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

Withania somnifera root extract prolongs analgesia and suppresses hyperalgesia in mice treated with morphine

Previous studies demonstrated that Withania somnifera Dunal (WS), a safe medicinal plant, prevents the development of tolerance to the analgesic effect of morphine. In the present study, we investigated whether WS extract (WSE) (100 mg/kg, i.p.) may also modulate the analgesic effect induced by acute morphine administration (2.5, 5, 10 mg/kg, s.c.) in the tail-flick and in the hot plate tests, and if it may prevent the development of 2.5 mg/kg morphine-induced rebound hyperalgesia in the low intensity tail-flick test. Further, to characterize the receptor(s) involved in these effects, we studied, by receptor-binding assay, the affinity of WSE for opioid (μ, δ, k), cannabinoid (CB1, CB2), glutamatergic (NMDA), GABAergic (GABAA, GABAB), serotoninergic (5HT2A) and adrenergic (α2) receptors. The results demonstrated that (i) WSE alone failed to alter basal nociceptive threshold in both tests, (ii) WSE pre-treatment significantly protracted the antinociceptive effect induced by 5 and 10 mg/kg of morphine only in tail-flick test, (iii) WSE pre-treatment prevented morphine-induced hyperalgesia in the low intensity tail-flick test, and (iv) WSE exhibited a high affinity for the GABAA and moderate affinity for GABAB, NMDA and δ opioid receptors. WSE prolongs morphine-induced analgesia and suppresses the development of morphine-induced rebound hyperalgesia probably through involvement of GABAA, GABA B, NMDA and δ opioid receptors. This study suggests the therapeutic potential of WSE as a valuable adjuvant agent in opioid-sparing therapies

Methoxyflavones from <i>Stachys glutinosa</i> with Binding Affinity to Opioid Receptors: In Silico, in Vitro, and in Vivo Studies

Fractionation of the bioactive dichloromethane extract from the aerial parts of <i>Stachys glutinosa</i> led to the isolation of four flavones, xanthomicrol (<b>1</b>), sideritoflavone (<b>2</b>), 8-methoxycirsilineol (<b>3</b>), and eupatilin (<b>4</b>), along with two <i>neo</i>-clerodane diterpenes, roseostachenone (<b>8</b>) and a new compound, 3α,4α-epoxyroseostachenol (<b>7</b>). In order to study structure–activity relationships, two methoxyflavones [5-demethyltangeretin (<b>5</b>) and tangeretin (<b>6</b>)] were synthesized by the methoxylation of xanthomicrol. The isolated compounds (<b>1</b>–<b>4</b>, <b>7</b>, and <b>8</b>) as well as the xanthomicrol semisynthetic derivatives (<b>5</b> and <b>6</b>) were evaluated for their binding affinity to the μ and δ opioid receptors. Xanthomicrol was the most potent binder to both μ and δ receptors, with a <i>K</i>_i value of 0.83 and 3.6 μM, respectively. Xanthomicrol administered intraperitoneally in mice at a dose of 80 mg/kg significantly reduced morphine-induced antinociception in the tail flick test. Our results suggested that xanthomicrol is a μ opioid receptor antagonist. Docking experiments were carried out to acquire a deeper understanding about important structural aspects of binding of xanthomicrol. In summary, these data suggest that xanthomicrol is a valuable structure for further development into a potential μ opioid receptor antagonist

Conscious Dwelling For Transdisciplinary Cityscapes

Through a transdisciplinary perspective, the book aims to read the complex urban dimension, in front of increasing density, soil consumption, abandoned places, and the recent pandemic which proved megacities particularly inadequate to provide healthy psychophysical conditions. Assuming bodily and emotional comfort as a reference horizon, it tends to inspire the design research beyond a paradoxical binary logic that separates public and private, outside and inside, far and near, mind and places. The first part deals with built spaces and addresses sustainable strategies not only to overcome an ecologic and systemic crisis but also to improve places liveability in our contemporary city. The second part deals with our perception of aesthetic spaces, welcoming the stimuli coming from neuro-aesthetics studies on affordances and atmosphere and encouraging the intersection between interior architecture and design culture and arts. The third part deals with relational spaces and how they influence human behaviour, starting from psychological, anthropological and philosophical perspectives. Fostering an innovative standpoint, the book benefits scholars, academics and practitioners focused on interior architecture and design, as well as other researchers involved in the relationship between people and places. The new challenge posed by the recent pandemic requires more than ever to rely on consciousness, culture and creativity to increase the intelligence of our surroundings, allowing our sense of belonging and improving our personal and mutual wellbeing