226 CALOTROPIS-STANDARDISATION AND BIOLOGICAL ASSAY [Vol. XXII, Part III,
METHODS
Standardi ation. The first standard prepared by the authors consists of a
mixture of ten different samples of the leaves of Calotropis gigantea, collected from
the city of Madras and its neighbourhood, dried at the room temperature of about
28°C. for about ten days, powdered to pass through a No. 60 sieve, dried in an electric
hot air oven between 55°C. and 60°C. for six hours, ampouled in sterile containers
and stored in the frigidaire. The moisture content of the standard powder was
determined and found to be 2.45 per cent.
Unit. The unit of the first standard was defined as the acitvity contained in
0.1 gm. of the standard calotropis leaf powder prepared and kept in this laboratory,
(The Department of Physiology, Madras Veterinary College, Madras.)
Biological Assay. The standard is now available for any one to compare an
unknown sample of leaf to see how far this sample differed in acitvity from that of
the standard. Among the various methods of comparison available namely the
frog, guinea pig and cat methods, the cat method as used for the Third International
Digitalis Standard by the World Health Organisation (7) was tried in this laboratory
and found to be very satisfactory. In this method, the mean of the lethal dose
of the sample being tested for a series of cats, expressed per Kg. body-weight was
compared with the mean obtained for the standard powder.
In brief the procedure adopted was as follows :—
Preparation and storage of tincture. The contents of the ampoule of the standard
were weighed to the nearest milligram and transferred to a conical flask of 50 ml.
capacity. For each gram of the powder 10 ml. of 80 per cent (by volume) alcohol
was added. The stopper was inserted and the mixture shaken for 24 hours at
room temperature (28°C.-30°C.) by mechanical means, which continuously brings
the solid material into fresh contact with the liquid phase. Immediately after-
wards it was filtered by pressure through the filter paper taking all possible pre-
cautions to avoid evaporation of the solvent. The tincture was then transferred
to a hard, amber coloured glass bottle with tight closure and kept in the frigidaire
and used within 30 days.
Preparation of diluted tincture. At the time of testing the standard tincture
was diluted with 0.9 per cent sodium chloride to make 1:20. It was prepared
fresh on the day of use and shaken before it is poured into the burette.
Selection of animal. Domestic cats free from gross evidence of disease and
weighing between 2 to 4 Kg. were chosen for the assay. Weight was determined
to the nearest 0.05 Kg. Those which upon examination appeared either obest,
ematiated, lactating or pregnant were not used. The cats were starved for 16
hours before use.
The cats were anaesthetised with chloralose at the rate of 110 mg. per Kg.
given intramuscularly dissolved in alcohol, two hours before use. The tincture
was then injected through the femoral vein from a burette and artificial respiration
through the tracheal canula was maintained throughout to ensure that the death
was not due to respiratory failure. Carotid blood pressure was recorded kymogra-
phically.