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Clinical pharmacy PharmD program Third level Phytochemistry-1 (PG-504)

These are positive with any compound containing a steroidal nucleus including cardenolides
and bufadienolides:

Liebermann’s test: Cardiac glycosides in glacial acetic acid + H 2 SO 4 → red, violet, blue to
green.

- 2 Reactions due to the ( - CH 2 - ) group of the lactone ring : characteristic for cardenolides
having a 5 - membered lactone ring.

Kedde’s test: Cardenolide+ 3,5-dinitrobenzoic acid + NaOH → violet.

Baljet’s test: Cardenolide + picric acid + NaOH → orange or red

- 3 Color reactions due to the 2 - deoxy sugar moiety

Keller - Killiani’s test: The glycoside is dissolved in glacial acetic acid that contain traces of
FeCl 3 , and then concentrated H 2 SO 4 is carefully added on the side of the test tube to form a
lower layer

→ a blue ring is formed

Quantitative determination (Standardization)

Factors affecting the content of cardiac glycosides in plant material are numerous including:

stage of development,

seasonal variations,

drying and storage conditions.

In addition, due to their high toxicity , cardiac glycosides - containing pharmaceuticals should be carefully
standardized before administration, several methods are used :

1. Colorimetric methods based on color tests e.g. Baljet's or
Kedde's

2. Biological method, these are the most widely used and are based
on determination of the minimum lethal dose that stops the heart
under specified conditions.

3. Immunoassay

4. By RP-HPLC (reversed phase high performance liquid
chromatography) using UV or fluorometric detectors

Structure-Activity Relationship (SAR)

153