The oil of the Cucurbita pepo Cucurbita species seeds is a rich beginning of phytosterols, phytoestrogens, polyunsaturated fatty acids, vitamin E and beta-carotene. All of these compounds have been shown to hold some positive influence on plasma lipoids and protecting the arterias from harm induced by hypercholesteremia. It is hence hypothesized that since PSO is a rich beginning of these cardioprotective compounds, so it will forestall the lifts in entire cholesterin and supply antiatherogenic action induced by hypercholesteremia.
The survey involved the usage of male and female Sprague – Dawley rats weighing between 250 to 380 g. Hypercholesterolemia was induced by feeding rats with nutrient incorporating 4 % cholesterin and 1 % cholic acid. Animals having this hypercholesterolemic diet ( CC ) were subdivided to have unwritten 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 Zhou and subdivided to have unwritten 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 yearss per hebdomad ( Monday to Friday ) for 10 hebdomads. All groups consisted of five to twelve animate beings. At the terminal of the intervention period all rats were anaesthetized with 15 % urethane and blood collected by cardiac puncture in EDTA tubings. Plasma was collected and assessed for entire cholesterin ( TC ) , low denseness lipoprotein cholesterin ( LDL-C ) , high denseness lipoprotein cholesterin ( HDL-C ) and trigylcerides ( TG ) . The thoracic aorta for each rat was collected and aortal ring readyings were mounted in organ baths incorporating Krebs – Henseleit solution, aerated with 95 % O and 5 % C dioxide. Vascular responsiveness was examined by precontracting the aortal rings with phenyleprine ( 3 A- 10 -7 M ) and measuring endothelium dependent relaxation utilizing acetylcholine ( 10 -4 – 10 -8 M ) and endothelium independent relaxation with Na nitroprusside ( 10 -7 – 10 -10 M ) . The liver of each rat was assessed for gross anatomical differences, so removed and fixed in 10 % formal saline for histological rating.
TC was increased four times in CC+OO group when compared to rats on normal diet, N+OO ( 208.26 A± 26.3 mg/dL vs. 52.53 A± 5.81 mg/dL ; P & lt ; 0.001 ) with no important alteration in TG. Further appraisal of the alteration showed that while there was no alteration in HDL-C, there was an octuple addition in LDL-C ( 192.76 A± 25.59 mg/dL in CC+OO vs 24.77 A± 5.38 mg/dL in N+OO ; P & lt ; 0.001 ) . This lift in TC induced by the hypercholestoremic diet was inhibited by PSO supplementation at the lower dosage ( 122.95 A± 13.3 mg/dL ; P & lt ; 0.05 ) and even greater protection was observed at the higher dosage ( 82.30 A± 11.33 mg/dL ; P & lt ; 0.01 ) . Similarly, the protection was reflected in the concentration of LDL – Degree centigrade at the lower dosage of PSO ( 94.09 A± 13.38 mg/dL ; P & lt ; 0.01 ) and even greater protection at the higher dosage ( 55.84 A± 9.13 mg/dL ; P & lt ; 0.01 ) . There was no important difference in TG or HDL-C in these PSO treated hypercholesterolemic groups. Additionally, PSO did non impact the concentration of any of the plasma lipoids in rats on normal diets.
Based on the TC and LDL-C consequences, fatty infiltration of hepatocytes was expected which would interrupt the cytoarchitectonics of the liver. Rats in the CC+OO group after histological scrutiny showed important break to the hepatic cytoarchitectonics and fatty devolution of hepatocytes. Hypercholesterolemic groups having PSO were protected from this infiltration ; this protective consequence did non look to be dose related. These consequences suggest that PSO supplementation inhibited the sum of cholesterin that was delivered from the diet to the liver. No difference was observed in the hepatic cytoachitecture of rats having PSO and on normal diet.
Phenylephrine induced contractions were non affected by any of the interventions. 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, bespeaking that the endothelium relaxant activity was compromised. This damage was prevented by PSO supplementation, with significance seen at the higher PSO dosage. The endothelium independent relaxations assessed with Na nitroprusside showed no difference between the groups.
It can be concluded from this survey that PSO has the potency to forestall diet-induced hypercholesteremia and the hurtful effects on fat infiltration of hepatocytes. Hypercholesterolemia is known to bring on coronary artery disease, evident in the early phases as vascular changes to endothelium dependent relaxation. Vascular changes were achieved in the survey and were prevented by PSO supplementation. These positive influences are most likely associated with the combination of compounds present which have been proven to supply protective benefits in hypercholesteremia.
Keywords: Hypercholesterolemia ; Pumpkin seed oil ; Vascular responsiveness.
The completion of this thesis would hold merely been a dream without the Godhead inspiration and intercession of God my male parent. You are still in the miracle working concern. Thank you.
I would wish to show my grasp to my supervisors: Dr. Maxine Gossell – Williams who, from my undergraduate experience inspired my involvement in Ethnopharmacology. I thank you for your counsel and huge forbearance. Particular thanks to Professor Oswald Simon for his parts. To Mr. Michael Gardener, thank you for your clip – the lone thing in the universe we ( still ) can non purchase and your adept advice.
My gratitude besides goes to Mr. Everton Thomas, Mr. Marc Grey and Mr. Hopeton Marshall for their priceless parts you have each made in helping above and beyond the call of responsibility.
To Miss. Kadish Johnson and Mr. Davis Haynes from the Anatomy Department for their aid with the histological subdivisions of this survey.
Dr. K. W. Wolff from the Electron Microscopy Department for his aid in bring forthing the photomicrographs for my thesis.
To the Scientific Research Council and Mr. Sheridan Hibbert for their aid in extraction procedure.
To Agriventures for providing all the works stuff used in the survey.
Margot Thompson and Tricia Melville who were my changeless incentives. Thank you.
To the academic and proficient staff ; every bit good as fellow pupils of the Pharmacology Department who are a fantastic human existences. Thankss
The most particular thanks goes to my spouse and best friend, my hubby, Barry for your company during those late darks in the lab, you gave me your unconditioned support and love through this procedure. Te Amo.
To all my household and friends I am everlastingly thankful for all your supplications and words of encouragement you have provided.
This undertaking was supported by the Office of Research & A ; 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 manner since the beginning of my surveies, who sacrificed to supply me with one of the best gifts – Education.
Table of Contentss
List of Figures xii
List of Traces xv
List of Tables xvi
List of Equipment and Chemicals xvii
List of Abbreviations xix
Chapter i?‰ – Introduction
1.1 Cholesterol 1
1.2 Hypercholesterolemia and coronary artery disease 6
1.3 Anatomy of the liver 8
1.4 Vascular responsiveness of the aorta 10
1.4.1 Phenylephrine 11
1.4.2 Acetylcholine 11
1.4.3 Sodium Nitroprusside 13
1.5 Rat theoretical account of hypercholesteremia 13
1.6 Pharmacological direction of hypercholesteremia 13
1.7 Alternate 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 I? – provitamin A 20
1.8 Pumpkin seed oil 20
1.8.1 Components of PSO 23
1.9 Hypothesis and Objectives 23
Chapter i?‰i?‰ – 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 Initiation of hypercholesteremia 28
Appraisal of the effects of PSO on hypercholesterolemic and normal rats 30
Body Weight 30
Measurement of biochemical parametric quantities 30
Collection of plasma
HDL – cholesterin
Vascular responsiveness 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 i?‰i?‰i?‰ – Consequences
3.1. Extraction of pumpkin seed oil 39
3.2 Assessment of organic structure weight throughout the intervention period 39
3.3 Analysis of rat plasma lipid degrees 41
3.3.1 Analysis of entire cholesterin concentration 41
3.3.2 Analysis of triglyceride concentration 43
3.3.3 Analysis of HDL – cholesterin concentration 45
3.3.4 Analysis of LDL – cholesterin concentration 47
3.4 Response of aortal rings to vasoactive substances 49
Determination of the dosage of PE used to bring forth a sub – maximum contraction of the aortal rings for vascular responsiveness surveies 49
The reactivity of aortal rings to precontraction with ( 3 A- 10 -7 M ) of PE 52
3.4.3 Relaxation responses to acetylcholine and sodium nitroprusside of aortal rings
precontracted with phenylephrine 54
3.4.4 Effectss of acetylcholine and Na nitroprusside on phenylephrine precontracted
aortal rings 56
Endothelium dependent responses of aortal rings precontracted with
Endothelium independent responses to aortal rings contracted with
Table of Contents ( continued ) page
Macroscopic appraisal of the livers of animate beings on a criterion
and hypercholesterolemic diet 64
3.6 Histological appraisal of the rat liver 66
Chapter i?‰V – Discussion & A ; ConClusion 74