EFFECTS OF AQUEOUS EXTRACT OF PUNICA GRANATUM SEED ON TRITON-X100 INDUCED HYPERCHOLESTEROLEMIA IN RATS
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TITLE PAGE
EFFECTS OF AQUEOUS EXTRACT OF PUNICA GRANATUM SEED ON TRITON-X100 INDUCED HYPERCHOLESTEROLEMIA IN RATS
BY
---
EE/H2013/01430
DEPARTMENT OF ----
SCHOOL OF ---
INSTITUTE OF ---
DECEMBER,2018
APPROVAL PAGE
This is to certify that the research work, "effects of aqueous extract of punica granatum seed on triton-x100 induced hypercholesterolemia in rats" by ---, Reg. No. EE/H2007/01430 submitted in partial fulfillment of the requirement award of a Higher National Diploma on --- has been approved.
By
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Supervisor Head of Department.
Signature………………. Signature……………….
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---
External Invigilator
DEDICATION
This project is dedicated to Almighty God for his protection, kindness, strength over my life throughout the period and also to my --- for his financial support and moral care towards me.Also to my mentor --- for her academic advice she often gives to me. May Almighty God shield them from the peril of this world and bless their entire endeavour Amen.
ACKNOWLEDGEMENT
The successful completion of this project work could not have been a reality without the encouragement of my --- and other people. My immensely appreciation goes to my humble and able supervisor mr. --- for his kindness in supervising this project.
My warmest gratitude goes to my parents for their moral, spiritual and financial support throughout my study in this institution.
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The present study was carried out to investigate the effects of oral administration of aqueous extract of Punicagranatum seed on Triton X-100 induced hypercholesterolemia in rats. Hypercholesterolemia was induced by single intraperitoneal injection of Triton X-100 and the rats were later treated with aqueous extract of Punicagranatum seed at different dosage levels (50,100 and 200mg/kg body weight) and 10ml/kg/day of standard drug (Astrovastatin) for seven days. Another group received 0.9% Normal saline (1 ml/kg/day). Animals were euthanize by anaesthetization at the end of the experiment to harvest serum used for the biochemical analysis. Aqueous extract of Punicagranatum seed extract prevented triton- induced increase in serum cholesterol, triglyceride (TG), low density lipoprotein (LDL) and decrease in high density lipoprotein (HDL) in a statistically significant manner (p < 0.05) and reversed the elevated serum LDL/HDL ratio comparably to Astrovastatin, The results demonstrated the amelioration of triton- induced hypercholesterolemia in rats by aqueous extract of Punicagranatum seed. Consequently, we can hypothesize that the part of Punicagranatum seed mediated therapeutic effects is associated with its antiatherogenic/hypolidemic component.
TABLE OF CONTENTS
Title page i
Approval page ii
Dedication iii
Acknowledgement iv
Table of Content v
List of Table ix
List of Figure x
Abstract xi
CHAPTER ONE: INTRODUCTION
- Background of the study 1
- Justification of the study 3
- Aim and Objectives 3
CHAPTER TWO: LITERATURE REVIEW
2.1 Punicagranatum fruits 5
2.1.2. Botanical description of Punicagranatum seed 10
2.1.3. Health benefits of Punicagranatum seed 10
2.2 Cholesterol 12
2.2.1. Hypercholesterolemia 15
2.2.2. Description of Hypercholesterolemia 15
2.2.3. Signs and symptons of Hypercholesterolemia 19
2.2.4. Causes of Hypercholesterolemia 20
2.2.5. Biomakers of hypercholesterolemia 22
CHAPTER THREE: MATERIALS AND METHODS
3.1. Materials 26
3.1.1. Punicagranatum fruits 26
3.1.2. Experimental animals 26
3.1.3. Reagent and assay kits 26
3.2. Methods 27
3.2.1 Preparation of aqueous extract of Punicagranatum seed 27
3.2.2. Phytochemical analysis 27
3.2.3. Induction of Hypercholesterolemia 31
3.2.4. Experimental Design 31
3.2.5. Preparation of Serum 32
3.3. Biochemical assay 33
3.3.1. Determination of Serum Total cholesterol 33
3.3.2 Determination of Serum High Density Lipoprotein 35
3.3.3. Determination of Serum Triglycerides 38
3.3.4. Determination of Low Density Lipoprotein 41
3.3.5. Determination of Very Low Density Lipoprotein 42
3.3.6. Determination of Artherogenic Index 42
3.3.7. Determination of Coronary Artery Risk 43
CHAPTER FOUR: RESULTS
4.1. Estimation of Total phenol content of aqueous extract of
Punicagranatum seed. 44
4.2. Estimation of radical scavenging 2,2diphenyl – 1- picrylhydrazyl
of aqueous extract of Punicagranatum seed. 44
4.3. Estimation of inhibitory activity using Lipase of aqueous extract of Punicagranatum seed 45
4.4 Total Cholesterol and Triglyceride cholesterol
4.5. High Density Lipoprotein and Very low density lipoprotein
4.6. Low density Lipoprotein
4.7. Atherogenic index and Coronary risk index
CHAPTER FIVE DISCUSSION
Conclusion 57
References 58
Appendix 68
LIST OF TABLE
Table 1: Classification of hypercholesterolemia 18
Table 2: Serum HDL – Concentration range 25
LIST OF FIGURE
Figure 1: Punicagranatum fruit (Al- Muammar 2012) 9
Figure: 2: Chemical structure of cholesterol 14
Figure 3: Hypercholesterolemia 17
Figure 4: Estimation of Total polyphenol content on aqueous extract of Punicagranatum seed 49
Figure 5: The IC50 value obtained by linear equation of Punicagranatum seed. 50
Figure 6: The IC50 value obtained by linear regression equation for
Punicagranatum seed. 51
Figure 7: Effect of Punica granatum aqueous seed on serum cholesterol
and triglyceride concentration in hypercholesterolemic rats. 52
Figure 8: Effect of Punica granatum aqueous seed on serum HDL
and VLDL concentration in hypercholesterolemic rats. 53
Figure 9: Effect of Punicagranatumaqueous seed on serum LDL
concentration in hypercholesterolemic rats. 54
Figure 10: Effect of Punica granatum aqueous seed on serum AI and CRI
concentration in hypercholesterolemic rats. 55
CHAPTER ONE
INTRODUCTION
1.1 Background of study
Hypercholesterolemia is a lipoprotein metabolic disorder characterized by increase in plasma low density lipoprotein (LDL-c) and very low density lipoprotein (VLDL-c) cholesterols or decrease in plasma high density lipoprotein cholesterol (HDL-c). It was reported to be one of the most important risk factors in the development and progression of atherosclerosis that lead to cardiovascular diseases (Rerkasem et al., 2008). Hypercholesterolemia poses a major problem to many societies as well as health professionals because of the close correlation between cardiovascular diseases and lipid abnormalities (Matos et al., 2005; Ramachandran et al., 2003).Hypercholesterolemia is highly prevalent in all the geopolitical zones of Nigeria. It ranges from 60% among apparently healthy Nigerians to 89% among diabetic Nigerians (Oguejiofor et al., 2012). Clinical trials have demonstrated that intensive reduction of plasma low density lipoprotein (LDL-c) levels could reverse atherosclerosis and decrease the incidence of cardiovascular diseases (Brown and Jessup, 1999; Ichihashi et al., 1998).
Punicagranatumcommonly known as pomegranate is a member of monogeneric family, punicacae and is mainly found in Iran which is considered to be its primary centre of origin (Facciola, 1990).Punicagranatum can be divided into several anatomical compartments including seed,juice,peal,leaf,flower,bark and root each possessing interesting pharmacological and toxicological activities. Punicagranatumhas extensively been used as a traditional remedy against acidosis,microbial infection,diarrhea (Kim and Choi, 2009).Punicagranatum seeds have also been shown to contain estrogenic compounds,oestrone and oestradiol (Kim and choi, 2009).The seeds are embedded in a white, spongy astringent pulp (Stover and Mercure, 2007). Punicagranatum is known for its high antioxidant capacity, it has been used for medicinal purposes for centuries (Syed et al 2007). Punica granatum products have also been reported to possess among others anti-inflammatory,antimicrobial and protective effects on liver function and lipid glucose metabolism(Medjakovic and jungbauer, 2013).According to Al-Muammar and khan 2015, the routine supplementation of Punica granatum may prevent or even correct obesity, diabetes and cardiovascular diseases.
1.2 Justification of study
Hypercholesterolemia is a problem faced by manysocieties since it constitutes one of the major risk factors for the development of cardiovascular diseases, such as atherosclerosis and its complications, acute infraction of themyocardium or hypertension (Robbins, 1991; Gerhardt and Gallo 1998; Gomeset al., 1998). In view of the adverse effects associated with hypercholesterolemia, the quest for natural products with lipid-lowering potential and with minimal or no side effect is needed.
1.3 Aim and Objectives of the study
The main aim of this research is to evaluate the effect of aqueous extract of Punica granatum seed on triton X-100 induced hypercholesterolemia in male albino rats.
In order to achieve the aim, the following specific objectives are followed.
- To estimate total phenol content in aqueous extract of Punicagranatum seed.
- To estimate invitro radical scavenging of power aqueous extract of Punicagranatum seed using 2, 2, diphenyl-1-picrylhydrazyl (DPPH) as substrate.
- To estimate inhibitory power of aqueous extract of Punicagranatum seed on lipase enzyme activity.
- To induce hypercholesterolemia in rats using Triton X-100.
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