Preparation of Knockout Extract for Determination of Really Active Compound Using MAb

Takuhiro      Uto; Indree      Tuvshintogtokh; Yukihiro      Shoyama

Abstract

The crude-rhizome extract of P. japonicus was loaded on the immunoaffinity column conjugated with antiginsenoside- Rb1 monoclonal antibody (MAb) and washed with the washing solvent, followed by elution solvent, to give fraction 2 containing higher concentration of compound 1. Compound 1 clearly indicated a dammarane saponin having protopanaxadiol as a framework and three sugars in a molecule suggesting that compound 1 is chikusetsusaponin III. Compound 2 was also determined as chikusetsusaponin VI compared to the staining color, its Rf value and the comparison with ginsenoside Rb1. We succeeded in one step purification of ginsenoside-Rb1 by immunoaffinity column conjugated with anti- ginsenoside-Rb1 MAb leading to the knock-out extract which will be useful for pharmacological investigation. The antibody was stable when exposed to the eluent, and the immunoaffinity column showed almost no decrease in capacity after repeated use more than 10 times under the same conditions. From the crude extract of licorice we isolated glycyrrhizin by one-step purification by the immunoaffinity column using anti-glycyrrhizin MAb. Washing fraction contained all components except for only glycyrrhizin and was named as the knockout extract. We confirmed the synergic effect of glycyrrhizin with some other components for the inhibition of nitric oxide (NO) production by blocking inducible nitric oxide synthase (iNOS) expression by using its knockout extract.

Keywords: Ginsenosides, immunoaffinity column, one-step purification, knockout extract, glycyrrhizin, crude-rhizome extract, P. japonicus, glycyrrhizin., metal chelate, alkaloids, ginseng, cross-reactivity, Panax ginseng