Development of Immunoassays for Ginsenosides in Ginseng

Abstract

Ginsenosides separated by silica gel thin-layer chromatography (TLC) blotted onto a PVDF membrane that was treated with a NaIO₄ solution and bovine serum albumin (BSA) resulted in a ginsenoside–BSA conjugate. The blotted spots were stained with anti-ginsenoside Rb1 (G-Rb1) and Rg1 (G-Rg1) monoclonal antibodies (MAbs). A newly established immunostaining method, namely, eastern blotting, was applied to determine whether ginsenosides, which are used in traditional Chinese medicine (TCM), contain protopanaxadiol and/or protopanaxatriol. This is a new method of separating the ginsenoside molecule into two functional parts using a simple and well-known chemical reaction. The sugar parts are oxidized by NaIO₄ to produce dialdehydes, which react with amino groups of the protein and covalently bind to the adsorbent PVDF membrane. The MAb binds to the aglycon part of the ginsenoside molecule for immunostaining. Double staining of ginsenosides using anti-G-Rb1 and anti-G-Rg1 MAbs in eastern blotting allows for complete identification of ginsenosides in the Panax species. The immunoaffinity concentration of G-Rb1 was determined using an immunoaffinity column conjugated with anti-G-Rb1 MAb that produced the knockout extract, which may be useful for pharmacological investigations. To concentrate and determine the amount of G-Rb1 in P. japonicus, the crude extract of P. japonicus was fractionated using an immunoaffinity column conjugated with anti-G-Rb1 MAb. Although G-Rb1 was expected to be a component of P. japonicus through enzyme-linked immunosorbent assay (ELISA) analysis, two ginsenosides, namely, chikusetsusaponins III and IV, which have protopanaxadiol as an aglycon, were identified using eastern blotting.