Fig. 1
Number of abdominal constrictions in mice during the acetic acid-induced abdominal constriction test after treatment with PEMC, acetylsalicylic acid (ASA; positive control), or vehicle ([C] negative control). Acetic acid was administered intraperitoneally 60 min before oral administration of treatment. n = 6 in each group. Numbers above the bars indicate the percentage of analgesia. ***p < 0.001 as compared to negative control treatment by one-way analysis of variance followed by Dunnett's post hoc test.
Fig. 2
Effect of PEMC in formalin-induced paw licking test. Data represents the mean ± SEM of six rats. The rats were pretreated with vehicle (10% DMSO), PEMC (100, 250, and 500 mg/kg, p.o.), acetylsalicylic acid (ASA, p.o.), or morphine (5 mg/kg, p.o.), 60 min before i.pl. injection of formalin. The asterisks denote the significance levels as compared to control, ***p < 0.001 by one-way ANOVA followed by Dunnett's post hoc test. ***Data differed significantly (p < 0.05) when compared to the 10% DMSO-treated group.
Fig. 3
Antinociceptive profile of PEMC assessed using the hot-plate test in mice. *p < 0.05 when compared with the control group at same the respective interval. Data are the mean ± SEM; n = 6 mice per group.
Fig. 1
Number of abdominal constrictions in mice during the acetic acid-induced abdominal constriction test after treatment with PEMC, acetylsalicylic acid (ASA; positive control), or vehicle ([C] negative control). Acetic acid was administered intraperitoneally 60 min before oral administration of treatment. n = 6 in each group. Numbers above the bars indicate the percentage of analgesia. ***p < 0.001 as compared to negative control treatment by one-way analysis of variance followed by Dunnett's post hoc test.
Fig. 4
Effect of PEMC on capsaicin-induced paw licking test in rats. Each column represents the mean ± SEM of six rats. The rats were pretreated with vehicle (control, 10% DMSO) or PEMC (100, 250, and 500 mg/kg, p.o.) 60 min before injection of capsaicin (1.6 µg/paw, 50 µl, i.pl.). The asterisks denote the significance levels as compared to control, ***p < 0.001 by one-way ANOVA followed by Dunnett's post hoc test.
Fig. 5
Effect of PEMC on glutamate-induced paw licking test in rats. Each column represents the mean ± SEM of six rats. The rats were pretreated with vehicle (control, 10% DMSO) or PEMC (100, 250, and 500 mg/kg, p.o.) 60 min before injection of glutamate (10 µmol/paw, 50 µl, i.pl.). The asterisks denote the significance levels as compared to control, ***p < 0.001 by one-way ANOVA followed by Dunnett's post hoc test.
Fig. 6
Effect of PEMC on nociception induced by phorbol 12-myristate 13-acetate (PMA) on rats. Each column represents the mean ± SEM time spent on paw-licking behavior of six animals. Animals were administered with vehicle (negative control), PEMC (100, 250, and 500 mg/kg, p.o.) or acetylsalicylic acid (ASA; 100 mg/kg, p.o.) 60 min before injection of PMA (50 ml solution containing 0.05 mg PMA/paw) in the right hind paw. Numbers above bars indicate percentage of analgesia. ***p < .001 as compared to the control group by one-way analysis of variance followed by Dunnett's post hoc test.
Fig. 7
The antinociceptive effect of PEMC against bradykinin-induced paw licking. Each column represents the mean ± S.E.M. of six rats. Vehicle (negative control; 10% DMSO, p.o.), PEMC (100, 250 and 500 mg/kg, p.o.) and acetylsalicylic acid (ASA: 100 mg/kg, p.o.). ***p < 0.001 when compared to control group.
Fig. 5
Effect of PEMC on glutamate-induced paw licking test in rats. Each column represents the mean ± SEM of six rats. The rats were pretreated with vehicle (control, 10% DMSO) or PEMC (100, 250, and 500 mg/kg, p.o.) 60 min before injection of glutamate (10 µmol/paw, 50 µl, i.pl.). The asterisks denote the significance levels as compared to control, ***p < 0.001 by one-way ANOVA followed by Dunnett's post hoc test.
Fig. 6
Effect of PEMC on nociception induced by phorbol 12-myristate 13-acetate (PMA) on rats. Each column represents the mean ± SEM time spent on paw-licking behavior of six animals. Animals were administered with vehicle (negative control), PEMC (100, 250, and 500 mg/kg, p.o.) or acetylsalicylic acid (ASA; 100 mg/kg, p.o.) 60 min before injection of PMA (50 ml solution containing 0.05 mg PMA/paw) in the right hind paw. Numbers above bars indicate percentage of analgesia. ***p < .001 as compared to the control group by one-way analysis of variance followed by Dunnett's post hoc test.
Fig. 7
The antinociceptive effect of PEMC against bradykinin-induced paw licking. Each column represents the mean ± S.E.M. of six rats. Vehicle (negative control; 10% DMSO, p.o.), PEMC (100, 250 and 500 mg/kg, p.o.) and acetylsalicylic acid (ASA: 100 mg/kg, p.o.). ***p < 0.001 when compared to control group.
Fig. 8
(A) Effects of L-arginine, L-NAME, and their combination on PEMC antinociception as assessed by acetic acid-induced abdominal constriction test. The letters represent the significance levels between compared groups; a
p < 0.001 when compared with control group; b
p < 0.001 when compared with PEMC-treated group; c
p < 0.001 when compared with L-NAME-treated group, by one-way ANOVA followed by Dunnett's post hoc test. (B) Effects of L-arginine, methylene blue, and their combination on PEMC antinociception as assessed by acetic acid-induced abdominal constriction test. The letters and symbol above the columns represent significance levels between compared groups; a
p < 0.001 when compared with control group, b
p < 0.05 when compared with MB-treated group, #
p < 0.05 when compared between selected group.
Fig. 9
Effect of PEMC on nociception in the acetic acid-induced abdominal constriction test in mice following pretreatment with potassium channels inhibitors. Each column represents the mean ± SEM number of constrictions observed in six animals. Vehicle (10% DMSO, p.o.), PEMC (500 mg/kg, p.o.), glibenclamide (GLIB: 10 mg/kg, i.p.), apamin (APA: 0.04 mg/kg, i.p.), tetraethylammonium chloride (TEA: 0.01 mg/kg, i.p.) The asterisks, *p < 0.05 and **p < 0.01, denote the significance levels as compared to their respective control group. Comparisons between groups were made by one-way analysis of variance followed by Dunnett's post hoc test.
Fig. 10
The involvement of several non-opioid receptor antagonists in PEMC-induced antinociception against acetic acid-induced abdominal writhing test in mice. Vehicle (V: 10% DMSO, p.o.), PEMC (PE: 500 mg/kg, p.o.), atropine (A: 10 mg/kg, i.p.) haloperidol (H: 20 mg/kg, i.p.), pindolol (P: 1 mg/kg, i.p.), caffeine (C: 3 mg/kg, i.p.), yohimbine (Y: 0.15 mg/kg, i.p.). a
p < 0.001 significantly different when compared to vehicle-treated group; b
p < 0.001 significantly different when compared to PEMC-treated group.
Fig. 11
Analysis of opioid receptor subtypes involvement in PEMC-induced antinociception against acetic acid-induced writhing test in mice. Control (C: 10% DMSO, p.o.), PEMC (500 mg/kg, p.o.), naloxone (NLX: 5 mg/kg, i.p.), naltrindole (NALT: 1 mg/kg, i.p.), β-funaltrexamine (β-FNA: 10 mg/kg, i.p.), nor-binaltorphimine (nor-BNI: 1 mg/kg, i.p.) and acetylsalicylic acid (ASA: 100 mg/kg, p.o.). **p < 0.01 and ***p < 0.001 when compared to the group treated only with PEMC.
Fig. 12
(A) The HPLC profile of PEMC at different wavelengths (i.e. 254, 300 and 366 nm). Several major peaks were detected at the retention time (R
T) of 13.77, 20.14, 21.98, 26.08 and 29.25 s at the wavelength of 330 nm. (B) The HPLC profile of PEMC at different wavelengths (i.e. 210, 254, 280, 300. 330 and 366 nm) supported by the UV spectra analysis of PEMC. The UV spectra demonstrated the presence of four major peaks labeled as P1 (R
T 13.772 min), P2 (R
T 20.171 min), P3 (R
T 21.592 min), and P4 (R
T 28.144 min), which were observed at their respective λ
max at the region of 211.9–272.0, 234.3–308.8, 240.1–270.8, 241.3–292.2 nm, respectively, suggesting, in part the presence of flavonoid-based compounds. (C) Comparison of the HPLC profile of PEMC against several pure flavonoids at the wavelength of 366 nm. Only comparison with pinostrobin and flavanone demonstrated matching HPLC profile to that of the extract.
Fig. 13
Schematic diagram of the proposed mechanisms of antinociceptive action demonstrated by PEMC. PEMC exerts antinociceptive activity at peripheral and central levels via modulation of several receptors and neurotransmitters systems. Ca2+: calcium ion; cGMP: cyclic guanosine monophosphate; GC: guanylate cyclase; GTP: guanosine triphosphate; Kv: voltage-gated potassium channel; KATP: ATP-sensitive potassium channel; SKCa: small conductance calcium-activated potassium channel; 5-HTR: serotonergic receptor; α2R: α2-adrenergic receptor; A1R: adenosine receptor; Glut: glutamate; PGE2: prostaglandin E2; EP1: prostaglandin receptor; B2: bradykinin receptor; NMDA: N-methyl-D-aspartate; PKC: protein kinase C enzyme; NOS: nitric oxide synthase; NO: nitric oxide; TRPV1: transient receptor potential cation channel subfamily V member 1.