Antinociceptive Potential of Ximenia americana L. Bark Extract and Caffeic Acid: Insights into Pain Modulation Pathways

Abstract

Background/Objectives: This study evaluated the antinociceptive effect of the Ximenia americana L. bark extract (HEXA) and its primary component, caffeic acid (CA), through in vivo assays. Methods: The antinociceptive properties were assessed using abdominal writhing, hot plate, and Von Frey tests. Additionally, the study investigated the modulation of various pain signaling pathways using a pharmacological approach. Results: The results demonstrated that all doses of the HEXA significantly increased latency in the hot plate test, decreased the number of abdominal contortions, reduced hyperalgesia in the Von Frey test, and reduced both phases of the formalin test. Caffeic acid reduced licking time in the first phase of the formalin test at all doses, with the highest dose showing significant effects in the second phase. The HEXA potentially modulated α₂-adrenergic (52.99%), nitric oxide (57.77%), glutamatergic (33.66%), vanilloid (39.84%), cyclic guanosine monophosphate (56.11%), and K⁺ATP channel-dependent pathways (38.70%). Conversely, CA influenced the opioid, glutamatergic (53.60%), and vanilloid (34.42%) pathways while inhibiting nitric oxide (52.99%) and cyclic guanosine monophosphate (38.98%). Conclusions: HEXA and CA exhibit significant antinociceptive effects due to their potential interference in multiple pain signaling pathways. While the molecular targets remain to be fully investigated, HEXA and CA demonstrate significant potential for the development of new analgesic drugs.

Keywords: HPLC; anti-inflammatory; mechanism of action; natural compounds; nociception; pain.

Figure 1

Antinociceptive effect of HEXA (50, 100, and 200 mg/kg) by acetic acid-induced abdominal writhing test (A); the hot plate test (B) and the hypernociception measure by Von Frey test induced by CFA (C). The arrow indicates the treatment day. One-way ANOVA followed by Tukey’s test. (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns = not significant when compared to the negative control group) for the acetic acid-induced abdominal writhing test. Two-way ANOVA followed by Tukey’s test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 when compared to the negative control group) for hot plate and hypernociception induced by CFA assay.

Figure 1

Antinociceptive effect of HEXA (50, 100, and 200 mg/kg) by acetic acid-induced abdominal writhing test (A); the hot plate test (B) and the hypernociception measure by Von Frey test induced by CFA (C). The arrow indicates the treatment day. One-way ANOVA followed by Tukey’s test. (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns = not significant when compared to the negative control group) for the acetic acid-induced abdominal writhing test. Two-way ANOVA followed by Tukey’s test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 when compared to the negative control group) for hot plate and hypernociception induced by CFA assay.

Figure 2

Evaluation of the antinociceptive effect of HEXA (50, 100, and 200 mg/kg) and CA (0.18 and 1.8 mg/kg) on the neurogenic phase (phase 1—(A,C)) and inflammatory phase (phase 2—(B,D)) against formalin-induced pain in mice. These values represent the arithmetic mean ± SE (Standard Error of the Mean) (n = 6/group). One-way ANOVA followed by Tukey’s test. (* p < 0.05; *** p < 0.001; **** p < 0.0001; ns = not significant when compared to the negative control group).

Figure 3

Signaling pathways underlying the antinociceptive response of HEXA (100 mg/kg) and CA (1.8 mg/kg) in the antinociceptive response: (A) vanilloid; (B) glutamatergic; (C) opioid; (D) L-Arginine/Nitric Oxide/cGMP; (E) cyclic guanosine monophosphate. (F) Participation of α2-adrenergic receptors; (G) K+ATP channels against formalin-induced pain in mice. Values present the mean ± SE (Standard Error of the Mean) (n = 6/group). One-way (ANOVA) followed by the Tukey test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 when compared to the negative control group; # p < 0.05; ## p < 0.01; #### p < 0.0001 when comparing agonist vs. antagonist + agonist or HEXA alone vs. antagonist + HEXA group; ns = not significant).

Figure 3

Signaling pathways underlying the antinociceptive response of HEXA (100 mg/kg) and CA (1.8 mg/kg) in the antinociceptive response: (A) vanilloid; (B) glutamatergic; (C) opioid; (D) L-Arginine/Nitric Oxide/cGMP; (E) cyclic guanosine monophosphate. (F) Participation of α2-adrenergic receptors; (G) K+ATP channels against formalin-induced pain in mice. Values present the mean ± SE (Standard Error of the Mean) (n = 6/group). One-way (ANOVA) followed by the Tukey test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 when compared to the negative control group; # p < 0.05; ## p < 0.01; #### p < 0.0001 when comparing agonist vs. antagonist + agonist or HEXA alone vs. antagonist + HEXA group; ns = not significant).

Figure 3

Signaling pathways underlying the antinociceptive response of HEXA (100 mg/kg) and CA (1.8 mg/kg) in the antinociceptive response: (A) vanilloid; (B) glutamatergic; (C) opioid; (D) L-Arginine/Nitric Oxide/cGMP; (E) cyclic guanosine monophosphate. (F) Participation of α2-adrenergic receptors; (G) K+ATP channels against formalin-induced pain in mice. Values present the mean ± SE (Standard Error of the Mean) (n = 6/group). One-way (ANOVA) followed by the Tukey test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 when compared to the negative control group; # p < 0.05; ## p < 0.01; #### p < 0.0001 when comparing agonist vs. antagonist + agonist or HEXA alone vs. antagonist + HEXA group; ns = not significant).

Figure 4

Participation of (A) cholinergic, (B) adenosinergic, (C) dopaminergic pathway, and (D) serotonergic system for the antinociceptive response of HEXA (100 mg/kg) and CA (1.8 mg/kg) against formalin-induced pain in mice. The values present the mean ± SE (Standard Error of the Mean) (n = 6/group). One-way (ANOVA) followed by the Tukey test (* p < 0.05; ** p < 0.01, *** p < 0.001; **** p < 0.0001 when compared to the negative control group; #### p < 0.0001 when comparing agonist vs. agonist + antagonist; ns = not significant).

Figure 4

Participation of (A) cholinergic, (B) adenosinergic, (C) dopaminergic pathway, and (D) serotonergic system for the antinociceptive response of HEXA (100 mg/kg) and CA (1.8 mg/kg) against formalin-induced pain in mice. The values present the mean ± SE (Standard Error of the Mean) (n = 6/group). One-way (ANOVA) followed by the Tukey test (* p < 0.05; ** p < 0.01, *** p < 0.001; **** p < 0.0001 when compared to the negative control group; #### p < 0.0001 when comparing agonist vs. agonist + antagonist; ns = not significant).