Running head: PRECLINICAL EVALUATIONS BLEND EXTRACT S. SYMPETALA P. OCCIDENTALIS Preclinical evaluations of the blend extract of Souroubea sympetala and Platanus occidentalis in rats: proposal of a potential novel treatment for post- traumatic stress disorder

Post traumatic stress disorder (PTSD) is characterized by an inability to extinguish fear memories. If left untreated, PTSD typically follows a chronic, unremitting course culminating in significant impairments of vocational, marital and social functioning. Current treatment approaches for PTSD generally yield only symptomatic improvement, possibly owing to the complicated nature of the underlying processes behind the disorder. Due to the less than optimal efficacy of current drug treatments available for PTSD, some attention has also been devoted to exploring the potential of complimentary alternative medicines (CAM). This thesis was aimed at investigating the use of a botanical blend ethanolic extract of a 1:1 Souroubea sympetala and Platanus occidentalis (Sycamore tree bark) as a potential novel treatment for PTSD, which may act by blocking the reconsolidation of fear memories. During reconsolidation, the formed memories are rendered labile during reactivation, thus providing a window of opportunity for pharmacologically manipulating formed fear memories. In the present thesis, the efficacy of reconsolidation blockade of short-term (4h) as well as long term (24h and 9 days) fear memories through treatment with the plant blend extract (7mg/kg), diazepam (1mg/kg) and vehicle was measured through the Fear Potentiated Startle paradigm. PRECLINICAL EVALUATIONS BLEND EXTRACT S. SYMPETALA, P. OCCIDENTALIS 5 Table of contents 0.1 Dedication.2 0.2 Acknowledgements.3 0.3 Abstract.4 0.4 Table of contents.5 0.5 List of abbreviations.7 0.6 List of figures.8 Introduction 1.0 Stress and fear memory.10 1.1 Post Traumatic Stress Disorder.11 1.2 Animal models of PTSD.16 1.2.1 Fear memory reconsolidation in PTSD.19 2.0 Complimentary Alternative Medicine.20 2.1 Botanicals: Betulinic acid, Souroubea sympetala and Platanus occidentalis.21 3.0 Rationale and objectives.23 Methods and materials 4.0 Materials.24 4.1 Plant material.24 4.2 Plant extraction.25 4.3 Animals.25 5.0 Methods.25 5.1 Drug administration.25 5.2 Behavior fear potentiated startle.26 5.2.1 Experiment 1.26 5.2. 2 Experiment 2.27 5.2. 3 Experiment 3.28 5.2. 4 Experiment 4.28 Results PRECLINICAL EVALUATIONS BLEND EXTRACT S. SYMPETALA, P. OCCIDENTALIS 6 6.1 Experiment 1.29 6.2 Experiment 2.31 6.3 Experiment 3.33 6.4 Experiment 4.35 Discussion.37 0.5 References.42 PRECLINICAL EVALUATIONS BLEND EXTRACT S. SYMPETALA, P. OCCIDENTALIS 7 List of abbreviations ACTH Adrinocorticotropic hormone AMY alphaand betaamyrin ANOVA Analysis of variance BE Betulinic acid CAM Complimentary alternative medicine CB1 Cannabinoid type-1 CER Conditioned emotional response CNS Central nervous system CRF Corticotrophin releasing factor CS Conditioned stimulus eCB Endocannabinoid EMDR Eye motion desensitization and reprocessing FPS Fear potentiated startle GABA Gamma-aminobutyric acid HPA Hypothalamic, pituitary, adrenal ITI Intertrial intervals PTSD Post-traumatic stress disorder SAD Substance abuse disorder SNRI Selective norepinephrine reuptake inhibitor SPS Single prolonged stress SSRI Selective serotonin reuptake inhibitor TFCBT Trauma focused cognitive behavior therapy US Unconditioned stimulus PRECLINICAL EVALUATIONS BLEND EXTRACT S. SYMPETALA, P. OCCIDENTALIS 8 List of figures Chapter 1 Fig. 1 FPS paradigm.17 Fig. 2 Conditioned emotional response paradigm.17 Chapter 2 Fig. 3 Souroubea sympetala.21 Fig. 4 Betulinic acid.22 Fig. 5 Platanus occidentalis.23 Chapter 5 Fig. 6a Experiment 1 – timeline.27 Fig. 6b Experiment 2 – timeline.27 Fig. 6c Experiment 3 – timeline.28 Fig. 6d Experiment 4 – timeline.28 Chapter 6 Fig. 7a Experiment 1 – timeline.30 Fig. 7b Graph of PR-STM after 4h following PR administration of treatment.30 Fig. 8a Experiment 2 – timeline.32 Fig. 8b Graph of PR-LTM after 24h following PR administration of treatment.32 Fig. 9a Experiment 3 – timeline.34 Fig. 9b Graph of PR-LTM after 9 days following PR administration of treatment.34 Fig. 10a Experiment 4 – timeline.36 Fig. 10b Graph of non-reactivated controls 24h after treatment.36 Fig. 10c Graph of non-reactivated controls 9 days after treatment.36 PRECLINICAL EVALUATIONS BLEND EXTRACT S. SYMPETALA, P. OCCIDENTALIS 9