Effect of Chloroform- Methanol Extract of Tetracarpidium conophorum Nuts : Current School News

Effect of Chloroform- Methanol Extract of Tetracarpidium conophorum Nuts (Walnuts) on Oxidative Stress Markers in Hydrogen Peroxide Induced Wistar Albino Rats

Filed in Biochemistry Project Topics, Current Projects by on September 21, 2020

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Effect of Chloroform- Methanol Extract of Tetracarpidium conophorum Nuts (Walnuts) on Oxidative Stress Markers in Hydrogen Peroxide Induced Wistar Albino Rats.

ABSTRACT

This work was done to ascertain the efficacy of the seed of Tetracarpidium conophorum on hydrogen peroxide induced oxidative stress in Wistar albino rats. Thirty five (35) male wistar albino rats weighing (120- 140g) were distributed into seven (7) groups of five rats each.

Groups 2-6 were administered with hydrogen peroxide (1.0 ml/kg), while group 1 served  as  the normal control while group 2 serves as positive control Groups 3, 4, 5 and 6 were treated with vitamin C (100 mg/kg), 200, 400 and 800 mg/kg of the  extract  respectively  for  five days, Group 7 was administered 800 mg/kg b.w of the extract only.

Blood was collected from the animals on the 7th day through occular puncture for assay  of  some  biochemical parameters.

The qualitative and quantitative analysis of the seed  extract  were  determined using standard methods and showed that the extract contained terpenoids (4.36 ± 0.06 mg/g), Tannins (1.89 ± 0.11 mg/g), alkaloids (20.31 ± 0.30mg/g) and cardiac glycosides (12. 45 ±

0.08 mg/g), anthraquinones, saponins and steroids were not detected in the extract. Vitamins constituents of the extract were vitamin A (10.55 ± 2.67 mg/ 100g), vitamin C (13.09 ± 0.23 mg/ 100g) and vitamin E (5.77 ± 0.08 mg/ 100g).

The mineral constituents indicated the presence of mg (133.59 ± 0.11 mg/ 100g), Ca (118.90 ± 0.01 mg/ 100g), Fe (3.67 ± 0.07 mg/ 100g), Zn (2.22 ± 0.01 mg/ 100g), Cu (1.54 ± 0.78 mg/ 100g). The acute toxicity test of the extract showed no toxicity up to 5000 mg/ kg b.w.

Serum  ALT  activity  significantly decreased (p< 0.05) in all the test groups compared to group 2. Serum AST and ALP activity decreased significantly (p< 0.05) in all the test groups except group 6 for ALP activity compared to the enzyme activities of normal and positive controls.

A significant decrease (p< 0.05) was observed in the serum MDA concentration of rats in  the  test  groups  when compared to the group 2. A significant increase (p < 0.05) was observed in the serum GPx activity of groups 4, 6 and 7 compared to the GPx activity of group 2 rats.

There was a significant increase (p < 0.05) in groups 4 and 7 compared to group 2. The serum cholesterol concentration showed a significant decrease (p < 0.05) in the test  groups  relative to  those of the controls.

There was a significant  decrease (p  < 0.05) in the  serum LDL and TAGs of rats in the test groups when compared to the controls. The serum HDL of groups  5  and  7  increased significantly (p < 0.05) compared  to  the normal control and the positive control.

The effect of the extract on lipid profile showed that it increased HDL at a concentration of 400mg/kg body. These antioxidant enzymes results support the claims made by several scientists that the plant could be used to scavenge free radicals in the system which often lead  to the risk of various diseases.

TABLE OF CONTENTS

Title Page………….……i
Certification Page……….iii
Acknowledgement ………iv
Abstract ……………………….v
Table of Contents ……vi
List of Tables ………….. xii
List of Figures ……… xiii
List of Abbreviations … xiv

CHAPTER ONE: INTRODUCTION

1.1.Background of the study ……….1
1.2.Tetracarpidium conophorum……………2
1.2.1. Scientific classification of Tetracarpidium conophorum……. 3
1.2.2. Medicinal, nutritional and industrial importance of Tetracarpidium conophorum ……4
1.2.2.1. Medicinal uses of Tetracarpidium conophorum ………..4
1.2.2.2. Nutritional uses of Tetracarpidium conophorum ….4
1.2.2.3. Industrial uses of Tetracarpidium conophorum ………………5
1.3. Oxidative stress ………………..5
1.3.1. Chemical and biological effects of oxidative stress …………..6
1.3.2. Oxidative stress and diseases …………7
1.3.3. Free radical generations/reactive oxygen species……..7
1.3.3.1. Sources of free radicals ……………7
1.4. Hydrogen peroxide ……..….9
1.5. Lipid peroxidation ……10
1.5.1. Malondialdehyde……….……11
1.5.2. Antioxidants ………………11
1.5.2.1 Enzymatic antioxidants …………12
1.5.2.2. Superoxide dismutase ……………..13
1.5.2.3 Catalase ……………..13
1.5.2.4 Glutathione ……………………….13
1.5.3. Antioxidant vitamins ………..14
1.5.3.1. Carotenoids ……………14
1.5.3.2. Vitamin C (Ascorbic Acid) ………………………15
1.5.3.3. Vitamin E …………………..16
1.5.4. Cholesterol ………………….17
1.5.5. Phytochemicals ………..18
1.5.5.1.Phytochemical constituents of plant……….…..19
1.5.6. Minerals and Vitamins constituents of Chloroform-methand extract of Tetracarpidium conophorum 22
1.5.7. The liver ……….24
1.5.7.1. Serum enzyme markers involved in hepatic disorder…4
1.6 Aim and objectives of the study …………….24
1.6.1 Aim of the study……….24
1.6.2 Specific objectives of the study……..25

CHAPTER TWO: MATERIALS AND METHODS

2.1. Materials ………………26
2.1.1. Plant Material………26
2.1.2. Animals……..26
2.1.3. Equipment……….…….26
2.1.4.Chemicals and Reagents………26
2.2 Methods ………….……27
2.2.1 Preparation of Plant Material …………..27
2.2.2 Extraction of Plant Material …27
2.2.3 Preparation of Reagent for phytochemical analysis…28
2.2.4 Qualitative phytochemical analysis of the seed of Tetracarpidium conophorum …….29
2.2.4.1 Test for Alkaloids …….29
2.2.4.2 Test for Flavonoids……….30
2.2.4.3 Test for Glycosides ………30
2.3.4.4 Test for Saponins…….….30
2.3.4.5 Test for Tannins ………30
2.3.4.6 Test for terpenoids and steroids……………31
2.2.5. Quantitative phytochemical analysis of the seed of Tetracarpidium conophorum …..31
2.2.5.1. Alkaloids determination ……………31
2.2.5.2 Flavonoids determination ………….31
2.2.5.3 Steroids determination …………32
2.2.6 Determination of vitamin contents of the seed of Tetracarpidium conophorum …….32
2.2.6.1 Vitamin A……………….32
2.2.6.2 Vitamin C ………………32
2.2.6.3 Vitamin E ……….33
2.2.7 Determination of mineral contents of the seed of Tetracarpidium conophorum ………33
2.2.8 Acute toxicity test if the chloroform methanol extract of the seeds of Tetracarpidium conophorum …34
2.2.9 Experimental Design and Induction of oxidative stress………34
2.2.10 Liver function test of rats treated with chloroform methanol extract of the seed of Tetracarpidium conophorum ………..…..35
2.2.10.1 Determination of Alanine aminotransferase (ALT)…….35
2.2.10.2 Determination of Aspartate amino transferase (AST) ……35
2.2.10.3 Determination of Alkaline phosphatase (ALP) …………36
2.2.11 Determination of lipid peroxidation (Malondialdehyde) ……..36
2.2.12 Determination of Glutathione Concentration………………37
2.2.13 Assay of catalase Activity ………..37
2.2.14 Assay of superoxide dismutase (SOD) Activity..……38
2.2.15 Determination of Total Cholesterol Concentration………38
2.2.16 Low density lipoprotein cholesterol (LDL-C) ……39
2.2.17 High density Lipoprotein ………….…..39
2.2.18 Determination of triacylglycerol concentration……….…..40
2.3 Statistical analysis ……….…..40

CHAPTER THREE: RESULTS

3.1. Qualitative phytochemical constitutents of chloroform-methanol extract of Tetracarpidium conophorum seeds….41
3.2 Quantitative phytochemical constitutents of chloroform- methanol extract of Tetracarpidium conophorum seeds……………..….41
3.3 Vitamin contents of chloroform- methanol extract of Tetracarpidium conophorum seeds 41
3.4. Mineral contents of chloroform- methanol extract of Tetracarpidium conophorum seeds……41
3.5.Acute toxicity (LD50) test of chloroform methanol extract of Tetracarpidium conophorum seeds ….44
3.6.Effect of chloroform- methanol extract of Tetracarpidium conophorum seeds on serum alanine aminotransaminase (ALT) of rats ………….. ..45
3.7. Effect of chloroform- methanol extract of the of Tetracarpidium conophorum on serum alkaline phosphatase (ALP) of rats … ….47
3.8. Effect of chloroform- methanol extract of Tetracarpidium conophorum seeds on serum Aspartate aminotransaminase (AST) of rats ……..….49
3.9. Effect of chloroform methanol extract of the seeds of Tetracarpidium conophorum on serum superoxide dismutase (SOD) of rats.…… 51
3.10 Effect of chloroform methanol extract of the seeds of Tetracarpidium conophorum on serum catalase activity of rats…………………. 53
3.11 Effect of chloroform- methanol extract of the seeds of Tetracarpidium conophorum on serum glutathione peroxidase (GPx) activity of rats ………………………….55
3.12. Effect of chloroform- methanol extract of Tetracarpidium conophorum seeds on serum malondialdehyde (MDA) concentration of rats………………….57
3.13. Effect of chloroform methanol extract of the seed of Tetracarpidium conophorum on serum Cholesterol of rats ….59
3.14. Effect of chloroform methanol extract of the seeds of Tetracarpidium conophorum on serum low density Lipoproteins (LDL) of rats ……..61
3.15. Effect of chloroform methanol extract of the seeds of Tetracarpidium conophorum on serum Triacyglycerol (TAGs) of rats …………………………………………….63
3.16. Effect of chloroform methanol extract of the seeds of Tetracarpidium conophorum on serum High density Lipoproteins (HDL) of rat……65

CHAPTER FOUR: DISCUSSION

4.1. Discussion ….67
4.2. Conclusion …….…70
4.3. Suggestion for further studies …………70
References …………………..71

INTRODUCTION

The use of various fruits, vegetables, nuts and various parts of plants in traditional medicine is as old as man (Evans, 2002).

This lies outside the mainstream of orthodox or Western medicine, it has been estimated that two thirds of the world population (mainly in developing countries) rely on traditional medicine as their primary form of health  care (Sumner, 2000).

The use of traditional medicine cannot fade out in the treatment and management of diseases in the African continent and this could be attributed to the socio- cultural, socio-economic, lack of basic health care and qualified personnel (Elujoba et al., 2005).

Plants contain active components such as anthraquinones, flavonoids, glycosides, saponins, tannins, etc which posses medicinal properties that  are harnessed  for  the treatment of different diseases (Chevalier, 2000).

The active ingredients for a vast number of pharmaceutically derived medications contain the healing properties known as the active principles and are found to differ from plant to  plant  (Chevalier,  2000).

Nuts  vary considerably in their nutrient content and are sources of vitamins, antioxidants, proteins, essential amino acids, etc. (Fasuyi, 2006).

They are included in meals mainly for their nutritional values. However, some are reserved for their medicinal values such as increase in brain health, decreased depression, increase in antioxidant  levels; thus, helping to  mop  up  free radicals which have been implicated in a number of diseases (Oladiyi et al., 2007).

REFERENCES

A.O.A.C (1970) Official Methods of Analysis of the Association of Analytical Chemists Washington DC.

A.O.A.C (1976) Official Methods of Analysis of the Association of Analytical Chemists Washington DC

A.O.A.C. (1995). Official Methods of Analysis of the Association of Analytical Chemists Washington DC.

Abell, L.L., Levey, B.B. and Kendall, F.E. (1952). Extraction of Cholesterol. Journal of Biological Chemistry, 195(1): 357-366.

Aebi, H.E. (1983). Catalase. In: Methods of Enzymatic Analysis. 3rd Edn.  (Bergmeyer,  H.U., et al. Ed)] Weinheim, Deerfield Beach, Florida. pp. 273-285.

Agte, V. V., Tarwadi, K.V., Mengale, S. and  Chiplonkar,  S.A.  (2000).  Potential  of indigenous green vegetables as natural sources of fortification of eight micronutrients. Journal of Food Comparative Analysis, 13:885-891.

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