Ginseng and its extract have been used as one of the most popular traditional medicines for the promotion of general health in Eastern Asian countries for thousands of years. The pharmacological and therapeutic effects of ginseng have been reported to have anti-aging, anti-diabetics, anti-stress, maintenance of homeostasis, and to effect on central nervous system and immune function.1) As its extensive effectiveness and usefulness have been well-noticed, numerous researchers have investigated about its constituents, chemical and physical characteristics as well as its pharmacological activities and their mechanisms. The constituents of ginseng are identified as saponins called ginsenosides, phytosterols, peptides, polysaccharides, fatty acids, polyacetylenes, vitamins, and minerals. Among them, the main biological components are more than 60 kinds of ginsenosides that have been isolated from Panax genus.
2)There have been reports on the productions and applications of monoclonal antibodies to ginsenoside Rb1 (G-Rb1), 3) G-Rg1, 4) and G-Rf 5) as well as polyclonal antibodies to GRg1 6) and G-Rf/G-Rg2. 7) They were developed to quantify or trace specific ginsenosides in the body fluids or in mixed ginseng extracts by immunoassay without cross reactivity.In this study, for the first time, we report the monoclonal antibody to G-Rg3. G-Rg3 is a type of protopanaxadiol having glucose (2→1) glucose attached at the C-3 hydroxyl group (Fig. 1). Both G-Rg3 and its metabolite G-Rh2 have already been sold as anti-cancer drugs in the Chinese and Taiwanese markets. 8) Although many researchers have revealed that G-Rg3 shows the effects such as anti-angiogenic activity, 9) anti-proliferative activity, 10) apoptosis 11) and protective actions to chemotherapy 12) both in vitro and in vivo, its mechanism has yet to be clearly established. Therefore, the proteins to 'give and take' the influences with G-Rg3 related to tumor inhibition draws attention to understand the mechanism of G-Rg3.Herein, we produce, purify and characterize monoclonal antibody to G-Rg3. In our investigation, we proved that the monoclonal antibody works specifically to G-Rg3, not G-Rh2. G-Rh2, having a glucose at the C-3 hydroxyl group, possesses very similar structure to G-Rg3 and also shows anticancer activity. Therefore, the monoclonal antibody to GRg3 is a very useful tool not only for the study in vitro and in vivo at the cellular level but also it would be a good immunological approach to quality control or quantitative determination of the mixed samples containing G-Rg3.
MATERIALS AND METHODSPreparation of Ginsenosides G-Rg3 (Fig. 1A) was preparatively purified from the steamed ginseng extract by high speed counter current chromatography.13) G-Rh2 (Fig. 1B) and G-Rg2 (Fig. 1C) were purchased from Chengdu Cogon Bio-tech Co., Ltd. (Chengdu, China).Synthesis of Antigen Conjugates Each ginsenosidebovine serum albumin (BSA) conjugate was synthesized by the previous method 7) with slight modification. After dissolving 4 mg NaIO 4 (Sigma, St. Louis, MO, U.S....