Effects Of Aqueous Extract Of Punica Granatum Seed On Triton-X100 Induced Hypercholesterolemia In Rats

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.

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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


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


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


Table 1:          Classification of hypercholesterolemia                                   18

Table 2:          Serum HDL – Concentration range                                        25


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


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.



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
  • 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.
  • To study the effect of aqueous extract of Punica granatum seed on serum lipid profile of hypercholesterolemic rats.


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