The Potential Antihypertensive and Antidiabetic

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The Potential Antihypertensive and Antidiabetic The potential antihypertensive and antidiabetic activities of stevia in preventing chronic cardiovascular disease in rat models of hypertension and diabetes: Comparison to the calcium channel antagonist verapamil Saquiba Yesmine A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Medical and Applied Sciences CQUniversity, Rockhampton, Queensland 2012 To my parents To my husband ii Acknowledgements This PhD journey has been made possible by the support and encouragement of many. I would like to thank them all for making this journey so rewarding and meaningful. Firstly, I would like to thank my supervisor Dr Andrew Fenning for his constant guidance, support and valuable comments during the course of this study. I thank him for his patience and spending many long hours in the ‘Fenning Lab’ with me during all my experiments. His encouragement and humour always made me feel comfortable and inspired me from the very first day. I thank my co-supervisor Dr. Fiona Coulson for her thoughtful advice, guidance and encouragement throughout the study. I gratefully acknowledge the support of an IDP (Australia) Post Graduate Research Scholarship that allowed me to undertake this study. I wish to thank my fellow PhD students Douglas Jackson, Rebecca Vella, Candice Pullen, Kylie Connolly, Alannah for their support and help in my experiments and sharing the fun time. I thank Maree Bennett and Laura Harbinson for their assistance during my experiments. I wish to acknowledge the assistance given by Damian Byrt, Graeme Boyle, Heather Smyth, Yvonne McDonald, Charmain Elder and all the technical staff of the Biomedical Sciences of CQUniversity with the spectrophotometer and in the chemistry lab. My sincere thanks are due to my friends Neeta, Ashfaque, Aman, Avro, Habibah for the help and support they gave me when I needed it most. In particular, I am most grateful to Neeta for her constant encouragement and spending many hours with me. I am most thankful to Nasrin Akthar Shimul and Nur Hassan Anupam for their help, care and support for me and my young son Rafat. My deep gratitude and thanks are to my family who have helped in so many ways to complete this project. To my loving parents and my brother Masud Khan, thank you for your constant support and encouragement. To my loving sister-in-law, Kiswar Afreen Khan, without her support, care, advice, and friendship it would not have been possible to complete this project. Whenever I felt down and low, she was always there to help me out. My deepest gratitude and thanks to Kishwar Afreen. I thank my nephew Sadid Khan for his help with his typing and compiling of my experimental data and results. I wish to thank my niece Anika Khan for her sweet and warm company throughout my study. Her encouraging critique and humour gave me a lot of mental strength to continue my study. Lastly, to my parents-in-law, my beloved husband Shumon Mohsin and my little boy Rafat whose understanding and sacrifice made this study possible. Thank you Shumon for your continuous encouragement, support and always having faith in me. Rafat, you should be happy now that your mother will be able to spend more time with you. iii Declaration by author I hereby declare that I am the sole author of this thesis. I authorize CQUniversity to lend this thesis to other institutions or individuals for the purpose of scholarly research. The work contained in this thesis has not been previously submitted, either in whole or in part, for a degree at CQUniversity or any other tertiary institution. To the best of my knowledge and belief, the material presented in this thesis is original, except where due reference is made in text. 26/07/2013 Copyright statement This thesis may be freely copied and distributed for private use and study; however, no part of this thesis or the information contained therein may be included in or referred to in any publication without prior written permission of the author and/or any reference fully acknowledged. 26/07/2013 iv Abstract Diabetes and hypertension are major contributors to cardiovascular disease which is the leading causes of death worldwide. The impact of diabetes on the cardiovascular system is high, with almost 75% of diabetic deaths being directly attributed to some form of heart or blood vessel disorder. Diabetes and hypertension induce significant damage to the heart, blood vessels and kidney. Stevia, Stevia rebaudiana Bertoni, a naturally sweet herb, has been used for several hundred years as an anti-diabetic and cardiotonic in South America. In limited human and rat trials, stevia reduced hypertension, improved insulin-sensitivity and decreased blood lipids following hypertension and diabetes. The current study aimed to identify any cardioprotective benefits of stevia in reversing and/or preventing further cardiovascular damage in animal models of type 1 diabetes and hypertension compared to the calcium channel antagonist verapamil. A mechanistic study on normal healthy tissues was also performed to establish a putative mechanism of action of stevia. The mechanistic study showed that stevia could reduce cardiac action potential duration (APD) at 20%, 50% and 90% of repolarisation, dilate vascular beds and decrease isolated ileum contraction induced by electrical field stimulation (EFS) and M3 receptor agonist carbachol. In short, our results suggested that the mechanism of action of stevia is multimodal since stevia showed beneficial modulatory effects on cardiovascular and gastrointestinal tissues which can be related to calcium channel antagonism, activation of M2 muscarinic receptor function, and via enhanced nitric oxide (NO) release. The second study examined the chronic effects of hypertension in the spontaneously hypertensive rat (SHR). Chronic dosing (8 weeks) of stevia (200 mg/kg/day) or verapamil (4 mg/kg/day) were compared in the SHR to establish if either compound could reduce the severity of cardiovascular remodelling and peripheral neuropathy following hypertension. Stevia treatment prevented maladaptive left ventricular changes, restored cardiac contractility and end systolic pressure and reduced diastolic stiffness in the hypertensive rats. Stevia also normalised liver and kidney weights compared to body weight/tibial length ratio in the SHR rats. Stevia reduced oxidative stress and inflammation as observed by significant increase in TAC and serum NO levels comopared to the unrtreated SHR. Verapamil improved systolic and diastolic functions, except for the heart rate which was significantly reduced in the SHR. The final study utilized the streptozotocin (STZ)-induced type 1 diabetic rat model with the effect of stevia and verapamil again investigated for an eight-week treatment period. The results showed that both stevia and verapamil treatment improved the maximal contractile and relaxation responses of thoracic aortic rings and mesenteric arteries, improved serum NO synthesis, total anti-oxidant capacity and malondialdehyde (MDA) levels following diabetes. Electrophysiological studies showed that stevia prevented prolongation of the action potential at 20%, 50% and 90% of repolarisation suggesting that stevia might restore sinus rhythm and reduce ventricular arrhythmogenesis following diabetes. Verapamil showed similar results in v these electrophysiological studies. In the isolated ileum, stevia prevented the gastrointestinal dysmotility following chronic diabetes. In summary, this study established that stevia can prevent the cardiovascular remodelling following diabetes and hypertension with putative mechanisms thought to be through multiple pathways such as calcium channel blockade, reduction in insulin resistance, increased NO synthesis and reduction of oxidative damage. Verapamil was also shown to be effective in these animal models acting as an anti-inflammatory, antioxidant and NO promoting treatment in addition to its primary pharmacological role. vi List of publications and presentations Publications 1. Yesmine, S, Connolly, K, Hill, N, Coulson, FR & Fenning, AS. (2012), Electrophysiological, vasoactive and gastromodulatory effects of stevia in healthy Wistar rats: accepted in Planta Medica (PLAMED-2012-05-0486-OP) 2. “Prevention of cardiovascular dysfunction and peripheral nerve damage by stevia in diabetic rats” Saquiba Yesmine, Maree Bennett, Douglas J. Jackson, Fiona R. Coulson , Andrew S. Fenning Submitted to the Journal of Ethnopharmacology in December 2011(under review) 3. Islam, S, Yesmine, S, Khan, SA, Alam, NH & Islam, S. (2008), A comparative Study of Thyroid Hormone Levels in Diabetic and Non-Diabetic Patients, South East Asian Journal of Tropical Medicine and Public Health; Vol 39, No. 5. Abstracts presented in refereed conferences 1. Yesmine, S, Connolly, K, Floyd, K, Coulson, FR & Fenning, AS. (2010), Vasoactive and gastrointestinal effects of Stevia, poster presentation at XXth World Congress of the international Society for Heart Research, Kyoto, Japan; May 13-16. 2. Yesmine, S, Hill, N, Coulson, FR & Fenning, AS. (2010), Evaluation of cardiac electrophysiological effects of Stevia, poster presentation at XXth World Congress of the international Society for Heart Research, Kyoto, Japan; May 13-16. 3. Yesmine, S, Bennett, M, Coulson, FR & Fenning, AS. (2009), Prevention of Vascular and Gastrointestinal Damage in Diabetic Rats by Stevia, Heart, Lung and Circulation, vol. 18, suppl. 3, pp. S311-S311. 4. Yesmine, S, Bennett, M, Coulson, FR & Fenning, AS. (2008), Prevention of vascular and gastrointestinal
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