Development of an Identification Test for Hampi (反鼻) by Using TLC and the Structural Analysis of the Indicator Components

Abstract

Hampi (反鼻) is an animal crude drug obtained from Gloydius blomhoffii H. Boie and G. brevicaudus Stejneger, which is obtained after removal of the skin and internal organs. It is compounded into many crude drug products, mainly for analeptic effect, and its annual transaction amount in Japan reaches ca. five tons. Therefore, the drug has been standardized by listing it in Non-JP Crude Drug Standards 2018, where Ptyas dhumnades Cantor is defined as the source animal together with G. blomhoffii and G. brevicaudus. In this paper, we report the development of an identification test by TLC in preparation for the listing. Two indicator spots of the test were purified from G. blomhoffii and P. dhumnades using repeated column chromatography. The chemical structures of those 2 spots were elucidated as (i) a mixture of phosphatidylethanolamine (1) and its analogues and (ii) a mixture of lysophosphatidylethanolamine (2) and its analogues based on the comparison of results of TLC, LC/MS, and NMR with those of authentic compounds.

The TLC used silica gel as chromatographic support and an ethyl acetate : ethanol (99.5) : water (1 : 1 : 1) mixture as the developing solvent. The identification test had a developing length of 7 cm, used ninhydrin reagent for visualization, and reported an R_f of 0.7 for both the mixture of phosphatidylethanolamine and its analogues, and the mixture of lysophosphatidylethanolamine and its analogues.