Post-traumatic stress disorder is a disorder characterized by an inability to extinguish traumatic memories and heightened reactivity to emotional stimuli. Due to the heightened resistance of traumatic memories to extinction, treatment for PTSD has been challenging and is limited to behavioral therapies targeted at reducing responsivity to threatening stimuli. Currently there are no standard pharmacological interventions that are specific to PTSD; rather, drugs used appear to target symptoms of some of the co-morbid conditions, such as anxiety (e.g. benzodiazepines) or depression (antidepressants) - which may also affect fear-memory. In this thesis, we explore the effects of natural health products (NHPs) including naturally occurring peptides and some medical botanicals on fear memory in order to explore the efficacy of natural products as potential pharmacological targets for fear-based disorders. Fear-conditioning has been used effectively in both rodents and humans to study fear-learning. Fear-conditioning is a learning paradigm during which an unconditioned aversive stimulus (such as foot shock) is paired with a neutral stimulus (such as light or tone), such that the neutral stimulus becomes associated with aversion. Fear-learning has several well-characterized stages, including acquisition, consolidation, reconsolidation, expression, and extinction that can be manipulated in order to study the pharmacological action(s) on the attenuation of learned-fear. Blockade of reconsolidation, the state during which formed memories are briefly rendered susceptible to change following recall, may provide a window of opportunity to pharmacologically diminish learned fear. In Chapter 1 of the thesis, we discuss fear-conditioning as a pre-clinical model of PTSD to explore the effects of novel pharmacological treatments on the reconsolidation process in rodents. We ultimately hope to provide a framework for translational work in humans for attenuating conditioned responses to trauma-related stimuli among humans with PTSD. In Chapter 2, we present evidence that systemic administration of gastrin-releasing peptide attenuates the reconsolidation of conditioned fear in rodents. Similarly, in chapter 3, we explore the effects of Δ9-Tetrahydrocannabinol (THC) and Cannabidiol (CBD) on the reconsolidation of learned-fear, and provide evidence that cannabinoid molecules may similarly prove effective at blocking the reconsolidation of conditioned fear memories. In chapter 4, we present evidence demonstrating that extracts of medical botanical Souroubea sympetala and its components may similarly block reconsolidation of conditioned fear-memory, and also exert more general anxiolytic-like activity in the elevated plus maze paradigm. Finally, in chapter 5 a general discussion considers the relative therapeutic potential for future human clinical trials of each of the three tested groups of compounds.

This thesis investigates the phytochemistry and pharmacology of two neotropical plants used traditionally to treat anxiety and stress, Souroubea sympetala (Marcgraviaceae) and Piper amalago (Piperaceae). A method of phytochemical analysis was developed to characterize S. sympetala extracts, identifying and quantifying four triterpenes, which were present in higher amounts in bark as compared to leaf. Subsequently, a standardized supercritical CO2 extraction procedure for S. sympetala was developed and compared favourably with conventional extraction methods in terms of its anxiety-reducing effects in a behavioural assay of anxiety and content of the active principle, betulinic acid (BA). All of these materials demonstrated anxiolytic properties. The pharmacological mode of action of S. sympetala raw plant, extracts and isolated active principle were examined in rodent behavioural models of anxiety. The extracts were shown to have affinity for the γ-amino butyric acid (GABA)a benzodiazepine (GABAa- BZD) receptor of the central nervous system in vitro, in a competitive binding assay. Pre-treatment of animals with the GABAa-BZD antagonist flumazenil, followed by plant extract and pure compound extinguished the anxiety-reducing effect, demonstrating that S. sympetala and BA act at the GABAa- BZD receptor in vivo. The effect of S. sympetala in stressed animals, specifically its cortisol-lowering ability was investigated in vitro and in vivo in rainbow trout. Both leaf extract and BA significantly lowered cortisol in response to an adrenocorticotropic hormone (ACTH) challenge in vitro and a standardized net restraint assay in vivo. The anxiety-reducing effect of P. amalago was examined and the bioactive principle identified by bioassay-guided fractionation. P. amalago extract significantly reduced anxiety-like behaviour in rats and demonstrated affinity for the GABAa-BZD receptor in vitro. The bioactive molecule was determined to be a furofuran lignan. Together these results provide a pharmacological basis for the traditional use of S. sympetala and P. amalago to treat anxiety and elucidate their mode of action and active principles. S. sympetala is now thoroughly characterized and represents an excellent candidate plant for development as a natural health product.

This thesis investigates the phytochemistry and pharmacology of two neotropical plants used traditionally to treat anxiety and stress, Souroubea sympetala (Marcgraviaceae) and Piper amalago (Piperaceae). A method of phytochemical analysis was developed to characterize S. sympetala extracts, identifying and quantifying four triterpenes, which were present in higher amounts in bark as compared to leaf. Subsequently, a standardized supercritical CO2 extraction procedure for S. sympetala was developed and compared favourably with conventional extraction methods in terms of its anxiety-reducing effects in a behavioural assay of anxiety and content of the active principle, betulinic acid (BA). All of these materials demonstrated anxiolytic properties. The pharmacological mode of action of S. sympetala raw plant, extracts and isolated active principle were examined in rodent behavioural models of anxiety. The extracts were shown to have affinity for the γ-amino butyric acid (GABA)a benzodiazepine (GABAa- BZD) receptor of the central nervous system in vitro, in a competitive binding assay. Pre-treatment of animals with the GABAa-BZD antagonist flumazenil, followed by plant extract and pure compound extinguished the anxiety-reducing effect, demonstrating that S. sympetala and BA act at the GABAa- BZD receptor in vivo. The effect of S. sympetala in stressed animals, specifically its cortisol-lowering ability was investigated in vitro and in vivo in rainbow trout. Both leaf extract and BA significantly lowered cortisol in response to an adrenocorticotropic hormone (ACTH) challenge in vitro and a standardized net restraint assay in vivo. The anxiety-reducing effect of P. amalago was examined and the bioactive principle identified by bioassay-guided fractionation. P. amalago extract significantly reduced anxiety-like behaviour in rats and demonstrated affinity for the GABAa-BZD receptor in vitro. The bioactive molecule was determined to be a furofuran lignan. Together these results provide a pharmacological basis for the traditional use of S. sympetala and P. amalago to treat anxiety and elucidate their mode of action and active principles. S. sympetala is now thoroughly characterized and represents an excellent candidate plant for development as a natural health product.