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The oil of the pumpkin Cucurbita species seeds is a rich source of phytosterols, phytoestrogens, polyunsaturated fatty acids, vitamin E and beta-carotene. All of these compounds have been shown to have some positive influence on plasma lipids and protecting the arteries from damage induced by hypercholesterolemia. It is therefore hypothesized that since PSO is a rich source of these cardioprotective compounds, then it will prevent the elevations in total cholesterol and provide antiatherogenic action induced by hypercholesterolemia.
The study involved the use of male and female Sprague - Dawley rats weighing between 250 to 380 g. Hypercholesterolemia was induced by feeding rats with food containing 4% cholesterol and 1% cholic acid. Animals receiving this hypercholesterolemic diet (CC) were subdivided to receive oral dosing of olive oil (CC+OO), PSO at 40 mg/Kg (CC+P40) and PSO at 80 mg/Kg (CC+P80). Three other groups of rats were fed normal rat chow and subdivided to receive oral dosing of olive oil (N+OO), PSO at doses of 40 mg/Kg (N+P40) and 80 mg/Kg (N+P80). Dosing with olive oil or PSO was done five days per week (Monday to Friday) for 10 weeks. All groups consisted of five to twelve animals. At the end of the treatment period all rats were anaesthetized with 15% urethane and blood collected by cardiac puncture in EDTA tubes. Plasma was collected and assessed for total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) and trigylcerides (TG). The thoracic aorta for each rat was collected and aortic ring preparations were mounted in organ baths containing Krebs - Henseleit solution, aerated with 95% oxygen and 5% carbon dioxide. Vascular reactivity was examined by precontracting the aortic rings with phenyleprine (3 Ã- 10 -7 M) and assessing endothelium dependent relaxation using acetylcholine (10 -4 - 10 -8 M) and endothelium independent relaxation with sodium nitroprusside (10 -7 - 10 -10 M). The liver of each rat was assessed for gross anatomical differences, then removed and fixed in 10% formal saline for histological evaluation.
TC was increased fourfold in CC+OO group when compared to rats on normal diet, N+OO (208.26 ± 26.3 mg/dL vs. 52.53 ± 5.81 mg/dL; P < 0.001) with no significant change in TG. Further assessment of the change showed that while there was no change in HDL-C, there was an eightfold increase in LDL-C (192.76 ± 25.59 mg/dL in CC+OO vs 24.77 ± 5.38 mg/dL in N+OO; P < 0.001). This elevation in TC induced by the hypercholestoremic diet was inhibited by PSO supplementation at the lower dose (122.95 ± 13.3 mg/dL; P < 0.05) and even greater protection was observed at the higher dose (82.30 ± 11.33 mg/dL; P < 0.01). Similarly, the protection was reflected in the concentration of LDL - C at the lower dose of PSO (94.09 ± 13.38 mg/dL; P < 0.01) and even greater protection at the higher dose (55.84 ± 9.13 mg/dL; P < 0.01). There was no significant difference in TG or HDL-C in these PSO treated hypercholesterolemic groups. Additionally, PSO did not affect the concentration of any of the plasma lipids in rats on normal diets.
Based on the TC and LDL-C results, fatty infiltration of hepatocytes was expected which would disrupt the cytoarchitecture of the liver. Rats in the CC+OO group after histological examination showed significant disruption to the hepatic cytoarchitecture and fatty degeneration of hepatocytes. Hypercholesterolemic groups receiving PSO were protected from this infiltration; this protective effect did not appear to be dose related. These results suggest that PSO supplementation inhibited the amount of cholesterol that was delivered from the diet to the liver. No difference was observed in the hepatic cytoachitecture of rats receiving PSO and on normal diet.
Phenylephrine induced contractions were not affected by any of the treatments. Endothelium dependent relaxation was significantly impaired in rats on the hypercholesterolemic diet (CC+OO), as responses to acetylcholine were attenuated when compared with N+OO rats, indicating that the endothelium relaxant activity was compromised. This impairment was prevented by PSO supplementation, with significance seen at the higher PSO dose. The endothelium independent relaxations assessed with sodium nitroprusside showed no difference between the groups.
It can be concluded from this study that PSO has the potential to prevent diet-induced hypercholesterolemia and the deleterious effects on fat infiltration of hepatocytes. Hypercholesterolemia is known to induce atherosclerosis, evident in the early stages as vascular alterations to endothelium dependent relaxation. Vascular alterations were achieved in the study and were prevented by PSO supplementation. These positive influences are most likely associated with the combination of compounds present which have been proven to provide protective benefits in hypercholesterolemia.
Keywords: Hypercholesterolemia; Pumpkin seed oil; Vascular reactivity.
The completion of this thesis would have only been a dream without the divine inspiration and intervention of God my father. You are still in the miracle working business. Thank you.
I would like to express my appreciation to my supervisors: Dr. Maxine Gossell - Williams who, from my undergraduate experience inspired my interest in Ethnopharmacology. I thank you for your guidance and immense patience. Special thanks to Professor Oswald Simon for his contributions. To Mr. Michael Gardener, thank you for your time - the only thing in the world we (still) cannot buy and your expert advice.
My gratitude also goes to Mr. Everton Thomas, Mr. Marc Grey and Mr. Hopeton Marshall for their invaluable contributions you have each made in assisting above and beyond the call of duty.
To Miss. Kadish Johnson and Mr. Davis Haynes from the Anatomy Department for their assistance with the histological sections of this study.
Dr. K. W. Wolff from the Electron Microscopy Department for his assistance in producing the photomicrographs for my thesis.
To the Scientific Research Council and Mr. Sheridan Hibbert for their assistance in extraction process.
To Agriventures for supplying all the plant material used in the study.
Margot Thompson and Tricia Melville who were my constant motivators. Thank you.
To the academic and technical staff; as well as fellow students of the Pharmacology Department who are a wonderful human beings. Thanks
The most special thanks goes to my partner and best friend, my husband, Barry for your companionship during those late nights in the lab, you gave me your unconditional support and love through this process. Te Amo.
To all my family and friends I am eternally grateful for all your prayers and words of encouragement you have provided.
This project was supported by the Office of Research & Publication and the Office of Graduate Awards at the University of the West Indies.
This thesis is dedicated to my parents Bendley and Ennis Melville who have supported me all the way since the beginning of my studies, who sacrificed to provide me with one of the best gifts - Education.
Table of Contents
List of Figures xii
List of Traces xv
List of Tables xvi
List of Equipment and Chemicals xvii
List of Abbreviations xix
Chapter ‰ - INTRODUCTION
1.1 Cholesterol 1
1.2 Hypercholesterolemia and atherosclerosis 6
1.3 Anatomy of the liver 8
1.4 Vascular reactivity of the aorta 10
1.4.1 Phenylephrine 11
1.4.2 Acetylcholine 11
1.4.3 Sodium Nitroprusside 13
1.5 Rat model of hypercholesterolemia 13
1.6 Pharmacological management of hypercholesterolemia 13
1.7 Alternative therapies 15
1.7.1 Phytosterols and Phytoestrogens 15
Table of Contents (continued) page
1.7.2 Polyunsaturated fatty acids 18
1.7.3 Tocopherols (Vitamin E) 19
1.7.4 Î² - carotene 20
1.8 Pumpkin seed oil 20
1.8.1 Components of PSO 23
1.9 Hypothesis and Objectives 23
Chapter ‰‰ - Materials and Methods
2.1 Collection and Authentication of Pumpkin seeds 25
2.2 Preparation of seeds 25
2.3 Supercritical Extraction (SFE) of PSO 25
2.4 Animals 27
2.5 Induction of hypercholesterolemia 28
Assessment of the effects of PSO on hypercholesterolemic and normal rats 30
Body Weight 30
Measurement of biochemical parameters 30
Collection of plasma
HDL - cholesterol
Vascular reactivity 33
Histological analysis of liver 34
Preparation of liver for histology
Table of Contents (continued) page
Preparation of slides for microscopy
2.7 Statistical analysis 37
Chapter ‰‰‰ - Results
3.1. Extraction of pumpkin seed oil 39
3.2 Assessment of body weight throughout the treatment period 39
3.3 Analysis of rat plasma lipid levels 41
3.3.1 Analysis of total cholesterol concentration 41
3.3.2 Analysis of triglyceride concentration 43
3.3.3 Analysis of HDL - cholesterol concentration 45
3.3.4 Analysis of LDL - cholesterol concentration 47
3.4 Response of aortic rings to vasoactive substances 49
Determination of the dose of PE used to produce a sub - maximal contraction of the aortic rings for vascular reactivity studies 49
The responsiveness of aortic rings to precontraction with (3 Ã- 10 -7 M) of PE 52
3.4.3 Relaxation responses to acetylcholine and sodium nitroprusside of aortic rings
precontracted with phenylephrine 54
3.4.4 Effects of acetylcholine and sodium nitroprusside on phenylephrine precontracted
aortic rings 56
Endothelium dependent responses of aortic rings precontracted with
Endothelium independent responses to aortic rings contracted with
Table of Contents (continued) page
Macroscopic assessment of the livers of animals on a standard
and hypercholesterolemic diet 64
3.6 Histological assessment of the rat liver 66
Chapter ‰V - Discussion & ConClusion 74