Comparison between Cacao and Ibuprofen as Adjuvants to Paracetamol on Serum iNOS Levels and Von Frey Scores in Acute Pain Model

Abstract

Acute post-traumatic pain induces peripheral inflammation and neuroinflammation, upregulating inducible nitric oxide synthase (iNOS) and decreasing mechanical pain thresholds. Multimodal analgesia targeting distinct pathways in the pain-inflammation cascade may enhance pain relief. Cacao, a natural source of polyphenols with anti-inflammatory properties, may modulate nitric oxide signaling similarly to nonsteroidal anti-inflammatory drugs (NSAIDs). This research aimed to compare the effects of crude ethanolic cacao extract and ibuprofen as adjuvants to paracetamol on serum iNOS levels and mechanical pain thresholds, assessed using Von Frey aesthesiometry, in a rat model of postoperative pain. A randomized, three-arm study was conducted with 18 male Wistar rats (6/group). Rats received paracetamol (15 mg/kg, control), paracetamol + ibuprofen (10 mg/kg), or paracetamol + cacao extract (1000 mg/kg) at T0, T+8, and T+16 hours after induced femoral fracture with fixation. Serum iNOS levels and Von Frey thresholds were assessed at T+24 hours. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test, with effect size (η²) analysis. Serum iNOS levels were significantly lower in both adjuvant groups compared to control (control 13.56 ± 1.59 ng/mL; ibuprofen 10.75 ± 0.54 ng/mL; cacao 9.28 ± 0.71 ng/mL; ANOVA p < 0.001). Cacao reduced iNOS more effectively than the control (p < 0.05) and ibuprofen (p = 0.021). Von Frey thresholds increased significantly in the adjuvant groups (control 9.52 ± 1.63 gf; ibuprofen 19.38 ± 4.15 gf; cacao 26.19 ± 8.01 gf; F = 15.07, p < 0.001). Effect sizes were large for iNOS (η² = 0.80) and moderate to large for Von Frey thresholds (η² = 0.67). Both ibuprofen and cacao, when used as adjuvants to paracetamol, reduced serum iNOS levels and increased pain thresholds within 24 hours in a postoperative pain model. Cacao combination showed the lowest serum iNOS level and highest alteration of Von Frey absolute change. Cacao demonstrated greater iNOS suppression and better pain tolerance, suggesting its potential as a natural, effective analgesic adjuvant. Further studies are needed for cacao extract standardization and clinical translation.