EFFECT OF FRACTION OF ABRUS PRECATORIUS SEED METHANOL EXTRACT ON PARACETAMOL-INDUCED LIVER DAMAGE IN RATS

ABSTRACT

The aim of this study was to investigate the prophylactic and curative effects of an alkaloid-rich fraction of Abrusprecatorius seedchloroform-methanol extract on paracetamol-induced hepatotoxicity in rats. The percentage yield of the methanol extract of Abrusprecatorius seeds was found to be 2.08% w/w. Further purification of the extract using Sephadex gel G15 to get a purer sample was done. The fractions were spotted on a TLC plate and was spread with Drangendoff’s reagent in which some fractions turned purple indicating the presence of alkaloids. The fractions that turned purple were pulled into a beaker and called fraction I which gave a percentage yield of17.75% and was used in this study. The qualitative phytochemical analysis of fraction Irevealed a wide range of phytochemicals such asalkaloids, flavonoids, saponins, glycosides, tannins and carbohydrates, steroids, terpenoids, and peptides which could be physiologically potent in ameliorating several diseases. The quantitative phytochemical analysis of fraction I of Abrusprecatoriusseed methanol extract showed the presence of alkaloids (5480 ± 184 mg/100g), flavonoids (215 ± 97 mg/100g), saponins (2.98 ± 1.33 mg/100g) and tannins (6.4 ± 0.72 mg/100g). Hepatotoxicity was induced using paracetamol (2500 mg/kg b.w.) orally. For prophylactic treatment (hepato-protective), administration of extract was done for 7 days before paracetamol induction and collection of blood was done after 24 hours of administration. Curative treatment (hepato-curative) was done after paracetamol induction at day 0 and treatment was done for 14 days. Blood was collected on days 8 and 15 for the analyses.Prophylactic and curative treatments with fraction I of Abrusprecatorius methanol extract at the dose of 100 and 200mg/kg b.w for group 4 and 5 produced a significant decrease (p˂0.05) in the activities of the liver marker enzymes (ALP, AST, ALT) and bilirubin levels in a dose- and time-dependent manner compared to the paracetamol untreated group 2 (positive control).Groups 3, 4, and 5 treated with 100 mg/kg b.w.silymarin (standard hepato-protective and curative drug),100mg/k.g. b.w. of fraction I and 200mg/k.g. b.w. of fraction I respectively before and after paracetamol induction caused a significant decrease (p˂0.05) in the serum urea and creatinine concentrations of both hepato-protective and hepato-curative groups compared to the positive control. Serum electrolyte concentrations showed a significant increase (p˂0.05) in the treated groups of both hepato-protective and curative when compared to the positive control.The MDA concentration decreased significantly (p˂0.05) in the treated groups and standard groups compared to the positive control after 24 hours (hepato-protective)and at day 8 and 15 (hepato-curative). Serum SOD activity of both protective and curative models, showed adose- and time-dependent significant increase (p˂0.05) in the treated groups compared to the positive control. The haematological parameters of the rats treated with fraction I of Abrusprecatorius methanol extract at various dosesshowed a significant increase (p˂0.05) in the PCV levels, Hb concentration and RBC count compared to the positive control. A dose- and time-dependentsignificant decrease (p˂0.05) was observed in the WBC count of all treated groups (hepato-protective and hepato-curative) compared to the positive control. The test groups that received fraction I of Abrusprecatoriusin both models showed a dose- and time-dependent effects on the biochemical markers used in the study similar to the standard drug. However,fraction I had more curative effect than protective but silymarinwas more potent.

 

 

TABLE OF CONTENTS

Title page                            –   –           –           –           –           –           –           –           –           i

Certification                       –   –           –           –           –           –           –           –           –           ii

Dedication                          –   –           –           –           –           –           –           –           –           iii

Acknowledgement             –   –           –           –           –           –           –           –           –           iv

Abstract                              –   –           –           –           –           –           –           –           –           vi

Table of contents                –   –           –           –           –           –           –           –           –           vii

List of figures-                    –   –           –           –           –           –           –           –           –           xii

List of tables-                     –   –           –           –           –           –           –           –           –           xiv

CHAPTER ONE: INTRODUCTION

1.1       General Description of AbrusprecatoriusLinn            –           –           –           –           1

1.1.1    An Overview of AbrusprecatoriusSeed         –           –           –           –           –           2

1.1.2    Taxonomy of Abrusprecatorius Linn  –           –           –           –           –           3

1.1.3    Importance of Abrusprecatorius Linnin Traditional Medicine           –           –           3

1.1.4    Pharmacological Uses of Abrusprecatorius    –           –           –           –           –           4

1.1.5    Toxicity of Abrusprecatorius  –           –           –           –           –           –           5

1.2       Phytochemicals           –           –           –           –           –           –           –           –           6

1.2.1    Alkaloids         –           –           –           –           –           –           –           –           –           6

1.2.2    Flavonoids      –           –           –           –           –           –           –           –           –           7

1.2.3    Tannins            –           –           –           –           –           –           –           –           –           7

1.2.4    Steroids           –           –           –           –           –           –           –           –           –           8

1.2.5    Glycosides      –           –           –           –           –           –           –           –           –           8

1.3       Acetaminophen (Paracetamol)            –           –           –           –           –           –           9

1.3.1    Pharmacokinetics           –              –              –              –              –              –              –              –              9

1.3.2    Metabolism of Paracetamol    –           –           –           –           –           –           –           10

1.3.3    Mechanism of Action             –           –           –           –           –           –           –           –           11

1.3.4    Toxicity of Paracetamol          –           –           –           –           –           –           –           11

1.4       The Liver         –           –           –           –           –           –           –           –           –           14

1.4.1    Anatomy/Physiology of the Liver      –           –           –           –           –           –           14

1.4.2    Liver Intoxication (Hepatotoxicity)    –           –           –           –           –           –           14

1.4.3    Mechanism of Liver Damage –           –           –           –           –           –           –           15

1.5       Liver Function Tests   –           –           –           –           –           –           –           –           15

1.5.1    Alkaline Phosphatase –           –           –           –           –           –           –           –           16

1.5.2    Aspartate Aminotransferase   –           –           –           –           –           –           –           16

1.5.3    Alanine Aminotransferase      –           –           –           –           –           –           –           17

1.5.4    Bilirubin          –           –           –           –           –           –           –           –           –           17

1.6       Kidney Function Tests            –           –           –           –           –           –           –           17

1.6.1    Urea    –           –           –           –           –           –           –           –           –           –           18

1.6.2    Creatinine        –           –           –           –           –           –           –           –           –           18

1.6.3    Sodium Ion     –           –           –           –           –           –           –           –           –           19

1.6.4    Potassium Ion –           –           –           –           –           –           –           –           –           19

1.6.5    Chloride Ion    –           –           –           –           –           –           –           –           –           19

1.7       Antioxidants   –           –           –           –           –           –           –           –           –           19

1.7.1    Superoxide Dismutase –          –           –           –           –           –           –           –           20

1.8       Lipid Peroxidation and Tissue Damage          –           –           –           –           –           20

1.9       Haematology   –           –           –           –           –           –           –           –           –           21

1.9.1    Haemoglobin Count    –           –           –           –           –           –           –           –           21

1.9.2    Packed Cell Volume (PCV)    –           –           –           –           –           –           –           22

1.9.3    Red Blood Cell (RBC) Count            –           –           –           –           –           –           –           22

1.9.4    White Blood Cell (WBC) Count        –           –           –           –           –           –           22

1.10     Aim of the Study        –           –           –           –           –           –           –           –           22

1.11     Specific Research Objectives  –           –           –           –           –           –           –           23

 

CHAPTER TWO: MATERIALS AND METHODS

2.1       Materials         –           –           –           –           –           –           –           –           –           24

2.1.1    Animals           –           –           –           –           –           –           –           –           –           24

2.1.2    Plant Materials            –           –           –           –           –           –           –           –           24

2.1.3    Drugs  –           –           –           –           –           –           –           –           –           –           24

2.1.4    Instruments/Equipment           –           –           –           –           –           –           –           24

2.1.5    Chemicals and Reagents         –           –           –           –           –           –           –           24

2.2       METHODS     –           –           –           –           –           –           –           –           –           25

2.2.1    Extraction of Abrusprecatorius Seeds            –           –           –           –           –           25

2.2.2    Determination of Extract Yield          –           –           –           –           –           –           26

2.2.3    Fractionation   –           –           –           –           –           –           –           –           –           26

2.2.4    Thin Layer Chromatography   –           –           –           –           –           –           –           26

2.2.5    Visible Spectroscopy  –           –           –           –           –           –           –           –           26

2.2.6    Determination of Fraction Yield        –           –           –           –           –           –           26

2.2.7      Qualitative Phytochemical Analysis of the DifferentFractions of Abrus

precatoriusSeedMethanol Extract      –           –           –           –           –           –           27

2.2.7.1 Test for Alkaloids       –           –           –           –           –           –           –           –           27

2.2.7.2 Test for Flavonoids     –           –           –           –           –           –           –           –           27

2.2.7.3 Test for Saponins        –           –           –           –           –           –           –           –           27

2.2.7.4 Test for Tannins          –           –           –           –           –           –           –           –           27

2.2.7.5 Test for Carbohydrates                       –           –           –           –           –           –           28

2.2.7.6 Test for Terpenoids and Steroids        –           –           –           –           –           –           28

2.2.7.7 Test for Peptides         –           –           –           –           –           –           –           –           28

2.2.7.8 Test for Glycosides     –           –           –           –           –           –           –           –           28

2.2.7.9 Test for Resins                        –           –           –           –           –           –           –           29

2.2.7.10Test for Reducing Sugars      –           –           –           –           –           –           –           29

 

2.2.8    Quantitative Phytochemical Analysis of the DifferentFractions of Abrus

precatoriusSeed Methanol Extract –   –           –           –           –           –           –           29

2.2.8.1 Determination of Alkaloids    –           –           –           –           –           –           –           29

2.2.8.2 Determination of Flavonoids  –           –           –           –           –           –           –           29

2.2.8.3 Determination of Saponins     –           –           –           –           –           –           –           29

2.2.8.4 Determination of Tannins       –           –           –           –           –           –           –           30

2.2.9    Toxicological Studies (Acute Toxicity Test)  –           –           –           –           –           30

2.2.10  Induction of Liver Damage    –           –           –           –           –           –           –           30

2.2.11  Experimental Design   –           –           –           –           –           –           –           –           30

2.2.12  Preparation of Sample Solutions         –           –           –           –           –           –           31

2.2.12.1Preparation of Normal Saline             –           –           –           –           –           –           31

2.2.12.2Preparation of Phosphate Buffer       –           –           –           –           –           –           31

2.2.12.3Preparation of Stock Solution                        –           –           –           –           –           31

2.2.12.4Preparation of Drug Solution             –           –           –           –           –           –           31

2.2.12.5Preparation of Serum Samples                       –           –           –           –           –           31

2.2.13  Assay of Alkaline Phosphatase Activity        –           –           –           –           –           32

2.2.14  Assay of Aspartate Aminotransferase Activity          –           –           –           –           32

2.2.15  Assay of Alanine Aminotransferase Activity             –           –           –           –           33

2.2.16  Determination of Total Bilirubin Concentration         –           –           –           –           35

2.2.17  Determination of Serum Urea Concentration             –           –           –           –           35

2.2.18  Determination of Serum Creatinine Concentration    –           –           –           –           37

2.2.19  Determination of Serum Sodium Ion Concentration  –           –           –           –           38

2.2.20  Determination of Serum Potassium Ion Concentration          –           –           –           39

2.2.21  Determination of Serum Chloride Ion Concentration                        –           –           –           39

2.2.22  Assay for Superoxide Dismutase (SOD) Activity                  –           –           –           40

2.2.23  Determination of Malondialdehyde   –           –           –           –           –           –           40

2.2.24  Determination of Haemoglobin Concentration          –           –           –           –           42

2.2.25  Determination of Packed Cell Volume (PCV)           –           –           –           –           42

2.2.26  Determination of Red Blood Cell (RBC) Count                    –           –           –           42

2.2.27  Determination of Total White Blood Cell (WBC) Count      –           –           –           43

2.3       Statistical Analysis      –           –           –           –           –           –           –           –           43

CHAPTER THREE: RESULTS

3.1       Percentage Yield of Extract   –           –           –           –           –           –           –           44

3.2       Result of Acute Toxicity Studies (LD₅₀)        –           –           –           –           –           46

3.3       Detection of Fraction              –           –           –           –           –           –           –           48

3.4       Percentage Yield of Fraction I and II            –           –           –           –           –           –           50

3.5       Qualitative phytochemical composition of fractions 1 and II of Abrus

precatorius seed extract          –           –           –           –           –           –           –           52

 

3.6       Quantitative phytochemical composition of fractions 1 and II of Abrus

precatoriusseed extract           –           –           –           –           –           –           –           54

 

3.7       Effect of fraction I of Abrusprecatoriusseed methanolextract on alkaline

phosphatase(ALP) activities in paracetamol-intoxicated rats.            –           –           56

 

3.8       Effect of fraction I of Abrusprecatoriusseed methanol extracton aspartateaminotransferase (AST) activities in paracetamol-intoxicated rats.          –           –           58

 

3.9       Effect of fraction I of Abrusprecatoriusseed methanol extracton alanine

aminotransferase(ALT) activities in paracetamol-intoxicated rats.    –           –           60

 

3.10     Effect of fraction I of Abrusprecatoriusseed methanol extracton total

bilirubin levelsin paracetamol-intoxicated rats            –           –           –           –           62

 

3.11     Effect of fraction I of Abrusprecatoriusseed methanol extracton serum urea

concentrationin paracetamol-intoxicated rats.            –           –           –           –           64

 

3.12     Effect of fraction I of Abrusprecatoriusseed methanol extract on serum

creatinineconcentration in paracetamol-intoxicated rats.        –           –           –           66

 

3.13     Effect offraction I ofAbrusprecatoriusseed methanol extracton the sodium

ionconcentration in paracetamol-intoxicated rats.      –           –           –           –           68

 

3.14     Effect offraction I ofAbrusprecatoriusseed methanol extract on the potassium

ionconcentration in paracetamol-intoxicated rats       –           –           –           –           70

 

 

3.15     Effect offraction I ofAbrusprecatoriusseed methanolextract on the chloride

ionconcentration in paracetamol-intoxicated rats.      –           –           –           –           72

 

3.16     Effect of fraction I of Abrusprecatoriusseed methanolextract on the superoxide dismutase (SOD) activities in paracetamol-intoxicated rats.          –           –           –           74

 

3.17     Effect of fraction I of Abrusprecatoriusseed methanol extract on the

malondialdehyde (MDA) concentration in paracetamol-intoxicated rats      –           76

 

3.18     Effect of fraction I of Abrusprecatoriusseed methanolextract on the

haemoglobinconcentration in paracetamol-intoxicated rats    –           –           –           78

 

3.19     Effect of fraction I of Abrusprecatoriusseed methanol extracton the packed

cell volume (PCV) concentration in paracetamol-intoxicated rats.    –           –           80

 

3.20     Effect of fraction I of Abrusprecatoriusseed methanolextract on the red

blood cell count (RBC) in paracetamol-intoxicated rats                    –           –           82

 

3.21     Effect of fraction I of Abrusprecatoriusseed methanol extract on the white

blood cell count (WBC) in Paracetamol-intoxicated rats       –           –           –           84

 

CHAPTER FOUR: DISCUSSION

4.1       Discussion       –           –           –           –           –           –           –           –           –           86

4.2       Conclusion      –           –           –           –           –           –           –           –           –           91

4.3       Suggestions for Further Studies         –           –           –           –           –           –           91

References

Appendices

 

CHAPTER ONE

1.0 INTRODUCTION

Plants, the first medicine of human being, have played a remarkable role in health care since the ancient times. Traditionally plant-based medicines still exert a great deal of importance to the people living in developing countries and also lead to the discovery of new drugs for a variety of diseases that threatens human health. Plants are the rich sources of organic compounds, many of which have been used for medicinal purposes. Medicinal plants are the plants whose parts (leaves, seeds, stems, roots, fruits, foliage etc), extracts, infusions, decoctions or powders are used in the treatment of different diseases of humans, plants and animals (Nweze et al., 2004). There is a wide spectrum of trees, plants and shrubs whose seeds, roots, barks and leaves are used by humans throughout the globe due to their nutritional or medicinal value (Doughari et al., 2009). The importance of herbs in the management of human ailments cannot be over emphasized.

Herbs play a major role in the management of various liver disorders along with other system associated diseases (Ebenyi et al., 2012). Medicinal plants such as Aloe vera,Eclipta alba, Phyllanthus niruri, Solanum Indicum, Maytenus emerginata and Aegle mameloes are well known for their hepato-protective effects (Parmar et al., 2010). Abrus precatoriusLinn is a leguminous plant of the Fabaceae family. Its seeds, roots and leaves are widely used for medicinal purposes in Africa and Asia (Yadava and Reddy, 2002). In Nigeria, the Igbos use the aqueous decoction of the seeds to treat a wide range of conditions including ulcer, infections, hypertension, diarrhoea, infarct and ogbanje (Nwodo and Alumanah, 1991).

1.1.General Description of Abrus precatorius Linn

Abrus precatorius, which belongs to the family of fabeceae is a plant that originated from Southeast Asia and now can be found in subtropical climate areas such as India, Sri Laka, Thailand, the Philippine Islands, South China, Tropical Africa and the West Indies (Vavaprasad and Varahalarao, 2009). It is a slender, perennial climber that twines around trees, shrubs, and hedges. The leaves are pinnate and glabrous, with many leaflets (12 or more) arranged in pairs. Flowers are small and pale violet in colour with a short stalk, arranged in clusters. The plant is best known for its seeds, which are used as beads and in percussion instruments, and which are toxic due to the presence of abrin. The plant is native to Indonesia and grows in tropical and subtropical areas of the world where it has been introduced. It has a tendency to become weedy and invasive where it has been introduced.

1.1.1 An Overview ofAbrus precatorius Seed

The name Abrus, meaning beautiful or graceful, used to describe the appearance of the seed. The seed is found in a variety of colours such as black, brown, white and most commonly, red with a glossy appearance with the black band at the end that attaches to the plant. The Abrus precatorius seed is known by a variety of names that include jequirity (India), Crab’s eye (Guam), Rosary  pea (Egypt), Precatory peabean(USA), Indian Liquorice (Nigeria) and GiddeeGiddee or Jumbie bead in Trinidad and Tobago (Hartley,  2010). The seeds of Abrus precatorius are much valued in native jewery for their bright coloration. Most beans are black and red, suggesting a ladybug, though other colors are available. The Tamils use Abrus seeds of different colors. The red variety with black eye is the most common, but there are black, white and green varieties as well. The seeds of Abrus precatorius are very consistent in weight. Formerly Indians used these seeds to weigh gold using a measure called a Ratti, where 8 Ratti = 1 Masha; 12 Masha = 1 Tola (11.6 Grams) (Hartley, 2010).

Figure 1: Abrus precatoriusseeds

(Source: Ali and Malek, 1996)

 

 

 

 

1.1.2Taxonomyof Abrus precatorius Linn

Table 1: Taxonomic Hierarchy of Abrus precatorius 

Kingdom	Plantae – plantes, planta, vegetal, plants
Subkingdom	Viridaeplantae – green plants
Infrakingdom	Streptophyta – land plants
Division	Tracheophyta – vascular plants, tracheophytes
Subdivision	Spermatophytina –  spermatophytes, seed plants, phenerogames
Infradivision	Angiospermae – flowering plants, angiosperms
Class	Magnoliopsida
Superorder	Rosanae
Order	Fabales
Family	Fabaceae – peas, legumes
Subfamily	Faboideae
Genus	Abrus
Specie	Abrus precatorius Linn – rosarypea, crab’s eye, jeguerity,     precatory bean, giddee giddee, Indian liquorice.

(Source; Ali and Malek, 1996)

1.1.3 Importanceof Abrus precatorius Linnin Traditional Medicine

In the Ayurvedic medicine, leaves of Abrus precatorius are used as laxative, expectorant and aphrodisiac medicines known as Coq’s eye. Seeds are said to be purgative, emetic, tonic, antiphlogistic, aphrodisiac and antiopthalmic. For the indigenous people they are potent phytomedicines, many of them in mixture with other plants. Their toxicity is underestimated (Anant and Maitreyi, 2012)

In Tanzania, traditional healers claim the competence in the treatment of epilepsy. Abrus precatorius can be found between 60 plants commonly used against this illness (Moshi et al., 2005). Also in Zimbabwe, extracts of 58 plants popularly known to be effective against schistosomiasis were tested in vitro against excysted cysticercoids. Extracts of stem and root of Abrus precatorius were under the ten most effective samples (Ndamba and Nyazema, 1994).

In China, the herb of Abrus precatorius is used as a folk-medicine for the treatment of bronchitis, laryngitis and hepatitis. Because of their platelet inhibiting activity, abruquinones are suspected to be the active substances (Kuo et al., 1995).

The leaf of Abrus precatorius has been used in Nigeria for the treatment of myriad of diseases including malaria, typhoid, cough, respiratory tract infections and hepatitis (Saganuwan and Onyeyili, 2010).The leaves are also considered useful in biliousness and in leucoderma, itching and other skin diseases. Its juice is employed as a cure for hoarseness, mixed with oil and applied to painful swellings. Dried leaves paste are used as a germicide to wounds in cattle. The seeds are deadly poisonous but it has been reported that the toxic form of abrin gets converted to mitogenic form upon long refrigerated storage. Usually seeds are of two types one is scarlet with black spot and the other variety is pure white and traditionally used againstleucoderma, wounds, alopecia, asthma, tubercular glands, leprosy, fever, ulcer and tumor. Seeds of Abrus precatorius Linn are applied locally in sciatica, stiffness or shoulder joint and paralysis. It is useful in dysentery and skin diseases (Anant and Maitreyi, 2012).

Roots of Abrus precatorius Linn are used as diuretics and in the preparations prescribed for gonorrhoea, jaundice and haemoglobinuric bile. Some of the parts are used in night blindness, inflamed gums, muscular pain and convulsion. It is also used for pain relief in groins, mucus in urine and grave land bone fracture in cattle (Anant and Maitreyi, 2012).

1.1.4Pharmacolical Uses of Abrus precatorius

.The seed extract of abrus precatorius have also been shown to possess several pharmacological properties. It has been shown to have antifertility effect (Rao, 2007); ureterotonic effect and antidiarrhoeal effect (Nwodo, 1991). Aqueous extract of A. precatorius seeds has also been shown to exert antimicrobial activities (Desai and Sirsi, 1966).

It has been reported that Methanol extracts of Abrus precatorius exhibited antibacterial activity towards almost all the bacterial microorganisms (Klebsilla pneumonia, staphylococcus aureus, streptococcus mitis and Micrococcus luteus) used in the study (Varaprasad and Varahalarao, 2009). Also,Abrus precatoriushas Abortifacient effect (Sethiet al., 1990); Agglutinin activity (Lin et al., 1981); Cytotoxic activity (Desai et al., 1966); Anti-inflamatory activity (Anam, 2001); Insecticidal activity (Hartzell and Wilcoxon, 1941); Hemagglutinin activity (Khan  et al., 1966); Spermicidal effect (Rajeshwari, 2011); Uterine relaxation effect and Uterine stimulant effect amongst many others (Nwodo and Botting, 1983). Although,Abrus precatorius has been shown to have so many pharmacological activities, the presence of toxic lectins in its seed limits its pharmacological utility.1.1.5 Toxicity of Abrus precatoriusLinn

Abrus precatoriusbeans are known as one of the most toxic plant parts worldwide. The human fatal dose is estimated as 0.1-1 µg/kg(Monago and Alumanah, 2005). The toxicity of the Abrus seed is associated with the presence of the toxic component, Abrin (a type of toxalalbumin) which is a mixture of at least five lectins, abrin A – D, and Abrus-agglutinin(Chaudhari et al., 2012). The abrins consist of two peptide chains connected by a disulfide bridge.

Abrin A consists of an A-chain with N-glycosidase activity, which inhibits protein synthesis, and lectin-like B-chain responsible for binding with cell-surface receptors and penetrating of abrin-A molecule into the cell (Ohba and Morowaki, 2004). The relative molecular weight of abrin A and C are around 64.000Da, that of two agglutinins 128.000Da (Hegde et al., 1991). For furtheridentification, the crystal structure was investigated. The abrin A crystals belong to the monoclinic space group P 2 (Tahirov, 1994). The sequence of amino acids of the B-chain in both abrin-A and abrin-B were elucidated by enzymatic digestion with trypsin. They consist of 268 amino acids and share 256 identical residues (Komira et al., 1993). This chemical structure is assumed to be responsible for its toxic effects.

Abrins disarrange the proteinbiosynthesis by interfering with the 60 S-ribosomes of animal cells irreversibly. The toxicity of these abrins is variable, but they are the most potent toxins of the world, comparable with the botulinus toxin. The fifth of them, Abrus agglutinin, is not very toxic against cells, but it exhibits agglutination toward animal erythrocytes. Also it has been shown that Abrus agglutinin causes a total haemolysis in blood groups followed by a haemorrhagic gastroenteritis (Khanet al., 1966).

Fatal incidents have been reported following ingestion of well-chewed seeds of Abrus precatorius. It has also been reported that poisoning has been experienced through a finger prick when stringing the seed. Symptoms of seed poisoning include severe gastroenteritis with pronounced nausea and vomiting, muscular weakness, tachycardia, cold sweat, bloody diarrhoea, dyspnoea, dehydration, loss of condition and recumbence (Anant and Maitreyi, 2012).

There is no physiological antidote. The treatment is essentially symptomatic. Since there is a long latent period associated with abrin poisoning, little value can be placed on induction of emesis or gastric lavage; these measures are useful only if ingestion has just occurred. Bismuth trisilicate may be given during poisoning by Abrus precatorius to reduce the level of gastrointestinal damage. If the emesis and/or diarrhoea become excessive, replacement fluids and electrolytes are advocated. If haemorrhage occurs, blood transfusion may be necessary(Khan et al., 1966).

1.2.0 PHYTOCHEMICALS

Phytochemicals (from the Greek word phyto, meaning plant) are biologically active, naturally occurring chemical compounds found in plants, which provide health benefits for humans (Mamta et al.,2013).They protect plants from disease and damage and contribute to the plant’s color, aroma and flavor. In general, the plant chemicals that protect plant cells from environmental hazards such as pollution, stress, drought, UV exposure and pathogenic attack are called as phytochemicals (Narasinga, 2003). Phytochemistry is the study of natural bioactive products found in plants that work with nutrients and dietary fibre to protect against diseases (Doughari et al., 2009). Recently, it is clearly known that they have roles in the protection of human health, when their dietary intake is significant. In wide-ranging dietary phytochemicals are found in fruits, vegetables, legumes, whole grains, nuts, seeds, fungi, herbs and spices. Broccoli, cabbage, carrots, onions, garlic, whole wheat bread, tomatoes, grapes, cherries, strawberries, raspberries, beans, legumes, and soy foods are common sources (Mathai, 2000).

These phytochemicals are present in a variety of plants utilized as important components of both human and animal diets, and they are found in different parts of the plant which include; fruits, flower, bark seeds, root and stem(Tiwari et al.,2011). They are chemical compounds formed during the plant normal metabolic processes. These chemicals are often referred to as ‘secondary metabolites’ of which there are several classes including alkaloids, flavonoids, glycosides, gums, coumarins, polysaccharides, phenols, tannins, terpenes and terpenoids .

1.2.1   Alkaloids

Alkaloids are natural products that contains heterocyclic nitrogen atoms, are basic in character. The name of alkaloids derives from the “alkaline” and it was used to describe any nitrogen-containing base (Mueller-Harvey and McAllan, 1992). These are the largest group of secondary chemical constituents made largely of ammonia compounds comprising basically of nitrogen bases synthesized from amino acid building blocks with various radicals replacing one or more of the hydrogen atoms in the peptide ring, most containing oxygen.

Alkaloids are significant for the protecting andsurvival of plant because they ensure their survival against micro-organisms (antibacterial and antifungal activities), insects and herbivores (feeding deterrens) and also against other plants by means of allopathically active chemicals (Molyneuxet al., 1996). The useof alkaloids containing plants as dyes, spices, drugs or poisons can be traced back almost to the beginning of civilization. Alkaloids have many pharmacological activities including antihypertensive effects (many indole alkaloids), antiarrhythmic effect (quinidine, sardine), antimalarial activity (quinine), andanticancer actions (dimeric indoles, vincristine, and vinblastine). These are just a few examplesillustrating the great economic importanceof this group of plant constituents (Wink et al.,1998). Some alkaloids have stimulant property as caffeine and nicotine, morphine are used as the analgesic and quinine as the antimalarial drug (Rao et al.,1978).

1.2.2   Flavonoids

Flavonoids are important group of polyphenols widely distributed among the plant flora. Structurally, they are made of more than one benzene ring in its structure (a range of C15 aromatic compounds) and numerous reports support their use as antioxidants or free radical scavengers (Kar, 2007). The compounds are derived from parent compounds known as flavans. They are organic compounds that have no direct involvement with the growth or development of plants, they are plant nutrients that when consumed in fruits and vegetables pose no toxic effect on humans, and are also beneficial to the human body. Flavonoids are poly-phenolic compounds that are ubiquitous in nature (Harborne and Baxter, 1999). More than 4,000 flavonoids have been recognized, many of which occur in vegetables, fruits and beverages like tea, coffee and fruit drinks (Pridham, 1960).

Flavonoids can be classified into five major sub groups, these include; flavones, flavonoids, flavanones, flavonols and anthocyanidines (Nijveldt et al.,2001). Flavones are characterized by a planar structure because of a double bond in the central aromatic ring. Quercetin, one of the best described, is a member of this group. Quercetin is found in abundance in onions, apples, broccoli and berries. Flavonones are mainly found in citrus fruit, an example is narigin. Flavonoid is involved in scavenging of oxygen derived free radicals (Harborne and Baxter, 1999). It has been identified as a potent hypolipidemic agent in a number of studies (Tapas et al., 2008). It has also been established that flavonoids from medicinal plants possess a high antioxidant potential due to their hydroxyl groups and protect more efficiently against free radical related diseases like arteriosclerosis (Kris-Etherton et al.,2002).

1.2.3 Tannins                                          

Tannins are polymerized phenols with defensive properties. Their name comes from their use in tanning, rawhides to produce leather. In tanning, collagen proteins are bound together with phenolic groups to increase the hide’s resistance to water, microbes and heat (Hans-Walter and Fiona, 2005). Two categories of tannins that are of importance are the condensed and hydrolysable tannins. Though widely distributed, their highest concentration is in the bark and galls of oaks (Hans-Walter and Fiona, 2005). They are phenolic compounds of high molecular weight. Tannins are soluble in water and alcohol and are found in the root, bark, stem and outer layers of plant tissue. They are acidic in reaction and the acidic reaction is attributed to the presence of phenolics or carboxylic group (Kar, 2007).

Many human physiological activities, such as stimulation of phagocytic cells, host-mediated tumour activity, and a wide range of anti-infective actions, have been assigned to tannins (Haslam, 1996). One of their biological actions is to compete with proteins through non-specific forces such as hydrogen bonding and hydrophobic interactions, as well as by covalent bond formation (Haslam, 1996). Thus, their mode of antimicrobial action may be related to their ability to inactivate microbial adhesions, enzymes, cell envelope, transport proteins etc.

1.2.4 Steroids

Sterols are triterpenes which are based on the cyclopentane hydrophenanthrene ring system (Harborne, 1998). Sterols were at one time considered to be animal substances (similar to sex hormones, bile acids, etc) but in recent years, an increasing number of such compounds have been detected in plant tissues. Sterols have essential functions in all eukaryotes. For example, free sterols are integral components of the membrane lipid bilayer where they play an important role in the regulation of membrane fluidity and permeability (Galm and Shen, 2007). While cholesterol is the major sterol in animals, a mixture of various sterols is present in higher plants, with sitosterol usually predominating. Sterols in plants are generally described as phytosterols with three known types occurring in higher plants: sitosterol (formerly known as ß-sitosterol), stigmasterol and campesterol (Harborne, 1998).

1.2.5 Glycosides

Glycosides in general, are defined as the condensation products of sugars (including polysaccharides) with a host of different varieties of organic hydroxyl (occasionally thiol) compounds (invariably monohydrate in character), in such a manner that the hemi-acetal entity of the carbohydrate must essentially take part in the condensation. Glycosides are colorless, crystalline carbon, hydrogen and oxygen-containing (some contain nitrogen and sulfur) water-soluble phyto-constituents, found in the cell sap. Chemically, glycosides contain a carbohydrate (glucose) and a non-carbohydrate part (aglycone or genin) (Kar, 2007). Glycosides are neutral in reaction and can be readily hydrolyzed into its components with ferments or mineral acids. Glycosides are classified on the basis of type of sugar component, chemical nature of aglycone or pharmacological action (Firn, 2010).

 

1.3Acetaminophen (Paracetamol)

Fig 2:N-(4-hydroxyphenyl)acetamide (Acetaminophen)(Macintyreet al.,2008).

Acetaminophen chemically known as N-acetyl-p-aminophenol, is a widely usedanalgesic and antipyretic agent with little anti-inflammatory effect (McDaid et al.,2010).Acetaminophen is a white, odorless, crystalline powderwith a slightly bitter taste. It has a molecular formula of C₈H₉NO₂and molecular weight of 151.16 g.It is the most widely used drug for pain relief. Paracetamol is the International Non-proprietary Name (INN) and British Approved Name (BAN), while acetaminophen is the United States Adopted Name (USAN) and Japanese Adopted Name (JAN)(Macintyreet al., 2008).

Paracetamol is classified as a mild analgesic. It is commonly used for the relief of headaches and other minor aches and pains and is a major ingredient in numerous cold and flu remedies. In combination with opioid analgesics, paracetamol can also be used in the management of more severe pain such as post-surgical pain and providing palliative care in advanced cancer patients. Though paracetamol is used to treat inflammatory pain, it is not generally classified as an NSAID because it exhibits only weak anti-inflammatory activity(Macintyreet al., 2008).

1.3.1 Pharmacokinetics

In order for increase effectiveness, paracetamol canbe administered rectally, orally and intravenously. While all three mode of administration can achieve adequate plasma concentrations, there are differences in absorption and time to reach the plasma peak levels. With rectal administration, absorption can be unpredictable with bioavailability ranging from 24% to 98% varying with factors such as formulation of suppositories number used and the particle size of the paracetamol (McDaid et al.,2010). Oral bioavailability is dose dependant: with larger doses, the hepatic first pass effect is reduced due to overwhelming of the liver enzymatic capacity; and therefore, bioavailability is increased. In this case, bioavailability is inconsistent and in overall reduced, due to incomplete dissolution of the suppository in the rectum. The absorption rate through this route of administration is elongated.

The analgesic activity is attributable to the small fraction that penetrates into the brain(McDaid et al.,2010). Paracetamol given at therapeutic doses binds to plasma proteins at less than 20%. In case of intoxication, this proportion may increase to up to 50%(Huber et al., 2009). Paracetamol is essentially metabolized in the liver by conjugation with glucuronic acid (55%) and sulfuric acid (35%). Hepatotoxic metabolites are produced in small amounts by the cytochrome P450 (isoenzyme CYP2E1). In the therapeutic plasma concentration range, this metabolite is detoxified by conjugation with glutathione(Macintyreet al., 2008). In case of intoxication the amount of this toxic metabolite increases and outweighs the amount of available glutathione, which can lead to hepatic failure and renal tubular necrosis. Metabolites are excreted through the kidneys in the urine. Only 2-5% of the dose is excreted in an unchanged form in the urine. As a consequence of its short elimination half-life (1-3h), 24 hours after the ingestion of a single dose of paracetamol, 98% of the dose is eliminated(McDaid et al.,2010).

1.3.2 Metabolism of paracetamol.

Fig. 2: Metabolism of paracetamol, Source: (Huber et al., 2009)

Paracetamol is metabolised primarily in the liver through three metabolic pathways into toxic and non-toxic products. These pathways are glucuronidation, sulfation and N-hydroxylation (Huber et al.,2009)

• Glucuronidation is believed to account for 40% to two-thirds of the metabolism of paracetamol.

• Sulfation (sulfate conjugation) may account for 20–40%.
• N-hydroxylation and rearrangement, then GSH conjugation, accounts for less than 15%. The hepatic cytochrome P450 enzyme system metabolises paracetamol, forming a minor yet significant alkylating toxic metabolite known as NAPQI (N-acetyl-p-benzo-quinone imine)(also known as N-acetylimidoquinone) NAPQI is then irreversibly conjugated with the sulfhydryl groups of glutathione(Macintyreet al., 2008).

1.3.3 Mechanism of action

Acetaminophen, also known as paracetamol, is a non-steroidal anti-inflammatory drug withpotent antipyretic and analgesic actions but with very weak anti-inflammatory activity. When administered to humans, it reduces levels of prostaglandin metabolites in urine but does not reduce synthesis of prostaglandins by blood platelets or by the stomach mucosa. Paracetamol has long been suspected of having a similar mechanism of action with aspirin due to their similarity in structure. Because acetaminophen is a weak inhibitor in vitro of both cyclooxygenase (COX)–1 and COX-2, the possibility exists that it inhibits a so far unidentified form of COX, perhaps COX-3(Graham and Scott, 2005). In animal studies, COX enzymes in homogenates of different tissues vary in sensitivity to the inhibitory action of acetaminophen. This may be evidence that there are 12 isoforms of the enzyme. Recently, a variant of COX-2 induced with high concentrations of non-steroidal anti-inflammatory drugs was shown to be highly sensitive to inhibition by acetaminophen. Therefore COX-3 may be a product of the same gene that encodes COX-2, but have different molecular characteristics(Dong et al., 2000).

Much investigation has centered on paracetamolinhibition of the COX enzyme because its analgesic and antipyretic effects are similar to those of aspirin, the archetype NSAID. However, paracetamol does not have significant anti-inflammatory activity nor does it inhibit production of the pro-clotting TXAs. Paracetamol does not appear to have a major effect peripherally, but its action appears to be mostly central. It seems reasonable to assume that although there may be some effect on COX enzyme, this effect is different from that seen with the NSAIDs(Graham and Scott, 2005).

1.3.4 Toxicity of Paracetamol

Hepatotoxicity is a direct liver injury caused by thetoxic metabolite of acetaminophen N-acetyl-p-bezoquinone imine (NAPQI).Acetaminophen is considered a predictable hepatotoxin,where biochemical signs of liver damage will become apparent within 24 to 48 hours after the time of overdose and produce a dose-related centrilobular necrosis in the liver (Lauraet al.,2003)

When taken in therapeutic doses, greater than 90% of acetaminophen is metabolized to phenolic glucuronide and sulfate in the liver by glucuronyltransferases and sulfotransferases and subsequently excreted in the urine. Of the remaining acetaminophen, about 2% is excreted in the urine unchanged; approximately 5% to 10% is metabolized by cytochrome P450, mainly the enzyme CYP2E1, to N-acetyl-p-benzoquinoneimine (NAPQI), a highly reactive, electrophilic molecule that causes harm by formation of covalent bonds with other intracellular proteins. This reaction is prevented by conjugation with glutathione and subsequent reactions to generate a water-soluble product that is excreted into bile(Kanchana andSadiq, 2011).

After an overdose of paracetamol, elevated levels of the toxic NAPQI metabolite are generated, which extensively deplete hepatocellular GSH and covalently modify cellular proteins resulting in hepatocyte death (Galal et al., 2012). With acetaminophen overdose, glucuronyltransferases and sulfotransferases are saturated, diverting the drug to be metabolized by cytochrome P450 and generating NAPQI in amounts that can deplete glutathione. If glutathione is not replenished, NAPQI will begin to accumulate in the hepatocytes.NAPQI can form covalent bonds with cellular proteinsand modify their structure and function resulting in inhibition of enzymatic activities (Prescott et al.,2006). Two of the enzymes that have been shown to be inhibited in paracetamol treated animals are glutathione peroxidase and thiol transferase. Inhibition of these enzymes renders the cell vulnerable to endogenous activated oxygen species with further oxidation of protein thiols (Prescott et al.,2006). Also this cellular disturbance leads to a decrease in calcium ATPase activities and an increase in levels of cytosolic calcium. Abnormal cellular calcium homeostasis can alter the permeability of the cell, causing the formation of blebs in the cellmembrane and loss of membrane integrity(Dong et al., 2000).

 

Fig 3; Toxic reactions of paracetamol(Source: Isao et al., 2004)

1.4The Liver.

The liver is the largest internal organ of the body weighing approximately 1.5kg in adults.It is a vital organ present also in vertebrates and some other animals.It lies below the diaphragm in the abdominal-pelvic region of the abdomen. The liver has a wide range of functions, including detoxification, protein synthesis, and production of biochemical necessary for digestion. It plays a major role in metabolism and has a number of functions in the body, including glycogen storage, decomposition of red blood cells, plasma protein synthesis, hormone production, and detoxification. It produces bile, an alkaline compound which aids in digestion via the emulsification of lipids. The liver is a highly specialized tissue that regulates a wide range of vital biochemical reactions, including the synthesis and breakdown of small and complex molecules, many of which are necessary for normal vital functions(Song etal., 2001).

1.4.1 Anatomy/Physiology of the Liver

The body of the liver is a reddish brown organ with four lobes of unequal size and shape. A human liver normally weighs 1.44–1.66 kg, and is a soft, pinkish-brown, triangular organ (Cotran et al., 2005). It is both the largest internal organ (the skin being the largest organ overall) and the largest gland in the human body. It is located in the right upper quadrant of the abdominal cavity, resting just below the diaphragm. The liver lies to the right of the stomach and overlies the gallbladder. It is connected to two large blood vessels, one called the hepatic artery and one called the portal vein. The hepatic artery carries blood from the aorta, whereas the portal vein carries blood containing digested nutrients from the entire gastrointestinal tract and also from the spleen and pancreas. These blood vessels subdivide into capillaries, which then lead to a lobule. Each lobule is made up of millions of hepatic cells which are the basic metabolic cells. Lobules are the functional units of the liver(Rajiv et al., 2012).

The liver plays a major role in metabolism which includes biosynthesis, degradation and storage of biochemical compounds. The various functions of the liver are carried out by the liver cells or hepatocytes.The liver performs over 500 metabolicfunctions, resulting in synthesis of products that are released into the blood stream(e.g. glucose derived from glycogenesis, plasma proteins, clotting factors and urea),or that are excreted to the intestinal tract (bile) (Song etal., 2001).

1.4.2Liver Intoxication (Hepatotoxicity)                         

This is implies a chemical – driven liver damage. The liver plays a central role in transforming and cleaning chemicals and susceptible to the toxicity from these agents. Certain mechanical agents when taken in overdoses and sometimes even when introduced within therapeutic ranges may injure the organ (Pablo etal., 1992). Other chemical agents such as those used in laboratories and industries, natural chemicals and herbal remedies can also induce hepatotoxicity. Chemical that cause liver injury are called hepatotoxins. The liver is a major organ for metabolism of foreign substances and also functionally interposed between the site of reabsorption and the systemic circulation. These conditions render the liver not only the most important organ for detoxification of foreign substances duct also a major target of their toxicity. More than 1000 drugs have been associated with idioscratic hepatotoxicity (Chau, 2008). Moreover, drug – induced hepatotoxicity contribute more than half of the cases of acute liver failure with paracetamol being the principal offending agent in western countries (Chau, 2008). Hepatotoxicity may be predictable or unpredictable. Predictable reactions typical are dose/related and occur with short latency (within a few days) after some threshold toxicity is reached. Paracetamol (acetaminophen) is a classic example. Conversely, idiosyncratic reactions occur with variable, sometimes prolonged latency (1 week to 1 year), with low incidence may not be dose-related (Chau, 2008).

1.4.3 Mechanism of liver damage

Drugs continue to be taken off the market due to late discovery of hepatotoxicity. Due to its unique metabolism and close relationship with the gastro – intestinal tract, the liver is susceptible to injury from drugs and other substances seventy-five percent (75%) of blood coming to the liver arrives directly from gastrointestinal organs and spleen via portal veins which bring drugs and xenobiotic in concentrated form(Song etal., 2001). Several mechanisms are responsible for either inducing hepatic injury or worsening the damage process. For instance, in the case of paracetamol-induced liver damage, when the concentration of the toxic metabolite N-acetyl-p-benzoquinoneimine (NAPQI) generated from paracetamol metabolism exceed that of the gluthatione store, the toxic metabolite begins to react with other intracellular macromolecules thereby causing damage to the liver cells(Galal et al., 2012).  Many other chemicals damage the mitochondria, an intracellular organelle that produces energy especially when there concentration exceeds that of the antioxidant level in the liver. Injury to the hepatocytes and bile duct could lead to accumulation of bile acids inside the liver. This promotes further liver damage.

1.5Liver Function Tests

Liver function tests a broad range of normal functions performed by the liver. The diagnosis of liver disease depends upon a complete history, complete physical examination and evaluation of liver function test and further invasive and non-invasive tests (Rajiv et al., 2012).The liver performs different kinds of biochemical, synthetic and excretory functions of the liver. An initial step in detecting liver damage is a simple blood test to determine the presence of certain liver enzymes in the blood. Under normal circumstance, these enzymes are resided in the cells of the liver. But when the liver is injured these enzymes are spilled into the blood stream (Sultana et al., 2004). Among the most sensitive and widely used of these liver enzymes are the aminitransfereses (ALT). These enzymes are normally contained within liver cells. When the liver is injured, the cells spill the enzymes into the blood stream, raisng the enzymes level in the blood and signaling their damage (Rajiv et al., 2012).

1.5.1 Alkaline phosphatase (ALP)

Alkaline phosphateses are a family of zinc metaloenzymes, with a serine residue at the active centre; they release inorganic phosphate from various organic orthophosphates and are present in nearly all tissues (Thapa and Anuj, 2007). ALP is produced in the lower bile duct, bone and gut and is widely distributed in the body. In liver, alkaline phosphatase is found histo-chemically in the microvilli of bile canaliculi and on the sinusoidal surface of hepatocytes. In liver, two distinct forms of alkaline phosphatase are also found but their precise roles are unknown. ALP is a hydrolase enzyme responsible for removing phosphate group from many types of molecules, including nucleotides, protein and alkaloids. Alkaline phosphatase lines the cells in the biliary ducts of the liver. ALP levels in plasma will rise with large bile duct obstruction, intrahepatic cholestasis or infiltrative diseases of the liver. It is present in the bone and placenta, so it is higher in growing children (as their bones are being remodelled) and elderly patients with Paget’s disease (Manson, 2004). Elevations occur as a result of both intrahepatic and extra-hepatic obstruction to bile flow. ALP is also raised in cirrhosis and liver cancers, but levels can be within the reference range or with a slight increase in acute hepatitis. The normal range is 39-120IU/L.

1.5.2 Aspartate aminotransferaes

Aspartate aminotransferaes (AST) is more widely distributed than ALT. it is present in the liver, heart, kidneys, skeletal muscle and red blood cells. AST levels are raise in shock. It is less specific for liver disease and is not included in liver function profile by all laboratories because the enzyme is not localized in the liver. AST levels are also raised in pregnancy and after exercise. Ratios between ALT and AST are useful to physicians in assessing the etiology of liver enzyme abnormalities and also useful in differentiating between causes of liver damage (Manson, 2004). ALT exceeds AST in toxic hepatitis, chronic active hepatitis and cholestatic hepatitis. The ratio is characteristically elevated in alcoholic liver disease (Thapa and Anuj, 2007).The AST and ALT levels are increased to some extent in almost all liver diseases. The highest elevations occur in severe viral hepatitis, drug or toxin induced hepatic necrosis and circulatory shock.

1.5.3 Alanine aminotransferease

The enzyme ALT is present in high concentration in the liver. It is also found cardiac and skeletal muscle(Manson, 2004). However, ALT is considered as specific marker of hepatocellular damage because levels are generally only significantly raised in liver damage. ALT is the heart and muscles in much lower concentrations – only marginal elevations occur in acute myocardial infarction. People with acute liver damage have particularly high ALT levels and those with chronic liver disease and obstructive jaundice have more modestly raised levels. Low ALT (and AST) levels suggest vitamin B6 deficiency. The levels of ALT abnormality are increased in conditions where cells of the liver have been inflamed or undergone cell death. As the hepatocytes are damaged, the ALT leaks into the blood stream leading to a rise in the serum level (Manson, 2004). Any form of hepatic cell damage can result in an elevation in the ALT. ALT is the most sensitive marker for liver cell damage (Manson, 2004). Elevations are often measured in multiples of the upper limit of normal (ULN). Reference range 5 to 40 IU/L (Reitman and Frankel, 1957).

1.5.4 Bilirubin

Bilirubin is an endogenous anion derived from haemoglobin degradation from the red blood cell (RBC). The classification of bilirubin into direct and indirect bilirubin is based on the original van der Beigh method of measuring bilirubin. Bilirubin is a yellow fluid produced in the liver when worn – out red blood cells are broken down at the end of their 120 day lifespan (Manson, 2004). Bilirubin is a major product of hemoglobin. During splenic degradation of red blood cells, hemoglobin is separated out from iron and cell membrane components. Haemoglobin is transferred to the liver where it undergoes further metabolism in a process called conjugation. Conjugation allows hemoglobin to become more water – soluble. The water insoluble bilirubin will be excreted into bile (Rajiv et al., 2012). In the blood, unconjugated (or indirect) bilirubin is carried by albumin to the liver. It is conjugated to make it more water soluble, before it is excreted in bile. Conjugated bilirubin is also called direct bilirubin. The concentration of bilirubin in the serum therefore reflects the balance between the amount produced by erythrocyte destruction and that removed by the liver. As the liver becomes irritated, the total bilirubin may rise.

1.6 Kidney Function Tests

The kidney is bean shaped organ that serve several essential regulatory roles in vertebrate animals. They are essential in the urinary system and also serve homeostatic functions such as regulation of electrolytes, maintenance of acid base balance, and regulation of blood pressure (via maintaining salt and water balance). They serve the body as a natural filter of the blood, and remove water soluble wastes, which are diverted into the urinary bladder. Kidney function tests are series laboratory tests that are done to ensure the functionality of the kidney. The tests include urea, creatinine and electrolytes (Bartels and Rohmen, 1972).

1.6.1Urea

Urea or carbamide is an organic compound with the chemical formula CO(NH₂)². The molecule has two —NH2 groups joined by a carbonyl (C=O) functional group.

Urea is synthesized in the body of many organisms as part of the urea cycle, either from the oxidation of amino acids or from ammonia. In this cycle, amino groups donated by ammonia and L-aspartate are converted to urea, while L-ornithine, citrulline, L-argininosuccinate, and L-arginine act as intermediates (Godfrey et al., 1997). Urea production occurs in the liver and is regulated by N-acetylglutamate. Urea is then dissolved into the blood (in the reference range 10-55mg/dl which is laboratory dependent) and further transported and excreted by the kidney as a component of urine. In addition, a small amount of urea is excreted (along with sodium chloride and water) in sweat.

The blood urea nitrogen (BUN) test is a measure of the amount of nitrogen in the blood that comes from urea. It is used as a marker of renal function, though it is inferior to other markers such as creatinine because blood urea levels are influenced by other factors such as diet and dehydration (Manson, 2004).

1.6.2 Creatinine

Serum creatinine (a blood measurement) is an important indicator of renal health because it is an easily measured bye-product of muscle metabolism that is excreted unchanged by the kidneys. Creatinine itself is produced via a biological system involving creatinine, phosphocreatine (also known as creatine phosphate), and adenosine triphosphate (ATP, the body’s immediate energy supply) (Godfrey et al., 1997).

Creatinine is removed from the blood chiefly by the kidneys, primarily by glomerular filtration, but also by proximal tubular secretion. Little or no tubular reabsorption of creatinine occurs. If the filtration in the kidney is deficient, creatinine blood levels rise. Therefore, creatinine levels in blood and urine may be used to calculate the creatinine clearance (CrCl), which correlates with the glomerular filtration rate (GFR) (Taylor, 1989). Blood creatinine levels may also be used alone to calculate the estimated GFR (eGFR). Men tend to have higher levels of creatinine than women because, in general, they have a greater mass of skeletal muscle. Measuring serum creatinine is a simple test, and it is the most commonly used indicator of renal function. A rise in blood creatinine level is observed only with marked damage to functioning nephrons. The reference range of serum creatinine is 1.2- 1.9 mg/dl (Bartels and Rohmen, 1972).

1.6.3 Sodium Ion

Sodium is the major cation of extracellular fluid. It plays a central role in the maintenance of the normal distribution of water and the osmotic pressure in the various fluid compartments. The main source of body sodium is sodium chloride contained in ingested foods. Only about one-third of total body’s sodium is contained in the skeleton since most of it is contained in the skeleton since most of it is contained in the extracellular fluids (Tietz, 1976). Hyponatremia is found in a variety of conditions including the following: severe polyuria, metabolic acidosis, Addison’s disease, diarrhoea, and renal tubular disease. Hypernatremia (increased serum sodium level) is found in the following conditions: hyperadrenalism, severe dehydration, and diabetic coma after therapy with insulin, excess treatment with sodium salts (Henry et al., 1974). Normal range is 100 – 130 mEq/L.

1.6.4 Potassium Ion

Potassium is the principle cation of the intracellular fluid. It is also an important constituent of the extracellular fluid due to its influence on muscle activity. Its intracellular function parallels that of extracellular function, namely influencing acid-base balance and osmotic pressure, including water retention (Henry et al., 1974). Elevated levels of potassium (hyperkalemia) are often associated with renal failure, dehydration shock or adrenal insufficiency. Decreased potassium levels (hypokalemia) is associated with malnutrition, negative nitrogen balance, gastrointestinal fluid losses and hyperactivity of the adrenal cortex (Tietz, 1976). The reference range is 4 – 7 mEq/L.

1.6.5 Chloride Ion

Chloride, a major anion, is important in the maintenance of the cation/anion balance between intra- and extracellular fluids. This electrolyte is therefore essential to the control of proper hydration, osmotic pressure, and acid/base equilibrium. Low serum chloride values are found with extensive burns, excessive vomiting, intestinal obstruction, nephritis, metabolic acidosis, and in Addisonian crisis. Elevated serum chloride values may be seen in dehydration, hyperventilation, congestive heart valve, and prostatic or other types of urinary obstruction (Skeggs and Hochstrasser, 1964). The reference range is 70 – 95 mEq/L.

1.7 Antioxidants

An antioxidant is a molecule that inhibits the oxidation of other molecules. Oxidation is a chemical reaction that involves the transfer of electrons from a substance to an oxidizingagent. Antioxidants are often reducing agents such as thiols, polyphenols or ascorbic acid (Seaver and Imlay, 2004). Antioxidants are intimately involved in the prevention of cellular damage which is the common pathway for a variety of diseases.Although, oxidation reactions are crucial for life, they can also be damaging, however insufficient levels of antioxidants or inhibition of the antioxidant enzymes (e.g. superoxide dismutase), causes oxidative stress which will subsequently lead to inflammation and cellular damage. Antioxidants are widely used in dietary supplements and has been investigated for the prevention of diseases such as cancer, coronary heart disease and other sickness(Bjelakovic et al.,2007).

1.7.1 Superoxide Dismutase

Superoxide dismutases (SOD, EC 1.15.1.1) are enzymes that catalyze the dismutation of superoxide (O₂−) into oxygen and hydrogen peroxide. Thus, they are an important antioxidant defense in nearly all cells exposed to oxygen. Superoxide is one of the main reactive oxygen species in the cell. Consequently, SOD serves a key antioxidant role. The physiological importance of SODs is illustrated by the severe pathologies evident in mice genetically engineered to lack these enzymes(Hijora et al., 2005).

Superoxide dismutase is an enzyme whose function is to protect against the potentially damaging activities of the superoxide radical generated by aerobic metabolic reactions. Two types of SOD have been found in all mammalian cells except erythrocytes. Cu, Zn-SOD was present in both the cytosol and the intermediate membrane space of the mitochondria, and Mn-SOD was present in the mitochondrial matrix (Bjelakovic et al.,2007).Non-sulfur Fe enzyme known as superoxide dismutase (SOD) catalyze disproportion of FeSOD is found in bacteria, especially the more primitive ones, the chloroplast of plants, a few protists and possibly eukaryotes. The homologous MnSODs and found in bacteria and mitochondria, and are believed to protect DNA from endogenous oxidative stress, whereas FeSOD may serve as a housekeeping enzyme and provide resistance to environmental oxidatives, SOD caused by the chemical progeny of O₂-: H₂O₂ and OH. Atomic absorption spectroscopy reveals that SOD monomer contains one metal ion and no other cofactors (Bjelakovic et al.,2007).

1.8Lipid Peroxidation and Tissue Damage

Lipid peroxidation is a known mechanism of cellular injury in human and is used as an indicator of oxidative stress in cells and tissues. Lipid peroxides derived from PUFA are unstable and decompose to form a complex series of compounds. These include reactive carbonyl compounds which is the most abundant is malondialdehyde (MDA) (Sarka and Rautary, 2009).

Lipid peroxidation is one of the molecular mechanism for cell injury and is associated with a decrease of cellular antioxidants such as glutathione, superoxide dismutase (SOD) and catalase (CAT) (Hijora et al., 2005). Free radicals are released by activated leucocytes which cause peroxidation of membrane lipids. There is a rupture of the liposomal membranes, the release of lysosomal enzymes, necrosis of the cell and destruction of parenchymal tissue. All these processes culminate in an increase in serum MDA levels. Hence, increased serum MDA could be used as a marker for the free radical mediated destruction of liver parenchymal cells. Liver disease is accompanied by an increased production of free radicals. MDA has the ability to interact with lipoproteins and so has received particular attention in pharmacological studies (Sarkar and Rautava, 2009).

1.9 Haematology

Blood is a highly specialized fluid-like connective tissues, which circulate in a closed system of vessels as a liquid with red colour, but forms a solid phase out of the system, which we call plug or blood clot. Some people would also say that blood is the river of life simply because it connects to all the tissues of the body and these tissues needs the blood to survive. Haematology is the science that studies about the blood, blood transfusion, blood-forming tissues, and its’ structure, function, disease, and the convenience between structure and the function. Haematology also includes the study of etiology, diagnosis, treatment, and prevention of blood diseases that affect the production of blood and its components, such as, haemoglobin, blood proteins, and the mechanism of coagulation (Lewis et al., 2002).

1.9.1 Haemoglobin Count

Haemoglobin contains the red pigment that gives the red cells their colour and also carries oxygen from the lungs to the tissues and carries carbon dioxide (the waste prod­ucts) from the tissues to the lungs. This test is primarily used to determine the presence of anaemia or, its reverse, polycythaemia, or to monitor a patient’s response to treatment. Capillary blood or EDTA anti-coagulated venous blood can be used. The hemoglobin content in a solution may be estimated by several methods: by measurement of its colour, its power of combining with oxygen or carbon monoxide and by its iron content(Yared et al., 2006).

Normal hemoglobin reference range: children 6 y – 12 y 6 -12 g/dl; Adult men 10-18 g/dl (Yared et al., 2006). A low haemoglobin level means that less oxygen is being delivered round your body, leading to symptoms of anaemia such as fatigue, breathlessness, pallor, and palpitations. The patient may need to have a blood transfusion to help relieve these symptoms. In this case the patient may need to have additional blood tests, in order to match the transfusion to your own blood as closely as possible(Cheesbrough, 2000).

1.9.2 Packed Cell Volume (PCV)                    

Packed cell volume is a measure of the proportion of blood volume that is occupied by red blood cells. It is normally about 45% in men and 40% in women(Yared et al., 2006). It is considered an integral part of a person complete blood count results along with hemoglobin concentration, white blood cell count and platelet count.Low levels of PCV can be seen in the case of anemia, inflammation, kidney damage, malnutrition and pregnancy. However increase in PCV may be seen in myeloproliferative disorder, chronic obstructive pulmonary disease (COPD), capillary leak syndrome and anabolic androgenic steroid (AAS)(Lewis et al., 2002).

1.9.3 Red Blood Cell (RBC) Count                    

Red blood cell count (RBC) measures the number of red cells in the blood.Red blood cells carry oxygen to the tissues and remove waste products from the body’s tissues. These cells also con­tain hemoglobin. Red blood cells are measured in millions per cubic mil­limeter (mil/uL) of blood. The normal range for red blood cell count is 4.6 – 6.2 x 10⁶ cells/µL (Lewis et al.,2002). A low count often accompanies anaemia, excess body fluid and blood loss. A high count is commonly seen in dehydration but could also mean some other complications such as polycythaemia, lung disease, alcoholism, smoking, kidney disease, dehydration, burns, sweating, diarrhea, carbon monoxide (co) exposure, etc while low RBC might indicate anaemia, sickle cell disease, cancer, peptic ulcer, lead poisoning, heavy menstrual bleeding etc depending on the aim of the test (Cheesbrough, 2000).

1.9.4 White Blood Cell (WBC) Count

White blood cell count (WBC) is a blood test carried out in the laboratory that measures the number of white blood cells per litre of blood. White cells protect against infection and allergies. High counts are seen during infection, after exercise and with stress. Low counts may be seen if there is suppression of the immune system. The normal range of WBC is 4.8- 10.0 × 10⁹/L (Yared et al., 2006). An increase above the normal range could imply some other complications such as, leukemia, inflammation, tissue damage, stress, malnutrition, burns, lupus, kidney failure, rheumatoid arthritis, tuberculosis, thyroid gland problems while low WBC count might indicate alcoholism, AIDS, enlarged spleen, viral infection, malaria, that the patient is undergoing chemotherapy, depending on the aim of the test.

1.10 Aim of the Study                                                                                                                      

Thisstudy is aimed at determining the possible effect of Fraction1 ofAbrus precatoriusseed methanol extract on paracetamol-induced liver damage.

1.11 Specific Research Objectives

• To determine the median lethal dose (LD₅₀) of the extract.
• To fractionatethe extract using Sephadex G15.
• To determine qualitatively and quantitatively the phytochemical constituentsof the

differentfractions of Abrus precatoriusseed methanol extract.

• Determination of the effect of Fraction 1 on liver marker enzymes.
• To determine the effect of Fraction 1 onsome haematological parameters.
• To determine the effect of Fraction 1 on some kidney markers.
• To determine the effect of the Fraction 1 on some antioxidant enzymes.

 

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