Effect of Diclofenac and Andrographolide Combination on Carrageenan-Induced Paw Edema and Hyperalgesia in Rats

Abstract

Studies into drug combination at low doses are a promising approach to the management of pain and inflammation. The aim of this study was to evaluate the anti-edema and anti-hyperalgesic effects of a combination of diclofenac and andrographolide. Male Sprague-Dawley rats were first treated with diclofenac or andrographolide alone (3-100 mg/kg), as well as a combination of the 2 drugs. Carrageenan was then injected into the right hind paw of rats, and changes in paw volume and sensitivity to mechanical (von Frey) and thermal (Hargreaves test) stimuli measured. Results showed drug combination produced synergistic effects at reducing paw edema especially at lower doses, with a Loewe synergy score of 13.02 ± 8.75 in SynergyFinder and a combination index of .41 ± .18 after isobolographic analysis. Again synergy scores for decreasing response to 1.0 and 3.6 g force application of von Frey filaments after drug combination were 10.127 ± 5.68 and 8.554 ± 6.53, respectively, in SynergyFinder. Synergistic effects were also seen after drug combination in the Hargreaves test with a synergy score of 5.136 ± 16.38. In conclusion, combination of diclofenac with andrographolide showed better pharmacologic effects after carrageenan injection and was more synergistic at low-dose combinations.

Keywords: andrographolide; carrageenan; combination; diclofenac; hyperalgesia.

Figure 1.

Effect of diclofenac (3—100 mg/kg, p.o.) and andrographolide (3—100 mg/kg, p.o.) on time course curves for percentage change in paw volume (a, c) and total edema (b, d) in the rat carrageenan-induced paw edema experiment. Total edema was calculated as AUC over the 6-hour period. % inhibition of total edema for both treatments was calculated and used to plot DRCs (e). Values are presented as mean ± S.E.M. (n = 5). *P < .05; **P < .01; ***P < .001; ****P < .0001 compared to vehicle-treated control group (Two-way ANOVA followed by Dunnett’s post hoc test). ##P < .01; ###P < .001 compared to vehicle-treated control (Cntrl) group (one-way ANOVA followed by Dunnett’s post hoc test).

Figure 2.

Surface plot in two- (a) and three- (b) dimensional views, showing synergy scores for effect of diclofenac (Diclo) plus andrographolide (Andro) combination (3—100 mg/kg, p.o., dose combination matrix) on paw volumes in the rat carrageenan-induced paw edema experiment. Loewe synergy scores (shown as δ-scores) were calculated from % inhibition of total edema in the dose combination matrix. Areas in red, white, and green show regions of synergy, additivity, and antagonism, respectively. Arrows indicate the regions of highest synergy for lowest dose of diclofenac. Asterisks indicate 1:1 ED₅₀ dose combinations used in isobolographic analysis.

Figure 3.

Surface plot in two- (a, c) and three- (b, d) dimensional views, showing synergy scores for effect of diclofenac (Diclo) plus andrographolide (Andro) combination (3—100 mg/kg, p.o., dose combination matrix) on % response to von Frey filaments in the rat carrageenan-induced paw edema experiment. Loewe synergy scores (shown as δ-scores) were obtained from % response of animals to filaments 5 (a, b) and 9 (c, d) in the dose combination matrix. Areas in red, white, and green show regions of synergy, additivity, and antagonism, respectively. Arrows indicate the regions of highest synergy.

Figure 4.

Surface plot in two- (a) and three- (b) dimensional views, showing synergy scores for effect of diclofenac (Diclo) plus andrographolide (Andro) combination (3—100 mg/kg, p.o., dose combination matrix) on %MPE of the Hargreaves test, in the rat carrageenan-induced paw edema experiment. Loewe synergy scores (shown as δ-scores) were calculated from % MPE in the dose combination matrix. Areas in red, white, and green show regions of synergy, additivity, and antagonism, respectively. Arrows indicate the regions of highest synergy.

Figure 5.

Effect of 1:1 combination of ED₅₀ doses (4.01, 8.01, 16.02, and 32.04 mg/kg, p.o.) of diclofenac (Diclo; 3.73 mg/kg, p.o.) and andrographolide (Andro; 28.31 mg/kg, p.o.) on time course curves for % change in paw volume (a) and total edema (b) in the carrageenan-induced paw edema experiment in rats. Total edema was calculated as AUC over the 6-hour period. DRCs for the drug combination and individual drugs (c) were plotted using % inhibition of total edema. The isobolograph (d) was plotted using experimental ED₅₀ value (Zmix) obtained from drug combination. Values are presented as mean ± S.E.M. (n = 5). *P < .05; **P < .01; ***P < .001; ****P < .0001 compared to vehicle-treated control group (Two-way ANOVA followed by Dunnett’s post hoc test). #P < .05; ##P < .01; ###P < .001; ####P < .0001 compared to vehicle-treated control (Cntrl) group (One-way ANOVA followed by Dunnett’s post hoc test).