This document describes the bioassay of digitoxin, which is extracted from the plant Digitalis lanata and used to treat conditions like congestive heart failure. It discusses two common bioassay methods - the guinea pig method and pigeon method. Both methods involve injecting extracts of the test and standard digitoxin samples intravenously into guinea pigs or pigeons and determining the lethal dose that causes heart arrest. The potency of the test sample is then calculated based on its lethal dose compared to that of the standard.

1. BIOASSAY OF
DIGITOXIN by
B. NAGA SIVA TEJA
16DU1R0003
B PHARMACY

2. CONTENTS
■ INTRODUCTION
■ PRINCIPLE OF BIOASSAYS
■ PURPOSE OF BIOASSAYS
■ DIGITALIS
■ BIOASSAY OF DIGITALIS
■ GUNIEA PIG METHOD
■ PIGEON METHOD
■ REFERENCES

3. INTRODUCTION
■ A bioassay is an analytical method to determine concentration or
potency of a substance by its effect on living cells or tissues.
■ Observation of the pharmacological effect on
– Living cells or tissues
– Microorganisms
– Animals

4. PRINCIPLE OF BIOASSAYS
■ To estimate the relative potency of a sample compound to a standard
compound.
■ Typical bioassay involves a stimulus (ex. drugs) applied to a subject (ex.
animals, tissues, plants) and a response (ex. death) of the subject is
triggered and measured.
SIMPLY
 Comparison of test substance with the international standard
preparation of the same and to find out how much of the test
substance is required to produce the same biological effect as that of
standard.

5. PURPOSE OF BIOASSAYS
■ To measure the pharmacological activity of chemically undefined
substance.
■ To ascertain the potency of a drug.
■ Helps to determine the specificity of a compound to be used i.e; penicillins
are effective against gram +ve sps rather than gram –ve sps.
■ Certain complex compounds like vit-b12 can only be analysed by bioassays.
■ For the substances that doesn’t have any other method of evaluation
available.

6. DIGITALIS
■ In Digitalis, Digitoxin is a purified cardiac glycoside extracted from the
plant Digitalis lanata.
■ Phytosteroid.
■ M.O.A:
– Inhibits the Na-K-ATPase membrane pump, resulting in an increase in
intracellular sodium and calcium concentrations.
– Increased intracellular concentrations of calcium may promote
activation of contractile proteins.
■ Used in treatment of
– CHF,
– Atrial Flutter,
– Cardiac Arrhythmias, etc.

7. BIOASSAYS OF DIGITALIS
■ It can be done in 2 methods
– Guniea pig method and
– Pigeon method.
Principle
Potency of the test sample is compared with that of the standard
preparation by determining the action on the cardiac muscle. Any other
equivalent method, which gives results similar to those obtained by this
method as also valid.

8. Standard Preparation and Units:
The standard preparation is a mixture of dried and
powdered digitalis leaves.
1 unit = 76 mg.
Preparation of Extract:
Exact amount of the powder is extracted with dehydrated
alcohol in a continuous extraction apparatus for six hours. The final
extract should contain 10 ml(5 ml. alcohol + 5 ml. water) per 10 g of
digitalis powder.
 It should be stored in between 5ºC and –5ºC.

9. GUNIEA PIG METHOD [END POINT METHOD]
■ Standard and test sample extracts are diluted with normal saline in such a way
that 1 g of digitalis powder is diluted to 80 ml.
■ A guinea pig is anaesthetized with a suitable anaesthetic.
■ It is dissected on the operation table.
■ The jugular vein is traced out by removing adhering tissues and cannulated by
means of venous cannula.
■ A pin is inserted in the heart, such that it gets inserted in the apex of the
heart.
■ In this way, we can observe the heart beats by up and down movements of the
pin.

10. Removal of hair Jugular vein detection
Injecting digitoxin Intravenously

11. ■ The injection is continued through venous cannula untill the heart is arrested in
systole.
■ The amount of extract required to produce this effect is taken as the lethal dose of
the extract.
■ Another set of 19 animals of the same species are used for this experiment and the
average lethal dose is determined.
■ It is not necessary to determine the lethal dose of the std. during each time of the
experiment.
■ But it should be occasionally checked.
■ The lethal dose of the test sample is determined in a similar way using minimum 6
guinea–pigs of the same strain.
 The potency of the test sample is calculated in relation to that of the std.
preparation
mean threshold dose of standard
mean threshold dose of test
Potency = × concentration of
standard

12. PIGEON METHOD
REQUIREMENTS OF PIGEONS :
 No. of pigeons : 6
■ They should be free from gross evidence of disease or emaciation.
■ The weight of the heaviest pigeon should not exceed twice the weight of the
lightest pigeon.
■ Food is withheld 16-28 hours before the experiment.
■ Pigeons are divided on the basis of their sex, weight and breed, into two
groups.

13. Intra venous injection at alar vein
Crop sac of pigeon

14. ■ They are anaesthetized with anaesthetic ether.
■ One side of the wing is dissected and the alar vein is cannulated by means of a venous
cannula.
■ Dilutions are made with normal saline. stoppage of heart is associated with a characteristic
vomiting response called ‘emesis’.
■ The milk from the crop sac of pigeons is being ejected out. This may be taken as the end point
response of digitalis.
■ Average lethal dose of each sample is determined; results are tabulated and calculated as per
guinea pig method.
■ The lethal dose per kg. of body weight is determined for each pigeon.
■ The potency of the test sample is determined.
mean threshold dose of standard
mean threshold dose of test
Potency =
× concentration of
standard

15. REFERENCES
■ Hoskins, W. M.; Craig, R. (1962-01-01). "Uses of Bioassay in
Entomology". Annual Review of Entomology. 7 (1): 437–464. .
■ Guidance for Industry: Potency Tests for Cellular and Gene Therapy
Products, Food and Drug Administration, January 2011, p. 7
■ Laska, E M; Meisner, M J (1987-04-01). "Statistical Methods and
Applications of Bioassay". Annual Review of Pharmacology and
Toxicology. 27 (1): 385–397.
■ An Noordwijk, Jacobus (1989). "Bioassays in whole animals". Journal
of Pharmaceutical and Biomedical Analysis. 7 (2): 139–145.