Studies on the Chemical Constituents of Periploca sepium Bge.——Ⅱ.Chemical and Biological Methods for the Determination of Cardiac Glycosides in the Plant

As reported previously, the stem-, root- and rhizome-barks of the plant Periploca sepium Bge.(Asclepiadaceae) contain cardiac glycosides and a highly active total glycosidal preparation has been prepared from it. In order to determine the distribution and seasonal variation of the cardiac glycosides in the different parts of the plant, it was necessary to study the assay methods. Spectrophotometric methods based on the Kedde's reaction and Baljet's reaction as well as biological method using frog as test animal were studied and detailed procedures proposed. The method based on Kedde's reaction is carried out as follows: 5ml of decolorized tincture of the plant material(prepared according to Rawson)is mixed with 5ml of the reagent (2% 3,5-dinitrobenzoic acid in methanol mixed with equal volume of 1 N potassium hydroxide solution just before use). Determine the extinction in 5—12 minutes at 530mμ. Use a mixture of 6.4% ethanol and the reagent (1:1) as blank.Calculate the percentage content from the standard curve prepared with a reference standard. Either a total glycosidal preparation or a stem-bark powder of definite glycosidal content may be used as the reference standard. It was found that (1) the reagent prepared with methanol as solvent is paler in colour and more stable than that prepared with ethanol; (2) the tincture after mixing with the reagent showed an absorption peak at 530 mμ and the colour is deepest and most stable in 4—12 minutes; (3)the concentration-absorption relation obeys Beer's law and the curve is a straight line. The deviation coefficient(P=0.05) of the mean extinction of two determinations was found to be ±4.25%. The method based on Baljet's reaction is carried out as above with the following modifications:(1)the reagent used is 1% picric acid aqueous solution mixed with 10% sodium hydroxide solution(9:1);(2)the tincture after mixing with the reagent showed an average absorption peak at 505 mμ(varied between 500—510 mμ with glycoside concentration) and the colour is deepest and most stable in 40—80 minutes; (3) the concentration-absorption relation does not strictly obey Beer's law and the curve is not a straight line. The deviation coefficient(P=0.05)of the mean extinction of two determinations was found to be±2.29%. The frog method of biological assay described in the Soviet Pharmacopoeia was found to be suitable with the following modifications: (1) the male frog, Rana nigromaculata, common in north China is used as test animal; (2) a reference standard preparation is determined for LD_min at the same time and the relative minimum lethal dose calculated.The deviation coefficient (P=0.05) was found to be: for single determination,±10.24%; for the mean of two determinations, ±7.24%. When plant material of low glycosidal content is used for the assay, the tincture prepared may be concentrated on a boiling water-bath without significant loss of potency: By comparing the conformity of results between the chemical and biological methods,it was found that the method based on Kedde's reaction is superior to that based on Baljet's reaction.