Quantitative analysis of the anti-inflammatory activity of orengedokuto II: berberine is responsible for the inhibition of NO production

Orengedokuto is a Kampo formula that has been used for removing “heat” and “poison” to treat inflammation, hypertension, gastrointestinal disorders, and liver and cerebrovascular diseases. We report here our analysis of the anti-inflammatory effect of the component crude drugs of orengedokuto and their constituents using the inhibition of nitric oxide (NO) production in the murine macrophage-like cell line J774.1. An initial comparison of NO production inhibitory activities of the extracts of the component crude drugs and their combinations revealed that the activity could be attributed to Phellodendron Bark and Coptis Rhizome. Berberine (1), the major constituent of these crude drugs, showed potent activity (IC₅₀ 4.73 ± 1.46 μM). Quantitative analysis of 1 in the extracts of all combinations of component crude drugs revealed that the amount of 1 in each extract of the combination of Scutellaria Root with either Phellodendron Bark and/or Coptis Rhizome was lower than that in the corresponding mixtures of the extracts of the individual crude drugs and that 1 was present in the precipitates formed during the decoction process. To the contrary, the differences in the amounts of 1 were smaller in the extracts containing Gardenia Fruit. These results indicated that the constituents of Scutellaria Root precipitated with 1 and that the constituents of Gardenia Fruit dissolved the precipitates. To identify the constituents affecting the solubility of 1, we fractionated the hot-water extracts of Scutellaria Root based on solubility tests of 1 to give baicalin (2), wogonin (3) and oroxyloside (4), which formed precipitates with 1.