Synergistic effect of baicalein, wogonin and oroxylin A mixture: multistep inhibition of the NF-κB signalling pathway contributes to an anti-inflammatory effect of Scutellaria root flavonoids

Scutellaria root, the root of Scutellaria baicalensis Georgi, is a crude drug used for inflammatory diseases. In our previous report, the combination of flavonoids contained in Scutellaria root have been found to inhibit PGE₂ production more strongly than individual flavonoids. Here, to investigate the mechanism of the synergistic effect, we examined the effects of an equimolar mixture (F-mix) of baicalein (1), wogonin (2) and oroxylin A (3) on the production of PGE₂ in LPS-treated J774.1 cells. Although 1 and 3 inhibited COX-2 activity, the F-mix showed no synergistic effect on COX-2 inhibition. Therefore, we investigated the steps leading to the activation of COX-2 protein. Compounds 1–3 and F-mix inhibited the expression of COX-2 protein. However, only 2 inhibited the expression of COX-2 mRNA among the flavonoids, and the F-mix showed no synergistic effect. Only 1 inhibited NF-κB translocation into the nucleus, and the F-mix showed no synergistic effect. Although 2 did not affect NF-κB translocation, it strongly inhibited NF-κB-dependent transcriptional activity, and the F-mix inhibited the activity slightly more than 2. Compounds 1–3 also inhibited NO production, and the F-mix showed a synergistic effect. However, the effects of each flavonoid on the expression of iNOS mRNA were not consistent with those on COX-2 mRNA. Because the flavonoids inhibit different steps in the production of PGE₂ and NO, and their mixture did not show apparent synergistic effects in each step, we conclude that the synergistic effect of the flavonoid mixture reflects the total effect of the flavonoids inhibiting different steps in the NF-κB signalling pathway.