One-step immunochromatographic separation and ELISA quantification of glycyrrhizin from traditional Chinese medicines

The bioactive constituent, glycyrrhizin or glycyrrhizic acid (GA), was purified from two traditional Chinese medicines (TCM), Shaoyao gancao tang and Dahuang gancao tang, and from crude extracts from licorice roots by means of immunoaffinity chromatography using anti-GA monoclonal antibody (MAb) and was quantified with an enzyme-linked immunosorbent assay (ELISA). Laboratory preparations included the synthesis of conjugate GA-human serum albumin (GA-HSA), the production of anti-GA-MAb, the optimization of the immunoaffinity column packed with the anti-GA-MAb coupled to hydrazide gel and the determination of the GA content in TCM and crude drugs from five different sources by ELISA and high performance liquid chromatography (HPLC). The experimental results reveal that the anti-GA-MAb coupled to Affi-Gel Hz gel results in a coupling efficiency of 95.2%, and the immunoaffinity chromatography gives a mean recovery of 97.6% of GA with a capacity of 33.5+/-2.40 microg/mL of immunoaffinity gel under the given conditions. The GA content of the crude extracts (ranging 74.8-114.6 microg/mg) from different sources by the ELISA method is much greater than that of the TCM (16.4-25.1 microg/mg) which is, in good agreement with the results of the HPLC method. Our report provides a rapid, reliable and sensitive approach for one-step separation and quantification of GA.