Cardenolides assay

Assay results with the two new 1,2-cis (ft-d) cardenolides show enhanced activity as compared with the two unnatural, a-D-rhamnosides. They have potencies that fall well within the range for those of the naturally occurring cardenolides. These results support the postulate that the a-D-glycosidic linkage in cardenolides containing D-sugars is unfavorable for cardiotonic activity. 

Practically all of the known cardenolides have been assayed by Chen and his coworkers the data were obtained in one laboratory under conditions as nearly identical as possible. It is for this reason that meaningful results can best be obtained from this method, particularly for comparison studies in terms of structure-activity relationships. Also, it is important to note that the potency figures for cats are applicable to man, when the drugs are administered intravenously. The data cited in Table I are all Chen assay values those obtained prior to 1961 may be found in an excellent compilation by Hoch.1 Cardenolides, for which assay figures have since been given by Chen, will be referenced accordingly. 

In order to estimate the productivity of a large number of clones, it is desirable to develop a simple assay technique to measure trace amount of the compound in a small amount of sample. Radioimmunoassay (RIA) [118] and HPLC [119, 120] have been carried out for the quantitative analysis of cardenolides. However RIA cannot be used in a usual laboratory, and HPLC requires much time for analysis. Therefore, the ELISA for cardenolides was established to investigate the production of cardenolides by hairy root cultures of D. lanata [116, 117]. 

Centrifuge the solution (1 min at 18500g) and dilute the supernatant with C-PBS (at least ten times) or evaporate the combined extract in vacuo, redissolve in a small volume of 70 % EtOH, and dilute with C-PBS (at least ten times). Less than 10 % EtOH dose not affect the reaction between the cardenolides and the first antibody. Diluted sample solutions will be subjected to ELISA. However, 3 to 5 different dilutions of each sample are usually required for the assay, because the reliable range is narrow as shown in Fig. 7. If ELISA is employed for the screening of the samples which contain higher concentrations of target compound, the stepwise dilution will not be needed. 

---