EFFECTS OF AQUEOUS EXTRACT OF PUNICA GRANATUM SEED ON TRITON-X100 INDUCED HYPERCHOLESTEROLEMIA IN RATS


EFFECTS OF AQUEOUS EXTRACT OF PUNICA GRANATUM SEED ON TRITON-X100 INDUCED HYPERCHOLESTEROLEMIA IN RATS  

ABSTRACT

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

Acknowledgementiv

Table of Content v

List of Table ix

List of Figurex

Abstract xi

CHAPTER ONE: INTRODUCTION

1.1 Background of the study 1

1.2 Justification of the study 3

1.3 Aim and Objectives 3

CHAPTER TWO: LITERATURE REVIEW

2.1Punicagranatum fruits5

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 Hypercholesterolemia20

2.2.5. Biomakers of hypercholesterolemia22

CHAPTER THREE: MATERIALS AND METHODS

3.1. Materials 26

3.1.1. Punicagranatum fruits26

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 analysis27

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

4.4Total 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.

1 To estimate total phenol content in aqueous extract of Punicagranatum seed.

2 To estimate invitro radical scavenging of power aqueous extract of Punicagranatum seed using 2, 2, diphenyl-1-picrylhydrazyl (DPPH) as substrate. 

3 To estimate inhibitory power of aqueous extract of Punicagranatum seed on lipase enzyme activity.

4 To induce hypercholesterolemia in rats using Triton X-100.

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

CHAPTER TWO

LITERATURE REVIEW

  2.1 Punica granatum fruit

Punicagranatum is a very useful and important pharmacological fruit. It is a fruit which bears deciduous shrub or small tree growing up to 8 meters tall (Facciola, 1990).

  The great thing of Punica granatum fruit is that the trees on which it grows can live for many years. Aside from being a great décor for a backyard it produces the delicious pomegranate fruit. Pomegranate fruit have a round, spherical shape.

Punica granatum (Pomegranate) is a small tree which measures between five and eight meters tall and mainly found in Iran, the Himalayas in northern India, China, USA and throughout the Mediterranean region (Facciola, 1990). The Punica granatum can be divided into several anatomical compartments including seed, juice, peel, leaf, flower, bark, and root with each possessing interesting pharmacological and toxicological activities. The edible fruit is a berry which is about 5-12 cm in diameter with a rounded hexagonal shape, thick reddish skin and around 600 seeds, each surrounded by a water-laden pulp (aril) ranging in colour from white to deep red or purple, the aril is the edible part of the fruit. The seeds are embedded in a white, spongy, astringent pulp (Stover and Mercure, 2007). According to the holy Quran, pomegranates grow in the gardens of paradise and the Quran has recited the Punica granatum twice as an example of God’s good creations.

The fruit of the Punica granatum has extensively been used as a traditional remedy against acidosis, dysentery, microbial infections, diarrhoea, helminth infection, hemorrhage and respiratory pathologies (Kim and Choi, 2009). Punica granatum seeds have also been shown to contain the estrogenic compounds, oestrone and oestradiol (Kim and Choi, 2009). Furthermore, the dried pericarp and the juice of the fruit are considered beneficial for treatment of colic, colitis, menorrhagia, oxy-uriasis, headache, diuretic, acne, piles, allergic dermatitis, and treatment of oral diseases (Ricci et al., 2006). Recent studies have shown new scientific investigations for the traditional uses of Punica granatum (Ricci et al., 2006).  

Punica granatum, known for its high antioxidant capacity, has been used for medicinal purposes for centuries (Syed et al., 2007). Syed et al. (2007) investigated their potential antiproliferative, anti-invasive and pro-apoptotic activities against various human cancer cell lineages and in animal models. To date, over 50 substances showing phytoestrogenic and antioxidant activities have been isolated from the seeds, juice and peel of the fruit and from the leaves and flowers of the tree. Dried peel from ripe pomegranate is used in the treatment of stomach-ache and has proved to be effective in preventing lipoperoxidation. Fruit extracts have succeeded in inhibiting herpes and influenza viruses as well as in suppressing the proliferation of human breast and prostate cancer cells. Reductions in bone erosion and in the severity of depression in ovariectomized rats were observed after administering pomegranate seed extract (Tran et al., 2010; Johanningsmeier 2011). Punica granatum products have also been reported to possess, among others, anti-inflammatory, antimicrobial and immunosuppressive activities and protective effects on liver function and lipid and glucose metabolism (Miguel et al., 2010; Medjakovicand Jungbauer, 2013). According to Al-Muammar and Khan (2015), the routine supplementation of pomegranate juice or its extracts may prevent or even correct obesity, diabetes, and cardiovascular diseases. As indicated in their review, decreasing energy intake, the intestinal absorption of dietary fats by inhibiting pancreatic lipase, and oxidative stress and inflammation might be important mechanisms for the antiobesity effects of pomegranate fruit as a whole (Al-Muammar and Khan 2015).

The fruit can be divided into three parts: seeds, accounting for about 3% total weight and containing 20% oil; juice, accounting for approximately 30% total weight and pericarp, including skin and inner membranous walls and accounting for approximately 67% total weight (Lansky and Newman, 2007). 

Punica granatum seeds are usually by products from the fruit processing. (Kyralanet al., 2009). The seeds have significant antioxidant capacity and a rich chemical composition (sugars, polyunsaturated fatty acids, vitamins, polysaccharides, polyphenols and minerals) (Syed et al., 2007). On, average, pomegranate seeds grown in Brazil, carbohydrate content was found to account for 43.97 %, followed by moisture content 38.30 % and high lipid content 14.06 % (Jardiniet al., 2007). Given the fact that great quantities of punicagranatum seeds are mostly wasted and that they can be processed to produce an attractive amount of a chemically-rich oil, which may be of great interest to the food industry as food or a nutraceutical ingredient.

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EFFECTS OF AQUEOUS EXTRACT OF PUNICA GRANATUM SEED ON TRITON-X100 INDUCED HYPERCHOLESTEROLEMIA IN RATS



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