Secondary Metabolites from Otanthus maritimus, Stachys glutinosa and Withania somnifera: Isolation, Structure Elucidation and Interactions with Cannabinoid and Opioid Systems

In our continuous search for plant secondary metabolites that bind to CB and/or opioid receptors, we selected four extracts that showed interesting affinity versus the above mentioned receptors . In particular: the DCM extract obtained from the aerial parts of Stachys glutinosa (SGE) was able to bind with a good affinity both MOR and DOR with a Ki of 10.3 and 9 μg/mL, respectively while the DCM extract from the leaves of Otanthus maritimus (OME) showed good binding affinity to CB1 (Ki = 2.2 μg/mL) and CB2 (Ki = 1.3 μg/mL) and moderate affinity to MOR and DOR. The third was an alkaloid fraction obtained from the MeOH extract of the roots of Withania somnifera (WSAE) that displayed appreciable affinity versus DOR (Ki = 25.5 μg/mL), CB1 (Ki = 23.5 μg/mL), CB2 (Ki = 20.3 μg/mL) and GABAA (Ki = 14 μg/mL). The in toto MeOH extract (WSE) bound with very low affinity to CB and opioid receptors but displayed interesting affinity to GABAA receptors (Ki = 14 μg/mL) Based on this results this study, carried out in collaboration with the group of Dr. Stefania Ruiu of CNR-­‐Institute of Translational Pharmacology of Cagliari, aimed to: 1. Isolate the secondary metabolites that were responsible of the observed binding affinity 2. Identify the compounds by spectrometric and spectroscopic methods 3. Evaluate the binding affinity of the isolated metabolites to opioid and cannabinoid receptors. 4. Evaluate the most potent and abundant compounds in antinociceptive experiments in mice. A total of 30 secondary metabolites were isolated from the extracts. Two semisynthetic derivatives were also prepared. The OME yielded two new alkylamides and one new neo-­‐lignan , along with thirteen known compounds. Among the constituents identified, 1-­‐[(2E,4E,8Z)-­‐tetradecatrienoyl]piperidine was the most potent binder to both CB1 and CB2 receptors with a Ki value of 0.8 M and 0.16 M, respectively. The molecular modeling approaches applied in this study put in evidence, important requirements for the activity of this series of compounds and gives several hints for the design of optimized CB2 ligands. From the SGE one new neo-­‐clerodane, 3,4α-­‐epoxyroseostachenol along four known flavones (eupatilin, sideritoflavone, xanthomicrol, and 8-­‐methoxycirsilineol) and one neo-­‐clerodane diterpene (roseostachenone), were isolated. In order to find a structure-­‐activity relationships, two metoxyflavones (5-­‐demethyltangeretin, and tangeretin) were synthesized by methoxylation of xanthomicrol. Our results showed that xanthomicrol, the main constituent of S. glutinosa aerial parts (2% of the dried extract) is the principal responsible of the observed opioid binding affinities of the extract, with a Ki value of 0.825 μM to μ opioid receptor. Since the μ receptor is thought to be primarily responsible for the mediation of opioid anti-­‐nociception, we evaluated the anti-­‐ nociceptive activity of xanthomicrol in the tail flick test. Our data demonstrated that pretreatment of xanthomicrol inhibited morphine-­‐induced anti-­‐nociception in the tail flick test, suggesting an antagonistic effect at μ opioid receptor. As regards WSE and WSAE, the results of the binding assays (Table 10) for the μ, CB and GABAA receptors suggested a possible anti-­‐nociceptive effect of W. somnifera. Starting from these preliminar data and from previous studies demonstrating that Withania somnifera prevented the development of tolerance to the analgesic effect of morphine,166 using behavioral appoaches we demonstrate for the first time the ability of WSE pre-­‐treatment to prolong analgesia and to prevent the development of rebound hyperalgesia in mice treated with morphine.