Association of terpinolene and diclofenac presents antinociceptive and anti-inflammatory synergistic effects in a model of chronic inflammation

Abstract

Pharmacological treatment of inflammatory pain is usually done by administration of non-steroidal anti-inflammatory drugs (NSAIDs). These drugs present high efficacy, although side effects are common, especially gastrointestinal lesions. One of the pharmacological strategies to minimize such effects is the combination of drugs and natural products with synergistic analgesic effect. The monoterpene terpinolene (TPL) is a chemical constituent of essential oils present in many plant species, which have pharmacological activities, such as analgesic and anti-inflammatory. The association of ineffective doses of TPL and diclofenac (DCF) (3.125 and 1.25 mg/kg po, respectively) presented antinociceptive and anti-inflammatory effects in the acute (0, 1, 2, 3, 4, 5 and 6 h, after treatment) and chronic (10 days) inflammatory hyperalgesia induced by Freund's complete adjuvant (CFA) in the right hind paw of female Wistar rats (170-230 g, n=6-8). The mechanical hyperalgesia was assessed by the Randall Selitto paw pressure test, which determines the paw withdrawal thresholds. The development of edema was quantified by measuring the volume of the hind paw by plethismography. The TPL/DCF association reduced neutrophils, macrophages and lymphocytes in the histological analysis of the paw, following a standard staining protocol with hematoxylin and eosin and the counts were performed with the aid of optical microscopy after chronic oral administration of these drugs. Moreover, the TPL/DCF association did not induce macroscopic gastric lesions. A possible mechanism of action of the analgesic effect is the involvement of 5-HT2A serotonin receptors, because ketanserin completely reversed the antinociceptive effect of the TPL/DCF association. These results suggest that the TPL/DCF association had a synergistic anti-inflammatory and analgesic effect without causing apparent gastric injury, and that the serotonergic system may be involved in the antinociceptive effect of this association.

Figure 1. A, Sodium diclofenac. B, Monoterpene terpinolene (4-isopropylidene-1- methylcyclohexene) (Opdyke, 1988).

Figure 2. Experimental design. CFA: complete Freund's adjuvant; TPL: terpinolene; DCF: diclofenac.

Figure 3. Effect of acute (A) and chronic (B) administration of diclofenac (DCF). Each point represents the mean±SEM of the paw withdrawal threshold in female Wistar rats (n=6-8) with mechanical hyperalgesia induced by Freund's complete adjuvant (CFA) (50 μL/paw) injection (ipl) subjected to the Randall Selitto test. **P<0.01 and ***P<0.001 compared to vehicle group (two-way ANOVA, Bonferroni test).

Figure 4. Effect of acute (A) and chronic (B) administration of terpinolene monoterpene (TPL). Each point represents the mean±SEM of the paw withdrawal threshold in female Wistar rats (n=6-8) with mechanical hyperalgesia induced by Freund's complete adjuvant (CFA) (50 μL/paw) injection (ipl) subjected to the Randall Selitto test. DCF: diclofenac. **P<0.01 and ***P<0.001 compared to vehicle group (two-way ANOVA, Bonferroni test).

Figure 5. Effect of acute (A) and chronic (B) administration of terpinolene monoterpene (TPL)/diclofenac (DCF) association. Each point represents the mean±SEM of the paw withdrawal threshold in female Wistar rats (n=6-8) with mechanical hyperalgesia induced by Freund's complete adjuvant (CFA) (50 μL/paw) injection (ipl) subjected to the Randall Selitto test. ***P<0.001 compared to vehicle group (two-way ANOVA, Bonferroni test).

Figure 6. Effect of acute (A) and chronic (B) administration of diclofenac (DCF) on the paw edema induced by Freund's complete adjuvant (CFA) in female Wistar rats (n=6-8). Each point represents the mean±SEM of the paw volume (mL) before CFA injection (i.e., baseline) or at the times indicated thereafter. *P<0.05, **P<0.01 and ***P<0.001 compared to vehicle group (two-way ANOVA, Bonferroni test).

Figure 7. Effect of acute (A) and chronic (B) administration of terpinolene monoterpene (TPL) on the paw edema induced by Freund's complete adjuvant (CFA) in female rats (n=6-8). Each point represents the mean±SEM of the paw volume (mL) before CFA injection (i.e., baseline) or at the times indicated thereafter. DCF: diclofenac. *P<0.05, **P<0.01 and ***P<0.001 compared to vehicle group (two-way ANOVA, Bonferroni test).

Figure 8. Effect of acute (A) and chronic (B) administration of terpinolene monoterpene (TPL)/diclofenac (DCF) association on the paw edema induced by CFA in female rats (n=6-8). Each point represents the mean±SEM of the paw volume (mL) before CFA injection (i.e., baseline) or at the times indicated thereafter. *P<0.05, **P<0.01 and ***P<0.001 compared to vehicle group (two-way ANOVA, Bonferroni test).

Figure 9. Histological analysis of the rat right hind footpad 11 days after injection of Freund's complete adjuvant (CFA) (40×) showing the number of immune cells (arrows). A, Contralateral paw B: Sham; C, vehicle; D, terpinolene monoterpene (TPL) (25 mg/kg, po); E, diclofenac (DCF) (5 mg/kg, po), and F, TPL (3.125 mg/kg, po)+DCF (1.25 mg/kg, po).

Figure 10. Differential leukocyte counts after 11 days of Freund's complete adjuvant (CFA) injection in the footpad of female Wistar rats (n=6). TPL: Terpinolene monoterpene; DCF: diclofenac. Data are reported as the mean±SEM of the number of cells/10 random fields. **P<0.01 and ***P<0.001 compared to vehicle group (one-way ANOVA, Tukey test).

Figure 11. Involvement of the serotonergic system in the analgesic effect of terpinolene monoterpene (TPL)/diclofenac (DCF) combination in the mechanical compression test (Randall Selitto). Twenty-four hours after induction of inflammation by Freund's complete adjuvant (CFA) in female Wistar rats (n=6-8), the groups were treated with ketanserin (3 mg/kg sc, 5-HT2A antagonist) and vehicle, and then 30 min later, with TPL (25 mg/kg, po), TPL (3.125 mg/kg, po)+DCF (1.25 mg/kg, po) or DCF (5 mg/kg, po) as positive control. Data are reported as mean±SEM. ^aP<0.001 and ^bP<0.05 compared to saline group; ^cP<0.001 compared to ketanserin + TPL group; ^dP<0.001 compared to TPL+DCF group (two-way ANOVA, Bonferroni test).