Antinociceptive Activity of Methanolic Extract of Clinacanthus nutans Leaves: Possible Mechanisms of Action Involved

Abstract

Methanolic extract of Clinacanthus nutans Lindau leaves (MECN) has been proven to possess antinociceptive activity that works via the opioid and NO-dependent/cGMP-independent pathways. In the present study, we aimed to further determine the possible mechanisms of antinociception of MECN using various nociceptive assays. The antinociceptive activity of MECN was (i) tested against capsaicin-, glutamate-, phorbol 12-myristate 13-acetate-, bradykinin-induced nociception model; (ii) prechallenged against selective antagonist of opioid receptor subtypes (β-funaltrexamine, naltrindole, and nor-binaltorphimine); (iii) prechallenged against antagonist of nonopioid systems, namely, α₂-noradrenergic (yohimbine), β-adrenergic (pindolol), adenosinergic (caffeine), dopaminergic (haloperidol), and cholinergic (atropine) receptors; (iv) prechallenged with inhibitors of various potassium channels (glibenclamide, apamin, charybdotoxin, and tetraethylammonium chloride). The results demonstrated that the orally administered MECN (100, 250, and 500 mg/kg) significantly (p < 0.05) reversed the nociceptive effect of all models in a dose-dependent manner. Moreover, the antinociceptive activity of 500 mg/kg MECN was significantly (p < 0.05) inhibited by (i) antagonists of μ-, δ-, and κ-opioid receptors; (ii) antagonists of α₂-noradrenergic, β-adrenergic, adenosinergic, dopaminergic, and cholinergic receptors; and (iii) blockers of different K⁺ channels (voltage-activated-, Ca²⁺-activated, and ATP-sensitive-K⁺ channels, resp.). In conclusion, MECN-induced antinociception involves modulation of protein kinase C-, bradykinin-, TRVP1 receptors-, and glutamatergic-signaling pathways; opioidergic, α₂-noradrenergic, β-adrenergic, adenosinergic, dopaminergic, and cholinergic receptors; and nonopioidergic receptors as well as the opening of various K⁺ channels. The antinociceptive activity could be associated with the presence of several flavonoid-based bioactive compounds and their synergistic action with nonvolatile bioactive compounds.

Figure 1

Effect of MECN on capsaicin-induced nociception in mice. Animals were treated with vehicle (10 mL/kg, p.o.), CAPZ (0.17 mmol/kg, p.o.), or MECN (100, 250, and 500 mg/kg, p.o.) 60 min before intraplantar administration of capsaicin (1.6 μg/paw prepared in normal saline; 20 µL) into the right hind paw. Each column represents the mean ± SEM of six mice. Statistical analyses were performed using 1-way ANOVA followed by Dunnett's post hoc test. ^∗∗∗p < 0.001 compared to the control group. Values in parentheses denote percentage of inhibition.

Figure 2

Effect of MECN on glutamate-induced nociception in mice. Animals were treated with vehicle (10 mL/kg, p.o.), ASA (100 mg/kg, p.o.), or MECN (100, 250, and 500 mg/kg, p.o.) 60 min before intraplantar administration of glutamate (10 umol/paw prepared in normal saline; 20 µL) into the right hind paw. Each column represents the mean ± SEM of six mice. Statistical analyses were performed using 1-way ANOVA followed by Dunnett's post hoc test. ^∗∗∗p < 0.001 compared to the control group. Values in parentheses denote percentage of inhibition.

Figure 3

Effect of MECN on PMA-induced nociception in mice. Animals were treated with vehicle (10 mL/kg, p.o.), ASA (100 mg/kg, p.o.), or MECN (100, 250, and 500 mg/kg, p.o.) 60 min before intraplantar administration of glutamate (0.05 μg/paw prepared in normal saline; 20 µL) into the right hind paw. Each column represents the mean ± SEM of six mice. Statistical analyses were performed using 1-way ANOVA followed by Dunnett's post hoc test. ^∗∗∗p < 0.001 compared to the control group. Values in parentheses denote percentage of inhibition.

Figure 4

Effect of MECN on bradykinin-induced nociception in mice. Animals were treated with vehicle (10 mL/kg, p.o.), ASA (100 mg/kg, p.o.), or MECN (100, 250, and 500 mg/kg, p.o.) 60 mins before intraplantar administration of bradykinin (10 nmol/paw prepared in normal saline; 20 µL) into the right hind paw. Each column represents the mean ± SEM of six mice. Statistical analyses were performed using 1-way ANOVA followed by Dunnett's post hoc test. ^∗∗∗p < 0.001 compared to the control group. Values in parentheses denote percentage of inhibition.

Figure 5

The involvement of various nonopioid receptor antagonists on MECN-induced antinociception in the acetic acid-induced abdominal constriction test in mice. Yohimbine (YOH; 0.15 mg/kg, i.p.), pindolol (PDL; 1 mg/kg, i.p.), caffeine (CAF; 3 mg/kg, i.p.), and haloperidol (HAL; 0.2 mg/kg; i.p.) were administrated 15 min before vehicle (10 mL/kg, p.o.) or MECN (500 mg/kg, p.o.). Each column represents the mean ± SEM of six mice. Statistical analyses were performed using 1-way ANOVA followed by Dunnett's post hoc test. ^∗∗∗p < 0.001 compared to the control group. ^#p < 0.05 and ^###p < 0.001 compared to 500 mg/kg MECN-treated group.

Figure 6

Effect of opioid receptor antagonists on MECN-induced antinociception in the acetic acid-induced abdominal constriction test in mice. β-funaltrexamine (β-FNA; 10 mg/kg, i.p.), naltrindole (NALT; 1 mg/kg, i.p.), or nor-binaltorphimine (nor-BNI; 1 mg/kg, i.p.) were administered 90 min, 15 min, and 30 min, respectively, before oral administration of vehicle (10 mL/kg) or MECN (500 mg/kg). Each column represents the mean ± SEM of six mice. Statistical analyses were performed using 1-way ANOVA followed by Dunnett's post hoc test. ^∗∗p < 0.001 and ^∗∗∗p < 0.001 compared to control group. ^###p < 0.001 compared to 500 mg/kg MECN-treated group.

Figure 7

Effect of glibenclamide, apamin, charybdotoxin, and tetraethylammonium chloride on MECN-induced antinociception in the acetic acid-induced abdominal constriction test in mice. Animals were pretreated with glibenclamide (GLIB; 10 mg/kg, i.p.), apamin (APA; 0.04 mg/kg, i.p.), charybdotoxin (CHAR; 0.02, i.p.), or tetraethylammonium chloride (TEA; 4 mg/kg, i.p.) 15 min before oral administration of either vehicle (10 mL/kg) or MECN (500 mg/kg). Each column represents the mean ± SEM of six mice. Statistical analyses were performed using 1-way ANOVA followed by Dunnett's post hoc test. ^∗∗∗p < 0.001 compared to control group. ^###p < 0.001 compared to 500 mg/kg MECN-treated group.

Figure 8

HPLC profile of MECN. Eight peaks were detected from the chromatogram of MECN at various wavelengths ranging between 210 and 366 nm. Interestingly, four peaks were detected at 366 nm, and each of them possessed the maximum wavelength (λ_max) value that represents flavonoid-based bioactive compounds. Comparison between chromatogram obtained from MECN against several pure flavonoids, namely, fisetin, quercetin, rutin, quercitrin, naringenin, genistein, pinostrobin, hesperetin, and dihydroquercetin, at 366 nm shows that none of the peaks of MECN matched any of the flavonoids peak.