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. In vitro 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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