In the present study, we performed to selectively deplete
glycyrrhizin from
Si-Ni-San, a traditional Chinese prescription that consists of 4 Chinese herbs including Radix Glycyrrhizae Uralensis, and examined its influence on the suppressing activity of
Si-Ni-San against
contact sensitivity in mice. An immunoaffinity column was made by covalently coupling the polyclonal antibody, obtained by the immunization with
glycyrrhizin-BSA conjugate, to CNBr-activated
Sepharose 4B. By using this column,
glycyrrhizin in
Si-Ni-San was selectively and almost completely depleted from the whole extract, which was confirmed by high-performance liquid chromatography (HPLC). Both 200 mg/kg of
Si-Ni-San and 10 mg/kg of
glycyrrhizin, the dose corresponding to its proportion contained in
Si-Ni-San, significantly reduced the ear swelling of
picryl chloride (PCl)-induced ear
contact sensitivity in mice and the inhibition by
Si-Ni-San was stronger than that by
glycyrrhizin. The adhesion activity to
type IV collagen of the isolated spleen cells from PCl-sensitized mice was significantly decreased by both
Si-Ni-San and
glycyrrhizin. However, the
glycyrrhizin-depleted sample of
Si-Ni-San (
Si-Ni-San(GL-)) only showed a slight inhibition on the cell adhesion. Furthermore, the spleen cells from PCl-sensitized mice produced more
matrix metalloproteinase (MMP)-2 and -9 than naive spleen cells did, and both
Si-Ni-San and
glycyrrhizin remarkably reduced MMP-2 and MMP-9 production. In contrast,
Si-Ni-San(GL-) only showed a slight inhibition. These results suggest that
glycyrrhizin may act as one of the active constituents of
Si-Ni-San in inhibiting delayed-type
hypersensitivity reaction via down-regulating the
MMP production and the cell adhesion to extracellular matrix. The present study also provides a new approach to recognize and validate an active constituent in traditional prescription through a selective depletion.