Evaluation of a new eastern blotting technique for the analysis of ginsenoside Re in American ginseng berry pulp extracts

Abstract

A new eastern blotting technique has been established for ginsenoside Re (G-Re) contained in American ginseng berry pulp extracts. G-Re in American ginseng berry pulp was extracted using 100% methanol, 100% ethanol, 50% aqueous methanol, and 50% aqueous ethanol. The combined crude extracts were applied onto a polyethersulfone membrane and developed using the methanol–water–acetic acid solvent system (45:55:1 v/v). Separated components were immunostained using anti-G-Re monoclonal antibody. G-Re was first specifically detected and then quantitatively analyzed using NIH Imaging software. We also confirmed that the most suitable solvent was 50% aqueous methanol for extracting G-Re from American ginseng berry pulp.

Introduction

The major active components of ginseng are thought to be ginsenosides, a typical group of triterpene dammarane saponins. Ginsenosides are distributed in various Panax species, including the root, leaf, and berry, and these parts have different pharmacological activities [1]. The ginseng root is mainly used for traditional Chinese medicines (TCMs). Xie et al. recently reported that ginsenoside Re (G-Re) which is the major component in American ginseng (Panax quinquefolium), has an anti-diabetic effect resulting in it becoming one of the top 10 selling natural health products in USA [2]. Diabetes mellitus is the sixth leading cause of death in both the USA and the rest of the world [3]. Furthermore, it has been reported that intake of American ginseng berry extract helps to reduce blood glucose and body weight in ob/ob mice [4], [5]. It is well known that the concentration of individual ginsenosides in different Panax species and extracts varies depending on the extraction procedure, subsequent treatment, and even the collection season [6]. Standardization and authentication of ginseng are therefore necessary in research and for public use.

In our continuous effort to produce monoclonal antibodies (MAbs) for naturally occurring bioactive compounds, we prepared anti-ginsenoside G-Rb1 [7], anti-ginsenoside Rg1 [8], and anti-G-Re [9], [10] MAbs. Furthermore, we applied these MAbs to enzyme-linked immunosorbent assay (ELISA) systems [11]. This led to the development of an eastern blotting technique, which makes use of a novel immunostaining methodology [12], [13], [14] that permits detection and visualization of small molecule compounds, such as natural glycosides.

The present study evaluates the most suitable solvent for G-Re in the preparation of American ginseng berry pulp extract and its identification using a new eastern blotting technique with anti-G-Re MAb and NIH Imaging software.