Bioassay of Digitalis PDF

Bioassay of Digitalis  Bioassay of Digitalis The activity of a sample prepared is determined by comparing its activity on the cardiac muscle of guinea pigs or pigeons with that of a standard preparation of prepared digitalis. Standard preparation and units: It is supplied in ampoul...

Bioassay of Digitalis  Bioassay of Digitalis The activity of a sample prepared is determined by comparing its activity on the cardiac muscle of guinea pigs or pigeons with that of a standard preparation of prepared digitalis. Standard preparation and units: It is supplied in ampoules / vials containing approximately 2.5 g. 1 mg contain 76 Units of the standard preparation. 1 Unit contains 0.01316 mg.  Preparation of extracts  The contents of a vial containing a suitable quantity of standard preparation are rapidly transferred to a stoppered weighing bottle & weighed.  The contents of a weighing bottle are transferred to a hard glass, glass stoppered container of at least 50 ml capacity.  10 ml of alcohol (80%) is added for each gram of powder.  The stopper is inserted and the container is shaken continuously for 24 hours at 20C-30C or for 48 hours at 10C-20C.  The mixture is then immediately centrifuged or filtered through a sintered glass filter with a precautions to avoid evaporation of the solvent.  The liquid is transferred to a dry, hard glass bottle, which is tightly closed and kept at a temperature between 5C to -5C.  The extract should be used within one month.  An extract of the test sample being tested is prepared in the same manner.  By Injection into Guinea Pigs  Select 12 guinea pigs randomly weighing between 200-600 g.  The weight of the heaviest and lightest animal should not differ by 100 g.  Divide them in 2 groups.  Mean body weight of each group should not differ by 10%.  One group is used for test and one for standard preparation.  The extract is injected slowly in to the vein of guinea pig which already anaesthetized with urethane & whose respiration may be maintained artificially.  The duration of injection may be between 20-40 minutes.  The injection is continued until the heart is arrested (this is determined from an electrical recording).  The volume of the extract administered is taken as the lethal dose.  The mean lethal dose (ml) per kg of body weight should determined for each group.  Result The result of the assay is calculated by referring Pakistan Pharmacopeia 1973. When 6 guinea pigs are used for standard and 6 for test sample, the allowed limits of error is 80-125 percent of the estimated potency.  By Injection into Pigeons  Select 12 pigeons or more randomly, divide them into 2 groups.  The pigeons must be free from any disease, obesity or emaciation.  The weight of the largest pigeon should not exceed twice the weight of the smallest.  The mean weight of the group should not differ by more than 30%.  One group used for standard and one for test sample being examined.  Food but not water is withheld for 16-28 hours before the test.  Dilute the extract of standard and test sample with saline in such away that the estimated lethal dose per kg body weight contained in 15 ml.  Anaesthetize the alar vein of the pigeon.  Inject the diluted extract into alar vein through cannula.  The dose is 1ml per kg of body weight and is administered within a few seconds, & repeat at 5 minutes intervals until the heart is arrests.  If the average no of doses of any given dilution required to produce death is less than 13 or more than 19, or if the average no. of doses for the two groups differ by more than four, the test is repeated by using freshly prepared extract.  The mean lethal dose (ml) per kg of body weight should determined for each group.  Results The result of the assay is calculated by referring Pakistan Pharmacopeia 1973. When 6 pigeons are used for standard and 6 for test sample, the allowed limits of error is 80-125 percent of the estimated potency.