Previous studies conducted in our laboratory demonstrates the ability of the MEMC to produce significant antinociceptive activity in both chemicals- and thermal-induced nociception test model indicating possible participation of central and peripheral antinociceptive mechanisms. The present study focuses further on the mechanisms of action involved in antinociception induced by crude methanol extract from the leaves of M. calabura. The results obtained revealed that the oral administration of MEMC produced significant dose-dependant inhibition of intraplantar (i.pl.) injection of bradykinin- and PMA-induced nociception.
Protein kinase C (PKC) has been reported to indirectly involved in the central sensitization of normally silent N-methyl D-aspartate (NMDA) glutamate receptors located in the postsynaptic neuron [32, 33], suggesting that the activation of PKC also play important role in the nociceptive transmission through a glutamatergic system. The i.pl. administration of PMA causes nociception, thermal hyperalgesia as well as mechanical allodynia in experimental animal model . Based on the above statements, our data further strengthen the involvement of MEMC in central mechanism of antinociception as reported previously . The activation of PKC occurs through interaction with intracellular lipid second messenger phosphatidylserine and diacylglycerol (DAG), and high level of calcium ions , which leads to the phosphorylation of many cellular components including the modulation of TRPV1 receptor [36–38]. Based on our findings, MEMC caused significant reduction in the nociceptive response induced by PMA in a dose-dependent manner, which in turn prevents the phosphorylation of TRPV1. This correlates well with our previous finding  where we demonstrated the possible involvement of TRPV1 in MEMC-induced antinociception through capsaicin-induced paw licking test. We, therefore, suggest that the MEMC antinociceptive activity involved partly the inhibition of TRPV1 receptor phosphorylation via attenuation of the PKC activation.
It is reported that PKC can also be directly activated by binding of bradykinin to its receptor . This view was supported by our results demonstrating the MEMC's ability to suppress the nociception caused by bradykinin. Bradykinin is a potent inflammatory peptide messenger which is generated from a protein precursor, kallidin, through the action of specific enzyme kallikrein. During injury or inflammation, bradykinin will be released from the damaged tissues, from mast cells, as well as produced in the blood where it serves as vasodilators and increases vessel permeability . This peptide is considered as one of the most potent pain-producing substance as it not only excites plenty of nociceptors, but also sensitizes them to other noxious stimuli through activation of B1 and B2 receptors [35, 39].
Bradykinin acted through G-coupled protein receptor, on dorsal root ganglion (DRG) sensory neurons, elicits marked increase in Ca2+, through activation of DAG and PKC pathway [40, 41]. Peripheral sensitization by bradykinin, which acted on the Aδ and C-fibers, evoke the release and synthesis of other second messengers, including prostaglandins, nitric oxide and neurokinins [40, 42, 43]. Pain induced through the introduction of bradykinin into the right hind paw of the experimental rat is significantly inhibited by oral administration of 250 and 500 mg/kg MEMC. It has been reported that the pain induced by bradykinin can be inhibited by cyclooxygenase (COX) inhibitor indomethacin , and therefore this type of pain is mediated by prostaglandins (probably PGE2). The ability of MEMC to inhibit bradykinin hyperalgesia correlates well with previous report , which proposed that MEMC antinociceptive activity seen in acetic acid-induced nociception may occurs through the inhibition of COX, as well as other mediators mentioned above or possibly by directly blocking the B2 receptors.
The noradrenergic receptor system involved greatly in descending modulation of pain pathways. Clonidine, a α2-adrenergic agonist, acting on the nerve endings of primary afferent fibers will inhibit the release of norepinephrine, glutamate and substance P, as well as pro-inflammatory cytokines resulting in sedative and analgesic actions [45, 46]. Our findings suggested the involvement of α2-adrenergic, but excluded the α1-adrenergic receptors since MEMC activity was significantly reversed, when challenged with yohimbine (α2-adrenergic antagonist). In addition, serotonergic receptor pathway correlates with that of noradrenergic system. Activation of serotonergic receptor will cause the release of noradrenaline which activate postsynaptic α2-adrenergic in the spinal cord leading to antinociception [47, 48], and pretreatment with pindolol (5-HT1A/1B receptor/β-adrenoceptor inhibitor) significantly reversed MEMC antinociceptive activity indicating its role in serotonergic system.
We also demonstrated the involvement of adenosinergic receptor system in MEMC-induced antinociception. Caffeine, a non-selective adenosinergic receptor antagonist significantly reduced the action of MEMC. Pharmacologically, caffeine blocked adenosine A1, A2A, A2B, and A3 receptor but with lower affinity [49, 50]. Adenosine receptor activation particularly A1 produces antinociception, by reducing PGE2[51, 52] and triggers NO/cGMP/PKG/KATP pathway  in acute pain, and increases pain threshold  as well as inhibit glutamate release  in chronic pain. Adenosinergic and serotonergic systems are closely involved as A1 receptor antagonist can block serotonin analgesic action . On the other hand, atropine (a cholinergic receptor antagonist) and haloperidol (a dopaminergic receptor antagonist) did not cause any significant changes in the number of abdominal constrictions, indicating lack of involvement of those receptor systems in MEMC antinociception.
The activation of 5-HT1A has been shown to promote the opening of K+ channels and closing of Ca+ channels through coupling negatively to adenylyl cyclase which lead to sensory transmission inhibition . Corroborating to the finding, we demonstrated that pre-treatment with glibenclamide (a specific ATP-sensitive K+ channel blocker), apamin (small conductance Ca2+-activated K+ channels), charybdotoxin (an inhibitor of large conductance Ca2+-activated K+ channels) and tetraethylammonium chloride (a non-selective voltage dependant K+ channel inhibitor) significantly reversed the antinociceptive effect of MEMC. The opening of ATP-sensitive K+ channel has been reported to participate in opioid-mediated antinociception, at the level of K+ and not opioid receptor , since specific ATP-sensitive K+ channel blockers (glibenclamide and gliquidone) shown to dose-dependently reduce the antinociceptive of morphine [59, 60]. This correlates well with previous study demonstrating the involvement of MEMC in opioid receptor system .
Previously, we have demonstrated the involvement of opioid receptor in MEMC antinociception using non-selective opioid antagonist, naloxone . In the present study, we elucidated the possible role of opioid receptor subtype in the modulation of MEMC antinociception using μ, δ, and κ opioid antagonists. Our findings demonstrated MEMC activity was significantly attenuated by all of the opioid subtypes' antagonists, suggesting the role of those receptors in the analgesic activity of MEMC. These receptors, found throughout the nervous system, spinal cord, midbrain and cortex, can mediate pain inhibition , and the report showed increased expression of δ opioid receptor when μ receptors were repeatedly activated , which accounted for the synergistic action seen in other studies [63, 64].
The phytochemical screening shows the presence of flavonoids, triterpenes, saponins steroids and tannins which is in line with previous report , and interestingly all of these bioactive constituents has been reported to be involved in antinociceptive activity [65–68]. Our HPLC analysis revealed the possible presence of flavonols, namely rutin, quercitrin and fisetin. The ability of quercitrin to inhibit the pro-inflammatory mediators involved in pain modulation, especially cytokines, has been reported . Rutin has been reported to produced antinociceptive activity by inhibiting both COX and lipooxygenase (LOX) pathways at high concentration . Various reports have demonstrated that these types of flavonoids possess significant antinociceptive and/or anti-inflammatory activities [71–73].