major factors in copper-induced toxicity in astrocytes, and RNSs can cause neuronal injuries (Reddy, 2008).

2.1.5 Generation of Free Radicals

2.1.5.1 Reactive Oxygen Species (ROS)

The generation of reactive oxygen species begins with NADPH being activated in the reaction catalyzed by oxidase and the production of superoxide anion (Babior, 1999).

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Reactive oxygen species can also be generated from O2 and H2O2 via respiratory burst by Fenton equation and Haber-Weiss equation (Esra, 2012).

Fenton equation:

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The reactive oxygen species can also be generated by myeloperoxidase-Halide H2O2 system. The enzyme myeloperoxidase in the presence of chloride ion. H2O2 is converted to hypochlorus which is an oxidant (Babior, 1999).

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2.1.5.2 Reactive Nitrogen Species (RNS)

Reactive nitrogen Species (RNS) are produced by the enzyme oxide synthase from arginine (Esra, 2012).

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NO' and O2' react together to produce peroxynitrite (ONOO-) a very strong oxidant10

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Peroxynitrite is a porent oxidant which attack a variety of biological molecules. Free radicals lead to diseases like cancer, heart disease, and osteoporosis. Free radicals also attack biomolecules mainly the polyunsaturated fatty acids (PUFA) of cell membranes. This is known as lipid peroxidation and this is destructive to cell membranes. The general process of lipid peroxidation as depicted below, where LH is the target of the polyunsaturated fatty acid and R' as the initializing free radical. Oxidation makes reactive oxygen to form fatty radical L' which reacts with oxygen to form fatty acid peroxy radical (LOO'). These fatty acid radicals oxidize polyunsaturated fatty acid molecules and initiate new chain reactions (Shahidi, 2010).

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Oxygen metabolism also generates 'OH and H2O2. The 'OH is highly reactive and reacts with biological molecules such as DNA, proteins and lipids.

2.1.5.3 Modulation of Free Radicals by Natural Antioxidants

Two types of antioxidants namely the enzymatic antioxidants and non- enzymatic antioxidants modulate free radical reactions. The body protect itself from Reactive Oxygen Species by using antioxidant mechanism. The antioxidants reduce the level of lipid hydro peroxide and H2O2 and are important in the prevention of lipid peroxidation and maintaining the structure and cell membrane function (Koruk, 2004).

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Superoxide Dismutase (SOD) is present in the mitochondria and cytosol and converts O2' into H2O2 in the presence of cofactors such as copper, zinc, manganese. The enzyme Catalase (CAT) converts H2O2 to water and oxygen. Gluthatione Peroxidase (GSHPx) found in cytoplasm and converts H2O2 in water (Stocker, 1991).

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Radical scavenging activity of SOD, CAT, and GSHPx .

2.1.6 Uses of Antioxidants in Technology

- Industrial uses

Antioxidants are frequently added to industrial products. A common use is as stabilizers in fuels and lubricants to prevent oxidation, and in gasoline to prevent the polymerization that leads to the formation of engine-fouling residues (Hamilton, 1955).

- Food preservatives

Antioxidants are used as food additives to help guard against food deterioration. Exposure to oxygen and sunlight are the two main factors in the oxidation of food, so food is preserved by keeping in the dark and sealing it in containers or even coating it in wax, as with cucumbers. However, as oxygen is also important for plant respiration, storing plant materials in anaerobic conditions produces unpleasant flavors and unappealing colors (Cort, 1974). Antioxidants are an especially important class of preservatives as, unlike bacterial or fungal spoilage, oxidation reactions still occur relatively rapidly in frozen or refrigerated food (Zallen, 1975). These preservatives include natural antioxidants such as ascorbic acid and tocopherols (Inverson, 1995).

2.1.7 Methods of Antioxidant Capacity Determination

There are numerous methods for antioxidant capacity determination of samples (plant extracts, foods, commercial antioxidants etc) which are based on distinct principles. Low- density lipoproteins oxidation, Oxygen Radical Absorbance capacity (peroxyl radical scavenging ), Total radical trapping Antioxidant power, Cupric Reducing Antioxidant power, deoxyribose assay ( hydroxyl radical scavenging ), Ferric reducing power ( metal reducing power ) 2,2 Azino-bis (3-ethylbenz thiaxolime 6- sulfonic acid) ( organic radical scavenging ), DPPH 2,2-Diphenyl- 1-picryhydrazyl), Thiobarbituric Acid Reactive Substances (quantification of the products formed in the lipid peroxidation), (Marinova and Batchvarov, 2011).

Antioxidant capacity assays can be classified mostly as either hydrogen atom transfer (HAT) or single electron transfer (SET) based assays. Most of HAT assays are usually kinetic based and deals with a scheme of competitive reaction where substrate and antioxidant compete for free radicals which are thermally produced through azo compounds decomposition. SET assays determine an antioxidant capacity through an oxidant reduction which undergoes colour change when reduced (Marinova and Batchvarov, 2011).

2.1.7.1 Review of methods for determination of antioxidant activity

Several methods have been developed to monitor the total antioxidant capacity in biological samples. These assays differ in how the different radicals and or target molecules are generated and in the way the end points are measured (Roginsky, 2005). In investigating the antioxidant, activity of Cleome viscosa in this research, standard 2, 2- diphenyl-1-picrylhydrazyl (DPPH) assay and DNA protection was employed. DPPH is a stable commercially available organic nitrogen radicals with a dark purple color which when reduced becomes yellow (MacDonald, 2006). The DPPH assay is popular in natural product antioxidant studies. One of the reasons is that the method is simple, less expensive and sensitive (Lewis, 2012). The use of DPPH to measure antioxidant properties of compounds dates back to the 1950s. The assay is a decolonization assay based on the theory that a hydrogen donor is an antioxidant (Wood, 2006). It measures the capacity of compounds that acts as radical (DPPH) scavengers by monitoring its absorbance at 517 nm with a UV spectrophotometer (Lewis, 2012). The ability of extract to protect DNA from damaging effects of hydroxyl radicals generated by Fenton’s reagent was assessed by DNA nicking assay (Lee, 2002).

2.2 HEPATOCELLULAR (LIVER DAMAGE)

Hepatotoxicity (from hepatic toxicity) implies chemical-driven liver damage. Drug- induced liver injury is the cause of acute and chronic liver disease.

The liver plays a central role in transforming and clearing chemicals and is susceptible to the toxicity from these agents. Certain medicinal agents when taken in overdoses and sometimes even when introduced within therapeutic ranges, may injure the organ. Other chemical agents, such as those used in laboratories and industries, natural chemicals (e.g., microcystins) and remedies can also induce hepatotoxicity. Chemicals that cause liver injury are called hepatotoxins.

More than 900 drugs have been implicated in causing liver injury (Friedman, 2003) and it is the most common reason for a drug to be withdrawn from the market. Hepatotoxicity and drug-induced liver injury also account for a substantial number of compound failures, highlighting the need for drug screening assays, such as stem cell-derived hepatocyte-like cells, that are capable of detecting toxicity early in the drug development process (Friedman, 2003). Chemicals often cause subclinical injury to the liver, which manifests only as abnormal liver enzyme tests.

Drug-induced liver injury is responsible for 5 % of all hospital admissions and 50 % of all acute liver failures (Ostapowicz, 2002).

Adverse drug reactions are classified as type A (intrinsic or pharmacological) or type B (idiosyncratic). Type A drug reaction accounts for 80 % of all toxicities (Davies, 1986). Drugs toxins that have a pharmacological (type A) hepatotoxicity are those that have predictable dose-response curves (higher concentrations cause more liver damage) and well characterized mechanisms of toxicity, such as directly damaging liver tissue or blocking a metabolic process.

As in the case of acetaminophen overdose, this type of injury occurs shortly after some threshold for toxicity to be reached.

Idiosyncratic (type B) injury occurs without warning, when the causing agents are non- predictable. Hepatotoxicity in susceptible individuals is not related to dose and has a variable latency period. This type of injury does not have a clear dose-response or temporal relationship, and most often does not have predictive models. Idiosyncratic hepatotoxicity has led to the withdrawal of several drugs from the market even after rigorous clinical testing as part of the FDA approval process; Troglitazone (Rezulin) and trovafloxacin (Trovan) are trovafloxacin are two prime examples of idiosyncratic hepatotoxins pulled from the market (Zimmerman, 1978).

Drugs continue to be taken off the market due to late discovery of hepatotoxicity. Due to its unique metabolism and close relationship with the gastrointestinal tract, the liver is susceptible to injury from drugs and other substances. It is estimated that 75 % of blood coming to the liver arrives directly from gastrointestinal organs and then spleen via portal veins that bring drugs and xenobiotics in near-undiluted form. Several mechanisms are responsible for either inducing hepatic injury or worsening the damage process. Many chemicals damage mitochondria, an intracellular organelle that produces energy. Its dysfunction releases excessive amount of oxidants that, in turn, damage hepatic cells. Activation of some enzymes in the cytochrome P-450 system such as CYP2E1 also leads to oxidative stress (Jaeschke, 2002).

Injury to hepatocyte and bile duct cells lead to accumulation of bile acid inside the liver. This promotes further liver damage. Non-parenchymal cells such as Kupffer cells, fat storing stellate cells and leukocytes (i.e. neutrophil and monocyte) also have a role in the mechanism ( Patel, 1998).

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Figure 2.3 (Drriad, 2011)

2.2.1 Mechanism of Hepatotoxicity

Mechanism of drug-induced hepatotoxicity is variable and complex. Some drugs are directly toxic and begin to exhibit hepatotoxic reactions, which are dose related within hours of exposure whereas others may produce liver injury only in susceptible people and symptoms appear after few days or weeks. These reactions are rarely allergic or more accurately described as idiosyncratic. Drug-drug interaction, though the drugs may not be hepatotoxic themselves, might also play a critical role in the propagation of toxicity (Yamazaki et al., 2005).

The pathogenesis of drug-induced toxicity mediated by reactive metabolites has been a center of research enthusiasm since spearheading examinations in the 1950s uncovered the connection between these metabolites and chemical carcinogenesis (Park et al., 2005). A major cause of hepatotoxic reactions may be drug-induced intrahepatic cholestasis, which often occurs during the drug discovery and development process. The vital roles of ROS in the cellular damage are widely investigated and it has been suggested that the covalent binding of ROS as well as reactive intermediates to macromolecules could likely contribute to the severe harmful drug reactions (Recknagel et al., 1989).

There are several studies that suggest the generation of reactive metabolites and free radicals from hepatotoxic drugs (Reknagel et al., 1989). Membrane lipid peroxidation is directly related to the depletion of tissue GSH (an intracellular antioxidant) leading to the altered functional integrity of these structures and if the damage is severe, it could be fatal (Ross, 1988). Membrane lipid peroxidation may lead to alteration in membrane fluidity and permeability, enhanced rates of protein degradation, and ultimately cell death (Garcia et al., 1997). The assumption is upheld by the way that oxidative damage to erythrocytes causes loss of membrane capacity by enhancing lipid peroxidation (LPO) and modifying the erythrocyte antioxidant framework (Vajdovich et al., 1995). The concentration of intracellular GSH, therefore, is the key determinant of membrane integrity and the extent of toxicant-induced hepatic cell injury (Ross, 1988).

2.2.2 Signs and symptoms for specific hepatotoxicity

1. Jaundice
2. Itching
3. Easily bruising

2.2.3 Drug metabolism in the liver

The human body identifies almost all drugs as foreign substances (xenobiotics) and subjects them to various chemical processes (metabolism) to make them suitable for elimination. This involves chemical transformations to reduce fat solubility and to change biological activity. Although almost all tissues in the body have some ability to metabolize chemicals, smooth endoplasmic reticulum in the liver is the principal "metabolic clearing house" for both endogenous chemicals, example, cholesterol, steroid hormones, fatty acids, proteins and exogenous substances, example, drugs and alcohol. The central role played by liver in the clearance and transformation of chemicals makes it susceptible to drug-induced injury (Donald, 2006).

Drug metabolism is usually divided into two phases: phase 1 and phase 2. Phase 1 reaction is thought to prepare a drug for phase 2. However, many compounds can be metabolized by phase 2 directly. Phase 1 reaction involves oxidation, reduction, hydrolysation and many other rare chemical reactions. These processes tend to increase water solubility of the drug and can generate metabolites that are more chemically active and potentially toxic. Most of phase 2 reactions take place in the cytosol and involve conjugation with endogenous compounds via transferase enzymes.

Chemically active phase 1 products are rendered relatively inert and suitable for elimination by this step.

A group of enzymes located in the endoplasmic reticulum, known as cytochrome P-450, is the most important family of metabolizing enzymes in the liver. Cytochrome P-450 is the terminal oxidase component of an electron transport chain. It is not a single enzyme, but rather consists of a closely related family of 50 isoforms; six of them metabolize 90% of drugs. There is a tremendous diversity of individual P-450 gene products, and this heterogeneity allows the liver to perform oxidation on a vast array of chemicals (including almost all drugs) in phase 1(Sket, 2001). Drug metabolism in the liver: transferases are: glutathione, sulfate, acetate, glucuronic acid. P-450 is cytochrome P-450 enzymes. 3 different pathways are depicted for drugs A, B, and C

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Figure 2.4 : Drug metabolism in the liver (Drriad, 2011)

2.2.4 Causes of Liver Damage

Liver cells may become temporarily inflamed or permanently damaged by exposure to medications or drugs. Some medications or drugs require an overdose to cause liver injury while others may cause the damage even when taken in the appropriately prescribed dosage.

Taking excess amounts of acetaminophen (Tylenol, Panadol) can cause liver failure. For patients with underlying liver disease or those who abuse alcohol, that daily limit is lower and acetaminophen may be contra-indicated in those individuals. Statins are drugs commonly prescribed to control elevated blood levels of cholesterol. Even when taken in the appropriately prescribed dose, liver inflammation may occur. This inflammation can be detected by blood tests that measure liver enzymes. Stopping the medication usually results in return of the liver function to normal.

There are numerous other medications that may cause liver inflammation. These includes antibiotics such as nitrofurantoin , amoxicillin and clavulanic acid. Methotrexate, a drug used to treat autoimmune disorders and cancers, has a variety of side effects including liver inflammation that can lead to cirrhosis. Disulfiram (Antabuse) is used to treat alcoholics and can cause liver inflammation. Many mushrooms are poisonous to the liver and eating unidentified mushrooms gathered in the wild can be lethal. The term "hepatitis" means inflammation, and liver cells can become inflamed because of infection. Hepatitis A is a viral infection that is spread primarily through the fecal-oral route when small amounts of infected fecal matter are inadvertently ingested.

Hepatitis B is spread by exposure to body fluids (needles from drug abusers, contaminated blood, and sexual contact) and can cause an acute infection, but can also progress to cause chronic inflammation (chronic hepatitis) that can lead to cirrhosis and liver cancer.

Hepatitis C causes chronic hepatitis. Hepatitis C is spread by exposure to body fluids (needles from drug abusers, contaminated blood, and some forms of sexual contact).

Chronic hepatitis C may lead to cirrhosis and liver cancer. Other viruses can also cause liver inflammation or hepatitis. Viral infections with infectious mononucleosis and cytomegalovirus can inflame the liver. (Edlin, 2015).

Non-alcoholic fatty liver disease describes the accumulation of fat within the liver that can cause inflammation of the liver and a gradual decrease in liver function. Hemachromatosis (iron overload) is a metabolic disorder that leads to abnormally elevated iron stored in the body. The excess iron may accumulate in the tissues of the liver, pancreas, and heart and can lead to inflammation, cirrhosis, liver cancer, and liver failure. Hemochromatosis is an inherited disease.

Wilson's disease is another inherited disease that affects the body's ability to metabolize copper which leads to cirrhosis and liver failure. In Gilbert's disease, there is an abnormality in bilirubin metabolism in the liver. It is a common disease that affects up to 7 % of the North American population. There are no symptoms and it is usually diagnosed incidentally when an elevated bilirubin level is found on routine blood tests (Edlin, 2015).

- Acetaminophen-induced hepatotoxicity

Acetaminophen toxicity remains the most drug induced cause of hepatic liver failure. Most of the death that results from the overdose of this drug usually occurs about 7 days after ingestion. It was reported in a combined data from 22 special medical centers in the United States that acetaminophen related liver injury was the leading cause of acute liver failure for the year 1998 through 2003 (Larson et al., 205). This study stated above also found that high percentage of cases of acetaminophen liver injury were associated with unintentional overdose, in which the patient mistakenly takes too much acetaminophen. The result of this was further strengthened by later study in 2007 (Bower et al., 2007). Usually the lowest dose that causes hepatotoxicity is believed to be in the range of 125­150 mg/kg (Larson, 2007). Even though, 90 % of patients suffering from drug-induced hepatotoxicity recover by proper treatment and management, liver transplantation remains the only remedy for those unable to recover (Sang and Min, 2013).

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Figure 2.5: Structure of acetaminophen

- The mechanism of action of paracetamol-induced toxicity

During drug metabolism in humans, greater than 80 % of acetaminophen undergoes glucuronidation, sulfation through direct conjugation by glucuronyltransferases and sulfotranferase respectively to excrete this toxic non-metabolite through the urine. When the enzyme involved in the conjugation reaction becomes saturated, a little fraction (5 % to 10 %) of acetaminophen is metabolized by CYP450 into N-acetly-p-benzoquinone imine (NAPQI). NAPQI is subsequently detoxified by glutathione and further excreted from the biological system. When the glutathione gets depleted in the system, NAPQI accumulate in the hepatocytes thereby interacting with thio-containg proteins leading to hepatic necrosis (Corcoran et al., 1985)

2.2.5 Diagnosis of Liver Damage

The precise diagnosis of liver disease involves a history and physical examination performed by a health care professional. Understanding the symptoms and the patient's risk factors for liver disease will help guide any diagnostic tests that may be considered. Sometimes history is difficult, especially in patients who abuse alcohol. These patients tend to minimize their consumption, and it is often family members who are able to provide the correct information.

Liver disease can have physical findings that affect almost all body systems including the heart, lungs, abdomen, skin, brain and cognitive function, and other parts of the nervous system. The physical examination often requires evaluation of the entire body. Blood tests are helpful in assessing liver inflammation and function. Specific liver function blood tests include:

- AST and ALT (transaminase chemicals released with liver cell inflammation).
- GGT and alkaline phosphatase (chemicals released by cells lining the bile ducts).
- Protein and albumin levels.

Other blood tests which may be considered, including the following:

- Complete blood count (CBC), patients with end stage liver disease may have bone marrow suppression and low red blood cells, white blood cells and platelets. As a result, patients with cirrhosis may have bleeding.
- INR blood clotting function may be impaired due to poor protein production and is a sensitive measure of liver function.
- Lipase to check for pancreas inflammation.
- Electrolytes, BUN and creatinine to assess kidney function; and
- Ammonia blood level assessment is helpful in patients with mental confusion to determine whether liver failure is a potential cause.

Imaging studies may be used to visualize, not only the liver, but other nearby organs that may be diseased. Examples of imaging studies include:

- CT scan (computerized axial tomography),
- MRI (magnetic resonance imaging), and
- Ultrasound (sound wave imaging, which is especially helpful in assessing the gallbladder and bile ducts.

Liver biopsy may be considered to confirm a specific diagnosis of liver disease. Under local anesthetic, a long thin needle is inserted through the chest wall into the liver, where a small sample of liver tissue is obtained for examination under a microscope (Wedro, 1996).

2.2.6 Prevention of liver damage

With a large variety of conditions that cause liver inflammation, it’s important to prevent the disease or all that can be done is to treat symptoms as they arise. With a diet for liver disease, lifestyle changes and early diagnosis, you have the best chance at preventing and managing liver disorder, liver failure and liver cancer. But much can be done to prevent liver disease that is the result of a viral infection, or alcohol and drug abuse, or diet choices (Sonderup, 2015). Here are some things that can be done to prevent liver disease caused by the above-mentioned things:

- Maintaining a healthy weight

Fat accumulation in the liver because of an unhealthy diet can cause serious liver damage. It is known as non-alcoholic fatty liver disease and often affects people who are overweight or obese, according to the journal today’s Dietitian. Weight loss is recommended, as well as regular exercise and a healthy, low-fat, high fibre diet.

- Excellent hygiene

Good hygiene habits will go a long way to preventing hepatitis A. The virus is spread by coming into contact with infected faeces. It is essential to wash your hands after going to the toilet, and after changing a baby’s nappy. You also need to wash your hands before working with food. Boil your drinking water if you are not sure that it is clean.

- Vaccinations

You can be "vaccinated" against hepatitis by being given an injection of donated blood that contains immunoglobulins or antibodies to the hepatitis A and B viruses. This is not effective if you have already been infected. A vaccine against hepatitis A is also now available and is often used as a preventative mechanism for staff in day-care Centre’s or in health settings.

- Avoid close contact

Hepatitis B and C are blood-borne viruses. Hepatitis B is particularly infectious. Close contact (rough play amongst children, direct contact with the blood of an infected person, sharing of razors, unsterile tattooing instruments, sharing of needles among drug abusers, from mother to baby, unsafe sex) must be avoided in order to prevent infection with both hepatitis B and C.

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