Evaluation of the Antinociceptive Properties of Hyptis crenata Pohl (Brazilian Mint) Thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy By Graciela Silva Rocha Institute of Neuroscience (ION) July 2013 Abstract This project aimed to investigate the main traditional use of the plant Hyptis crenata (HC) and evaluate its activity in a pre-clinical trial. A traditional use survey was carried out in two municipalities in Brazil, interviewing 20 people. The results showed that the main use of HC is for pain relief (19/20). The main method used for its preparation was decoction extract (11/20). The antinociceptive activity of HC decoction extract was evaluated showing that HC 15 mg/kg (p.o.) and HC 150 mg/kg (p.o.) increased delay in withdrawal response in the Hargreaves thermal withdrawal test by 29% and 28% respectively and decreased writhing occurrences induced by acetic acid by 70% and 71% (p<0.05), when compared to vehicle. These treatments were examined in the acetic acid writhing test for their effects on c-fos protein expression. The results showed that HC 150 mg/kg (p.o.) decreased c-fos expression in the hypothalamic paraventricular nucleus by 40% (p<0.05). This study also evaluated the HC doses 1 mg/kg (p.o.), 5 mg/kg (p.o.) and 45 mg/kg (p.o.) to assess the dose-response effect. HC dose- dependently induced antinociception in both animal models from 0 mg/kg (p.o.) to 15 mg/kg (p.o.), until a plateau response occurred between 15 mg/kg and 45 mg/kg (thermal withdrawal test p=0.0002 and writhing inhibition p=0.4725). Then, the decoction extract was fractionated and tested in an attempt to identify which HC compounds were active in inducing the antinociceptive effect. The hexane fraction had the highest activity (per dose) compared to the other fractions, indicating that it was enriched with the active compounds. Also, a preliminary COX inhibition assay was carried out; the results indicating possible COX-2 inhibition for HC treatments. Additionally, an investigation through behavioural analyses of whether HC and its fractions were affecting basal behaviour, such as muscle relaxation and sedation, showed no change of such behaviour. Overall, these data support the antinociceptive effect of Hyptis crenata and that there are specific compounds responsible for this effect. © Newcastle University 2013 i Declaration I certify that this thesis is the result of my own investigations and that no part of it has been submitted for any degree other than the Doctor of Philosophy at the University of Newcastle upon Tyne. All references to the work of others are acknowledged. Graciela Silva Rocha © Newcastle University 2013 ii Acknowledgements I would like to express my sincere gratitude to my supervisors Professor Colin Ingram and Dr Kirsten Brandt, first of all for believing in me since the beginning and advising and supporting me on everything. In addition my thanks go to: Prof. Heleno Dias Ferreira from the University of Goias herbarium, Brazil, for verifying Hyptis crenata Pohl identification. For the people of Vila Bela and Porto Esperidiao communities in Brazil who took part in the traditional use survey. Professor Evandro Dall’ Oglio and people from the natural products laboratory at UFMT-Brazil, for sending the plant material. I would like to thank the staff at the Agriculture Building for making my work environment so pleasant and assisting with their advice. My special thanks go to Brian Brown, Wendy Bal, Fiona Maclachlan and Chris Bulman. Thanks must also be expressed to Prof. Christopher J. Seal for giving me some GC-MS advices. To Dr Edward Okello for his great support and advice during the project, especially for the COX inhibition assay. To Dr Matthew Leach, Dr Johnny Roughan, Professor Paul Flecknell and Amy Dickinson for their advice and help in the animal experiments. Especially Johnny, who was always available to help even if it was his busiest day. To Dr Sasha Gartside and Barbara-Anne Robertson for their crucial help with the c-fos expression experiment. To Dr Tom Smulders for his assistance and advice regarding the brain sectioning. To Dr Richard McQuade for his much appreciated suggestions and advice. To Llwyd Orton for his help on fluorescence microscopy pictures. © Newcastle University 2013 iii To Dr Michael Carroll for his chemistry explanations which were given so clearly and patiently and also to Dave Dunbar for the LC-MS analyses and for being so friendly. I would also like to thanks my colleagues and friends from the agriculture school and medical school, especially Helio Lima Neto for giving up his salad box when I didn’t have time to buy one. Also to Andrew Baron, Ann Fitchett, Cigdem Kurt, Claire Savy, Deniz Sarica, Joanne Wallace, Kulapa Supongpan, Ollie Szyszka, Yolande Seddon, Rafael Olea, Richard Austin, Ruth Starr-Keddle and Socrates Stergiades for the good times spent together. Also my dear friends Alan Wild, Alessandra Dantas, Azahara Mesa, Erica Teixeira, Eric Tempel, Joyce Wild, Melanie Ingleton, Peggy Sung, Sonia Dantas and Thalita Rocha for giving me support and making me feel at home during the difficult times of adaptation to a new country and culture. Finally, I could not have done any of this without the support, love and encouragement I have received from my family, specially my parents Clovis and Maria, my grandparents, my sisters, Elem and Jana, and my extended lovely family. My special thanks go to Paul Donald for being so loving, caring and for being by my side when I needed it most, for patiently proof reading and correcting my various drafts and helping me tidy up the data tables. Also to his lovely mother Helen Donald for her words of encouragement to me. © Newcastle University 2013 iv Abbreviations B.C. Before Christ c-fos Immediate early gene family of transcription factors CNS Central nervous system COX Cyclo-oxygenase enzyme DAPI 4',6-diamidino-2-phenylindole DCM Dichloromethane DLPAG Dorsal lateral periaqueductal grey DMPAG Dorsomedial periaqueductal grey FITC Fluorescein GABA Gamma-aminobutyric acid GC-MS Gas chromatography mass spectrometry GPCR G-protein-coupled receptors HC Hyptis crenata HF Hyptis fruticosa HP Hyptis pectinata HPLC High-performance liquid chromatography HS Hyptis suaveolens ID Indomethacin LC-MS Liquid chromatography-mass spectrometry LPA Lysophosphatidic acid mRNA Messenger RNA © Newcastle University 2013 v PAG Periaqueductal grey PBS Phosphate-Buffered Saline PFA Paraformaldehyde PGE1 Prostaglandin E1 PGE2 Prostaglandin E2 PVA Paraventricular thalamic nucleus, anterior PVN Paraventricular nucleus of the hypothalamus R.T. Retention time SA Salicylic acid SO Supraoptic nucleus THC Tetrahydrocannabinol VLPAG Ventrolateral periaqueductal grey n= Number © Newcastle University 2013 vi Table of Contents Chapter 1. Introduction and Literature Review ............................................................ 1 1.1 Traditional Use of Medicinal Plants ................................................................. 2 1.1.1 Historical background of herbal medicine ................................................ 2 1.1.2 Current use of plants as medication .......................................................... 3 1.2 Research on Medicinal Plants ........................................................................... 5 1.2.1 Pain relief drugs developed based on medicinal plants ............................ 5 1.2.2 The process of evaluation of plant activities and their pharmacological properties ................................................................................................................... 7 1.3 Pain .................................................................................................................... 8 1.3.1 Describing and classifying pain – Acute and chronic pain ....................... 9 1.3.2 The physiology of pain and pathways ....................................................... 9 1.3.3 Nociceptors ............................................................................................. 11 1.3.4 Neurotransmitter receptors and Pain ....................................................... 15 1.3.5 Receptors with potential targets for pain relief ....................................... 16 1.3.5.1 Cannabinoid receptor .......................................................................... 16 1.3.5.2 Glutamate and GABA receptors ......................................................... 16 1.3.5.3 Lysophosphatidic acid (LPA) ............................................................. 17 1.3.5.4 Muscarinic acetylcholine receptors ..................................................... 17 1.3.5.5 Opioid receptors .................................................................................. 17 1.3.5.6 Serotonin receptors.............................................................................. 18 1.3.5.7 Somatostatin and galanin receptors ..................................................... 18 1.3.6 Gender and genetic differences involved in pain perception and modulation .............................................................................................................. 18 1.3.7 Pain management and mechanisms of pain relief ................................... 19 1.3.7.1 Opioids ................................................................................................ 20 1.3.7.2 Non-opioids ......................................................................................... 22 1.4 The Genus Hyptis Jacq: Chemical and Biological Studies