Phytomedicinal approach toward the treatment of CNS disorders: behavioural and biochemical evaluation of the effects of a methanolic dry root extract of Withania somnifera and its isolated metabolite, Docosanyl Ferulate, in rodent models of anxiety and drug addiction

The use of phytomedicinal remedies has increased during the last decades thanks to the relative low cost, low toxicity, and potential synergic effects of plant-derived medications. Withania somnifera (WS), commonly known as Indian Ginseng, is a medical plant belonging to the Indian Traditional Medical System, the Ayurveda. In preclinical studies, WS has shown an impressive variety of therapeutic effects in several pathological conditions. Among these effects, the anxiolytic and antiaddictive properties of this plant are the ones discussed in this work of thesis. Both effects have been attributed to a GABAA -mimetic activity of WS. This motivated the search for the single isolated compounds responsible for the GABA mimetic component of the plant. Accordingly, our group performed binding studies on a methanolic dry root extract of WS (WSE) and recently identified several components of the extract endowed with GABAergic activity. Among these, the Ferulic Acid ester, Docosanyl Ferulate (DF), resulted the most potent GABAA agonist, being able to enhance the GABAA receptor inhibitory postsynaptic currents with an IC50 value of 7.9 μM. This thesis consists of a behavioural and biochemical evaluation of the effects of this newly isolated compound in rodent models of anxiety and substance use disorder. Considering that agonism on the GABAA receptor is the mechanism of action of benzodiazepines, the most used and prescribed anxiolytic drugs in the world, a behavioural comparison between DF and Diazepam (DZP) as reference compound has been performed in order to assess the eventual anxiolytic activity of DF. The treatment with DF exerted an anxiolytic effect at the Elevated Plus Maze in adult CD1 mice at the doses of 0.25 and 2 mg/kg and the effects of this last dose did not differ statistically from those induced by DZP (2 mg/kg). In addition, the anxiolytic effects of DF were prevented by a pre-treatment with the GABAA receptor antagonist Flumazenil. Intriguingly, at variance from DZP, DF did not carry motor, mnemonic and addictive side effects and did not potentiate ethanol-induced depressant activity. Moreover, considering the importance of the GABAA mimetic component of WSE in its antiaddictive effects, we verified whether DF could share with the whole extract the property of preventing the acquisition and expression of ethanol- and morphine-induced Conditioned Place Preference (CPP). Interestingly, DF prevented the acquisition, but not the expression of both substances-induced CPP, probably through a preventive effect on ethanol- and morphine-induced activation of the Extracellular signal-regulated kinase (ERK) in the Shell subregion of the Nucleus Accumbens (AcbSh) in adult CD1 mice. Finally, a very detailed characterization of the role of ethanol’s metabolism in ethanol-mediated activation of the mesolimbic pathway was performed. The results pointed out the μ receptors-mediated effect of ethanol-derived salsolinol in the posterior Ventral Tegmental Area (pVTA) as responsible for the ethanol-mediated increases in the dopaminergic traffic in the AcbSh of rats. Hence, considering that WSE can prevent ethanol-induced activation of the mesolimbic pathway and increase in the dopaminergic traffic of AcbSh in rats, we verified whether also DF could share this property and if the salsolinol-mediated mechanism of action could be involved in its action. Remarkably, DF prevented alcohol-induced formation of salsolinol in the pVTA and the consequent increase of DA traffic in the AcbSh. In conclusion, these results confirm the efficiency of WSE as potential treatment for anxiety and substance use disorder and point out DF as a new potent, safe and interesting molecule to be further characterized in order to better understand its therapeutical potential. Moreover, this thesis suggests, for the first time, a plausible mechanism of action for the ethanol-mediated activation of the mesolimbic pathway in vivo