Methoxyflavones from Stachys glutinosa 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 Stachys glutinosa led to the isolation of four flavones, xanthomicrol (1), sideritoflavone (2), 8-methoxycirsilineol (3), and eupatilin (4), along with two neo-clerodane diterpenes, roseostachenone (8) and a new compound, 3α,4α-epoxyroseostachenol (7). In order to study structure-activity relationships, two methoxyflavones [5-demethyltangeretin (5) and tangeretin (6)] were synthesized by the methoxylation of xanthomicrol. The isolated compounds (1-4, 7, and 8) as well as the xanthomicrol semisynthetic derivatives (5 and 6) 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 Ki 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.

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, GABAB, NMDA and δ opioid receptors. This study suggests the therapeutic potential of WSE as a valuable adjuvant agent in opioid-sparing therapies.

N-Alkyl dien- and trienamides from the roots of Otanthus maritimus with binding affinity for opioid and cannabinoid receptors.

Two new thienylheptatrienamides (1, 5) and one new neo-lignan (12), together with thirteen known compounds (2, 3, 4, 6-11, 13-16) were isolated from the roots of Otanthus maritimus. The structures of the new compounds were elucidated on the basis of extensive 1D and 2D NMR experiments as well as high resolution mass spectrometry. All the isolated amides (1-10), the known pontica epoxide (11) and the new neo-lignan (12) were evaluated for their binding affinity to the CB1 and CB2 as well as to the µ and δ opioid receptors. Some alkylamides showed moderately high binding affinity for CB2 receptors and 1-[(2E,4E,8Z)-tetradecatrienoyl]piperidine (10) resulted the most active one with a Ki value of 160 nM. As far as we know, this is the first example of a tertiary alkylamide that binds CB2 receptors with significant potency. Compounds that showed the highest affinity for cannabinoid receptors (6-8, 10) were much less potent against opioid receptors. Primary structure-activity relationship is discussed. Docking experiments were carried out with the aim to understand the key interactions of the most active compounds with CB2 receptor.