New staining methodology: Eastern blotting for glycosides in the field of Kampo medicines

Ginsenosides separated by silica gel TLC blotted to a polyvinylidene difluoride (PVDF) membrane treated with a NaIO₄ solution followed by bovine serum albumin (BSA) resulted in a ginsenoside-BSA conjugate on a PVDF membrane. The blotted spot were stained by antiginsenoside Rb1 (GRb1) and Rg1 (GRg1) monoclonal antibodies (MAbs). The newly established immunostaining method, Eastern blotting, was applied for the determination of ginsenosides possessing protopanaxadiol and/or protopanaxatriol in the Kampo medicines. In this method, we developed a new way to separate the ginsenoside molecule into two functional parts using a simple and well-known chemical reaction. The sugar parts were oxidized by sodium periodate to give dialdehydes, which reacted with amino groups of the protein and covalently bound to the adsorbent PVDF membrane. The MAb bound to the aglycone part of the ginsenoside molecule for immunostaining. Double staining of Eastern blotting for ginsenosides using anti-GRb1 and GRg1 MAbs promoted complete identification of ginsenosides in Panax species. This method was validated for immunocytolocalization of ginsenosides in fresh ginseng root. In addition, Eastern blotting for the detection of glycyrrhizin (GC) was also investigated. GC can be clearly determined by Eastern blotting in the Glycyrrhiza species. It is also possible for GC in rat serum to be surveyed by Eastern blotting by simple pretreatment as a further application.