Analysis of the synergistic effect of glycyrrhizin and other constituents in licorice extract on lipopolysaccharide-induced nitric oxide production using knock-out extract

The pharmacological evidence for synergism between natural compounds is not fully elucidated. In this study, we investigated the synergistic function of one target compound in medicinal plant extract by using knock-out (KO) extract, which is one target compound-eliminated extract from whole crude extract. Licorice is the most important ingredient used in the traditional Chinese medicine (TCM) and the Japanese Kampo medicine, and one of the major active components of licorice is glycyrrhizin (GC). To identify the potential role of GC, we prepared GC-removed extract (GC–KO extract) from licorice extract (LE) using immunoaffinity column conjugated with anti-GC monoclonal antibody (MAb), which could eliminate 99.5% of GC from LE. LE inhibited nitric oxide (NO) production and inducible NO synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated RAW264 murine macrophage cells. However, treatment of GC alone could not show the suppression of NO production and iNOS expression. Interestingly, the inhibitory effect of GC–KO extract was significantly attenuated compared with LE. Furthermore, the combined treatment with GC–KO extract and GC could improve the attenuated inhibition. Taken together, our results indicate that GC may exert synergistic suppression of iNOS expression when coexisting with the other constituents contained in LE, and KO extract is a useful approach for determination of real pharmacological functions of natural compound in the phytochemical mixture.

► Knock-out (KO) extract is one target compound-eliminated extract from crude extract.
► Glycyrrhizin (GC) is one of the major active components of licorice extract (LE).
► GC–KO extract is prepared from LE using anti-GC immunoaffinity column.
► GC may synergistically suppress iNOS expression with the other constituents in LE.
► KO extract is a useful approach to determine the real functions of natural compound.