Bioassay of Digitalis, d-tubocurarine , Oxytocin

Bioasssay & methods involved in testing Digitalis,d-tb,Oxytocin

1. BIOASSAY OF DRUGS [DIGITALIS ,d- tubocurarine ,OXYTOCIN, ] PRESENTED BY : HEENA PARVEEN , Dept. Pharmacology, Assistant Professor.

2. BIOASSAY OF DIGITALIS Principle: The potency of test sample is determined by comparing its activity on cardiac muscle with that of standard preparation of digitalis under the conditions of suitable method of bioassay. Standard Preparation : It consists of mixture of dried & powdered digitalis leaves Preaperation of Test extract : Extracting Digitalis powder with Dehydrated alcohol in a continuous extraction apparatus for 6 hours. The final extract should contain 10ml (5ml Water+ 5ml Alcohol) per 10gm of digitalis powder. 1Unit = 76mg

3. Assay Methods: Guinea pig method Cat method Pigeon method

4. Guinea pig method Test and standard extracts are diluted with normal saline in 1:80 ratio( i.e ; 1g extract in 80ml Saline). A guinea pig is anaesthetised with suitable anaesthetic . It is then dissected to cannulate its Jugular vein using venous cannula . A pin is insereted in to apex of the heart. The standard preparation is injected through the venous cannula until the heart stops. Amount of extract utilized to produce this effect is taken as Lethal dose. Another 19animals are used to determine mean Lethal dose.

5. Mean lethal dose of test sample is then determined in a similar manner using atleast 6 guinea pigs of same strain. The potency of unknown substance is calculated as ‘ Concentration = Mean lethal dose of standard X Concentration of of test sample Mean lethal dose of standard standard sample

6. Cat Method A cat is anaesthetised using Chloralose & its BP is noted. The standard preparation is slowly infused in to the animal until cardiac arrest occurs & the blood pressure falls to zero. The amount of extract required to produce this effect is taken as Threshold dose. The procedure is repeated using test extract & then the potency of test sample relative to standard is calculated.

7. Pigeon Method A Minimum of 6 disease free pigeons are used for testing each sample. They are fasted for 16-28hrs before the experiment. They are anaesthetized with anaesthetic ether to dissect one side if their wings & to cannulate their A lar vein through venous cannula . The standard preparation is injected through the cannula until the heart stops & emesis is produced.

8. The volume of fluid extract required to produce emesis is take as Lethal dose. The mean lethal dose of both standard & test preparation is determined & the potency of test sample relative to standard is calculated.

9. Bioassay of d- tubocurarine Rabbit Head-drop Method: Frog Rectus Abdominis muscle preparation

10. Rabbit Head-drop Method: Principle: d- tb is an Non-depolarizing neuromuscular blocking drug which mainly acts as an antagonist at cholinergic receptors of motor end plate. Hence, it blocks neuromuscular transmission &causes MOTOR PARALYSIS. It is injected into the marginal ear vein of a rabbit’s ear until the neck muscles are paralysed & rabbit drops its head . The total amount of test sample required to produce head drop is compared with that of the standard preparation.

11. Procedure: Healthy rabbits weighing 2kgs are selected . A minimum of 4 rabbits are used for testing each sample. Each rabbit is placed in a holder with its head protruding outside, such that the head is freely movable. Standard preparation of d- tb (0.012% w/v saline) is infused in to the marginal vein at a rate of 0.4ml/min until the head drop s seen. The mean dose of standard preparation that produces head drop is determined. Mean dose of test sample which produces head drop in rabbits is similarly determined & compared with that of stadard .

12. Precautions: Neostigmine methyl sulphate (0.05mg) & Atropine sulphate should be immediately injected in to the marginal vein to avoid Respiratory distress. Cross-over test [ Rabbits which receive std prpn on 1 st day should be treated with test sample on 2nd day & vice versa ] should be carried to minimize biological error.

13. Frog Rectus Abdominis muscle preparation Principle: d- tb is a neuromuscular blocking agent which antagonizes the actions of Ach by blocking nicotinic cholinergic receptors in the skeletal muscles. Hence, the actions of Ach are inhibited in the presence of d- tb . The CRC of Ach shifts to the right in the presence of d- tb .

14. Procedure: A frog is pithed & laid on its back on a dissecting board to which it is pinned . The skin covering the abdomen is removed to expose the rectus abdominis muscle. Rectus abdominis muscle is then cut, tied with a thread at its two ends & suspended in organ bath containing Ringer solution under a tension of 1g. The tissue is oxygeated to keep it alive & allowed to relax for 30-45 mins during which the tissue is washed with a fresh quantum of ringer solution atleast thrice.

15. Contractions due to Ach using atleast 4 increasing doses are recorded using Frontal writing lever. d- tb (1-2 u g/ml) is added to the reservoir containing ringer solution & allowed to act for 30mins. The CRC of Ach I the presence of d- tb is repeated. The % inhibition in the response of Ach due to d- tb is calculated & a log DRC for standard drug is plotted. The potency of the test sample is calculated from the standard curve.

16. Bioassay of Oxytocin Depression of BP in Chicken Contraction of Rat uterus Measurement of Milk-ejection pressure in a lactating rat

17. Principle: Potency of the test sample is compared with that of the standard preparation. Standard preparation : The standard preparation is the 4 th International standard for oxytocin estd I 1978, consisting of Freeze-dried synthetic oxytocin peptide with human albumin & citric acid or any other suitable preparation , the potency of which has been determined in relation to the International standard.

18. Procedure: Depression of BP in chicken: A young healthy adult cockerel weighing 1.2-2.3 kg is selected & anaesthetised with a suitable anaesthetic . The gluteus primus muscle of one thigh is cut & retracted to expose the popliteal artery & cural {Brachial}vein. The popliteal artery & cural vein are cannulated . The standard preparation is diluted with saline before use & then two doses of preparation are injected into the vein through the cannula at an interval of 3-10 mins .

19. The decrease in Bp is noted. The test sample is also diluted with saline to obtain response similar to that of standard & two test doses are selected such that the ratio between them is same as the ratio between two standard doses . The test doses are injected in to the cannulated vein & the decrease BP is recorded. A minimum of 6responses are measured for each sample & calculated using standard statistical methods.

20. Contraction of Rat uterus: Female rats weighing 120-200g are selected. About 18-24hrs before the assay,100micro gram of Oestradiol benzoate is injected i.m to attain oestrous / proestrous stage. The rat is sacrificed & one horn of uterus is isolated & suspended in an organ bath containing solution with the following composition : Components Composition (% w/v) Nacl 0.663 Kcl 0.045 Cacl2 0.007 NaHCo3 0.256 NaH2Po4 0.003 MgCl2 0.010 C6H12O6(Dextrose) 0.050 Na2HPo4 0.029

21. The bath is maintained at a temperature of 32 o C. The tissue is oxygenated to keep alive &allowed to relax for 30min under a tension of less than 2g. Two doses of standard preparation suitably diluted with the above solution are added at an interval of 3-5 mins . The contractions produced by the addition of two standard doses are recorded. The test sample is also suitably diluted with above solution & two test doses with the same interdose ratio are selected ,added,& then contractive doses are recorded. A minimum of 6 responses are measured for each sample & calculated using standard statistical methods.

22. Measurement of Milk-ejection pressure in lactating Rat: A lactating rat in 3-21 days after parturition & wighing about 300g are selected. It is seperated form the litter & 30-60mins later, it is anaesthetised with phenobarbitone sodium. It is then toed to the operating table maintained at 37oC by its hind legs leaving the front legs free. The trachea is cannulated with a short polyethylene tube with an internal diametre of 2.5mm to facilitate artificial respration . An external jugular/femoral vein is then cannulated with a polyethylene tube of internal diametre of 0.4mm.

23. The skin surrounding the inguinal & abdominal teats is shaved & tip of one of the teat is excised . A polyethylene tube of internal diametre0.3mm& external diametre 0.6mm is inserted into the primary teat duct which opens onto the cut surface & tied firmly in place with a ligature . This tube is connected to a suitable strain guage transducer. The whole system is the filled with 3.8% w/v sodium citrate or saline solution containing 50units of Heparin per ml to prevent milk clotting. The strain guage is clamped to apply a slight tension to the teat & to preserve its natural alignment.

24. The guage is connected to a potentio -metric recorder which gives a full scale deflection for an increase in milk ejection pressure of about 5.3kpa . Standard & test samples are being examined are prepared in saline solution. Two doses of standard preparation are selected such that an increase in milk-ejection pressure is about 1.35kpa for the lower dose & about 2.7kpa for the higher dose. The two doses are injected into the cannula & the milk ejection pressure are recorded. Two doses of test preparation with same inter-dose ratio are selected, injected in to the cannula & response is recorded. A minimum of 4responses of each preparation are measured & calculated by standard statistical methods.

25. TH9K UH!