Anti-nociceptive mechanisms of Melilotus officinalis Linn. ethanoic extract in mice: Involvement of opioidergic, nitrergic and muscarinic receptors

Pain is a physiologic protective function with regard to external or internal harmful stimulus or tissue damage. The aim of the current study was to determine anti-nociceptive activity of Melilotus officinalis (Linn.) extract on formalin-induced pain in mice.
In experiment 1, adult male mice were injected (i.p) with saline, ethanoic extract of Melilotus officinalis (Linn.) (EEMO) (100, 200 and 400 mg/kg) or morphine (5 mg/kg). In experiment 2, mice were injected with saline, EEMO (400 mg/kg), naloxone (2 mg/kg) and co-injection of EEMO (400 mg/kg) + naloxone (2 mg/kg). In experiment 3, animal received i.p injection of saline, EEMO (400 mg/kg), L-NAME (10 mg/kg) and EEMO (400 mg/kg) + L-NAME (10 mg/kg). In experiment 4, mice were injected (i.p) with saline, EEMO (400 mg/kg), atropine (1 mg/kg) and co-administration of EEMO (400 mg/kg) + atropine (1 mg/kg). Then, the time spent for paw licking was determined the in first and second phase after formalin injection. Then, licking and biting time of the injected paw was recorded after formalin injection in first and second phases.   
According to the results, EEMO in a dose dependent manner significantly diminished licking and biting time of injected paw (pain response) in comparison with the control group (P<0.05). Co-injection of the naloxone + EEMO significantly amplified pain response compared to the EEMO group (P<0.05). Co-injection of the L-NAME + EEMO significantly decreased pain response in comparison with the EEMO group (P<0.05). Pretreatment with atropine significantly enhanced pain response in comparison with the EEMO group (P<0.05).
These findings suggest that anti-nociceptive activity of the EEMO is mediated via opioidergic, nitrergic and muscarinergic systems in mice.