Biological tests and Bioassay of Oxytocin

In this slide contains introduction, role, mechanism, and assay of oxytocin.
Presented by: P.PAVAN KALYAN (Department of pharmaceutical analysis ).
RIPER, anantapur

1. 1 BIOLOGICAL TESTS AND ASSAY OF OXYTOCIN A Seminar as a part of curricular requirement for I year M. Pharm I semester Presented by P.Pavan kalyan ( Reg. No . 20L81S0705 ) Under the guidance/Mentorship of shakir basha Associate Professor Dept. of Pharmaceutical Analysis

2. 2 Introduction to Oxytocin Role of Oxytocin Oxytocin - mechanismm of action Biological assay of Oxytocin Contents

3. 3 Introduction Oxy -Rapid Tocos –Labor Oxytocin synthesized in both sexes ( male and female ),well recognised physiological effects only in women. Pituitary gland consist posterior lobe which produce Oxytocin and diuretic hormone .

4. 4 It stimulates the contraction of the uterine smooth muscle and memory gland. Oestrogen progesterone and prolactin – responsible for production of milk by mammary gland but ejection require Oxytocin. It facilitates the contraction of uterus. Role of Oxytocin

5. 5 Neuropeptide made in hypothalamus that stimulates contractions , that expel the infant from uterus. Responsible for milk letdown and triggered by the nipple stimulation of suckling. Oxytocin called love and binding hormone. It has a very special affect on mothering . Psychologically , Oxytocin promotes a feeling of well being and tranquility. It enables the growing sense of love and attachment to the infant. The more the infant suck the more Oxytocin is produced. Oxytocin – Mechanism of action

6. 6 Principle potency is determined by comparing it’s activity - depression of BP - Contraction of uterus - Milk ejection of pressure - Vasopressor activity With standard preparation of Oxytocin. Standard Preparation It consisting free dried synthetic Oxytocin peptide with human albumin , citric acid ( 12.5 units ). Biological assay of Oxytocin

7. 7 Method – A . Depression of the BP in the chiken Test animals : cockerel ( young male chiken ) Weight : 1.2 – 2.3 kg Method – B By contraction of the rat uterus Test animals : female rat Weight : 120- 200 g

8. 8 Method – C . Milk ejection pressure in lactating rat Test animals : lactating rat ,3 – 21 days after parturition Weight : 300 g Method – D Vasopressor activity Test animals : male rat Weight : 300 g

9. 9 Depression of the B.P in chiken Test animals : cockerel (young male chiken) 1.2- 2.3 kg Anesthetized cock- prolonged and remove popliteral artary and crural vein. Cannulate the popliteral artery and record B.P response . Cannulate the crural vein or branchial vein. Prepare standard solution with saline injection 0.1 -0.5 ml. Inject 2 doses of standard solution in to cannulate vein and record B.P response. Dose should be cause decrease in B.P. Method – A

10. 10 Interval between 2 injections, between 3-10 min depends on the rate at which B.P return normal. Dil.test preparation with saline so as to get same response as standard. The ratio between standard and test should be equal. If animal rapidly becomes insensitive to repeated injection the solution another must used . Measure all responses and calculated results of the assay by standard statistical methods.

11. 11 Contraction of the rat uterus Test animals : female rat ,120 – 200 g Inject 100 micro gram of oestradiol benzoate IM in to female rat before the assay. Kill rat and suspend one horn of uterus in organ bath containing a solution of following Nacl, Kcl ,Cacl 2,Na2HCo3 ,NaHPo4, NaH2Po4 ,Mgcl2 , dextrose. Temp at 32’C. Bath liquid required dose between 10-50 units / ml . Oxygenate solution with mix of 95% of O2 and 5% of CO2 record contraction of muscle. Method – B

12. 12 Record contraction produces by addition of two dose of std.preparation. Dose should be added at regular interval (3-5 minutes). Similarly record the contraction of test preparation as a standard. Ratio between two doses of test and two doses of standard should be equal.this ratio kept constant through out the assay. Measure all response and calculate results of assay by standard statistical methods.

13. 13 Milk Ejection pressure in lactating rat test animals : lactating rat,3-21 day after parturition,300 g. Separate from litter and 30-60 minutes later anesthetized (IP pentobarbitone Na ). Tie rat to an operation table,at 37 degrees ,by its hind legs leaving front legs free. Cannulate trachea with a short PE tube of i.d 2.5 mm in such a manner so as to ensure a free airway.apply artificial respiration only if necessary. Shave the skin surrounding the inguinal and abdominal teats and excise the tip of one teat,preferally the lower inguinal teat. Method – C

14. 14 Insert a P.E tube i.d 0.3 mm and e.d 0.6 mm,to a deapth sufficient to obtain appropriate measurement of pressure ( 3-10mm depth),in to the primary teat duct. Connect this cannula with a suitable strain gauge transducer. Prepare 3.8 % w/v of Na citrate/ saline , contain 50 units of heparin Na/ml to prevent the clotting of milk. Then inject 0.005-0.2 ml of this solution in to teat duct through transducer. Then slight tension is applied to the teat and connect the gauge to a potentiometric recorder for an increase in milk ejection pressure of 5.3 kpa.

15. 15 Prepare a solution of standard and test preparation in saline solution, so that the volume to be injected is between 0.1-0.4 ml . Choose two doses of standard preparation such that the increase in the milk ejection pressure is about 1.35 kpa for lower dose and about 2.7 kpa for high dose. Choose two doses of the test preparation with matching effects of doses of the standard preparation as closely as possible. Inject four doses (2 doses of standard and 2 doses of test ) at intervals of 3-5 minutes.

16. 16 2 doses of standard and 2 doses of test , should be given according to randamized block or a Latin square design and at least four responses to each recorded. Measure all responses and calculate results of the assay by standard statistical methods.

17. 17 Vasopressor activity Test animals : male rat ,300 g Vasopresser activity of test with standard preparation of arginine vasopresin (Freeze dried syn.arginine vasopressin peptide acetate with human albumin and citric acid). Inject slowly in to the tail vein of male albino rat weight 300 g – solution of a suitable adrenoreceptors blocking agent,(10 ml/kg body weight ), solution prepared by 5 mg of phenoxy benzamine HCl in 0.1 ml of ethanol (95% ), adding 0.05ml of 1 M HCl and dil to 5 ml with saline. After 18 hr ,anaesthetize rat- that will maintain prolonged and uniform B.P. After 45-60 min ,tie the rat on its back to the operating table by its hind legs . Method – D

18. 18 Cannulate trachea with short P.E of E.D 2.5 mm and dissect carotid artery ready for cannulation. Then cannulate the femoral vein close to the inguinal ligament. Dissect femoral vein towards inguinal ligament from corresponding artery. Tie a short P.E cannula of E.D about 1 mm in to femoral vein by two ligatures. At this stage inject through venous cannula 200 units of heparin, dissolved in saline for 100 gm of body weight. Then tie in a carotid cannula of E.D about 1 mm and connect to saline contain heparin with a Hg manometer of I.D about 2- 3 mm. No artificial respiration is necessary.

19. 19 Dil.extract of standard and test preparation with saline,that volume to be injected is between 0.1 to 0.5 ml. Choose 2 doses of the standard preparation such that elivation of the B.P is about 4 kpa for lower dose And 7 kpa for higher dose. Choose 2 doses of test preparation with same inter dose ratio matching effects of dose of standard preparation. Inject doses at intervals of 10 – 15 minutes. 2 doses of std and 2 doses of test Preparation should given in randamized block / latin square design and 4- 5 responses to each record. Measure all responses and calculate results of the assay by standard statistical methods.

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