EFFECTS OF HYDRO ALCOHOL EXTRACT OF GREEN AMARANTH (AMARANTHUS HYBRIDUS LINN.) LEAVES ON BIOCHEMICAL INDICES OF THIOACETAMIDE INDUCED HEPATIC DAMAGE IN RATS

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ABSTRACT

 

Vegetables are widely used as herbal remedies for combating liver diseases. This study evaluated the phyto contents and in vitro antioxidant capacity of various solvent fraction of the crude extract as well as some biochemical changes following exposure to the hydro-alcohol extract of Amaranthus hybridus in thioacetamide- induced hepatic damage in rats. The plant sample was extracted with 70% ethanol. Preliminary photochemistry and in vitro antioxidant potential of the plant extract was performed using standard methods.  The hepatoprotective effect of HAAH extract was measured in rat model of thioacetamide- induced liver damage for 70 days. Animals were divided into six (6) groups, and were exposed to thioacetamide (300mg/l) in drinking water throughout experimental period, except group that served as control. Liver function status test performed to access the possible effect of HAAH on liver damage. Oxidative stress conditions were measured using malondialdehyde, nitric oxide and glutathione levels. The effects protective activity of HAAH on free radical scavenging were assayed using enzymatic antioxidants. Histology of the liver tissue was performed to access if the extract protected liver architecture from damage. Fractionation of crude plant extract was achieved using solvent partitioning system, and various fractions assayed for antioxidant potential to confirm most potent fraction. The most active fraction was characterised using GC-MS spectrometry. Result showed that the plant extract was rich in phenolic compounds (140.12+0.01 GAEequ.mg/g), flavonoids (3.39+0.02 mgQE/g) and Vitamin C (2.86+0.01mg/100g). Amino acid profiling of the plant extract showed that the extract contained 16 amino acids, which include aromatic amino acids phenylalanine (3.34%), tyrosine (6.08%) and many essential amino acids. Ivitro antioxidant studies showed a dose dependent activity for the plant extract that was comparable to standard vitamin c. Results for liver function status assay showed significant (p<0.05) reduction in the liver enzymes, increase in total proteins in the groups administered HAAH compared to negative control. There was significant (p<0.05) increase in enzymatic antioxidant activities in liver homogenates in group administered the plant extract compared to negative control. MDA was significantly (p<0.05) lower in the group treated with plant extract when compared to negative control, while nitric oxide inhibition and GSH concentration were higher in group treated with the plant extract compared to negative control. Histology findings corroborated result obtained from the liver function status assays. Methanol fraction of crude extract showed highest antioxidant potential, and GC-MS profiling of the methanol fraction showed 6 compounds having benzene ring backbone, like those found in most phenolic acids. From the result of this study, it can be concluded that methanol fraction of HAAH contains compounds that may be responsible for the observed hepatoprotective and antioxidant properties.






TABLE OF CONTENTS

Title Page                                                                                                                    i

Declaration                                                                                                                  ii

Certification                                                                                                                iii                                                                        Dedication                                                                                                                  iv

Acknowledgements                                                                                                    v

Table of Contents                                                                                                       vi

List of Tables                                                                                                              xii

List of Figures                                                                                                             xiii

List of Plates                                                                                                               xiv

Abstract                                                                                                                      xv

 

CHAPTER 1: INTRODUCTION

1.1       Background of the Study                                                                               1

1.2       Aim of the Study                                                                                            3

1.3       Objectives of the Study                                                                                  3

1.4       Justification of the Study                                                                               3

 

CHAPTER 2: LITERATURE REVIEW

2.1       Liver                                                                                                                5

2.1.1    Liver structure and function                                                                           5

2.1.2    Hepato-toxicity                                                                                               6

2.2       Thioacetamide Metabolism and Toxicity                                                        6

2.2.1    Thioacetamide                                                                                                 6

2.2.2    Thioacetamide metabolism and toxicity                                                         7

2.3       Amaranthus hybridus Linn                                                                             8

2.3.1    Biology of Amaranthus hybridus                                                                    8

2.3.2    Proximate composition of Amaranthus hybridus Linn.                                  9

2.3.3    Mineral composition of Amaranthus hybridus Linn. leaves                           9

2.3.4    Vitamin composition of Amaranthus hybridus leaves                                    10

2.3.5    Amino acid profile of Amaranthus hybridus leaves                                       10

2.3.6    Phytochemical composition of Amaranthus hybridus leaves              10

2.3.7    Medicinal uses of Amaranthus hybridus                                                         11

2.3.7.1 Antioxidant potentials of Amaranthus hybridus and other Amaranthus spp.            12

2.3.7.2 Anti-inflammatory and anti-nociceotive properties of Amaranthus hybridus13

2.3.7.3 Anti-cancerous properties of Amaranthus hybridus                                       14

2.3.7.4 Hepatoprotective ability of Amaranthus hybridus                                          14

2.3.7.5 Antimicrobial properties of Amaranthus hybridus                                          15

2.3.7.6 Antimalarial properties of Amaranthus hybridus                                            15

2.3.7.7 Active components of Amaranthus hybridus                                                 16

2.4       Polyphenols                                                                                                     16

2.4.1    Plant based phenolics with antioxidant and anticancer properties                 16

2.4.1.2 Beans extract                                                                                                  17

2.4.1.3 Coffee and cocoa                                                                                            17

2.4.1.4 Honey and propolis extract                                                                             17

2.4.1.5 Onions                                                                                                             18

2.4.1.6 Soy extracts                                                                                                    18

2.4.1.4 Potatoes                                                                                                          18

2.4.2    Phenolic antioxidants isolated from plants                                                     18

2.4.2.1 Flavonoids                                                                                                      18

2.4.2.2 Anthocyanins                                                                                                  19

2.4.2.3 Gallic acid                                                                                                       19

2.4.2.4 Chalcones                                                                                                        19

2.4.2.5 Ellagic acid                                                                                                     19

2.5       Rutin: a major flavonoid in Amaranthus hybridus Linn.                                20

2.5.1    Hepatoprotective and anticancer properties of rutin                                      20

2.6       Effects of some medicinal plants and compounds on thioacetamide

             induced toxicity                                                                                             23

 

CHAPTER 3: MATERIALS AND METHODS

3.1       Materials                                                                                                         25

3.1.1    Plant material procurement                                                                             25

3.1.2    Experimental animals                                                                                      25

3.1.3    Chemicals                                                                                                        26

3.1.4    Equipment                                                                                                       27

3.2       Methods                                                                                                          28

3.2.1    Plant material extraction                                                                                 28

3.2.2    In vitro antioxidant assays                                                                              28

3.2.2.1 Hydrogen peroxide scavenging potential                                                      28

3.2.2.2   1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging potentials                     29

3.2.2.3 Nitric oxide (NO) scavenging activity                                                           29

3.2.2.4   2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical

  scavenging activity                                                                                       30

3.2.2.5 Reducing power potential of the extract                                                        31

3.2.3    Quantitative phytochemical screening                                                            31

3.2.3.1 Determination of total flavonoids                                                                  31

3.2.3.1 Determination of anthocyanins                                                                       32

3.2.3.3 Determination of total phenolics                                                                    32

3.2.3.4 Vitamin A and E estimation                                                                           33

3.2.3.5 Vitamin C estimation                                                                                      33

3.2.3.6 Amino acid quantification of Amaranthus hybridus extract                          33

3.2.4    Determination of lethal dose of extract (LD50)                                              35

3.2.5    Animal study design                                                                                       35

3.2.6    Blood and organ collection                                                                             36

3.2.7    Biochemical analyses                                                                                      36

3.2.7.1 Liver function status assays                                                                            36

3.2.7.1.1 Serum alanine transaminase (ALT) and aspartate transaminase

   activity (AST)                                                                                              36

3.2.7.1.2 Serum alkaline phosphatase (ALP) activity                                                 38

3.2.7.1.3 Serum total bilirubin                                                                        39

3.2.7.1.4 Serum total protein                                                                                      39

3.2.7.1.5 Serum albumin                                                                                             40

3.2.7.1.6 Serum conjugated bilirubin (CB) and total bilirubin (TB)                           41

3.2.7.2 Antioxidant enzyme activity assay                                                                42

3.2.7.2.1 Superoxide dismutase (SOD) activity                                                         42

3.2.7.2.2 Catalase activity                                                                                           43

3.2.7.2.3 Glutathione peroxidase (GPx)                                                                     44

3.2.7.3    Determination of oxidative stress markers                                                  44

3.2.7.3.1 Malondialdehyde (MDA)                                                                            44

3.2.7.3.2 Nitric oxide scavenging activity                                                                 45

3.2.7.3.3 Reduced glutathione (GSH)                                                                        45

3.2.7.4    Determination of calcium concentration                                                      46

3.2.7.5    Determination of liver hydroxyproline concentration                                 46

3.2.7.6    Determination of cholesterol concentration                                                 47

3.2.7.7    Histology examination                                                                                 47

3.2.8       Extract solvent partitioning                                                                         49

3.2.9       GCMS analysis of active fraction of extract                                               49

3.2.10     Statistical analysis                                                                                        50

CHAPTER 4: RESULTS AND DISCUSION

4.1       Results                                                                                                           51

4.1.1    Total phenolics, flavonoids and anthocyanin concentrations in crude

 extract                                                                                                                        51

 

4.1.2    Vitamins A, C and E concentrations in crude extract                                   52

4.1.3    Amino acid profile of crude extract of HAAH extract                                 53

4.1.4    DPPH scavenging potential activity of HAAH extract                               55

4.1.5    Reducing power potential of HAAH extract                                                56

4.1.6    ABTS scavenging potential of HAAH extract                                              57

4.1.7    Nitric oxide scavenging potential of HAAH extract                                    58

4.1.8    Acute toxicity (LD50) test of HAAH extract                                                59

4.1.9    Mean weekly body weight of the rats                                                           60

4.1.10  Relative liver weight of the rats                                                                     61

4.1.11  Antioxidant enzymes activities in the experimental animals                         62

4.1.12  Oxidative stress indicators in the experimental animals                                63

4.1.13  Hydroxyproline concentration in the experimental animals                          64

4.1.14  Cholesterol concentration in the experimental animals                                  65

4.1.15  Calcium concentration in the experimental animals                                        66

4.1.16  AST/ALT and GLOB/ALB ratios concentration in the experimental

 animals                                                                                                           67

4.1.17   Liver function status assay in the experimental animals                               68

4.1.18   Histology of the liver tissue                                                                          69

4.1.18.1 Histology of liver of positive control group                                                 69

4.1.18.2 Histology of liver of normal control group                                                   70

4.1.18.3 Histology of liver of negative control group                                                71

4.1.18.4Histology of liver in HAAH400 group                                                          72

4.1.18.5 Histology of liver in HAAH200 group                                                         73

4.1.18.6 Histology of liver in HAAH100 group                                                         74

4.1.19    Total phenolics and flavonoids content of solvent fractions obtained from

  crude extract                                                                                                 75

4.1.20    Vitamin A, C and E solvent fractions obtained from crude extract             76

4.1.21     DPPH nitric oxide and hydrogen peroxide scavenging capacity of various    solvent fractions obtained from crude extract                                                77

 

4.1.22     Reducing power potential of various solvent fractions obtained from

   crude extract                                                                                                            78

4.1.23     GCMS spectra of compounds in methanol fraction of crude extract          79

4.2          Discussion                                                                                                    82

 

CHAPTER 5: CONCLUSION AND RECOMMENDATIONS

5.1       Conclusion                                                                                                      97

5.2       Recommendations                                                                                          97

References                                                                                                      97

Appendix                                                                                                        114





LIST OF TABLES

 

2.1:      Proximate composition of Amaranthus hybridus leaves                                 9

2.2:      Phenolic compounds content of Amaranthus hybridus                                  11

3.1:      List of chemicals and their manufacturers                                                      26

3.2:      List of equipment and their model                                                                 27

3.3       Protocol                                                                                                           48

4.1:      Total phenolics, flavonoids and anthocyanin contents of plant extract         51

4.2:      Showing vitamins A, C and E contents of plant extract                                52

4.3:      Quantification of amino acid present in crude extract of HAAH                  54

4.4:      DPPH scavenging potential of plant extract                                                 55

4.5:      Reducing power potential activity of plant extract                                        56

4.6:      ABTS scavenging capacity of plant extract                                                  57

4.7:      Nitric oxide scavenging capacity of plant extract                                         58

4.8:      Acute toxicity text of plant extract                                                               59

4.9:      Relative liver weight of the animals                                                              61

4.10:    Antioxidant enzymes activities of the rats                                                     62

4.11:    Oxidative stress indicators of the rats                                                            63

4.12:    Hydroxyproline concentration in test and control rats                                   64

4.1.13: Cholesterol concentration in test and control rats                                          65

4.1.14: Calcium concentration in test and control animals                                         66

4.1.15: AST/ALT and ALB/GLOB ratios in test and control rats                             67

4.1.16: Result of liver function status assay                                                               68

4.1.17: Total phenolics and flavonoids obtained from crude extract after

partitioning                                                                                                      75

4.1.18: Concentration of vitamin A, C and E obtained from crude extract after partitioning                                                                                                          76

4.1.19: DPPH, nitric oxide and hydrogen peroxide Scavenging capacity of various fractions of crude extract after partitioning                                                         77

4.1.20: Reducing power potential of various fractions obtained from crude extract partitioning                                                                                                     78

4.1.21: Compounds detected by GC-MS                                                                   81






 

LIST OF FIGURES

 

2.1:      Metabolism of thioacetamide                                                                         7

2.2:      Structure of rutin                                                                                            21

4.1:      Chromatogram showing amino acids present in crude extract of HAAH      53

4.2:      Body weight changes of the rats                                                                    60

4.3:      Chromatogram showing compounds in methanol fraction of extract            79






LIST OF PLATES

 

4.1:                  Histology of liver of positive control                                                  69

4.2:                  Histology of liver of normal control                                                   70

4.3:                  Histology of liver of negative control                                                 71

4.4:                  Histology of liver in HAAH400 group                                               72

4.5:                  Histology of liver in HAAH200 group                                               73

4.6:                  Histology of liver in HAAH4100 group                                             74







 

CHAPTER 1

INTRODUCTION

 

1.1         BACKGROUND OF THE STUDY

Liver toxicity and damage caused by toxins and drugs have been well documented in literature (Delgado-Montemayor et al., 2015; Ejiofor et al., 2017). Liver is the chief organ involved in metabolism of xenobiotics, hence it makes it a prominent point and target for toxicity. Most xenobiotics induce liver damage by promoting oxidative stress in the liver (Kadiiska et al., 2000).

 

Thioacetamide (TAA), a widely used hepatotoxicant is a thiono-sulfur containing and water soluble that could orally be administered (Salguero-Palacious et al., 2008) or intraperitoneally (Baskaran et al., 2010). Toxicity of thioacetamide begins after it’s metabolic activation (Chilakapati et al., 2007). It undergoes bioactivation by CYP2E1 and FAD mono-oxygenases (Chilakapati et al., 2005). The activation of thioacetamide leads to formation of reactive species derived from thioacetamide S- Oxide (Chilakapati et al., 2007). The reactive species (RS) resulting from TAA metabolism binds to cellular components and induce oxidative stress (Pallottini et al., 2006). Lotkova et al. (2007) reported increased lipid peroxidation, glutathione depletion and reduction in SH- thiol groups following TAA metabolism in biological systems. They free radical generated TAA metabolites also cause necrosis, induces apoptosis and inflammation of liver tissue (Moronvalle-Halley et al., 2005).

 

Plants are rich source of bioactive compounds that have desirable health benefits and have been used traditionally to prevent or manage chronic diseases (Yogalakshmi et al., 2010).

 

The plant Amaranthus hybridus Linn. is widely cultivated because of its nutritional importance (Kavita and Puneet, 2017). Interest is increasing in the use of vegetables to combat hepatotoxicity because of it rich antioxidants and phytochemical compounds. Previous studies have reported the high nutritional content of Amaranthus hybridus (Fasuyi, 2006; Odhav et al., 2007). A. hybridus is used traditionally for the treatment of liver infections and pains associated with the knee, it laxative, diuretic and cicatrisation properties has been reported by Nacoulma, (1996). Studies by Ejiofor et al. (2017) reported the ameliorative effect of methanol crude extract of Amaranthus hybridus on oxidative stress induced by cadmium in rat model. Fernand et al. (2012) reported Amaranthus hybridus contains a major flavonoid known as rutin. The in vitro antioxidant activity of the plant crude extract has shown positive result against free radicals such as DDPH (Fernand et al., 2012; Muniz-Marquez et al., 2014).

 

Polyphenols (phenolics and flavonoids) are classified as dietary antioxidants, they are useful because they possess protective ability against reactive species which are involved in the pathogenesis of various diseases such as cancer, liver diseases, cardiovascular diseases etc. Plant based antioxidants are supportive towards human’s natural antioxidant defence system. Studies have also linked increased dietary antioxidant consumption to low risk of degenerative diseases (Wang et al., 2008; Yang et al., 2011; Fernanda et al., 2015).

 

As liver diseases keep multiplying rapidly on a global scale, there is need to search for polyphenols from plants owing to their medicinal and nutritional properties. Dark green vegetables have shown promising trends in the search for plant based phenolics, and since Amaranthus hybridus is a vegetable, it is important to investigate if it extract may offer hepatoprotective activities.


1.2       AIM OF THE STUDY 

To identify the bioactive compounds in, and determine the antioxidant and hepatoprotective ability of hydro-alcohol extract of Amaranthus hybridus Linn. leaves against thioacetamide- induced oxidative stress and liver damage in rats.

 

1.3       OBJECTIVES OF THE STUDY

        i.            To extract phenolics from Amaranthus hybridus using hydro-alcohol as solvent

      ii.            Determination of total phenolics, anthocyanin, flavonoids and antioxidant vitamin contents of hydro-alcohol extract of Amaranthus hybridus leaves   

    iii.            Determination of amino acid profile in hydro-alcohol extract of Amaranthus hybridus leaves  

    iv.            Determination of the in vitro antioxidant potential hydro-alcohol extract of Amaranthus hybridus leaves

      v.            To induce oxidative stress and liver damage in rats using thioacetamide

    vi.            Determination of the hepatoprotective and antioxidant potentials of the Amaranthus hybridus leaves extract using biochemical indices

  vii.            Fractionation of the hydro-alcohol extract of Amaranthus hybridus leaves using solvent partitioning system

viii.            Determination of total phenolics and total flavonoids in the obtained fractions

    ix.            Determination of antioxidant vitamin concentrations in the obtained fractions.

      x.            Determination of in vitro antioxidant capacity of the obtained fractions

     xi.            GC-MS identification of compounds in most active fraction  

 

1.4       JUSTIFICATION OF THE STUDY

The liver is a very important organ which is involved in the metabolism of drugs and other compounds and is highly susceptible to damage and oxidative stress. Toxicants and chemicals are gotten from every day activities, through foods, water, air and skin contacts. Owing to the critical functions of the liver, it is important that the liver is fully protected from damage. One major source of liver damage is through oxidative stress initiated by reactive oxygen species, following metabolism of most xenobiotics or toxicants. Globally, cases of hepatotoxicity that starts from mild cases and proceeds to hepato-carcinoma is increasing and of major concern, leading to scientists and researchers searching for new compounds or remedies that can be combined with already existing liver enhancing drugs or administered alone to achieve hepatoprotective effects and efficiency. The search for liver protective and enhancing drugs and compounds usually would require inducing toxicity on the liver followed by testing for hepatoprotective ability of the test compounds. Plants however are known to have enormous phytocompounds with various medicinal properties. One point of interest in search of hepatoprotective compounds are phenolics. Phenolics are known to possess antioxidant activities and are good scavengers of free radicals responsible for most hepatic damage. This study tries to investigate if phenolic rich extract of Amarantus hybridus possess hepato-protective and antioxidant activity.

 


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